Adult-derived human liver stem/progenitor cells (ADHLSC) are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC) derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity.ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays.Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10.Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles. The liver is composed of parenchymal and non-parenchymal cell populations. Complex and well-organized interactions between such cell types allow a perfect coordination of the liver functions for preservation of the systemic homeostasis. Indeed, the liver is concomitantly managing numerous important functions such as metabolism, protein synthesis and detoxification. Hepatocytes are the main parenchymal cell type and represent the most important functional one. Liver non-parenchymal cells include epithelial bile duct cells, non-epithelial Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells (HSCs) Spindle shaped HSCs are located in the space of Disse between hepatocytes and sinusoidal endothelial cells Our group previously obtained stem/progenitor cells from healthy adult human liver (ADHLSC). These expandable cells present a hepato-mesenchymal phenotype and have the potential to differentiate into hepatocyte-like cells both in vitro and in vivo In the current study, we carried out an extensive comparison between HSCs and ADHLSCs in order to assess the unique identity of ADHLSCs and to identify tools that can be used to differentiate both populations. To this end, we compared these mesenchymal cells after isolation from the same liver by following their phenotype, genotype and behavior in vitro from passage 5 until passage 11.We report several characteristics similar to both cell types but shed light on significant gene expression profile and functional differences. This study confirms the unique characteristics of ADHLSCs and demonstrates their secretion potential of cytokines that could be of therapeutic and immuno-modulatory importance.The protocol and experiments were approved by the ethical committees of the St-Luc Hospital and faculty of Medicine of Université Catholique de Louvain. An agreement from the Belgian Ministry of Health was obtained for the Hepatocytes and Hepatic Stem Cells Bank. A written and signed informed consent has been obtained for each human liver used in the current study.Four donors were used in the current study . ADHLSC 2. The viability of recovered cells was evaluated using trypan blue exclusion assay.Both cell types were cultured using DMEM containing 4.5 g/L glucose (Invitrogen) supplemented with 10% Fetal Calf Serum (PAA) and 1% Penicillin/Streptomycin (Invitrogen), at 37°C in a fully humidified atmosphere (5% CO2). When reaching 80% confluence, cells were lifted with 0.05% trypsin-EDTA (Invitrogen) and replated at a density of 5000 cells/cmCells were fixed using paraformaldehyde 3.5%, for 15 min at room temperature. Endogenous peroxidase was eliminated using hydrogen peroxide 3.3% for 3 minutes. All steps were performed at room temperature. Cells were permeabilized using D-PBS containing 1% Triton X-100 (Sigma) for 10 minutes. Non-specific immuno-staining was prevented by 1 h incubation in D-PBS containing 1% Bovine Serum Albumin (Sigma). Thereafter, cells were incubated with primary antibody for 1 h . After wExtracted Protein concentrations were determined using the BCA protein assay kit (Thermo Scientific). Fifty micrograms of protein samples were boiled for 10 min at 100°C and prepared for loading by adding dithiotreitol and bromophenol blue. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes. Blots were blocked with 5% milk powder in TBS with 0,2% tween (TBS-T). Following a washing step with TBS-T, membranes were incubated overnight with following primary antibodies and dilutions: CK18 (1∶350 [DAKO]), CK19 (1∶350 [DAKO]). Antibodies were diluted in blocking buffer. After additional TBS-T washes, membranes were incubated with secondary antibody for 1 h (dilution: 1/20.000). The antigens were detected by enhanced chemiluminescence using ECL substrate.RNA was quantified with a Nanodrop1000 (Thermo Scientific) and RNA integrity evaluated with a Bioanalyzer 2100 (Agilent Technologies). 500 pg–50 ng total RNA were amplified with an Ovation Pico SL V2 Kit (NuGENE) and labelled with an Encore Biotin Module (NuGENE) following standard procedures. HG-U219 plates were hybridized with an automated array processing GeneTitan (Affymetrix).Affymetrix gene expression data were normalized using the robust multi-array algorithm For the detection of differentially expressed genes, a linear model was fitted to the data and empirical Bayes moderated statistics were calculated using the limma package from Bioconductor http://www.geneontology.org). Only genes that could be associated with a unique Entrez Gene ID were used. Among those, only the genes representing a fold change of 1.5 or greater and a moderated p-value<0,05 were selected. The hypergeometric distribution was used to evaluate the probability of randomly observing the enrichment for each GO term Functional analysis of gene expression data was conducted using the R/Bioconductor package GOstats and the GO database  was employed to identify biological pathways significantly associated with the ADHLSC. In comparison to other strategies for analysis of molecular profiling data that focus on high scoring individual genes, GSEA does not employ a significance threshold and evaluates microarray data at the level of gene sets defined based on prior biological knowledge. This approach has been reported to yield robust results even when dealing with heterogeneous samples with subtle sample class differences. For the current analysis, gene sets were extracted from the full Molecular Signature Database (MSigDB v.4-0). Additionally the subset of canonical pathway is analyzed alone. Analyses were based on a Signal2noise metric and a weighted scoring scheme with 1000 permutations on gene sets. Only gene sets with more than 15 genes were included in the analysis.Total RNA was extracted from 1.5 million cells for each cell population using Tripure isolation reagent (Roche). cDNA was generated from 1 µg of RNA using the Thermoscript™ RT kit (Invitrogen) according to manufacturer's instructions. qPCR was carried out in duplicate using TaqMan Gene expression Master Mix (Applied Biosystems) and pre-designed TaqMan probes and primers obtained from Applied Biosystems. The amplification was performed using the StepOnePlus Real-time PCR machine. The housekeeping gene cyclophilin A (PPIA) was used as a reference gene for normalization and water was used as a negative control.5 cells/ml. For intracellular immunostaining, cell permeabilization was performed with cytofix/cytoperm for 20 minutes at 4°C (BD Pharmingen). Cells were then washed and incubated for 30 minutes at 4°C with the antibodies . Hepatocyte Growth Factor (HGF) and Transforming Growth Factor beta 1 (TGFβ1) levels in the culture supernatants were assayed by using Quantikine Elisa Kits from R&D Systems. The experiments were performed according to the manufacturer's instructions. For TGFβ1 Elisa, samples were activated by acidification followed by neutralization in order to activate latent TGFβ1 to immune-reactive TGFβ1 detectable by the Quantikine TGFβ1 immunoassay. The measurement of the absorbance at 450 nm was done with a Victor X4 plate Reader (PerkinElmer). For HGF and TGFβ1 kits, a reading at 570 nm was subtracted to the 450 nm reading to correct the optical imperfections of the plates.We also used a 27plex kit  and the Luminex technology  to investigate the secretome of both liver cell types. The principle of the technique is based on color-coded beads and enables to detect up to 100 cytokines simultaneously. The primary antibody directed against the target protein is conjugated with the dyed beads. After several washes to remove unbounded proteins, a secondary biotinylated antibody is added to the reaction. Streptavidin-phycoerythrin (Streptavidin-PE) is then added to bind the biotinylated antibody. By measuring the relative fluorescence intensity, the antigen-antibody reaction can be measured. The assays were performed following the manufacturer's instructions.Briefly, after the pre-wetting of the plate, 50 µl of the beads were added in each well and washed twice. 50 µl of the samples (serum free culture supernatants recovered after 24 hours of culture) were added to the plate. The plate was shaken during 30 seconds and then incubated for 45 minutes on a plate shaker at 120 rpm at room temperature. The plate was washed three times with the Bio-Plex wash buffer and 25 µl of the detection antibody was added in each well and incubated for 30 minutes on a plate shaker at 120 rpm. The plate was then washed three times with the Bio-Plex wash buffer and 50 µl of the Streptavidin-PE solution was added in each well. The plate was shaken during 30 seconds and incubated for 10 minutes on a plate shaker at 120 rpm. Finally, after three washes of the plate with the Bio-Plex wash buffer, the beads were resuspended with 125 µl of Bio-Plex Assay Buffer. The plate was read by the Luminex machine and the data were analyzed using Bio-Plex Manager 6.0.t test for two groups' comparison. Differences were considered significant when p values *p<0.05, **p<0.01, ***p<0.001.Results, others than those of microarray studies, are expressed as mean ± standard error of the mean (SEM). Statistical differences were determined by Student's For each liver donor, HSC and ADHLSC were cultivated under the same culture conditions and concomitantly followed. The fibroblastic morphology displayed by both cell types remained stable over the different studied passages . The popWe investigated the mesenchymal phenotype of both cell types (n = 4) by exploring the expression of several specific appropriate markers using flow cytometry. Both cell types were immuno-positive for most of the membrane markers widely used to characterize mesenchymal stem cells. This was the case for CD73, CD90, CD29, and CD44 for which expression levels remained stable for the analyzed passages . No signThis first comparative characterization only confirmed the mesenchymal phenotype of ADHLSCs and HSCs but did not allow us to discriminate both cell populations. We thereafter studied the gene expression profile of ADHLSCs and HSCs using Affymetrix HG-U219 gene chips expression. The analysis was performed on 7 different samples for each cell population (4 samples from the four donors at Passage 5+3 samples from only 3 donors at Passage 7). These experiments allowed us to point out significant signature differences  after screening of 13925 genes . We notiWhen comparing MMP expression