NR5A1 have been reported as a frequent cause of 46,XY disorders of sex development (DSD) associated to a broad phenotypic spectrum ranging from infertility, ambiguous genitalia, anorchia to gonadal dygenesis and female genitalia. Here we present the clinical follow up of four 46,XY DSD patients with three novel heterozygous mutations in the NR5A1 gene leading to a p.T40P missense mutation and a p.18DKVSG22del nonframeshift deletion in the DNA-binding domain and a familiar p.Y211Tfs*83 frameshift mutation. Functional analysis of the missense and nonframeshift mutation revealed a deleterious character with loss of DNA-binding and transactivation capacity. Both, the mutations in the DNA-binding domain, as well as the familiar frameshift mutation are associated with highly variable endocrine values and phenotypic appearance. Phenotypes vary from males with spontaneous puberty, substantial testosterone production and possible fertility to females with and without Müllerian structures and primary amenorrhea. Exome sequencing of the sibling’s family revealed TBX2 as a possible modifier of gonadal development in patients with NR5A1 mutations.Mutations in  The term “Disorders of sex development” (DSD) comprises a broad spectrum of clinical conditions affecting the gonadal and genital development. DSD is divided into three categories, namely numerical chromosomal abnormalities, 46,XX DSD and 46,XY DSD . VariousNR5A1 encoding Steroidogenic factor-1 (SF-1) located on chr 9q33.3 (OMIM 184757) [SRY and SOX9 [NR5A1 mutations were described in 46,XY DSD patients. The phenotypical spectrum encompasses hypospadias [NR5A1 mutations were found in 46,XX patients with premature ovarian failure and primary ovarian insufficiency [NR5A1 mutations were described in 46,XY DSD patients with normal adrenal function at the date of examination.One gene emerging in the last years to be causative in 10–20% of 46,XY DSD is  OMIM 18457 [SRY aospadias , ambiguoospadias –15, or aospadias –19, but ospadias . Furtherficiency , 23, 24.ficiency –27. Exceficiency –32 and sficiency , 33 mostNR5A1 mutations. Beside 46,XY DSD patients with low concentrations of AMH and detectable Müllerian structures [NR5A1 mutations as well. In most cases testosterone is low in the neonatal period while the phenotype ranges from ambiguous genitalia to female external genitalia with or without well-developed Wolffian structures [NR5A1 and offer new explanations for phenotypic variation.Gonadotropins and sex hormone levels vary a lot in patients with ructures , 34 and ructures , 35, theructures , 36, 37.ructures , 38 or oructures . Moreoveructures , 39, 40.ructures , 15. HerWritten informed consent was obtained from all patients and family members studied. The study was approved by the Ethical Committee of the University Hospital of Lübeck, Lübeck, Germany (AZ: 08–081).Ambiguous genitalia were noted at birth and a thorough evaluation was initiated at the age of 4 weeks. Initially, a female sex assignment was favored. The clinical description noted normal weight (3250 g) and length (54 cm) and unsuspicious internal and neurological examination except for genital appearance. Prominent labioscrotal folds were noted with palpable gonads, the phallic structure measured 1.3 cm. The urethral and vaginal openings were not described. On urogenitoscopy, a normal bladder was seen, a small vagina, unclear uterine structures, but no colliculus seminalis. On laparoscopy, hypoplastic testicular appearing gonads were seen without Müllerian remnants. On histology, the gonads showed a neonatal testicular appearance with germ cells. Karyotype was 46,XY. Endocrine evaluation showed a high-normal ACTH of 54 pg/mL (reference < 60) with a cortisol of 26 ng/mL, which was interpreted as subnormal. LH was 3.9 IU/L, FSH 13.2 IU/L and interpreted as normal to slightly elevated (FSH reference < 11.0 IU/L), while testosterone was 20 ng/dL and seen also as subnormal. Endocrine testing was performed and revealed a normal response in the ACTH test with a rise in cortisol from 8.2 ng/mL to 54.3 ng/mL. On résumé, a male gender was favored and the child had been reassigned accordingly.2 body surface area and after 72 hours, testosterone had increased to 242 ng/dL. This was seen as proper testosterone response, although the low inhibin B was seen as a possible failure of Sertoli cells. At the age of 11;3 years, inhibin B was below the detection limit, and at 11;11 years, FSH elevated to 20.9 IU/L, thus a progression of testicular dysfunction was presumed. At that time testosterone was 162 ng/dL with LH 5.2 IU/L. Therefore, additional testosterone gel therapy with initially 25 mg testosterone gel and later 37.5 mg was initiated to foster pubertal development. At the age of 15;8 years, the adolescent presented with a height of 180.5 cm, weight 64.3 kg. Genital appearance male according to Tanner stage 4, pubic hair Tanner stage 6. Testes were palpable with a volume of 4–6 cc. LH was 7.6 IU/L, FSH 18.2 IU/L, testosterone 508 ng/dL under supplementation with 37.5 mg testosterone gel. At this time, ACTH was 39.2 pg/mL, cortisol 154 ng/mL, which was interpreted as normal adrenal function and further molecular studies were initiated with the consent of the family. An ACTH-test with 250 μg Synacthen was performed at the age of 16 yrs. with basal cortisol at 182 ng/mL and 319 ng/mL after 60 minutes, thus corresponding with a good adrenal response. At the age of 18 years the patient had a low-normal testosterone production of 383 ng/dL (without testosterone supplementation), but increased gonadotropins . A spermiogram revealed azoospermia and the patient opted for a trial of testicular sperm extraction (TESE) and cryopreservation to preserve fertility.During childhood, several surgical procedures were performed to reconstruct male genital appearance including hypospadias repair, orchidopexy and scrotal fusion. At the age of 10;7 years, clinical appearance was described with a testicular volume of 3–4 cc on both sides and a Tanner stage G2. Hormonal evaluation showed LH 1.03 IU/L, FSH 8.8 IU/L and testosterone 82.6 ng/dL. Inhibin B was 19 ng/L . An hCG-Test was performed with 5.000 IU/mth week of gestation, a thickened neck fold had been documented by routine ultrasound triggering subsequent chorionic villus biopsy. This excluded trisomy 21 and revealed a 46,XY karyotype. Unexpectedly, a second ultrasound in the 30th week of gestation did not reveal evidence for male external genitalia. Delivery occurred in the 41st week of gestation by emergency Caesarian section because of maternal fever due to urinary tract infection. External genitalia of the child were ambiguous with rugated labioscrotal folds, partial labioscrotal fusion and a micropenis of 1.3 cm in length thus confirming the prenatal ultrasound. Only in the left urogenital fold, a gonad of 0.5 cc was initially palpable. At the age of about 2 weeks, ultrasound revealed no evidence for an uterus and the 46,XY karyotype was confirmed. Basal androstenedione was 6 ng/dL , testosterone was < 1 ng/dL , and dihydrotestosterone was < 1 ng/dL . 72 hours following hGC stimulation with 1,200 IU , androstenedione rose to 40 ng/dL, testosterone to 8 ng/dL and DHT to < 1 ng/dL, demonstrating massively compromised gonadal androgen biosynthesis. LH was < 0.5 IU/L, FSH was 7.4 IU/L. No salt losing crises occurred in the newborn period or at any time later on. Plasma steroid profiling combined with a Synacthen test (125 μg) revealed the following basal and stimulated values (0 min / 60 minutes): cortisol 11 ng/mL  / 187ng/mL ; aldosterone 0.28 ng/mL  / 0.45 ng/mL  demonstrating normal adrenal steroid biosynthesis. Further normal basal and stimulated values, respectively, were determined for progesterone, 11-deoxycorticosterone, corticosterone, 17OH-progesterone, and 11-deoxycortisol. All steroid hormones had been determined by multi-steroid RIA following extraction and chromatographic separation through Sephadex columns. Due to the presumed gonadal androgen biosynthesis defect in the absence of Müllerian structures and because the parents wished genetic counseling, molecular analyses of genes involved in gonadal androgen biosynthesis has been initiated. However, gene sequencing of WT1, LHCGR including exon 6A, CYP17A1, HSD17B3 and SRD5A2 revealed no mutations. In order to establish the DSD diagnosis, a combined examination by vaginoscopy and laparoscopy had been initiated. Vaginoscopy revealed a short urogenital sinus while laparoscopy confirmed the previous ultrasound findings demonstrating absence of Müllerian duct derivates. Two testis-like structures could be found in both inguinal channels but no epididymis and no ductus deferens was present on either side  and length (50 cm). At birth a bipartite scrotum and a small phallus size of 20 mm length with chordae and scrotal hypospadias was noted. Both testes were descended and at day 2 a testosterone value of 187 ng/dL was measured. Abdominal sonography revealed no vagina or uterus. Subsequent chromosomal analysis revealed a normal 46,XY karyotype. Endocrine evaluation at 3.5 months revealed normal LH (2.6 IU/L) and FSH (3.7 IU/L) and inhibin B (144 ng/L) values. A stimulation test with hCG revealed normal basal/stimulated values for testosterone (232/735 ng/dL) and DHT (80/469 ng/dL). Hormone values for 17-hydroxpregnenolone (2257 ng/dL), DHEA (116 ng/mL), androstenedione (90 ng/mL), and hydroxyprogesterone (185 ng/dL) were unremarkable. Sex assignment was discussed with the parents and a male sex assignment was favored and the child received hypospadias repair. Sequence analysis of the ospadias  and partPatient 4 is the elder sister of P3 and came to attention in our clinic because of primary amenorrhea at the age of 14;5 years. She presented without pubic hair or breast development . A 46,XY karyotype was detected. LH (31.6 IU/L) and FSH (90.7 IU/L) values were highly elevated and testosterone (16 ng/dL) and inhibin B (<10 ng/L) values very low or even below the detection level. ACTH was 25.16 pg/mL and cortisol was 38.5 ng/mL both values within the normal reference ranges. On ultrasound a very small uterus of prepubertal shape and size was suspected. A laparoscopy was performed, which confirmed the presence of a small uterus and small dysgenetic gonads. The gonadal remnants were removed. The histology revealed rudimentary testicular tissue with fibrosis, some ducts and also Leydig cells. Also epididymal tissue and tubular tissue was identified .Puberty was induced with estradiol. At the age of 15;10 years, she received 2 mg of estradiol valerate. Breast development corresponded to pubertal stage Tanner 4. The uterus was now 4 cm long . GonadotThe AR mutation previously detected in her brother was unlikely the reason for the observed phenotype of gonadal dysgenesis and a search for the genetic cause of the familiar DSD by exome sequencing was initiated.A summary of the endocrine values of patient 1–4 and clinical presentations is given in NR5A1 including the exon-intron boundaries were amplified and sequenced by direct cycle sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3130 Genetic Analyser (Applied Biosystems).Genomic DNA was extracted from peripheral blood leukocytes using the Qiaquick DNA kit . Exons 1–7 of Exome capture and sequencing were performed as described previously . The exoNR5A1 expression plasmid and the STAR and AMH-luciferase reporter genes were kindly provided by Dr. Yukihiro Hasegawa, Tokyo and have been described previously [A>C and c.51_65del were generated by a two-step mediated PCR. In the first step two intermediate PCR products were amplified using NR5A1 wt vector as template and PCR primers CMV_forw: 5’-gatccggtactagaggaactgaaaaac-3’ and NR5A1_a118c_AS: 5’- tgttgttctgcaccgGgcgcttgaagaagcc-3’ for PCR 1 and primers NR5A1_a118c_S: 5’- ggcttcttcaagcgcCcggtgcagaacaaca-3’ and SF1_Ex4intAS_RP: 5’- gcagcacgtagtccggtg-3’ for PCR 2 or mutation c.118A>C and primers CMV_forw and NR5A1_del51_65_AS: 5’- tccgtagtggtagccgcacacggggc-3’ for PCR 1 and primers NR5A1_del51_65_S: 5’- gccccgtgtgcggctaccactacgga-3’ and SF1_Ex4intAS_RPfor PCR 2 for mutation c.51_65del. The resulting fragments were gel purified, annealed and amplified in a third PCR using primer pair CMV_forw and SF1_Ex4intAS_RP for both mutations. These amplicons were again purified and subcloned into the NR5A1 expression vector using the restriction sites EcoRI and AfeI for c.118A>C and EcoRI and BsrGI for c.51_65del. The correct sequence was verified by Sanger sequencing.The c-myc-tagged eviously . The mut2 at 37°C. Cells were transfected using 30 ng of wt or mutant NR5A1 construct or empty vector, 200 ng of the respective AMH- or STAR-reporter plasmid and 10 ng of pGL4.74 Renilla luciferase (Promega) to normalise for transfection efficiency and 0.72 μL FuGene HD (Promega). After 24 hours luciferase activity was determined using the Dual Luciferase Assay System (Promega). Experiments were performed in triplicates and at least 3 independent experiments were carried out. For comparison of experiments performed at different days, activity of wt-NR5A1 was set 100%. HeLa cells (DSMZ no. ACC 57) were obtained from Leibnitz-Institute DSMZ, Göttingen (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH).Transient transfection assays were performed in 24 well plates. 