Osteosarcoma is the most common bone cancer in children and adolescents. Previously, we have found that cyclin-dependent kinase 11 (CDK11) signaling was essential for osteosarcoma cell growth and survival. Subsequently, CDK11 siRNA gene targeting, expression profiling, and network reconstruction of differentially expressed genes were performed between CDK11 knock down and wild type osteosarcoma cells. Reconstructed network of the differentially expressed genes pointed to the AR as key to CDK11 signaling in osteosarcoma. CDK11 increased transcriptional activation of AR gene in osteosarcoma cell lines. AR protein was highly expressed in various osteosarcoma cell lines and patient tumor tissues. Tissue microarray analysis showed that the disease-free survival rate for patients with high-expression of AR was significantly shorter than for patients with low-expression of AR. In addition, AR gene expression knockdown via siRNA greatly inhibited cell growth and viability. Similar results were found in osteosarcoma cells treated with AR inhibitor. These findings suggest that CDK11 is involved in the regulation of AR pathway and AR can be a potential novel prognostic marker and therapeutic target for osteosarcoma treatment. Osteosarcoma is the most common primary malignant bone cancer. It is also the most common bone cancer in children and young adultsCyclin-dependent kinases (CDKs) are the family of serine/threonine protein kinases and control progression through the cell cycle. Among cell cycle proteins CDKs are essential. High activation and expression of CDKs have been found in different types of cancer, and are becoming popular and potentially useful therapeutic targets3479101110121310In this study, CDK11 siRNA targeting, gene expression profiling, and gene network reconstruction of differentially expressed genes were performed to investigate the difference in signaling between CDK11 knockdown and wild type in osteosarcoma cells. In an effort to validate and further understand the AR connection, we experimentally examined the expression and relationship between CDK11 and AR in osteosarcoma cell lines and patient tissues. The functional roles of AR in growth/viability and migration of osteosarcoma cell lines were evaluated. Furthermore, we determined the effects of AR specific inhibitor bicalutamide  on osteosarcoma cells and interrogated the underlying mechanism of CDK11 and AR gene network in osteosarcoma.2 at 37 °C. Bicalutamide  is a synthetic, non-steroidal AR antagonist and a pure antiandrogen used in the treatment of prostate cancer, hirsutism, and other androgen-dependent conditions. Doxorubicin was purchased from APP pharmaceuticals, LLC, .The human osteosarcoma cell lines, U-2OS , SaOS, MNNG/HOS, and MG63 were purchased from the American Type Culture Collection  in 2014. The human osteosarcoma KHOS  cell line was provided by Dr. Efstathios Gonos  in 2008. KHOS and U-2OS are two commonly used cell lines in osteosarcoma research. The human osteoblast cell lines NHOst and HOB-c were purchased from LonzaWalkersville  and PromoCell GmbH  respectively in 2013. The osteosarcoma cell lines were cultured in RPMI-1640  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin . The osteoblast cell lines were cultured in osteoblast growth medium (PomoCell) with 10% fetal bovine serum. All cells were maintained in a humidified atmosphere containing 5% COCDK11 knockdown in osteosarcoma cell lines was carried out with human CDK11 siRNA purchased from Life Technologies . The siRNA sequence targeting CDK11 corresponded to the coding region (5′-AGAUCUACAUCGUGAUGAAtt-3′) of the CDK11 gene. Nonspecific siRNA oligonucleotides (Life Technologies) were used as controls. The siRNA oligonucleotides were dissolved in nuclease-free water at a concentration of 100 mM. Osteosarcoma KHOS or U-2OS cells were transfected with Lipofectamine RNAiMax reagent (Life Technologies) according to the manufacturer’s recommendations. Total RNA was collected from these cells using TRIzol Reagent (Life Technologies) 48 hours after CDK11 siRNA transfection.2 (100). These normalized and filtered values were taken forward into the differential expression analysis. Raw microarray data has been deposited in GEO database (GEO accession number: GSE73422).CDK11 knockdown in osteosarcoma cell lines was carried out with human CDK11 siRNA. Total RNA was processed and hybridized to Affymetrix Genechip U133 Plus 2.0 arrays  by the Gene Array Technology Center at Harvard Medical School This array exhibits coverage of the human transcriptome with over 47,000 transcripts. We normalized the microarrays using the gcrma package from the R Bioconductor suiteWe generated a dataset of expression differences (henceforth named Diff_Data) by subtracting the expression values of the CDK11 siRNA sample from the values of the non-specific siRNA sample. The starting expression data did not appear to follow a normal distribution, but Diff_Data exhibits a much better approximation of a normal distribution. We calculated the mean and standard deviation of Diff_Data and calculated z-scores for each point in Diff_Data. With a standard metric to compare probes, we extracted all probes with a score above or below 3 or −3, respectively. We then generated an overlapping hit list by taking the intersection of the lists from KHOS and U2OS. In the event of multiple probes matching the same gene, their expression values were averaged. For visualization of the data, we generated heatmaps showing the expression values of the hits using the programming language R . The genThe common differentially expressed genes in both KHOS and U-2OS upon CDK11 knockdown were used as seeds to construct gene networks using GeneGO. Two of the constructed networks included CDK11. Both of these networks were combined and filtered for genes expressed in bone and neoplasms. The resulting network was further trimmed for irrelevant members. The final network pointed to AR as an important node .Osteosarcoma TMA were generated as described previously18CDK11 or AR knockdown in osteosarcoma cell lines was conducted by transfection of human CDK11 siRNA (Life Technologies) or AR siRNA . Transfection of siRNA was performed using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. Nonspecific siRNA oligonucleotide (Life Technologies) was used as a negative control.® 800CW or IRDye® 680LT were purchased from LI-COR . Western blot analysis was performed as previously reportedOsteosarcoma cells were lysed with RIPA Lysis Buffer  plus complete protease inhibitor cocktail tablets . The components of RIPA lysis include buffer 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40 and 1 mM EDTA. Protein concentrations were measured using the Bio-Rad Protein Assay kit . Antibodies against AR (1:1000 dilution), CDK11 (1:1000 dilution), and β-actin (1:2000 dilution) were purchased from Cell Signaling Technology, Santa Cruz, and Sigma-Aldrich, respectively. Secondary antibodies IRDye#LS010). The reporter construct relies on RenSP luciferase driven by the AR promoter. Wild type CDK11 in pCMV6 vector (RC216465) was purchased from Origene . pEGFP-N3 plasmid was purchased from Clontech  and served as control. KHOS and U-2OS cell lines (3*104 cells per well) were seeded in 96-well plates. One day post seeding, cells were cotransfected with CDK11 plasmid (20 ng or 40 ng) and GoClone AR reporter . To test the effect of CDK11 knockdown, KHOS and U-2OS cells were transfected with CDK11 siRNA (10 nM or 20 nM) during cell seeding. Twenty-four hours later, GoClone reporter carrying wild type AR promoter  was transfected in those cells. Luciferase activity with LightSwitch Assay Reagents was measured with a plate luminometer 48 hours after reporter transfection.AR promoter luciferase reporter assay was performed according to the manufacturer’s protocol  was added to each well and the cells were cultured for 4 hours at 37 °C. Absorbance at a wavelength of 490 nm was measured on a SPECTRAmax Microplate Spectrophotometer from Molecular Devices .Effects of AR siRNA or AR inhibitor bicalutamide on osteosarcoma or osteoblast cell growth and viability were assessed The colony forming unit assay (CFU) assay was used to validate the effect of AR siRNA or AR inhibitor on viability and differentiation of osteosarcoma cells. KHOS or U-2OS cell lines were transfected with AR siRNA (40 nM), nonspecific siRNA (40 nM) or dosed with bicalutamide (5 μM), and DMSO (5 μM) as described above. 