SULF2 methylation in gastric cancer.Biomarkers capable of discriminating the patients who are likely to respond to certain chemotherapeutic agents could improve the clinical efficiency. The sulfatases(SULFs) play a critical role in the pathogenesis of a variety of human cancers. Here, we focused our investigation on the prognostic and predictive impact of SULF2 was analyzed in 100 gastric cancer samples. The in vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan and pemetrexed were determined by histoculture drug response assay(HDRA). Additionally, 56 gastric cancer patients treated with a modified FOLFOX regimen were retrospectively analyzed to further evaluate the prognostic and predictive impact of SULF2 methylation in gastric cancer.Promoter CpG island methylation of SULF2(SULF2M) was detected in 28 patients, while the remaining 72 patients showed unmethylated SULF2. Samples with SULF2U were more sensitive to cisplatin than those with SULF2M, while samples with SULF2M were more sensitive to irinotecan than SULF2U. There were no association between SULF2 methylation and in vitro sensitivity to docetaxel, gemcitabine and pemetrexed. SULF2 methylation was found to have a significant association with cisplatin efficacy and irinotecan efficacy. Among the 56 patients receiving the modified FOLFOX regimen, a significant association was observed between survival and SULF2 methylation status. Multivariate analysis revealed that SULF2 methylation was an independent prognostic factor of overall survival in gastric cancer patients treated with platinum-based chemotherapy.Methylated SULF2 methylation is negatively associated with cisplatin sensitivity in vitro. SULF2 methylation may be a novel prognostic biomarker for gastric cancer patients treated with platinum-based chemotherapy. Gastric cancer is one of the most frequent causes of cancer-related death worldwide SULFs in gastric cancer, however, are non-conclusive, and the prognostic or predictive value of SULFs for chemosensitivity prediction remains unknown.Heparan sulfate proteoglycans (HSPGs) are coreceptors for heparin-binding growth factors and cytokines distributed on the cell surface and in the extracellular matrix. Two isoforms of the extracellular heparan sulfate 6-O-endosulfatases (SULFs), SULF1 and SULF2, have been discovered in mammals. Both proteins are secreted to the cell surface to modulate the sulfation of HSPGs SULF2, the gene encoding the SULF2 endosulfatase, and its association with in vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan, and pemetrexed in 100 human gastric cancer samples. To this end, we performed a series of in vitro sensitivity tests on freshly-removed gastric tumor tissues and evaluated the possible use of SULF2 methylation status for predicting the chemotherapeutic efficacy of there agents. Then, we retrospectively analyzed the SULF2 methylation in a cohort of 56 gastric cancer patients treated with a modified FOLFOX regimen and concluded that SULF2U serves as an independent prognostic biomarker in gastric cancer patients treated with a modified FOLFOX regimen.In this study, we have analyzed the promoter CpG island methylation of All research involving human participants have been approved by the Human Research Protective Committee of Drum Tower Hospital Affiliated to Medical School of Nanjing University and written informed consent was obtained from all patients.in vitro evaluation of chemosensitivity by histoculture drug response assay (HDRA) Enrolled patients were those undergoing the gastrectomy in the Department of General Surgery of the Drum Tower Hospital, Nanjing, China during the period from August 2010 to October 2011. Eligibility criteria for enrollment into the study included the following parameters: (1) age >18 years; (2) histologically confirmed gastric adenocarcinoma, mucinous or signet ring cell adenocarcinoma; (3) no previous or concomitant malignancies other than gastric cancer; (4) no previous history of chemotherapy or radiotherapy, either adjuvant or palliative; and (5) adequate function of all major organs. Tissue samples were extracted from 100 freshly-removed gastric tumors. Each tumor tissue was divided into two parts: (1) one part was kept in 4°C Hanks’ balanced salt solution with 1% penicillin/streptomycin after collection, and then sent to the laboratory within 15 min in 4°C, for in vitro sensitivity and resistance were 20 ug/ml for cisplatin 2. After histoculture, 100 µl of type I collagenase  and 100 µl of 3--2,5-diphenyl-2H- tetrazolium bromide (MTT) solution  were added to each culture well and incubated for another 16 hours. After extraction with dimethyl sulfoxide, the absorbance of the solution in each well was read at 540 nm.HDRA procedures were performed as previously reported by Furukawa and colleagues in vitro efficacy rate was also estimated based on the cut-off inhibition rate as follows: efficacy rate (%) = number of sensitive cases/total number of cases.The absorbance per gram of cultured tumor tissue was calculated from the mean absorbance of tissue from 8 parallel culture wells, and the tumor tissue weight was determined before culture. The inhibition rate of each anti-cancer agent was calculated by using the following formula: Inhibition rate (%) = (1–T/C) ×100, where T is the mean absorbance of treated tumor / Weight and C is the mean absorbance of control tumor / Weight. The cut-off inhibition rates used to distinguish the sensitive and the resistant cases were adopted at 30%, 40%, 50%, and 60% for each drug tested. The −2 plus folinic acid 200 mgm−2 as a 2 h infusion on day 1, followed by a 22 h infusion of 5-FU 2000 mgm−2 on days 1 and 2, every two weeks. These 56 patients were further followed up and their survival time was calculated from the date of diagnosis to the date of the last follow-up or death from any cause.Of all patients, 56 with Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤2 received a modified FOLFOX (a combination of 5-FU and platinum) chemotherapy regimen after resection of primary tumors as follows: oxaliplatin 85 mgmThree 7-µm sections were prepared from primary tumor blocks that contained at least 80% tumor cells. After hematoxylin-eosin staining, the cancerous parts were microdissected and transferred into a microcentrifuge tube. DNA was isolated routinely and then was chemically modified by sodium bisulphite to convert all unmethylated cytosines to uracils while leaving methylcytosines unaltered SULF2 using the ABI Prism 7300HT Sequence Detection System (Applied Biosystems). Each PCR reaction contained genomic DNA 2 µl, SYBR Green PCR Mix  10 µl, water 7.7 µl, and primers 0.15 µl (10 µmol/l). The PCR conditions were 95°C for 10 min, followed by 45 cycles at 59°C for 30 s, 72°C for 30 s and 95°C for 30 s. Primers for SULF2 methylated PCR  were as follows: forward 5′ TAAGTGTTTTTTTTATAGCGGC 3′, reverse 5′TACCGTAATTTCCGCTATC 3′. Primers for SULF2 unmethylated PCR  were as follows: forward 5′ GTTTATAAGTGTTTTTTTATAGTGGT3’, reverse 5′TACCATAATTTCCACTATCCCT 3′. Each batch of reaction included a positive control from Methyltransferase (M.SssI)-treated human genomic DNA (fully methylated), a negative control from DNA samples which had been confirmed as unmethylated and another negative control without DNA. All tests were performed in duplicate. Results were validated for selected samples through combined bisulfite modification and bisulfite sequencing.MSP was performed to determine the methylation status of in vitro chemosensitivity efficacy were analyzed using the Fisher’s exact probability test. All statistical tests were two-sided, and a P value of less than 0.05 was considered as statistical significance. Statistical analysis was performed using the SPSS, version 16.0.Values were expressed as means ± standard deviation. Differences in inhibition rates between groups were evaluated using the t-test. The possible associations of SULF2 methylation with clinical characteristics and SULF2 (SULF2M) was detected in 28 patients, while the remaining 72 patients showed unmethylated SULF2 . The RT-PCR amplification curves of SULF2M and SULF2U were shown in SULF2 methylation and any of the patients’ characteristics, including sex, histology, tumor site, stage, histological grade and lymph node metastasis.The characteristics of all patients are shown in In vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan and pemetrexed was successfully tested in all the samples. The mean inhibition rates for each tested drug are listed in SULF2U were more sensitive to cisplatin than those with SULF2M , while samples with SULF2M were more sensitive to irinotecan than SULF2U  and irinotecan efficacy . No other association between clinical characteristics and survival was found . A significant association was observed between survival and SULF2 methylation status. The median overall survival was 309 days (95% CI = 236 to 382 days) for patients with SULF2M, and 481 days (95% CI = 418 to 490 days) for those with SULF2U . A significant association was observed between survival and tumor site  the rate of SULF2M in human gastric cancer is around 30%; (ii) the first evidence for the SULF2U is associated with cisplatin sensitivity in cancer; (iii) evidence for the SULF2M is associated with irinotecan sensitivity in gastric cancer; (iv) a retrospective study and validation for SULF2 methylation in a cohort of 56 patients with gastric cancer, which allowed us to discover that SULF2U is an independent prognostic biomarker in gastric cancer patients treated with a modified FOLFOX regimen.Although previous studies have demonstrated the involvement of SULF2 in cancer pathogenesis, its value for chemosensitivity prediction remains unclear. This study focuses on the promoter CpG island methylation of SULFs were inconclusive. In one study, only 13 early-stage breast and gastric cancers, most of which were stage I, were analyzed by semi-quantitative RT-PCR for SULF1 expression SULF1 was prevalent in these two types of cancer. In another study, the expression of both SULF1 and SULF2 mRNA was determined by real-time RT-PCR for a large cohort of gastric cancer tissues, finding that SULFs were expressed at higher levels in gastric cancer as compared with normal tissues. In the current study we found by examining the SULF2 methylation in 100 gastric cancer samples, that the rate of SULF2M was approximately 30%, which indicated that predominate gastric cancer were SULF2U. Recent studies by integrated genomic analyses revealed that SULF2 acts as a downstream effector of p53, and that activation of p53 could lead to the SULF2U and up-regulation of SULF2. The possible link between p53 and SULF2 in growth factor signaling pathway suggested a possible role for SULF2 in cancer development and cancer patients’ outcome SULF2 unmethylation and SULF2 up-regulation needs to be further tested in these samples. The different expression levels and methylation status of SULF2 between tumor and normal tissue also need to be further verified.In previous studies of gastric cancer, the findings on methylation status and expression levels of SULF2 methylation as chemosensitivity predictor are scarce. Methylation of the SULF2 promoter has been associated with better survival of resected lung adenocarcinoma patients, and also with a marginal improvement in survival of advanced NSCLC patients receiving standard chemotherapy  SULF2M were 134-fold more sensitive to topoisomerase I inhibitor than those with SULF2U. In the current study, we have demonstrated that gastric tumors with SULF2M are more sensitive to irinotecan than those with SULF2U. Furthermore, we demonstrated for the first time that SULF2 methylation is also a potential predictive biomarker for cisplatin efficacy. Gastric tumors with SULF2U were more sensitive to cisplatin than those with SULF2M, and gastric cancer patients with SULF2U showed lower mortality when receiving platinum-based chemotherapy. Although several predictive biomarkers have been identified for cisplatin, such as ERCC1 SULF2 unmethylation increases tumor sensitivity to cisplatin may lie on ubiquitin conjugating enzymes (UBE). It has been reported that SULF2 methylation results in increased expression of UBE SULF2M induced cisplatin resistance.Studies on SULF2 methylation and cisplatin sensitivity may partially explain this phenomenon. Treatment of gastric cancer with DNMT inhibitors could result in demethylation of SULF2 and consequently increase the sensitivity to cisplatin. Thus, in addition to the predictive impact, our data also supports the inclusion of a DNMT inhibitor in current treatment protocols for at least a subset of gastric cancer patients.A significant synergistic effect of cisplatin and DNA methyltransferase (DNMT) inhibitors 5-aza-dC (DAC) on cell viability was observed in the cisplatin-resistant AGS cell line but not in the cisplatin-sensitive MKN28 cell line SULF2 methylation is negatively associated with cisplatin sensitivity in vitro. SULF2 methylation is a potential prognostic biomarker for gastric cancer patients treated with platinum-based chemotherapy.In conclusion, our study provides novel evidences that Figure S1The RT-PCR amplification curves of SULF2M and SULF2U. The red curve stands for amplification of SULF2M, and the blue curve stands for amplification of SULF2U. (TIF)Click here for additional data file.