between the two cell types, we noticed an elevated expression of MMP2, MMP3 and MMP14 in ADHLSCs and an increased expression of MMP16 in HSCs .In order to obtain a functional annotation of our gene expression results, we used the Gene Set Enrichment Analysis (GSEA) method. We have used GSEA to identify those most relevant “canonical” pathways and Gene ontology categories enriched in ADHLSC. Results are included in Interestingly, HSCs exhibited significantly higher levels of neuronal markers such as NGF, neurotrophin 3, NDNF, and NEDD 2 & 9 than ADHLSCs . Such siHSC but not ADHLSC expressed hepatic cytokeratins like cytokeratin 18 and cytokeratin 19 at the protein level, as demonstrated using western blotting . These oAll together, these findings clearly show that cultured HSCs and ADHLSCs are two different cell populations with distinct gene expression profiles.After phenotypic and genotypic characterization, we carried out a functional analysis of both cell populations by assessing their secretome profile in the corresponding conditioned culture medium of cells originated from 3 different donors at passages 5 &7. This was performed on supernatants collected 24 hours after incubation with serum free medium. As collagen is known to be secreted by activated HSCs, we evaluated its secretion by measuring the pro-collagen type-I C-Peptide in the culture supernatants. No significant difference was observed between HSCs and ADHLSCs . We therHGF is a hepatocyte mitogen, which has several crucial physiological functions including organ protection and regeneration. Following liver injury, HGF is known to be secreted by distant organs such as spleen, lungs and kidneys as well as by sinusoidal cells such as Kupffer cells and HSCs. Moreover, HGF has anti-inflammatory properties. We observed a significant difference in HGF production between HSCs and ADHLSCs. The liver stem/progenitor cells seemed to secrete HGF about three times more than HSCs .In the second part of this functional analysis, we focused on cytokines implicated in the inflammatory response. Indeed, HSC are known to secrete several pro-inflammatory cytokines while ADHLSC seem to exhibit immuno-modulatory properties. Twenty-seven cytokines were analyzed, using a multiplex technology. The cytokines were classified as growth factors, chemokines, pro-inflammatory cytokines, anti-inflammatory cytokines and those with dual roles. Among the growth factors analyzed, the main differences were noticed for VEGF and PDGFbb for which significant higher levels were detected in ADHLSCs . The incThese findings confirm the singularity of both cell populations and support the expression profile differences demonstrated using gene expression analyses.The aim of the current study was to establish an in-depth comparison of ADHLSCs and HSCs, cells of human liver origin exhibiting similar fibroblastic morphology and both reported as stem/progenitor cells. By comparing these cell populations successfully isolated from the same donors, we i) demonstrate that ADHLSC and HSC are two distinct liver cell types displaying significant differences in gene expression profiles as well as functional features, and ii) confirm the stem/progenitor properties of ADHLSC.Using flow cytometry and immuno-cytochemistry, we confirmed the mesenchymal phenotype of both ADHLSCs and HSCs. Both cell populations were also negative for hematopoietic and epithelial protein markers rendering the demonstration of their singularity difficult.To assess a thorough analysis of both cell types, we used microarray analysis and screened more than 10.000 genes on the 4 different samples of ADHLSC and HSC. Top 100 differentially expressed genes were identified. The main differences between ADHLSCs and HSCs are seen at the level of the chemokines expressed. Such chemotactic molecules which regulate the infiltration of immune cells to sites of inflammatory injuries, are initially all expressed in the liver. With respect to the first family, CCL7 seems to be the highly expressed one in ADHLSC in accordance with its documented role in development as demonstrated in neuronal differentiation The second CXC chemokine family displays well-documented neutrophil chemotactic, angiogenic, and mitogenic properties HGF, a pleitropic cytokine of mesenchymal origin, participates in the regulation of proliferation, differentiation and chemotactic migration of mesenchymal stem cells In our study, we did detect protein expression of cytokeratins 18 & 19 in only HSC as demonstrated using western blotting which is in accordance with gene expression profiling data. The data are supported by the increased expression of desmoplakin, which anchors these intermediate filaments to desmosomes After its interaction with dystroglycan, dystrophin forms a glycoprotein transmembrane complex required for spatial organization of laminin on the cell surface and for basement membrane assembly. Such complex is up regulated during spontaneous activation of HSC in culture MMP3 (stremolysin), which degrades collagen types II, III, IV, IX, and X, proteoglycans, fibronectin, laminin and elastin, was the highly up-regulated MMP in ADHLSC compared to others MMPs like MMP2 and MMP14. In contrast, MMP3 is down-regulated in other MSCs like adipose-tissue MSC, as compared to fibroblasts The functional analysis of ADHLSCs and HSCs allowed us to support the hypothesis that both cell populations also behave differentially in basal conditions. Obviously, the secretion profile of each cell type is different and supported the microarray data, emphasizing that ADHLSCs secrete cytokines of therapeutic and immuno-modulatory importance.In the liver, TGF-β is initially produced in non-parenchymal liver cells, whereas absent in fully differentiated epithelial cells Collagen is one of the predominant structural proteins of the liver extracellular matrix and is involved in stem cell proliferation and differentiation These secretion profile analyses also suggest that ADHLSC secreted cytokines and growth factors may be of therapeutic and immuno-modulatory importance in the context of their transplantation development.VEGF has been shown to promote liver fibrosis by stimulating the activation, proliferation, and chemoattraction of HSCs In conclusion, our study demonstrates that ADHLSCs and HSCs represent two distinct hepatic cell populations. Even if they share numerous similar characteristics, we highlighted significant differences supporting their singularity mainly at the gene expression and secretion profile levels. This study also provides additional features supporting the potential therapeutic development of ADHLSC for liver cell-based therapy.


Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes.Drosophila retinal architecture is laid down between 24–48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles we showed that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore we demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions.By using quantitative real time PCR to examine rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level.These findings suggest a general mechanism by which  Differential adhesion plays a central role in morphogenesis, allowing cells to sort themselves out from initially unpatterned populations as they become committed to specific fates Irre Recognition Module or IRM roughest (rst) and kin-of-irre (kirre) paralogue genes of Drosophila C. elegans SYG-1 C. elegans SYG-2 rst or kirre show only very subtle muscle defects, whereas embryos deficient for both genes are also almost completely deficient in myoblast fusion hbs and snsAdhesive properties of cells are critically dependent on a highly diverse group of cell surface adhesion molecules, or CAMs, that can specifically bind to similar or different CAMs present either in other cells or in the extracellular matrix. At any given time each cell expresses a characteristic subset of CAMs that define both the specificity and mechanical strength of these interactions. Additionally a significant number of CAM families are composed of transmembrane glycoproteins whose intracellular domains also interact with the cell cytoskeleton, thus being able to mediate complex changes in cell shape upon binding or dissociating from their ligands. Among these, an important, albeit comparatively little studied group is the D. melanogaster the adult eye is made of about 750 repetitive hexagonal units called ommatidia, each containing eight photoreceptors, four lens-secreting cone cells, three morphologically and functionally distinct groups of pigment cells , and three mechanosensory bristles. Except for the latter, the specification of these cell types from a previously unpatterned epithelial monolayer - the eye imaginal disc - and their assembly into ommatidial units proceeds sequentially, starting around mid-third larval instar, and can be visualized as a wave of cell differentiation, the morphogenetic furrow, that sweeps the disc from posterior-to-anterior. This process concludes during the first half of pupal development, when some of the still unspecified interommatidial cells (IOCs) are recruited to become pc2 and pc3 cells while the remaining ones undergo apoptosis. The choice between life and death in this specific context seems to rely on the relative position of IOCs, which sort themselves so as to maximize their apical contacts with the border of the already specified pc1 cells . Nevertheless, some rst mutants that appear to almost completely lack Rst protein have compound eyes that are either normal or show only very mild roughness rst activity .The final steps of Drosophila compound eye morphogenesis constitute an excellent model to investigate the functional interplay between IRM molecules in the context of cell fate specification. In Drst, a relatively unstable, semi-dominant regulatory mutation in the rst gene, showed that the right timing of Rst redistribution in IOCs appears to be critical not only for the correct elimination of surplus IOCs but also for the process of pc2 and pc3 differentiation, and strongly indicated that precise temporal regulation of rst transcription might be one of the key requirements for its correct functioning in the final steps of eye morphogenesis rst function by analysing its transcriptional dynamics in the developing retina by quantitative real-time PCR, both in wild type and mutant alleles, during IOC sorting and in the stages immediately preceding and following it. Our results support the model previously put forward by Araujo et al locus transcriptional rate. Additionally we show that rst paralogue kirre appears to function redundantly with it in this process, similarly to what has been previously shown for both genes in myoblast