Danio rerio) sense organs—neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish ( Oxidative stress is a pathological state when reactive oxygen species (ROS), products of normal cellular metabolism, are overproduced and antioxidant defenses are not sufficiently efficient . H2O2 differs from the mentioned factors. The difference is that the involvement of heavy metals, aminoglycosides, cisplatin, or noise leads to ROS formation, whereas H2O2 is a ROS itself. Moreover, H2O2 unlike previous agents is an important endogenously produced molecule functioning under physiological conditions . PACAP-38 is a neuropeptide widely distributed in the nervous system and peripheral organs acting through three distinct receptors: PAC1-R, VPAC1-R, and VPAC2-R  was used in the present study. Valuable biological characteristics make the zebrafish an excellent animal research model partially replacing the mammalian models. The zebrafish inner ear and lateral line consist of individual sense organs—neuromasts composed of hair cells, which resemble hair cells of mammalian inner ear . Embryo solution (E3 medium) is a breeding medium containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4. The embryos were kept in the incubator at the 28.5 °C and 14 h light:10 h dark photoperiod without feeding until 5 dpf.In the study, 5 dpf Tubingen and Turku zebrafish strain was used. Adult fish were maintained at 28 °C with a 14-h light:10-h dark photoperiod and fed three times daily ad libitum with dry food and 2O2 solution . Firstly, the exposure time and H2O2 dose were selected after several experiments aimed to create the most proper conditions for caspase-3 activation. The establishment of the most effective H2O2 concentration was achieved by dose-dependent manner study. Animals were divided into five H2O2 exposure groups: 0.1, 0.5, 1.5, 2.5, and 5 mM. Exposures lasted 1 h. For the purpose of our study, the 1.5 mM H2O2 was chosen , the same which was used for breeding. To induce the oxidative stress resulting in caspase-3 activation in hair cells, the larvae were incubated in HImmunohistochemistry was performed in both experiments. The zebrafish neuromasts are located superficially, therefore, whole-mount immunohistochemistry was sufficient to obtain the distinct and specific staining.The 5 dpf larvae immediately after the experimental treatment were fixed with 4 % PFA over night (o/n) at 4 °C. Next, 4 % PFA was replaced with phosphate-buffered saline  for 3 h and kept at 4 °C. After PBS prerinsing, the specimens were washed three times for 30 min in PBS with 0.3 % Triton X-100,  on slow shaking. After this time, all the groups were set in preincubation buffer containing PBST with 1 % dimethyl sulfoxide (DMSO), 4 % normal goat serum (NGS), and 0.1 sodium azide (blocking solution) o/n at 4 °C. Subsequently, the specimens were incubated with primary antibodies.To visualize the neuromasts, an antiacetylated α-tubulin antibody was used. To label the apoptotic cells, an antiactive caspase-3 antibody was used. The activation of p-38 MAPK in the hair cells was detected using an antiphospho-p-38 antibody . To obtain the desirable image ×20 and ×40 objectives were used. Stacks of images were composed into one to obtain maximum intensity projection images with ZEN 2009 software . The quantification was made by counting hair cells visibly affected by active caspase-3 on the images using z-stack. Z-stack tool enabled quantifying every single hair cell noting its 3D structure.3) of phosphorylated form of p-38 MAPK in the whole neuromast. For this purpose, the Imaris software  was used. The measurement threshold was determined by selecting the best value which labeled only specific stain in the neuromast body, not the background. The most specific threshold value was 30 units. Using the set threshold, every single neuromast was analyzed and the value of phosphorylated p-38 volume was generated by Imaris software in cubic micrometers (µm3).The visualization of phospho-p-38 MAPK was achieved using Leica TCS SP2 AOBS confocal microscopy system . To obtain the desirable image, ×40 objectives and 4.0 digital magnifications were used. Like previously, stacks of images were composed into one to obtain maximum intensity projection images with Leica Confocal software (Leica Microsystems). The morphology of phospho-p-38 staining was different than that of caspase-3 and the cell borders were hardly distinguishable; therefore, in this experiment, more relevant measurement technique was based on determining the volume  and the group consisting of the larvae incubated in 100 nM PACAP-38 for 1 h to check whether PACAP-38 has any influence on the behavior. Each experiment was repeated three times using the same behavioral tracking system. After exposure time, each solution used in the experiment was replaced with E3 medium. The behavioral procedure was applied according to Cario et al. .The statistical analyses were performed using GraphPad Prism, version 5.0. Each analysis concerning data with Gaussian assumption was made using one-way ANOVA test, One-way analysis of variance with Tukey multiple comparisons tests as a posttest. Data analyses not assuming Gaussian distribution were based on one-way ANOVA test, Kruskal–Wallis test with Dunn’s multiple comparisons test as a posttest. The error bars are reported as mean ± SEM of the mean. Significance level was set at 2O2 dose-dependent manner toxicity toward the neuromasts, following H2O2 concentrations were chosen: 0.1, 0.5, 1.5, 2.5, and 5 mM  appearance was 1.5 mM  hair cells within the neuromast comparable with 1.5 mM exposure, however the hair cells rosette remained much more disintegrated, resulting in higher amount of hair cells occurring outside the neuromast  and caspase-3 (Casp3) colocalization is hardly detectable, pointing to the caspase-3 properties in cleaving cytoskeletal proteins including α-tubulin. Based on this experiment, the most effective H2O2 concentration was 1.5 mM, therefore it was used for the purpose of the present study.To demonstrate the H2O2-induced hair cell death, the appropriate dose and incubation time for H2O2 had to be established. The best H2O2 concentration which evoked the caspase-3 activation was 1.5 mM. The minimum exposure time resulted in caspase-3 IR hair cells was 1 h; 15 min—the other exposure time point used in our study was found to be insufficient to trigger caspase-3 activation (data not shown). These two experimentally established parameters were combined and used in the subsequent study. 1 h 5 dpf zebrafish exposition to 1.5 mM H2O2 resulted in a marked increase in the number of caspase-3 IR hair cells in comparison to that found in the control group. 5 dpf larvae coincubation with 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 alone resulted in a nearly two-fold decrease in the number of caspase-3 IR hair cells in PACAP-38-treated group in comparison to H2O2-exposed group . Exposure to 1.5 mM H2O2 for 15 min resulted in nearly three-fold increase in phospho-p-38 MAPK volume in the hair cells compared to the control value  and the group incubated with 100 nM PACAP-38 for 1 h  . Likewise, exposure to 1.5 mM H2O2 for 15 min significantly decreased all studied behavioral parameters as compared to control levels. In this case, 100 nM PACAP-38 1 h preincubation was sufficient to improve the investigated behavioral elements impaired by H2O2. First, PACAP-38 treatment resulted in increased percentage of movement events. The time of larval centroid displacement in H2O2-exposed group preincubated with PACAP-38 was about one-third longer (SE = 1.08) than that observed in H2O2-exposed group (SE = 0.58) and did not differ from that determined in the control group (SE = 1.17)  and H2O2-exposed group preincubated with 100 nM PACAP-38 (SE = 0.08). Compared to H2O2-exposed group (SE = 0.04), PACAP-38 preincubation contributed to one-third increase in swimming speed . No differences in the active velocity (mm/s) between all the groups were observed    (SE = 0.06) in relation to the H2O2-exposed group (SE = 0.14) demonstrating similar values to the control (SE = 0.06) (Fig. 2O2 exposed group (Fig. Zebrafish neuromasts as mechanoreceptors are, inter alia, responsible for detection of water movements, thus they influence the behavior and movement parameters. Their dysfunction can strongly affect behavioral traits. A 50 min tracking of 5 dpf larval fish indicated that PACAP-38 has an ameliorative influence on basic movement behavior impaired by HReactive oxygen species underlie many injuries and critical diseases, including age-related hearing loss (Darrat et al. 2O2 leads to apoptotic events in the zebrafish hair cells through caspase-3 and p-38 MAPK activation. H2O2 dose-dependent manner study demonstrated that the minimum effective concentration of H2O2, during 1 h exposure, leading to caspase-3 activation is 1.5 mM. Higher concentrations resulted in greater neuromast disruptions, where in addition to the caspase-3 appearance, the whole neuromast morphology was abnormal. The range of the H2O2 concentration resulted in caspase-3 activation specifically and directly in the neuromast hair cells was quite small, because already at concentration of 5 mM, the apoptosis started to appear in other organs, especially within the skin of the trunk and head. Other studies confirm caspase-3 and p-38 MAPK involvement in hair cells oxidative damages (Wei et al. 2O2 causes their depolymerization (Lee et al. The present study has revealed that H2O2 exposure. PACAP-38 antiapoptotic abilities have been previously shown in rat cerebellar granule neurons (Vaudry et al. The aim of this study was to investigate the ameliorative influence of PACAP-38 on hair cells during oxidative stress. We analyzed both PACAP-38 action at the cellular level and the behavioral aspects of its accomplishment. PACAP-38 unveiled antiapoptotic properties through a marked reduction in caspase-3 activation upon H2O2-treated group, PACAP-38 treatment resulted in a nearly two-fold decrease in the number of caspase-3 IR hair cells upon H2O2 exposure. The impact of PACAP-38 on active caspase-3 reduction has been demonstrated several times in the cells of various origins (Vaudry et al. 2O2 exposure. The relationship between reactive oxygen species and p-38 MAPK has been well established earlier (Zhuang et al. SOD-1 and CAT, promoting cell survival (Gutierrez-Uzquiza et al. Our study has revealed the involvement of PACAP-38 in protection of hair cells, mostly via the antiapoptotic pathway. In comparison to the H2O2 dose-dependent manner study, for present behavioral assay, the 1.5 mM H2O2 concentration was chosen. 