100 cells were seeded into each well of a 12-well plate for 7–10 days. Medium was changed every 3 days. Colonies were fixed with methanol for 10 minutes, following staining with crystal violet (0.5%) for 10 minutes. A stereo microscope and a scoring grid were used to identify and count individual colonies.5 cells per well were plated into 12-well plates and transfected with AR siRNA (40 nM). Three parallel lines were made in confluent cell cultures with a 200 μl tip. After suspended cells were washed away with serum-free medium, the cultures were fed with complete medium again. The wounds were observed at 0, 8, and 24 hours after scratching separately, and photographed via a microscope  at each time point using a 10 X objective. The distance between the two edges of the wound width was quantified at 10 random sites in each image. The cell migration distance was defined as the wound width at 0 h time point minus the wound width at each time point and then divided by two.In order to identify the potential role of AR in the metastasis of osteosarcoma, cell migration was assayed by multiple scratch wound assay. 1*10® 96-Well Hanging Drop Plates (3D Biomatrix) according to the manufacturer’s protocol.Three dimensional culture (3D) growth in suspension is an improved method over 2D culture and a more accurate model to determine the behavior of cancer cell growth. KHOS and U-2OS cell lines were used and cell spheroid formation was established in HDP1096 Perfecta3DP values < 0.05 was regarded as statistically significant. The log-rank test was used to compare the differences in survival curves. Prognostic factors associated with overall survival or disease free survival (DFS) were investigated according to the Cox proportional hazards regression model, in a stepwise manner. Immunohistochemical staining was evaluated with the χ2 test to analyze the relationship between AR expression and CDK11 expression in clinical-pathological parameters of osteosarcoma.GraphPad PRISM 5 software  was used for statistical analyses. Results are expressed as mean ± SD and CDK11 expression is known to play a crucial role in tumor cell growth and viability1021CDK11 and AR have been shown to be involved in the regulation of cell viability and apoptosis in some human cancers23P = 0.047). Patients with high-expression of AR responded well to preoperative chemotherapy, but a with a trend towards a worse 5-year survival  exhibited immunostaining in the cell nucleus, ranging from no staining , 1+ staining , 2+ staining , 3+ staining , 4+ staining , and 5+ staining ; 27 specimens were not counted due to tissue loss on the TMA slide. The median age for the patients in the TMA was 30 years (range from 6 to 72), and predominantly male (62.1% of patients). Among the available specimens, less than 25% of positive staining were classified as AR-low patients (58.6%); while intense staining (over 25%) were classified as AR-high patients (41.4%). We then compared the expression levels of AR in primary osteosarcoma specimens from cases with different responses to preoperative chemotherapy. There were significant differences in AR expression between good and poor response to preoperative chemotherapy . Kaplan-Meier analysis showed that osteosarcoma patients had a lower disease free survival in the high CDK11 or AR expressions group compared with patients in the low CDK11 or AR expressions group (P < 0.001 and P = 0.012) (P = 0.013). However, AR expression was not an independent predictor for either 5-year overall survival or disease-free survival, although it showed a trend (We further examined the relationship between CDK11 and AR expression in the available TMA. We found a close relationship between CDK11 and AR staining patterns in cells with positive nuclei staining . The Spe= 0.012) . To dete= 0.012) . Multiva a trend .We took advantage of a luciferase reporter assay system to further identify whether CDK11 proteins are involved in the transcriptional activity of AR. To characterize the functional role of AR in osteosarcoma cell growth and viability, AR siRNA was transfected in U-2OS and KHOS cell lines. Cells without any treatments or cells transfected with non-specific siRNA were used as control. Western blot analysis confirmed low-expression of AR in osteosarcoma cells transfected with AR siRNA and showed the AR expression closely correlated with cell viability . Both inin vivo environment, a 3D cell culture was set-up. After 7 days of 3D growth in suspension, the diameter of spheroids formed by bicalutamide was smaller than DMSO or blank controls . As some factors such as osteosarcoma subtypes and stage of disease might also affect the above results, larger numbers of osteosarcoma patients in other populations should be further studied.In humans, androgen secretion is regulated by gender and age. In men, androgens play a key role in the development of the testis and prostate and secondary sexual characteristics including bone mass increase+ breast cancer35Prostate cancer depends on AR signaling and has been well studied within the context of this cancer. In fact, metastatic prostate cancer cells are able to maintain AR signaling throughout this cancer development. The role of AR in osteosarcoma is not understood. In this study, by differential gene expression we found several genes that might be the AR downstream target genes in osteosarcoma, such as cyclin G2, CDC20, Caspase-8, JNK. Most of those genes have been reported to be involved in AR signaling and cancer prostate cancer. Those data suggest that, AR signaling might also play an important role in osteosarcoma. Androgen-deprivation therapy for prostate cancer was described several decades ago and remains common practicehttp://www.cancerrxgene.org/). Bicalutamide and doxorubicin suggested synergistic effects in the inhibition of viability in osteosarcoma cell lines. Bicalutamide with tamoxifen have a synergistic inhibitory influence on prostate cancer growth and insulin-like growth factor 1 expression in prostate cancer patientsin vivo conditions. After 7 days of culture in 3D environment, the diameter of spheroids formed was significantly inhibited by bicalutamide compared with blank or DMSO control groups. Thereby, AR inhibitors may be effective in osteosarcoma treatment both in vivo and in vitro. Given that multiple agents targeting AR are currently in preclinical or clinical development, the results from this study may lead to another tool in the chemotherapy for osteosarcoma clinical trials.Prior to the approval of enzalutamide , bicalutamide was considered as the standard-of-care anti-androgen for the treatment of prostate cancer. Relative to the non-steroidal anti-androgens flutamide and nilutamide, bicalutamide has the highest affinity for the ARIn conclusion, the identification of a link between CDK11 and AR transcriptional regulation provides a means for prioritizing key targets in potential osteosarcoma therapeutic interventions. High expression of AR has been shown in osteosarcoma cell lines and tissues, and its inhibition attenuates cell growth and viability. A correlation between AR and survival of osteosarcoma patients implicates the high expression of AR as a potential biomarker for prognosis. We speculate that a CDK11-AR