For step-and-shoot type delivery of intensity-modulated radiation therapy (IMRT), beam stability characteristics during the first few monitor units need to be investigated to ensure the planned dose delivery. This paper presents the study done for Siemens ONCOR impression plus linear accelerator before commissioning it for IMRT treatment. The beam stability for 6 and 15 MV in terms of dose monitor linearity, monitor unit stability and beam uniformity is investigated in this work. Monitor unit linearity is studied using FC65G chamber for the range 1-100 MU. The dose per MU is found to be linear for small monitor units down to 1 MU for both 6 and 15 MV beams. The monitor unit linearity is also studied with portal imaging device for the range 1-20 MU for 6 MV beam. The pixel values are within ±1σ confidence level up to 2 MU; for 1 MU, the values are within ±2σ confidence level. The flatness and symmetry analysis is done for both energies in the range of 1-10 MU with Kodak diagnostic films. The flatness and symmetry are found to be within ±3% up to 2 MU for 6 MV and up to 3 MU for 15 MV. Intensity-modulated radiation therapy (IMRT) is becoming the commonly chosen treatment modality because of its dose conformity to the concave tumor volumes, sparing surrounding normal organs. Siemens ONCOR Impression Plus linear accelerator with photon beam energies of 6 and 15 MV equipped with double-focused multileaf collimator (MLC) has been installed in our clinic. These accelerators are designed to deliver intensity-modulated fields using step-and-shoot technique. In this type of IMRT delivery, multiple segments are the result of the optimization process to conform the dose to the target volume. These segments are delivered in a stack arrangement. Each beam segment is delivered separately with the radiation beam turned off (paused)[The operation of the linear accelerator delivering low monitor unit segments needs to be investigated for every linear accelerator prior to commissioning the linear accelerator for IMRT. Many aut1max in the SP34 slab phantom.[1Dose monitor linearity and monitor unit stability at small monitor units for 6 and 15 MV were examined using a FC65G ionization chamber connected to DOSE1 electrometer. The measurements were done at 100 cm SSD with the chamber at dMonitor unit stability was studied with the same setup as described above. For the collimator setting of 10 cm × 10 cm, the ionization was recorded for 10 MU. It was compared with the cumulative ionization of 5 MU measured two times; 2 MU measured five times, 1 MU measured ten times. SimilarlFilm dosimetry was used to study dose uniformity for low monitor units. Beam flaFor low-MU linearity, ionization readings were recorded in the range of 1-100 MU. Similar study done for 15 MV is shown in Figures Figures For 15 MV , the varFor 15 MU, as shown in Flatness symmetry study was done with Kodak diagnostic films. Flatness was calculated using ‘variation over mean’ (80%) method. The symmetry value was obtained with ‘point difference quotient’ method. For 6 MVFor 15 MV beam, inplane and crossplane flatness for 1 MU is 4.11 and 4.88% respectively. For 2, 3 5 and 10 MU, the crossplane flatness values are within 2%. The inplane flatness is within 3% for 2, 5 and 10 MU; while it is 3.97% for 3 MU .The symmetry analysis for 15 MV  shows thFor 6 MV beam, the stability of the beam at low MU in terms of cGy/MU is within ±2%, assuming all other factors are proportional while converting the charge to cGy. The stability in terms of flatness and symmetry is also acceptable and is within ±3% up to 2 MU. The stability of 15 MV beam in terms of flatness and symmetry is within ±3% above 3 MU.However, it is suggested that segments with monitor units less than 5 MU should be avoided. The inverse planning system used at our center takes care of prevention of small MU segments such as 5 MU from the treatment plan. So for the range of monitor units used for patient treatment, the Siemens ONCOR Impression Plus linear accelerator has been delivering stable beams for step-and-shoot IMRT treatments.


Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish.The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the  In vertebrates, major parts of the craniofacial skeleton derive from cranial neural crest cells (cNCCs) that previously migrated into the pharyngeal arches tfap2α starting at 12 hours post fertilization (hpf). tfap2α mutants show defects both in pre-migratory cNCC specification and in cartilage development Col2a1 in mouse , while in zebrafish both sox9a and sox9b are required for col2a1 expression. However, they play both distinct and overlapping functions in zebrafish chondrogenesis dlx2-expressing cNCC. After migration of the cNCCs into the pharyngeal arches, sox9a is expressed in cNCCs under control of dlx2aox9b is only expressed in the pharyngeal endoderm sox9a in the jef mutant leads to a complete absence of cartilage sox9b mutation causes an absence of ceratobranchials and a reduction of the two most anterior pairs of arches. Sox9a is essential for endochondral ossification, while sox9b has an additional role in formation of some dermal bones Runx2b expression Differentiation of cNCCs into mature chondrocytes is controlled by a number of transcription factors. In zebrafish, pre-migratory cNCCs express casanova (cas) and bonny and clyde (bon) mutants lack endodermal markers and present severe cartilage defects van gogh (vgo) mutants make pharyngeal endoderm but fail to form pouches, which leads to an absence of cNCC segmentation into distinct elements vgo and cas mutants restores the cartilaginous phenotype In addition to these endogenous patterning cues, cNCCs also receive external signals from surrounding cells and extracellular matrix, such as Fgf, BMP and Shh signals e.g. mitosis, differentiation and apoptosis Egr1 is expressed during embryogenesis in cartilage, bones, teeth, salivary glands, nasal glands, developing vibrissa, tendons and skeletal muscles egr1 gene is composed of two exons and one intron in situ hybridization before gastrulation gr1 expression was observed in the pharyngeal region until at least 48 hpf.The early growth response (EGR) family contains four highly conserved zinc finger (C2H2 type) transcription factors that mediate the cellular response to many stimuli inducing Egr1 knockout mice display sterility for both sexes and are smaller due to defects in adenohypophysis development and particularly LHβ expression Egr1 KO mice present increased bone resorption, a reduced bone mass and an increased production of the osteoclastogenic cytokine M-CSF (macrophage Colony-Stimulating Factor) in stromal cells of the bone matrix egr1 leads to a reduction of the eye size, while the retina and lens lack appropriate differentiation Egr1 is known to play various roles such as tumor promotion in prostate fsta), a known BMP antagonist. We also show that BMP signaling is required around 30 hpf in the neural crest cells for runx2b expression in chondrocytes and proper pharyngeal cartilage formation, while BMP signaling is absent in the pharyngeal region upon egr1 knock-down.In this study, we report that the transcription factor Egr1 plays a specific role in pharyngeal cartilage formation during zebrafish embryogenesis, while it is expressed in pharyngeal endoderm. We show that Egr1 is part of a regulatory cascade, together with the endodermal factors Runx3 and Sox9b, that suppresses expression of follistatin A (Danio rerio) were reared in a recirculating system from Techniplast, Italy at a maximal density of 7 fish/l. The water characteristics were as follows: pH = 7.4, conductivity = 500 µScm-1, temperature = 28°C. The light cycle was controlled . Fish were fed twice daily with dry powder (ZM fish food®) with size adapted to their age, and once daily with fresh Artemia salina nauplii (ZM fish food®). Larvae aged less than 14 days were also fed twice daily with a live paramecia culture. Wild type embryos from the AB strain were used and staged according to Kimmel Tg(hsp70l:dnBmpr-GFP)Zebrafish .Breeding: the day before breeding, 2 males and 2 females were placed in breeding tanks out of the recirculating system, with an internal divider to prevent unwanted mating. On the day of breeding, fish were placed in fresh aquarium water and the divider was removed to allow mating. Eggs were collected every 30 minutes and raised in E3  were synthesized by Gene Tools  and are complementary to the 5′ sequence near the translation start site or to the splice junctions. MO stock solutions were prepared as suggested by Gene Tools. Tetramethylrhodamine dextran  was added at a concentration of 0.5% to sort correctly injected embryos a few hours after injection. The morpholino sequences are as follow:egr1: GGATTTAGTGCTTACCTCCAGCAAG.MO splicing egr1: TGCAGCCATCTCTCTGGAGTGTGCT.MO translation runx3: TGCTCGGGTCTACGGGAATATGCAMO translation fst1a: CTGACGTTTAGCATCCTTAGCATGMO translation CCTCTTACCTCAGTTACAATTTATA (Gene-Tools®).MO standard control: gr1 cDNA fragment starting at the ATG start codon and spanning the entire coding region was obtained by PCR-amplification from the cDNA clone egr1 mRNA was synthesized using mMessage mMachine Sp6 (Ambion®) and injected alone or co-injected with a morpholino at the one-cell stage with a microinjector (Narishige®).An eegr1 spl was examined by RT-PCR using SuperScript® from Invitrogen . mRNA of 50 injected embryos was extracted for each experiment. The primers used were: Zf-Egr1 forward: 5′-CAGTTTGATCACCTTGCTGG-3′; Zf-Egr1 reverse: 5′-GGAAGACGTGGAAGAGGAAG-3′.Efficiency of the splicing morpholino MOin situ hybridization were raised in presence of 0.003% of 1-phenyl-2-thiourea until the desired stage, fixed overnight in 4% of PFA at 4°C and stored in 100% methanol at −20°C until use. Visible in situ hybridizations (ISH) were performed as described Injected embryos for Whole-mount fluorescent immunohistochemistry was described in in situ hybridization were taken by Nikon® Eclipse 90i microscope and NIS-Elements microscope imaging software. Fluorescent images were acquired with a Leica® SP2 confocal microscope.Pictures of visible Cartilage was stained with Alcian Blue 8 GX (Sigma®). Four days old embryos were fixed in PFA 4% for 2h at room temperature, rinsed with PBST and finally stained overnight with 10 mM Mg Cl2/80% ethanol/0.04% Alcian Blue solution. Embryos were rinsed with 80% ethanol/10 mM MgCl2. Pigments were bleached in H2O2 3%/KOH 0,5% for 1 h. 25% glycérol/0,1% KOH, 50%Glycerol/0,1% KOH.hsp70l:dnBmpr-GFPTransgenic embryos 10 mM stock solution of dorsomorphin  was diluted in DMSO . Embryos at desired stage were placed into multiwell plates with dorsomorphin diluted in E3 rearing medium at the desired concentration during a specific time period. DMSO alone was used as control. Embryos were then rinsed several times with E3, raised in E3 and finally fixed at the desired stage.egr1 transcripts or against a control sequence (MOcon). Two different morpholinos against egr1 were used, one inhibiting egr1 splicing (MOegr1 spl) and the other preventing translation of egr1 mRNA (MO egr1 tr). Injection of MO egr1 spl efficiently inhibited egr1 splicing in embryos, as judged by RT-PCR of 2 days post fertilization (dpf) embryos  directed against  embryos , whereasPharyngeal and cranial cartilages were stained with Alcian Blue in 4 dpf embryos. Remarkably, in embryos injected with 8 ng of MOegr1 tr, branchial cartilages were completely absent, while Meckel’s cartilage and the palatoquadrate were significantly reduced and the ceratohyal and hyosymplectic misshapen  . A similegr1 mRNA together with MO egr1 or MOcon. Co-injection of 75 pg of egr1 mRNA together with 8 ng MOegr1 tr rescued the development of pharyngeal and cranial skeleton in 87% of co-injected embryos   , while bpression . In contpression . Thus, fegr1 morphant