1.5 mM H2O2 concentration is an appropriate and relevant model, restricted only to the neuromast hair cells, therefore, it can be assumed that the protective effect of PACAP is mostly directed toward neuromast hair cells. PACAP-38 administration after 1 h H2O2 exposure did not change behavior in a statistically significant way. This indicates that despite the active caspase-3 inhibition by PACAP-38, the neuromast functional properties can be affected by general ROS toxicity. ROS toxicity may also concern whole organisms, precluding larval normal behavior. However, the caspase-3 and phospho-p-38 MAPK immunostainings after 1.5 mM H2O2 exposure were specifically detectable only within the neuromast hair cells, emphasizing that the H2O2 dose applied was specifically selected. In turn, after shorter time of H2O2 exposure, PACAP-38 preincubation resulted in an improvement of investigated behavioral parameters, which corresponded with the control values. The lack of differences in behavior between PACAP-38 and the control group allows to hypothesize that any impact of PACAP-38 on the locomotor parameters results from its protective functions, not from inherent properties to increase the mobility. However, there is an evidence suggesting an arousal effect of PACAP-38 in zebrafish with PACAP-38 overexpression (Woods et al. 2O2 at specific doses effectively and selectively affects neuromast hair cells. It has been reported that lateral line hair cell disruptions cause inability in current water flow orientation (Buck et al. 2O2 toxicity is predominantly directed toward neuromasts and PACAP-38 performs ameliorating effect. However, it should be emphasized that impairment of the movement may not only be the result of neuromast disturbances, because it can be caused by several other factors including muscular or nervous system toxicity.Behavioral analyses completed in the present study have revealed PACAP-38 ameliorative effect on the basic movement parameters, undoubtedly supporting the protective role of PACAP in hair cells against oxidative stress. Based on the H2O2-induced reduction in movement. In our opinion, the results obtained contribute to the knowledge, which hopefully will allow the design of potentially new-generation antioxidant drugs that could be used for the treatment of oxidative stress damaged hearing. Moreover, another, not less important, advantage of present study seems to be an obvious conclusion that the zebrafish should be considered as an excellent research tool for studying the auditory system and that the resulting findings can be treated as fully related to those obtained in mammals.In conclusion, the present study has disclosed the protective effect of PACAP-38 on oxidative stress damage in zebrafish hair cells. Our in vivo findings reaffirm results of previous in vitro investigations suggesting PACAP-38 ability to prevent hair cells from apoptosis. We revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but somewhat unexpectedly had no influence on p-38 MAPK pathway. The behavioral analyses validated the PACAP-38 protective role, demonstrating that PACAP-38 treatment rescued H


N-acetyl-glucosamine and CO2 induce a morphogenetic conversion between white (W) and opaque (O) cells in MTL (mating-type locus) homozygous and heterozygous (a/α) strains of the human fungal pathogen Candida albicans. Here, we identify 8 histone-modifying enzymes playing distinct roles in the regulation of W/O switching in MTL homozygous and heterozygous strains. Most strikingly, genetic removal of the paralogous genes RPD3 and RPD31, both of which encode almost identical orthologues of the yeast histone deacetylase (HDAC) Rpd3, reveals opposing roles in W/O switching of MTLa/α strains. We show that Rpd3 and Rpd31 functions depend on MTL genotypes. Strikingly, we demonstrate that Rpd3 and Rpd31, which are almost identical except for a divergent C-terminal extension present in Rpd31, exert their functions in distinct regulatory complexes referred to as CaRpd3L and CaRpd31S complexes. Moreover, we identify the Candida orf19.7185 product Ume1, the orthologue of yeast Ume1, as a shared core subunit of CaRpd3L and CaRpd31S. Mechanistically, we show that the opposing roles of Rpd3 and Rpd31 require their deacetylase activities. Importantly, CaRpd3L interacts with the heterodimeric transcriptional repressor a1/α2, thus controlling expression of WOR1 encoding the master regulator of W/O switching. Thus, our work provides novel insight about regulation mechanisms of W/O switching in MTLa/α strains. This is the first example of two highly conserved HDACs exerting opposing regulatory functions in the same process in a eukaryotic cell.Chromatin modifications affect gene regulation in response to environmental stimuli in numerous biological processes. For example,  RPD3-like histone deacetylases  are conserved from unicellular eukaryotes to mammals. Specifically, the genome of the human fungal pathogen Candida albicans, the most frequent cause of invasive fungal infections of high morbidity and mortality, harbors two almost identical paralogous HDACs, Rpd3 and Rpd31. We show here for the first time that Rpd3 and Rpd31 acquired functional divergence related to a distinct C-terminal domain. Rpd3 and Rpd31 associate with different complexes in the control regions of the master regulator gene WOR1, which is required for white-opaque (W/O) morphogenesis, respectively. The ability to switch is important for fungal pathogenesis, since it enables distinct host niche colonization. This work is to the best of our knowledge the first description of two paralogous HDACs playing opposing functional roles in the same developmental process. Our work adds a new angle concerning the molecular understanding of HDACs in the regulation of cell fate decisions. Candida spp. enables their rapid adaptation to host niches and allows for colonization or infection  and opaque (O) phases  of histones dynamically modulate chromatin architecture and function, which play pivotal roles in numerous biological processes in response to environmental stimuli , 2. In fnfection . Candidapathogen , can caupathogen . The abialbicans , 7. Morealbicans , C. albi) phases . W cells) phases . Althoug) phases , 11. Mor) phases , 13.MTL homozygous mating-type  strains of C. albicans  or CO2, suggesting that phenotypic switching competency is a common trait shared by all MTL types of C. albicans  switching was presumed to occur exclusively in albicans . The het W state . Howeveralbicans . Of notealbicans . At leasalbicans –18. Furtlbicans –, 19, 20.lbicans –, as welllbicans – and bioflbicans –. However to date , 18.a/α cells to override the inhibition of WOR1 by the a1/α2 repressor complex and thus facilitate W/O switching. Of note, emerging evidence suggests the epigenetic integration of extracellular and intrinsic signaling events  showed similar effects in MTL homozygous and heterozygous strains (dot1Δ/Δ strains lacking an H3K79 methylase (sas2Δ/Δ mutant devoid of a histone acetyltransferase (HAT)  , and thee mutant  showed itive HAT  and the nsferase , complet strains . Notablyus cells . Removal strains . Most sufunction . Since trpd3Δ/Δ and rpd31Δ/Δ mutants showed consistent results in Lee’s glucose medium and Lee’s GlcNAc medium. While lack of Rpd3 strongly decreased switching, loss of Rpd31 dramatically increased the W/O conversion, showing that the opposing roles of Rpd3 and Rpd31 are medium independent . However, the WT MTLa/a control strain, as well as both rpd3Δ/Δ and rpd31Δ/Δ mutants, showed 100% W/O switching both on Lee’s glucose medium and Lee’s GlcNAc medium when incubated in 5% CO2. Because the strong inducing impact of CO2 may mask intrinsic rpd3Δ/Δ and rpd31Δ/Δ phenotypes, we performed W/O switching assays on synthetic complete (SC)-glucose medium with 5% CO2. The result showed that rpd31Δ/Δ deletions in both MTLa/a and a/α strains increased W/O switching frequencies compared to those of the isogenic WT strains . Thus, our data demonstrate that RPD3 regulates W/O switching differently in MTL homozygous strains versus heterozygous strains . Rpd3 and Rpd31 share 74% identical residues, but a unique and major difference lies in the C-terminal stretch of about 170 residues, starting from amino acid 407  served as controls. Without induction, 31.4% ± 6.0% of W cells of the WT/pNIM1-WOR1 strain switched to the O phase, whereas the rpd3Δ/Δ/pNIN1-WOR1 strain showed 10-fold-lower switching frequencies of about 3.8% ± 1.5%. However, upon induction with 50 µg/ml doxycycline, both the WT/pNIM1-WOR1 and rpd3Δ/Δ/pNIM1-WOR1 strains regained 100% W/O conversion. There were no differences in the switching of control strains between inducible and noninducible conditions. Furthermore, cell morphologies upon ectopic WOR1 expression were verified by light microscopy . WOR1 in WT within one doubling time first increases and then decreases again. However, while showing expression patterns similar to those in the WT, average WOR1 levels were significantly reduced in rpd3Δ/Δ cells at all time points compared to the WT control. Of note, rpd31Δ/Δ cells displayed a distinct WOR1 expression pattern, showing sustained elevated expression up until 4 h. These data provide strong evidence that the loss of Rpd31 increases the probability of Wor1 to exceed the threshold levels required to activate its positive feedback loop, whereas the converse is true for the rpd3Δ/Δ cells. These data are entirely consistent with the observed altered W/O switching frequencies of rpd3Δ/Δ and rpd31Δ/Δ strains and present a mechanistic explanation for the opposing regulatory phenotypes.Notably, Wor1 is stochastically expressed