relationship will be particularly useful in targeting oncogenic pathways utilizing a combinatorial therapeutic strategy in the treatment of osteosarcoma.How to cite this article: Liao, Y. et al. Androgen receptor is a potential novel prognostic marker and oncogenic target in osteosarcoma with dependence on CDK11. Sci. Rep.7, 43941; doi: 10.1038/srep43941 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Enzalutamide-resistant LNCaP and CWR-R1 cells, but not LAPC-4 and VCAP cells, demonstrated increased castration-resistant and metastatic growth in vivo. Global gene expression analyses between short-term enzalutamide treated vs. enzalutamide-resistant cells identified both AR pathway and non-AR pathway associated changes that were restored upon acquisition of enzalutamide resistance. Further analyses revealed very few common gene expression changes between the four resistant cell lines. Thus, while AR-mediated pathways contribute in part to enzalutamide resistance, an unbiased approach across several cell lines demonstrates a greater contribution toward resistance via pleiotropic, non-AR mediated mechanisms.Enzalutamide (MDV3100) is a second generation Androgen Receptor (AR) antagonist with proven efficacy in the treatment of castration resistant prostate cancer (CRPC). The majority of treated patients, however, develop resistance and disease progression and there is a critical need to identify novel targetable pathways mediating resistance. The purpose of this study was to develop and extensively characterize a series of enzalutamide-resistant prostate cancer cell lines. Four genetically distinct AR-positive and AR-pathway dependent prostate cancer cell lines  were made resistant to enzalutamide by long-term culture (> 6 months) in enzalutamide. Extensive characterization of these lines documented divergent  Since the 1940′s the standard of care for the treatment of advanced prostate cancer has focused on the inhibition of the Androgen Receptor (AR) signaling axis. In the normal prostate AR is necessary for the function, survival, and differentiation of prostatic tissue . In contThe current clinical paradigm for the treatment of prostate cancer, even in the castration-resistant state, remains focused on the blockade of AR signalling. Whether prostate cancer (PC) cells adapt to enzalutamide through alterations to AR directly (e.g. mutation or splice-variation), or whether there are distinct non-AR adaptive mechanisms of resistance is a fundamental question in understanding the development of enzalutamide resistance. Most of the work studying AR antagonist resistance has focused on activating mutations on the AR ligand binding domain (LBD) and splice variants, particularly the constitutively active AR- variants that lack the ligand binding domain –16. SuchR). We compared differences in proliferation, viability, resistance to docetaxel, and in vivo growth to the parental and cells treated short-term (48 hours) with enzalutamide. We also characterized changes in gene expression longitudinally during the evolution of resistance. These studies revealed heterogeneous characteristics acquired by each cell line as well as key differences in AR pathway adaptation. Our data shows that although AR-mediated pathways contribute to enzalutamide resistance, an unbiased approach across several cell lines indicates that there may be a significant contribution from pleiotropic, non-AR mediated mechanisms.Previous data has supported a role for alternate mechanisms of resistance that may contribute to enzalutamide resistance. For example, expression and activity of the Glucocorticoid Receptor (GR) and the pluripotent stem cell transcription factor Sox2 have been shown by our group and others to promote enzalutamide resistance –22. We hTMPRSS2-ERG gene fusion . However, the LNCaP-EnzR cells demonstrated robust castration-resistance, whereby tumor initiation of the LNCaP-EnzR cells was 95% compared to only 15% in parental cell lines, and median tumor initiation was accelerated by over one month  injection of luciferase-expressing cells into castrated male SCID mice followed by bioluminescence imaging showed significantly increased metastatic colonization of EnzR cells  , scraped, and sonicated . Protein was quantified by BCA assay (Thermo-Fisher Scientific), and 30 μg of protein were loaded per lane. Antibodies used were: anti-AR ; anti-AR ; anti-AR ; anti-AR Variant 7 ; anti-Beta Actin ; anti-glyceraldehyde-3-phosphate dehydrogenase ; and anti-Lamin A/C . Secondary antibodies and Nitrocelluose membranes from Licor  from were used and data captured using a Licor Odyssey system  as previously described , then fixed in 4% Paraformaldehyde (EMS), 1.5% Bovine Serum Albumin  and 0.5% Triton-X (Sigma-Aldrich) in CB for 15 minutes. Cover slips were then washed 3 times in Phosphate Buffered Solution , and incubated with 1:50 AR N-20 (Santa Cruz) antibody and 2 μM Rhodamine conjugated Phalloidin (Invitrogen) in 1.5% BSA  and 0.5% Triton-X (Sigma-Aldrich) in CB overnight at 4°C. Coverslips were then washed again 3 times in PBS, and incubated for 1 hour at 4°C in 1:1000 alexa-488 conjugated goat-anti-rabbit (Invitrogen), 1.5% BSA (EMD) and 0.5% Triton-X (Sigma-Aldrich) in CB. Coverslips were then washed again 3 times in PBS, and mounted onto microscope slides (Fisher Scientific) with Fluormount Aqueous Mounting media (Sigma-Aldrich) with 1:10,000 DAPI solution (Invitrogen). Cells were then imaged using a Nikon Ti-E microscope with a Lumen 200 Pro light source  and an HQ2 cooled CCD camera  controlled via Metamorph acquisition software , and analyzed with Image J software .Cells grown on ultra-clean glass cover slips  were washed in Cytoskeletal Buffer , and TMPRSS2  transcripts were quantified using Power SYBR® Green Master Mix (Invitrogen) using custom primers  and quality tested using an Agilent Bioanalyzer 2100 . For standard Q-RT-PCR, extracted RNA was converted to cDNA by reverse transcription using SuperScriptmers See . StandarRNA from samples in biological quadruplicates was purified and quality tested as described in Q-RT-PCR methods. Gene expression profiling was performed using HumanHT-12 v4 Expression BeadChip Kit  by the University of Chicago Functional Genomics Core. To minimize batch effect, experiments containing the same cell line were placed on the same chip. Data was then quantile normalized using BRB-Array tools and a 1.5-fold change filter was applied to the samples. Differentially expressed genes were selected using the Significance Analysis of Microarrays (SAM), which performs appropriate tests for multiplicity. Genes that met a 5% false discovery rate (FDR) were retained. Datasets used for this study were deposited into GEO under accession number GSE78201.In vivo tumor formation of parental and EnzR cells were conducted via a sub-cutaneous inoculation of one million cells  in 4–6 week old male athymic nude mice  using a 75% Matrigel and 25% HBSS solution (BD Biosciences). To measure tumor take in a castrated host, host mice were surgically castrated one week prior to cell inoculation. Intracardiac injections were performed in castrated 4–6 week old C.B.-17 SCID mice  castrated at least one week prior to injection. 500,000 Luciferase expressing LNCaP and LNCaP EnzR cells, or 250,000 CWR-R1 and CWR-R1 EnzR cells, were suspended in 100 μl of PBS (Invitrogen), and injected into the left ventricle of the mice. Metastatic colonization was visualized via Optical Imaging at least once a week post-injection until endpoint was met. Animals were imaged 10 minutes after IP injection of a bolus of Luciferin (Invitrogen) at saturating levels (37.5 mg/kg), once per week following intracardiac injection. Optical Imaging was performed at the integrated Small Animal Imaging Research Resource, Optical Imaging subcore at the University of Chicago on a PerkinElmer IVIS Spectrum with Living Image 4.4 Software.All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Chicago Institutional Animal Care and Use Committee . All surgery was performed under Ketamine/Xylazine or Isoflurane anesthesia, and all efforts were made to minimize suffering. t-tests performed with normalization to control analyses to obtain p-values. For survival analysis, Log-rank (Mantel-Cox) tests were utilized. Growth data was analyzed with n = 6 using multiple pair-wise student t-tests.Data was analyzed using GraphPad Prism software version 5.0 f  software, experiments were performed in triplicate to determine mean standard error, and student's