embryos by in situ hybridization. Expression of early neural crest markers ap2a3 was not affected in egr1 morphants at 24 hpf  and 48 hpf  egr1 morphants .Runx2b is a transcription factor known to play a key role in chondrocyte maturation and is expressed in mesenchymal cartilage condensations in the viscerocranium starting at 40 hpf orphants . At 48 hegr1 spl , revealiegr1 expression is dispensable for early specification and migration of cNCCs, but is required for proper late chondrogenesis in pharyngeal arches.We conclude that egr1 expression pattern in the developing pharyngeal region. Whole-mount in situ hybridization against egr1 confirmed its expression in the pharyngeal region starting at 30 hpf and persisting until at least 48 hpf  and fli1 , which is expressed in pre-cartilage condensations and endothelium fli1 in the cartilage condensations while egr1 expression is located in stripes separating cartilages  and sox9a  mutant embryos, devoid of all molecular endodermal markers egr1 transcripts were observed at 48 hpf in the pharyngeal region as well as in different brain regions and in the heart. In contrast, in 48 hpf homozygous cas embryos , while expression is maintained in the brain, the duplicated hearts and increased in the fin buds.To determine precisely in which tissue  , reminiscent of the situation observed in egr1 morphants.At stages beyond 26 hpf, expression of the Sox9b transcription factor is localized in pharyngeal epithelium and endoderm; this factor indirectly regulates gr1 is expressed in pharyngeal endoderm, we investigated potential regulatory connections between the sox9b and egr1 genes. At 42 hpf, egr1 morphants do not express sox9b in the pharyngeal pouches and its expression in the brain is altered  (not shown). At 48 hpf, sox9b mRNA is still absent in the branchial arches of egr1 morphants, while a decreased expression relative to controls is observed in the two first pharyngeal arches   (sox9b mutant (b971sox9b) embryos, the egr1 expression pattern remains intact in the pharyngeal endoderm and epithelium as compared to wild type control siblings  . The obs11, 84%) . Converssiblings . Our resrunx3 is another gene expressed in pharyngeal endoderm and required for runx2b expression in the ventral pharyngeal chondrocytes egr1 and sox9b genes. Therefore, we decided to investigate the contribution of the runx3 gene to the regulatory cascade in pharyngeal endoderm by performing epistasis experiments.In zebrafish, in situ hybridization, we showed that runx3 expression at 48 hpf was similar in endodermal pouches of egr1 morphants  to that in control embryos      . We also73, 91%) . Importa47, 89%) . Consist98, 86%) . Finallyintained  indicatirunx3 morphants by alcian blue staining at 4 dpf, we observed that co-injection ofrunx3 mRNA caused a partial rescue in 89% of the larvae , however its co-injection with MO runx3 caused a partial rescue of the anterior part of the viscerocranium in 62% of the larvae (egr1 expression.), indicating that Runx3 is indeed acting through activation of When we tested the specificity of the effects observed in e larvae , while re larvae , while re larvae , while regr1 expression, which in turn is required for sox9b expression and, finally Sox9b presumably triggers an extracellular signal leading to runx2b expression in post-migratory cNCC and chondrogenesis.In conclusion, our results show that in pharyngeal endoderm Runx3 is required for egr1 morphants. Shh is expressed in pharyngeal endoderm gr1 morphants compared to control embryos at 48 hpf. Among the BMP factor family, bmp2a, bmp2b, bmp4, bmp5 and bmp7 are all expressed in pharyngeal endoderm. By in situ hybridization in Egr1 depleted embryos, no significant alteration of expression was observed in the pharyngeal region for any of these genes (data not shown).To determine which extracellular signal is controlled by the endodermal regulatory cascade, we decided to analyze different candidate ligands of the HH and BMP signaling pathways in casanova mutants, devoid of endoderm, sox9b mRNA was undetectable   (The gene coding for the secreted TGFß/BMP antagonist follistatin A (Fsta) is expressed in presumptive cephalic mesendoderm at 8 hpf 96, 95%) ,B and fs61, 93%) ,D in theegr1 morphants, a clear over-expression of fsta  relative to controls was observed at two days of development    , or of S79, 97%)  displayefstaTo further characterize a putative function of Follistatin A in cartilage development, we decreased its expression by injecting a splicing morpholino against Taken together, these results indicate that the endodermal cascade of transcription factors Runx3, Egr1 and Sox9b is required to reduce the level of fsta expression in the pharyngeal region.hand2 and edn1 at 24 hpf in egr1 and fsta morphants. Expression of both ventral markers was maintained in the microinjected embryos  of type 1 larvae, 23% (47/197) of type 2 larvae and 6% (12/197) of larvae with a complete loss of viscerocanium (type 3), while after treatment between 27 hpf and 37 hpf, we observed only 6% (14/231) of type 1 larvae, 77% (177/231) of type 2 larvae and finally 17% (40/231) of type 3 larvae . Treatmehsp70l:dnBmpr-GFP)w30. We performed a heat shock at 37°C during 30 minutes at 29 hpf and compared the head cartilage at 4 dpf of the transgenic larvae to that of their non-transgenic siblings ((hsp70l:dnBmpr-GFP)w30 was performed after 48 hpf, almost all larvae presented normal cartilage  (type 1). These results clearly confirm that BMP signaling is required for pharyngeal cartilage formation between 27 hpf and 37 hpf.To confirm this requirement for BMP signaling at late stages, we also used an inducible dominant negative BMP receptor-GFP transgenic line , while it remained expressed in the ethmoid plate and cleithrum. Upon heat shock of (hsp70l:dnBmpr-GF)w30 transgenic animals at 29 hpf, we observed the same percentage  of embryos expressing runx2b only in the cleithrum at 48 hpf.The cartilage defects observed upon BMP inhibition are quite similar to those obtained in ed 37 hpf . Half ofrunx2b expression at 2 dpf, similar to the defects observed upon blocking of the Runx3-Egr1-Sox9b cascade in the endoderm.Taken together, these observations indicate that inhibition of BMP signaling between 29 and 37 hpf causes cartilage defects at 4 dpf and prevents fli1-GFP embryos. We used fli-GFP expressing embryos in order to visualize the cNCC through the presence of GFP. The embryos were injected with a control morpholino or the egr1 splicing morpholino and fixed at 32 hpf to perform immunohistochemistry  in pharyngeal endoderm and sox9b mutants or morphants display a dramatic reduction of pharyngeal cartilage at 4 dpf and a lack of runx2b expression at 48 hpf, while exogenously expressed Sox9b partially rescued the mutant phenotype unx2b expression in cNCC cells.We have shown that all three factors are coexpressed in pharyngeal endoderm starting at about 30 hpf and that each of them is absolutely required for pharyngeal cartilage formation. These results are in agreement with previous studies concerning the function of Sox9b and Runx3 in zebrafish. Expression of ox9b in endoderm and runx2b in cNCCs. Expression of egr1 in endodermal pouches was deduced from single and double fluorescent in situ hybridization experiments, co-expression of egr1 with cNCC markers such as dlx2a (not shown) or sox9a was never observed. The complete absence of egr1 mRNA in the pharyngeal region of cas mutants, lacking endoderm, further supports this conclusion. The defects were observed following gene knock-down using translation or splicing morpholinos and the specificity of these defects for Egr1 depletion was shown by the rescue experiments.Here, we introduce a new player by showing that the endodermal transcription factor Egr1 is required for cartilage formation and expression of segr1 and sox9b, while runx3 expression was not affected in egr1 morphants or sox9b mutants. Egr1 depletion decreases sox9b expression only, while conversely egr1 expression is not affected in sox9b mutants. Finally, sox9b mutants display normal expression of egr1 and runx3. We further show that the defects observed in runx3 morphants can be partially rescued by expression of exogenous runx3 or egr1 mRNA, clearly indicating that Runx3 is located upstream of Egr1. Taken together, these results establish a regulatory cascade where Runx3 activates expression of Egr1, which itself then activates sox9b transcription. This cascade is not required for pharyngeal endoderm formation or the survival of pharyngeal endodermal cells, as was previously shown for Runx3 morphants egr1 or runx3 morphants or sox9b mutants, expression of the endodermal markers nkx2.3 and sox17 is not altered compared to controls. In conclusion, we describe a regulatory cascade that operates mainly in the pharyngeal endoderm and controls expression of runx2b in cartilage mesenchyme as well as cartilage differentiation and morphogenesis. This control exerted by endodermal transcription factors is obviously mediated by an extracellular signaling pathway that remains to be described.Additional experiments revealed that Runx3 depletion led to decreased expression of both runx2b expression increases after 24 hpf and is most crucial during the period between 27–37 hpf. This result was confirmed by inducing the expression of a dominant-negative Bmp receptor runx3egr1 and sox9b gene expression in pharyngeal endoderm.One of the signaling pathways involved in cartilage and bone formation is the TGFß/BMP pathway egr1 morphants, we did not detect any decrease of expression for any of these genes. During development, many processes require inhibition of BMP signaling by secreted BMP antagonists follistatin a (fsta) in the pharyngeal region at 48 hpf in egr1 morphants. Such a strong over-expression of Follistatin A would obviously lead to a perturbation of BMP signaling in the entire pharyngeal region and cause defects similar to the BMP inhibition experiments discussed above. Indeed, when we tested activation of the BMP pathway using antibodies against phospho-Smad1/5/8, we observed that egr1 knock-down dramatically reduces BMP signaling in the pharyngeal region. Pathway activation was abolished not only in the post-migratory cNCC clusters, but also in the neighboring tissues, potentially causing additional perturbations in skeletal morphogenesis.When we tested the expression of various extracellular signaling molecules  in Sox9 knock out mice Clearly, the function of BMP signaling in craniofacial development is conserved in vertebrates, including mouse and human, and the importance of antagonists such as Follistatin is also well documented. Different threshold levels of Bmp4 were shown in mouse to regulate various genes involved in craniofacial skeletal morphogenesis fsta, which needs to be tightly controlled so that the Bmp ligands can bind to their respective receptors located on cartilage precursors cells  in dorso-ventral patterning by specifying expression of dorso-ventral markers hand2 and edn1 at 24 hpf. This is consistent with the onset of expression after 24 hpf of the regulators Runx3, Egr1 and Sox9b in pharyngeal endoderm and with the observed lack of fsta overexpression in egr1 morphants at 24 hpf. An earlier function for control of BMP signaling by Follistatin A was previously shown in dorso-ventral patterning of the retina fsta expression was shown to be increased at 13 hpf upon knock-down of the TALE-class homeodomain transcription factor Meis1, leading to a decrease of BMP signaling in the optic vesicle. At 19 hpf, fsta expression was highly increased in meis1 morphants in all its expression domains fsta expression specifically in the pharyngeal region at later stages is taken over by the endodermal regulatory cascade described here.Interestingly, manipulation of the endodermal regulatory cascade