fusion during embryogenesis, thus providing an explanation for the weak phenotype often observed in loss-of-expression rst alleles. Finally we provide evidence that both genes are co-regulated, having complementary transcriptional dynamics and that RNAi mediated silencing of rst leads to a dramatic increase of kirre mRNA levels around the time of IOC redistribution. Taken together, these results refine and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of compound eye morphogenesis in insects are controlled at the transcriptional level.Previous work from our group that resulted in the phenotypic and molecular characterization of Drst flies differ from other previously described rst mutant alleles by having “glassy”- looking compound eyes with a brownish-red color . Taken together these data lead to the intriguing implication that, at least in some circumstances, rst expression might be dispensable for correct ommatidial patterning.The analyses of the two owing it . RWT8, fDrst revertants having normal compound eyes despite lacking rst expression in the pupal retina could be easily resolved by assuming that at least some rst functions during eye development could be equally performed by its paralogue kirre, similarly to what has been demonstrated for myoblast fusion in the embryo kirre specific primers. A comparison between kirre mRNA temporal profiles of wild type and Drst is shown in rst and remained relatively constant throughout the time period analysed. Nevertheless significant departures from the basal level did occur, with the lowest levels coinciding with the peak of rst mRNA expression, at about 24% APF and highest around 34% APF when rst mRNA concentration approached again basal levels. The same qualitative profile was displayed by Drst retinae although here also, peak levels were higher than in wild type. In contrast, quantification of kirre mRNA in the retinae of the two Drst revertants w deletion which removes both rst and kirre genes + kirre+rst/− kirre−rst are viable and have normal compound eyes, as do the − kirre+rst/− kirre+rst homozygote RTW6 flies. However, trans-heterozygotes 67k30Df(1)w/RTW6, genotipically − kirre−rst/− kirre+rst display mild but characteristic ommatidial defects present in other rst mutants, including irregularly spaced bristles and fused ommatidia (inset). The relatively weak phenotype observed could be a consequence of the high levels of kirre mRNA produced in the retina by the remaining genomic copy of kirre, but could also be an indication that the eye phenotype rescue seen in RTW6 revertants was only in part due to kirre redundant function. To test this possibility we used an UAS-kirre RNAi construct driven by GMR Gal4 to knock down kirre function in RTW6 retinae. As shown in kirre expression alone at the time of IOC sorting can bypass the requirement for rst function in this process and provide strong support for the idea that both genes could act redundantly also during eye development.The drastic quantitative and qualitative changes in kirre mRNA levels seen in Drst revertants in the absence of normal rst mRNA levels during the 19–34% APF time window, together with their almost perfectly complementary timing of expression, leads naturally to the suggestion that transcription of both genes might be co-regulated and, somehow sensitive to each other's RNA or protein levels. Moreover rst and kirre both map next to each other in the genome and are transcribed in opposite directions rst regulatory region, it was conceivable that they could reflect a “lucky accident” caused by a favorable reorganization of regulatory sequences in the DNA of RTW6 and RTW8 rather than a true physiological response. We therefore asked whether by deliberately lowering rst mRNA peak levels in wild type retinae we could bring about a corresponding increase in kirre mRNA. To this end we quantified the levels of rst and kirre mRNA at 24% APF in wild-type retinas that expressed either one of two independently generated rst RNAi constructs under the control of a GMR-Gal4 driver  suggesting that at least in some developmental contexts rst and kirre might not be fully redundant.Perhaps the most interesting finding presented here is the evidence for compensatory co-regulation of rst and kirre mRNA concentrations and its asymmetrical nature. A possible, although unlikely explanation for this asymmetry could reside in the much higher absolute levels of rst mRNA present during the temporal period examined, making small changes in relative concentration easier to detect with confidence. Also we have neither investigated the effect of overexpression of one gene on the concentration of the other, nor whether similar coordination in mRNA expression is taking places in other developmental contexts where complementary or redundant functioning of rst expression levels and adjust those of kirre accordingly. Here again mRNA, rather than protein concentration, may play the main role, since in CTrst low levels of rst activity caused by the truncated intracellular domain and destabilization of the protein from the membrane leads only to a minimal, if any, increase of kirre mRNA levels at 24% APF. Also it is conceivable that rather than upregulating kirre transcription, a decrease in degradation could be taking place, leading to an accumulation of kirre mRNA molecules. Experiments designed to further test this possibility as well as to map rst mRNA sequences that might be relevant for this putative regulatory feedback loop between rst and kirre are currently underway. Whatever the case, these findings suggest a general mechanism by which the expression both genes could be fine tuned to optimize their redundant roles during development.Finally, an intriguing aspect of the compensatory response reported here is that it implies some kind of sensor mechanism capable to post-transcriptionally read UAS-rstRNAi27223, UAS-rstRNAi951, UAS-kirreRNAi3111 and UAS-kirreRNAi27277 were obtained from the Vienna Drosophila RNAi stock center Drst revertant stocks RTW6/FM7i and RTW8 were isolated in our laboratory as spontaneous reversions to wild-type eye phenotype in a homozygous Drst stock on a Canton S background that additionally were Drst dominant suppressors. Genomic organization of RTW6 in the region where the Drst rearrangement was mapped RTW8 appears to have resulted from a 1 kb internal deletion in the rearranged area. Both mutants are homozygous viable but RWT6/RWT6 females are sterile. All other wild type and mutant stocks used in this study were previously described Df(1)67k30/RTW6 flies were generated by selecting the non-Bar female progeny from a cross between RTW6/Y males and Df(1)67k30/FM7 females. Flies specifically expressing rst or kirre RNAi in pupal retinae resulted from the progeny of crosses between UAS-RNAi stocks described above and GMR-Gal 4 flies RTW6/Y; GMR-Gal4/UAS-kirre-RNA3111 were obtained by first constructing RTW6/FM7i; CyO/UAS-kirre-RNA3111 females, crossing them to GMR-Gal4 homozygote males carrying the transgene in the second chromosome and selecting for non-Bar, non Curly males. Flies were examined under a Leica MZFLIII dissecting stereomicroscope equipped with Leica DFC 500 digital camera and acquired images were processed using a Leica Application Suite V3.1.0 software.Fly manipulations were performed in vials containing standard Drosophila food at 25°C in a temperature-controlled room. RNAi transgenic stocks lmmunohistochemistry of staged pupal retinae with monoclonal antibody 24A5 5′– TGCCACCGAGGATCGCAAAG; QPCR-RST-Reverse CG4125∶4 Type exon 5′– GCAGCGGTATCACCGTGTAC; QPCR-KIRRE-Forward CG3653∶6 5′- CGGTGCACAAAATGCAGTTAC and QPCR-KIRRE-Reverse CG3653∶6/7 5′- GCTAACTCAGGAAGCGAAAT. All reactions were run at 95°C for 10 minutes, then 40–45 cycles of 95°C for 15 seconds and 60°C for 1 minute. RpL32A was used as reference and amplified using primers QPCR-RPL32A-Forward CG7939 5′– TCGACAACAGAGTGCGTCGC and QPCR-RPL32A-Reverse CG7939 5′– CTTGAATCCGGTGGGCAGCA. To check amplification efficiency, calibration curves for both target and reference genes were performed. Reactions without template were run in parallel for all plates as internal controls. A melting curve analysis was made after each run to confirm the specificity of the amplification. The ΔΔCq method rst expression between wild type and mutants. rst transcript levels were normalized with RpL32A transcript levels in the same cDNA templates and used as indicators of the expression levels. Data points represent the average of independent biological samples  with error bars corresponding to standard deviations.The two retinae from a single staged pupa were dissected in PBS and while one retina was processed for immunohistochemistry with anti-Rst as a control, the other was immediately transferred into Trizol reagent (Invitrogen) for RNA extraction according to the manufacturer's specification. Genomic DNA was removed by treatment with DNase I (Promega). Concentration and purity of total RNA samples were determined with NanoDrop spectrophotometer . Only Samples with optical density absorption ratio A260/A280 between 1.8 and 2.2 were taken for cDNA synthesis. The High-Capacity Reverse Transcription Kit (Applied Biosystems) was used for the reverse-transcription reaction with the concentration of 200–400 ng RNA sample in accordance with the manufacturer's protocol. RT-qPCR reactions were performed mostly in three biological and technical replicates in a 96-well plate (Applied Biosystems) using the Gene Amp® 7500 Sequence Detection System (PE Applied Biosystems) with Sybr Green Rox Plus (LGC Biotechnology) and Sybr Green PCR Master Mix (Applied Biosystems), for the experiments involving RNAi silencing. The primers sequences were: QPCR-RST-Forward CG4125∶3 Type exon