Klinefelter Syndrome (KS) is the most common chromosomal disorder in men leading to non-obstructive azoospermia. Spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. We evaluated AR variations and polymorphism length in 135 non-mosaic KS patients, aimed to find possible correlation with clinical features, sex hormones and sperm retrieval. Among 135 KS patients we found AR variations in eight subjects (5.9%). All variations but one caused a single amino acid substitution. Four variations P392S, Q58L, L548F, A475V found in six patients had been previously described to be associated with different degrees of androgen insensitivity. Moreover we observed in two patients Y359F and D732D novel variations representing respectively a missense variation and a synonymous variation not leading to amino acid substitution. All the Klinefelter patients with AR gene variations were azoospermic. Spermatozoa were retrieved with TESE for two men (40%), sperm retrieval was unsuccessful in other 3 patients. This is the only study reporting AR variations in KS patients. Relevant clinical differences not emerged between AR mutated and not AR mutated KS patients, but does each variation play an important role in the trasmission to the offspring obtained by ART in this patients? Patients with Klinefelter syndrome  are characterized by eunuchoid body proportions, gynecomastia, small firm testes and azoospermia.Klinefelter syndrome (KS) is the most frequent sex chromosome disorder, occurring in about 1 boy in 600 and diagnosed in 11% of azoospermic men2 . Testicular sperm recovery is estimated in 50% of adult KS men by means of TESE and used as the sperm source for intracytoplasmic sperm injection (ICSI)3.Testicular biopsies from Klinefelter patients show mixed areas with Sertoli-cell-only tubules and sclerotic or hyalinized tubules as well as interstitial Leydig cell hyperplasia. However, scattered areas with focal spermatogenesis can be seen even in non-mosaic patients4. AR, as a ligand-activated transcription factor, is activated by binding either of the androgenic hormones testosterone or dihydrotestosterone5. In humans, AR is a 110 kD protein composed of 919 aminoacids that is encoded by the AR gene located on the X chromosome at Xq11-127. AR gene polymorphisms and mutations cause a wide spectrum of phenotypic abnormalities in 46, XY subjects. Androgen insensitivity syndrome (AIS) comprises two clinical subgroups: complete AIS (CAIS) and partial AIS (PAIS)8. Missense mutations lead to partial androgen insensitivity syndrome (PAIS), characterized by the presence of abnormal genital development in a 46,XY individual with normal testis development and partial responsiveness to age-appropriate levels of androgens. There are no available data on the prevalence of PAIS in the general population. CAIS is a disorder of sex development (DSD) characterized by the presence of female external genitalia, ambiguous genitalia or variable defects in virilization in a 46,XY individual with absent or partial responsiveness to age-appropriate levels of androgens. The CAIS phenotype is associated with an AR mutation that completely disrupts AR function; target cells do not respond to testosterone or dihydrostosterone (DHT). The estimated incidence is between 1/20,000 and 1/99,000 live male births. In the AR gene, four different types of mutations have been detected to generate defective AR: (I) single point mutations resulting in aminoacid substitutions or premature stop codons; (II) nucleotide insertions or deletions most often leading to a frame shift and premature rumination; (III) complete or partial gene deletions; and (IV) intronic mutations causing alternative splicing9. Defective AR may include loss or gain of function AR alterations leading different implication in spermatogenic function. The human AR gene contains two polymorphic (CAG)n (polyGln/polyQ) and (GGC)n (polyGly/polyG) repeat sequences in exon 1. The number of polyQ and polyG repeats is 21.6 ± 3.3  and 17.4 ± 1.4  respectively, as reported in fertile controls in a previous work of our group10.KS patients can have normal total testosterone values, but there are many KS presenting hypogonadism and poor virilization. Androgens play an important role in the prenatal and pubertal virilization of the external genitalia in 46,XY fetuses. The effect of androgens is mediated by the androgen receptor that plays a critical role in male sexual differentiation, development and maintenance of secondary male characteristics and the induction and maintenance of spermatogenesis11.PolyQ tract length has been reported to affect AR activity. Shorter polyQ tract can enhance the critical intramolecular N-C terminal interaction of AR, allowing the response to lower androgen concentrations associated with higher levels of specific p160 coactivatorsThe framework already complex of AR gene is made even more complicated in KS patients, given that: (1) there are two X chromosomes, (2) the X chromosomes may be the same or different depending on the type inheritance, (3) inactivation of the X chromosome.In the present study we evaluated AR mutations and CAG/GGC polymorphism in KS patients in order to find possible correlations with clinical features, sex hormones and sperm retrieval.We retrospectively evaluated the presence of AR mutations in 135 non-mosaic KS patients referred at the Unit of Andrology and Reproduction Medicine of Padova for fertility problems. Six AR variations were found in eight infertile men affected by non mosaic Klinefelter Syndrome. AR variation frequency was of 5.9% (8/135).Table www.androgendb.mcgill.com) and shown to be associated with different degrees of androgen insensitivity. Two patients (patient 2 and 4) carrying the same AR variation P392S, other two patients (patient 3 and 5) carrying the same AR variation Q58L. Variation Y359F (found in patient 1) and variation D732D (found in patient 8) represent novel finding of missense and synonymous variation (not leading to amino acid substitution) respectively. Five patients  had heterozygous variations, while three showed homozygosity for the variation . Of the six variations, four (66%) were localized in the TAD protein domain, one (16%) in the DBD protein domain and one (16%) in the LBD protein domain (LBD) . All the Klinefelter patients with AR gene variation were azoospermic and no immature germ cells were found in semen, even after centrifugation. Among these patients, five undergone testicular biopsy and the results of sperm retrieval obtained by bilateral surgery are reported in table 12. Despite non-mosaic Klinefelter patients show the same karyotype, they have variable phenotypic characteristics13. The reason for this heterogeneous clinical finding is unclear, but it has been suggested that it may be caused by various genetic and epigenetic effects15. Moreover, this heterogeneous clinical variability could may be caused by low androgen levels or to a reduced androgen sensitivity. On the same basis, a lower fertility potential has been supposed in those patients with more phenotypic severity, however this theory has not been confirmed yet. Despite low testosterone levels are associated with reduced fertility in 46, XY