at very low levels, with levels varying even among individual cells in the W phase. Wor1 decorates its own DNA regulatory region, forming a positive feedback loop to activate its transcription . Therefocis-acting DNA sites  with specific and hitherto unrecognized roles in W/O switching in MTL heterozygous strains. These data demonstrate a novel role for these epigenetic modifiers in the integration of environmental stimuli (GlcNAc and CO2) into the cell fate decisions. Notably, we also provide the first evidence for a novel mechanism governing the interplay of epigenetic control and transcriptional repression involving orthologous but functionally distinct HDAC proteins. Specifically, we demonstrate that W/O morphogenesis in MTL heterozygous strains of C. albicans is critically and inversely controlled by the paralogous HDACs Rpd3 and Rpd31. Remarkably, our data are the first report of two HDACs with the same enzymatic function showing opposing regulatory functions in a single biological process such as W/O morphogenesis.Eukaryotes possess numerous histone-modifying enzymes to modify nucleosomes and chromatin states to regulate various biological processes in response to environmental stimuli. Here, we show how histone modifications can aid the integration of environmental signals to control W/O morphogenesis in the most prevalent human fungal pathogen, C. albicans harbors two paralogous genes, orf19.2834 (RPD3) located on chromosome R and orf19.6801 (RPD31) on chromosome III. The genes encode two RPD3-like class I HDACs, which are conserved from unicellular yeast cells to humans , as identified in this study, CaRpd31S includes exclusively Rpd31 and at least one other subunit, Rco1. Remarkably, the CaRpd3L complex drives W/O switching, whereas the CaRpd31S complex acts as a negative regulator of W/O conversion in C. albicans.o humans , 38. We cis-acting motifs  in this clade harbor only one orthologue in their genome, sharing a higher sequence conservation with CaRpd31 rather than CaRpd3. Of note, diploid Candida species are more pathogenic than haploid species, and several gene families implicated in pathogenesis are highly enriched for gene duplications , we speculate by analogy that CaRpd31S decoration is restricted to the coding region of WOR1 to inhibit its intragenic transcription. Unfortunately, and despite intense efforts, we were unable to prove a direct interaction of either Rpd3 or Rpd31 with DNA (data not shown), making chromatin immunoprecipitation sequencing (ChIP-seq) efforts futile and obsolete. This may be caused by high off rates and/or the presence of low-affinity transient complexes or may simply be due to indirect interactions of Rpd31 or Rpd3 with DNA mediated by other complex components. Interestingly enough, we also identify the putative histone acetyltransferase Spt10 as another key regulator of W/O switching, implying that the dynamic interplay of acetylation and deacetylation in the WOR1 gene controls the stochastic output of Wor1 expression in individual cells. Of note, our data do not indicate a significant increase in the acetylation in the WOR1 coding region in rpd31Δ/Δ cells compared to the WT. However, this may be due to the fact that WOR1 expression is extremely low in W cells and may in fact not reach detectable levels under normal culture conditions in mixed cell populations. This could also explain why the W morphostate is the default growth phase in MTL heterozygous cells. In any case, our date uncover for the first time opposing functions of two HDACs acting in the same biological process. These data exemplify the tight communication and dynamic interplay of epigenetic and transcriptional control in the regulation of developmental cell fate decisions in eukaryotic cells.Taken together, our data unequivocally demonstrate distinct and opposing regulatory functions of Rpd3 and Rp31 in W/O switching in /α cells . Rpd3 bist cells . Therefost cells , we propcription , 45. In cription . Interescription . Of noteStrains were routinely grown on YPD  medium. Lee’s glucose medium, Lee’s GlcNAc medium, and synthetic complete (SC) medium used for W/O switching assays were prepared exactly as previously described , 49.SAT1-flipper cassette  and PHO23 and ROC1 were deleted in strain J4-2.1 (WT) by using the Candida maltosa LEU2 (CmLEU2) and Candida dubliniensis HIS1 (CdHIS1) markers as described by Noble and Johnson  tagging cassettes, the 3′ part of the coding sequence and the terminator region of the corresponding gene were fused with the 9myc and the SAT1-FLP . For thecassette . For theid pNIM1 . Transfoescribed . Deletio2 at 25°C for 5 days. The switching frequency on Lee’s medium was analyzed on day 5. For cells grown on SC medium, pure white cells were replated on fresh SC medium on day 5 and cultured with 5% CO2 at 25°C for another 5 days before the switching frequency was analyzed. Pure opaque cells obtained in the W/O switching assays were used to perform the O/W assay using the conditions indicated in the text. At least 350 colonies for each strain were analyzed.The white-opaque (W/O) switching assays were carried out exactly as previously described , 15. Bri600) of 2.0, harvested, washed, and resuspended in 0.5 ml of lysis buffer . Then cells were broken with glass beads (425 to 600 mm [Sigma-Aldrich]) on a FastPrep instrument by shaking 5 times at 6 m/s for 30 s. Whole-cell-free extracts were incubated with protein G-coupled Dynabeads (Invitrogen) containing a covalently attached mouse monoclonal antibody (4A6) recognizing the myc epitope. After washing the beads twice with lysis buffer and once with phosphate-buffered saline (PBS), bound proteins were eluted and resolved on a 10% SDS-PAGE gel followed by silver staining  to identify proteins.Immunoprecipitation experiments were performed as described previously , with mistaining  or immun600 of 0.15 in fresh YPD medium and then were grown at 30°C to an OD600 of 1. Cells were cross-linked with 1% formaldehyde and quenched by 125 mM glycine, and ChIP was carried out essentially as described previously (MTLα2-myc ChIP was performed with an antibody against myc (4A6). All ChIP experiments were performed at least with three biological replicates.Overnight cultures were diluted to an ODeviously . For hisWOR1 expression, overnight cell cultures were washed and diluted to an OD600 of 0.5 in fresh Lee’s GlcNAc medium and cultured at 25°C at 220 rpm in a rotary shaker. Cells were collected at time zero and at 1, 2, 3, and 4 h, respectively. RNA was isolated and qPCR performed as previously described (PAT1 using the threshold cycle (ΔΔTC) method (For the qPCR quantification of escribed . Data we) method .Figure S1 C. albicans Rpd3 (480 aa) and Rpd31 (577 aa) with S. cerevisiae Rpd3 (433 aa). The gene sequences were retrieved from http://www.candidagenome.org and http://www.yeastgenome.org, respectively, and subjected to ClustalX software for alignment. Download Sequence comparison of Figure S1, TIF file, 10.3 MBFigure S2 rpd3Δ/Δ and rpd31Δ/Δ in another clinical strain with a different genetic background (JX1250). Strains were grown on YPD plates at 30°C for 2 days. Then cells were plated on SC-glucose medium at 25°C with 5% CO2. After 5 days, cells from white colonies were replated on fresh SC-glucose medium at 25°C with 5% CO2 for 5 days. Download Confirmation of phenotypes of Figure S2, TIF file, 3.8 MBFigure S3 rpd31Δ/Δ::RPD31-T2-3HA, rpd31Δ/Δ:: RPD31-3HA, and untagged control cells were fractionated through a 10% SDS-PAGE gel, followed by immunoblotting using an antibody against the HA tag. The solid arrow indicates partial Rpd31-T2-3HA, and the dotted arrow indicates Rpd31-3HA. Download Truncated Rpd31-T2-3HA and Rpd31-3HA are expressed at similar levels. Equivalent amounts of protein extracts prepared from 11 different transformants of the Figure S3, TIF file, 11.6 MBFigure S4 RPD3-9myc-tagged a/α and a/a cells showed the same silver-staining profiles. C-terminally epitope-tagged Rpd3-9myc a/α (J126-7) and a/a (J148-1) strains and the untagged strain J4-2.1 were used for affinity purification of complexes. Immunoprecipitation was performed as described in the legend to Figure S4, TIF file, 4.4 MBFigure S5 C. albicans Rco1 (688 aa) and S. cerevisiae Rco1 (688 aa) share conserved functional domains. (A) Schematic representation of ScRco1 and CaRco1 proteins. The sequences were analyzed with SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). PHD domains are highlighted in blue, and pink boxes indicate low-complexity regions. (B) Sequence alignment of two PHD fingers. The three conserved residues in PHD1 and PHD2 are highlighted. Download Figure S5, TIF file, 8.6 MBTable S1 C. albicans strains used in this study.Table S1, DOCX file, 0.1 MBTable S2 Plasmids used in this study.Table S2, DOCX file, 0.1 MBTable S3 Primers used in this study.Table S3, DOCX file, 0.1 MBTable S4 S. cerevisiae and C. albicansComparison of orthologues involved in Rpd3L and Rpd3S complexes in Table S4, DOCX file, 0.1 MBTable S5 Epitope tagging does not change the W/O switching of parent strains.Table S5, DOCX file, 0.1 MB