In Duchenne muscular dystrophy (DMD), loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology.mdx) mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group): (1) vehicle control; (2) 5 mg/kg/day LANZO; (3) 5 mg/kg/day prednisolone; and (4) combined treatment of 5 mg/kg/day prednisolone (PRED) and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan) and functional outcomes (grip strength and Rotarod) were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions.We designed a preclinical trial to investigate the effects of lansoprazole (LANZO) administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx mice. Our findings warrant future investigation of the clinical efficacy of LANZO and prednisolone combined treatment regimens in dystrophic pathology.Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in  Dystrophin and other proteins of the dystrophin-associated complex serve as scaffolding proteins that regulate the association of proteins, such as ion channels, with the sarcolemma. Loss of dystrophin is thought to destabilize the myofiber membrane, in part, by altering the flux of ions and other proteins through the sarcolemma mdx mice The progression of dystrophic pathology occurs as multiple mechanical and inflammatory insults contribute to chronic degeneration-regeneration remodeling cycles. Therapies aimed at reducing muscle inflammation, i.e. glucocorticoids, remain the standard-of-care for dystrophic pathology. Despite the acknowledged efficacy and widespread use of glucocorticoids, they do not fully ameliorate dystrophic pathology. Diets high in NaCl have been shown to attenuate myofiber necrosis and serum creatine kinase levels in +/K+-ATPase) pumps located on the apical surface of gastric parietal cells. LANZO H+/K+-ATPase binding inhibits parietal cell secretion of hydrogen ions into the gastric cavity. LANZO is widely used in the treatment of gastroesophageal reflux disease (GERD) and peptic ulcers. In addition to reducing gastric acidity, LANZO has been observed to attenuate inflammation in the gastric mucosa mdx mice and proinflammatory macrophage accumulation has been shown to lyse dystrophic fibers in an NO-dependent manner mdx mice. We hypothesized that LANZO administration would attenuate the dystrophic skeletal muscle phenotype via altering ion influx and attenuating proinflammatory and apoptotic signaling cascades underlying the dystrophic pathology.Lansoprazole, 2-[(4-trifluoroethoxy-3-methyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole, is a 369.36 kDa substituted benzimidazole that acts as a gastric proton pump inhibitor (PPI). Lansoprazole (LANZO) reduces stomach acidity by binding to hydrogen-potassium adenosine triphosphatase  8–10 week-old female mice were purchased from Jackson Laboratories . All mice were acclimated for 7 days prior to study initiation and individually housed in a vented cage system with 12 h alternating light-dark cycles. Mice received standard mouse chow and purified water ad libitum.This protocol was approved by the Children’s National Medical Center Institutional Animal Care and Use Committee (IACUC) guidelines (Protocol # 1206) and thereby all animals were treated according to local IACUC standards. C57BL/10ScSn-Dmdmdx/J  vehicle control group receiving saline solution ; (b) experimental group treated with 5 mg/kg/day LANZO; (c) experimental group treated with 5 mg/kg/day prednisolone; and (d) combined treatment group receiving 5 mg/kg/day prednisolone and 5 mg/kg/day LANZO. Saline and LANZO were administered via oral gavage, and prednisolone was administered via oral drops. Each of the treatment regimens was administered 5 days per week (Monday-Friday) for 3 months. All functional, behavioral and histological assessments were acquired and analyzed in a blinded fashion.mdx mice based on previous studies demonstrating an exacerbation of the dystrophic phenotype in mdx mice with treadmill exercise mdx mice (http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.001.pdf). All exercise was performed between 0800 h and 1200 h and completed 1–2 days prior to functional testing.Treadmill exercise was performed on all mice before and during the 3-month treatment regimen to unmask the mild dystrophic phenotype of Rotarod testing was performed according to methods we have described previously Forelimb and hindlimb grip strength was assessed at the beginning and end of the trial in order to evaluate changes in motor strength in response to the different treatment regimens. Forelimb and hindlimb grip strength measurements were performed using a Grip Strength Meter . Grip strength was quantified by averaging five successful hindlimb and forelimb strength measurements each recorded within a 2 min time interval in order to calculate absolute grip strength. All strength measures were then normalized to individual animal body mass to calculate normalized grip strength, expressed in kgf/kg, as described previously Open field behavioral activity was measured using an open field Digiscan apparatus  in order to assess locomotor activity as described previously At the end of the study, all mice were euthanized via carbon dioxide exposure, and organ and tissue samples were collected for histological evaluation. After weighing, a portion of each of the dissected tissues  was kept in formalin for H&E staining. The remaining portion of each tissue was embedded in OCT compound and frozen in isopentane chilled in liquid nitrogen. Sections were imaged using a Nikon E800 microscope and taken at a magnification of 20x. Five non-overlapping field images representative of the overall tissue histology were captured using digital imaging computer software . All of the digital images were uploaded into Image J Software  for analysis using a cell counter software plug-in. The number of inflammatory foci and fibrosis were assessed in the diaphragm between treatment groups. Diaphragm fibrosis was assessed by use of Picro sirius red staining with a Weigert's haematoxylin nuclear counter stain on diaphragm cross-sections. In gastrocnemius cross-sections the total number of degenerating, regenerating and inflammatory foci per field were quantified in order to assess differences between treatment groups. The average number of fibers sampled in the 5 non-overlapping cross-sections was 170, with a range of 88–300 fibers per field. Fibers displaying a loss of striations/homogenous appearance of fiber contents were counted as degenerating fibers. Regenerating fibers were counted as fibers with basophilic cytoplasm, large peripheral, or central nuclei with prominent nucleoli. All cell counts per image field (degenerating/regenerating fibers and inflammatory foci) were assessed in a blinded manner as described previously Blood for creatine kinase (CK) assay was collected by cardiac puncture into preservative-free Eppendorf tubes immediately after animal sacrifice. Blood serum was isolated by centrifugation for CK activity determination by standard enzymatic assay preformed according to the manufacturer’s instructions . Enzymatic activity was calculated by measuring light absorption on a spectrometer set at 340 nm every minute for a total of 2 min at 37°C. All serum samples were run in duplicate for CK quantification.In vitro force measurements were performed according to methods we have described previously 2–5% CO2. Muscles were secured to the lever arm of a servomotor/force transducer by the distal tendon  and the proximal tendon was connected to a stationary post in the bath. The muscles were stimulated at optimal length for 300 ms. Increasing electrode frequencies were used until a plateau was achieved and recorded as the maximal force. Specific force was obtained by dividing the maximal force by the EDL cross sectional area. Additionally, the muscles were subjected to a protocol of 4 lengthening contractions separated by 1 min intervals of rest. The muscles were lengthened over 10% of their length.Post-hoc pairwise group comparisons were performed where ANOVA models showed a significant F-test and the resulting group p-values were adjusted for multiple comparisons using the Sidak method. For measurements not meeting the assumption of normality (e.g. baseline open field measures), a non-parametric Kruskal-Wallis test was used to compare medians among groups. Data displaying significant p-values from the Kruskal-Wallis was further analyzed for between group differences via Wilcoxon rank-sum pair-wise comparisons. Resulting p-values were again adjusted for multiple comparisons using the Sidak method. Rotarod data was analyzed as a time to fall analysis using a log rank test of the equality of survivor functions between treatment groups. CK values were log-transformed to conform to normality. Histology measurements were compared between treatment groups using Poisson regression models for count data where appropriate. Longitudinal analysis of the repeated lengthening contractions used a mixed-effects linear regression model where mouse ID# was a random coefficient and where each treatment was compared to control only. Nominal statistical significance was set at α = 0.05.All analyses were performed using Stata V11 . Results are represented as mean±SEM or median (range), where appropriate. Normality was assessed for each measurement using the Shapiro-Wilk normality test. For measurements meeting the assumption normality, analysis of variance (ANOVA) was used to compare means among groups . mdx mice gain more weight overtime than wild type littermate controls Mice in each of the treatment groups gained body mass over the course of this three-month pre-clinical trial . AlthougIn vitro maximal and specific muscle force was compared between treatment groups. Maximal force was not significantly different among treatment groups (p = 0.179), although there was a non-significant 13% and 15% increase in maximal force in LANZO and combined treatment groups, respectively, relative to the vehicle control group at the trial’s end . Although no statistically significant between group differences were observed, mice in the LANZO and combined treatment groups displayed a 19% and 18% increase in specific force, respectively, relative to the vehicle control at the trial’s end . Therefore, baseline absolute forelimb grip strength was used as a covariate in ANCOVA models when determining between group differences in post-study grip strength measures. At the end of the pre-clinical trial, mice in the combined treatment group displayed a 10% higher absolute hindlimb grip strength relative to the vehicle control (p<0.01) (Forelimb and hindlimb grip strength testing was performed before and after the 3-month study and on untreated (non-exercised) C57Bl/10 mice, which served as a normal reference group. In (p<0.01) . Additio(p<0.01) . Normali(p<0.01) . However(p<0.01) .No statistically significant differences in post-treatment or changes in open field behavioral activity measures, including: total distance traveled, vertical activity, horizontal activity, movement time and rest time were detected between treatment groups . Of noteMuscle histology was performed on gastrocnemius cross sections at time of sacrifice to quantify the number of degenerating/regenerating fibers and the number of inflammatory foci. Histology was compared between groups via Poisson regression on the median for count data, where appropriate. A decrease in the median number of degenerating fibers was observed in the prednisolone and combined treatment groups relative to mice receiving vehicle control  and LANZO treatment groups  A. No siThere were no significant between group differences in the mean number of inflammatory foci or fibrosis per diaphragm cross