males, there is no similar evidence in KS patients.47 XXY karyotype is the most common chromosomal disorder in men and accounts for 11% of all men with azoospermia. More than 95% of individuals are unable to have biological children by natural means18 in association with a wide array of phenotypic abnormalities19, but only few data are available for Klinefelter patients. To our knowledge, only one study has investigated the presence of AR mutations in a little group of Klinefelter subjects, showing no mutations in this cohort20. It is possible, however, that mutations in these patients remained undetected because the low sensitivity of the SSCP method (Single Strand Conformation Polymorphism) used for the analysis or because the little number of included patients.Mutations in the AR gene have been reported in infertile males21. Four variations P392S, Q58L, L548F, A475V had been previously described27, while the detection of Y359F and D732D variations represents a novel finding.In our study, 135 Klinefelter subjects were evaluated by Sanger sequencing for variations in the AR gene and we found nucleotide variations in 8 of them (5.9%). Of the six variations identified, four (66%) were localized in the TAD domain, one (16%) in the DBD domain and one (16%) in the LBD domain. Mutations in the LBD or DBD domains have been reported in more severe forms of androgen insensitivity syndrome while variations in the TAD domain appear to be often associated with mildest phenotypes, such as isolated male infertilityBeside testicular failure and sex hormones abnormality, Klinefelter syndrome is frequently associated with alterations of lipid, glucose and bone metabolism. In our study the analysis of these parameters showed no difference in testicular volumes, sex hormones and anthropometric parameters of KS patients with and without AR variations. Also metabolic features were similar in two groups, except for PTH levels. Despite in the normal range, circulating PTH resulted significantly higher in patients without AR variations compared with mutated KS subjects. No difference was observed regarding Vitamin D, calcium and phosphorus levels. PTH significantly lower in the group of KS patients with AR mutation may simply be an expression of an important numerical difference between the two groups.30.Despite our findings suggest that AR seems not to affect spermatogenesis, sex hormones and metabolic pattern in KS, this is the first report showing AR variations in these patients. On the other hand, we cannot exclude that AR variations in Klinefelter Syndrome may have an impact on the fertility because of the following reasons: (i) different types of mutation  usually leads to different phenotypes; (ii) X inactivation status could influence phenotypic expression of the AR mutated X. As KS patients have two X chromosomes, the mutational inactivation of AR gene on the mutated allele may be compensated by the presence of the wild type AR gene on the transcriptional active X allele; (iii) homozygous or heterozygous status of the mutation  may have different impact on the testicular functionTo further point out the complexity of this issue, we underline that two patients with the same heterozygous variation (Q58L) showed a different outcome at testicular biopsy. Patient 5 had successful sperm retrieval while patient 3 had not. We can speculate that this discrepancy depends on a different inactivation pattern of the extra X allele. It is possible to suppose that in the patient 5 the X-inactivation acts by silencing the X-allele carrying the mutation, while in the patient 3 the silencing affects the wild type allele. In addition, patient 1, carrying in homozygous state the Y359F variation (not previously described in literature) and showing particularly small testicular volumes (each testicle was <2cc), had bilateral sperm retrieval. In this patient, since the effect of the variation was not masked by the X-inactivation of the extra X allele (homozygous), it is possible to suppose that this variation may have not an impact or even a protective effect on spermatogenesis.In conclusion, AR variations can be identified in a significant percentage of KS patients. From this study we cannot drive final conclusions regarding the role of AR variations in KS subjects, however it is possible that these alterations may have a significant impact on the clinical feature and spermatogenic impairment of patients. Therefore, we hope that further studies involving larger cohort of subjects will be performed to clarify these aspects to evaluate the presence of AR gene variations in KS patients and to study the relevance of X-inactivation in mutated subjects aimed to extend the knowledge on this topic. Finally, the knowledge of AR variations could be useful to better determine the reproductive risks during the genetic counselling of those KS patients undergoing assisted reproduction. Their female offspring carriyng AR variation maight transmit the mutated allele to the 50% of her offspring, resulting in a failure of normal masculinization of the external genitalia in chromosomally male individuals (Morrys Syndrome). This failure of virilization can be either complete or partial depending on the amount of residual androgen receptor function, the partial androgen insensitivity syndrome ranging from mildly virilized female external genitalia to mildly undervirilized male external genitalia.The study has been approved by the Ethics Committee of the University Hospital of Padova (Protocol number 2357P) and each participant gave his written informed consent. The study has been conducted in accordance with the principles expressed in the Declaration of Helsinki.The patient provided written consent to diagnosis and anonymous divulging of medical data for scientific and research purposes, according to the local ethics committee. Patients with more than one supernumerary X chromosome, mosaicisms, or with endocrine dysfunction different from hypogonadism, and subjects assuming testosterone replacement therapy or any other drug were excluded from study. Subjects evaluation included complete medical history , physical examination , lipid and glucose metabolism , complete blood count (CBC), serum insulin, PSA, hormone levels , phosphocalcic metabolism , and analysis of microdeletions of the Y chromosome. Each subject performed two seminal examination and, in case of azoospermia, a testicular biopsy. Finally, we compared KS subjects with and without AR mutations.Plasma glucose was determined by enzymatic test ; glycated hemoglobin was determined with HPLC procedure ; total cholesterol was measured by enzymatic colorimetric test ; HDL cholesterol, and triglyceride concentrations were measured by enzymatic colorimetric test . Creatinine was determined by the Jaffe’ method. Calcium and phosphate were analyzed by enzymatic colorimetric test . PSA was determined by chemiluminescent immunoassay .For all the parameters, the intra- and inter-assay coefficients of variation (CV) were\8 and 10%, respectively.Serum concentrations of total T and E2 were determined by competitive chemiluminescent enzyme immunoassay, using commercial kits . Serum concentrations of SHBG, FSH, and LH were determined by chemiluminescent enzyme immunometric assays (Immulite 2000). Detection limits and intra-assay and interassay coefficients of variation were 15 ng/dL (0.5 nmol/L), 7.2%, and 8.2% for T; 15 pg/mL (55 pmol/L), 9.9%, and 16% for E2; 0.02 nmol/L, 2.7%, and 5.2% for SHBG; 0.1 IU/L, 3.2%, and 4.1% for FSH; and 0.05 IU/L, 3.4%, and 7% for LH.31.CDUS scrotal evaluation was performed by two expert sonologists using an AplioTM XV echo color Doppler device  with a high resolution multifrequency linear probe (6–13 MHz). The testicular volume was estimated using the ellipsoid formula (length × width × height × 0.52), and the varicocele was