Noonan syndrome (NS) and LEOPARD syndrome (LS) cause congenital afflictions such as short stature, hypertelorism and heart defects. More than 50% of NS and almost all of LS cases are caused by activating and inactivating mutations of the phosphatase Shp2, respectively. How these biochemically opposing mutations lead to similar clinical outcomes is not clear. Using zebrafish models of NS and LS and mass spectrometry-based phosphotyrosine proteomics, we identified a down-regulated peptide of Fer kinase in both NS and LS. Further investigation showed a role for Fer during development, where morpholino-based knockdown caused craniofacial defects, heart edema and short stature. During gastrulation, loss of Fer caused convergence and extension defects without affecting cell fate. Moreover, Fer knockdown cooperated with NS and LS, but not wild type Shp2 to induce developmental defects, suggesting a role for Fer in the pathogenesis of both NS and LS. PTPN11 encoding for Src-homology domain 2 (SH2) containing phosphatase 2 (Shp2) PTPN11 and patients display similar symptoms as NS. An acronym of the symptoms, Lentigines, Electrocardiographic conduction anomalies, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retarded growth and Deafness gave this syndrome its name, LEOPARD syndrome (LS)(OMIM 151100) DrosophilaNoonan syndrome (NS) (OMIM 163950) is a congenital disorder that manifests itself in heart defects, short stature, webbed neck, hypertelorism and an increase in the occurrence of juvenile myelomonocytic leukemia (JMML) and other malignancies. The most common causes for NS are mutations in The phosphatase Shp2 consists of two N-terminal SH2 domains that are able to bind to tyrosine phosphorylated targets of Shp2, a protein tyrosine phosphatase (PTP) domain, and a C-terminal tail with a proline rich domain and two tyrosines that are able to bind Grb2 when phosphorylated. Under basal conditions, the N-SH2 domain blocks access of the PTP domain to its substrates, resulting in an inactive conformation of Shp2 in utero and have gastrulation defects, showing malformations of the notochord and posterior truncations spina bifida and secondary neural tubes resulting from gastrulation defects Homozygous mice defective for Shp2 die As NS and LS Shp2 are thought to act biochemically opposite, yet give rise to similar clinical symptoms, we sought to identify potential common targets of NS and LS Shp2 in a zebrafish model. Pinpointing disease associated Shp2 signaling shared by NS and LS, may contribute to the understanding of the underlying mechanisms of NS and LS pathogenesis and the development of therapeutic strategies for both NS and LS. Using a comparative phosphoproteomics approach, phosphotyrosine (pTyr)-containing peptides of NS- and LS-Shp2 expressing zebrafish embryos were isolated, identified and compared to peptides of WT embryos. A peptide corresponding to the autophosphorylation site of Fer kinase was identified as one of the most down-regulated peptides in developing NS and LS zebrafish. Further investigation revealed a role for Fer in C&E cell movements during gastrulation. Downregulation of Fer cooperated with NS and LS to induce developmental defects, suggesting a genetic interaction between Fer and the NS and LS variants of Shp2.Only embryos up to 4 days post fertilization (dpf) were used for these experiments, which do not require approval of the animal experiments committee according to national and European law.ba2Tg(−4.9sox10:EGFP) zebrafish were kept under standard conditions. Embryos were staged as described by Westerfield In situ hybridizations were done as described before bmp2b, chd, cyc, gsc, krox20, myod, ntl, pax2 and six3. For the fer probe, Fer-pBSK- was digested with NotI for the antisense probe and with ApaI for the sense probe, respectively. T7 and T3 RNA polymerase were used to generate digoxigenin labeled RNA (Roche). Embryos were fixed at the indicated stages with 4% paraformaldehyde (PFA). Embryos were treated from 24 hpf onwards with 0.2 mM 1-phenyl 2-thiourea (Sigma) in E3-medium to block pigmentation.Wild type and fer was amplified from cDNA of 1 dpf WT embryos and cloned into EcoRI/XhoI sites of pCS2+ and pBSK- and verified by sequencing using: Fer_out_Fw: 5′-GAGAGAGACTGCGTGCCTTG-3′, Fer_out_Rv: 5′-ATGAGATTCTGAGGGCGAAA-3′ and Fer_EcoRI_Fw: 5′-GGCGAATTCATGGGGTTCGGCCGGGAC-3′, Fer_XhoI_Rv: 5′-CGGCTCGAGTTAGGGATCACCTGGATG-3′. The mutation Y716F was introduced into fer by site-directed mutagenesis using Fer_Y716F_Fw: 5′-TCCTGATGAGGAGAAGATGCCGTCGTC-3′ and Fer_Y716F_Rv: 5′-GACGACGGCATCTTCTCCTCATCAGGA-3′.5′-GCGCCATTGCTTTGCAAGAATTG-3′) was described before and was used as recommended by the manufacturer 5′-GGCTTCTGGTGATCTGTTGAAATA-3′) was used at 1.0 ng/µl (high concentration) or 0.5 ng/µl (low concentration). Fer MO e9i9 (5′-GGGATTACACTTACTGACGAAGAGC-3′) was used at 2.5 ng/µl (high concentration) or 1.0 ng/µl (low concentration). For co-injection, suboptimal concentrations of both MOs were combined.Fer splicing morpholinos (MOs) were designed for several intron-exon boundaries and ordered from GeneTools . The p53 MO (Embryos were anesthetized with tricaine methane sulfonate (Sigma) and fixed in 4% PFA at 4 dpf. After several hours of fixing, embryos were washed for 10 min in 50% ethanol. Embryos were stained overnight at 4 degrees in staining solution (0.04% Alcian blue (Sigma), 70% ethanol and 50 mM magnesiumchloride) and washed in 0.2% Triton X-100 (Sigma). Afterwards, embryos were bleached to remove pigmentation  and washed with 0.2% Triton X-100. Images were quantified by using ImageJ software.Total RNA of 3 dpf Nacre MO and Fer MO injected embryos was isolated. Approximately 30 embryos were dissolved in 1.0 ml TriZol (Invitrogen). The homogenate was centrifuged . 0.2 ml Chloroform was added to the supernatant, vortexed for 15 sec, incubated for 3 min at room temperature (RT) and afterwards centrifuged . 0.5 ml isopropanol was added to the upper phase, incubated for 10 min at RT and centrifuged . The pellet was washed with 75% ethanol and after vortexing centrifuged . The pellet was dried for 5 min and dissolved in 100 µl deionized water. Subsequently, the solution was incubated for 10 min at 37°C, precipitated in 250 µl 100% ethanol and 10 µl 3 M sodium acetate and stored at −80°C.5′-TTTCTGTCCCCATTGCAAAG-3′, Fer_Rv_exon_7: 5′-GATTCTGCTGGGTTATTAGC-3′, Fer_Fw_exon_6: 5′-GAACCTAATGTAGAATTTGATGC-3′, Fer_Fw_exon_8: 5′-CGGAGGCCAAACTCATGGCACA-3′ and Fer_Rv_exon_10: 5′-CGCACCAGAAAGTCTCCCTG-3′. Gadph was used as a loading control for the RT-PCR reaction.For the reverse transcription reaction (RT-PCR) with M-MLV-RT (Promega) 1 µg of total RNA was used. PCR of cDNA was performed using standard protocol of GoTaq (Promega) using Fer_Fw_intron_5: Embryos were injected at the 1-cell stage with 150 pg wild type Shp2, 150 pg Shp2-D61G or 50 pg Shp2-A462T RNA, respectively. Shp2 RNA injected embryos were co-injected with enhanced green fluorescent protein (GFP) RNA used for screening of injection efficiency. Embryos expressing GFP were selected, manually dechorionated and collected at 26 hpf to 28 hpf. Embryos were then deyolked and washed using deyolking buffer (1/2 Ginzburg Fish Ringer) without calcium (2×10 µl/embryo) snap frozen and stored at −80°C for further usage Lysate supernatants equivalent to 3 to 4 mg total protein per condition were generated in principle as described previously IP was performed as described 18- AQ, 3 µm ; 40 cm * 50 µm inner diameter, packed in house) at ∼100 nl/min and eluted using a 3-h gradient from 0 to 40% solvent B (0.1 M acetic acid in 8∶2 (v/v) acetonitrile/water). The eluent was sprayed via distal coated emitter tips (New Objective) butt-connected to the analytical column. The LTQ Orbitrap XL was operated in data-dependent mode, automatically switching between MS and MS/MS. Full-scan MS spectra (from m/z 350 to 1500) were acquired in the Orbitrap with a resolution of 60,000 at m/z 400 after accumulation to target value of 500,000. The three most intense ions at a threshold above 500 were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35% after accumulation to a target value of 10,000.As described in 2 spectra were processed and quantified with Proteome Discoverer (version 1.3.0.399). Runs were searched against a concatenated forward-decoy version of the Zebrafish Uniprot database  with Mascot . The database search was performed with the following parameters: a mass tolerance of ±50 ppm for precursor masses and ±0.6 Da for CID fragment ions, allowing two missed cleavages, cysteine carbamidomethylation as fixed modification. Light, intermediate and heavy dimethylation of peptide N-termini and lysine residues; methionine oxidation; phosphorylation on serine, threonine and tyrosine were set as variable modifications. The enzyme was specified as trypsin. The phosphorylation site localization of the identified phosphopeptides was performed by the phosphoRS algorithm 3.1 implemented in Proteome Discoverer using a 75% cut-off. For phosphopeptides that did not have a phosphorylation site with a pRS score above 75%, the site was counted as “ambiguous”. The dimethyl-based quantitation method was chosen in Proteome Discoverer, with mass precision requirement of 2 ppm for consecutive precursor mass measurements. A 0.5 min retention time tolerance was applied for isotope pattern multiplets and allowed spectra with maximum 1 missing channels to be quantified. Mascot results were further filtered with the following criteria: (i) mass deviations of ±10 ppm; (ii) Mascot ion score of at least 20; (iii) a minimum of 6 amino-acid residues per peptide; and (iv) position rank 1, which results in a peptide FDR<1%. Phosphopeptides that were found to be differentially phosphorylated were manually validated.All MSQuantified phosphopeptides were normalized against the median of all non-phosphopeptides. For peptides with phosphorylation sites with a pRS score above 75%, only these phosphopeptides were used for quantification. For peptides with only pRS scores below 75% all phosphopeptides were used for quantification, but the phosphorylation site was counted as “ambiguous”. Log2 ratios of heavy/light and medium/light of more than 1 or less than −1 were accepted as significant changes in phosphorylation levels.Phosphorylation sites of each peptide were compared to the total protein sequence of the corresponding zebrafish protein to identify the phosphorylation site. These sites were then compared to human protein in Phosphosite.org. For ambiguous phosphorylation sites (pRS<75) the sequence was compared to Phosphosite.org to identify the most commonly identified site. Non-annotated peptides were BLASTed against the zebrafish proteome to identify the protein.2O, light) for the WT embryos, deuteroformaldehyde  for the NS embryos and 13C deuteroformaldehyde , for LS embryos. Labeled peptides were then combined and the mixture was processed further. The labeled peptide mixtures were enriched for pTyr by immunoprecipitation, using PY99 agarose beads To identify novel downstream targets of disease associated Shp2 that are affected similarly in NS and LS, we performed a mass spectrometric analysis on zebrafish embryos as outlined in 1 spectra and normalized to the total ratio of all (non-phosphorylated) peptides as input. The input corresponding to the LEOPARD Shp2 expressing embryos (heavy labeled) was 2.35 fold higher than wild type (light labeled), whereas Noonan (medium labeled), was 1.38 fold higher than wild type. Whereas many peptides were identified, only a small number of phosphopeptides were identified and of suitable quality for quantification  (peptide “c”) (Log2: 1.50 fold), Y185 (Log2: 1.16 fold) (peptide “f”) and T183 (Log2: 1.18 fold) (peptide “e”) of a MAPK12/MAPK14 protein (zgc:171775). Additionally, we identified a phosphopeptide that was decreased in LS including Y666 of receptor tyrosine kinase-like orphan receptor 2 (ROR2) (peptide “j”) (Log2: −1.15 fold).Multiple peptides that were affected in a similar way (upregulation or downregulation in both NS and LS) were identified (lower-left and upper-right quadrant). To gain further understanding in how activating and inactivating mutations in NS and LS, respectively lead to similar outcomes, we chose to focus on this group of peptides. Two of the main hyperphosphorylated peptides in both NS and LS corresponded to PZR, an earlier identified target of Shp2 2 spectrum of the Fer phosphopeptide is given in http://www.phosphosite.org). In addition, the peptide was immunoprecipitated with anti-pTyr antibodies, which indicates that this tyrosine is likely phosphorylated. Interestingly, this tyrosine is the autophosphorylation site of Fer At the other end of the spectrum, the most decreased phosphopeptide in NS and LS corresponded to Fer kinase . The MSThe Fujinami poultry sarcoma (fps)/Feline sarcoma (fes) related kinase (Fer) is a widely expressed, 94 kDa non-transmembrane protein tyrosine kinase (PTK) that is associated with cell migration, tumor growth and was first described in 1986 Homo sapiens to Danio rerio  which is the Fer autophosphorylation site, phosphorylation of which results in activation of Fer kinase activity io rerio , all codin situ hybridization (ISH) was performed on zebrafish embryos, using a fer-specific probe  induced developmental defects including reduced body axis length, heart edema and craniofacial defects at 3 dpf . These dt of p53 . Taken tba2Tg(−4.9sox10:EGFP) transgenic line, which expresses GFP in neural crest-derived cartilage in the head n = 40), injection of high levels of Fer MO e5i6 induced an increased angle of the ceratohyal , as did injection of high levels of Fer MO e9i9  which were both significant (p<0.005 Student's t-test) (n = 57 (n.s.) and a ratio of 0.93±0.01 (p<0.001). Low dose Fer MO e9i9 injected embryos showed an angle of 88±1, n = 50 (n.s.) and a ratio of 0.89±0.01 (n.s.). Co-injection of both MO's at low doses together resulted in a drastic increase of craniofacial defects. Quantification of the craniofacial defects of these embryos showed an angle of 123±6, n = 70 (p<0.001) and a ratio of 0.79±0.01 (p<0.001)  . The rat t-test) . Embryosp<0.001) .We tried to rescue the Fer MO-induced developmental defects by expression of mRNA encoding full length Fer. Unfortunately, despite many attempts, we failed to rescue the Fer MO induced developmental defects . Expressin situ hybridization (ISH) using probes for myoD, staining the somites and krox20 staining rhombomeres 3 and 5, a verified method to quantify C&E defects in developing zebrafish embryos . Whereas Fer knockdown caused developmental defects , partialUsing a comparative phosphoproteomics approach focused on pTyr-containing proteins, we identified a phosphopeptide corresponding to Fer kinase as the main decreased phosphopeptide in zebrafish embryos expressing NS or LS mutant Shp2 compared to WT. Fer knockdown induced developmental defects in zebrafish embryos that are reminiscent of defects induced by NS and LS Shp2. Epistatic interaction analyses suggested a genetic interaction between Fer kinase and mutant Shp2.in vivo gives more relevant insights into the etiology of the disease. Therefore, we performed for the first time a comparative phosphoproteomics experiment in zebrafish using pTyr immunoprecipitation and stable isotope dimethyl labelling.Previously, we used comparative phosphoproteomics in zebrafish to identify differences between wild type and Fyn/Yes knockdown embryos fps/fes/fer homologues exist in sponges (Fes/FER_SR), D. melanogaster (dfps85D) and C. elegans (FRK-1) C.elegans, yet its kinase activity seems to be redundant in development fer and ptpn11, since loss of Fer contributes to the NS and LS phenotype Click here for additional data file.Figure S2Attempted rescue of Fer MO and overexpression of Fer. Embryos were injected at the 1-cell stage with 2.5 ng Fer MO e9i9 either alone or in combination with increasing amounts of synthetic Fer mRNA . Co-injection of Fer mRNA did not rescue the Fer knockdown phenotype. B. embryos were injected at the 1-cell stage with Fer WT mRNA. Injection of Fer mRNA by itself induced craniofacial defects, shorter length and heart edema.(TIF)Click here for additional data file.Table S1Raw MS data. Tab1 “PD_Output” shows raw, non-normalized data from Proteome Discoverer, which is used for normalization on all nonphosphopeptides. “numberOFphospho”: number of phosphoresidues present in the peptide. “numberOFlocalized”: number of localized phosphoresidue (pRS>75). “peptideSite”: localized site. Tab2 “Aggregation” shows data averaged per rows based on the following values . “SITES” tab shows all normalized, quantified, localized, protein assigned peptides. “SITES with lacking protein” tab shows all normalized, quantified, localized, peptides, including non-protein assigned peptides. “quantified only pRS<75” shows all normalized quantified peptides for which no phosphosite could be assigned. “all quant phosphopepts combined” shows a combination of “SITES with lacking protein” and “quantified only pRS<75” with duplicate peptides for which one site is assigned and the other is not removed. “(XLSX)Click here for additional data file.


The results showed that phoxim treatments led to brain damages, swelling and death of neurons, chromatin condensation, and mitochondrial damage. Normal nerve conduction was severely affected by phoxim treatments, as revealed by: increases in the contents of neurotransmitters Glu, NO, and ACh by 63.65%, 61.14%, and 98.54%, respectively; decreases in the contents of 5-HT and DA by 53.19% and 43.71%, respectively; reductions in the activities of Na+/K+-ATPase, Ca2+/Mg2+-ATPase, and AChE by 85.27%, 85.63%, and 85.63%, respectively; and increase in the activity of TNOS by 22.33%. CeCl3 pretreatment can significantly reduce such damages. Results of DGE and qRT-PCR indicated that CeCl3 treatments significantly upregulated the expression levels of CYP4G23, cyt-b5, GSTs-σ1, ace1, esterase-FE4, and β-esterase 2. Overall, phoxim treatments cause nerve tissue lesions, neuron death, and nerve conduction hindrance, but CeCl3 pretreatments can promote the expression of phoxim resistance-related genes in silkworm brains to reduce phoxim-induced damages. Our study provides a potential new method to improve the resistance of silkworms against OP pesticides.CeCl Therefore, it is also used as a model insect to study the toxicology of pesticides and pest control and serves as an environmental indicator. Organophosphorus (OP) based pesticides are one of the most widely used pesticides. The main mechanism of action of OP pesticides is to irreversibly bind acetylcholinesterase (AChE) and inhibit its activity, which leads to the accumulation of neurotransmitter acetyl choline (ACh) in synaptic clefts then insect convulsion and eventual death, because nervous excitement cannot be terminated2Silkworm 3 pretreatment could increase the growth and survival rate of B. mori under phoxim-induced toxicity by increasing antioxidant capacity and improving protein and carbohydrate metabolism3 pretreatment could decrease oxidative damage of B. mori caused by nucleopolyhedrovirus infection via increasing antioxidant capacity3 pretreatment could alter the gene expression pattern and relieves the damages in the midgut of B. mori caused by phoxim3 can relieves the damages caused by phoxim, protect B. mori silk gland and remit the reduction in body weight and cocooning rate through changing gene expression patterns and reducing oxidative stressRare earth elements (REEs) are widely used in various industries due to their diverse physical and chemical properties3 can relieve the brain damage in B. mori caused by phoxim is still unknown. In this study, brain was used as the study object to further explore the mechanism of relief from toxic symptoms caused by phoxim under CeCl3 pretreatment.Brain is the main part of central nervous system and the important target of phoxim. Whether CeClB. mori larval brain sections in 3-treated group , nitric oxide (NO), ACh, 5-hydroxytryptamine (5-HT), and dopamine (DA) did not change significantly. However, in the phoxim-treated group, contents of Glu, NO and ACh were all significantly increased, by 63.65%, 61.14%, and 98.54%, respectively (P < 0.01), while the 5-HT and DA contents were significantly decreased by 53.19% and 43.71%, respectively (P < 0.01)  from those of the control group; as a contrast, phoxim treatments significantly reduced the activities of Na+/K+-ATPase, Ca2+/Mg2+-ATPase, and AChE by 85.27%, 85.63%, and 85.63%, respectively , while significantly increasing the activity of TNOS by 22.33% (P < 0.01)  . These r < 0.01) ; these r3’s and phoxim treatment, we used the digital gene expression (DGE) method to detect the differences in gene expression. The CeCl3-treatment group , cytochrome b5 (cyt-b5), GSTs-σ1, acetylcholinesterase type 1 gene (ace1), esterase-FE4, β-esterase 2, and catalase (CAT) were increased 12.583, 8.623, 16.462, 5.843, 6.714, 2.583, and 2.793 fold, respectively. With CeCl3 treatment, CYP4G23 was increased by 3.472 fold, while phoxim treatment increased CYP4G23’s level by 6.363 fold, ace1’s level by 19.453 fold, and esterase-FE4’s level by 3.671 fold  microplusMyzus persicae (SULZER)3 treatments increased the expression levels of AChE, β-esterase 2, and esterase-FE4 , and serine-pyruvate aminotransferase (AGT)  , these eet al. attempted to hydrolyze the phosphodiester of OP using Cerium complexes with saccharides and revealed that the complexes were able to degrade methamidophos, omethoate and dimethoateCerium ion has some special features, including functioning as an antibiotic3 pretreatment could reduce phoxim-induced nerve damages. CeCl3 enhanced the expression of detoxification enzymes, esterases, P450s, and GSTs, which participate in phoxim metabolism; CeCl3 could also upregualte the genes related to free radical clearance and decrease the expression of apoptosis genes to protect neurons in the brain. As a result, CeCl3 may enhance phoxim metabolism in silkworms to protect their nerve systems. This study illustrated the potential mechanisms of CeCl3’s effect to enhance phoxim metabolism in silkworm brains and provided a theoretical basis to use CeCl3 as an additive to improve silkworms’ pesticide tolerance in sericulture.In this study, we found that CeClB. mori (strain: Qiufeng × baiyu) maintained in our laboratory were reared at 27 ± 2 °C on mulberry leaves under a 12 h light/12 h dark cycle.The larvae of 3  was purchased from Shanghai Chem. Co. (China).Phoxim was purchased from Sigma-Aldrich (USA). CeClLC50) of phoxim in B. mori was 7.86 μg/mL, and at 4 μg/mL, B. mori showed poisoning symptoms without death13 was used, which were administered to fifth-instarlarvae, the optimum concentration of CeCl3 was 0.5 mg/L for growth of these larvaeB. mori larvae were feed with CeCl3-treated leaves (leaves were dipped in 0.5 mg/L CeCl3 solution for 1 min and dried in the air) and normal leaves respectively three times a day until the 2nd day of fifth-instar. Then a part of the larvae of these two groups were feed with phoxim-treated leaves (leaves were dipped in 4 μg/mL phoxim for 1 min and dried in the air). Each treatment was performed three times. Forty-eight hours after phoxim treatments, 100 fifth-instar larvae was selected randomly