sectional field. However, the mean number of inflammatory foci was decreased ∼22% in the diaphragm of mice receiving combined treatment relative to vehicle control . AdditioSera was obtained via cardiac venipuncture at time of sacrifice and analyzed in duplicate via enzymatic assay. Mice in the combined treatment group displayed a ∼64% reduction in mean serum CK relative to vehicle control (p<0.05) . Mice in+/K+-ATPase proton pump restores resting Na+ and K+ gradients and helps maintain muscle excitability for repeated contractions mdx mice. The main finding of our study was that combined LANZO and prednisolone treatment improved some components of the dystrophic phenotype in dystrophin deficient mdx mice. Attenuation of the dystrophic phenotype in the combined treatment group relative to the vehicle control was indicated by observations of: 1) a decreased number of degenerating fibers and inflammatory foci per gastrocnemius cross sectional field; 2) attenuated declines in normalized forelimb and hindlimb grip strength; 3) attenuated declines in maximal in vitro EDL force in response to repeated lengthening contractions. LANZO administration decreased declines in body mass and reduced the number of muscle inflammatory foci. LANZO administration also appeared to improve specific and maximal gastrocnemius muscle force, although this did not reach statistical significance. The effects of LANZO and prednisolone were not additive, mice in the combined treatment group did display greater improvements in muscle force in response to repeated eccentric contractions and a reduced number of muscle inflammatory foci relative to the prednisolone treatment group.Skeletal muscle excitability is regulated by ionic gradients. Following membrane depolarization, activation of the Na+/K+-ATPase in vivo inhibition of proton pumps extragastrically has not been thoroughly investigated. We hypothesize that the mechanisms by which combined LANZO and prednisolone treatment improve the dystrophic phenotype in mdx mice may include: 1) reduced inflammatory cell recruitment to the skeletal muscle; 2) altered ion conductivity of skeletal muscle Na+/K+-ATPase and chloride ion channels; 3) decreased apoptotic signaling cascades promoting myonecrosis.The beneficial effects of dual LANZO and prednisolone administration are likely multifold. LANZO belongs to a class of proton pump inhibitors (PPIs) that become active in weakly acidic environments. LANZO has been suggested to bind to other classes of proton pumps, including the Naet al. observed that PPI administration promotes B cell apoptosis in acute B cell lymphatic leukemia via altering tumor lysosomes and extracellular pH et al. who observed improved tumor outcomes in animals receiving combined treatment of LANZO and chemotherapeutics +/K+-ATPases are inhibited by disulfide binding to cysteine residues on their α subunits +/K+-ATPase, the Na+/K+-ATPase is a class IIC P-type ATPase which is expressed in skeletal muscle. LANZO has also been observed to inhibit the Na+/K+-ATPase, albeit at a higher IC50 than it inhibits the H+/K+-ATPase +/K+-ATPase and Na+/K+-ATPase pumps are highly homologous and display species conservation in their catalytic α subunit cysteine residues (LANZO binding sites) +/K+-ATPase pump resides at the sarcolemma and t-tubule system, where it functions to restore membrane potential following contraction via ATP-mediated Na+ extrusion and K+ import. It is conceivable that LANZO may improve components of the dystrophic phenotype by binding to the skeletal muscle Na+/K+-ATPase and altering ion influx. Dystrophic fibers display an accumulation of intracellular calcium, which promotes myonecrosis mdx mice + that may occur via LANZO-mediated Na+/K+ ATPase pump inhibition may reduce intracellular Ca2+ influx. A reduction in intracellular Ca2+ would thus reduce myofiber lysis and improve the dystrophic phenotype in mdx mice. Such alterations in Ca2+ ionic flux due to LANZO administration may also affect macrophage polarization and NO production as macrophage production of NO is enhanced in the presence of Ca2+ ionophores + -mediated Ca2+ extrusion, an increase in intracellular Na+ levels may increase intracellular Ca2+ by decreasing the electrogenic potential of the Na+/Ca2+ exchanger. This is supported by findings from Allen et al. who observed that elevated intercellular Na+ promotes Ca2+ influx in myocytes + and Ca2+ influx and warrant further investigation. In dystrophic fibers, decreases in membrane resistivity increase membrane conductivity of a variety of ions including Cl−− conductance alter skeletal muscle membrane excitability − ion flux in fibroblasts surrounding myofibers of mdx mice has also been shown to worsen the dystrophic phenotype in vitro muscle force in response to repeated eccentric contractions in mice given combined LANZO and prednisolone treatment could thus be due to altered Cl−, Na+ or Ca2+ conductance and subsequent enhanced muscle excitability in response to LANZO administration. Declines in in vitro muscle force in response to eccentric contractions have been attributed to failure in E–C coupling and thus our findings support a potential role for LANZO in improving dystrophic muscle excitability Off-label use of LANZO has been more thoroughly investigated in the treatment of cancer, wherein LANZO has been suggested to have anti-inflammatory and anti-metastatic effects. De Milito mdx mice by reducing neutrophil recruitment and respiratory burst and by skewing of muscle macrophages away from a proinflammatory M1 macrophage phenotype. This is supported as populations of iNOS expressing muscle macrophages have been suggested to contribute to muscle cell lysis in mdx mice and shifts in muscle macrophage activation from a proinflammatory to anti-inflammatory/regenerative phenotype improve dystrophic pathology mdx mice Another putative beneficial effect of LANZO on the dystrophic phenotype may be due to its anti-inflammatory properties. Macrophage pre-treatment with LANZO decreases nitric oxide (NO) synthesis and cell viability in response to stimulation by LPS +/K+-ATPase pumps and chloride channels. The beneficial effects of dual LANZO and prednisolone administration were not additive. Glucocorticoids, like prednisolone, upregulate Na+/K+-ATPases +/K+-ATPase activity, due to prednisolone treatment, and further decreasing neutrophil and macrophage cytoxcity. Additional investigation of the dose-dependent effects of combined LANZO and prednisolone treatment are thus warranted as higher doses of LANZO may be more efficacious in the treatment of dystrophic pathology.LANZO irreversibly binds to cysteine moieties and its serum half-life is ∼1.5 hours. Due to the rapid catabolism of LANZO by the gastric environment and metabolism by the liver, the effects of LANZO administration are likely less pronounced in the skeletal muscle relative to the gastric epithelia. Therefore, we hypothesize that dual LANZO and prednisolone administration was efficacious due to partial inhibition of Namdx mice relative to prednisolone treatment alone. Complications of DMD may include other symptoms that may be improved with LANZO administration, such as delayed gastric emptying, gastric inflammation, smooth muscle fibrosis and dilation Our findings suggest that combined LANZO and prednisolone treatment provides slightly improved therapeutic efficacy in treating the dystrophic skeletal muscle phenotype in Table S1Changes in body mass and functional outcomes before and after treatment. The mean change  in each parameter tested is presented for all treatment groups.(TIFF)Click here for additional data file.


The mgtCBR transcript includes a long leader region harbouring two short open reading frames (ORFs). Translation of these ORFs is anticipated to impact the formation of particular stem-loop structures and control transcription of the coding region by an attenuation-like mechanism. We previously reported that ORF mgtM enables Salmonella to promote transcription of the mgtC and mgtB coding regions when experiencing a rise in cytoplasmic ATP levels. We now show that the proline codon-rich ORF mgtP mediates an increase in transcription of the mgtC and mgtB coding regions under conditions predicted to decrease the levels of proline-charged tRNAPro. The high ATP and low proline signals act independently in an additive form. Replacing conserved mgtP proline codons with codons specifying other amino acids abolished the response to proline limitation but had no effect on the response to ATP. Substitution of conserved adenine nucleotides in mgtM abolished the response to ATP but had no effect in the response to proline limitation. This provides a singular example of a leader mRNA with tandem attenuators responding to different signals.The  Transcription attenuation is a bacterial regulatory mechanism that entails the formation of either of two alternative based-paired RNA structures in the leader region of a transcript: one promoting transcription termination and one advancing transcription elongation into the coding region. Which RNA structure forms is determined by growth conditions that favour (or hinder) the normal coupling of transcription of the leader region with translation of a short open reading frame (ORF) located within the leader . NormallmgtCBR operon from Salmonella enterica serovar Typhimurium specifies the virulence protein MgtC, which is required for survival inside macrophages .We analysed the rt codon . This ORm-loop E . BecausemgtC transcription start site . These data indicate that Mg2+ can modify the structure of this portion of the mgtCBR leader, which provides support for the genetic experiments suggesting that the mgtCBR leader functions as a Mg2+-sensing RNA . Taken together with our previous findings  in the region overlapping and adjacent to mgtP  still responded to proline limitation by enhancing the mRNA levels of the mgtC coding region  is dependent on the mgtP Pro codons  transcribing the mgtCBR leader when cytosolic ATP levels are low because RNAP may pause at the conserved adenine nucleotides hindering transcription elongation into the coding region. By contrast, mgtCBR transcription and mgtM translation could become uncoupled at high ATP levels, which would advance transcription into the coding region.The g region . This wag region . It was mgtP start codon mutant decreased expression of the mgtC coding region mgtCBR lemgtCBR leader act independently and that the effect of the two inducing conditions is additive   from those involved in extracytoplasmic Mg2+ homeostasis .Transcription initiation of the MgmgtB and mgtA genes because both of them encode P-type ATPases  using 14028s genomic DNA as a template. The resulting PCR