50.000 HeLa cells/ well were grown over night in Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F-12 Ham  supplemented with 10% foetal bovine serum (FBS) in 5% COTo analyse protein expression HeLa cells were transfected and cultured as described before. After a 24 hours incubation they were washed twice with phosphate buffered saline (PBS) and lysed with 100 μl/well ice-cold Mammalian Protein Extraction Reagent  including protease inhibitor cocktail . The cells were incubated on a shaker on ice for 5 minutes, scraped and homogenized using Qiashredder columns. The protein extracts were diluted 1:1 with 2x sample buffer  and denaturated for 10 minutes at 95°C. The proteins were separated by SDS-Page using a 10% discontinuous TGX Stain Free polyacrylamide gel (BioRad). After UV-activation of the gel proteins were blotted onto a nitrocellulose membrane (Bio-Rad) and total protein was visualized by ChemiDoc Touch Imaging System (BioRad) The membrane was blocked in 5% Skim Milk (BD) in PBS with 0.1% Tween-20 (Sigma-Aldrich) over night at 4°C. Myc-tagged SF-1 was detected with the primary antibody anti-c-myc-peroxidase  in a 1:1000 dilution for one hour at room temperature. Specific bands were detected by ChemiDoc Touch Imaging System (BioRad) or Fusion SL chemiluminescence imaging system (Vilber Lourmat) using Western Lightning Plus ECL (Perkin Elmer) as substrate.NR5A1 expression vector using 2.88 μl FuGene HD (Promega)/well. 24 hours later nuclear and cytoplasmic extracts were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents and Halt Protease Inhibitor Single-Use Cocktail EDTA-Free (both Thermo Scientific). Electrophoretic mobility shift assays (EMSA) were performed using Light Shift Chemiluminescent EMSA Kit (Thermo Scientific) according to manufacturer’s instructions. Nuclear extracts were incubated with 5’-biotin labelled oligonucleotides containing a SF-1 responsive element of the mouse AMH promoter without or with either a 200 fold excess of unlabelled target DNA or unlabelled mutated target DNA. Samples were separated by non-denaturating polyacrylamide gel electrophoresis, blotted onto a nylon membrane , UV-crosslinked and detected using Fusion SL chemiluminescence imaging system (Vilber Lourmat). All sequences of oligonucleotides comprising the SF-1 responsive element were previously described [300.000 HeLa cells per well were cultured in 6 well plates as described before and transfected with either 960 ng/well of the WT, mutant or empty escribed  and are The newly identified variants were annotated using ANNOVAR .http://evs.gs.washington.edu/EVS/), the Exome Aggregation Consortium Browser [Allele frequencies of these variants were estimated using the data of the exome sequencing project (6500 samples) , Seattle, WA  in exon 2 of the NR5A1 gene was identified (18DKVSG22) within the loop of the first zinc finger of the protein  and was later also found in their elder 46,XX sister .Exome sequencing of the family of patient 3 and patient 4 revealed a common heterozygous 7 bp deletion (c.630-636del) in exon 4 of X sister . Patiente father . We alsoin-vitro, HeLa cells were co-transfected with the mutant or wild type NR5A1 expression construct and a NR5A1 responsive STAR- or AMH-reporter gene and transient expression assays were carried out. Both SF-1 mutants display an extremely reduced transactivation capacity on both reporter genes  compared to the wild type . For missense mutations NR5A1 p.T40P and TBX2 p.N214S all pathogenicity prediction algorithms i.e. SIFT, PolyPhen2, LRT, MutationTaster or FATHMM, predict these mutations as deleterious, probably damaging or disease causing. The newly invented scaled Combined Annotation Dependent Depletion (CADD) scores (V1.3) [18D_22Gdel.All three s (V1.3)  are 26.8NR5A1 gene are listed in the Human Gene Mutation Database (HGMD) and have been described mostly associated with 46,XY DSD, but also associated with hypospadias, cryptorchidism, infertility or anorchia. In 46,XX individuals, mostly in mothers or sisters of 46,XY DSD patients an association to premature ovarian insufficiency (POI) have been observed [NR5A1 mutations. So far almost all NR5A1 mutations associated with DSD are described as heterozygous mutations. Mice with a homozygous targeted deletion of the Nr5a1 gene displayed a lack of adrenal glands and gonadal agenesis and died at postnatal day 8 because of the lack of corticosteroids [Today more than 100 different mutations in the observed . Recentlobserved –27. The steroids . Only thsteroids , 49, 50.STAR and AMH promoter. The severe functional deficit of both mutant proteins may explain the phenotype of ambiguous genitalia observed after birth but hardly predicts pubertal outcome or gender assignment. Promoters in NR5A1 target genes show several variations of the consensus binding site (Py)CA AGGTCA and it has been shown for the NR5A1 mutant G35E that mutations in the DNA-binding domain can lead to a differential binding and activation of these target genes [The mutations of P1 and P2 lie within the N-terminal zinc finger of the highly conserved DNA-binding domain and have not been described before. The predicted deleterious, probably damaging or disease causing nature of mutation p.T40P and the disruptive effect of the nonframeshift deletion of five amino acids within the loop of the first zinc finger in mutation p.D18_G22del was confirmed experimentally by a severe loss of DNA-binding or transactivation capacity as demonstrated by EMSA and two different NR5A1-dependent reporter gene assays using fragments of the human et genes . Amino aet genes  and intrNR5A1 mutations [Two natural NR5A1 mutations adjacent to T40 have been described, p.R39P and p.V41P. Both are associated to 46,XY girls with clitoromegaly and primary amenorrhea and low testosterone values , 54. Patutations , 38, 55.NR5A1 microdeletion indicate a common paternal allele in P3 and P4. Interestingly, a very similar NR5A1 deletion  was found in another family with 3 affected members [T-box factor 2 (TBX2) found in P4 and her mother. TBX2 is a transcription factor expressed in the mouse at E13.5 in the interstitium as well as in the Wolffian and Müllerian ducts and may have a role in Leydig cell development, Wolffian duct differentiation or Müllerian duct regression [The reason for the high intra-familiar phenotypic variability observed between P3 and P4 and also in other families still remains elusive , 56. Furgression .NR5A1 mutations in rare cases [Possible fertility proven by paternal transmission of re cases , 58 may NR5A1 have been observed in men with infertility or hypospadias as well as in 46,XY individuals with ambiguous genitalia or anorchia, but also in 46,XY girls with primary amenorrhea with and without Müllerian structures. Very recently it has been shown that this spectrum of DSD phenotypes is extended also to 46,XX ovotesticular DSD (OTDSD) and 46,XX males with testicular DSD (TDSD) [SOX9 expression in 46,XY DSD or an insufficient repression of SOX9 in 46,XX OTDSD/TDSD [Gonadal dysgenesis in the clinical context has been defined by the presence of Müllerian structures in 46,XY DSD. Mutations in D (TDSD) , 26. MorD (TDSD) , 26, 55.DSD/TDSD –27. AlsoIn contrast to mice, where Nr5a1 is sexually dimorphic expressed in somatic cells at the early stage of testis and ovary development human NR5A1 is upregulated in the undifferentiated gonad, maintained in the period of fetal development in both sexes and may contribute to the development of testes and ovaries , 59. TheNR5A1 have been detected in three 46,XY DSD patients as well as in two 46,XX girls. Interestingly, the same homozygous R92Q mutation was detected in a 46,XY DSD patient and a 46,XX girl from two different families [NR5A1 mutations. Therefore we measured ACTH and cortisol values or performed ACTH stimulation tests and monitored basal and stimulated plasma steroid values. Our patients showed normal cortisol values or a good response to ACTH and no evidence for adrenal insufficiency. Nevertheless, adrenal function should be followed in carriers of NR5A1 mutations, because additional factors that contribute to phenotype in the rare cases described are unknown and adrenal insufficiency may develop over time.Adrenal insufficiency associated to heterozygous and homozygous mutations in families , 32. As families , 30. Thefamilies . These rS1 TableThe oligonucleotides contain either a core SF-1 binding motif of the mouse AMH promoter (mMIS) or a mutated SF-1 binding motif mMIS_mut. The SF-1 binding motif is highlighted in bold. Oligonucleotide sequences have been described previously .(DOCX)Click here for additional data file.S1 FigRetracted testis-like right and left gonads out of the inguinal channels, respectively. No epididymis or ductus deferens could be detected on either gonad.(TIF)Click here for additional data file.S2 FigImmature Sertoli-cell-only tubules with small or absent lumina and reduced tubular diameter. No Leydig cells were detected in the interstitium.(JPG)Click here for additional data file.S3 FigCYP17A1 staining revealed clusters of vacuolated Leydig-cell-like cells in fibrotic stroma of the gonad A) overview 40x, B) 400x magnification. A very low testosterone value of 16 ng/dl in P4 indicates non-sufficient testosterone synthesis despite high CYP17A1 expression. C) Other parts of the gonad reveal epididymal and tubular tissue (HE stain).(TIF)Click here for additional data file.S4 FigA) before and B) after estrogen treatment.(TIF)Click here for additional data file.S5 FigLHX2 and OLFML2A alleles from the father and, therefore most likely, also the same NR5A1 alleles from the mother as well as the father.Nearest informative SNPs in the exome data are located 0.3 Mb upstream and 0.48 Mb downstream of the micro deletion and revealed that both siblings carry the same (TIF)Click here for additional data file.