from each group to collect brain tissues that were frozen at −80 °C for further study.Phoxim stock solution was prepared by 10 × dilution using acetone. For the treatment, phoxim stock solution was dissolved in water to obtain a concentration of 4 μg/mL. The lethal concentration , placed onto glass slides, and stained with hematoxylin-eosin for 15 min. Stained samples were observed and photographed using an optical microscope .Five larvae’s brain tissues of each group were fixed in freshly prepared 0.1 M sodium cacodylate buffer with 2.5% glutaraldehyde and 2% formaldehyde, before being treated at 4 °C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2-7.4) for 2 h. Staining was performed overnight with 0.5% aqueous uranyl acetate. After serial dehydration with ethanol , the specimens were embedded in Epon 812 and sliced. Ultrathin sections were treated with uranyl acetate and lead citrate, and observed with a HITACHI H600 TEM . The damages of brain was determined by observing the changes in nuclear morphology, .2+-ATPase, Ca2+/Mg2+-ATPase, Na+/K+-ATPase, and TNOS in the brain were measured spectrophotometrically with commercial kits , targeting the oxidation of oxyhaemoglobin to methaemoglobin by nitric oxide.To determine enzymatic activities, brain tissues were homogenized in 0.15 M NaCl. The homogenate of brains was centrifuged at 3,000 g for 15 min at 4 °C, A portion of supernatant was used to measure the activities of different enzymes. The activities of AChE, CaThe homogenate of brains was centrifuged at 12,000 g for 20 min at 4 °C. The concentrations of DA, 5-HT, and ACh were measured spectrophotometrically with commercially kits .Glu concentrations were measured using commercial kits , and standard curves were generated by using standard Glu stock solutions. Sample Glu levels were detected using a spectrophotometer at 340 nm and expressed as μmol/g prot. The concentration of NO in the brain was measured using a commercial kit . The OD value was determined by using a spectrophotometer . NO results were read with OD values at 550 nm. The results were calculated using the following formula: NO (μmol/L) = (Asample – Ablank)/(Astandard – Ablank) × 20 (μmol/L).Trizol reagent was used to extract the total RNA from brain samples  and treated with DNase to remove potential genomic DNA contamination. The quality of the RNA was quantitated spectrophotometrically at 260 and 280 nm.et al.For RNA library construction and deep sequencing, equal quantities of brain RNA samples (n = 3) were pooled for the control group and the treatment group, respectively. Approximately 6 μg of RNA representing each group was submitted to Solexa (now Illumina Inc.) for sequencing. Detailed methodology was according to the method described by Gu actin3. qRT-PCR was performed using the 7500 Real-time PCR System (ABI) with SYBR Premix Ex TaqTM  according to the manufacturer’s instructions. The qRT-PCR analysis was carried out following the method described in the studies of Peng et al. and Wang et al.4The specific primers for the 7 genes of interest are listed in How to cite this article: Wang, B. et al. Molecular Signatures of Reduced Nerve Toxicity by CeCl3 in Phoxim-exposed Silkworm Brains. Sci. Rep.5, 12761; doi: 10.1038/srep12761 (2015).


South African cassava mosaic virus-[South Africa:99] (SACMV). To date, there is limited gene regulation information on viral stress responses in cassava, and global transcriptome profiling in SACMV-infected cassava represents an important step towards understanding natural host responses to plant geminiviruses.Cassava mosaic disease is caused by several distinct geminivirus species, including 2 fold cut-off (p <0.05), comparative analysis between the six normalized cDNA libraries showed that 4181 and 1008 transcripts in total were differentially expressed in T200 and TME3, respectively, across 12, 32 and 67 days post infection, compared to mock-inoculated. The number of responsive transcripts increased dramatically from 12 to 32 dpi in both cultivars, but in contrast, in T200 the levels did not change significantly at 67 dpi, while in TME3 they declined. GOslim functional groups illustrated that differentially expressed genes in T200 and TME3 were overrepresented in the cellular component category for stress-related genes, plasma membrane and nucleus. Alterations in the expression of other interesting genes such as transcription factors, resistance (R) genes, and histone/DNA methylation-associated genes, were observed. KEGG pathway analysis uncovered important altered metabolic pathways, including phenylpropanoid biosynthesis, sucrose and starch metabolism, and plant hormone signalling.A RNA-seq time course  study, monitoring gene expression in SACMV-challenged susceptible (T200) and tolerant (TME3) cassava landraces, was performed using the Applied Biosystems (ABI) SOLiD next-generation sequencing platform. The multiplexed paired end sequencing run produced a total of 523 MB and 693 MB of paired-end reads for SACMV-infected susceptible and tolerant cDNA libraries, respectively. Of these, approximately 50.7% of the T200 reads and 55.06% of TME3 reads mapped to the cassava reference genome available in phytozome. Using a logMolecular mechanisms for TME3 tolerance are proposed, and differences in patterns and levels of transcriptome profiling between T200 and TME3 with susceptible and tolerant phenotypes, respectively, support the hypothesis that viruses rearrange their molecular interactions in adapting to hosts with different genetic backgrounds.The online version of this article (doi:10.1186/1471-2164-15-1006) contains supplementary material, which is available to authorized users. Manihot esculenta Crantz, is a tropical crop that is important for food security and income generation for many poor farmers in several Asian and African countries. Fresh tubers of cassava are suitable for consumption by both humans and animals, and provide the most important dietary source of calories for more than a billion people in about 105 countries, providing an estimated one third of calorie intake  [NCBI-AF155806] (SACMV) [Cassava mosaic disease (CMD) is the most important biotic constraint of cassava production in sub-Saharan Africa , 6. CMD  (SACMV) . SACMV hBeet curly top virus (BCTV) in Arabidopsis plants, tissue recovered from infection showed hypermethylated BCTV DNA, and AGO4 was required for recovery [Pepper golden mosaic virus (PepGMV) [In past years there have been high levels of resistance/tolerance to CMD found in several Nigerian cassava landraces including TME3 –11. By urecovery . Symptom(PepGMV) , and has(PepGMV) .Plants have developed both highly specialized defence responses to prevent and limit disease. Many disease responses are activated locally at the site of infection, and can spread systemically when a plant is under pathogen attack –20. ThisArabidopsis thaliana, Solanum lycopersicum and Nicotiana benthamiana[Tomato yellow leaf curl virus (TYLCV) has been shown to interact with a NAC domain protein in a yeast two-hybrid system, where overexpression of the NAC transcription factor causes enhanced viral replication [Following virus infection, transcriptional reprogramming takes place at a global level, both temporally and spatially within the plant leaves and other organs, and depending on the collective outcome, a resistance or susceptible response is initiated , 33–35. thamiana, 38, 39 thamiana. Geminivthamiana, 41, 42.thamiana, 31, 32 lication .Arabidopsis with geminiviruses, namely Cabbage leaf curl virus (CaLCuV) at 12 dpi [Tomato yellow leaf curl Sardinia virus (TYLCSV) was investigated [Tomato yellow leaf curl virus (TYLCV) resistance. Approximately 70 different cDNAs, representing genes preferentially expressed in a resistant (R) tomato line compared to a susceptible line from the same breeding program, were identified. Furthermore, a hexose transporter gene LeHT1 was shown to be up-regulated upon infection in R plants and its silencing in R plants led to the collapse of resistance [Gene expression technologies, such as microarrays represent a well-established technology and have been widely exploited in the last years leading to a vast amount of gene expression information, particularly in the area of host-pathogen interactions , 44–46. t 12 dpi , and mort 12 dpi . More restigated . In anotstigated , a reversistance . In anotsistance . Upon TYIn recent years, the introduction of next-generation sequencing (NGS) has provided new and innovative ways to speed up the identification of large numbers of genes in many plant and animal species, particularly those under biotic and abiotic stresses , 52, 53.Xanthomonas axonopodis[http://www.phytozome.net/cassava) [Arabidopsis (http://www.arabidopsis.org/) can be used to predict the function of cassava genes. Cassava belongs to the family Euphorbiaceae, and its genome comprises an estimated 770 Mb [http://cassava.igs.umaryland.edu/cgi-bin/index.cgi) can be mapped to the genome, making this a powerful tool for functional genomic studies. To date 30,666 protein-coding loci have been predicted, and the cassava genome can easily be aligned to soybean, castor bean, Arabidopsis, and rice. In addition to the cassava draft genome, there are also a number of additional cassava EST resources available through different databases  . Moreoved 770 Mb . A draftd 770 Mb . The geniewed in ). Some oAfrican cassava mosaic virus (ACMV), and 3,210 differentially expressed genes were identified, with the study focusing on photosynthesis-related gene expression [Agrobacterium mock-inoculated controls was successfully generated. RNA-Seq data generated from the SOLiD platform was assembled and reference-based mapping against the cassava genome was performed. In total, 4181 and 1008 differentially expressed genes (DEGs) were identified in T200 and TME3, respectively, across all 3 time points, and their biological functions were established through gene ontology (GO) annotation and Kegg pathway analysis. Real-time qPCR was used to validate RNA-seq data and genes of interest selected for further analysis. Comparisons of expression patterns between T200 and TME3 at three time points post inoculation , compared to mock inoculated tissue, demonstrated that differential responses to SACMV infection occur between the susceptible T200 and tolerant TME3 cultivars, and also between time points. TME3 had a significantly lower number of altered transcripts compared with T200. Comparisons were made to a previous study, conducted by Pierce and Rey, 2013 [Arabidopsis-SACMV pathosystem, and results uncovered similar and different global patterns or trends in differentially expressed genes between the two hosts.Most recently, a NGS (Illumina Solexa) gene profiling study was performed on cassava infected with pression . Despitepression . SOLiD ), while at 32 and 67 dpi, 2.19 × 103 and 4.43 × 105 SACMV molecules of DNA-A/ng TNA were detected. In comparison, for tolerant cultivar TME3, viral loads of DNA-A were significantly lower (p < 0.05) than those detected in T200 where no virus was detected at 12 dpi, and 1.79 × 102 and 3.23 × 104 SACMV molecules of DNA-A/ng TNA were present at 32 and 67 dpi, respectively   and percentages were calculated for each F3 and F5 mapping combination for T200 and TME3 libraries  using the BioProject accession number: PRJNA255198 [Sequence reads were obtained using the SOLiD v4 sequencing platform in order to generate a gene expression profile of T200 and TME3 infected with SACMV. The sequencer was run in the paired end mode with 50 bp forward (F3) and 35 bp reverse (F5) tags. Forward and reverse pairs were mapped to reference genome NA255198 .In