products were digested with SmaI and XbaI and cloned into plasmid pACYC-′lacZ digested with the same enzymes. The sequence of the resulting constructs was verified by DNA sequencing.Polymerase chain reaction (PCR) fragments corresponding to nucleotides 148–219 of the mgtP was constructed as follows: a PCR fragment corresponding to the mgtP ORF generated by PCR with primers 8587 and 8589 using 14028s genomic DNA as a template, was digested with HindIII and BamHI and cloned into pUHE 21-2lacIq digested with the same enzymes. The sequence of the resulting constructs was verified by DNA sequencing.Plasmid pgfp plasmid with the PhoP-dependent mgtCBR promoter and the wild-type mgtC leader, and its derivative, the A44–46→T substitutions in the mgtC leader were constructed as described .ptGFP, a mgtCBR promoter, the wild-type mgtCBR leader and first two codons of mgtC fused in frame to the gfp gene was constructed as follows: a PCR fragment generated with primers 1746 and 10111 using 14028s genomic DNA as a template and digested with EcoRI and BamHI was cloned into plasmid ptGFP digested with the same enzymes.ptGFP303, a plasmid with the PhoP-dependent mgtCBR leader region were constructed by cloning PCR fragments generated by two rounds of PCR reactions. For the substitution in the position at 211–214 in the mgtC leader to hinder formation of stem-loop C, a first PCR fragment was generated with primers 1746 and 10113, and a second fragment was generated with primers 10112 and 10111 using 14028s genomic DNA as a template. A third PCR reaction was performed with primers 1746 and 10111 using the two PCR-generated DNA fragments as templates. The resulting PCR product was cloned into ptGFP using the same restriction enzymes used for construction of ptGFP303. All other substitutions were generated in a similar way using the following primer pairs: mgtP (CUG) (1746/8344 and 8347/8117) using 14028s genomic DNA as a template and mgtP (CUG) re211-214 (1746/10113 and 10112/10111) using ptGFP mgtP (CUG) plasmid as a template. DNA sequencing was used to verify the nucleotide sequences of all constructs.Derivatives of ptGFP303 with nucleotide substitutions in the Salmonella strain deleted for the proB gene was generated by the one-step gene inactivation method (R cassette was PCR amplified from plasmid pKD3 using primers 11729 and 11730 and the resulting PCR product was integrated into the 14028s chromosome to generate EL350 (proB::CmR). A P22 phage lysate grown in strain EL350 was used to transduce EG9652 (purB877::Tn10) Salmonella selecting for chloramphenicol resistance to generate EL333 (proB::CmRpurB877::Tn10).A n method . A CmR cmgtCBR leader. For strain EL379, we used the fusaric acid method as described , 8118/8348 and 8349/7308 (for UAA2), 8118/8699 and 8698/7308 (for UAA3), 8118/8809 and 8808/7308 (for UAA4), 8118/8704 and 8703/7308 (for UGA5) or 8118/9853 and 9852/7308 (for UAG2) and 14028s genomic DNA as a template in the first PCR reaction. For the second PCR reaction, we mixed the two PCR products from the first PCR reaction as templates and amplified the DNA fragment with the expected substitutions using primers 8118 and 7308. The resulting PCR products were purified and integrated into the EG18798 chromosome and selected against TetR in media containing fusaric acid to generate strains EG18799, EG18801, EG19251, EG19272, EG19253 and EG19840, which were TetS AmpS.To create mutations with the start codon or stop codons at different positions in mgtC leader were constructed by a multiple step PCR process. Strain EL296 was constructed by inserting a CmR cassette in the yicL gene, which is 278 nt upstream from mgtC transcription start site. The CmR cassette was amplified from plasmid pKD3 using primers 4801 and 4802 and the resulting PCR products were integrated into the 14028s chromosome to generate EL296 (yicL::CmR). Then, we prepared DNA fragments containing a CmR cassette and the proper nucleotide substitutions in the mgtC leader using two primer pairs and EL296 genomic DNA as a template: 10077/8826 and 8827/7308 for the mgtPPro10,11,12→Gly substitution; 10077/11732 and 11731/7308 for the mgtPPro12→Gly substitution; 10077/11734 and 11733/7308 for the mgtPPro10,11→Gly substitution; 10077/11736 and 11735/7308 for the mgtPPro10→Gly substitution; and 10077/11727 and 11726/7308 for the A44–46→T substitution in the mgtCBR leader. The two resulting DNA fragments from the first PCR reactions were mixed and used as PCR templates to amplify DNA fragments containing CmR cassette and the proper nucleotide substitution using primers 10077 and 7308. The resulting DNA fragments were purified and integrated into the 14028s chromosome by the one-step inactivation method  as described  and 500 µM Mg2+ with or without 1 mM proline and growth continued for 45 min. Bacteria were stabilized using RNAprotect Bacteria Reagent (Qiagen) and RNA was isolated for further analysis.The proline limitation experiment was performed as described .Experiment was performed as described  and 500 µM Mg2+ with or without 1 mM proline in the presence of 25 µM or 250 µM adenine and growth continued for 1 h. Bacteria were stabilized using RNAprotect Bacteria Reagent (Qiagen) and RNA was isolated for further analysis.Proline and adenine auxotrophic strains were grown overnight in N-minimal medium containing 10 mM MgmgtC, mgtB, mgtA, phoP and rrs genes were measured by quantification of cDNA using SYBR Green PCR Master Mix  and appropriate primers  and monitored using a Fast ABI7500 machine . Data were normalized to the levels of 16S ribosomal RNA amplified with primers 6970 and 6971.Total RNA was isolated using RNeasy Kit (Qiagen) according to the manufacturer’s instructions. The purified RNA was quantified using a Nanodrop machine (NanoDrop Technologies). cDNA was synthesized using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). The mRNA levels of the 2 concentrations for 4 h at 37°C with shaking. The activity was determined as described  from the DNA template amplified from wild-type 14028s and primers 10336 and 6140 for the mgtC leader 196–385. To probe the structures at different Mg2+ concentrations, 1 pmol of 5′-end-labelled mgtC leader RNA was incubated in buffer  with 1, 5 or 20 mM Mg2+ for 40 h at room temperature. Reactions were quenched with urea gel loading buffer II (Ambion) and analysed on a 10% denaturing polyacrylamide gel.Experiments were carried out as described  with the


Although the expression of cell signaling proteins is used as prognostic and predictive biomarker, variability of protein levels within tumors is not well studied. We assessed intratumoral heterogeneity of protein expression within primary ovarian cancer. Full-length proteins were extracted from 88 formalin-fixed and paraffin-embedded tissue samples of 13 primary high-grade serous ovarian carcinomas with 5–9 samples each. In addition, 14 samples of normal fallopian tube epithelium served as reference. Quantitative reverse phase protein arrays were used to analyze the expression of 36 cell signaling proteins including HER2, EGFR, PI3K/Akt, and angiogenic pathways as well as 15 activated (phosphorylated) proteins. We found considerable intratumoral heterogeneity in the expression of proteins with a mean coefficient of variation of 25% (range 17–53%). The extent of intratumoral heterogeneity differed between proteins (p<0.005). Interestingly, there were no significant differences in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins. In comparison, we assessed the variation of protein levels amongst tumors from different patients, which revealed a similar mean coefficient of variation of 21% (range 12–48%). Based on hierarchical clustering, samples from the same patient clustered more closely together compared to samples from different patients. However, a clear separation of tumor versus normal tissue by clustering was only achieved when mean expression values of all individual samples per tumor were analyzed. While differential expression of some proteins was detected independently of the sampling method used, the majority of proteins only demonstrated differential expression when mean expression values of multiple samples per tumor were analyzed. Our data indicate that assessment of established and novel cell signaling proteins as diagnostic or prognostic markers may require sampling of serous ovarian cancers at several distinct locations to avoid sampling bias. Ovarian cancer is the second most common gynecological malignancy and the one with the highest mortality in the Western world A number of cell signaling proteins have been previously identified as possible therapeutic targets or as potential prognostic or predictive biomarkers. These include VEGF, VEGFR, PDGFR, EGFR or HER2, PI3K, Akt, mTOR and others The overall goal of this study was to assess the level of heterogeneity of cell signaling protein expression in serous ovarian cancer. We analyzed 36 cell signaling proteins, including 15 phosphorylated proteins representing proliferation and angiogenesis related pathways. We additionally investigated the potential impact of heterogeneity on detection of differential protein expression between tumor and normal tissue or between tumor subgroups.In comparison, we assessed the physiological variation of protein expression in normal serous epithelial tissue between different patients. Fallopian tube epithelium from healthy individuals and from uninvolved contralateral tubes of cancer patients was utilized as a reference tissue, since previous studies have demonstrated that serous cancers are molecularly most closely related to tubal epithelium. Fallopian tube epithelial cells have been suggested as the cells of origin for at least some high-grade serous carcinomas For the analysis of large numbers of samples and target proteins as applied in this study, conventional immunoblot methodology is not suitable as one would need more than 3500 Western blot lanes to conduct a single analysis of all samples and antibodies. The reverse phase protein array (RPPA) allows the simultaneous analysis of multiple samples for the expression of several proteins under the same experimental conditions A total of 88 FFPE samples from 13 patients with primary high-grade serous ovarian cancer were studied. 