The prognosis of this tumor is favorable, and it is characterized by slow progression. 10–30% of these tumors recur after 4–7 years of the primary surgery and the 5-year survival rate from the first recurrence is 55%, for the incompletely resected patients. At this time, complete resection is the only prognostic factor for better outcome, and establishing a novel strategy for identification and/or treatment of recurrent tumors is crucial. After the discovery of heterozygous FOXL2 status in 5 matched primary and recurrent samples by immunohistochemistry, and TaqMan allelic discrimination assay to address the role of FOXL2 in potential mechanisms of recurrence.We analyzed the clinical data of 56 aGCT patients to find a marker of recurrence. In particular, we compared the c.402C>G FOXL2 mutation and the FOXL2 protein expression.The clinical data analysis was consistent with complete resection as an indicator of disease eradication, though the sample size was limited. The genetic analysis showed all the samples, including recurrent tumor samples up to 14 years after the primary surgery, expressed heterozygous c.402C>G FOXL2 mutation in recurrent aGCTs. This finding adds further credence to the concept that the c.402C>G FOXL2 mutation is oncogenic and integral to this disease.This report describes the preservation of heterozygous  In studies with larger cohorts6]7]9][7][9]6][[9][7][9][7][9]6], 80–90% Despite an overall favorable 10-years survival rate for aGCT, the prognosis after relapse is still poor. In previous studies, 30% (3/10)  of recurc.402C>G missense mutation in the FOXL2 gene in 97% of aGCTs tumors, this strongly implied a driver potential for this mutation in aGCT. Tum. Tum27].Tumor areas were enriched by manual macro-dissection with a scalpel from 30μm thick sliced FFPE sections to remove non-tumor tissues; a pathologist-marked serial H&E-stained section was used as a guide. Samples were deparaffinized and genomic DNA was purified using QIAamp DNA FFPE Tissue Kit (Qiagen) following the manufacturer’s protocol.FOXL2 c.402C>G mutation using primers: 5’-GCGCAAGGGCAACTACTG-3’ (forward) and 5’-CGGTAGTTGCCCTTCTCGAA-3’ (reverse), along with wild type specific probe (5’-FAM dye- CATGTCTTCCCAGGCCG-NFQ (non-fluorescent quencher)) and mutation specific probe (5’-VIC dye-CATGTCTTCGCAGGCCG-NFQ) included in the genotyping master mix. Reactions were performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems). Reaction volume of 5 μl was used for each replicate well including 2.5 μl 2X TaqMan master mix (Life Technologies), 0.125 μl (40 ×) custom synthesized allelic discrimination primer/probe mix (Life Technologies), 1 μl DNA (standard input of 20 ng was adjusted), and water. After denaturation at 95°C for 10minutes, DNA was amplified over 40 cycles  . In a. In a7].c.402C>G FOXL2 mutation was found in 89% (33/37) of primary and 80% (4/5) of metastatic aGCTs . Pat. Patc.40c.402C>G FOXL2 mutation, between primary and recurrence, even in with late recurrence 14 years after primary surgery. One patient had three distinct recurrence events, with consistent results (FOXL2 IHC and mutation) in all specimens. No previous reports have focused on the FOXL2 status in pairs of primary-recurrent tumors, however our result is concordant with the finding that 3 of 4 index cases and 16 of 69 in first series in original description of c.402C>G FOXL2 by Shah and colleagues[c.402C>G FOXL2 mutation and the FOXL2 protein expression were preserved in all evaluated primary, metastatic lesions, and recurrent samples is consistent with this mutation being critical for long-term maintenance of aGCTs, as opposed to strictly tumor initiation, and may also reflect the genetically stable characteristics of aGCTs.All the recurrent samples also showed similar FOXL2 level of staining and heterozygous olleagues. The finc.402C>G FOXL2 mutation as an apparent driver of aGCT in primary and matched recurrent tumors. The importance of the complete resection is evident in our cohort, despite its limited sample size. Larger cohorts, long term follow up and molecular analysis will be required to better this disease.This is the first report to identify heterozygous S1 Table(TIFF)Click here for additional data file.S2 Table(TIFF)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.


Expression of NC-B27 was determined by immunohistochemistry and flow cytometry using HC10 and HD6 antibodies. These data are the extension of the data presented and discussed in “Non-conventional forms of HLA-B27 are expressed in Spondyloarthritis joints and gut tissue”  Data is presented showing expression of non-conventional (NC) heavy chain forms of B27 in synovial tissues from SpA patients. Data is presented showing the expression patterns of NC-B27 in joint, gastrointestinal and lymphoid tissues from B27 transgenic (TG Specifications TableValue of the data•Data presented in this article confirm the role of NC-B27 in SpA pathogenesis in both human and transgenic rats.•This data serves as a benchmark for future studies on the pathogenic role of NC-B27 in SpA.•The data is valuable for future studies on development of novel treatment strategies for SpA.11 rats with M.tb-induced SpA and in healthy WT and B7 TG controls (1 rats in a spontaneous and M.tb-induced SpA before and after disease onset (The immunohistochemistry data show expression of NC-B27 forms (HC10 and HD6 staining) in synovial tissues from B27+ve SpA patients , and in controls , Fig. 7.se onset , Fig. 1022.1+ SpA patients, including 1 with Ankylosing Spondylitis (AS) fulfilling the New York classification criteria Human synovial tissue samples were obtained with informed consent and appropriate ethical permission, from B272.21 HLA-B27/Huβ2m 1. A higher proportion of these B27 TG1 male rats spontaneously develop arthritis  and spondylitis  without symptoms of gut inflammation M. tuberculosis (hereafter referred to as “M.tb-induced arthritis and spondylitis”) 1 rats with spontaneous or induced SpA at age 4–15 weeks. For M.tb-induced arthritis and spondylitis 1 rats were immunized with 30–45 μg of heat-inactivated M.tb in incomplete Freund׳s adjuvant 1 HLA-B7/Huβ2m TG (B7 TG) and Lewis wild type (WT) animals +/− 200 μg of heat-inactivated M.tb in IFA  were used as controls. All animals were bred and housed at the animal facility of the AMC, University of Amsterdam, Netherlands. All animal procedures were carried out in compliance with Institutional Standards for Human Care and Use of Laboratory Animals.B27 transgenic (TG) rats first generated by Hammer and colleagues spontaneously develop inflammatory gut and joint disease 2.3The HC10 antibody stains many or all heavy chain forms  of most human HLA-B and some HLA-A alleles, but does not cross react with rat MHC 2.4+ substrate-chromogen  and counterstained using Mayer׳s hematoxylin. Slides were visualized using an LSM Zeiss confocal microscope, scanned using AperioCS2 Scanner and analyzed using Aperio ImageScope software .Human SpA and RA, and rat paraffin-embedded synovial tissue samples were prepared as previously described 2.5®780 (eBioscience). Flow cytometric analysis was performed with BD FACS Canto and data were analyzed using FlowJo Software (TreeStar). Staining was performed in triplicates. Error bars were calculated based on SD mean of the values if 3≥ animals per group. P values were determined using nonparametric Mann–Whitney test.Splenocytes and LNs were freshly isolated and immediately stained as described previously Arthritis Research UK, United Kingdom Grant no. 19,611, and by an EMBO travel award. This work was supported by the Oxford National Institute of Health Research (NIHR) Biomedical Research Center, the Oxford NIHR Biomedical Research Unit (PB).OR was supported by


Type 2 diabetes (T2D) is a high-risk factor for Alzheimer’s disease (AD) due to impaired insulin signaling pathway in brain. Capsaicin is a specific transient receptor potential vanilloid 1 (TRPV1) agonist which was proved to ameliorate insulin resistance. In this study, we investigated whether dietary capsaicin could reduce the risk of AD in T2D. T2D rats were fed with capsaicin-containing high fat (HF) diet for 10 consecutive days (T2D+CAP). Pair-fed T2D rats (T2D+PF) fed with the HF-diet of average dose of T2D+CAP group were included to control for the effects of reduced food intake and body weight. Capsaicin-containing standard chow was also introduced to non-diabetic rats (NC+CAP). Blood glucose and insulin were monitored. The phosphorylation level of tau at individual sites, the activities of phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) and glycogen synthase kinase-3β (GSK-3β) were analyzed by Western blots. The results revealed that the levels of phosphorylated tau protein at sites Ser199, Ser202 and Ser396 in hippocampus of T2D+CAP group were decreased significantly, but these phospho-sites in T2D+PF group didn’t show such improvements compared with T2D group. There were almost no changes in non-diabetic rats on capsaicin diet (NC+CAP) compared with the non-diabetic rats with normal chow (NC). Increased activity of PI3K/AKT and decreased activity of GSK-3β were detected in hippocampus of T2D+CAP group compared with T2D group, and these changes did not show in T2D+PF group either. These results demonstrated that dietary capsaicin appears to prevent the hyperphosphorylation of AD-associated tau protein by increasing the activity of PI3K/AKT and inhibiting GSK-3β in hippocampus of T2D rats, which supported that dietary capsaicin might have a potential use for the prevention of AD in T2D. Alzheimer’s disease(AD) is a devastating neurodegenerative disorder and exhibits two hallmark brain lesions—neurofibrillary tangles and senile plaques . NFTs arDiabetes is one of the most common metabolic diseases worldwide and type 2 diabetes (T2D) accounts for 90–95% of all diabetes. T2D is characterized by peripheral insulin resistance that leads to glucose intolerance and hyperglycemia. Cognitive impairment and dementia have become the new complications of T2D, with the elongation of T2D patients' life expectancy. It’s concluded that T2D patients are at about 60% greater risk for the development of dementia compared with those without diabetes –9. Down-2+-permeable nonselective cation channel highly expressed in dorsal root ganglion cells and primary sensory afferents, where it is involved in thermal nociception in addition to the burning sensation we feel upon eating spicy food [Capsaicin is the major pungent ingredient in hot chili peppers and is a highly selective agonist for the transient receptor potential vanilloid 1 (TRPV1) . TRPV1 iicy food . TRPV1 hicy food . Growingicy food  and TRPVicy food . In the icy food . A recenicy food . HoweverWe hypothesized that dietary capsaicin might have a beneficial effect on preventing AD or AD-like changes in the hippocampus of T2D rats. Therefore, the T2D rats model by feeding with high fat diet and a single intraperitoneal injection of streptozotocin (STZ) were used. The phospho-sites of tau protein, the activities of PI3K/AKT and GSK-3β were examined after dietary capsaicin exposure, in order to elucidate whether PI3K/AKT plays a critical role in the regulating effect of dietary capsaicin on tau phosphorylation.A total of 60 male Sprague-Dawley rats, aged between 10 and 12 weeks, weighing 200–250 g, were purchased from the Animal Center of Tongji Medical College, Huazhong University of Science and Technology . Animals were individually housed on a light/dark cycle 12h/12h (lights on at 21:00h) in a temperature (23°C) and humidity (50 ± 10%) controlled room. All procedures were approved by the Animal Care and Use Committee of the Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.The procedures of this experiment are shown in Food was taken away during 9:00h-21:00h (dark cycle) and the blood glucose and plasma insulin were monitored at 20:00h before the light cycle. After examination, the food was put back and the light cycle began. The blood sample was considered to be fasting status. Rats were considered hyperglycemic if the fasting blood glucose level was higher than 16 mmol/L . The rats with HF diet and STZ injection did not develop hyperglycemia within three days after the STZ injection were excluded from further study. Thus, the final numbers of T2D rats were 32.Three days after the intraperitoneal injection, rats were further randomly grouped. T2D rats were divided into three groups: T2D+CAP group, T2D+PF group, and T2D group. Rats in T2D+CAP group were fed with 0.01% capsaicin –23 contaBlood glucose levels were measured by using a 3-μl drop of blood obtained by nicking the tail blood vessel and a One Touch Ultra Link Blood Glucose Meter . Plasma insulin was determined by sandwich enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instruction . Insulin resistance was assessed by homeostasis model assessment-insulin resistance (HOMA-IR) using the formula of HOMA-IR = fasting insulin (mIU/L) × fasting blood glucose (mmol/L) / 22.5 .Just before the light cycle (21:00h), rats were sacrificed by decapitation