general, for the TME3 tolerant library, an average of 23.41% of both the forward and reverse reads mapped to the reference sequence, 22.74% of the forward F3 reads mapped, but only 6.50% of the reverse F5 read mapped. Furthermore, 47.19% of F3 + F5 reads did not map at all. Similarly, for T200, an average of 23.79% of both the forward and reverse reads mapped to the reference sequence, 22.19% of the forward F3 reads mapped but only 5.91% of the reverse read mapped. For T200, 48.11% of F3 + F5 reads did not map at all. The difference in F3 versus F5 mapping results from the actual SOLiD sequencing protocol which leads to a much higher percentage of F3 mapped reads compared to F5. Because the F5 reads are of lower quality, the aligner (Lifescope) preferentially uses the F3 quality scores in mapping to the reference genome. The fraction of unmapped reads may be due to the incompleteness of current cassava genome assembly in which thousands of scaffolds are still not interconnected, and also the lower quality of the F5 reads, as mentioned previously. Despite incomplete assembly of the cassava genome, the current status of gene annotation for the assembled scaffolds on phytozome is reliable, which makes this assembly useful for RNA sequencing alignment and analysis.Arabidopsis database , the total number of differentially expressed genes (DEGs) were identified as SACMV- responsive genes for T200  infection, and similarly, in the Arabidopsis-SACMV study [In order to quantify the differential expression of genes at 12, 32 and 67 dpi in susceptible T200 and tolerant TME3 landraces, the tag count for all genes at 12, 32 and 67 dpi versus the tag counts at the same time points in mock-inoculated samples were computed. This allowed the change in expression between SACMV-infected and mock-inoculated leaf tissue samples to be calculated at all three time points for both landraces. After statistical filtering of the data , at each time point  for each cultivar. Transcripts were sorted into GoSlim term categories for molecular function, biological processes, and cellular component, and comparisons with a microarray expression study performed in SACMV-infected Arabidopsis  [Arabidopsis) and platform (NGS or microarray), both pathosystems displayed similar trends in differential gene function categories representing the highest number of transcripts  and cytoplasmic component  for Arabidopsis . For biological processes, cell organization and biogenesis, responses to stress and biotic/abiotic stimuli, and other metabolic and cellular processes were all highly represented categories  vocabulary available on TAIR 10  36 dpi)  was unde6 dpi  wa2 ratio of target gene expression level to UBQ10 expression level measured in the same cDNA sample. Therefore, expression levels are presented as the relative Log2 ratio of the infected cassava leaf tissue sample compared with the control mock-inoculated sample at each time point. Results showed that computational predictions of differential expression were validated. Although, in general, RT-qPCR was expectedly more sensitive, all fifteen genes showed correlated Log2 gene expression patterns (up or down regulated), in agreement with those observed in SOLiD sequencing data.To validate the SOLiD RNA-seq data, RT-qPCR was performed on fifteen (12 from T200 and 3 from TME3) genes that were significantly changed upon SACMV infection . The expression levels for cellulose synthase, cyclin p4, PHE-ammonia lyase, plant invertase, thaumatin PR protein, cytochrome P450, JAZ protein 10, Rubisco methyltransferase, WRKY70, MAPK3, cyclin 3B, histone H3/H4, pectin methylesterase (PME3), lipoxygenase (LOX3) and TIR-NBS-LRR  T200 and TME3 data . Details of metabolites and p-values are depicted in Table Arabidopsis showed a similar pattern of low numbers mapping to metabolic pathways at 14 dpi, while at 24 and 36 dpi, 5.6% and 7.1% of altered genes mapped to metabolic pathways  [et al. (2011) [Rice stripe virus, but not in a susceptible variety, suggesting that PAL plays a defence response. Similarly, the silencing of a pathogen-inducible UDP-glycosyltransferase in tobacco resulted in the depletion of UDP-glycosyltransferase in tobacco which enhanced oxidative stress and weakened resistance of silenced tobacco plants to TMV infection [Arabidopsis study [One of the most interesting discoveries, which have not been extensively reported in cassava before, was the mapping of several flavanoid and phenylpropanoid genes involved in T200 infection, which were prominently altered at 32 dpi and maintained at 67 dpi. Genes mapping to these pathways included flavonol synthase (cassava4.1_011509m.g), UDP-glycosyltransferase (cassava4.1_005848m.g), chalcone synthase  and phenylalanine ammonia lyase . Furthermore, these genes were all found to be highly induced with expression ratios in the range of Logathogens , 75. Phe. (2011)  demonstris study , PAL andis study .Transcript quantification showed that T200 had a far greater change in the number of differentially expressed genes as well as the magnitude of expression changes across time points compared with TME3  gene in T200, is interesting in itself as it compares with previous cloned Rx, RT4-4 and N resistance genes which also contain TIR domains. The down-regulation of TIR-NBS-LRR implies that TIR-NB-LRR receptor activation in cassava T200 is repressed and therefore SACMV may be avoiding detection and inhibition by plant defence response, therefore promoting virus replication and movement. Furthermore, suppression of TIR-NBS-LRR could negatively affect other signalling pathways downstream of TIR-activation such as the mitogen-activated protein kinase pathway. Collectively, the high number of repressed R genes at 32 and 67 dpi in T200 strongly supports a significant role in susceptibility to SACMV.The identification and characterization of R genes has long been under scrutiny, where 7 major classes have been identified . To date virus X, RT4-4 gManihot glaziovii[CMD1 and CMD2 have been located on linkage maps of cassava, these genes have not yet been identified and mapped to any scaffolds of version 4.1 of the cassava draft genome presently available through phytozome, and therefore the potential role of these two genes in CMD resistance remains to be elucidated. In summary, the remarkable lack of R gene response in the tolerant TME3 landrace at 12 dpi, in comparison with the highly susceptible T200 where most R genes were down-regulated, and a notable up-regulation of eight R gene homologues at 32 and 67 dpi in TME3, support a role for these R genes in the recovery of TME3 to SACMV infection.Resistance to CMD from wild-species such as laziovii was showlaziovii, 82, andlaziovii. Subsequlaziovii to underlaziovii. Howeverlaziovii, they idAfrican cassava mosaic virus (ACMV) or Sri Lankan cassava mosaic virus (SLCMV) [Pepper golden mosaic virus (PepGMV), there was no difference between the number of differentially expressed genes between recovered and symptomatic leaves compared to mock-inoculated, and a higher number of genes were up-regulated compared to down-regulated. This was not the case in SACMV-infected TME3, where a high number of transcripts were repressed at 32 and 67 dpi. Within the set of altered defence response genes in pepper, there appeared to be little difference between recovered and symptomatic leaves, but rather a new set of genes were identified including genes involved in histone modification, supporting a role for TGS in recovery [Nicotiana benthamiana[2 = 1.24 vs. Log2 = −1.22) and histone H4 (Log2 = 1.65 vs. Log2 = −1.76) were also found to be induced, while in recovered pepper leaves from PepGMV [Previous studies, such as cassava infected with either  (SLCMV) , have sh (SLCMV) , and the (SLCMV) , 87, and (SLCMV) , 89). Inrecovery . Severalrecovery –92. Histrecovery , while hrecovery . Up-regurecovery . In a sithamiana, histonem PepGMV  these weTo support a role for RNA silencing or methylation in the susceptible and tolerant phenotypes of T200 and TME3, respectively, NGS sequencing and quantification of small silencing RNA (vsRNA) populations (21–25 nt) targeting SACMV genomic DNA A and DNA B components in infected T200 vs. TME3  was performed (unpublished results). Normalized data revealed that the number of vsRNAs targeting SACMV DNA components in T200 was consistently higher compared with TME3. In both T200 and TME3 there was a significant increase in vsRNAs against DNA A and DNA B from 12 to 32 dpi despite persistence of symptoms and virus replication. However in T200 at 67 dpi there was a massive decrease in vsRNAs targeting DNA A and B, which led to a significant increase in virus replication and symptom severity, while in comparison, in TME3 the levels of vsRNAs increased, associated with a recovery phenotype (unpublished results). Although siRNA populations can range in length between 21- and 26 nt, the 24-nt siRNA range, produced by DCL3 , 97 cleaArabidopsis and these function with chromatin remodelling proteins to inactivate gene expression and control chromatin structure mediated by CpG methylation [2 = 2.478) which could bind to methylated CpG regions on SACMV DNA-A and B, therefore inhibiting replication. This could be one of the reasons accounting for lower viral titres and the recovery phenotype observed in TME3 at 67 dpi as compared with T200.Twelve methyl-CpG-binding domain proteins (MBD) have been identified and characterized in hylation , 99. OneDCLs, RdRPs or AGOs, in either T200 or TME3, Raja et al. 2008 [Arabidopsis mutants defective in a number of genes that are key players in the RdDM pathway  results in hyper-susceptibility to infection with the geminiviruses Cabbage leaf curl virus (CaLCuV) and Beet curly top virus (BCTV).The recovery phenotype is observed in TME3 from ~55 dpi onwards (in this study sampled at 67 dpi), and we conclude that evidence collectively points to durable resistance or tolerance in TME3, mediated by concomitant early suppression of genes (likely to be involved in creating a supportive cellular environment for replication), persistent RNA silencing maintenance of genes required by SACMV as evidenced by a significantly lower number of altered transcripts throughout infection, and by methylation-associated TGS of SACMV DNA-A and B. This is also evident by a decline in virus load and symptoms at recovery. While in this study, there was little evidence for altered gene expression in RNA silencing associated transcripts such as al. 2008  elegantlP. syringae pv. Maculicola 1 (RPM1), Mildew Resistance Locus O  and Non-host Resistance to P.S. Phaseolicola 1 (NHO1) resistance proteins; transcription factors such as WRKY; and heat shock proteins (HSPs) which are involved in defence  genes were shown to be increased upon infection by SACMV primarily at 32 and 67 dpi in T200  and MAP kinases are involved in plant defence response. The MAPK signalling pathway is evolutionary conserved, and MAP kinases primary role is to transfer sensors to cellular responses [Arabidopsis[Nicotiana tabacum resulted in compromised N-gene-mediated resistance to Tobacco mosaic virus. Furthermore, RRSI, a gene that confers resistance to bacterial pathogen Ralstonia solanacearum encodes a TIR-NBB-LRR protein with a C-terminal WRKY motif (WRKY52). This additional WRKY structural feature of RRS1 could indicate a direct relationship between Avr-recognition and the downstream transcriptional activation of defence genes [Arabidopsis[Studies in esponses . A MAPK esponses , 106. Twbidopsis where MAbidopsis–110. MAPbidopsis, and is bidopsis, 112. Inbidopsis. WRKY TFbidopsis. A studybidopsis demonstrce genes . In thisbidopsis. AtWRKY1bidopsis, 