14 samples of normal fallopian tube epithelium, including 10 healthy individuals and 4 uninvolved contralateral tubes from cancer patients were used as reference. A schematic of the patient cohort and selection of samples is depicted in For all 13 cases, 5–9 tissue samples were available. In ten patients with bilateral tumors, samples were obtained from both ovaries.H&E stained sections of all paraffin embedded samples were reviewed by an experienced pathologist (S.A.) to characterize the histological subtype, percentage of viable invasive tumor cells, fibrosis or necrosis, percentage of infiltrating inflammatory cells, and frequency of mitoses.All tissue samples from the same patient were processed simultaneously. Protein extraction was performed as previously described Table S1. RPPAs were generated using the SpotBot® Extreme Microarray Spotter according to the manufacturer’s instructions . For every lysate and dilution (adjusted (2 mg/ml), 1∶2, 1∶4, 1∶8, 1∶16, extraction buffer), 2 replicates were applied onto a nitrocellulose coated glass slide , which produced 12 data points per sample.The expression levels of 36 proteins including 15 phosphorylated proteins were determined by RPPA. Antibodies and experimental conditions are summarized in Immunodetection was performed similar to Western blot analysis and as previously described coefficient of variation (CV). The CV, defined as the ratio of the standard deviation to the mean multiplied by 100, provides a relative measure for variation independent of the absolute values, and therefore allows comparing the variation of proteins with different absolute expression levels.Intratumoral heterogeneity as well as the range of protein expression amongst different patients (inter-patient variation), and the variability of protein expression between fallopian epithelium from healthy individuals were assessed using the Intratumoral heterogeneity was assessed separately for each protein by calculating the CV of all primary tumor samples from the same patient. As summary statistic, the root-mean-square (RMS) average of the CVs th and 75th quartiles, and whiskers (1.5 times the interquartile range) for each patient.The variation among tumors from different patients was assessed for each individual protein by calculating the CV of mean expression values among the different patients. Results are displayed graphically using box-plots showing the median expression value, 25The Friedman test was used to compare CVs for different proteins and the Mann Whitney test was used to compare protein expression between unrelated sample groups at a two-sided 5% level of significance. All statistical analyzes were performed using IBM Statistics .To compare protein expression between normal and tumor tissues and to visualize variation between samples from individual patients, non-supervised hierarchical clustering was performed by using Cluster and TreeView software All samples showed a tumor cellularity of >70% and <10% inflammatory cells. There were no significant differences in epithelial tumor cell content and tumor stroma or inflammatory cell infiltrates between samples from the same patient. In addition, no regional differences were identified in mitotic activity between samples from the same patient.All 36 proteins analyzed in this study showed considerable intratumoral heterogeneity with a mean CV of 25% (range 17–53%) . The extThe variation of protein expression among different patients was similar to the extent of intratumoral heterogeneity with a mean CV of 21% (range 12–48%). The inter-patient variability differed significantly among the proteins analyzed. While Hif1α, PDGF and PI3K demonstrated low variation between all patients analyzed, p4EBP1, pp44/42 MAPK and p1068EGFR showed a very high inter-patient variability. Variation between different patients was not significantly different for phosphorylated (mean CV 25%) and non-phosphorylated (mean CV 18%) proteins (p = 0.072). Inter-patient variation of protein expression was also assessed for normal serous epithelium of fallopian tubes from healthy individuals and for morphologically normal contralateral tubes of cancer patients. The inter-patient variability of tubal epithelium from healthy individuals  was similar to inter-patient variation of contralateral tubes from cancer patients . Overall, variation of normal serous epithelium among different individuals was in a similar range compared to variation between tumors from different patients (21%). Variability was not different between phosphorylated and non-phosphorylated proteins in either type of reference tissue. Results are summarized in To assess variation between individual tumor samples we performed non-supervised hierarchical clustering of all 88 individually taken tumor samples from 13 patients based on the expression of all 36 proteins analyzed Figure 3Figure S1).We assessed the influence of heterogeneity on molecular classification of distinct tissue samples as cancerous or normal based on hierarchical clustering. There was no clear separation of tumor samples versus normal serous epithelium when using all individual tumor samples per case (data not shown). In contrast, analysis of mean expression values for each tumor resulted in very good separation of normal and tumor tissues  were not detected when analyzing randomly selected single samples per case. Ten proteins, namely pB-Raf, EGFR, HER2, 4E-BP1, pPTEN, pVEGFR, VEGF, VEGFR, pS6RP, and VHL were identified as differentially expressed by all four approaches. PI3K and pHER2 were identified as differentially expressed only in single samples. P-values for all proteins identified as differentially expressed in at least one approach are summarized in We found considerable intratumoral heterogeneity in the expression of protein biomarkers related to cell signaling pathways in high-grade serous ovarian carcinoma. All 36 proteins analyzed by reverse phase protein array (RPPA) showed a marked heterogeneity in primary ovarian cancer with a mean coefficient of variation (CV) of 25% (range 17–53%) within an individual tumor. A similar degree of variation was found among different patients with a mean CV of 21% (range 12–48%). Our results suggest that significant sampling bias can be introduced when analyzing single samples of a primary tumor for the assessment of protein biomarkers. Although all 36 candidate proteins showed considerable intratumoral heterogeneity, the overall extent of heterogeneity was different for specific proteins with p4EBP1, pp44/42 MAPK and VEGF showing highest variability and EGFR, JNK/SAPK and p38MPK showing least variability. There was no difference in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins, suggesting that activated forms of cell signaling proteins can be assessed with similar reliability.A previous study in breast cancer detected a similar degree of intratumoral heterogeneity in protein expression (mean CV 31%) The presence of different tumor cell clones is a potential source for intratumoral heterogeneity in protein expression. A recent study demonstrated considerable clonal intratumoral heterogeneity of ovarian cancer based on the self-renewal and tumorigenic differentiation of tumor cells derived from a single ovarian clear cell carcinoma Previous studies reported a lower degree of intratumoral variation of protein expression in ovarian cancer. Two studies from the 1990s found low variation of protein expression between different areas of 9 primary ovarian carcinomas To further assess the variation between individual tumor samples we performed non-supervised hierarchical clustering. Although we had observed a similar extent of heterogeneity within one tumor and between tumors from different patients based on coefficient of variation, samples from the same patient clustered more closely together compared to samples from different patients. This might indicate that despite a high degree of heterogeneity in quantitative protein expression values, a ranking-based approach such as clustering may be less affected. Similarly, a comprehensive analysis of proteins in a signaling network, taking into account relative expression and ranking of individual proteins, might be less affected by heterogeneity.Expression of cell signaling proteins is also being used as a diagnostic biomarker to distinguish tumor from associated normal tissue. We found no significant difference in cell signaling protein expression between ovarian cancer and normal serous epithelium. However, when expression values of several samples per tumor were combined, a significant difference in expression between cancer and normal epithelium was detected for the majority of proteins. This highlights the importance of multiple sampling. A practical approach in future studies may be to pool multiple samples from different regions of an individual tumor prior to analysis.In comparison, we also assessed the physiological variation of protein expression in normal serous tubal epithelium. For technical reasons and limited availability of normal tissue samples, we could not compare different samples of the same patient. Between normal serous epithelium from different patients we observed a similar variation (mean CV 27-31%) compared to the variation between ovarian cancers from different patients (mean CV 21%).To rule out bias due to technical variations we previously assessed the technical reproducibility of protein quantification. High reproducibility of protein measurements from independent extractions and from independent RPPA analyses was demonstrated Limitations of the current study include the number of cases and individual tumor samples (n = 88). We analyzed macroscopic heterogeneity, whereas differences on a cellular level were not assessed. An important strength is the highly homogeneous patient group comprising exclusively high-grade serous ovarian cancers known to share common pathogenetic pathways, as well as the extensive sampling of the tumors in 5–9 different areas. We analyzed different regions within each primary ovarian cancer and did not compare primary and metastatic lesions.In conclusion, intratumoral heterogeneity can lead to sampling bias, and reliable assessment of cell signaling protein expression in ovarian cancer for assessment of prognostic or predictive biomarkers should include sampling of the primary tumor in several different locations.Figure S1Comparison of tumor versus normal tissue by non-supervised hierarchical clustering of 13 tumors and 14 samples of normal serous epithelium, based on mean protein expression values per tumor. Different patients are color-coded as indicated in the figure legend. Main clusters identified by the software are named clusters A and B. High relative expression of proteins is shown in red and low expression in green color. Grey spaces indicate missing data points.(DOC)Click here for additional data file.Table S1Antibodies and conditions used for protein detection.(DOC)Click here for additional data file.Table S2Intratumoral heterogeneity and variation between patients for the expression of 36 proteins assessed by reverse phase protein arrays .(DOC)Click here for additional data file.