without anesthesia, and the brains were rapidly removed, the hippocampus were dissected on an ice-cooled board and stored at -80°C for Western blotting analysis.3VO4, 50 mM NaF, 1.0 mM PMSF, and 2.0 μg/ml each of aprotinin, leupeptin and pepstatin. The homogenates were centrifuged at 16,000 rpm for 15 min, and the supernatants were stored in aliquots at -80°C until use. The tissue extracts were separated in SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membrane . Nonspecific binding sites on the membrane were blocked by 5% bovine serum albumin in 0.1% TBS/Tween-20 (TBST) for 1 h. The membrane was then incubated with specific primary antibodies (Brain tissue samples were homogenized in a buffer containing 40 mM Tris-HCl (pH 7.0), 1% Triton X-100, 0.2% SDS, 1.0 mM sodium deoxycholate, 1.0 mM EGTA, 1.0 mM EDTA, 1.0 mM Natibodies  overnighpost hoc test, or Student’s t test using the program GraphPad Prism 5.0 . Results are expressed as mean ± SD, and P < 0.05 was considered statistically significant.Data were analyzed by ANOVA, followed by Within 3 days after the STZ injection, 32 of the 40 HF-diet fed rats (80%) developed T2D, as assessed by blood glucose in fasting status (>16 mmol/L) . The remth day of capsaicin exposure  to control for the effects of reduced food intake and body weight. In our study, there were no significant improvements of blood glucose, plasma insulin and HOMA-IR between T2D and T2D+PF groups after capsaicin exposure, which indicated direct beneficial effects of capsaicin on glucose metabolism and insulin resistance. No significant changes of blood glucose, plasma insulin and HOMA-IR between NC+CAP and NC groups after capsaicin exposure, which suggested no effects of capsaicin on glucose metabolism and insulin resistance on normal diet condition.It has been reported that an increasing in tau hyperphosphorylation at several AD-associated hyperphosphorylation sites in the brains of type 2 diabetic patients  and rodeAKT and GSK-3β are important protein kinases in the insulin signaling pathway in brain . Upon biWhen we compared these two kinases between NC and T2D rats, we found that phosphorylation of both AKT and GSK-3β was significantly lower in T2D rat brains than NC group, whereas the total levels of these two kinases were unchanged . These rDuring the last three decades, tremendous progress was made in our understanding of the mechanisms of AD. However, discovery of new pharmacological approaches, especially of disease-modifying drugs, has not been successful to date . Lifestyth day of capsaicin exposure. After capsaicin exposure, T2D+CAP rats showed improved glucose metabolism compared to T2D rats but rats in T2D+PF group didn’t get such improvement compared to T2D group, which indicated that improvements in glucose metabolism and insulin sensitivity were directly the result of capsaicin itself rather than energy intake reduction. However, no significant body weight decrease was seen in T2D+CAP group or T2D+PF group in the end of 10-day exposure of capsaicin compared to T2D group, which may due to the short term exposure of capsaicin.Evidences supported that capsaicin was indicated in effects of obesity and improving glucose homeostasis in type 2 diabetes. Dietary capsaicin improved glucose homeostasis and insulin sensitivity, reduced body weight, attenuated inflammation of adipose tissue and liver, enhanced fatty oxidation in high-fat diet fed obese mice  and imprThere is no cure for AD at the present time. Although amyloid-β peptide is the primary trigger of AD according to the amyloid cascade hypothesis , neurofi2+/PI3K/AKT signaling is still unclear. Further studies are needed to demonstrate the specific molecular mechanism.Impairment of brain insulin signaling has been implied in the development of AD , 42. As In conclusion, this study supports the view that dietary capsaicin could partly restore the impaired insulin signaling pathway in brain, whereas ameliorate AD-associated hyperphosphorylated tau protein in T2D. No impairment in brain insulin signaling pathway was seen when capsaicin applied to non-diabetic rats fed with standard chow diet. Therefore, the present study provides possible supporting evidence for the role of dietary capsaicin reducing the risk of AD in T2D.S1 FigWestern blot analysis of tau protein, AKT, GSK3β in rat hippocampus.(PDF)Click here for additional data file.S1 TableBody weight changes of rats in different groups on week -12, day -3, day 0 and day 10.(PDF)Click here for additional data file.S2 TableEnergy intake of rats in different groups on week -12, day -3, day 0 and day 10.(PDF)Click here for additional data file.S3 TableFasting blood glucose and fasting plasma insulin levels of rats in different groups on week -12, day -3, day 0 and day 10.(PDF)Click here for additional data file.


Further investigation indicated that EZH2 protein significantly accelerated the cell cycle transition from the G0/G1 to S phase. TOP/FOP-Flash reporter assay revealed that EZH2 significantly activated Wnt/β-catenin signaling and the target genes of Wnt/β-catenin pathway were up-regulated, including β-catenin, cyclin D1, and c-myc. Moreover, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays confirmed that EZH2 inhibited the expression of glycogen synthase kinase-3β (GSK-3β) and TP53 through physically interacting with motifs in the promoters of the GSK-3β and TP53 genes. Additionally, blockage of the Wnt/β-catenin pathway resulted in significant inhibition of cell proliferation, and activation of the Wnt/β-catenin pathway resulted in significant enhancement of cell proliferation, as induced by EZH2. Taken together, our data demonstrate that EZH2 promotes cell proliferation and tumor formation in cervical cancer through activating the Wnt/β-catenin pathway by epigenetic silencing via GSK-3β and TP53.Enhancer of zeste homolog 2 (EZH2), a catalytic core component of the Polycomb repressive complex 2 (PRC2), stimulates the silencing of target genes through histone H3 lysine 27 trimethylation (H3K27me3). Recent findings have indicated EZH2 is involved in the development and progression of various human cancers. However, the exact mechanism of EZH2 in the promotion of cervical cancer is largely unknown. Here, we show that EZH2 expression gradually increases during the progression of cervical cancer. We identified a significant positive correlation between EZH2 expression and cell proliferation  Cervical cancer is the fourth most common tumor type and the fourth leading cause of cancer death among women worldwide, and its incidence has increased in recent years . Nearly EZH2, a key component of the PRC2 complex, which is a transcriptional repressor that helps to maintain cell identity during development by promoting chromatin modifications , has beein vitro and in vivo by inhibiting Wnt/β-catenin signaling, which has key roles in embryonic development [However, the molecular mechanisms of EZH2 in cervical carcinoma are largely unknown. In the present study, we demonstrated that EZH2 was up-regulated during the development and progression of cervical cancer and that the down-regulation of EZH2 using CRISPR-Cas9 knockout technology and shRNA in cervical cancer cells inhibited cell growth both elopment  and manyelopment –27.in situ (CIS), and cervical carcinoma (CC) tissues. EZH2 staining was observed in the nuclei of positive cells in all of the different cervical tissues  in the cervical cancer tissues , cervical carcinoma s Figure . The num tissues . Analysip<0.01). These results suggest that EZH2 is highly expressed in cervical cancer and that it may promote the development and progression of cervical carcinoma.Furthermore, EZH2 protein expression was detected by western blotting in 8 normal cervical specimens and 8 primary cervical carcinoma specimens Figure . The aveNext, using immunohistochemistry and western blot assay, we found that EZH2 showed different expression levels in five cervical cancer cell lines, including HeLa, SiHa, C33A, CaSki and HT-3 . To furtin vivo.To determine whether EZH2 affects tumor formation ability, female nude mice (6 mice per group) were injected subcutaneously with EZH2-modified cervical cancer cells. Tumor growth was monitored in terms of tumor volume every three days, and the net weights of the sacrificed mice were recorded upon termination of the experiment. The results showed that HeLa-CRISPR-EZH2 and HeLa-shEZH2 cells formed tumors that were much smaller Figure ; P<0.01 To determine whether the tumor promotion of EZH2 is involved in cell proliferation, the expression of Ki67, a well-known cell proliferation marker, was examined in EZH2-modified cervical cancer cells from tumor xenograft tissues and in control cells by immunohistochemical staining. Both Ki67 and EZH2 staining were weaker in the xenograft tissues formed by EZH2-silenced HeLa and SiHa cells than those formed by control cells, respectively Figure . Furtherin vitro.Next, HeLa and SiHa cells with knock down of EZH2, as mediated by both CRISPR/Cas9 (HeLa-CRISPR-EZH2 and SiHa-CRISPR-EZH2) and shEZH2 (HeLa-shEZH2 and SiHa-shEZH2), showed significantly reduced cell growth and viability than the control cells , as determined by cell growth curve assay and MTT assay Figure , P<0.05.To investigate the mechanism of the EZH2 protein-mediated enhancement of cervical cancer cell proliferation, cell cycle analysis of EZH2-modified HeLa and SiHa cells and control cells was performed by fluorescence-activated cell sorting (FACS). The percentage of S phase cells significantly decreased from 42.7% for HeLa-CRISPR-ctrl cells to 31.6% for HeLa-CRISPR-EZH2 cells Figure , P<0.01,p<0.05; SiHa, p<0.01). However, the luciferase signal was increased by approximately 2-fold in the HeLa-EZH2 cells compared with the control cells (p<0.05). These results suggest that EZH2 expression is positively related to the activity of the Wnt/β-catenin pathway in cervical cancer.A previous study has described EZH2-activated Wnt/β-catenin signaling in gastric cancer  and hepap<0.05). Consistent with the activation of Wnt/β-catenin signaling, the expression of the β-catenin, cyclin D1, and c-myc proteins in the EZH2-overexpressing HeLa cells was significantly increased compared with that in the control cells (p<0.05). However, the accumulation of β-catenin in cytoplasm and its translocation into the nucleus consistently occurred following activation of the Wnt/β-catenin pathway, leading to the transcription of downstream target genes [Next, the expression of β-catenin and the target genes of the Wnt/β-catenin pathway, including cyclin D1 and c-myc, were detected by western blotting using EZH2-silenced HeLa and SiHa cells and control cells Figure . The relet genes . The expp<0.01, Figure p<0.01, Figure p<0.05, Figure To further confirm that the Wnt/β-catenin pathway is the pathway by which EZH2 promotes the proliferation of cervical cancer cells, XAV939, which is an inhibitor of Wnt/β-catenin that acts by stimulating β-catenin degradation via stabilizing axin, a component of the β-catenin destruction complex , and CHIp<0.01, Figure We next elucidated the mechanism whereby EZH2 activates the Wnt/β-catenin pathway in cervical cancer. EZH2 is known to be a key component of the PRC2 complex, which functions as a transcriptional inhibitor by binding to target genes , 34. On in vivo. Five pairs of primers were designed to amplify the promoters of GSK-3β and TP53. Immunoprecipitation using a EZH2 antibody revealed the enrichment of EZH2, with binding to the promoters at the TP53-G2 and GSK-3β-G1 and G2 positions  assay to determine whether the EZH2 protein binds to specific regions of the GSK-3β and TP53 promoters s Figure . These rs Figure . Taken tTo validate the correlation between the expression of EZH2 and Wnt/β-catenin signaling-related proteins in cervical cancer specimens, EZH2, β-catenin, cyclin D1, and c-myc expression levels were detected by IHC staining Figure . NotablyThe overexpression of EZH2 was first found to be associated with carcinogenesis in prostate cancer  and breain situ tissues and then to cervical cancer tissues  were purchased from the American Type Culture Collection . HeLa, SiHa, and C33A cells were cultured in Dulbecco's Modified Eagle's Medium , and CaSki and HT-3 cells were cultured in RPMI-1640 (Sigma-Aldrich) and McCoy's 5A medium (Sigma-Aldrich), respectively, supplemented with 10% heat-inactivated fetal bovine serum . All cell lines were maintained at 37°C with 5% COin situ (CIS) tissues, and 62 squamous cervical cancer (SCC) tissues were collected from the First Affiliated Hospital of Xi'an Jiaotong University Medical College from 2009 to 2014. All of the procedures followed approved medical ethics practices. None of the patients had received chemotherapy, immunotherapy, or radiotherapy before specimen collection. The histological classifications and clinical staging were in accordance with the International Federation of Gynecology and Obstetrics classification system.A total of 40 normal cervical (NC) tissues, 41 cervical cancer Immunohistochemical staining was