When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic  Targeted genome modifications can be efficiently achieved using the bacterial immunity-derived system CRISPR-Cas9 –4. HowevDNA transposase enzymes can recognize short, inverted repeat (IR) DNA sequences, excise DNA flanked with IRs (transposon) and integrate it into a new location . Transpomariner family transposition . Tn5 and Mu were shown to be active for in vivo integration, for example, in the widely used EZ-Tn5™system, but published protocols require electroporation  carrying the gene of interest and flanked with short IRs (5΄-tatcaggtgtacaagtatgaaatgtcgttt-3΄ for Mos1 transposition or 5΄-taccaggtgtgtcggtaattcctttccggttttt-3΄ for Mboumar-9 transposition) was incubated with 70 nM purified transposase (protein:DNA molar ratio 10:1) for 1 h at 30°C in buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 100 mM NaCl, 10% v/v glycerol, 2 mM dithiothreitol (DTT), 200 μg/ml acetylated bovine serum albumin, 10 mM MnClFor bacterial and mammalian cells chemical transfections the whole reaction volume (20 μl) was used, whereas for electroporation 2 μl of the reaction was used.2 and incubated at 37°C with 250 rpm agitation until OD600 reached 0.48 for Escherichia coli DH10B. The culture was transferred to a chilled 250 ml centrifuge bottle and incubated on ice for 10 min. The cells were harvested for 5 min at 4000 g at 4°C. The pellet was resuspended in 40 ml of cold TFB1 buffer  by gently pipetting and incubated on ice for 5 min. Cells were then pelleted for 10 min at 1400 g at 4°C. The pellet was gently resuspended in 4 ml of cold TFB2 buffer  per 100 ml of culture and incubated on ice for 15 min. Aliquots of 100 μl were dispensed into pre-chilled 1.5 ml microcentrifuge tubes. Tubes were stored at −80°C after snap freezing in liquid nitrogen.A single colony was used to inoculate 5 ml Lysogeny Broth (LB) from a freshly streaked plate, and incubated overnight at 37°C with 250 rpm agitation. The culture was diluted 1:200 into 100 ml pre-warmed LB with 20 mM MgClE. coli DH10B were prepared using the RbCl method, as described above. The transposition reaction (20 μl) was added to the cells thawed on ice, tubes were gently flicked and incubated on ice for 30 min. After 90 s heat shock in a 42°C water bath, the tubes were returned to ice for 2 min and 400 μl of SOC medium (room temperature) was added. The tubes were incubated for 80 min at 37°C and 200 rpm shaking prior to plating on LB agar containing kanamycin 50 μg/ml. The colonies were scored after 20 h incubation at 37°C.Chemically competent ® TOP10 Chemically Competent E. coli (Invitrogen)  fusion of Mos1 was assembled with pET30a expression vector (Novagen) using the PaperClip DNA assembly method . A flexiequences .E. coli BL12 Gold (DE3). Protein expression was induced in Terrific Broth with IPTG at a final concentration of 1 mM at OD600 = 0.6 for 21 h at 220 rpm agitation and 25°C. Cells were collected at 8000 g for 1 h at +4°C. Five grams of cell pellet was resuspended in 50 ml buffer containing 20 mM NaPO4 pH 7.4, 500 mM NaCl, 5 mM MgCl2, 1 Kunitz DNase, 400 μg/ml lysozyme, 2 tablets Complete Protease inhibitor cocktail (Roche) and incubated for 1 h at +4°C with agitation. The cell suspension was homogenized by passing through a needle and cells were broken down in a Cell Disruptor (1.1 kW TS) at 25 kpsi. Cell debris was pelleted at 50 000 g for 1 h at 4°C. Supernatant was filtered through a 5 μm filter followed by a 0.45 μm filter (Millipore) and imidazole was added to a final concentration of 2 mM, prior to loading onto a HiTrap IMAC FF 1 ml column charged with Ni2+. The column was pre-equilibrated with 20 mM NaPO4 pH 7.0, 300 mM NaCl, 10 mM Imidazole. The injection flow rate was 1 ml/min. The column was washed with two column volumes (CV) of equilibration buffer and bound proteins were eluted with a linear gradient from 20 mM NaPO4 pH 7.0, 300 mM NaCl, 10 mM Imidazole to 20 mM NaPO4 pH 7.0, 300 mM NaCl, 500 mM Imidazole over 10 CV. Fractions (1 ml) were collected and analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide gels. Fractions containing bands of the expected size were pooled and concentrated to 250 μl using a Vivaspin® 6 mwco 10 000 Da. Further purification and analysis was performed on a Superdex 200 10/300 GL column at 0.5 ml/min flow rate. The final protein purity was estimated by ImageLab Software to be 75–80%.Sequence verified plasmids were transformed into 2O, incubated with the depurination solution (0.2 M HCl) for 20 min with gentle shaking and rinsed with dH2O. The DNA was denaturated by 2 × 15 min incubation with 0.5 M NaOH, 1.5 M NaCl with gentle shaking and rinsed with dH2O. The solution was neutralized by 30 min incubation in 0.5 M Tris pH 7.5, 1.5 M NaCl with gentle shaking, rinsed with dH2O and finally equilibrated for 30 min in 20× Saline Sodium Citrate (SSC) solution  with gentle shaking.Genomic DNA (1 μg) was separated on a 1% (w/v) agarose gel after overnight endonuclease digestion with EcoRI New England Biolabs (NEB). The gel was washed twice with dH2O and cross-linked for 90 s under UV .Capillary transfer was performed overnight at room temperature: the membrane was then rinsed with dH® 700, IDT) with 120 rpm agitation. The membrane was washed for 5 min at room temperature in 2× Saline-Sodium Phosphate-Ethylenediaminetetraacetic acid (SSPE), 5 min at 40°C min 2× SSPE, 2× for 15 min at 50°C in 2× SSPE/1% SDS, 2× for 15 min at 50°C in 0.2× SSPE. Fluorescent dye was visualized on a LI-COR Imaging System using Odyssey software.Pre-hybridization was performed in a plastic bag at 55°C for 1.5 h in 8 ml of hybridization solution with 120 rpm agitation. Hybridization was performed at 55°C for 15 h in a plastic bag containing 5 ml of hybridization solution with 3 μl of 100 μM fluorescently labeled 28 bp Mos1 IR .E. coli DH10B chemically competent cells. After recovery the total volume was plated out on LB agar containing kanamycin (50 μg/ml) and carbenicillin (100 μg/ml). Plasmid DNA was isolated from the resistant clones and analyzed by restriction digest and Sanger sequencing.For cloning of genomic DNA 5 μg of genomic DNA and 1 μg of pBSKS(+) was digested with EcoRI (NEB) in a final volume 50 μl overnight at 37°C. The enzyme was heat inactivated and 100 ng of pBSKS(+) was ligated with 400 ng of genomic DNA with T4 DNA ligase (NEB) in final volume 10 μl at 4°C overnight. The whole reaction was transformed into Inverse PCR: ∼1 μg of genomic DNA was digested with 1 μl of EcoRI (NEB) in 20 μl for 3 h at 37°C. The endonuclease was heat inactivated for 20 min at 65°C. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min.2O to 20 μl. The initial denaturation step was 98°C for 30 s, followed by 30 cycles of 98°C for 30 s, 56°C for 20 s and final elongation of 72°C for 7 min. The whole PCR reaction was analyzed on a 1% (w/v) agarose gel. The brightest bands were cut out, gel purified (Qiagen) and sequenced.PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dHin vitro transposition  under the regulation of the SV40 promoter was used. We deleted the SalI site from pBSKS(+) with site directed mutagenesis using primers 5΄-gcttatcgataccgacgacctcgaggggg-3΄ and 5΄-ccccctcgaggtcgtcggtatcgataagc-3΄ creating pBSKS(+)ΔSalI. Then we subcloned the kanR gene with the IRs sequences from pEPMosRR (digested with XbaI) and from pEPMboLL (digested with SacI) into pBSKS(+)ΔSalI using the appropriate sites. The kanamycin resistance cassette was cut out of both transposons with SalI. The neomycin resistance cassette of 1.6 kb was amplified from the pEGFP-C1 vector with the primers 5΄-tccatagtcgacagtcctgaggcggaaagaacc-3΄ and 5΄-tccatagtcgacatgagtaacctgaggctatggc-3΄ introducing SalI recognition sites. The resulting products were cloned using the SalI sites into the prepared vectors with two right IRs of Mos1 or two left IRs of Mboumar-9 transposons, creating pMosNeo and pMboNeo plasmids. The correct order and absence of mutations of the resulting constructs were confirmed by sequencing.® CCL-2) was maintained in DMEM (Dulbecco's Modified Eagle Medium) , containing 10% fetal bovine serum  and 1% penicillin-streptomycin .HeLa cell line  was maintained in DMEM , containing 10% fetal bovine serum  and supplemented with 0.1 mM Minimum Essential Medium (MEM) non-essential amino acids .HEK293-H cell line (Gibco5 cells per well). For transfection, cells of 60–70% confluence were used. The growth medium was aspirated, and cells were washed with 2 ml of Dulbecco's Phosphate-Buffered Saline (DPBS) , then with 2 ml of Opti-MEM® Reduced Serum .Cells were inoculated into 6-well plates, one day before transfection in several dilutions (0.8–3.2 × 10® with 3–5 μl of either Lipofectamine® 2000 or GeneJuice® and 1 tube with 230 μl of Opti-MEM® plus the whole volume (20 μl) of the transposition reaction were prepared, and incubated at room temperature for 5 min. . The solution containing DNA was added drop-wise to the tubes containing transfection reagent (with no vortexing or mixing). The tubes were incubated at room temperature for 30 min and each 500 μl was added drop-wise to 1.5 ml of DMEM in each well. The plate was rocked gently and incubated for 24 h prior to medium changing and/or splitting the cells for selection. For selection G418 was added to a final concentration of 1200 μg/ml for HeLa cells or 1000 μg/ml for HEK293-H cells after 72 h after splitting the cells.For each transfection reaction, 1 tube containing 245 μl of Opti-MEM®, No. 12605-010) and the reaction was quenched with 0.5 ml DPBS. Cells were counted with a Countess™ Automated cell counter (Invitrogen) according to the manufacturer's protocol.Selection was performed for 10–14 days (until visible colonies were formed). Colonies were fixed in ice-cold methanol for 5–10 min, stained with 0.1% (w/v) brilliant blue in methanol for 15 min and washed with DPBS until a clear background was achieved. For cell counting, cells were dissociated for 5 min with 0.2 ml TrypLE Express (Gibcog for 5 min at room temperature. Genomic DNA was isolated with a Wizard® Genomic DNA purification Kit (Promega). After overnight digestion of 2.5 μg of genomic DNA with HindIII-HF, the restriction enzyme was heat inactivated for 20 min at 80°C. DNA was phenol extracted, ethanol precipitated and dissolved in 20 μl of water, pre-warmed to 70°C. Self-ligation was performed overnight in 100 μl total volume using 150 ng of digested genomic DNA and T4 DNA Ligase (NEB) according to the manufacturer's protocol. Ligated DNA was phenol extracted, ethanol precipitated and dissolved in 20 μl of water pre-warmed to 70°C.HeLa cells from one well of a 6-well plate were trypsinized and pelleted at 400 2O to 50 μl. The initial denaturation step was 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 63°C for 30 s, 73°C for 3 min and final elongation of 73°C for 5 min. Fifteen microliters of the PCR reaction was analyzed on a 1% (w/v) agarose gel. The brightest bands were cut out, gel purified (Qiagen) and cloned into pJET plasmid using the CloneJET PCR Cloning Kit (ThermoFisher Scientific) according to the manufacturer's protocol. Carbenicillin resistant colonies were selected overnight. Plasmid DNA was isolated