assessed in standing position after the Valsalva’s maneuver in accordance with Sarteschi’s classificationMutation analysis of AR gene was performed on genomic DNA, extracted from peripheral blood leucocytes using a commercial DNA isolation kit according to the manufacturer’s instructions .32. Polymerase chain reactions were performed under standard conditions with the addition of 8% dimethyl sulfoxide in the reaction. Exon 1 was amplified to generate partially overlapping fragments while exons 2–8 were amplified using intronic primers. All PCR fragments were then analyzed by directly sequencing on ABI Prism sequencer (Applied Biosystems). Sequence analysis was performed using the gap4 software of the Staden package (http://staden.sourceforge.net/). Protein sequence comparison was performed using CLUSTALW computational analysis  (data not shown). Prediction of the possible impact of amino acid substitution on the structure and function of the protein was performed using the online free software Polyphen-2 (http://genetics.bwh.harvard.edu/pph)33. Aminoacid numbering is based on NCBI reference sequence NM_000044.2.DNA was amplified by polymerase chain reaction with previously published primers covering exons 1–8Data in tables are presented as mean ± standard deviation (SD) of the mean. Descriptive statistics were used to summarize all demographic and clinical characteristics of the patients.An appropriate statistical test was used to assess the relationship of various clinical characteristics with outcome. A paired ‘t’-test was used to compare means of all quantitative variables measured between the two studied groups. P-value smaller than 0.05 was considered as statistically significant.


Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl−/HCO3− exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3− and outward Cl− gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3− for stomatin-deficient patients. Similarly, kinetics of Cl− efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO EPB72) encodes a member of highly conserved and ubiquitously expressed proteins. Models of the predicted protein structure showed a short cytosolic N-terminal head, a strongly hydrophobic 28-amino acid stretch presumably encoding an amphipathic helix embedded in the cytosolic leaflet of the bilayer and a large cytosolic C-terminal domain composed of beta sheets and alpha helices. A part of this last domain is common to stomatin-like and related proteins and is referred as SPFH -domain478Stomatin, also known as protein 7.2 or as the major component of band 7, is a 31-kDa integral membrane protein expressed at the membrane of red blood cells (RBCs)1−/HCO3− exchange across the plasma membrane of both RBCs and alpha intercalated cells in the kidney , is a transmembrane protein that facilitates ClWe recently developed a stopped-flow based-assay in order to investigate the bicarbonate transport across the RBC membrane or plasma membrane of eukaryotic cells expressing recombinant AE1Furthermore, the availability of rare stomatin-deficient RBC samples from several patients presenting RBC membrane disorders and corresponding to previously described mutations in either RhAssociated Glycoprotein (RhAG) (F65S) for overhydrated stomatocytosis (OHSt)In the present study, chloride/bicarbonate exchange mediated by AE1 was examined in both stomatin-deficient RBCs and HEK293 cells expressing recombinant AE1 and overexpressing stomatin, by measurement of rapid fluxes of bicarbonate and chloride. Furthermore, the interaction between AE1 and stomatin, in both RBCs and HEK293 cells was demonstrated by proximity ligation assays (PLA).3− entry was applied to ghosts derived from four OHSt and one 7.2(−)CHC patients. Since the pHi measurement, using a fluorescent probe, is very sensitive, inconstant residual endogenous carbonic anhydrase resulting from the ghost preparation could non-homogeneously enhance the chloride/bicarbonate exchange. However, after the addition of 2 mg/ml of bovine carbonic anhydrase, HCO3− entry could be then accurately compared between several ghost samples (discussed in ref. −1 and Stom-positive: 4.89 ± 0.18 s−1). This decrease can be strictly correlated to a diminution of the AE1 activity, since similar expression levels of AE1 were previously demonstrated in Stom-deficient and Stom-positive RBCsIn order to investigate the impact of stomatin deficiency in RBCs on the activity of AE1, a stopped-flow assay based, as previously described3− (P’HCO3-) across the RBC membranes of different samples, not only the constant k but also V and SA, the volume and the surface area of the ghosts respectively, should be determined. Because the apparent permeability must satisfy the equation P’HCO3− = k.V/SA and because, ghosts being considered as spheres, this equation could be simplified into P’HCO3− = k.r/3, the radius r was estimated for each ghost sample  and adapted from a method previously described36Cl  −1 and Stom-positive: 2.59 ± 0.34 s−1), a significant decrease of 42% could be calculated  and stomatin-deficient (OHSt) RBCs were subjected to a proximity ligation assay (PLA) for protein interaction between AE1 and stomatin, as described in experimental procedures. The pictures  show theIn order to test the effect of overexpression of stomatin on the AE1 activity, recombinant HEK293-AE1 cells were transfected with plasmid pCEP4-stomatin. The transfection and tetracycline induction system  gave rise to four types of cells: endogenous stomatin-expressing cells, with or without tetracycline-induction of AE1 (AE1+ Stom+ or AE1-Stom+), and recombinant stomatin-expressing cells, with or without tetracycline-induction of AE1 (AE1+ Stom++ or AE1-Stom++). As shown by flow cytometry experiments using anti-AE1 antibody (BRIC6) directed against an extracellular epitope of the protein, AE1 was, as expected, only expressed after tetracycline-induction , using the stopped-flow approach, as previously described−1). While a similar background of 0.03 ± 0.01 s−1 was obtained in both AE1-Stom+ and AE1-Stom++, a significant increase of the AE1 activity (30.3%) was revealed in cells overexpressing stomatin (AE1+ Stom+: 0.23 ± 0.02 s−1 and AE1+ Stom++: 0.33 ± 0.03 s−1), indicating an impact of the expression level of stomatin on the Cl−/HCO3− exchange mediated by AE1  were subjected to a proximity ligation assay (PLA) for protein interaction between AE1 and stomatin. The pictures  show maxet al.in situ of an interaction between stomatin and the erythroid chloride/bicarbonate exchanger (eAE1). Regarding a decrease of PLA efficiency in RBCs compared to other cells, in particular recombinant HEK293 studied here, one explanation may lie in the presence of hemoglobin which is known to inhibit reactions of DNA polymerizationin situ interactions or at least proximity (<40 nm) between membrane proteins and cytoskeleton partners or other membrane proteins. This new approach on RBCs provides a qualitative method of describing the interactions within RBCs membrane complexes.The present findings provide a new insight on the participation of stomatin in the AE1 multimolecular membrane complexes at the erythrocyte membrane. Using mass spectrometry of the isolated cross-linked stomatin complexes from erythrocytes, Rungaldier Which among the different forms of AE1  are involved in AE1-stomatin complexes cannot be easily identified. Previous work on the expression of recombinant AE1 in HEK293 cellsAmong stomatin partners, GLUT1 was the first described with modifications of its activity depending on its interaction with stomatinThe activity of recombinant AE1 was also tested in HEK293 cells overexpressing recombinant stomatin. In these cells which express endogenous stomatin and carbonic anhydrase, kinetics of pHi changes could be assessed by stopped-flow by submitting the cells to a bicarbonate gradient, as