performed as previously described . EZH2 stFor the immunocytochemistry experiments, cells were cultured on coverslips, fixed with 4% paraformaldehyde for 30 minutes at room temperature, and permeabilized with 0.2% Triton X-100 for 15 minutes at room temperature, and they were then incubated with the primary antibodies described above. An antibody against EZH2 was obtained from Cell Signaling Technology , and additional antibodies against Ki67, β-catenin, cyclin D1 and c-myc were obtained from Santa Cruz Biotechnology .Western blot analyses were performed as previously described with lysates from fresh tissues and cells . The priHuman full-length EZH2 (NM_152998.2) cDNA was amplified by reverse transcription polymerase chain reaction using mRNA extracted from HeLa cells. The primer sequences were designed as follows:EZH2-F: 5-′ CCGCTCGAGGCCACCATGGGCCAGACTGGGAAGAAATCTG-3′; and EZH2-R: 5-′ CGCGGATCCTCAAGGGATTTCCATTTCTCTTTCG-3′The EZH2 DNA fragment was subsequently cloned into the Xho I and BamH I  sites of an internal ribosome entry site vector, pIRES2-AcGFP1 , to generate the pIRES2-AcGFP1-EZH2 recombinant plasmid.http://crispr.mit.edu/) hosted by the Feng Zhang laboratory  to obtain the minimum number of off-target sites in the human genome (The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Plasmid#42230) containing SpCas9 and single guide RNA was obtained from a non-profit plasmid share repository belonging to Feng Zhang  . Suitabln genome . The resn genome .To construct a small interfering RNA expression vector that expressed EZH2-specific short hairpin RNA (shRNA), the following four EZH2 sequences were used, which were obtained from GenePharma Co., Ltd. : GCAGCTTTCTGTTCAACTTGATTCAAGAGATCAAGTTGAACAGAAAGCTGCTTTTTTG, GGTAAATCCAAACTGCTATGCTTCAAGAGAGCATAGCAGTTTGGATTTACCTTTTTTG, GCAAAGCTTACACTCCTTTCATTCAAGAGATGAAAGGAGTGTAAGCTTTGCTTTTTTG, and GCTGATGAAGTAAAGAGTATGTTCAAGAGACATACTCTTTACTTCATCAGCTTTTTTG. All transfection experiments were performed using Lipofectamine 2000 reagent  according to the manufacturer's instructions. Transfected cells were treated with G418  for approximately 3 weeks, and drug-resistant colonies were collected, expanded, and identified.4) were seeded in triplicate with 1.5 ml of media into 35-mm tissue culture dishes for 7 days. The numbers of cells were counted after harvesting every two days using a hemocytometer under light microscopy. Cell viability assays were performed by applying 3--2,5-diphenyl tetrazolium bromide  dye to cells that were seeded in 96-well plates with 1000 cells in each well, as described in a standard protocol. Then, the absorbance at 490 nm was measured (Bio-Rad).Cells  according to the manufacturer's protocol. Cells were harvested and fixed in 75% ethanol overnight at 4°C. Thirty minutes before FACS analysis, the cells were treated with RNase A and were then stained with propidium iodide (Sigma-Aldrich). Cell cycle distribution was analyzed using a FACSCalibur flow cytometer with Mod-Fit LT software.6) were injected into the subcutises on the dorsa of 4-6 week-old female BALB/c-nude mice . The tumor volume (V) was determined by the length (a) and width (b) as V=ab2/2. The experimental protocols were evaluated and approved by the Animal Care and Use Committee of the Medical School of Xi'an Jiaotong University. At the end of the experiment, the mice were killed by cervical vertebral dislocation, and the weights of the tumors were measured after they were dissected out.The experimental protocols used were evaluated and approved by the Animal Care and Use Committee of the Medical School of Xi'an Jiaotong University. Cells in the exponential growth phase were harvested for inoculation. Tumor cells (1×104) in 24-well plates, and the activities of both firefly and Renilla luciferase reporters was determined at 48 hours after transfection using a Dual Luciferase Assay Kit  according to the manufacturer's instructions. The TOP-Flash reporter activity is presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. All experiments were performed three times in triplicate.TOP-Flash reporter and pTK-RL plasmids were transiently co-transfected into tumor cells  and pTK-RL plasmids were co-transfected into cells. Following incubation for 48 h, cell monolayers were harvested by resuspension in passive lysis buffer. Luciferase activity was determined with a luminometer (Promega). The efficiency of transfection was normalized by paired Renilla luciferase activity using a Dual Luciferase Reporter Assay System (Promega). The specific activity was displayed as the fold change of the experimental group versus the control group.Quantitative chromatin immunoprecipitation (qChIP) assays were performed using an EZ-ChIPTM Assay Kit  according to the manufacturer's protocol. Regions of interest were amplified from precipitated samples by real-time polymerase chain reaction (PCR). Each sample was assayed in triplicate, and the amount of precipitated DNA was calculated as a percentage of the input sample . The priStatistical analysis was performed with SPSS 18.0 software . All data were expressed as the group mean ± standard deviation of the mean (SD). A P value of <0.05 was considered statistically significant. For comparison among the groups, the chi-square test or one-way ANOVA was performed.


For each immune mediator, the relative change (RC) in the mean level was modeled by robust log-normal regression and correction for multiple testing was performed; (3) Results: Two associations were identified; there was a negative association between 25(OH)D (10 nmol/L increase) and leptin ), 0.98 ), and a positive association between 25(OH)D (10 nmol/L increase) and the chemokine, chemokine (c-x-c motif) ligand (CXCL) 8 (RC (95% CI), 1.07 ); (4) Conclusion: CXCL8 and leptin have significant associations with levels of 25(OH)D in the newly diagnosed cohort. These results do not indicate a strong influence of 25(OH)D on the peripheral immunological or inflammatory signature. (1) Background: We aimed to examine if 25-hydroxyvitamin D (25(OH)D) was related to the peripheral immunological and inflammatory signature both at birth, and in newly diagnosed patients with childhood type 1 diabetes (T1D) and their healthy controls; (2) Methods: The birth cohort consisted of 470 patients and 500 healthy controls. Dried blood samples were collected from the neonates in the period 1981–1999. The newly diagnosed cohort consisted of 460 patients and 453 siblings. Serum samples were collected in the period 1997–2005. A variety of peripheral immune mediators were measured and compared to total 25(OH)D levels (25(OH)D Type 1 diabetes (T1D) is an endocrine disease that results from autoimmune destruction of the pancreatic insulin-producing β-cells, leading to a loss of insulin secretion and symptomatic hyperglycemia. Changes in the complex interplay between genetic, epigenetic, and environmental factors are thought to be involved in T1D etiopathogenesis ,3. Vitamin D is in the spotlight, as many in vitro and animal studies indicate that vitamin D has anti-inflammatory activity and may also prevent or halt the destruction of β-cells ,5. FindiWe conducted two separate case-control studies—both study samples are described extensively elsewhere including coding of covariates ,8,9,10. Blood sampling (dried blood spots (DBS)) was performed within a week following birth during the years 1981 to 1999. For each identified clinical case (a neonate who subsequently develops T1D before the age of 18 years) one control was selected based on having measurements on both peripheral immune mediators and 25(OH)D. Clinical cases were identified from the Danish Patient Register and further validated against the Danish Registry of Childhood and Adolescent Diabetes (DanDiabKids) . Our ranSince 1981 the DBS have been stored at −20 °C/−4 °F in the DNSB and this biobank comprises almost 100% of the Danish population born since 1982 .A random sample of 500 clinical cases was collected from a biobank linked to the DanDiabKids. Blood samples were taken less than three months after the onset of T1D during the years 1997 to 2005. The onset date was defined as the date of the first insulin injection. The control group comprised a random sample of 500 siblings selected from an eligible sample of approximately 2000 siblings with blood samples stored in the biobank and a sibling diagnosed with T1D. The chosen control sample was independent of participation of a sibling with T1D in the study. Randomization aimed at covering all sample months (January 1997 to December 2005). A last inclusion criterion was that all individuals had to be between 0 to 18 years of age at time of blood sampling. Serum samples from the DanDiabKids were stored at −80 °C/−112 °F during the entire study period from 1997 to 2005.® technology were used to quantify the following cytokines: interleukin (IL)-1β, IL-4, IL-6, chemokine (c-x-c motif) ligand 8 (CXCL8), IL-10, IL-12 (p70), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 (active form) (TGFβ), leptin, and adiponectin. In addition, we also quantified c-reactive protein (CRP), mannose-binding lectin (MBL) and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) [Multiplexed sandwich immunoassays and the flowmetric Luminex xMAPsTREM-1) . In all sTREM-1) . BiomarksTREM-1) . Quality control of the analysis were made using mouse IL-6 as an internal analyte added to the extraction buffer to detect pipetting errors, and biotinylated beads to detect signal errors . CalibrThe serum samples were analyzed using the commercially available high-capacity Luminex xMAP technology combined with a 15-plex and 3-plex developed in-house for the simultaneous determination of 18 biomarkers, as described in Skogstrand et al. . The sec2 and 25(OH)D3 in 3.2 mm samples  taken from DBS cards. Sample preparation and analysis was performed using liquid chromatography–mass spectrometry (LC-MS) according to a modified method [In short, 25(OH)D status was assessed by measuring 25(OH)Dd method , describd method . 3 and 3 nmol/L for 25(OH)D2. The 25(OH)D levels from the DBS are full blood concentrations. Most 25(OH)D molecules in the bloodstream are protein bound and to approximate and report sera concentrations we corrected the original levels using the formula: serum 25(OH)D = full blood 25(OH)D × (1/(1 − 0.61)), where 0.61 is the haematocrit fraction for capillary blood [The lower limit of quantification (LLOQ) was 4 nmol/L for 25(OH)Dry blood .Vitamin D status was measured as serum 25(OH)D by high-performance liquid chromatography (HPLC) . Detecti2 + 25(OH)D3) was modeled by a robust log-normal model regression, which takes into account: (1) that measurements are potentially both left and right censored; and (2) correlation within assay. To account for correlation within cluster inference was based on a working independence generalized estimation equation (GEE) approach. For the birth cohort the clusters are equivalent to assay. For the newly diagnosed cohort, the cluster was equivalent to family/sibling ID.For each peripheral immune mediator the relative change (RC) in the mean level by 10 nmol/L increase in total 25(OH)D DFor the birth cohort, the following risk factors are included in the model: 25(OH) levels, sex, case status (T1D or control), gestational age, mothers age, birth weight, season and calendar year group. For the newly diagnosed cohort the following risk factors are included in the model: 25(OH) level, sex, case status (T1D or sibling), age at blood sampling, season and calendar year group. The coding of these variables is presented in Simultaneous evaluation of risk factors on all peripheral immune mediators was done using the model stacking approach detailed in Pipper et al. . SubsequOverall functional misspecification by including 25(OH)D as a trend (linear variable) was assessed by a lack-of-fit test. Specifically, we included a quadratic term of 25(OH)D and tested its significance by a robust Wald test. All analyses are made using the statistical software package R version 3.2.0  and the add-on packages survival, ggplot2, and multcomp.Both studies were performed in accordance with the Helsinki II Declaration. Furthermore, both studies were approved by the Danish Ethical Committee (H-4-2013-049 and H-KA-20070009). All of the patients and their parents or guardians gave informed consent.The birth cohort contains 970 individuals with a complete set of covariates (470 patients and 500 controls). In the birth cohort, the median/first and third quartile (Q1–Q3) 25(OH)D level was 26.3/17.1–38.8 nmol/L for patients and 25.7/17.0–37.8 for controls. The newly diagnosed cohort contains 913 individuals with a complete set of covariates (460 patients and 453 controls). In the newly diagnosed cohort the median/Q1–Q3 25(OH)D level was 62.0/40.1–95.5 for patients and 57.9/38.0–89.0 nmol/L for controls. It is noteworthy to mention that we have already shown that 25(OH)D levels are not associated with later risk of childhood T1D in the birth cohort and levels between newly diagnosed patients with childhood T1D and their healthy siblings do not differ either [We were unable to detect any linear association of 25(OH)D on the 14 examined peripheral immune mediators. The numeric results including a graphical overview (forest plot) are presented in p = 0.25).We also examined if non-linear associations existed between 25(OH)D and the peripheral immune mediators in the birth cohort, but found no sign of such associations (padjusted = 0.02), and a negative association between 25(OH)D (10 nmol/L increase) and leptin (RC (95% CI), 0.98 , padjusted = 0.01). The other 16 peripheral immune mediators were not associated with 25(OH)D. The numeric results including a graphical overview (forest plot) are presented in We only found a statistical significant positive association between 25(OH)D (10 nmol/L increase) and CXCL8 (RC (95% CI), 1.07 , p = 0.26).As for the birth cohort, we also examined if a non-linear associations between 25(OH)D and the peripheral immune mediators existed in the newly diagnosed cohort, but no proof of such associations were found D levels were compared with these immune and inflammatory factors, and two peripheral immune mediators, i.e., CXCL8 and leptin, were associated with 25(OH)D in the newly diagnosed cohort. These results do not indicate a strong role of 25(OH)D as an immune modulator when using peripheral immune mediators as a proxy for the child’s immunological fingerprint. 