and sequenced to establish the locations of neomycin cassette integrations in mammalian cells.To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 on a Deltavision Elite microscope with Photometrics Coolsnap CCD camera and objective ×60 PlanApoN NA 1.42 Oil. Images of the cells were taken every 20 min during 61 time points and throughout 10 positions on a dish. Data were analyzed using ImageJ v10.2. Twelve Z-stacks (1 μm), summed and the pixel intensity of a 5 μm circle of the cytoplasm and a 5 μm circle of the nucleus were compared (excluding the points of saturation for the camera). The averages of 10 positions on a dish are presented and standard deviations are shown as shadowed error bars.Prior to transfection, cells were inoculated into a MatTek glass bottomed dish (P35GC-1.5-14-C). Following transfection as described above, imaging was performed at 37°C and 5% COg for 10 min in 4°C. Subsequently, 250 μg of total protein extracts or recombinant purified proteins  were separated on 4–12% NuPAGE SDS-PAGE gels using MOPS running buffer (ThermoFisher) and were transferred onto a nitrocellulose membrane . The membrane was blocked overnight at 4°C with 1:10 Western Blocking Reagent (Roche) in Tris-buffered saline, Tween 20 (TBST) buffer  Tween 20). The next day, the membrane was incubated for 1 h at RT with primary rabbit polyclonal anti-GFP  solution in 1:20 Western Blocking Reagent diluted in TBST. After washing in TBST, the blots were incubated with the secondary anti-rabbit antibody  conjugated to horseradish peroxidase and were detected with WesternSure PREMIUM Chemiluminescent Substrate detection reagent using C-DiGit Blot Scanner (LI-COR).Total protein samples were isolated from cells by sonication in buffer D  and centrifugation at 5000 in vitro hop assay, in which a kanamycin resistance cassette, flanked by IRs, moves from a donor to a recipient plasmid upon addition of transposase and divalent cations  are selected and propagated. During optimization of this protocol for Mos1 we noticed that if transposase protein was not inactivated prior to transfection, kanamycin resistant colonies were obtained . All the clones analyzed contained between one and three integrations 3 Figure  and B. Insposase .in vivo transposition can occur (Figure E. coli, can indeed penetrate mammalian cells during transfection and concentrate in the nucleus retaining its integrity.During imaging of live cells, the transposase was visible in the cell cytoplasm 80 min after transfection Figure . After 3r Figure . To confr Figure . Our resmariner family are able to facilitate integration of target DNA into the genome of living prokaryotic or eukaryotic cells in vivo, without expression of the transposases inside the host cells. Transposase together with transposon DNA were successfully introduced into bacterial or mammalian cells by electroporation or transfection. It has been shown that co-transfection of restriction enzymes increases the recombination rate in eukaryotic cells (in vitro were introduced into cells by electroporation. Electroporation of proteins into cells is a well known procedure (mariner transposases, or it may be that other systems such as EZ-Tn5™ are also effective following chemical transformation procedures. Electroporation is difficult to automate; thus protocols, which do not require electroporation offer the potential for automated high-throughput generation of insertion libraries. Mariner transposases can integrate transposons in any TA dinucleotide present in the genome (Taken together, our results show that two transposases of the ic cells ,45, but ic cells  or Mu (4ic cells  transposrocedure ; howevere genome ,48, provAs with other protein-based transposase systems, this method allows screening of prokaryotic and eukaryotic host organisms without the need for specific promoters, vectors or delivery tools . This ismariner transposition in vivo.Moreover, as our method does not require DNA encoding the transposase, it overcomes the problem of unstable integration in the host cells, due to prolonged transposase expression. The absence of the transposase gene precludes its recombination with the host DNA, which could result in constant expression of transposase leading to undesired genomic rearrangements. Since the transposase concentration can be finely tuned by titration, overproduction inhibition should not be a limitation for ™, with the added advantage that electroporation is not required, opening the possibility for automated high-throughput integration systems. The one-step method described here is an open source tool, which has the potential to be universally applied to facilitate research in basic microbiology, molecular biology and biotechnology, as well as providing a one-step gene delivery and integration tool for the generation of libraries, sequencing and gene therapy.In summary, Mos1 and Mboumar-9 offer a functional alternative to other widely used commercial systems such as EZ-Tn5Supplementary DataClick here for additional data file.


TBX1, CRKL and/or MAPK1 haploinsufficiency. However, there are some individuals with clinical features resembling the syndrome who are eventually diagnosed with genomic disorders affecting other chromosomal regions. The objective of this study was to evaluate the additive value of high-resolution array-CGH testing in the cohort of 41 patients with clinical features of 22q11.2 deletion syndrome and negative results of standard cytogenetic diagnostic testing (karyotype and FISH for 22q11.2 locus). Array-CGH analysis revealed no aberrations at chromosomes 22 or 10 allegedly related to the syndrome. Five (12.2 %) patients were found to have other genomic imbalances, namely 17q21.31 microdeletion syndrome (MIM#610443), 1p36 deletion syndrome (MIM#607872), NF1 microduplication syndrome (MIM#613675), chromosome 6pter-p24 deletion syndrome (MIM#612582) and a novel interstitial deletion at 3q26.31 of 0.65 Mb encompassing a dosage-dependent gene NAALADL2. Our study demonstrates that the implementation of array-CGH into the panel of classic diagnostic procedures adds significantly to their efficacy. It allows for detection of constitutional genomic imbalances in 12 % of subjects with negative result of karyotype and FISH targeted for 22q11.2 region. Moreover, if used as first-tier genetic test, the method would provide immediate diagnosis in ∼40 % phenotypic 22q11.2 deletion subjects.Chromosome 22q11.2 deletion syndrome, one of the most common human genomic syndromes, has highly heterogeneous clinical presentation. Patients usually harbor a 1.5 to 3 Mb hemizygous deletion at chromosome 22q11.2, resulting in pathognomic The online version of this article (doi:10.1007/s13353-016-0366-1) contains supplementary material, which is available to authorized users. Chromosomal rearrangements of 22q11.2 are implicated in several genomic disorders, microdeletion 22q11.2 being the most common recurrent syndrome with a prevalence of ∼1:4000–1:6000 live births  on 22q11.2 has been reported strongly associated with the syndrome  were suspected of 22q11.2 deletion syndrome in view of the Tobias criteria (see below) , including a conotruncal anomaly and Fallot’s tetralogy in 51 % (19/37) and 16 % (6/37), respectively. Characteristic facial features associated with 22q11.2 deletion syndrome were described in 33 patients (80 %); the most frequent being micro(retro)gnathia (58 %), unusually shaped ears (55 %), long nose with broad bridge (52 %) and short palpebral fissures (30 %). Learning difficulties and/or developmental delay were reported in 24 individuals (59 %), whereas 17 % and 5 % of cases were diagnosed with cleft palate and velopharyngeal insufficiency, respectively. Moreover, a total of 16 patients (39 %) had swallowing difficulties. Other anomalies, such as short stature and renal abnormalities were found in 17 % and 15 % of patients, respectively. Only 5 % (2/38) of patients had primary immunodeficiency while none presented with persistent hypocalcaemia.The patients were evaluated by two clinical geneticists in view of the Tobias criteria  following the instructions provided by manufacturer with modifications as previously described . In the individual 22 array-CGH analysis revealed a 2.7 Mb terminal deletion of chromosome 6p25.3p25.2, the minimal region of chromosome 6pter-p24 deletion syndrome (MIM#612582), covering a total number of 18 genes, among which FOXC1 is proposed as the candidate gene correlating with the reported phenotype. Also, two individuals had interstitial aberrations at chromosome 17. Patient 15 presented with an 1.5 Mb duplication at 17q11.2, whereas a small deletion of 0.5 Mb at 17q21.31 was found in patient 18. The findings correspond with clinical diagnosis of NF1 microduplication syndrome (MIM#613675) and Koolen–de Vries syndrome (MIM#610443) respectively. A comparison of overlapping and distinct clinical features between the detected genomic disorders and the phenotype associated with 22q11.2 deletion is presented in Table No genomic aberrations at chromosomes 10 and 22 purportedly related to the syndrome were detected. Five patients (12.2 %) were found to have a submicroscopic genomic imbalances of the mean size of 1.5 Mb  and their apparently unaffected mother  with a 0.5 Mb deletion at 17q21.31 presented mild dysmorphic features and lacked blepharophimosis, ptosis, or abnormal hair pigmentation and texture usually observed in children with Koolen-de Vries syndrome. The girl (#29) with a 2.1 Mb deletion at chromosome 1p36 did not have structural abnormalities of the brain, vision or hearing impairment or other malformations usually present in children with the syndrome.In this study, 41 patients with clinical signs of 22q11.2 deletion syndrome and with negative standard diagnostic analysis were further evaluated using array-CGH. Five individuals (12.2 %) were diagnosed to harbor pathogenic genomic imbalances, including four known recurrent genomic disorders (Table NAALADL2 (MIM*608806), that is thought to be associated with the congenital defects, dysmorphic features and/or development delay Below is the link to the electronic supplementary material.