116, waotif WRKY2. This abidopsis. Upon pabidopsis. Resultssalicylic acid (SA) are present in plants in basal amounts, yet act in a well-balanced and regulative manner during plant growth and development [Arabidopsis-SACMV interaction study, since marker genes for JA were more prevalent and highly expressed throughout the course of infection compared to SA.Hormones, such as ethylene (ET), jasmonic acid (JA), abscissic acid, gibberellins and elopment . Any chaelopment . Howeverelopment , 122. JAelopment . In brieelopment , 116, waelopment  also repTobacco mosaic virus[Cauliflower mosaic virus and hindered long-distance movement of the virus. SACMV infection in cassava T200 appears to be supported by evasion of basal host defence via overall negative regulation of JA and ET signaling pathways and lack of host elicitation of SA pathway dependent resistance.ET is influential in mediating the outcome of synergism or antagonism between JA and SA signalling. ET is able to bypass key regulator genes such as NPR1 in SA signalling during SA/JA crosstalk therefore preventing suppression of JA signalling , 122. ETic virus has beenic virus showed tGibberellin-regulated family proteins  , calreticulin and plasmodesmata-callose binding protein. A number of previous studies have implicated a number of cellular components and proteins that are localised to the plasmodesmata (PD) and that play a role in either cell-to-cell communication or movement of molecules across the PD . SACMV iXanthomonas axonopodis pv. manihotis [Arabidopsis[Arabidopsis and tobacco. Additional analysis of the phylogenetic relationship between cassava TIR-NBS-LRR domains, and Arabidopsis, rice, castor bean, tomato and other plant species, is ongoing in our laboratory and will also prove interesting. Homology between these genes could provide some insight into the evolutionary conservation of these R genes.This is the first virus-responsive transcriptome study in cassava following the infection of a cassava geminivirus over three time points post infection, and it will prove interesting to compare these results in future with cassava in response to other pathogens, such as the bacterial pathogen anihotis , 151. Cobidopsis, 47, andBegomovirus, and several species, including SACMV, have been identified in regions of South Africa and some neighbouring countries including Zimbabwe, Mozambique and Swaziland. Understanding the mechanisms underlying CMD could facilitate control strategies to combat begomoviruses, either through genetic modification approaches or through breeding programs, which could result in conferring resistance or a degree of tolerance. The knowledge from this study will serve as a useful genetic resource for relevant cassava researchers globally. A systems biology approach is required to build geminivirus-interaction models, and complementary studies on small RNA population responses in T200 and TME3 (have been completed but is not the remit of this study), and further gene identification and verification of candidate gene functions, can lead to achieving this goal. Additional metabolome and proteome data will in future be needed to develop a comprehensive interactome model for geminivirus infection in host plants.In summary, CMD is a devastating disease caused by at least nine species of −2 sec−1. At the appearance of roots (approximately 10 days), plantlets were transferred into Jiffy® pellets  which were placed on a tray that was covered with plastic film and placed in a controlled growth chamber . Plantlets were gradually acclimatized by adding slits to plastic film. Acclimatized plantlets were allowed to grow until they reached a 4–6 leaf stage.Cassava T200 and TME3 landraces were micro-propagated by nodal cutting culture on Murashige and Skoog (MS) medium  supplemeAgrobacterium tumefaciens Agl. The two transformed cultures containing DNA-A and DNA-B were cultured separately in Luria Bertani (LB) Broth supplemented with carbenicillin (100 μg.ml−1) and kanamycin (100 μg.ml−1). Wild-type Agrobacterium Agl1 cultures served as a negative control for inoculations and was inoculated into LB broth supplemented with carbenicillin (100 μg ml−1). Cultures were grown overnight at 30°C until optical densities of 1.8-2.0 (OD600) were reached. From each of the three cultures, 5 ml was sub-inoculated into 30 ml fresh LB Broth, containing the correct combination of antibiotics as previously described. Cultures were once again grown overnight at 30°C until cultures reached optical densities of 1.8-2.0 (OD600). For each culture, 25 ml aliquots were pelleted by centrifugation at 13000xg, washed in sterile distilled water and subsequently resuspended in 5 ml LB Broth. Agl1-SACMV DNA-A and Agl1-SACMV DNA-B were resuspended and combined to form a homogenous mixture of Agl1- SACMV DNA-A and Agl1- SACMV DNA-B cells. T200 and TME3 plantlets were wounded along the stem with a hypodermic needle and each plantlet was inoculated with 100 μl the Agl1DNA-A/DNA-B suspension using a 1 ml Hamilton syringe. Control plants were mock-inoculated with 100 μl wild-type untransformed Agrobacterium Agl1inoculum.Agroinoculation of T200 and TME3 cassava plantlets was achieved by a protocol adapted from Hayes et al. . InfectiSACMV-infected and mock-inoculated plants were monitored over a 67 day period. Newly developed symptomatic leaf tissue from apical leaves was collected from each plant (n = 6) at each time point i.e. 12, 32 and 67 dpi, and pooled. Leaves 2–3 under the apex were selected as geminiviruses are known to replicate in actively dividing cells . Time poFor each time point , the leaves closest to the apex were harvested from six plants. Total nucleic acid (TNA) was isolated from these SACMV infected and mock-inoculated leaves using a modified CTAB-based extraction method . Fifty mSystemic infection in cassava leaf tissue for T200 and TME3 at 12, 32 and 67 dpi was confirmed by conventional PCR. 50 μl PCR reaction were set up and contained 0.4 μM of each primer, 200 μM dNTPs, 2 units DreamTaq DNA polymerase , 1x DreamTaq Buffer , and nuclease-free water to a final volume of 50 μl. A 550 bp fragment of the core coat protein (CCP) on SACMV DNA-A was amplified using degenerate forward primer: (V524) 5′ GCCHATRTAYAGRAAGCCMAGRAT 3′ and reverse primer: (C1048) 5′ GGRTTDGARGCATGHGTACANGCC 3′. Approximately 500 ng of the total nucleic acid (TNA) template was added to the reaction mixture. Reactions were cycled in a MyCycler™ Thermal Cycler (Bio-Rad) at 95°C for 5 minutes to activate the Taq DNA polymerase, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, primer extension at 72°C for 45 seconds, and a final extension step of 72°C for 5 minutes. DNA-A of SACMV cloned into pBluescript vector (50 ng) was used as positive control for PCR reactions. Amplification products were examined by electrophoresis on a 1.2% agarose TAE gel containing 10 μg/ml ethidium bromide.Plus SYBR Green I (Roche), 1 μl forward coat protein primer (10 μM) 5′ACGTCCGTCGCAAGTACGAT3′, 1 μl reverse coat protien primer (10 μM) 5′ATTGTCATGTCGAATAGTACG 3′ and 14 μl nuclease-free water. A 150 bp fragment was amplified and quantified using the following amplification conditions: 95°C for 10 min, followed by 35 cycles of 95°C for 10 sec, 60°C for 10 sec, and 72°C for 15 sec. A single fluorescence measurement was taken at the end of each extension step during the PCR amplification cycle. A melting curve (65°C-95°C) with a heating ramp rate of 0.1°C/s and a continuous fluorescence measurement was conducted after the amplification and quantification cycle. A 166 bp PCR product of ubiquitin was amplified from 100 ng of the same TNA samples used for viral quantification which served as an internal loading control. Primers used were previously tested in cassava. Primer sequences used were UBQ10 (fwd): 5′ TGCATCTCGTTCTCCGATTG 3′ and UBQ10 (rev): 5′ GCGAAGATCAGTCGTTGTTGG 3′ previously described in Moreno et al. [t-test.Determination of the viral titre in T200 and TME3 plants was achieved by use of qPCR on TNA extracted from both cultivars at time points 12, 32 and 67 dpi. TNA samples was all standardised to a concentration of 100 ng/μl. Duplicates of each sample were prepared as well as a no template control (NTC) of nuclease-free water. For each sample, a 20 μl reaction was set up in LightCycler capillaries containing 1 μl of 100 ng of leaf tissue TNA was added to 4 μl LightCycler ® FastStart DNA Mastero et al. . Data we) for 25 minutes at 4°C. RNA pellets were washed with 5 ml ice-cold 75% ethanol. RNA Pellets were dried at 37°C for 5 minutes. The pellet was resuspended in 100 μl preheated (55°C) RNase-free water and 1 μl RNase inhibitor (Fermentas). Concentrations were determined using the NanoDrop™ 1000 spectrophotometer  and RNA integrity was assessed using an Agilent 2100 Bioanalyzer.Total RNA was extracted on SACMV-infected and mock-inoculated leaf tissue using a modified high molecular weight polyethylene glycol (HMW-PEG) protocol . One gracDNA libraries were generated at the Functional Genomics Center UNI ETH Zurich, Switzerland. Briefly, 12 ug of total RNA for each sample was used to generate cDNA libraries. RNA was fragmented and subjected to hybridization and ligation using the SOLiD Total RNA-Seq Kit (Applied Biosystems) according to the manufacturer’s instructions. cDNAs were selected by size on a polyacrylamide gel before and after the library amplification. A total of 12 libraries were multiplexed using the SOLiD RNA Barcoding Kit (Applied Biosystems) and pooled in an equimolar ratio. The samples were then diluted and used for emulsion PCR. Beads containing a multiplex of 12 samples were deposited onto a single flow cell. Libraries were sequenced operating on 50 bp forward and 35 bp reverse paired-end sequencing chemistry on the ABI SOLiD V4 system.http://www.phytozome.net/cassava.php, Manihot esculenta 147, version 4.1) using the Lifescope software from LifeTech. As a result, SAM/BAM alignment files were prepared, sorted and indexed using samtools (http://samtools.sourceforge.net/). In the secondary data analysis phase, the BAM data were matched with the genome annotations available in Phytozome as a GTF/GFF3 file, which describes genes, transcripts and their exons with the genomes coordinates. The alignments were then transformed to counts using rnaSeqMap library (v.2.7.12) of Bioconductor [2-fold cut-off with a p-value of <0.05 to select for highly expressed transcripts.The SOLiD v4 sequencer was used for the generation of sequence reads and was run in paired-end mode (50 + 35 bp). For each time point, differential gene expression data was achieved by normalization against mock-inoculated. This resulted in two csfasta and two quality files per sample. The reads generated for each library were mapped to the genome assembly (onductor  (releaseonductor  from thehttp://www.idtdna.com/Primerquest/Home/Index). In brief, 1 μg of DNase-treated total RNA was reverse transcribed using the Improm-II-reverse transcriptase kit  according to manufacturer’s instruction. RNA, dNTPs and Oligo dT18 primer were denatured for 10 min at 70°C; then kept at 25°C for 5 min before the reverse transcription master mix was added. Reverse transcription was performed at 42°C for 1 hour followed by a 10 min incubation step at 70°C. Control reactions were set up without the addition of reverse transcriptase and used as negative controls in the real-time PCR study. RT-qPCR experiments were conducted on the Lightcycler 1.5 for all genes using the appropriate primer pair for each reaction  that were found to be differentially expressed were selected based on the SOLiD RNA-seq results  and analysed using real-time quantitative RT-PCR. One of the criteria used to select genes, was the differential expression observed in at least 2 of the 3 time points in T200 and TME3 SACMV-infected leaf tissue. Primers for each gene were designed using software available online through Integrated DNA technologies . These files can be accessed using BioProject accession: PRJNA255198  [http://Pairing statistics for cassava F3 and F5 Tags.(DOCX 15 KB)Additional file 1:Manihot esculenta −147- annotated transcriptome_genes.(XLSX 2 MB)Additional file 2:List of all differentially expressed genes in T200 at 12 dpi.(XLSX 6 MB)Additional file 3:List of all differentially expressed genes in T200 at 32 dpi.(XLSX 6 MB)Additional file 4:List of all differentially expressed genes in T200 at 67 dpi.(XLSX 4 MB)Additional file 5:List of all differentially expressed genes in TME3 at 12 dpi.(XLSX 6 MB)Additional file 6:List of all differentially expressed genes in TME3 at 32 dpi.(XLSX 6 MB)Additional file 7:List of all differentially expressed genes in TME3 at 67 dpi.(XLSX 6 MB)Additional file 8:Comparisons of number of differentially expressed genes between 12, 32 and 67 dpi in T200.(XLSX 468 KB)Additional file 9:Comparisons of number of differentially expressed genes between 12, 32 and 67 dpi in TME3.(XLSX 103 KB)Additional file 10:Transcript quantification for mock-inoculated T200 and TME3 leaf tissue.(XLSX 82 KB)Additional file 11:Comparative analyses of Kegg metabolic pathways differentially expressed in SACMV-infected Arabidopsis, cassava T200 and TME3.(XLSX 18 KB)Additional file 12:SACMV-responsive R gene homologues and histone-related genes in T200 and TME3.(XLSX 11 MB)Additional file 13:Primers used for qPCR validations.(DOCX 13 KB)Additional file 14:


Eucalyptus grandis × E. urophylla during a field survey in southern China in July 2010. This hybrid can accumulate as much as 13,549 mg/kg DW Mn in its leaves. Our results from Scanning Electron Microscope (SEM) X-ray microanalysis indicate that Mn is distributed in the entire leaf and stem cross-section, especially in photosynthetic palisade, spongy mesophyll tissue, and stem xylem vessels. Results from size-exclusion chromatography coupled with ICP-MS (Inductively coupled plasma mass spectrometry) lead us to speculate that Mn associates with relatively high molecular weight proteins and low molecular weight organic acids, including tartaric acid, to avoid Mn toxicity. Our results provide experimental evidence that both proteins and organic acids play important roles in Mn detoxification in Eucalyptus grandis × E. urophylla. The key characteristics of Eucalyptus grandis × E. urophylla are an increased Mn translocation facilitated by transpiration through the xylem to the leaves and further distribution throughout the leaf tissues. Moreover, the Mn-speciation profile obtained for the first time in different cellular organelles of Eucalyptus grandis × E. urophylla suggested that different organelles have differential accumulating abilities and unique mechanisms for Mn-detoxification.Manganese (Mn) is an essential micronutrient needed for plant growth and development, but can be toxic to plants in excess amounts. However, some plant species have detoxification mechanisms that allow them to accumulate Mn to levels that are normally toxic, a phenomenon known as hyperaccumulation. These species are excellent candidates for developing a cost-effective remediation strategy for Mn-polluted soils. In this study, we identified a new passive Mn-hyperaccumulator  Manganese (Mn) is an essential nutrient for plant growth and development, known to act as a direct cofactor for about 35 plant enzymes so far . For exaPhytolacca acinosa Roxb [Schima superba L[Polygonum hydropiper L [Polygonum perfoliatum L [Polygonum pubescens Blume [Eucalyptus grandis × E. urophylla can hyperaccumulate Mn from Mn-polluted soils. It is a prominent species originally identified in Brazil [Eucalyptus grandis × E. urophylla, and the possible detoxification and accumulation mechanisms.Of the 492 presently known metal-hyperaccumulator species , 15, onlosa Roxb  and Schisuperba L. Three aopiper L , Polygonliatum L  and Polyns Blume , have ben Brazil  and impon Brazil  due to iSoil and plant samples were collected from fields near abandoned mines open to public with no permission requirements.Eucalyptus grandis × E.urophylla from the mining area were collected from the lower canopy of 1–2 year-old plants. For each replicate, leaves from different stems were mixed together to obtain samples of similar weigh. Leaf and stem samples of Eucalyptus grandis × E.urophylla with the similar age were also collected from an uncontaminated area on the campus of Xiamen University, Xiamen, Fujian Province, Southeastern China . This site has a subtropical continental climate with an average annual temperature of 20.6°C, average rainfall of 1315 mm/year and average insolation of 1953 h/year. All plant samples were washed thoroughly with Milli-Q water and frozen at –20°C prior to analysis. Soils were sieved with 100 mu mesh and air-dried before digestion.The Liancheng mining area is located in Liancheng, Fujian Province, Southeastern China , having a subtropical continental climate with an annual average temperature of 18.7°C, an average rainfall of 1191 mm/year and an average insolation of 1950 h/year. The Liancheng mining area is famous for its high production of Mn, Pb, and Zn. There are two main mine tailing sites, including the “Mn site” and the “Pb/Zn site” which were named by the abundance of Mn and Pb/Zn, respectively. During the field survey, we collected samples from the dominant plant species, including leaves, stems and host soils from both sites. Here we report data only for the Mn site. The Mn content of 23 plant species and their host soils was determined . Mature 3/HClO4 (5:1) and diluted with Milli-Q water prior to determining metal content using ICP-MS. Soil samples were pulverized, digested with a mixture of HNO3/HF/HClO4 (5:1:1) and analyzed by ICP-MS for Mn, Pb, As, Cu, Cd, Zn, and Co content [-1; auxiliary gas flow 1.1 L min-1; nebulizer gas flow was 0.9 L min-1; isotope monitored was 55Mn; Dwell time was 100 ms.Leaf and stem samples were dried at 105°C for 2 days, weighed, digested using a mixture of HNO content . Here we3)2, 2.5 mM KNO3, 0.5 mM MgSO4, 0.5 mM KH2PO4, 40 μM Fe-EDTA, 25 μMH3BO3, 2.25 μM MnCl2, 1.9 μM ZnSO4, 0.15 μM CuSO4, and 0.05 μM (NH4)6Mo7O24, pH 5.8 to 6.0) for another 30 days. In addition to using 2.25 μM MnCl2, we used 5, 5 × 102, 5 × 103, 1 × 104 and 2 × 104 μM MnCl2, respectively, for one-week Mn treatments [3/HClO4 (5:1) and diluted with Milli-Q water for Mn content measurement using ICP-MS.Seeds obtained from NanHu Flower and Tree Market, Xiamen China, were surface sterilized with 70% EtOH for 1 min (twice), 100% EtOH for 1 min (twice) and washed with sterilized Milli-Q water (twice). The seeds were then germinated on one quarter-strength Murashige and Skoog (MS) agar plates for 4 weeks. Seedlings were transferred to half-strength Hoagland solution , 1 mM EDTA, 1 mM SDS, 20 mM β-mercaptoethanol to the powder. Next, four layers of gauze were used to filter the solution to remove residue. After centrifuging at 20,000 g at 4°C for 15 min, 100% ethanol was added to the supernatant to precipitate the proteins. To dissolve the pellets, a reasonable volume of 50 mM NH4HCO3 (pH 7.8) was added and centrifuged at 25,000 g at 4°C for 15 min. The supernatant was filtered using a cartridge with a 0.45-μM cellulose membrane and stored at - 20°C. A 100 μL aliquot of sample was used for HPLC/ICP-MS. The operating conditions were the same as described in [-1 Tris + 100 mmol L-1 NH4HCO3 (pH = 7.8); flow rate was 0.75 mL min-1; injection volume was 100 μL; detection was UV (214 and 280 nm) and ICP-MS; molecular standards were Glutathione , β-lactoglobulin  and ovalbumin . (B) The operating conditions for the ICP/MS was the same as described above.A soluble protein isolation method was used as described in , 33 withribed in : (A) HPLThe cell wall fraction was isolated from leaves using the method described in  with min2, 10 mM NaCl, 2 mM EDTA, 0.5 mM KH2PO4 and 2 mM sodium ascorbate). The slurry was filtered through four layers of gauze. The suspension was centrifuged at 1,000 g at 4°C for 2 min. The pellet (chloroplast) was suspended and osmotically broken in 5 mM MgCl2 for 30 seconds. Suspension buffer was added  to the broken chloroplast fraction and centrifuged at 3,000 g at 4°C for 5 min. Chloroplast membrane was in the supernatant. The pellet (thylakoid) was resuspended in buffer containing 2 mM MES, 5 mM MgCl2 and 15 mM NaCl) and stored on ice for 1.5 h. We then took a small amount of thylakoid, and used 80% acetone to extract chlorophyll and measure chlorophyll as described in [The chloroplast isolation method described in  was usedribed in . Next, wWe use the vacuole protein isolation protocol described in  with theLysis buffer I  Ficoll 400, 20 mM EDTA, 5 mM HEPES-KOH, pH 7.5) at 42°C was prepared. The protoplast suspension prepared above was diluted 4-fold in lysis buffer I for 15 min. The suspension was loaded at the bottom of a centrifuge tube and covered with a mixture  , which is two times the volume of the above suspension, then covered with buffer II, which is one time the volume of the suspension. We centrifuged the gradient at 1,500 g for 20 min, and vacuoles were collected at the interface of the first and second layers. The vacuoles were concentrated by centrifugation at 1,800 g for 20 min, and then used to extract soluble protein as described above for HPLC/ICP-MS analysis. The operating conditions for the HPLC/ICP-MS were the same as described above.3PO4 and 3% methanol, (pH 2.5); flow rate was 0.7 mL min-1; injection volume was 100 μL and detection was UV (210 nm).Organic acids were extracted using the method described in . A 100mgNeolitsea wushanica and Polli japonica, which contained less than 500 mg/kg Mn, most of the dominant plants accumulated Mn in the range of 1,000 to 5,000 mg/kg, which is much higher than those growing in normal soil, which accumulated 20–500 Mn μg/g dry weight [Pinus massoniana Lamb, 10,849 mg/kg in the leaves of Polygonum chinense L, 13,549 mg/kg in the leaves of Eucalyptus grandis × E.urophylla, and 14,365 mg/kg in the roots of Dennstaedtia wilfordii. Of these four plants, the hybrid Eucalyptus grandis × E. urophylla is a woody plant species having higher biomass. A 1–2 year old Eucalyptus grandis × E. urophylla growing at the Mn site is shown in , was only 640 ± 34 mg/kg DW and 372 ± 34 mg/kg  making its Mn tolerance ability similar to another Mn hyper-accumulator previously found in China, Phytolacca acinosa Roxb [Eucalyptus grandis × E. urophylla plants, the acid-extractable Mn ranged from 63.2 to 178.6 mg/kg. We are aware that the acid-extractable Mn in soil is much higher than the bioavailable Mn. However, if the threshold Mn concentration is 10,000 μM (corresponding to 549.4 mg/kg) based on the results of the hydroponic exposure experiments, the maximum Mn tolerance capacity of Eucalyptus grandis × E. urophylla has not yet been reached in the soil. Based on these findings, Eucalyptus grandis × E. urophylla should be a good candidate for remediating soils with extremely toxic amounts of Mn and may also be a good candidate for phyto-mining of Mn.Seeds of symptoms ; howeversymptoms . When exsymptoms . The criosa Roxb . In a prosa Roxb . Based oEucalyptus grandis × E. urophylla using ICP-MS. The Mn distribution in different tissues is shown in  > stem (157 mg/kg) > root (52 mg/kg). When treated with 500 μM Mn (corresponding to 28 mg/kg), the stems (3662 mg/kg) accumulated a similar amount