leaves intact the early function of BMP signaling in dorso-ventral patterning, as indicated by the maintained expression of the ventral markers sox9a, whose expression is maintained in pharyngeal cartilage precursor cells and required for their differentiation sox9a expression. In contrast, the key regulatory gene for chondrogenesis and osteogenesis, runx2b is dramatically down-regulated in egr1 morphants. Its expression in pharyngeal cartilage precursor cells normally starts at 34 hpf fsta down-regulation for efficient activation by BMP signaling as described here. runx2b expression is down-regulated despite the normal expression of sox9a, conversely it is not affected in sox9a mutants Our results indicate that BMP signaling is required in cNCC cells after their migration to their ventral position in the pharyngeal arches to induce their differentiation into hypertrophic chondrocytes, but also to allow their subsequent migration leading to morphogenesis of the different head cartilages. Additional extracellular signals will also play a role, such as Fgfs or Hhs. At 24 hpf, cNCC cells express the early differentiation marker runx2b in presumptive head cartilage, but left nearly intact its expression in the developing cleithrum, a dermal bone. Similar observations were made in the sox9a mutants sox9a expression and fsta inhibition are mainly required for endochondral (replacement) bone formation.Interestingly, inhibition of BMP signaling by increased Follistatin A expression in our experiments led to a decreased expression of fsta gene, thereby allowing the correct activation of BMP signaling in cNCC cells required for their differentiation and morphogenesis of pharyngeal and basicranial cartilage. This cascade starts by increased expression of runx3, followed by activation of egr1 expression and finally sox9b expression in the pharyngeal endoderm.In conclusion, we show here that a regulatory cascade is active in pharyngeal endoderm that represses expression of the Figure S1casanova mutants, lacking endoderm, do not express egr1 at 48 hpf. Single in situ hybridization for egr1 in casanova mutants. Lateral  and dorsal  views, anterior to the left. Scale bars 100 µm.  Wild-type or heterozygous +/−cas express egr1 in the pharyngeal endoderm (pe).  Homozygous −/−cas do not express egr1 in the pharyngeal region, but in the pectoral fins and in the two hearts of −/−cas embryos. Pectoral fin (pf), heart (h).(TIF)Click here for additional data file.Figure S2casanova mutants, lacking endoderm, do not express sox9b or fsta at 48 hpf. Lateral views of in situ hybridizations (A–D) with indicated markers, anterior to the left. Scale bars 100 µm. Homozygous −/−cas do not express fsta  nor sox9b  compared to wild-type or heterozygous +/−cas. Pharyngeal endoderm (pe), otic vesicle (ov).(TIF)Click here for additional data file.Movie S1Double fluorescent in situ hybridization for fli1  and egr1  in 47 hpf zebrafish embryos. Lateral view, anterior to the left, the movie proceeds from the left side going to the center of the embryo. fli1 is expressed in cranial neural crest cells  and in endothelium. fli1 displays the characteristic expression pattern in “stripes” of the pharygeal cartilage precursor cells. egr1 is expressed more inside of the embryo in the pharyngeal endoderm. fli1 and egr1 mRNAs never colocalize.(MP4)Click here for additional data file.Movie S2Double fluorescent in situ hybridization for sox9a  and egr1  in 72 hpf larvae. Lateral view, anterior to the right, the movie proceeds from the right side going to the center of the embryo. sox9a is expressed in pharyngeal cartilage cells and egr1 is expressed in pharyngeal endoderm. sox9a and egr1 mRNAs never colocalize and the egr1 expression domains surrounding the sox9a expression domains can be clearly observed.(MP4)Click here for additional data file.Movie S3Double fluorescent in situ hybridization for sox9b  and egr1  at 72 hpf. Lateral view, anterior to the right, the movie proceeds from the right side going to the center of the embryo. sox9b and egr1 are expressed in pharyngeal endoderm cells and colocalize in the same expression domains.(MP4)Click here for additional data file.Movie S4Double fluorescent in situ hybridization for fli1  and egr1  at 4 dpf. Lateral view, anterior to the right, the movie proceeds from the right side going to the center of the embryo. fli1 is expressed in pharyngeal cartilages and in endothelium. Egr1 is expressed in the phrayngeal endoderm and oral epithelium. fli1 and egr1 mRNAs never colocalize.(MP4)Click here for additional data file.


Hepatitis C is prevalent among thalassemia patients in Iran. It is mainly transfusion mediated, in particular among patients treated before 1996 when blood screening was introduced.The current study aimed to investigate why patients still seroconvert to anti-HCV in Iranian thalassemia centers.During 2006-2007 sera were sampled from 217 anti-HCV positive thalassemia patients at nine thalassemia centers in Tehran and Amol city, where 34 (16%) patients had been infected after 1996. The HCV subtype could be determined by sequencing and phylogenetic analysis of partial NS5B and/or 5׳NCR-core region in 130 strains.1a (53%) was predominant followed by 3a (30%), 1b (15%), and one strain each of 2k, 3k and 4a. Phylogenetic analysis revealed 19 clades with up to five strains diverging with less than six nucleotides from each other within subtypes 1a and 3a. Strains in seven clades were from nine patients infected between 1999 and 2005 and similar to strains from eight patients infected before 1996, indicating ongoing transmission at the centers. Further epidemiological investigation revealed that 28 patients infected with strains within the same clade had frequently been transfused at the same shift sitting on the same bed. An additional eight patients with related strains had frequently been transfused simultaneously in the same room.The results suggest nosocomial transmission at these thalassemia centers both before and after the introduction of blood screening. Further training of staff and strict adherence to preventive measures are thus essential to reduce the incidence of new HCV infections. Thalassemia major and thalassemia intermedia are both common transfusion dependent anemias in Iran. There are around 18000 known thalassemia patients in the country , which iThe current study aimed to characterize HCV strains from patients at thalassemia centers in Tehran in Tehran province and in Amol City in Mazandaran province to investigate possible nosocomial transmission at these centers.Two hundred seventeen thalassemia patients positive for anti-HCV were included in the study. Out of these, 112 patients were from seven thalassemia centers in Tehran, Zafar adult thalassemia clinic, Childrens׳ medical center , Sodeh clinic, Aliasghar Hospital, Mofid Hospital, Special Medical center, Boali Hospital and from Shahid Bahonar hospital in Karaj City. The other 105 patients were from Amol thalassemia center at Imam Reza hospital in northern Iran. All staff members in Amol and Zafar adult thalassemia centers were anti-HCV negative. 100 µl plasma was added to 500 µl lysis buffers (0.5% SDS and 10mM EDTA) and 500 µl water saturated phenol. RNA was precipitated with isopropanol. The pellet was washed with 70% ethanol and dissolved in 20 µl distilled water. cDNA synthesis and semi-nested PCR were performed with primers hep101, hep102 and hep105 as previously described  to give The products obtained from the NS5B and 5'UTR-core regions were purified using GFX PCR DNA and Gel band purification kit . The sequencing reaction was carried out with the ABI PRISM TM Big Dye TM Terminator Cycle Sequencing Reaction Kit  using purified PCR products as templates. PCR products amplified within the NS5B region were sequenced with hep105, and those amplified within the 5'UTR-core region were sequenced with primers univ-1 and 186. The obtained sequences were aligned with 347 sequences of the corresponding region retrieved from Gen Bank  patients had been infected after 1996, most of them, 26 (76 %), were infected after 2000 . Fourtee Strain 752 in clade 11 was from a patient infected in 1999 and identical to strain 794 from a patient infected before 1996. Seven clades were formed by strains only from Amol (clades 1, 5, 6, 7, 8, 10 and 12; The frequencies of subtype 1b and 3a in hepatitis C infected thalassemia patients from Tehran were 25% and 22%, respectively, which were in contrast with the findings from the thalassemia centers in Amol, where 1a and 3a were the most prevalent subtypes. In addition, data from earlier Iranian studies showed subtypes 1a and 3a were the most common ones in the country with geographical differences in their distribution, and 1a was frequent among patients infected through blood transfusion -11. Seve


Increased expression of EZH2 correlates with aggressive clinical behavior in various malignancies. In this study, we aim to investigate the clinical and prognostic values of EZH2 expression and activity in tumor tissues and improve the risk stratification in patients with renal cell carcinoma after surgery. We analyzed EZH2 expression and its activity as indicated by H3K27me3 levels comprising 373 patients with renal cell carcinoma in our institute. Outcome was assessed as overall survival and disease free survival using Kaplan-Meier analysis. Prognostic values of EZH2 and H3K27me3 expression for clinical outcomes were evaluated by Cox regression analysis. We used receiver operating characteristic to calculate diagnostic accuracy. High EZH2 expression correlates with poor overall survival in all patients, especially in advanced RCC, which is an independent prognostic factor in disease free survival and overall survival. Compared with EZH2, H3K27me3 expression is not an independent prognostic factor. The expressions of H3K27me3 and EZH2 are not completely consistent, which might be due to complicated interaction of Polycomb Repressor Complex 2. A combination of EZH2 expression and TNM stage could have better prognostic value than do TNM stage or EZH2 expression alone in both sets for disease free survival and overall survival. These results imply that evaluating intratumoral EZH2 density might improve prognostic value to the TNM staging system and inform treatment decisions for patients with late-stage renal cell carcinoma.  