In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I) agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis), as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL), which was reversed by coinjection of brilliant blue G (BBG), a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death.P2X Previls (Kir) . DHPG, als (Kir) . Activatls (Kir) , 7–12.7 receptor (P2X7R), one of purinergic (P2) receptors, is an ATP-sensitive ligand-gated cation channel. Activation of P2X7Rs leads to ion channel open, which is permeable for small cations . Repeated or prolonged activation of P2X7Rs under some pathological conditions may result in the formation of a nonselective channel/pore, through which large molecules up to 600–800 Da can pass, thus ultimately leading to cell death [7Rs were expressed in retinal ganglion cells (RGCs), and lots of evidence has demonstrated that activation of P2X7Rs may contribute to glaucomatous RGC death [7Rs in cultured rat brain astrocytes enhanced metabotropic purinergic receptor P2Y2 mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signaling pathways [7R expression in RGCs through ATP release.ATP, released predominantly from glial cells, plays important roles in modulating a variety of physiological and pathological processes by activating Gq-coupled purinergic receptors –20. P2X7ll death –23. It wGC death –26. In apathways . In the All experimental procedures described here were in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals and the guidelines of Nantong University on the ethical use of animals. For intravitreal injection experiments, Sprague-Dawley rats (100–300 g) were obtained from Nantong Laboratory Animal Company and maintained under conditions of a 12 h light/dark cycle. Postnatal 1 d Sprague-Dawley rats were used for primary retinal neuronal cell culture. During this study, all possible efforts were made to minimize the number of animals used and their suffering.μM (S)-3,5-dihydroxyphenylglycine (DHPG), 10 μM ATP, or 10 μM BBG dispersed in 2 μL of 0.9% saline was injected into the vitreous space through a postlimbus spot using Hamilton microinjector (Hamilton) under a stereoscopic microscope. The needle was inserted 2 mm behind the temporal limbus and directed to the optic nerve. Eyes that received only an injection of saline in the same manner served as a control group. The eyes with cataract, endophthalmia, or other damage after injection were excluded.Rats were anesthetized with an intramuscular injection of a mixture of ketamine (25 mg/kg) and xylazine (10 mg/kg). The pupil was dilated with tropicamide drops, and 10 6 and cultured in a neurobasal medium , supplemented with 2% B27 and 2 mM glutamine, in a humidified 5% CO2 incubator at 37°C. RGCs were identified by using antibody against Brn3a, a RGC marker. Experiments were performed on the 8th day of neurons in culture.Primary retinal neuronal cell culture was prepared as described previously  with minμL of 2% fluorogold  was injected into both superior colliculi and dorsal lateral geniculate nuclei. After seven days, rats from different groups were sacrificed and eyeballs were enucleated and placed in 4% paraformaldehyde for 4 h. The whole retina was then carefully dissected, flattened, and mounted with the vitreous side up on slides. Photographs were captured using a fluorescent microscope  and FG-labeled RGCs were counted in a masked fashion by the same investigator using automated particle counting software in ImagePro Version 6.0 . The number of labeled cells in 12 photographs of each retina  at 1/6, 3/6, and 5/6 of the retinal radius was summed together and expressed as mean RGC densities/mm2 for each group.To assess the effect of DHPG on RGCs in vivo, retrograde labeling of RGCs was performed. After anesthetization, the rats were placed in the stereotactic apparatus  and the brain surface was exposed by perforating the parietal bone to facilitate dye injection. 2 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined by the BCA method (Pierce). Protein samples  were subjected to 10% SDS-PAGE using a Mini-Protean 3 electrophoresis system (Bio-Rad) and electrotransferred to polyvinylidene fluoride membranes using a Mini TransBlot electrophoretic transfer system (Bio-Rad). The membranes were blocked with 5% skimmed milk at room temperature for 1 h and then incubated with rabbit antibody against-P2X7R . The blots were washed with TBST and incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG  for 1 h at room temperature and visualized with enhanced chemifluorescent reagent ECL  and exposed to X-ray film in the dark. The experiments were performed in triplicate, and the protein bands were quantitatively analyzed with Image J Analysis software.Western blot analysis was conducted as previously described with some modifications . After wμm thickness on a freezing microtome . The slices were collected and mounted on chrome-alum-gelatin-coated slides. After washing with 0.01 M PBS (pH 7.4), the sections were blocked in 6% bovine serum albumin  in PBS plus 0.1% Triton X-100 at room temperature for 2 h and then incubated with a rabbit antibody against-P2X7 receptor antibody (1 : 400) or mouse monoclonal against-GFAP  antibody at 4°C for 48 h. Immunoreactive proteins were visualized by incubating with FITC-conjugated goat anti-mouse IgG . The samples were mounted with anti-fade mounting medium with DAPI  and the immunofluorescence images were visualized with a Leica confocal laser scanning microscope .Immunohistochemistry was performed following the procedure described in detail previously . After r7 receptor antibody (1 : 400) at 4°C overnight. On the following day, the appropriate second antibodies and 4,6-diaminodiphenyl-2-phenylindole  were added in a dark room and incubated for 2-3 h. After washing, preparations were mounted and detected by a fluorescent microscope .For immunocytochemistry, cells were washed in PBS and fixed with 4% fresh PFA solution for 40 min at room temperature. Cells were rinsed in PBS, permeabilized with 0.1% Triton X-100, and incubated in PBS containing 1% BSA for 2 h to block the nonspecific binding sites. Triplicate wells were incubated with monoclonal antibodies against Brn3a , or a rabbit antibody against-P2XP < 0.05.All data were expressed as means ± SE. Differences among groups were analyzed by one-way ANOVA, followed by multiple comparison tests (LSD). All statistical analyses were carried out by the aid of SPSS 17.0 software package and significance level was set at + channels [7R expression in RGCs by using immunohistochemistry and Western blotting. Firstly, we confirmed DHPG-induced Müller cell gliosis as our previous report [μM, 2 μL) indeed induced an increase in GFAP expression in the retinal section obtained from 2 weeks after the injection (a2), as compared with that in the normal physiological solution- (NS-) injected retinal section (a1). We then examined changes in P2X7R protein expression after the DHPG injection. As shown in Figures 7R proteins were expressed in cells of the ganglion cell layer (GCL) and the inner nuclear layer (INL), as well as in the outer plexiform layer (OPL) ((b1) and (d1)). P2X7R expression showed a remarkable increase in retinal section obtained from the rat at 2 weeks after the DHPG injection, especially in the GCL , a specific P2X7R antagonist, was intravitreally coinjected with DHPG (10 μM). Retinal slice was made 2 weeks after the injection for immunohistochemistry. As shown in Figures 7R expression in the cells of GCL in retinal section was significantly reduced by BBG injection ((b3) and (d3)). Similar observations were obtained in other five eyes. Western blotting revealed that total P2X7R protein in the DHPG-injected retinas was profoundly increased to 185.0 ± 6.0% of the control, and the protein level was reduced to 121.0 ± 8.0% of the control by coinjection of BBG   was intravitreally injected every 7 days, and retinal sections or whole retinal extracts were made at different times after the injection for immunohistochemistry and Western blotting, respectively. As shown in 7R proteins were seen in the retinal section obtained from the NS-injected rat (control) ((a1) and (c1)), especially in the GCL and the INL. P2X7R expression was significantly increased in the sections obtained from ATP-injected rats started from 1 week ((a2) and (c2)) and through 6 weeks after the injection ((a3)–(a5) and (c3)–(c5)), as compared with the control (a1). Consistently, Western blot experiments showed that P2X7R protein levels were significantly increased after ATP treatment  at 1 week after the ATP-injection, while it considerably increased at 2 weeks after the injection  and through 6 weeks  (7R protein expression in the cells of GCL.We then tested whether ATP treatment may induce changes in P2Xreatment . The ave6 weeks) . These r7R expression in primary cultured RGCs. The culture was treated with ATP (100 μM) for different times , and P2X7R expression was examined by immunocytochemistry and Western blotting. 