Dark septate endophytic (DSE) fungi represent a frequent root-colonizing fungal group common in environments with strong abiotic stress, such as (semi)arid ecosystems. This work aimed to study the DSE fungi colonizing the plants of semiarid sandy grasslands with wood steppe patches on the Great Hungarian Plain. As we may assume that fungi colonizing both invasive and native species are generalists, root associated fungi (RAF) were isolated from eight native and three invasive plant species. The nrDNA sequences of the isolates were used for identification. To confirm that the fungi were endophytes an artificial inoculation system was used to test the isolates: we considered a fungus as DSE if it colonized the roots without causing a negative effect on the plant and formed microsclerotia in the roots. According to the analyses of the ITS sequence of nrDNA the 296 isolates clustered into 41 groups. We found that 14 of these 41 groups were DSE, representing approximately 60% of the isolates. The main DSE groups were generalist and showed no specificity to area or season and colonized both native and invasive species, demonstrating that exotic plants are capable of using the root endophytic fungi of the invaded areas. The DSE community of the region shows high similarity to those found in arid grasslands of North America. Taking into account a previous hypothesis about the common root colonizers of those grasslands and our results reported here, we hypothesize that plants of (semi)arid grasslands share common dominant members of the DSE fungal community on a global scale. Endophytes, which consist of living organisms that colonize plant tissues during some period of their life cycle yet cause no symptoms of tissue damage to their hosts Dark septate endophytes (DSE) are found worldwide and comprise a group of root-colonizing endophytic fungi that belong to a few orders of the phylum Ascomycota Recently, Mandyam and Jumponnen In the mycorrhizal status studies of plant communities of the Great Hungarian Plain, the majority of the plant species was found to be colonized by DSE fungi These results prompted us to study the compositional diversity of DSE fungi colonizing the plants of these semiarid sandy areas. We aimed to test the area specificity, the seasonality and whether the DSE fungi were generalist plant colonizers in the area. To test the latter, we sampled invasive plants in addition to the native indigenous hosts. The effects of soil microbiota on invasive plants are generally established Festucetum vaginatae) and mixed woody steppe (Junipero-Populetum) that are characteristic at each site. The first two areas are protected and belong to the Kiskunság National Park. The three areas are at 100–150 m altitude, the sum of sunny hours/year is 2000–2100, the mean annual precipitation is between 500 and 600 mm/year and the mean annual temperature is 10–12°C The samples were collected from three sites on the Great Hungarian Plain (Hungary); Bugac (N 46°39′ E 19°36′), Fülöpháza (N 46°52′ E 19°24′) and Tatárszentgyörgy (N 47°3′ E 19°24′) . The maiAilanthus altissima; common ragweed, Ambrosia artemisiifolia; and common milkweed, Asclepias syriaca) and eight native species . These plant species were chosen because they represent different life forms and they are characteristic components of these plant communities. To test season- and area-specificity, samples were collected from 10 marked Juniperus communis trees from each of the three areas in three seasons  of 2008 and 2009.The roots of eleven plant species were collected in the area, including three invasive species .Between 2005 and 2009, samples were taken three to four times per year from randomly chosen specimens. Invasive species were sampled in the native plant communities or in their close proximities. For example, ragweed generally colonizes disturbed areas and dirt roads but does not invade stable communities. For some species, only a few specimens were sampled  were cut from different regions of each root sample. The segments were surface-sterilized by washing in 2% sodium hypochlorite (90 s), 70% ethanol (60 s) and sterile distilled water (180 s). Then, the segments were cut into four pieces and placed on MMN media Hyphae growing out of the root segments were isolated to separate plates. Endophytes from the same root sample that showed similar colony morphology were considered identical. The isolates were kept on MMN media in the dark at 18°C, and transferred to new plates every three months.Taq polymerase (Fermentas), the ITS and LSU regions of nrDNA were amplified using ITS1F-ITS4 and LR0R-LR5 primer pairs, respectively, as described previously Total DNA was extracted from different isolates from each root sample using CTAB as described previously http://blast.ncbi.nlm.nih.gov/Blast.cgi) The sequences were compiled from electropherograms using the Pregap4 and Gap4 programs Multiple alignments of our sequences and datasets complemented by sequences from GenBank were made using MAFFT 6 Q-INS-i In vitro tests were performed with the representatives of each group by the analyses of ITS sequences of the fungal isolates (Allium porrum) to test whether an isolate could be categorized as a DSE fungus. Seeds were surface-sterilized with 30% H2O2 for 1 min, then washed twice in sterile tap water for 10 min. Surface-sterilized seeds were germinated and watered on sterile filter paper for approximately seven days. Seedlings were laid to MS medium isolates  using lein vitro experiments were prepared for microscopic analyses similarly to the method described in Kovács and Bagi Root samples from the field samples and All the field-collected root samples of the eleven plant species studied were colonized by intraradical melanized septate hyphae and microsclerotia . In addiNearly 200 samples were collected from the eleven species from the three sampling areas . In totain vitro tests, 59 representative isolates of the 41 groups were chosen  and Fusarium oxysporum (group 27). Both groups were isolated from several native and non-native plants from all sites  were considered DSE. These groups contained 142 of the 241 isolates (∼60%). Henceforth, these groups will be referred to as DSE groups 1–14 . The anaJuniperus communis trees of the three sampling sites were categorized as DSE fungi. All but one of the isolates belonged to DSE-1, -2 and -3 groups , groups with few isolates (number of isolates indicated by slashes after the groups) originated only from either invasive  or native (DSE-4/3/ and DSE-8/3/) plant species . The DSESandy areas in the Carpathian Basin of Danube-Tiscia interfluve, especially those of the open grasslands, represent semiarid regions with semidesert characteristics Auricularia . Unfortunately, the isolate was lost before we could perform any experiments with the fungus.Studies using molecular diversity screening methods generally detect basidiomycetes, mostly Agaricales, as common members of RAF communities e.g., . We founAlthough there is an increasing interest in DSE fungi, there are no clearly defined criteria to determine whether a fungus is DSE. Such criteria would be useful in avoiding confusion caused by inconsequent terminology as has occurred in some mycorrhizal examples The DSE isolates identified in this study belong to those ascomycete groups that are the most frequent root-colonizing endophytes, such as Pleosporales, Hypocreales and Helotiales.Cadophora formed the largest (44 isolates) DSE group (DSE-1); Cadophora is a widely distributed, common root colonizer, especially C. finlandia  Cadophora group is dubious and it may represent more than one taxon arid habitats.Using ITS clone libraries of RAFs of Curvularia species was used in experiments with its natural host Dichanthelium lanuginosum originating from an area with strong geothermal activity Curvularia. Based on the available sequence-similarity information given in the original article, our isolates are likely different from the fungus isolated by Redman et al. A mutual increase of heat tolerance was demonstrated when an endophytic Microdochium strains were isolated in prairie ecosystems of Kansas, United States, and the fungus was used in experimental studies about effects on plants and enzyme activities Microdochium varied according to different hosts, but the fungus is considered endophytic. The singleton group DSE-11 is a Microdochium showing high similarity with the isolate used in the aforementioned experiments Fusarium that includes endophytic lineages The DSE-14 group is composed of isolates of the widely distributed plant pathogen genus Embellisia sp., that was isolated solely from native plants. The puzzling question of host specificity could be addressed on several levels Periconia isolates and concluded that DSE fungi have ‘broad host range’, but there was a considerable difference in the characteristics and effects of the conspecific isolates. Mandyam et al. There are many results suggesting that DSE fungi are generalists and colonize several hosts The seasonality of DSE fungi has been addressed in previous works , Fülöpháza (b) and Tatárszentgyörgy (c).(PDF)Click here for additional data file.Figure S2Minimum Evolution tree of the ITS sequences of 241 isolates originating from roots of indigenous (O) and invasive (▴) plants. Name of the groups is shown in brackets. Isolates used in the artificial inoculation experiments are marked (X). Tree was inferred using the Maximum Composite Likelihood model and pairwise deletion at gaps using MEGA 4.0. Bootstrap values obtained from 1000 replicates are shown as percentages and not shown below 70%. Bar = 2 changes/100 characters.(PDF)Click here for additional data file.Figure S3The maximum likelihood (ML) tree of the ITS sequences of representatives of group DSE-1 and similar sequences from GenBank. Sequences obtained in this study are shown in bold. Pyrenopeziza revincta (AJ4302224) was used as outgroup. Accession number, isolation source and geographic origin of sequences from public databases are shown. NJ bootstrap (not shown below 70%) values are above and the Bayesian posterior probabilities as percentage (not shown below 90%) are below the branches. Abbreviations: uncultured (u.), clone (c.). Bar = 0.05 expected change on one nucleotide.(PDF)Click here for additional data file.Figure S4The maximum likelihood (ML) tree of the ITS sequences of representatives of group DSE-3 and similar sequences from GenBank. Sequences obtained in this study are shown in bold. Massarina corticola (FR668004) was used as outgroup. Accession number, isolation source and geographic origin of sequences from public databases are shown. NJ bootstrap (not shown below 70%) values are above and the Bayesian posterior probabilities as percentage (not shown below 90%) are below the branches. Abbreviations: uncultured (u.), clone (c.). Bar = 0.1 expected change on one nucleotide.(PDF)Click here for additional data file.Figure S5The maximum likelihood (ML) tree of the ITS sequences of representatives of group DSE-8 and similar sequences from GenBank. Sequences obtained in this study are shown in bold. Corollospora intermedia (EU557363) was used as outgroup. Accession number, isolation source and geographic origin of sequences from public databases are shown. NJ bootstrap (not shown below 70%) values are above and the Bayesian posterior probabilities as percentage (not shown below 90%) are below the branches. Abbreviations: uncultured (u.), clone (c.). Bar = 0.1 expected change on one nucleotide.(PDF)Click here for additional data file.Table S1The list of the 241 isolates with their name, group, accession number of ITS sequences, host plant, sampling site and season of the collection.(PDF)Click here for additional data file.Table S2Closest BLAST matches of representative isolates of DSE groups. Name, source, provenance country, query coverage and max ident of sequences from GenBank are indicated. Abbreviations: uncultured (u.), clone (c.), strain (st.), Host (H), Isolation Source (IS), Tissue Type (TT), Environmental Sample (ES).(PDF)Click here for additional data file.