Kidney and renal pelvis cancer were estimated to account for 65,150 new cases and 13,680 cancer-related deaths in the USA in 2013 .Histone modification is a crucial epigenetic modification. Recent findings suggested that SETD2, EZH2, and MLL2 methyltransferases, and UTX and JARID1C demethylases played crucial roles in the development of RCC ,6. HowevIn recent studies, overexpression of EZH2 is related to poor prognostic in cancers of the prostate, breast, melanoma, endometrium, and pancreas -13. HoweEthical approval was granted by the Clinical Research Ethics Committee of Zhongshan Hospital of Fudan University . Signed informed consent was obtained from all patients for the acquisition and use of patient tissue samples and anonymized clinical data.We enrolled patients from the Department of Urology, Zhongshan Hospital, Fudan University, and whose tumors were pathologically proven RCC. This database, started in 2005, is designed to prospectively study RCC patients undergoing nephrectomy. It includes patients’ baseline clinical and outcome data. Fuhrman grading system and TNM stage were according to 2004 WHO criteria and the American Joint Committee on Cancer 2010 TNM classification respectively -16. The We analyzed the EZH2 and H3K27me3 expression by immunohistochemistry. The immunohistochemical procedure (IHC protocol F) was performed on an automated immunostainer . Primary antibodies against EZH2  and H3K27me3  were used in the procedure. Two observers (Shuai. J and Lei. Z), unaware of the clinical features and outcomes of patients, evaluated slides. As previously described, scores were calculated by multiplying the staining intensity and extension ranging from 0 to 12 for each tumor or non-tumor tissue score . BrieflyWestern blot was carried out as previously described . PrimaryDFS is defined as the interval between curative surgery and recurrence or metastasis. OS is defined as the interval from date of curative surgery until death from any cause. The patients were cencerd if they were still alive until the last follow-up. We assessed disease-free survival and overall survival using Kaplan-Meier method and log-rank test.2 test, Fisher’s exact method, or CMH (Cochran-Mantel-Haenszel) χ2. Cox univariate analysis was performed. Those parameters with a p-value< 0.1 in the univariate analyses were included in a Cox multivariate proportional hazards regression model. We used ROC curves to assess sensitivity, specificity and respective areas under the curves (AUCs) with 95% (CI) for the prediction of survival. We used Logistic stepwise regression to estimate the EZH2, H3K27me3 and TNM diagnostic accuracy. The variable was removed if the p-value was greater than 0.05. A new variable predicted probability (p) for RCC was obtained by Logistic stepwise regression on the basis of an equation. All equations were provided in the We analyzed associations between EZH2 or H3K27me3 expression and clinical characteristics with χA total of 373 patients with RCC from two sets were included in this study. Clinical characteristics were much the same in each cohort. Most of the patients were men, and had clear-cell histology, Fuhrman grade II, and TNM stage I or II diseases. The median follow-up was 77 months (IQR 72-83) in the training set and 75 months (IQR 72- 82) in the validation set . RepreseDFS was our first outcome of interest. Ten patients in the training set and 11 patients in the validation set were excluded for distant metastasis before surgery or death from other causes . In bothTNM stage is the most common used clinical prognosticator for RCC patients. When compared with TNM stage, the predictive value of the EZH2 expression is much the same using ROC analysis in both sets   table 3table 3 . To deveEZH2, as a histone methyltransferase, catalyzes the trimethylation of the lysine 27 of histone H3 (H3K27me3). Our findings demonstrate that EZH2 is an independent poor prognostic factor for RCC patients undergoing nephrectomy. To our knowledge, it is the first study analysis the prognostic significance of EZH2 and H3K27me3 expression in RCC. Pathologic stages have proved to be the single most important prognostic factor for RCC. However, same cancer stage might still have diverse clinical outcomes. In this study, EZH2 expression is found to be predictive of survival of RCC patients independently. The prognostic value of TNM stage is further improved when EZH2 levels and TNM stage are combined as a predictive model. Moreover, EZH2 expression has a similar outcome predictive ability to TNM stage in ROC analysis and has good discriminatory power as a supplementary risk factor in patients with late-stage RCC. Evaluating EZH2 and H3K27me3 expression can be a clinically applicable procedure to discriminate patients with different prognosis. It could also lead to a more personalized treatment for RCC patients after curative surgery. Traditionally, cancer has been acknowledged as a genetic disease that is driven by the sequence of oncogenes or tumor suppressor genes alteration. However, epigenetic changes including histone modification, DNA methylation, and miroRNA regulation, are increasingly evident as a tumor developmental factor -21. OnceOur data showed that EZH2 has a better predictive outcome than H3K27me3 not only in late stage patients but also in all patients. Although several integrated staging systems (ISSs) have evolved and improved for prediction of prognosis and outcome for RCC, TNM staging system is still the most used clinical prognosticator and the essential guide to the evaluation of therapeutic options but is done on the basis of anatomical information ,29. MetaOur results prospectively suggest that high intratumoral EZH2 expression independently predicts poor postoperative outcome of RCC patients. Integration of intratumoral EZH2 expression levels into current TNM staging systems might improve prognostic value to patient survival and contribute to personalized therapy.Table S1New variable predicted probability (p) for RCC survival.(DOCX)Click here for additional data file.Table S2Clinical characteristics of patients according to the EZH2 and H3K27me3 expression in the training set (n=187).(DOCX)Click here for additional data file.Table S3Clinical characteristics of patients according to the EZH2 and H3K27me3 expression in the validation set (n=186).(DOCX)Click here for additional data file.Table S4Patients were excluded form DFS analysis.(DOCX)Click here for additional data file.Table S5Training set univariate analyses of factors associated with overall survival and disease free survival.(DOCX)Click here for additional data file.Table S6Validation set univariate analyses of factors associated with overall survival and disease free survival.(DOCX)Click here for additional data file.Table S7EZH2 expression positively correlates with H3K27me3 in both sets.(DOCX)Click here for additional data file.


Thus the relation between ADA1 and asthma could be mediated by events occurring during the early extrauterine life. Moreover the increased prevalence of allergic diseases in western populations parallels the widespread practice of phototherapy during the neonatal period. These observations prompted us to reevaluate our previous data and show new observations.Several studies suggest a protective role of bilirubin against oxidative damage during the neonatal period. ADAData from 2729 previously studied subjects, from 53 subjects studied at birth and after 30 years and from a survey of phototherapy frequency in four Italian Hospital including 7392 newborns are reported.1*2 allele carriers are less represented among asthmatic subjects than in controls (p=0.0004). ADA1*2 allele carriers among newborns undergoing phototherapy for hyperbilirubinemia is higher when compared to newborns not undergoing this treatment (p=0.006). In infants treated by phototherapy, the maximum bilirubin level attained during the first few days of life positively correlated with the ADA1*2 allele dose (p=0.001). Among subjects studied at birth, allergic rhinitis and/or conjunctivitis are more frequent among those treated with phototherapy than among those not treated (p=0.046).ADA1*2 allele through an increase of bilirubin level in the neonatal period protects infants from oxidative stress and favours Th2→Th1 switching thus preventing allergic manifestations in later periods of life.These observations support our hypothesis that ADA We also show (iii) a survey of the frequency of phototherapy in several hospitals.1 site controls ADA activity. Two codominant alleles, ADA1*1 and ADA1*2 are present and are associated with different enzymatic activity. The corresponding three common ADA1 phenotypes (11 is 15% more active than ADA1 2/1 and 30% more active than ADA1 2 (Adenosine deaminase (ADA) is a polymorphic enzyme present in all ammalian tissues . ADA is enotypes  have difn ADA1 2 .The ADA tissue enzymes consist of one or more molecules of RBC ADA and one molecule of adenosine deaminase complexing protein (ADPC) .ADPC is identical with CD26 (a T-cell activator molecule). ADA expression may be connected with T-cell activation , 16.ADA catalyses the irreversible deamination of adenosine to inosine. In purine metabolism, the classical function of the ADA enzyme is considered to be the regulation of intra-and extracellular levels of adenosine. ADA is present on the surface of many cell types, including lymphocytes where it acts as an ectoenzyme . A co-st1 polymorphism could modulate adenosine receptor activity in the respiratory tract via the signal transduction pathway of adenosine and other mediators.Current interest is focused on the effects produced by adenosine activation via surface adenosine receptors. Four adenosine receptors  exist on the surface of different cell types . In the Mice studies have shown a relationship between ADA and an inflammatory phenotype in the lungs, similar to human asthma. This includes extensive mast cell degranulation, eosinophilia, activation of alveolar macrophages, increase in baseline airway resistance and AHR –23.Some observations suggest that ADA activity may influence the expression of Th cytokine genes .We have reconsidered the following group samples:291 asthmatic children (sample 1) and 1417 control-children (sample 2) from the Italian population (120 asthmatic adults (sample 3) and 116 control adults (sample 4) from the Chinese Han population    (sample 7), 388 from Penne  (sample 8), 381 from the University of Rome La Sapienza (sample 9) and 1083 from the University of Rome Tor Vergata (sample 10) were considered . We have shown no difference between starch gel electrophoresis and DNA analysis.ADAphoresis  in samplChi square test of independence was performed by SPSS programs (25). Three way contingency table analysis was carried out according to Sokal and Rohlf .1 phenotypes in asthmatic patients and in controls. In both Italian and Chinese populations a significant associations between asthma and ADA1 phenotype (genotype) is observed. Carriers of ADA1*2 allele are less represented among asthmatics versus controls suggesting a protective role of this allele against asthmatic phenotypes.Table 1*2 allele among newborns treated with phototherapy for hyperbilirubinemia and among newborns not treated. The proportion of subjects carrying the ADA1*2 allele is significantly higher in newborn treated with phototherapy versus untreated newborns. This indicates that ADA1*2 allele is positively associated with higher bilirubin levels in the neonatal period.Table 1*2 allele dose.Table Table Table 1*2 allele and asthma and a positive correlation between ADA1*2 allele and bilirubin levels in the neonatal period. Parallel to these correlations are two epidemiological data: the widespread practice of phototherapy in the neonatal period and the rise of asthma and other allergic manifestations in Western populations. Preliminary data reported in Table The data shown in Tables 1*2 allele and asthma is mediated in part by the relationship between ADA1*2 and bilirubin level in the newborn. Our data support the hypothesis that ADA1*2 favouring high level of bilirubin has a beneficial effect on oxidative damage and in turn protects from allergic manifestations later in life.Because of the protective role of bilirubin from oxidative damage during the neonatal period, we speculate that the relationship between ADASubstances that contribute to antioxidant defence protect from asthma : it is p2→Th1 subpopulations during the neonatal period has not been fully studied. Increasing bilirubin level ADA1*2 allele could decrease oxidative stress favouring Th2→Th1 switch thus explaining the negative correlation between ADA1*2 and asthma.The role of oxidative stress on the switch Th2→Th1 switch cannot be excluded.The positive relationship between phototherapy and allergy observed in the small sample of subjects studied at birth and after 30 years supports our hypothesis. The decrease of bilirubin level induced by phototherapy favours the oxidative stress resulting in an increased susceptibility to allergy. However, the possibility of a direct negative effect of light on oxidative stress and on ThThe long term iatrogenic consequences of phototherapy in the population are not yet known. An epidemiological study on a larger sample of subjects examined at birth and after few decades is needed.