7R expression did not show a significant change at 2 h after ATP treatment (a1); however, it remarkably increased from 12 h to 48 h after ATP treatment ((a2)–(a5)). 7Rs were mainly expressed in Brn3a-positive RGCs , but the P2X7R protein level considerably increased to 373 ± 23.7% , 669.3 ± 12.2%  and 710.2 ± 17.1% of the control  at 12 h, 24 h, and 48 h after ATP treatment, respectively , ATP , or DHPG + BBG  was intravitreally injected every 7 days, respectively, and the numbers of FG-labeled RGCs were counted at 2 weeks and 6 weeks after the injection. As shown in 2 in control (n = 6), while it declined to 2110 ± 90/mm2  and 2037 ± 104/mm2  in the DHPG- and ATP-injected retinas at 2 weeks after the injection, and further to 1438 ± 90/mm2  and 1367 ± 64/mm2  in the DHPG- and ATP-injected retinas at 6 weeks, respectively  at 2 weeks, and 1799 ± 148/mm2  at 6 weeks after coinjection of BBG with DPHG . Regarding the mechanisms underlying ATP-induced changes in P2X7R expression, one possibility is that under pathological conditions prolonged stimulation of P2X7Rs by ATP may increase intracellular Ca2+ concentration and then trigger intracellular Ca2+-dependent signaling pathways, thus increasing P2X7R expression [Our previous study has demonstrated that activation of mGluR I induced Müller cell gliosis by inhibiting Kir Kon model . The mGlon model , 6, suggpression , for ourcker BBG . Secondls of GCL . Thirdly in RGCs . It shoucker BBG  and DHPGcker BBG , we specpression , 30.7Rs were also expressed in amacrine cells and glial cells [7R protein expression in the retinal sections, as shown in Figures 7R proteins was mainly in the GCL and inner nuclear layer (INL)  , and ATPrat RGCs . We spec7R expression aggravated RGC apoptosis. Therefore, the present study provides evidence that under pathological conditions ATP, released by activated Müller cells, may induce RGC apoptotic death through two pathways, directly acting on P2X7Rs and upregulating P2X7R expression.RGC apoptotic death is a common feature in retinal neurodegenerative diseases, such as glaucoma. It is noteworthy that overactivation of P2X7Rs induced upregulation of P2X7R expression in the cells of GCL , in addi7R blocker BBG. These results provide direct evidence that activated Müller cells in some retinal diseases and injuries contribute to RGC death via elevated ATP/P2X7R activity [7R activity may be potentially useful for the therapeutic management of RGC death in retinal diseases and injuries, such as glaucoma.Another important finding in the present study is that both DHPG and ATP intravitreal injection resulted in a reduction in the number of FG-positive RGCs, and the DHPG effect was partially rescued by coinjecting the P2Xactivity –39. On t


Signaling molecules such as cytokines regulate spermatogenesis duringthe maturation of germ cells and sperm apoptosis. Tumor necrosis factor alpha (TNFα) isone of the most-documented cytokines that is involved in spermatogenesis. We investigated the association of the TNFα -308 G/A single nucleotide polymorphism with spermabnormalities in Iranian males.This case-control study included 180 infertile men who re-ferred to Yazd Research and Clinical Center for Infertility and 100 healthy normospermic controls. Infertile men were classified into four groups of azoospermia (n=91), oligospermia (n=26), teratospermia (n=30) and asthenoteratospermia (n=33). After spermanalysis, DNA was extracted from blood and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out for the genotyping of TNFα-308 G/A.The A allele was significantly associated with sperm abnormality in our population [ 95% confidence interval (CI)=2.31]. In addition, the Aallele was also associated with azoospermia =2.484), oligospermia =2.51) and teratospemia =3.385) butnot with asthenoteratospermia (P=0.623).Our data suggest that this single nucleotide polymorphism (SNP) maybe associated with the risk of sperm abnormality in infertile men of Iranian origin. It is estimated that about 15% of couples globallysuffer from infertility. Male infertility constitutes50% of causes among which genetic factorsare mainly responsible . Other cTNFα gene as a single copy gene is locatedon chromosome 6p21.3 within the major histocompatibilitycomplex (MHC) gene cluster , oligospermia ,teratospermia  and asthenoteratospermia groups. This study wasapproved by the Ethics Committee of Shahid SadoughiUniversity of Medical Sciences, Yazd, Iran.Written informed consent was obtained from eachindividual. All semen analysis and clinical examinationswere done according to the World HealthOrganization guidelines method for genotyping of TNFα -308G/A. TheF: 5'-AGGCAATAGGTTTTGAGGGCCAT-3' andR: 5'-TCCTCCCTGCTCCGATTCCG-3' primerswere used to amplify a107 bp fragment of theTNFα promoter that included this SNP. PCR wascarried out in a total volume of 25 μl containing3-5 μl genomic DNA, 1 μl of each primer (10 μM)and 12.5 μl of PCR Master Mix and dH2O. The condition of DNA amplificationwas an initial denaturation step at 94°C for 5 minutes,followed by 35 cycles of 94°C for 40 seconds,60°C for 1 minute and 72°C for 40 seconds,and a final extension step at 72°C for 5 minutesand hold at 4°C. Subsequently, the PCR productswere digested with NcoI restriction enzyme (14hours at 37°C) and the specific bands were identifiedusing 2% agarose gel electrophoresis in 1XTris/Borate/EDTA (TBE) buffer and visualizedunder the ultraviolet (UV) light. When digested,the PCR fragment was cleaved into two fragmentswith sizes 87 bp and 20 bp.The frequency of alleles and genotypes were comparedwith a 2×2 contingency table using Chisquaredand Fisher’s exact test. Fisher’s exact testwas used when sample sizes were small in eachcategory. We considered P<0.05 as a statisticallysignificant and 95% confidence interval (CI) forcalculating odds ratios (OR).We used the SPSSstatistical software  for all statistical analyses.TNFα gene  group, 27.4% in the AZ group [OR (95% CI)=2.535], 26.9% in the O group [OR (95% CI) =2.97], 30% in the T group [OR (95% CI)=2.86] and 15.2% in the AT group. The AG genotype was identified in 22% of the N group, 32.9% of the AZ group [OR (95% CI)=1.74], 34.6% of the O group [OR (95% CI)=1.87], 43.3% of the T group [OR (95% CI)=2.71] and 24.2% of the AT group.TNFα SNPs with different diseases such as colorectal cancer, pre-eclampsia, prostate cancer and Crohn’s disease (Genetic variation such as SNPs in TNFα promoter region may affect its expression. There are several studies that have investigated the association of  disease , 22. The disease  in whichIn this study, we analyzed the TNFα -308 G/A SNP to identify its possible association with sperm abnormality in Iranian males. To the best of our knowledge, this study has not been undertaken in Iranian males. Our findings indicate that this SNP is significantly associated with azoospermia, oligospermia and teratospermia. In other words, this SNP is among many genetic factors that may lead to a decreased count of sperm and abnormal morphology in our cases.Similar to our study, Tronchon et al.  also fouTNFα variation with different sperm abnormalities in the Iranian population.Based on the results of previous studies, TNFα is known to affect spermatogenesis by changing the structure of the blood-testis barrier and apical ectoplasmic specialization of Sertoli cells, which may lead to abnormal spermatogenesis . MoreoveOur study shows that there is a positive association between TNFα -308 G/A SNP and different sperm abnormalities in the Iranian population. Given that the A allele leads to increased expression of TNFα, anti-TNFα agents could be a useful treatment for male infertility.