of Mn as leaves (3792 mg/Kg), and both were approximately 15 times higher than roots (249 mg/Kg). When exposed to elevated Mn levels, especially 10,000 and 20,000 μM (corresponding to 549.4 and 1098.8 mg/Kg), we unexpectedly observed that the stems accumulated much higher Mn  than the leaves  and roots . These observations indicate that young Eucalyptus grandis × E. urophylla plants accumulate more Mn in the stems when exposed to extremely toxic Mn levels to protect the young leaves, which is an important site for photosynthesis. Nevertheless, when the plants mature and have enough leaves to increase their Mn storage capacity and Mn detoxification, the Mn is transported and stored in the leaves, which explain why the leaf Mn concentration (13549 mg/kg) was much higher than that in the stems (5231 mg/kg) of the 1–2 years old Eucalyptus grandis × E. urophylla collected at the mining area. This could imply that the Mn stem-accumulation of Eucalyptus grandis × E. urophylla during its early development is a useful strategy to protect seedlings from Mn stress. However, while our results are suggestive, it is unclear if the development stage has a strong effect on metal accumulation and additional experiments on different plant species are warranted.We measured Mn concentrations in the roots, stems and leaves of Mn-stressed shown in . The plaEucalyptus grandis × E. urophylla using cross sections of samples collected from the control area and the Mn site. SEM pictures of the Mn site samples are shown in  [Eucalyptus grandis × E. urophylla, and was relatively high in the photosynthetic palisade and spongy tissue, both of which are highly vacuolated and may play an important role in storing excess Mn [2+ from vacuoles and chloroplasts in Arabidopsis thaliana L. and the ysl4ysl6 double knock-out mutant is more tolerant to excess Mn than wild type plants, but the physiological role of AtYSL4 and AtYSL6 in Mn homeostasis remains unclear [Compartmentalization of foliar Mn is quite different among metal hyper-accumulators. Large palisade mesophyll cells having high vacuolar Mn content are reported to be the main site of Mn storage for some hyper-accumulators, such as oides L. –49. One e-layers , 50. Morhillii L , 47. Furnnus L.) , 52. In xcess Mn . Mn is axcess Mn . However unclear , 53. AddP. acinosa L., suggesting that Mn is transported along with the transpiration stream [Eucalyptus grandis × E.urophylla, the major Mn sink in the stem was primarily the stem vessel, which is composed of dead cells and the cell wall composed primarily of polysaccharides and proteins. They have numerous functional groups, such as –COOH, –OH and –SH as well, playing a crucial role in metal binding [Eucalyptus grandis × E. urophylla.A decrease in Mn concentration from the midrib to the margin was observed within leaves of n stream . Howevern stream . In Euca binding . In thisEucalyptus grandis × E. urophylla , while Mn associated with LMW compounds (peak 2) in samples from the Mn site remarkably increased, suggesting that excess Mn from the Mn site tends to bind the low molecular weight compounds rather than the HMW proteins in the leaves. In the case of the stems (The water-soluble fractions collected from leaves and stems were used for size-exclusion chromatography (SEC) coupled with UV detection (λ = 254 nm) and ICP-MS in sequence. SEC/UV/ICP-MS chromatograms of the water-soluble fractions from leaves of the control and Mn site are shown in . Two majhe stems , althoughe stems . In comphe stems  but thatPhytolacca americana L, a hyper-accumulator that can accumulate large amounts of cadmium and Mn. Cd treatments increased phytochelatin synthesis, however excess Mn treatments did not result in a significant increase in non-protein thiols or phytochelatin concentrations in the leaves or roots, but did increase the oxalic acid concentration in the leaves [Eucalyptus grandis × E. urophylla played a significant role in detoxifying excess Mn in addition to the HMW proteins; however, the exact identity of the LMW compounds and the HMW proteins requires further investigation.Vacuolar compartmentalization or complexion of heavy metals by organic ligands has been reported as the main detoxification mechanisms in plants , for exae leaves . Our rest = 25 min, MW > 45K Da), LMW organic compounds  and a new Mn-containing peak    This sma peak 2)  because  peak 2) . It was 4CaO5 cluster that catalyzes the splitting of water and the production of O2 [Eucalyptus grandis × E. urophylla. PS II complex assembly is a complex process and happens in several membranes of the chloroplast. It was reported [2+ is taken up into the periplasmic space and incorporated by the PS II assembly factors Prat A complex (36 KD), and then Mn-Prat A would is delivered to the chloroplast plasma membrane, binding to the precursor form of D1  and forming a Mn-Prat A-pD1 complex. Next, YCF48 is joined and converted into the RC47 complex (RC47-Mn) and several low-molecular-mass PS II subunits bind CP47, resulting in a functional PS II-Mn-core. Among these process, proteins have a molecular weight varying from 12–76 KD were added step by step [Eucalyptus grandis × E. urophylla taken from the Mn site. In the isolated PS II fraction, the Mn-containing HMW proteins (peak 1) and the LMW organic compounds (peak 2) were found as well as medium molecular weight proteins (peak 3) that had been observed in the cell wall protein fraction  were found to be the predominant component, while the Mn-containing HMW proteins (peak 1) and the medium molecular weight proteins (peak 3) were low abundance. These results suggest that most of the proteins associated with Mn are fixed in the organelle structure and cannot be translocated into the vacuole, on the other hand, the LMW organic compounds associated with Mn could easily be transported into the vacuole and play an very important role in Mn detoxification.Manganese is critical for the photosynthetic apparatus , includion of O2 , 64. Thireported  that Mn2fraction . SimilarWe extracted organic acids from the leaf and stem samples from both the control and Mn site with liquid nitrogen and dilute HCl. The acid-extractable fractions, which were believed to contain organic acids, were subjected to RP-HPLC. The results obtained  indicateEucalyptus grandis × E. urophylla during early development is a helpful strategy to protect the seedlings from Mn stress.When compared with the elution time of the organic acid standards , the lowEucalyptus grandis × E. urophylla was identified during a field survey in southern China in July 2010. This plant can accumulate as much as 13,549 mg/kg DW Mn in its leaves. SEM X-ray microanalysis suggested that Mn is distributed throughout the entire leaf and stem cross-section, especially in photosynthetic palisade, spongy mesophyll tissue, and stem xylem vessel; SEC/ICP-MS and RP-HPLC studies led us to speculate that Mn might be associated with relatively high molecular weight proteins and low molecular weight organic acids, such as tartaric acid to prevent Mn toxicity. We have provided experimental evidence that proteins and organic acids play important roles in Mn detoxification in Eucalyptus grandis × E. urophylla. The Mn-speciation profile obtained for the first time in different cellular organelles of Eucalyptus grandis × E. urophylla suggested that different organelles have their own accumulating abilities and unique mechanism for Mn-detoxification. Tartaric acid in the stem induced by Mn stress allows the stem to store more Mn during early developmental stages, and facilitates Mn translocation by transpiration through xylem to the leaves to further distribute Mn throughout the leaf tissues. The information gained in the current study and similar mechanistic studies are important for future applications of Eucalyptus grandis × E. urophylla as a phytoremediation tool for Mn polluted soils.A inducible Mn-hyperaccumulator


The male-to-female sex ratio for medulloblastoma (MB) is approximately 1.5∶1, female gender being also a favorable prognostic factor. This study aimed at evaluating the impact of gender on MB tumorigenesis.In vitro activity of 17β-estradiol (E2), DPN , PPT  or DHT  was evaluated in three human MB cell lines. D283 Med cells were transplanted into athymic mice.A significant expression of ERβ, with little or no ERα, and low AR (androgen receptor) was found in MB cell lines. The compounds tested did not affect cell proliferation. In vivo, we observed a significantly lower growth of D283 Med in nude female mice compared to males. At microscopic examination, tumors from females showed a shift towards differentiation, as evaluated by lower nestin, and higher NSE (neuron-specific enolase) and GFAP  expression compared to males. Tumors from females also showed lower Ki67 and p53 expression. The wild-type ERβ protein (ERβ1) was lost in male tumors, while it was a permanent feature in females, and a strong negative correlation was found between Ki67 and ERβ1 expression. Conversely, tumor levels of ERβ2 and ERβ5 did not significantly differ between genders. Increased levels of cyclin-dependent kinase inhibitor p21 were observed in females, suggesting that estrogen may decrease tumor growth through blocking cell cycle progression. An inhibition of the insulin-like growth factor I (IGF-I) signaling was also evident in females.We provides mechanistic evidence supporting the idea that ERβ1 signaling may have pro-differentiation and tumor suppressive function in medulloblastomas. Once thought to be irrelevant regarding steroid hormones action, the cerebellum is now recognized to be affected by these hormones, with the majority of studies focusing on the role of estrogen. Indeed, both ERα and ERβ are expressed in the cerebellum and their expression varies with age Medulloblastoma (MB), a highly malignant primitive neuroectodermal tumor of the cerebellum, represents about 20% of all childhood primary CNS tumors. The peak age at presentation is 3–6 years, with only 25% of patients between 15 and 44 years of age. In adults, the disease is much less common, comprising only 1% of primary brain tumors +/−Ptch1), we previously showed a protective action of estrogen during early stages of MB development; indeed, susceptibility to MB development was significantly increased in OVX +/−Ptch1 females, and restored to levels observed in control mice after estrogen replacement In line with these epidemiological findings, using a well characterized mouse model of radiation-induced MB  was found in three human MB cell lines . Cell proliferation was not affected by 17β-estradiol (E2), DPN , PPT  or DHT . The medulloblastoma cell lines D283 Med (derived from a 6-year-old boy) and Daoy (derived from a 4-year-old boy) were purchased from the European Collection of Cell Cultures . UW228 cell line  5 per well), Daoy (8.0×104 per well), and UW228 (8.0×104 per well) cells were seeded in 6-well plates in complete culture medium. Cells from cultures in log phase of growth were used. After overnight incubation, the medium was changed to phenol-red free medium supplemented with 5% CS-FBS  and containing various concentrations of 17β-estradiol , 2,3-bis(4-hydroxyphenyl)-propionitrile , 4,4′,4″- trisphenol  or 5 alpha-dihydrotestosterone . The substances were dissolved in absolute ethanol and diluted in the appropriate culture medium immediately before use. Control cells received the same amount of diluent. The medium was renewed after 48 hours. At 96 hours of incubation, cells were harvested by trypsinisation, and viable cells were counted using Nucleocounter . All experiments were performed at least three times in duplicate. To validate our experimental