2D) can alter, e.g., up-regulate, its expression, which may help to clear infections by recruitment of specific immune cells [We demonstrated that CXCL8 levels in our newly diagnosed cohort rises with 1%–13% for every 10 nmol/L increase in 25(OH)D. CXCL8, also known as IL-8, is a chemokine which affects leukocyte migration positively and thereby promotes inflammation . Interesne cells ,24. In ane cells ,25. Our ne cells . FurtherWe also found leptin levels significantly reduced with 0%–4% for every 10 nmol/L increase in 25(OH)D in the newly diagnosed cohort. Leptin is an adipokine, secreted primarily from adipose tissue. The overall action of leptin in the immune system is to activate leukocytes and mediate inflammation . In a reThe effect sizes of the abovementioned significant associations are both small, and may not have profound immunological importance. Importantly, we do not find any association between physiological 25(OH)D levels and well-known Th1, Th2 or Treg cytokines that have been associated with either β-cell destruction or protection e.g., IL-1β, IFNγ, TNFα, IL-10 and TGFβ ,31. This study has a number of strengths. Firstly, the sample size in both cohorts includes over 900 individuals—both cohorts consist of a patient group (childhood T1D) and a healthy control group . Secondly, both cohorts are population-based and patients with childhood T1D are thoroughly validated and controls are randomly selected. Thirdly, quantification of both 25(OH)D and a broad spectrum of peripheral immune mediators, initially measured for testing other hypotheses, gave us the opportunity to conduct the current study using these unique cohorts. Our study also has some limitations. Firstly, a single measurement of both 25(OH)D and peripheral immune mediators may not detect long-term differences or reflect dynamic changes. Secondly, sample storage time may influence assay measures due to degradation, however calendar year of blood sampling was included in both models to control for this. Thirdly, one may ask if these measured peripheral immune mediators, in some part, mirror or affect the tissue-specific immunological micro-milieu in the islet of Langerhans in the pancreas. Or, it could be the case that we just looking at a more general immunological cross-sectional fingerprint and its association with 25(OH)D. Either way, we find a very small influence of 25(OH)D on these peripheral immune mediators. We have examined a wide range of peripheral immune and inflammatory mediators in both neonates who subsequently develop childhood T1D, and in a separate cohort of newly diagnosed patients with childhood T1D. Two immune factors, i.e., CXCL8 and leptin, have significant associations with levels of 25(OH)D in the newly diagnosed cohort. These results indicate that vitamin D does not appear to play a major role as an immune-modulator of the peripheral immune system. To further understand a possible role of vitamin D on the human immune system, studies need to be performed in more complex systems that include genetics, repeated measures of functional immune cell populations and gene-immune mediator interactions.


SCLO1B1 gene (Beta(SE) = -0.088(0.006)P = 1.65 x 10−51, P < 1 x 10−51), the allele previously associated with increased risk of statin associated myopathy is associated with higher hexadecanedioate levels. However this SNP did not show association with BP or hypertension. The top SNP in the second locus rs6663731 mapped to the intronic region of CYP4Z2P on chromosome 1 (0.045(0.007), P = 5.49x10-11). Hexadecanedioate levels also correlate with adipose tissue gene-expression of the 3 out of 4 CYP4 probes (P<0.05) and of alcohol dehydrogenase probes (Beta(SE) = 0.12(0.02); P = 6.04x10-11). High circulating levels of hexadecanedioate determine a significant effect of alcohol intake on BP (SBP: 1.12(0.34), P = 0.001; DBP: 0.70(0.22), P = 0.002), while no effect is seen in the lower hexadecanedioate level group. In conclusion, levels in fat of ADH1A, ADH1B and CYP4 encoding enzymes in the omega oxidation pathway, are correlated with hexadecanedioate levels. Hexadecanedioate appears to regulate the effect of alcohol on BP.The dicarboxylic acid hexadecanedioate is associated with increased blood pressure (BP) and mortality in humans and feeding it to rats raises BP. Here we aim to characterise the molecular pathways that influence levels of hexadecanedioate linked to BP regulation, using genetic and transcriptomic studies. The top associations for hexadecanedioate in a genome-wide association scan (GWAS) conducted on 6447 individuals from the TwinsUK and KORA cohorts were tested for association with BP and hypertension in the International Consortium for BP and in a GWAS of BP extremes. Transcriptomic analyses correlating hexadecanedioate with gene expression levels in adipose tissue in 740 TwinsUK participants were further performed. GWAS showed 242 SNPs mapping to two independent loci achieving genome-wide significance. In rs414056 in the  Hypertension represents a major global disease burden, but discovering pathways for blood pressure (BP) regulation has been challenging. A number of recent studies by our group and others have found several metabolites to be correlated with BP –4. CircuWe hypothesized that identifying the genetic contribution to circulating levels of hexadecanedioate and genes whose expression is highly correlated to this compound should reveal some of the pathways defining the regulation and pathology of how hexadecanedioate affects BP.The study participants were twins enrolled in the TwinsUK Registry, a national register of adult twins recruited via media campaigns without selecting for any particular disease and phenotype .Here we dissected the hexadecanedioate genome-wide association scan (GWAS) data that was previously generated and published as part of our GWAS-metabolomics study. BrieflyWe tested the multiple single nucleotide polymorphisms (SNPs) associated with hexadecanedioate for association with BP in the International Consortium for Blood Pressure (ICBP) and withThe association of hexadecanedioate with gene-expression levels in fat and lymphoblastoid cell line (LCL) was tested in 740 females from the TwinsUK cohort using random intercept linear regression after adjusting for age, BMI, metabolite batch, expression batch and family relatedness. Gene expression was analysed with the Illumina Human HT-12 V310 as previously reported[Post-genomic functional analysis was undertaken using the Database for Annotation, Visualization and Integrated Discovery (DAVID) . This isPathway analysis was carried out on the microarray results using a list of genes corresponding to probes with p values less than P<0.00002 (n = 52).2, BMI and family relatedness.We assessed the association between alcohol intake (measured via Food Frequency Questionnaire) and BP stratifying by hexadecanedioate levels by using random linear regression adjusting for age, ageThe study was approved by St. Thomas' Hospital Research Ethics Committee. All participants provided informed written consent.http://www.twinsuk.ac.uk/data-access/accessmanagement/).TwinsUK metabolomics, and phenotypic data are publicly available upon request on the department website (The flowchart of the study design is depicted in P<5x10-8)[SLCO1B1) on chromosome 12 and a cytochrome 4 cluster on chromosome 1 which contains the genes CYP4A11, CYP4B1 and CYP4Z2P. The locus plots of SLCO1B1 and the CYP4 cluster are presented in The results of the GWAS study from our previously published metabolomics GWAS are preseP<5x10-8). All theSLCO1B1 gene on chromosome 12 (Beta(SE) = -0.088(0.006)P = 1.65 x 10−51), encoding an Val174Ala amino acid change. This SNP did not show association with BP[P = 0.0347) which remains essentially unchanged if adjusted for SBP (-0.156(0.07), P = 0.0319).Among the top SNPs, rs4149056 is a non-synonymous polymorphism on the n with BP. The hexSLCO1B1 showed nominal association with HTN (Beta(SE) = 0.06(0.03), P = 0.02) in the GWAS of BP extremes , P = 5.49x10-11). No association with HTN was detected for the SNPs mapping to the cytochrome 4 cluster. However, circulating levels of hexadecanedioate correlate with adipose tissue gene-expression levels of probes mapping to CYP4B1 and CYP4Z2P . The top association is with the Alcohol Dehydrogenase 1B (Class I), Beta Polypeptide (ADH1B) gene on chromosome 4. SNPs on the gene are also associated with circulating hexadecanedioate levels in our metabolomics GWAS [Gene expression in 740 abdominal fat and LCL samples from the TwinsUK cohort were tested for association with hexadecanedioate levels. 23 gene transcripts showed significant association with hexadecanedioate levels in fat after adjusting for multiple testing  = 1.30(0.29),P = 1.1x 10−5) and DBP (0.74(0.19), P = 7.5x 10−5) supporting our previous findings. Overall alcohol consumption is significantly associated with SBP and DBP after adjusting for covariates (SBP:0.54(0.19), P = 6.2x10-3) and DBP . However when stratifying for hexadecanededioate levels, the association between alcohol and BP is present in the individuals in the top tertiles of hexadecanedioate circulating levels (SBP: 1.12(0.34), P = 0.001; DBP: 0.70(0.22), P = 0.002). On the other hand the level of alcohol intake has no influence on the association between hexadecanedioate levels and BP in those with low hexadecanedioate levels , P = 3.2x10-4).Because genetic variants at the uced HTN , we exple levels . When weThis result suggests that high levels of hexadecanedioate may be indicating a dysfunction or a saturation of enzymatic pathways within the liver related to alcohol metabolism.P values remained significant  tissues in which their functions regarding blood pressure are more important . The alcohol dehydrogenase 1B (class I), beta polypeptide (ADH1B) is however the strongest gene whose expression is associated with hexadecenedioate after adjusting for multiple testing. Although the effect of hexadecanedioate on BP remains unvaried after adjustment for alcohol intake [Our gene-expression analyses show that circulating levels of hexadecanedioate are nominally correlated with adipose tissue levels of probes mapping to SLCO1B1, an association previously reported [The strongest genetic association seen with hexadecanedioate maps to reported , 17 in areported .SLCO1B1 gene encodes OATP1B1,  which is mainly expressed on the sinusoidal membrane of human hepatocytes. Substrates of OATP1B1 include endogenous organic anions such bilirubin, estradiol, prostaglandin 2, leukotrienes C4 and thyroxine, and structurally diverse drugs, such as statins, antibiotics (Rifampicin) antivirals (Saquinavir) and some anti-hypertensive drugs [The alsartan).The functional variants that causes reduced function of OATP1B1 and identified as increasing the risk of statin myopathy (rs414056 and rs4363657 and variants in linkage disequilibrium with them) are the most strongly associated with higher levels of hexadecanedioate.SLCO1B1 with HTN or BP in the two GWAS that we tested [SLCOB1 reflects circulating levels in large part determined by hepatic uptake of the compound but this association may not be related to intracellular levels of hexadecanedioate.We find no convincing evidence of an association between variants in e tested , 11.ThisCYP4 subfamilies or by two other CYP450 enzymes, and the electron donor NADPH. The next step is the oxidation of the hydroxyl group to an aldehyde by NAD+ and is catalysed by alcohol dehydrogenase, whose subunits are encoded by genes ADH1A, ADH1B nad ADH1C[Hexadecanedioate is a product of omega-oxidation, a secondary fatty acid oxidation pathway. In the first step, an hydroxyl group is introduced