previously describedIn conclusion, from the analysis of both stomatin-deficient RBCs and recombinant cells overexpressing stomatin, the present study gives an evidence of not only a physical link between AE1 and stomatin, as previously suggested2 fragment of goat anti-mouse and donkey anti-goat immunoglobulins from Beckman Coulter . BCECF-AM  and pyranine  fluorescent probes, as well as carbonic anhydrase from bovine erythrocytes and the AE1 inhibitor DIDS  were purchased from Sigma-Aldrich . Chloride indicator SPQ (6-methoxy-N-(3-sulfopropy1) Quinolinium) was obtained from Invitrogen .Rabbit polyclonal antibody anti-AE1 was kindly provided by Dr C. Wagner . Mouse monoclonal antibody anti-AE1 (BRIC6) came from the International Blood Group Reference Laboratory . Mouse monoclonal antibodies anti-ankyrin R (clone N388A/10) and anti-AQP1 (clone 1/A5F6) were from Neuromab  and Bio-Rad  respectively, while goat polyclonal anti-stomatin (M-14) and rabbit polyclonal anti-CAII antibodies were purchased from Santa Cruz Biotechnology . Secondary antibodies used in flow cytometry analysis were phycoerythrin (PE) or fluorescein isothiocyanate (FITC)-conjugated F(ab’)RBCs used for ghost preparations correspond to blood samples described in a previous studyAAGCTTGCCGCCACCATGGCCGAGAAGCGGCA-3′ and antisens 5′-GGGAATTCGCGGCCGCCTAGC CTAGATGGCTGTG-3′. Primers contain restriction sites Hind III and Not I, respectively (underlined sequences). Using Phusion HF DNA polymerase , PCR amplification assay was performed as followed: 98 °C 30 sec, 98 °C 10 sec, 58 °C 10 sec, 72 °C 30 sec (30 cycles), 72 °C 30 sec. PCR product was then cloned into pCEP4 expression vector. The final construction was proof-sequenced  before starting experiment.Stomatin was amplified by PCR from human bone marrow cDNAs  using the following primers; sens 5′-GGGAATTC3, 5 μg/ml blasticidin and 25 μg/ml zeocin. AE1 expression was induced by addition of 1 μg/ml tetracyclin 16 h before experiments. The HEK293-AE1 cell line was transfected with plasmid pCEP4-stomatin in presence of FuGENE 6  and Opti-MEM (Invitrogen). Stable expression of recombinant stomatin was obtained by addition of 300 μg/ml hygromycin (Invitrogen) to the culture medium. Expression of AE1 at the surface of non-permeabilized HEK293 cells and of stomatin after cell permeabilisation was detected with a FACSCanto II  after staining with the relevant primary antibodies. Cells were gated on dot plots (SSC-A vs FSC-A), as shown in The tetracycline-inducible AE1 transfected cell line HEK293-AE13 gradient, thus creating entering HCO3− and exiting Cl− gradients. The pH-dependent fluorescence changes of BCECF and pyranine were monitored at a 485 nm and a 465 nm excitation wavelength (30 °C), respectively, and the emitted light was filtered with a 520 nm cut-off filter. The second assay, which is based on chloride concentration changes and carried out on ghosts derived from RBCs, was adapted from a method previously described by Illsley and Verkman− via diffusion-limited collisional quenching. The resealed ghosts were then mixed vol/vol with the bicarbonate buffer  in the reaction chamber at 20 °C, thus again creating entering HCO3− and exiting Cl− gradients. The kinetics of fluorescence increase, corresponding to the probe dequenching by decrease of the chloride concentration in the ghosts, were monitored at a 365 nm excitation wavelength and the emitted light was filtered with a 450 nm cut-off filter. Data from three to eight time-courses corresponding to kinetics of fluorescence increases were averaged and the experimental curves fitted to a mono-exponential function to estimate either the alkalinisation or the probe dequenching rate constants k (s−1), using the simplex procedure of the Biokine software package . The mono-exponential equations are : (HCO3−)tint = (HCO3−) ext. (1-exp(−kt)) and (Cl−)text = (Cl−) int.(1-exp(−kt)), where k is the rate constant of the exponentials.The first assay which is based on intracellular pH changes was performed on recombinant HEK293 cells expressing AE1 and on ghosts derived from RBCs, as previously describedHCO3− = k x V/SA, where k is the alkalinisation rate constant obtained from stopped-flow experiments.Ghosts prepared as described above were incubated in the appropriate chloride buffer supplemented with 0.5 mg/ml eosin-5-maleimid  for 1 h. For each sample, 300 ghosts were analysed under a LSM 710 microscope (Zeiss), with a fluorescence excitation wavelength at 450 nm. Mean radius value thus determined was used to calculate volume (V), surface area (SA) and apparent permeability P’ following the formula P’ in situ detection of protein interactions. HEK293-AE1 cells, expressing or not recombinant stomatin, were cultured on polylysine coverslips, induced or not for AE1 expression as described above. Cells on coverslips were fixed, permeabilized, incubated with antibodies raised in different species and then submitted to different mixtures for ligation and amplification according to the manufacturer’s instructions , as previously described. Antibodies used were rabbit anti-AE1 (1:4000) and goat anti-stomatin (1:50) antibodies. In the present study, we adapted the PLA to RBCs. RBCs were washed in PBS, fixed with 1% formaldehyde/0.025% glutaraldehyde, permeabilized in 1% octylglucopyranoside, then projected on polylysine coverslips by cytospin centrifugation (5 min at 800 rpm). Coverslips were then blocked in background reducing reagent  and treated as described above. Antibodies used here were rabbit anti-AE1 (1:4000) and goat anti-stomatin (1:25) or mouse anti-ankyrin R (1:2), anti-AQP1 (1:100) and anti CAII (1:60) antibodies. Cells and RBCs were analysed by confocal microscopy. Red dots indicating an interaction are produced when the two tested protein are <40 nm apart. They were quantified by ImageJ software.Proximity ligation assay (PLA) was used for How to cite this article: Genetet, S. et al. Stomatin modulates the activity of the Anion Exchanger 1 . Sci. Rep.7, 46170; doi: 10.1038/srep46170 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Inducing anoxia using argon (Ar) gas may cause less aversion or stress and thus be preferable to using CO2. Therefore, the aim of this research was to evaluate the effects of 100% CO2, 100% Ar or CO2 and Ar combined (60% Ar/40% CO2) on piglet welfare during euthanasia. The results from this research suggest that using CO2, Ar or a 60% Ar/40% CO2 mixture causes stress to piglets prior to loss of consciousness and hence alternative methods of euthanasia need to be evaluated.There are times on a swine farm when pigs become ill or injured and must be euthanised. Blunt force trauma to the head is currently the most commonly employed method for on-farm euthanasia of pre-weaned piglets. When performed correctly, loss of consciousness is rapid, but the potential for delivery of sub-lethal blows, along with aesthetic unacceptability to many operators, has led to the need for alternative methods to be developed. The practice of using carbon dioxide (CO2), 100% argon (Ar) or a mixture of 60% Ar/40% carbon dioxide (Ar/CO2). Two studies (n = 5 piglets/treatment/study) were conducted: (1) behavioural and physiological data were collected from conscious piglets during exposure to test gases via immersion in a pre-filled chamber and (2) electrophysiological data were collected from lightly anaesthetised, intubated and mechanically ventilated piglets exposed to the same test gases. Based on the duration of escape attempts and laboured breathing, piglets exposed to 100% CO2 experienced more stress than piglets exposed to 100% Ar prior to loss of consciousness, but there appeared to be no advantage of mixing Ar with CO2 on indices of animal welfare. However, spectral analysis of the electroencephalogram revealed no changes consistent with nociception during exposure to any of