An ideal intracanal medicament should be able to eliminate any remaining intracanal microorganism. The aim of this study was to compare the antimicrobial effects of Bioglass 45S5 with calcium hydroxide on Enterococcus (E) faecalis in-vitro.Direct exposure test (DET) was used to evaluate the antimicrobial effect of Bioglass 45S5, calcium hydroxide and normal saline (control group) on 80 paper cones contaminated with E. faecalis suspension. All samples were aseptically transferred into BHI culture medium to quantify microbial concentration in periods of 1, 24, 48 and 72 hours. Turbidity of the culture medium was measured via optical density (OPD) method with a spectrophotometer (wavelength=540nm). Results were then analysed statistically using student t-test.Mean difference of optical density between Bioglass 45S5 and calcium hydroxide appeared insignificant within 1 hour of the test period (P>0.05); however calcium hydroxide showed significantly greater antimicrobial properties after 24 hours (P<0.05). Antimicrobial effect of both materials displayed significant increases with time.Although both Bioglass 45S5 and calcium hydroxide exhibited antimicrobial effects against E. faecalis, neither attained complete eradication of bacteria. However, calcium hydroxide seemed to have superior disinfecting effect. Bacteria and their by-products play a major role in periradicular diseases and its progress; in fact, they are the main interfering factors in the periapical repair process 2]. The . The 2].Recent microbiologic studies demonstrate wide-spread presence of resistant microorganisms such as E. faecalis and Candida Albicans in persistent canal infections and secondary infections in endodontic failures 8]9][10[9][108][[10[9]105]6][10[6][105][[10[6]. Thi. Thi13].Bioglass S53P4 is thought to be the most effective glass on pathogenic microorganisms . WaltimoThe aim of the current in vitro study was to compare the antimicrobial effect of bioactive glass 45S5 (Nova Bone) and calcium hydroxide on E. faecalis using a direct exposure test (DET).This experimental study comprised of eighty paper cones contaminated with E. faecalis (ATCC 19818). Sterile absorbent points #50 were floated in a 10(8) CFU/mL concentration of E. faecalis suspension for 5 minutes. The paper cones were then regarded as the study samples. All experimental procedures were carried out under strict aseptic conditions. The contaminated paper cones were randomly divided into four test and control groups as outlined below.Test group 1 (n=20): 5.1 g bioactive glass 45S5 powder , CaO (24.5%), Na (24.5%), P2O5 (20.6%)) mixed with 4mL sterile distilled water to form a toothpaste consistency paste.2  mixed with 12mL sterile distilled water to form a paste with the same consistency as the Bioglass mixture.Test group 2 (n=20): 6.2 g calcium hydroxide Ca(OH)The control negative group (n=20) was exposed to normal saline and autoclaved, whereas control positive samples were only exposed to normal saline. Direct exposure test (DET) was used for the evaluation of antimicrobial effects of study samples. Each paper cone was separately placed in a sterile petri plate completely covered with experimental materials or saline (control groups). Plates were placed in a 37°C, 100% humidity incubator. A total of 5 samples were aseptically removed from each group at 1, 24, 48, 72 hours intervals and irrigated with normal saline to completely eliminate materials. Each paper cone was then inserted into sterile lab tubes containing 5mL BHI (brain heart infusion broth) and placed in a 37°C incubator for 48 hours. To evaluate the microorganisms in BHI culture medium, turbidity was assessed with a spectrophotometer that measured optical density (OD/100) (at a wavelength of 540nm).The optical density of culture medium is directly related to the total of existing bacterial in the medium. Subculture of blood agar solid medium was used to identify the target microorganism and prove the existence of E. faecalis; hence, 0.01mL was pulled out of the liquid culture medium with a sterile loop and subcultured. The morphology of the colonies of E. faecalis was evaluated both macro and microscopically (gram staining) after 24 hours. All data were statistically analysed to verify normal distribution; results were then analysed using Student t-test. The level of significance was set at P=0.05.According to kolmogrov-smirnov test, all data exhibited normal distribution. The absence of microorganism growth in the negative control group proved the aseptic and sterile working condition. Growth of microorganisms in the positive control group indicated bacterial existence within the incubation periods. Therefore, uncertainty regarding any unwanted disturbing factors during the experiment could be eliminated.There was no significant difference between Bioglass and calcium hydroxide after 1 hour; however, after 24, 48 and 72 hours intervals calcium hydroxide showed superior antimicrobial properties (P<0.05). Antimicrobial effect of both materials increased with longer time intervals, both groups displayed significant decrease of optical density (OD/100) after 72 hours .Many in vitro studies have been carried out on the antimicrobial efficacy of different materials 11]18][[18][11][[[18][11]It has been shown that the efficacy of Bioglass 45S5 increases after the first 24 hours ; this isPrevious studies revealed more efficient results for antimicrobial effect of Bioglass S53P4 compared to Bioglass 45S5 19]. Zeh. Zeh19].A recent study reported that antiseptic effect of bioactive glass (BAG) S53P4 powder was stronger than that of calcium hydroxide. The formulation of Bioglass 45S5 is: Sio2 (45%), Na2o (24.5%), CaO (24.5%), P2O6 (6%), whereas, Bioglass S53P4 is Sio2 (53%), Na2o (23%), CaO (20%), P2O4 (4%).This effect was apparently not exclusively pH-related. The presence of dentin powder in suspension increased the efficacy of S53P4 against a multiplicity of oral microorganisms . The sizAntimicrobial characteristics of Ca(OH)2 is due to the release of OH ions into the environment, the increase of pH and therefore destruction of the microorganism cell wall 17]28] [28] 17][ [28] 17]A recent study, has reported that the combined effect of dentin powder and BAG is neither a matter of osmolarity or even cellular agglutination, nor is it linked to binding of bacteria to dentin powder in the presence of BAG. It was proved that only combinations of solid BAG and dentin powder and not their supernatants in suspension have an additive effect. Dentin powder with its complex surface acted as a recipient for ions in solution, and hence acted as a catalyst for the dissolution of the glass in aqueous suspension. It was this ionic flow between the glass and dentin powder that appeared to interfere with bacterial viability . It shouThe current study demonstrated that the antimicrobial effect of Bioglass 45S5 against E. faecalis was lower than Ca(OH)2, although it tends to increase with time. Considering all desirable properties of Bioglass, the material could well be incorporated into endodontics practise as an intracanal medicament in the future. Further research is recommended to find more effective formulations of Bioglass.


The whole sequence is atom economic and the application of a multicomponent reaction assures diversity.An Ugi four-component reaction of propargylamine with 3-formylindole and various acids and isonitriles produces adducts which are subjected to a cationic gold-catalyzed diastereoselective domino cyclization to furnish diversely substituted spiroindolines. All the reactions run via an  The importance of nitrogen containing heterocyclic molecules in chemical biology is undisputed. The synthesis of such biologically interesting heterocycles is generally target-oriented, inspired by nature or randomly directed. In all these cases the design of a synthetic sequence to produce a library of diversely substituted molecules is the first and most important step. The basic concept of diversity-oriented synthesis (DOS) involves short reaction sequences, a strong focus on bond construction, and functional group compatibility –3. ReactAs an efficient activator of alkynes, gold has recently attracted a lot of attention –36. Many1a), propargylamine (2a), phenylacetic acid (3a) and tert-butylisonitrile (4a) in methanol at 50 °C gave the Ugi-adduct 5a with an excellent yield of 94%. With compound 5a in hand we were keen to apply the previously developed conditions for intramolecular hydroarylation [5a with 5 mol % of Au(PPh3)SbF6 in chloroform at room temperature produced the desired spiroindoline 6a in a moderate yield of 55% along with some unidentified byproducts  instead of TFA did not improve the outcome Cl (5 mol %) and AgSbF6 (5 mol %) were loaded along with chloroform (2 mL). Ugi product 5 (0.2 mmol) was added followed by TFA (1 equiv), and the reaction mixture was stirred at rt. After completion, the reaction mixture was partitioned between EtOAc (100 mL) and 2 N K2CO3 solution (2 × 50 mL). The organic layer was washed with brine (50 mL), dried over magnesium sulfate, and evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (10% diethyl ether in dichloromethane) to afford compound 6a–q.To a screw capped vial Au(PPhFile 1Experimental section.