onto the omega carbon. This reaction is carried out by certain members of the nad ADH1C. The thinad ADH1C.CYP4A11, CYP4B1 and CYP4Z2P on chromosome 1 as shown in The second strongest genes to be statistically associated with hexadecanedioate are the Cytochromes CYP4B1 has been shown to be involved in prostaglandin metabolism[etabolism through In rodent models, decreased expression of CYP4A results in increased epithelial sodium channel (ENaC) activity and salt-sensitive hypertension.Increased CYP4Z2P- along with the functional CYP4Z1-3'UTR expression has been shown to promote tumor angiogenesis in breast cancer partly via miRNA-dependent activation of PI3K/Akt and ERK1/2. This isADH1B(0.12(0.02), P = 6.04x10-11). In addition also gene expression levels of ADH1A are associated with hexadecanedioate levels (0.09(0.02), P = 1.19x10-6). These genes encode the second enzyme in the omega oxidation making this association logical. It further suggests that study of the relationships between alcohol, hypertension and hexadecanedioate is needed. We also find significant associations between circulating levels of hexadecanedioate and 3 SNPs on the ADH1B gene (P<0.0005) as shown in ADH1B is implicated in hexadecanedioate regulation. Though we find no association between SNPs on ADH1B in hypertension and no association has been reported in Caucasians, a role for ADH1B in hypertension has been found in Japanese men[The gene whose expression is most strongly associated with regards to hexadecanedioate is anese men. Here weBoth cross-sectional and prospective epidemiological studies have established a relationship between hypertension and alcohol consumption , 25. ExcOur finding that alcohol intake has a much stronger effect in individuals with high hexadecanedioate may have therapeutic implications for the treatment of alcohol-induced hypertension suggesting that the strategies for reducing blood pressure may be different depending on the subject’s hexadecanedioate levels.ADH1B, SLCOB1 and CYPB4A11 and BP may be due to their link to hexadecanedioate. SLCOB1 appears not to be associated with BP in spite of its very strong association with hexadecanedioate levels. Our data lend support to the use of intermediate phenotypes, in this case, a metabolite that contributes to BP regulation, to understand some of the pathways involved in BP regulation and cardiovascular risk.In conclusion, all three genes identified in this study as strongly associated with levels of hexadecanedioate have been previously linked to hypertension, but this association is not strong, or not present in Caucasians. Some of the effects previously reported  between S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Canine influenza virus (CIV) has the potential risk to spread in different areas and dog types. Thus, there is a growing need to develop an effective vaccine to control CIV disease. Here, we developed three vaccine candidates: 1) a recombinant pVAX1 vector expressing H3N2 CIV hemagglutinin (pVAX1-HA); 2) a live attenuated canine adenovirus type 2 expressing H3N2 CIV hemagglutinin (rCAV2-HA); and 3) an inactivated H3N2 CIV (A/canine/Guangdong/01/2006 (H3N2)). Mice received an initial intramuscular immunization that followed two booster injections at 2 and 4 weeks post-vaccination (wpv). The splenic lymphocytes were collected to assess the immune responses at 6 wpv. The protective efficacy was evaluated by challenging H3N2 CIV after vaccination (at 6 wpv). Our results demonstrated that all three vaccine candidates elicited cytokine and antibody responses in mice. The rCAV2-HA vaccine and the inactivated vaccine generated efficient protective efficacy in mice, whereas limited protection was provided by the pVAX1-HA DNA vaccine. Therefore, both the rCAV2-HA live recombinant virus and the inactivated CIV could be used as potential novel vaccines against H3N2CIV. This study provides guidance for choosing the most appropriate vaccine for the prevention and control of CIV disease. In 2004, canine influenza virus (CIV) was first reported in Florida  . CIV oriCIV, mainly including H3N8 , 7, 8 anOver recent decades, influenza A virus vaccines have been developed to contain several viral strains, and different vector types have been used for delivery. The most common human influenza A vaccine contains an inactivated virus –20. FurtIn an attempt to develop a vaccine against CIV, a canarypox-vectored EIV H3N8 vaccine expressing hemagglutinins of A/equine/Kentucky/94 (vCP1529) and A2/equine/Ohio /03 (vCP2242) and an equine herpesvirus type 1 recombinant EIV H3N8 vaccine expressing hemagglutinins of A/canine/PA/10915-07 were evaluated against H3N8 CIV in dogs , 35. In Figure 4  titer ≥ 2n = 3/group) was detected using a CCK-8 kit at 6 wpv. The stimulation index (SI) revealed prominent increases in lymphocytes in mice vaccinated with pVAX1-HA, rCAV2-HA, and inactivated vaccine  -2, IL-4, IL-10 and interferon (IFN)-γ) in mice vaccinated with pVAX1-HA, rCAV2-HA, and inactivated CIV were significantly increased compared with mice vaccinated with empty plasmid, adjuvant or PBS  Figure .The rectal temperature of mice was measured every day post-challenge. However, a relatively constant temperature of 36.8 ± 0.2°C was maintained (data not shown). At 5 days post-challenge (dpc), lungs were collected for pathology. Gross lung lesions (hemorrhages and tumidness) were characterized in all control groups Figure . The lunIn Figure The percent of weight change was based on the starting weight on 0 dpc. In this trial body weight loss was not noted in mice from the rCAV2 and inactivated groups, but slight body weight loss was noted in mice from the pVAX1-HA DNA group. However, significant body weight loss was noted in mice in the no vaccination control groups , and the body weight loss rate exhibited its lowest point on 6 dpc Figure .A hallmark of the influenza virus is the remarkable variability of its major surface glycoproteins. HA is one such surface glycoprotein that has been used in recombinant vaccines against influenza because it is a dominant antigen that can enhance the immune response to influenza . The HA TM Gel01 ST was used as adjuvants for both inactivated and live virus vaccines [As previously reported, the plasmid pVAX1 serves as a promising approach for antigen preparation in vaccine development. This DNA vaccine vector system is ideal for high-level recombinant protein production in CHO cells . Additiovaccines , 44 to eOf note, we observed that the HI titers of the rCAV2-HA group were increased compared with the pVAX1-HA group but reduced compared with the inactivated group at the same time post vaccination  of CIV-challenged mice, which was consistent with the previous study . We founOur results demonstrated that a live recombinant virus (rCAV2-HA) and an inactivated vaccine generated significant levels of immune activation in mice. Additionally, all three candidate vaccines displayed heightened protective efficacy against CIV H3N2 challenge, whereas limited protection was provided by the pVAX1-HA DNA vaccine. Therefore, both the rCAV2-HA live recombinant virus and the inactivated CIV could be used as potential novel vaccines against the current epidemic of CIV H3N2. Moreover, the time of challenge with H3N2 CIV after vaccination was 2 wpv. If the time of challenge was extended, a stronger immune response may be activated that would provide better protection from virus. In addition, a potential limitation should be noted. These CIV vaccines were only evaluated in mice, and the further studies in dogs should be explored.2. The avian-origin CIV strain A/canine/Guangdong/01/2006 (H3N2) was used for the inactivated vaccine and challenge experiment [MDCK cells  were maintained in Eagle's Minimum Essential Medium with 10% fetal bovine serum (FBS) and an antibiotic solution . All cells were incubated at 37°C and 5% COperiment . The ppoperiment .A total of 96 specific pathogen-free female BALB/c mice (six weeks old) were produced by the Center for Laboratory Animal Science of Guangdong and used for this study. All animal studies were conducted under guidelines approved by the Animal Care and Use Committee of the South China Agricultural University.® 2000 Transfection reagent (Invitrogen). The expression of HA mRNA and protein in MDCK cells were detected by RT-PCR and indirect immunofluorescence assays (IFA). The pVAX1-HA plasmid used for vaccination was extracted from E. coli DH5α using a no-endotoxin plasmid extract kit .The HA gene fragment amplified from the CIV genome was inserted into the pVAX1 vector to construct the recombinant plasmid pVAX1-HA, which was transferred into MDCK cells using Lipofectamine® 2000 Transfection reagent, and the cells were cultivated at 37°C for 72 h. Subsequently, the recombined ppoly2-CAV2-ΔE3-HA virus (rCAV2-HA) was collected and identified based on adenovirus cytopathologic characteristics as assessed by electron microscopy. HA protein expression in MDCK cells was verified by Western blotting, and the rabbit anti-H3N2 HA monoclonal antibody (obtained from our Lab) and IRDye 680RD goat  anti-rabbit IgG  were used. The viral titer of rCAV2-HA and CIV were quantified by TCID50 in MDCK cells as previously described [In addition, we generated the recombinant canine adenovirus type 2 virus (rCAV2-HA) expressing H3N2 CIV HA protein as previously described . Brieflyescribed . The virescribed  and asseescribed . Inactivn = 12 per group) and were intramuscularly injected with vaccines as follows: Group 1 (pVAX1-HA group), 100 μg protein pVAX1-HA in 0.1 mL; Group 2 (rCAV2-HA group), rCAV2-HA (104 TCID50) with 20 μL adjuvant in 0.1 mL; Group 3 (inactivated group), inactivated CIV (104 TCID50) with adjuvant in 0.1 mL; Group 4 (pVAX1 group):, 100 μg protein pVAX1 in 0.1 mL; Group 5 (CAV2 group): CAV2 (104 TCID50) with 20 μL adjuvant in 0.1 mL; Group 6 (adjuvant group), 0.1 mL adjuvant; Group 7 (PBS group), 0.1 mL PBS; Group 8 (NI group), no immunization; Blank control group (BC), no immunization and no challenge.The procedure for the immunization experiment is presented in Mice received an initial intramuscular immunization that was defined as 0 wpv. Then, mice received a booster immunization at 2 and 4 wpv. Mice were intramuscularly injected with an equal volume into each calf muscles.At two, four, six, and eight weeks after the initial vaccination, serum samples were collected from the tail vein of all mice to evaluate the HI antibody and cytokine expression levels. Sera were heat-inactivated (at 56°C for 30 min) and treated with a receptor-destroying enzyme (RDE) (1:3) before the test. The HI assay was performed as described previously .5 cells/well). Subsequently, 100 μL/well of medium containing HA protein (10 μg/mL), PBS (negative control) or Concanavalin A  (positive control) was added at 37°C and 5% CO2 for 72 h. The proliferative activity was measured using a CCK8 kit . The SI was calculated as the ratio of the average OD450 value of the wells containing antigen-stimulated cells to the average OD450 value of the wells containing only cells with the medium. All assays were performed in triplicate. Passive secretion of cytokines (interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ) from collected supernatants was detected using specific ELISA kits (R&D). Procedures were performed per the manufacturer's instructions. For each plate, the OD450 was transformed to a concentration by applying a linear regression formula, which was calculated from the standards of each kit.Briefly, splenocyte suspensions were isolated from mice (3/group) by pushing the spleens through a wire mesh one day prior to the challenge. RBCs were removed using the RBC lysis solution (Sigma), and splenocytes were supplemented with RPMI-1640 medium including 20% FBS and an antibiotic solution . Cells were plated in 96-well flat-bottom plates at 100 μL/well  were anesthetized with isoflurane (3%) and were intranasally inoculated with 0.1 mL CIV (105 TCID50) at 6 wpv. The body weight was monitored and recorded on 0, 3, 6, 9, and 14 dpc. The rectal temperature was measured every dpc. On 5 dpc, 3 mice were euthanized by carbon dioxide inhalation in a gas chamber. After carbon dioxide treatment, heart rates, respiratory rates, and pupillary light reflexes were tested to confirm death. Lungs were collected rapidly and then immersed in 10% neutral formalin buffer overnight to prevent autolysis. The lungs were then sectioned and stained with HE for histopathological analysis. In addition, the virus titers in respiratory tract tissues were assessed by the plaque assay [Mice  and p < 0.01 (**) were considered statistically significant and highly significant, respectively.All data were processed and analyzed using SPSS13.0 Data Editor . Lymphoproliferation, cytokine production and viral titers were compared between groups using one-way ANOVA. Statistical analysis was performed using the unpaired Student's All animal care and experimental procedures were performed under an approved protocol in compliance with CDC Institutional Animal Care and Use Guidelines.


Cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8) assay and cleaved caspase expression, respectively. Osteoblast differentiation was evaluated by Alizarin Red S staining and osteogenic marker gene expressions. Protein expression was detected by Western blot analysis. Dex upregulated the expression of Y1 receptor in MC3T3-E1 cells associated with reduced osteogenic gene expressions and mineralization. Blockade of Y1 receptor by shRNA transfection and BIBP3226 significantly attenuated the inhibitory effects of Dex on osteoblastic activity. Y1 receptor signaling modulated the activation of extracellular signal-regulated kinases (ERK) as well as the expressions of osteogenic genes. Y1 receptor agonist inhibited ERK phosphorylation and osteoblast differentiation, while Y1 receptor blockade exhibited the opposite effects. Activation of ERK signaling by constitutive active mutant of MEK1 (caMEK) abolished Y1 receptor-mediated Dex inhibition of osteoblast differentiation in MC3T3-E1 cells. Taken together, Y1 receptor regulates Dex-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling. This study provides a novel role of Y1 receptor in the process of GC-induced suppression in osteoblast survival and differentiation.High dose glucocorticoid (GC) administration impairs the viability and function of osteoblasts, thus causing osteoporosis and osteonecrosis. Neuropeptide Y1 receptor (Y1 receptor) is expressed in bone tissues and cells, and regulates bone remodeling. However, the role of Y1 receptor in glucocorticoid-induced inhibition of osteoblast differentiation remains unknown. In the present study, osteoblastic cell line MC3T3-E1 cultured in osteogenic differentiation medium was treated with or without of 10 Glucocorticoids (GCs) are extensively used as immunosuppressive and anti-inflammatory drugs for various disorders including autoimmune diseases and inflammatory ,2. ExcesNeuropeptide Y (NPY), a 36-amino-acid peptide abundantly expressed in the central nervous systems, is found to play an important role in the regulation of bone metabolism as well as the modulation of food intake and energy balance . Among tCrosstalk between NPY system and glucocorticoid is found in the regulation of various functions in different cells ,19. More−7 M being the most effective concentration. Application of 10−7 M dexamethasone to MC3T3-E1 cells for 48 h caused a significant increase of Y1 receptor mRNA expression  to the culture media and found that RU486 significantly reduced glucocorticoid-induced Y1 receptor expression in MC3T3-E1 cells  treatment in osteogenic differentiation media. The results of real-time PCR demonstrated that the expression of Y1 receptor was upregulated by dexamethasone in a dose-dependent manner A, with 1pression B in parapression C,D. Simipression E. To invcontrols G. Fewer controls H,I in coE1 cells J. To test whether Y1 receptor inhibition influenced Dex-induced suppression of osteoblast differentiation in MC3T3-E1 cells, we silenced the Y1 receptor using shRNA interference. The level of Y1 receptor mRNA was significantly decreased after treatment with shRNA plasmid targeting Y1 receptor, suggesting a high efficiency of shRNA interference A. The reFurthermore, the inhibitory effects of Dex on the expression of RUNX2 and osteocalcin (OCN), two osteogenic marker genes, were reversed by Y1 receptor shRNA A,B. Aliz31, Pro34]-NPY and antagonist BIBP3226. BIBP3226 at a concentration of 10−6–10−9 M significantly attenuated the Dex-induced inhibition of cell proliferation at 24 h  expression by glucocorticoid  upregulated the expression of osteogenic genes and enhanced bone formation, whereas a high dose of Dex (>10−8 M) significantly inhibited osteogenic differentiation and mineralization capacity in osteoblasts -NPY increased the baseline and Dex-induced RANKL/OPG ratio in osteoblastic cells, whereas BIBP3226 had the opposite effects. The modulation of RANKL/OPG ratio by the Y1 receptor also suggested the potential involvement of the Y1 receptor in bone resorption. Published studies have shown that the knockout of the Y1 receptor increased bone formation in C57/BL mice through the peripheral nervous system , whereasone loss . In this31, Pro34]-NPY on the osteoblastic activity in MC3T3-E1 cells. These results verified the involvement of ERK signaling in osteogenesis, and further suggested that the Y1 receptor, through targeting ERK signaling, played a role in glucocorticoid-induced suppression of osteoblast activity. To explore the mechanisms by which the Y1 receptor mediated the induction of Dex mentioned above, we focused on the response of ERK and p38 signaling to Y1 receptor modulation. The ERK and p38 signaling pathways mediate various biological reactions in osteoblasts ,24. The The application of glucocorticoid receptor (GR) blocker RU486 abolished the upregulation of Y1 receptor expression in cell cultures after Dex treatment. It has been demonstrated that the glucocorticoid receptor is the master modulator that mediates the biological effects of glucocorticoid on the osteoblasts . Loss of4 cells/well) and incubated at 37 °C (5% CO2/95% air). Induction of osteogenic differentiation was performed as previously described -NPY , and 10−7 M BIBP3226  in a culture medium containing 10−7 M Dex.The pre-osteoblast cell line MC3T3-E1 (subclone 4), obtained from ATCC, was cultured in α-MEM  medium containing 10% fetal bovine serum . Cell lines have been authenticated by short-tandem repeat (STR) analysis , and the cells were negative for mycoplasma as routinely detected by PlasmoTest . MC3T3-E1 cells were initially cultured in six-well plates (6 × 10escribed . After gThe shRNA plasmid targeting Y1 receptor gene was purchased from Santa Cruz (#36098-SH). According to the manufacturer’s instructions, subconfluent MC3T3-E1 cells were transfected with Y1 receptor shRNA plasmid or scrambled control by using Lipofectamine 2000 . Knockdown of Y1 receptor gene expression was confirmed by real-time PCR and Western blot analysis. For selection of stably transfected cells, Puromycin  was added to the culture for five days. To activate the ERK signaling pathway, the cDNAs coding constitutively active MEK-1 mutant  were subcloned into pUSE vectors ,36. MC3T3 cells/well) were incubated in osteogenic differentiation media in the presence or absence of 10−7 M dexamethasone for 24 h. A 10-μL volume of CCK-8 reagent was then added to each well. After incubating the mixture for another 1 h at 37 °C, the absorbance value of each sample was spectrophotometrically determined at a wavelength of 450 nm using a microplate reader . Cell proliferation and viability rate were expressed as fold changes relative to the vehicle groups. All experiments were conducted in triplicate. The cell proliferation and viability rates were determined by using a cell counting kit , according to the manufacturer’s instructions. Briefly, MC3T3-E1 cells seeded in 96-well plates  and quantified by absorbance measurement at the wavelength of 260 nm. To obtain cDNA, quantified RNA was reverse transcribed by using TaKaRa PCR Kit . The specific primers for mouse Y1 receptor, NPY, RUNX2, OCN, OPG and RANKL, as well as their gene numbers, are shown in v/v) Tween 20)) for 2 h at room temperature before incubation with primary antibodies for Y1 receptor , phosphorylated ERK , phosphorylated p38 , total ERK , total p38 , Cleaved Caspase-3 , Cleaved Caspase-9 , and GAPDH  at 4 °C overnight. The membranes were then washed thrice with TBST and incubated with HRP-conjugated secondary antibody  for 1 h. The target protein expression was detected using the Western Chemiluminescent HRP Substrate Kit , with GAPDH as the endogenous control. Quantitative densitometric values of the detected bands were quantified using the NIH Image J Software.Total cell lysate was collected using ice-cold RIPA lysis buffer  according to the manufacturer’s instruction. The protein content was then determined using Bicinchoninic Acid (BCA) Protein Determination Kit (Beyotime) with BSA as a standard protein. Each sample of 40 µg total protein was loaded onto 8%–12% SDS-PAGE and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% nonfat dry milk , as previously described . Brieflyt-test or one-way ANOVA followed by a post hoc Fisher’s least significant difference (LSD) test. Statistical analyses were performed using SPSS software , with p values less than 0.05 considered statistically significant. We performed all the independent experiments at least three times and presented the data as the mean ± SEM. Statistical differences were determined by the appropriate Student’s In this study, we demonstrated that Y1 receptor signaling was regulated by glucocorticoid treatment. Upregulation of the Y1 receptor by glucocorticoid led to inactivation of ERK signaling, thus decreasing osteoblastic proliferation and differentiation in MC3T3-E1 cells. Blockade of the Y1 receptor can effectively antagonize the inhibitory effects of Dex on osteoblastic cells. The present study is an effort to understand the complex molecular mechanisms of glucocorticoid-induced osteoporosis, and to provide new insights into Y1 receptor regulation of the glucocorticoid-induced suppression of proliferation and differentiation in osteoblasts. This study further suggests the great value of Y1 receptor downregulation in promoting osteoblast-mediated bone formation and even reversing glucocorticoid-induced osteoporosis.