conditions, the proliferation of MCF-7 cells (ECACC) was assessed following treatment with E2, DPN, PPT and DHT. Also, the proliferation of Daoy cells after GANT58  and IGF-1  treatments was evaluated to confirm that the growth of MB cells can be modulated in our culture conditions. Substances were dissolved according to manufacturer's instructions.D283 Med  and female (n = 12) athymic mice [Athymic Nude-nu], 6 weeks old, were obtained from Charles River S.r.l. , and housed under controlled conditions. The UKCCCR guidelines 7 D283 Med cells in phenol red-free/growth factor-reduced Matrigel . Inoculated animals were observed daily with tumors measured at least twice per week using vernier calipers. Tumor weight was calculated from two dimensional measurements (mm) 2/2. Upon health decline , they were euthanized to minimize suffering. All remaining animals were sacrificed 7 weeks after tumor inoculation (i.e. day 49 of the study). At necropsy, all tumors were removed, weighed and fixed in 4% buffered formalin for histology, or stored at −80°C for immunoblot analysis.At eight weeks of age all mice received 0.1 ml subcutaneous injection of 3×10Tumor tissues were homogenized in cold RIPA lysis buffer containing protease plus phosphatase inhibitors. Samples were incubated on ice for 30vmin, centrifuged, and the supernatant protein quantified by the Bradford assay (Biorad Laboratories Inc.). Equal amounts of protein (40 µg/sample) were separated by SDS polyacrylamide gel , and transferred onto PVDF membranes . The membranes were blocked for 1 hour with 5% (w/v) nonfat dry milk in Tris Buffered Saline containing 0.1% Tween 20 (TBST) and incubated with primary antibodies: ERα ; ERβ ; AR ; p-21 ; cyclin D1 ; Cyclin E ; anti-phospho-p44/42 mitogen-activated protein kinase (p-p44/42 MAPK) (Thr202/Tyr204) , and anti-total-p44/42 MAP kinase ; β-actin , in TBST with 5% albumin bovine serum, at 4°C overnight. After washing three times with TBST, the membranes were labeled with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Specific proteins were visualized with the ECL Western blotting system according to the manufacturer's instructions . β-actin Western blot analysis was performed as a control of equal sample loading.Immunohistochemical analysis was carried out on 3-µm thick paraffin sections as described We utilized human healthy liver to confirm specificity of the ER primary antibodies used, showing that in this tissue, ERβ1 and ERβ2 isoforms are not expressed, while specific nuclear ERβ5 immunoreactivity is detected. This pattern of ERβ isoforms immunoreactivity is consistent with previous descriptions Scoring of ERβ1, ERβ2 and ERβ5 was evaluated as previously reported p values ≤0.05 were considered statistically significant. Analyses were performed using GraphPad Prism version 5.0 for Windows .Tumor growth data were analyzed by repeated-measures ANOVA, followed by the Bonferroni method as post-test. The remaining data were analyzed for homogeneity of variance using an F test. If the variances were heterogeneous, log or reciprocal transformations were made in an attempt to stabilize the variances. If the variances remained heterogeneous, a non-parametric test such as the Mann–Whitney U test was used. Correlations between variables were identified employing the Spearman's rank correlation. P values are for two-sided tests; For each MB cell culture model, immunoblot analyses were used to evaluate the expression of ERα, ERβ and AR. While ERα was undetectable, total ERβ protein expression was identified in each cell line. A low AR expression was found in D283 Med and Daoy, but not in the female-derived MB cell line UW228 .2, DPN, PPT or DHT . In addition, both the ERβ- and the ERα-selective agonists used  did not influence viable cell numbers, up to 1000 nM , two ERβ splice variant isoforms  p<0.001, p<0.05)  in females and males respectively, showing a significantly lower proliferative potential of lesions from female mice when compared to males p<0.001, . Notably p<0.05) .Although immunostaining does not allow to distinguish wild-type from mutated p53, expression of p53 is often used as a surrogate marker for alterations in the functional status of p53. Indeed, a recent study using the same antibody as our, showed that, in clinical medulloblastoma samples, high levels of detectable p53 protein may correlate with point mutations Using WB analysis, we next investigated the expression of the p53-downstream target, p21 (a cell-cycle arresting factor) versus 5.3±0.4, mean ± SEM, p = 0.09; versus 2.13±0.6, mean ± SEM, p<0.05; Since the insulin-like growth factors (IGF) and their cognate receptors (IGFRs) have been shown to play an important role in MB formation and growth p<0.05, p<0.001, for both parameters, Finally, nestin, NSE and GFAP were used as immunohistochemical markers to compare the degree of tumour differentiation between female and male xenotransplanted mice. Immunohistochemical analysis showed significantly lower nestin levels in tumors from females compared to males p<0.05, , along wDespite the male preponderance of medulloblastoma and reports of female gender as a significant favorable prognostic factor, there has been little investigation into hormonal influences on tumor development. To the best of our knowledge data from experiments described here are the first to support a role of gender in medulloblastoma growth, thus reflecting the clear gender effect in MB susceptibility in both children and adults, and indicating that this body of accumulated evidence, and the conclusions drawn from it, warrant close reevaluation.Indeed, we found that the growth of D283 Med xenografts was significantly lower in nude female mice when compared to males, tumors from males also exhibiting clear features of anaplasia and a higher proliferative index. Consistent with these data, we observed a more differentiated phenotype, with significantly reduced malignancy, in tumors from females, as shown by a decreased stem cell population (nestin has been identified as a stem like cell marker for medulloblastoma) To shed light on the mechanisms responsible for a potential influence of sex on the biology of MB we examined hormone receptor expression profile in tumors from both male and female mice. We demonstrated that tumors from both groups were virtually negative for ERα and AR. On the other hand, we found that the expression of ERβ isoforms was differentially regulated in tumors from males and females. Indeed, while ERβ2 and ERβ5 were consistently expressed in all tumors independently of gender, ERβ1 was completely lost in tumors from males, but maintained in females. ERβ1 is the full-length receptor coded by exons 1–8. ERβ2–5 share exons 1–7 with ERβ1, but display unique sequences instead of exon 8. These differences in the C-terminal part of ERβ2–5 determine a truncation of the ligand binding domain and ablation of the ligand-dependent activation function AF-2. Therefore, ERβ1 is thought to be the only fully functional isoform that is able to bind ligand, whereas ERβ2, -β4, and -β5 do not form homodimers and have no innate activities of their own, but may modulate estrogen action when dimerized with ERβ1 To obtain a more complete view of the role of ERβ1 (and ERβ2/ERβ5) in MB growth, we wanted to investigate whether their differential expression in males and females was associated with the regulation of protein involved in cell proliferation and/or apoptosis. To this end, we analyzed p53 protein expression using immunohistochemistry and found that tumors from males had a significantly higher p53 labeling index when compared to females, this suggesting p53 dysfunction in male xenografts. Notably, in line with our preclinical results, abnormalities in this cell cycle pathway have been reproducibly associated with anaplasia and poor outcomes in clinical studies We went on to investigate the regulation of important protein governing proliferation in the G1-S phase. We found no differences between genders in Cyclin E and D1 expression. These results are at least partially in agreement with literature data showing that in colon cancer cell lines ERβ did not modulate Cyclin A and E, while inducing Cyclin D1 Ptch1+/− females compared to OVX, could be the results of a functional interaction between estrogen- and IGF-I-mediated pathways, since we demonstrated an increase in p-IGF-IR in tumors from OVX, in association with induction of the ERK/MAPKs cascade We previously suggested that the lower susceptibility to MB development observed in intact and E2-replaced OVX in vitro exposure (i.e. 5 to 7 days) or to the fact that estrogen's action may be partly indirect, involving cells of the tumor microenvironment.On the whole, these results support the hypothesis that the molecular mechanisms by which gender affects medulloblastoma may involve the ERβ signaling, and reinforce the growing body of evidence that an imbalanced ERβ expression might play a pivotal role in the development and progression of many tumor types In line with our data, Belcher and colleagues Notably, however, a protective role of estrogen has also been underlined for other brain tumors, with very recent studies suggesting a potential role of ERβ protein expression as an independent favorable prognostic factor in gliomas, and the use of ERβ agonist as therapeutic agent in this disease In summary, our study has shown for the first time a significant difference in medulloblastoma growth between sex in a preclinical mouse model of the human disease, thus reflecting the greater prevalence of MB in males compared to females, regardless of age, tumor histology and region of the world. We also provide mechanistic evidence supporting the idea that ERβ1 signaling may have pro-differentiation and tumor suppressive function in medulloblastomas, thus suggesting that functional activation of the ERβ pathways may be a potential therapeutic option for MB. However, additional studies are needed to dissect the molecular mechanisms by which estrogens affect tumorigenesis in the brain.Figure S1Staining pattern of the ERβ isoforms in human liver. In order to confirm the specificity of the ERβ antibodies used in the study, we immunostained sections of human healthy liver, showing that ERβ1 and ERβ2 isoforms are not expressed, while specific nuclear ERβ5 immunoreactivity is detected (magnification 20x and 40x). This pattern of ERβ isoforms immunoreactivity is consistent with previous descriptions (TIF)Click here for additional data file.Figure S2Proliferation studies. Effects of E2, the ERβ-selective agonist DPN, the ERα-selective agonist PPT and DHT  on the growth of MCF-7 cell line. Cells were seeded in phenol-red free medium supplemented with 5% charcoal stripped FCS, containing various concentrations of substances. Concentrations are expressed in nanomolar. Control cells received the same amount of diluent. The medium was renewed after 48 hours. At 96 hours of incubation viable cells were counted using Nucleocounter. All results are expressed as the mean ± SEM derived from at least three different experiments.(TIF)Click here for additional data file.Figure S3Proliferation studies. Effects of GANT58 and IGF-1 on the growth of Daoy cell line. Cells were seeded in phenol-red free medium supplemented with 5% charcoal stripped FCS, containing various concentrations of substances. Concentrations are expressed in micromolar for GANT58 and in ng/ml for IGF-1. Control cells received the same amount of diluent. At 48 hours of incubation viable cells were counted using Nucleocounter. Data are representative of one experiment done in triplicate (mean ± SEM).(TIF)Click here for additional data file.