Among the inherited disorders of blood, thalassaemia and Sickle Cell Diseases contributes to a major bulk of genetic diseases in India. Our aim was to verify the role of the Xmn1 polymorphism as a modulating factor in sickle cell patients and frequency of the polymorphism in Indian sickle cell patients. 60 sickle homozygous and 75 sickle beta thalassemia patients were included and 5 ml blood sample was collected from them. Screening of sickle patients was done by HPLC. An automated cell analyzer SYSMEX (K-4500 Model) was used to analyze the Complete Blood Count of patients. Xmn1 polymorphism analysis was done by PCR-RFLP and one-way ANOVA test was applied to analysis of variance between groups. Among the sickle patients 27 were heterozygous (+/−) and 19 were homozygous (+/+) while 30 were heterozygous (+/−) and 24 were homozygous (+/+) in sickle β-thalassemia patients. Extremely significant differences  of hematological parameters seen among patients with Xmn1 carrier and without the Xmn1 carrier. In our cases the clinical symptoms were barely visible and higher HbF level with Xmn1 carriers were found. Presence of Xmn1 polymorphism in sickle cell patients with higher HbF were phenotypically distinguished in the sickle cell patients. We conclude that the phenotypes of Indian sickle cell patients were greatly influenced by Xmn1polymorphism.X The sequence variation has been shown to increase Hb F levels in β-thalassaemia anemia.mn1 polymorphism on the phenotype of Indian sickle cell patient; thus our aim was to evaluate the role of the Xmn1 polymorphism as a modulating factor in sickle cell patients and its frequency.The C-T substitution at position −158 of the Gy globin gene, referred to as the Xmn1-γ polymorphism, is a common sequence variant in all population groups, present at a frequency of 0.32 to 0.35.+ while 46 patients were HbSβ0). About 5 ml blood sample was collected from hematology outpatient department AIIMS; after taking their consent. Study was approved from institutional ethical committee. Clinical evaluation was done during physical examination  as well as laboratory evaluation. Age of onset of splenomegaly and jaundice was <10 year and the presence of anemia was evaluated through hemogram analysis. Complete blood count and red cell indices were measured by automated cell analyzer . Quantitative assessment of hemoglobin Hb F, Hb A, Hb A2 and Hb S and diagnosis of HbSS and HbSβ-thalassemia was performed by high performance liquid chromatography . DNA extraction done by phenol chloroform method and DNA quantification done by nano drop spectrophotometer. Xmn1 polymorphism analysis was done by PCR-RFLP method as per Suttonmn1polymorphism was used to for comparison of clinical features.Subjects were 60 sickles homozygous and 75 sickle beta thalassemia patients (29 patients were HbSβmn1 polymorphism. Fourteen (23.33%) patient in sickle homozygous and 21(28%) patients in sickle β-thalassemia were normal for Xmn1 polymorphism. The frequency of the Xmn1 polymorphism was higher among sickle homozygous than sickle β-thalassemia patients. Clinical severity were improved with homozygous (+/+) Xmn1 polymorphism in sickle cell anemia as well as sickle β-thalassemia patients. Reticulocytes, haemoglobin and red cell indices were higher in Xmn1carriers than non carriers and found extremely statistically significant . The explanation to the improvement of RBCs in Xmn1 carriers is that the anemia was less and overall red cell indices and Hb value was improved. Hyperpigmentation was found in a few cases. Splenomegaly, gall stone, painful crisis, jaundice and frequency of blood transfusion in relation with HbF and Xmn1 polymorphism were lesser in Xmn1 carriers in comparison to non-carriers and found statistically significant . Details of haematological parameters and clinical parameters of HbSS and HbSβ-thalassemia are given in Sixty sickle homozygous  and 75 sickle β-thalassemia  patients were characterized. Out of 60 sickle homozygous patients; 27 45%) were heterozygous (+/−) and 19 (31.67%) were homozygous (+/+) while 30(40%) were heterozygous and 24(32%) were homozygous in sickle β-thalassemia for X5% were hmn1 site with elevated HbF expression has been previously published. The role of increased HbF response as an ameliorating factor has become evident in patients who were mildly affected despite being homozygotes or compound heterozygotes for β0 or β+ thalassaemia.mn1 and frequency of blood transfusion was reported in thalassemic patients.21Fetal hemoglobin (HbF) genes are genetically regulated and the level of HbF and its distribution among sickle erythrocytes is highly variable. Hb F is the major genetic modulator of the hematological and clinical features of sickle cell disease with the Senegal and Saudi-Indian haplotype which gives its beneficial effects to the pateints.mn1 site with the Arab Indian haplotype is thought to be associated with high fetal hemoglobin concentration and confer a benign course of the disease. However, the clinical presentation of sickle cell disease in different regions in our country is highly variable.mn1 carriers while non carriers of Xmn1polymorphism were found worsened. Our cases of sickle cell patient showed the presence of C-T variation at position −158 in the Gγ gene, affect hematologically as well as clinically and increased production of HbF. Xmn1 carriers of HbSβ-thalassemia patients had higher HbF than HbSS patients. The clinical features of HbS-β thalassemia are extremely variable, ranging from a completely asymptomatic state to a severe disorder similar to homozygous sickle cell disease. This heterogeneity is likely to be due to the presence of different β-thalassemia alleles or interaction with modulating genetic factors like associated α-thalassemia and/or a gene for raised HbF production (Xmn1 polymorphism).mn1 site polymorphism is also likely to influence phenotypemn1 polymorphism absence reduction is associated with acquired HbF elevation.mn1polymorphism and phenotypic effect on Indian sickilers. However a study on HbEβ-thalassemia report the phenotypic effect of Xmn1 polymorphismmn1 polymorphism in thalassemia intermedia patients.mn1 polymorphism and IVS 1-1 mutation leads to a milder phenotypic presentation causing a delay in onset of blood transfusions but dose not effect the amount of blood received /kg/year. However α-thalassemia also influence on the level of HbF in patients with sickle cell disease.mn1 polymorphism found higher amongst sickle cell anemia patient in comparison to sickle β thalassemia. Sickle homozygous and sickle β-thalassemia patients showed clinical variation and this could be due to the association of Xmn1polymorphism, either in homozygous or heterozygous state. Presence of Xmn1polymorphism in sickle patients with higher HbF that improve phenotypic presentation in the sickle cell patients. A study from Western Iran in β-thalassemia patients report the presence of Xmn1 polymorphic site on both chromosomes (+/+) the level of Hb F tended to be increased compared to the absence of Xmn1 ( / ) and the presence of this polymorphic site caused a positive influence on Hb F production and the Gγ percent which could improve the clinical symptoms of β-thalassemia patients.mn1polymorphism.The strong association of X


POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5′ upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5′ upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function.POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human  Dlx5-Dlx6 genes was found by the study of five affected members displaying hearing loss and craniofacial defects. The affected individuals shared a deletion of 5,115 bp. Bioinformatic analysis of this sequence indicated the presence of several HCNR, which in a transgenic mouse reporter assay, drove expression in the inner ear and developing bones The inner ear of vertebrates is one of the most complex sensory organs of the head and host two senses, the sense of hearing and the sense of balance. From an ectodermal layer adjacent to the hindbrain, the otic placode, a spheroid organ is generated by invagination/cavitation followed by a series of developmental processes such as patterning, cell diversification and morphogenesis. In the ventral portion of the inner ear, the cochlea or auditory organ emerges as an outpocketing of the otic vesicle, while in the dorsal portion of the otic vesicle the vestibular organs, semicircular canals and endolymphatic duct are developed. In each sensory organ, the main functional unit is composed by the hair-cells, the supporting cells and the sensory neurons that connect the hair-cells to the central nervous system POU4F3 (Brn3c) is specifically expressed in hair-cells and mutations in POU4F3 causes DFNA15, an autosomal dominant form of progressive hearing loss POU3F4 (Brn4) causes deafness type 3 (DFN3), characterized by a conductive hearing loss that results from stapes fixation and progressive sensorineural deafness POU3F4 is located in the X chromosome (Xql3-q22) being one of the most frequent causes of X-linked deafness. In rats, the Pou3f4 gene is expressed during embryonic development in the brain, the neural tube, and the otic capsule at E15.5 and E17.5 days Pou3f4 might be required for epithelial-mesenchymal interactions taking place during development POU3F4 gene was identified where several microdeletions also caused DFN3 phenotype, suggesting that regulatory regions were present in that region POU3F4 enhancer within a HCNR (HCNR 81728) was described to induce reporter expression in the otic mesenchyme. This enhancer lay within the smallest microdeletions shown to cause DFN3 Xenopus coding region, at position 970 Kb (HCNR 81675) and 170 Kb (HCNR 82478) of the pou3f4 coding unit that present otic vesicle enhancer activity in a zebrafish transgenesis assay. Here, we have analyzed the spatio-temporal activity of these enhancers, as well as the signaling pathways that initiate their activity. We found that the HCNRs display distinct temporal patterns of activation and; while HCNR 81675 is regulated by the retinoic acid (RA) signaling, the HCNR 82478 is regulated by the Fgf pathway and the HCNR 81728 enhancer is under the control of Hh. Finally we present direct evidence that the distal enhancer is bound in vivo by Sox2 and Pax2 transcription factors. Altogether, these data suggest that the regulatory apparatus of the pou3f4 is multiple, complex and integrate distinct developmental inputs.The POU proteins are transcription factors that bind to DNA through their POU-specific and POU-homeodomain regions and play essential roles during development. Several members of the POU family are expressed in the inner ear. The gene Zebrafish transgenic fishes have been maintained at the PRBB Animal Facility. Our Animal Facility in accordance with national and European regulations is registered as animal research center with the number B9900073. Veterinary welfare supervision and daily water check-ups are conducted  to ensure the animals good health status. Temperature, humidity, light intensity and noise control in the room are strictly monitorized to guarantee animal welfare. Zebrafish embryos have been sacrificed after being anesthetized with 0.016% tricaine when necessary. The experimental zebrafish procedures have been performed following the protocols (CEEA-PRBB ref JMC-07-1001-CPC and MM-08-1108BAE) approved by the Ethical Committee for Animal Research (CEEA) from Barcelona Biomedical Research Park (PRBB) according to the European Union regulations.pou3f4 HCNR 81675 and HCNR 82478 GFP zebrafish lines were obtained as described in http://pipeline.lbl.gov/cgi-bin/gateway2). Subsequently genome fragments were amplified by PCR from Xenopus tropicalis, cloned into the PCR8/GW/TOPO vector and stable zebrafish lines generated by the Tol2 transgenesis system.pou3f4 HCNR 81675 and HCNR 82478 GFP embryos were staged according to morphology and somite pair number. For life GFP imaging, tricaine-anesthesized embryos were mounted in slides with glycerol and images were taken under the microscope.Whole-mount in-situ hybridization (WISH) was performed according to standard protocols HCNR 81675 and HCNR 82478 GFP transgenic embryos were chorion punctured and incubated with different pharmacological inhibitors in system water, from 5.5, 7.5 or 9.5 hours post-fertilization (hpf) stage until 18–20 hpf or 36–40 hpf respectively. The battery of inhibitors included: 30 and 50 µM SU5402  to inhibit Fgf signaling, 100 µM DAPT  to inhibit Notch signaling, 20 µM DEAB (Sigma) to block RA synthesis, 30 µM Dorsomorphin (Biomol) to inhibit Bmp signaling and 45 µM cyclopamine A (CyA) to inhibit Hh signaling. DMSO diluted at 1/200 in system water or EtOH 95% was used as the carrier control treatment.pou3f4 HCNR 81675 developed embryos were frozen in tissue-tek OCT and sectioned (12 µm). Slides were fixed in −20°C methanol for 10 minutes and blocked-permeabilized in 10% fetal bovine serum (FBS), 3% bovine seric albumin (BSA) (Sigma) in 0.1% PBT (0.1% tween-20) for 90 minutes at room temperature. Slides were stained with rabbit anti-GFP (Takara) at 1∶400 overnight at 4°C and donkey anti-rabbit Alexa 488 (Molecular Probes) at 1∶400 for 90 minutes at room temperature, both in 0.1% PBT, 10% FBS, 3% BSA. Sections were mounted in mowiol for imaging. For double immunostaining, transgenic GFP was able to be detected after all the processing, thus pou3f4 HCNR 81675 embryos were sectioned, blocked-permeabilized and stained with mouse anti acetylated-tubulin (Sigma T6793) or rabbit anti-Pax2 (Zymed laboratories ref.71-6000) at 1∶400 overnight at 4°C and goat anti-mouse Alexa 546 or goat anti-rabbit Alexa 594 (Molecular Probes) at 1∶400 for 90 minutes at room temperature. Nuclei were stained with 4′6-diamidino-2-phenylindole  for 5 minutes and mounted in mowiol.For immunostaining after WISH, pou3f4 HCNR 81675 embryos were injected into the otic vesicle with a micromanipulator with the FM® 4-64FX reagent (Invitrogen) at 1∶5 dilution in water, and then left in system water for 15 minutes, fixed in 4% paraformaldehyde (Sigma) and frozen in tissue-tek OCT (Sakura) for sectioning and imaging.Alive 3 day-old pou3f4 HCNR 81675 sequence, human and Xenopus sequences were analysed in rVISTA 2.0 (http://rvista.dcode.org/) that predicts evolutionarily conserved transcription factor binding sites. Moreover, the sequence of Xenopus was also analysed by Transfac 7.0 in the Gene Regulation portal (http://www.gene-regulation.com/index.html). Pax2 and Sox2 binding sites found in the pou3f4 HCNR 81675 were subjected to site-directed mutagenesis. Based on the Transfac motif and literature research, we mutated the core nucleotides of the Pax2 and Sox2 binding sites by designing primers that contained the Pax2 binding core GTGAATAG mutated into TCAAACAT or the Sox2 binding core ACAAAA mutated into GTGCTC. Mutant primers were used to introduce these point mutations into the pCR8/GW/TOPO TA® - HCNR 81675 construct using the Quik Change® XL Site-Directed Mutagenesis Kit (Stratagene). Transgenic zebrafish containing either Pax2 or Sox2 mutant binding sites as well as the double mutant for Pax2 and Sox2 motifs were generated using the ZED vector and Tol2 transposon/transposase transgenesis method In order to predict conserved TFB sites in the Xenopus embryos were dissected and used for Chromatin immunoprecipitation assay. ChIP analysis was performed with minor modifications as described previously Xenopus pou3f4 HCNR 81675 were designed , whereas primers for a CNR in the Xenopus haemoglobin-2 locus with no predicted Pax2 and Sox2 binding sites according to rVISTA and Transfac databases were used as negative control .ACA, reverse Pictures were acquired in a Leica DRM microscope using a Leica DFC300 FX camera and the Leica IM50 software. Adobe Photoshop CS2 software was used for photograph editing.Xenopus and zebrafish to identify several highly conserved non-coding regions (HCNR) in the 1 Mb 5′ upstream region of the pou3f4 transcription start site with enhancer activity in the developing inner ear pou3f4 transcriptional start site  to detect GFP mRNA was performed. WISH in the same stage embryos revealed that, in both ectively . Thus, apou3f4 early expressed enhancers activate reporter GFP expression at different developmental stages in zebrafish, we next assayed whether the spatial pattern of expression in the inner ear also presented particularities. From 13 hpf to 24 hpf, GFP driven by HCNR 81675 is observed in almost the entire otic placode , the RA (DEAB) and Bmp (Dorsomorphin) pathways, smaller otic vesicles were observed since all three pathways play essential roles in otic placode formation pea3, neuroD, krox20, msx1 and ptc1 was done in inhibitor-treated embryos to confirm that each signaling pathway was abrogated at our working concentrations . These results were further confirmed by double labelling experiments. Double immunostaining with anti-GFP antibody and anti-Pax2a showed co-localization of both proteins in the same domain  observed in embryos transgenic for the HCNR 81675 enhancer, correlated with abrogation of  embryos . All togpax2a and sox2 are co-expressed in a similar domain than that promoted by the HCNR 81675 enhancer and retinoic acid inhibition results in loss of both, GFP and pax2a and sox2 expression, we then checked for Pax2 and Sox2 binding sites in the HCNR 81675 genomic sequence. TRANSFAC analysis of human and Xenopus HCNR 81675 sequences revealed a high number of conserved putative Transcription Factor Binding Sites (TFBS). Interestingly for the work presented here, a putative Sox motif and two Pax2/5/8 motifs where found in the sequence . PCR primers were designed to include the region of Pax2 and Sox2 binding sites. Otic vesicles from stage 30 to 34 Xenopus embryos were dissected and the chromatin was immunoprecipitated with either rabbit anti-Pax2 and rabbit anti-Sox2 antibodies or rabbit IgG as control. pou3f4 HCNR 81675 DNA was precipitated by the anti-Pax2 and anti-Sox2 antibodies but not by the isotopic antibody. Moreover, no binding of these two proteins in the Xenopus haemoglobin promoter which lacks Pax2 and Sox2 binding sites was detected, confirming the specificity of Pax2 and Sox2 binding to the endogenous Xenopus HCNR 81675  syndrome not only affect the POU3F4 coding sequence POU3F4 gene. Accordingly, we and others have recently identified several pou3f4 inner ear enhancers within this genomic region Hearing impairment is one of the most prevalent sensorineural defects in humans and in the last years many human ear disorders have been linked to mutations in over a hundred different genes POU3F4 expression mainly to the periotic mesenchyme in transgenic lacZ mouse embryos e POU3F4 coding unit several other evolutionary conserved sequences (from human to Xenopus) were identified Xenopus HCNR 81675 and HCNR 82478 specifically drive reporter GFP expression to the otic epithelium and at later stages to the inner ear sensory patches. HCNR 81675 directs reporter gene expression only to the otic vesicle, while HCNR 82478 acts as a general enhancer driving pou3f4 expression to all the tissues where it is expressed such as the otic vesicle, midbrain-hindbrain boundary, mesonephros and spinal cord. Both new otic enhancers are at each side of the previously identified POU3F4 enhancer pou3f4 expression to the otic territory indicating that Pou3f4 is a crucial transcription factor for inner ear development and that its expression needs very fine-tuned regulation. This is further exemplified by its distinct temporal activity. In stable transgenic fishes, GFP in the otic vesicle is switched on at different developmental stages, being the enhancer at HCNR 81675 active before the one at HCNR 82478, and this one before the regulatory element located in HCNR 81728. Moreover, transcriptional activation directed by the HCNR 82478 enhancer starts in the mesonephros, followed by activity in the midbrain-hindbrain boundary and finally to the otic vesicle. Later on, at 24–32 hpf GFP is no longer detected in the mesonephros and in contrast it is activated in the spinal cord.Previous work published by Ahn and colleagues described a human HCNR that specifically directs Hox and Irx gene clusters or the Sox2 gene Sox2, Kondoh and colleagues have nicely dissected out the genomic regulatory apparatus of the chicken Sox2 locus. Eleven enhancers were identified and from those, three and two enhancers control the expression of Sox2 in the spinal cord and otic placode, respectively.It has been well reported that developmental genes are very tightly regulated and thus in their loci several scattered enhancers are contained around or in the gene that regulate. Examples are found in the locus of the fgf3 and fgf8 results in complete ablation of the otic placode, while in chick and mice Fgfs from the mesoendoderm primarily control the process fgf expression POU3F4 mesenchymal enhancer; second, previous reports in mice indicated that Pou3f4 cooperates with Tbx1 during cochlear development Pou3f4 expression is reduced in conditional Tbx1 mutants pou3f4 expression, acting separately over distinct enhancers located at different HCNRs.Inner ear development is highly complex and many distinct signaling pathways regulate it. Fgf signaling for example is used throughout ear development to control distinct processes, initially is essential for the induction of the otic primordium from the ectoderm. In zebrafish embryos, loss of pou3f4 HCNR 81675 co-localized with the pax2a and sox2 expression domains in the otic vesicle. Since Sox and Pax but not Tbx binding sites were found in the HCNR 81675 sequence, we hypothesized that both Pax2 and Sox2 might be activating the enhancer in the zebrafish transgenic line. This was confirmed by chromatin IP and site-directed mutagenesis of the Pax2 and Sox2 binding sites that showed that Pax2 and Sox2 TF are bound directly to the enhancer and regulate GFP expression in vivo. Altogether, our results suggest that early pou3f4 expression directed by the HCNR 81675 enhancer may be regulated by retinoic acid and Sox2 and Pax2 transcription factors. Our mutagenesis analysis of the HCNR 81675 enhancer plus the fact that GFP is only found in domains of pax2a and sox2 co-expression, indicate that Pax2 and Sox2 transcription factors alone are not sufficient to activate this enhancer and act in a cooperative manner over the genomic locus. This would be similar to what has been reported by the cooperative interaction of Pax6 and Sox2 in the δ-crystallin and N3 Sox2 enhancers GFP driven by pou3f4/Pou3f4 gene is expressed in the midbrain-hindbrain boundary, mesonephros and spinal cord, as well as the periotic mesenchyme in Xenopus and mouse embryos pou3f4 expression in the periotic mesenchyme and the activation of the pou3f4 enhancers in the otic epithelium in zebrafish. First, endogenous pou3f4 transcripts might be present in the otic epithelium at lower and undetectable levels by in situ hybridization; secondly developmental differences of expression might appear when enhancers are extracted from their genomic context. The latter hypothesis of an improper regulation of foreign enhancers is favoured by the fact the human enhancer described by Ahn et al. 2009 when inserted in mice Endogenous POU3F4 gene and their regulation may contribute to the further understanding of the function of POU3F4 in inner ear and its implications in DNF3.In conclusion, the description of the spatiotemporal activity of novel enhancers of Figure S1HCNR 81675 activity is not dependent on RA, Fgf, Notch, Bmp and Hh at 7.5 and 9.5 hpf. (A–N) GFP is observed after treatment of HCRN 81675 transgenic embryos with pharmacological inhibitors of signaling pathways at 7.5 hpf (A–G) and 9.5 hpf (H–N). Orientation is anterior to the left and dorsal up.(TIF)Click here for additional data file.Figure S2Abrogation of different signaling target genes after treatment with specific signaling inhibitors. (A–J) In situ hybridization for the Fgf, Notch, Retinoic Acid, BMP and Sonic Hedgehog target genes pea3 (A–B), neuroD (C–D), krox20 (E–F), msxC (G–H) and ptc1 (I–J) to confirm inhibitor activity at our working concentrations. Dorsal view, orientation is anterior to the left.(TIF)Click here for additional data file.Figure S3HCNR 82478 activity is dependent on Fgf signaling when treated at 5.5 and 7.5 hpf. (A–N) GFP is inhibited after treatment of HCRN 82478 transgenic embryos with 30 µM SU5402 at 5.5 hpf (A–G) and 50 µM SU5402 at 7.5 hpf (H–N). Orientation is anterior to the left and dorsal up in all images.(TIF)Click here for additional data file.Figure S4Endogenous expression pattern of pou3f4/Pou3f4 in Xenopus and mouse. (A–B) In situ hybridization for pou3f4 mRNA in Xenopus embryos of stage 35 (A) and stage 42 (B). Note that the endogenous expression is detected at the periotic mesenchyme at stage 42, whereas at stage 35 pou3f4 is still not expressed. (C–D″) In situ hybridization for Pou3f4 mouse mRNA in mice embryos of stage E8.5 (C) and E16.5 (D). In mice, also Pou3f4 is expressed at the otic mesenchyme at later stages, shown in insets . Transverse sections shown in all panels.(TIF)Click here for additional data file.