P<0.05), and both positively correlated with pain (P<0.05). The expression levels of phosphorylated –ERK (p-ERK), phosphorylated-cAMP-response element binding protein (p-CREB), and P2X3 in endometriotic stromal cells (ESCs) were all significantly increased in comparison to the initial levels after treated with interleukin (IL)-1β (P<0.05) or adenosine triphosphate (ATP) (P<0.05), respectively, and did not increase after the ESCs were pre-treated with ERK1/2 inhibitor. Additionally, P2X3 and calcitonin gene related peptide (CGRP) were co-expressed in endometriotic lesions. These obtained results suggest that P2X3 might be involved in endometriosis pain signal transduction via ERK signal pathway.The purinergic receptor P2X ligand-gated ion channel 3 (P2X3) is crucially involved in peripheral nociceptive processes of somatic and visceral pain. Endometriosis pain is considered as a kind of inflammatory and neuropathic pain. However, whether P2X3 is involved in endometriosis pain has not been reported up to date. Here, we aimed to determine whether P2X3 expression in endometriotic lesions is involved in endometriosis pain, which is regulated by inflammatory mediators through extracellular regulated protein kinases (ERK) signalling pathway. We found that P2X3 expressions in endometriosis endometrium and endometriotic lesions were both significantly higher as compared with control endometrium ( Endometriosis is defined by the presence of functional endometrium outside the uterine cavity, resulting in dysmenorrhea, dyspareunia, pelvic pain, and infertility . Pain isIn women with endometriosis, inflammatory mediators including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, prostaglandins (PGs) and nerve growth factor (NGF) have been demonstrated to be elevated in peritoneal fluid and/or endometriotic lesions –15. In tATP has been identified to be a transmitter of signals in the synapse, and 15 different types of ATP receptors have been found so far, which were divided into two categories: P2X and P2Y. P2X3 is a subtype of P2X receptors, a family of ligand-gated ion channels activated by extracellular ATP. Although all P2X subtypes are found on sensory neurons, P2X3 is selectively expressed on the non-peptidergic small diameter sensory neurons –26. ReceIn the present study, P2X3 protein expression in the eutopic and ectopic endometrium of women with endometriosis was firstly determined and compared with control endometrium from women without endometriosis, and the correlation of P2X3 protein expression and pain was analysed. Secondly, P2X3 and calcitonin gene related peptide (CGRP) protein expression on the sensory nerves in human endometriotic lesions were revealed. Thirdly, P2X3 mRNA and protein expression in endometriotic stromal cells (ESCs) were detected when the cells were respectively treated with IL-1β, ATP and ERK inhibitor. Finally, the possible regulative mechanism was discussed.Between June 2013 and July 2014, a total of 65 women undergoing laparoscopic surgery for endometriosis, infertility and tubal ligation were recruited in this study. Each patient gave her written informed consent to participate in the study, and the study was approved by the Human Ethics Committee of Women’s Hospital, School of Medicine, Zhejiang University (No. 20140045). All the participants were able opt out of the study in the whole duration. The indications for women with endometriosis  were endometrioma, pain and infertility. Of the 48 women with endometriosis, 27 women (56.3%) had pain symptoms, 13 (27.1%) women had ovarian endometriosis, 15 (31.3%) had peritoneal endometriosis and 20 (41.7%) women had deeply infiltrating endometriosis. For women without endometriosis , tubal ligation was the only indication, and no woman complained of pain. The severity of pain was documented using a standardized questionnaire with a visual analog scale (VAS). The pain scale was subdivided into ten grades. “No pain” was indicated at the left side of the scale and “the maximum pain you could imagine” was designated at the right side of the scale. However, we did not identify pain types in this study. Additionally, none of the patients received sex-hormone therapy six months before surgery.et al. [We routinely collected three endometrial samples from all women with and without endometriosis immediately after surgery. The first specimen was fixed immediately in 10% neutral-buffered formalin for 24 hours for immunohistochemical staining analysis before processing and embedded in paraffin according to a standard protocol. The second specimen was immersed in liquid nitrogen for western blot analysis before storing at -80°C. The third specimen was quickly placed in Dulbecco’s modified Eagle Medium/F-12 at 4°C for primary ESCs culture. Endometrial histology was dated according to the general classifications of Noyes et al. .et al. [Immunohistochemical staining with P2X3 was performed as previously described by Huang et al. , 37. BriThe expression of P2X3 was classified according to the following grading system as previously described , 37. ScoIsolation of ESCs and endometriotic epithelial cells (EPCs) was performed as following. Briefly, ectopic endometrium was mechanically dispersed with a scalpel, and then enzymatically digested with 0.1% collagenase type 1 and 0.05% DNAse for 1h. Debris was removed using a 100 mm nylon cell strainer. The epithelial cells and stromal cells in the filter were aparted with a 40 mm nylon cell strainer. Epithelial cells were remained in the 40mm nylon while stromal cells were in the filter. Then the cells were cultured in 6-well plates with Dulbecco’s modified Eagle Medium/F-12 (DMEM/F12) (1:1) containing 10% fetal calf sera. Immunofluorescence and immunohistochemical analysis of cytokeratin  and vimentin  expressions were performed to confirm the purification of the isolated endometriotic stromal cells.Immunofluorescence double staining was performed as previously described . Briefly4/well in 1 ml culture medium in 24-well culture plates 24 h before experiment. Then ESCs were incubated with Ca2+ free PBS before tested. After treated with IL-1β (50ng/ml) , the cell supernatants were collected at the different time point. Before measured, samples were centrifuged for 5 min at a speed of 12000r/min. Then samples were placed in closed-bottom 96-well white polystyrene plates  in a Varioskan Flash  immediately. Several concentrations of ATP standard (0.1 nM-100 nM) with PBS were measured before analysis of each experimental sample set. Varioskan Flash reader settings were delay: 2 sec, integration: 10 sec.Release of ATP was determined directly using the firefly luciferin-luciferase assay . Briefly, endometriotic stromal cells were seeded at a concentration of 2×106 cells per well. Twenty-four hours later, ESCs were treated with IL-1β  or ATP, and harvested after 15 min, 30 min, 1 h, 2 h and 24 h incubation respectively for real-time PCR analysis of P2X3 mRNA levels and western blotting analysis of P2X3, p-ERK and p-CREB. In the meantime, the ESCs were treated with IL-1β or ATP after PD98059, an ERK1/2 inhibitor  pre-incubated for 45 min. Afterward, the ESCs were harvested after 15 min, 30 min, 1 h, 2 h and 24 h incubation respectively for mRNA levels of P2X3 and protein expression levels of P2X3, p-ERK, and p-CREB.The ESCs grown to confluence were detached with trypsin and incubated in 24-well plates at a density of 10-ΔΔCt.Trizol reagent  and Prime ScriptTM RT reagent Kit  were used to extract the total RNA in endometrial tissues or ESCs and reverse transcription. Then cDNA was amplified using GoldStar MasterMix . To detect the distribution of P2 receptor types in ectopic, eutopic and control endometrium, specific primers were synthesized from Generay Biotech Co., Ltd.  and the sequences were listed in The frozen tissues or cells were disintegrated on ice in lysis buffer . After centrifuging at 12,000g for 5 minutes at 4°C, the supernatants were collected and the total concentrations of protein were determined by BCA protein assay kit . 30 μg of total protein was separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then electrotransferred onto polyvinylidene diflouride membrane . After blocking in 5% bovine serum albumin  for 1 hour at room temperature, the membranes were incubated with anti-P2X3 antibody , anti-p-ERK1/2 , anti-p-CREB , and anti-GAPDH antibody  overnight at 4°C. The membranes were further incubated for one hour with a secondary antibody against rabbit/mouse IgG and labelled with horse-radish peroxidase . The immune-complexes were detected by ECL detection kit . The relative protein levels were quantified on band volume with respect to GAPDH expression as assessed by Image J software .P<0.05 was considered a significant difference.Statistical analysis was carried out using the Statistical Package for the Social Sciences Version 17.0 . The continuous variables were expressed as mean±standard deviations (SDs). Mann-Whitney test was conducted for numerical variables analysis. Categorical variables were analyzed by Fisher’s exact test. Pearson correlation analysis was used to determine the correlations between P2X3 protein levels and VAS score in women with endometriosis. P>0.05), except for pain symptoms . In turn, the frequency and score of P2X3 expression in endometriotic lesions were 77.8% (21/27) and 4.2±2.8 in women with pain (n = 27), and 33.3% (7/21) and 1.7±2.2 in women without pain (n = 21), respectively. The differences of P2X3 expression frequency and score between women with and without pain both reached statistical significance . Moreover, P2X3 expression scores in the eutopic and ectopic endometrium of women with endometriosis were both significantly correlated with VAS score , peritoneal endometriosis (P = 0.000) and deeply infiltrating endometriosis (P = 0.000), respectively, but no significant differences of P2X3 expression frequency in ovarian endometriosis, peritoneal endometriosis or deeply infiltrating endometriosis between women with and without pain were found  and 3.3±1.4 in women with pain (n = 27), and 33.3% (7/21) and 2.0±1.9 in women no pain (n = 21), respectively. P2X3 expression frequency and score were both significantly higher in endometriosis women with pain than those in endometriosis women without pain . However, no significant difference of P2X3 protein levels between eutopic and ectopic endometrium in women with endometriosis was found , continually increased at 2 min (139.6±6.6 nM), reached the peak value at 3 min (175.0±6.9 nM), and then decreased gradually. The concentrations of ATP in ESCs after treated with IL-1β for 2 min and 3 min, but not for 1 min, 4 min (145.0±17.7 nM) and 5 min (141.2±12.9 nM) were significantly higher than the initial levels , 2h (1.37 ± 0.22) and 24 h (2.99 ± 0.20) compared with the initial levels . WesternP<0.001), but significantly higher at 15 min (2.74±0.42), 30 min (3.22±0.57) and 1h (2.35±0.46) when compared with the initial levels  and 24 h , 30 min (2.33±0.27) and 1 h (2.53±0.61) after treatment . The expP2X3 mRNA expression levels in ESCs did not increased at any time but decreased at 2 h (0.78±0.14) after treated with ERK1/2 inhibitor and ATP . The expIn order to determine whether P2X3 is expressed on sensory nerves in endometriotic lesions, and the correlation of P2X3 protein expression and nerve fibers within the lesions, we used sensory neuronal marker CGRP to reveal sensory nerve fibers. Immunofluorescence double staining showed a co-expression of P2X3 and CGRP in endometriotic lesions, which were mainly seen in endometrial grandular epithelial cells, suggesting P2X3 is expressed on CGRP-positive sensory nerve fibers in human endometriotic lesions .P2X3 is as a subtype of ligand-gated P2X channels belonging to the member of purinergic receptors, which is are expressed not only on neuronal cells but also on most non-neuronal cells –26. In tEndometriosis pain is thought to be a kind of inflammatory, nociceptive and neuropathic pain , 39. HerActually, in acute and chronic pain models, inflammatory mediators induce nociceptive behavior, hyperalgesia and allodynia, which need to activate P2X3 receptor –30. As aIt has been shown that MAPK, one of the main protein phospho-regulating effectors that mediate nociceptive sensitization, is involved in endometriosis pain signal pathway by mediating inflammatory mediators –34. AlthInterestingly, when we used ATP rather than IL-1β to treat ESCs, it showed similar profiles of p-ERK, p-CREB and P2X3 expressions in ESCs to IL-1β. Actually, endogenous ATP, a ligand of P2X3 purinoceptors, is a powerful candidate molecule responsible for the molecular signature of neuronal sensitization and spontaneous aberrant firing in a variety of pain-related diseases . GeneralIn summary, our preliminary results showed that increased P2X3 expression in endometriotic lesions is correlated with endometriosis pain, and P2X3 might be involved in endometriosis pain signal transduction via ERK signal pathway. Further studies are needed.S1 FigGels showed PCR products of the estimated molecular weights corresponding to P2X and P2Y. Totally 7 P2X receptor  and 7 P2Y  receptors were detected in ectopic (A), eutopic (B) and control endometrium (C). PCR product sizes are indicated. GAPDH was shown as positive control.(TIF)Click here for additional data file.S1 TableLength (bp) of the PCR product are given for each primer pair.(DOCX)Click here for additional data file.