the three gas treatments. Based on the behavioural response to gas exposure, all gases tested caused signs of stress prior to piglets losing consciousness and hence alternative methods of euthanasia need to be evaluated. The aim of this research was to evaluate the welfare of pre-weaned piglets euthanised using three different gas treatments: 100% carbon dioxide (CO In study one, behavioural and physiological data were collected from conscious piglets during exposure to test gas mixtures via immersion in a purpose-built euthanasia chamber pre-filled with the test gas. In study two, electrophysiological data were collected from lightly anaesthetised, intubated and mechanically ventilated piglets exposed to the same test gas mixtures via an endotracheal tube. Electrophysiological data were collected in the first study; however, electroencephalograph (EEG) data were incomplete due to contamination of the recordings caused by voluntary (escape behaviour) and involuntary (convulsions) skeletal muscle activity which necessitated the need to repeat the study. In the second study, piglets were lightly anaesthetised and given a neuromuscular blocking agent (atracurium) to inhibit skeletal muscle activity and prevent subsequent contamination of EEG recordings. In both study one and two, healthy and viable pigs were used as we did not want to confound health status, such as respiratory disease, with the effects of gas euthanasia .ad libitum access to water and commercial creep feed until the time of testing. Immediately prior to testing each piglet was transported individually to the laboratory in an enclosed pet carry-cage.All piglets were commercial white-line (Landrace × Large white) entire males, sourced from a local commercial pig farm. At approximately 17 days of age, pre-weaned piglets were collected from the farm in the morning of the test day. Following transportation in a closed vehicle to the university, the animals were group housed in a 28 °C temperature-controlled ventilated room, with deep straw bedding over non-slip concrete flooring. Piglets had 2, n = 5), 100%  or a mixture of 60% Ar/40% carbon dioxide . Fifteen male piglets, approximately 17 days of age (range 14 to 20 days) and weighing 4.2 ± 1.14 kg (mean ± SD), were sourced from seven different litters and were randomly assigned to receive one of three test gas treatments: 100% carbon dioxide (COA purpose-built plastic 60 L chamber (558 length × 390 height × 327 width mm) was used to administer the gas treatments. The lid and one side of the chamber contained a clear perspex viewing window to provide unobstructed viewing and video recording of piglet behaviour throughout the procedure. Gas inlet and outlet hoses were located at one end of the chamber. The gas outlet hose was attached at a higher level than the inlet hose to facilitate displacement of ‘lighter’ air. A sealed port was situated in the lid of the chamber to allow instrument cables to be connected to the chart recorder.2, Ar and medical grade air  connected to the chamber. Gas was introduced at a rate of 10 L/min (20% of the chamber volume) for 10 min prior to the animal being introduced and was maintained throughout the test period at this flow rate. On each occasion the chamber was pre-filled with the appropriate test gas, sourced from cylinders of compressed COPrior to placement in the chamber, each piglet was instrumented as follows: A custom-made adjustable fabric belt was placed around the piglet’s abdomen to monitor respiration rate. The belt was fitted with a saline-filled pressure cuff connected to a force transducer, which in turn connected to a chart recorder to allow continuous recording of chest excursions related to respiration. Subcutaneous 27-gauge stainless steel needle electrodes  were positioned to record EEG and electrocardiograph (ECG) activity. A five-electrode montage was used to record EEG from both the left and right cerebral hemispheres, with inverting electrodes placed parallel to the midline over the left and right frontal bone zygomatic processes, non-inverting electrodes over the left and right mastoid processes and a ground electrode placed caudal to the occipital process. ECG was recorded using a base-apex configuration. A small amount of contact adhesive was used to prevent potential displacement of the electrodes during data recording. Both EEG and ECG signals were fed via breakout boxes to separate amplifiers . The signals were amplified with a gain of 1000 and a band-pass of 1.0–500 Hz and digitised at a rate of 1 kHz . The digitised signals were recorded on an Apple Macintosh personal computer for analysis off-line at the conclusion of the experiment. During instrumentation, piglets remained relatively calm.Following instrumentation, the lid of the pre-filled chamber was quickly lifted, the piglet was placed on its feet in the centre of the chamber and the lid quickly secured. During gas exposure, behaviour was continuously recorded until death using two digital video recorders  positioned outside the chamber to record dorsal and lateral views. Death was confirmed by isoelectric EEG and ECG in conjunction with respiratory arrest. In case of the piglet injuring themselves or equipment failure, pentobarbital was on hand as an alternative method of euthanasia. A 5 mL blood sample was collected into an EDTA-vacutainer by jugular venepuncture immediately prior to the piglet being placed in the chamber and once death was confirmed. Plasma was immediately separated by centrifugation and stored at −80 °C for later analysis of cortisol and epinephrine concentrations. Plasma samples were tested in duplicate according to the manufacturer’s instructions, using commercially available cortisol  and epinephrine  enzyme immunoassay test kits.Raw EEG, ECG and respiration data were recorded continuously for 10 min prior (baseline) to and during gas exposure. EEG was visually inspected and classified into one of three categories: active, transitional or isoelectric . EEG wasVideo recordings from both camera views were examined and the behaviour of each animal was scored according to the ethogram defined in 2, n = 5), 100% argon , or a mixture of 60% argon/40% carbon dioxide .Fifteen male piglets, approximately 17 days of age (range 14 to 22 days) and weighing 4.6 ± 0.89 kg (mean ± SD), were obtained from nine different litters and were randomly assigned to receive one of three test gases: 100% carbon dioxide (CO2O maximum inspiratory pressure) with end-tidal CO2 maintained in the normocapnic range (40–50 mmHg). Halothane delivery was reduced to achieve an end-tidal tension of 1.2 ± 0.5%, which was maintained throughout. Stainless steel needle electrodes were positioned as described in study one to monitor EEG and ECG activity. After electrode placement, a 22-gauge venous cannula was inserted in an auricular vein for the administration of a neuromuscular blocking agent (atracurium) to inhibit skeletal muscle activity and prevent subsequent contamination of electrophysiological recordings. Atracurium  was given at a rate of 1 mg/kg. A digital rectal thermometer was used to continuously monitor body temperature. The animal was placed on top of a warm-water heating blanket device  to help maintain body temperature throughout. A Doppler plethysmograph was positioned over the radial artery of the foreleg to monitor blood flow, and to identify the point at which blood flow ceased due to cardiac failure. When end-tidal halothane was stable at 1.2 ± 0.5% and end-tidal CO2 was stable in the normocapnic range, baseline EEG/ECG was recorded for 10 min.In preparation for testing, a piglet was placed into a custom-built perspex chamber and anaesthesia was induced with 5% halothane vaporised in air (4 L/min). Once adequate anaesthesia was achieved , endotracheal intubation was carried out using a 3.5–5.0 mm cuffed endotracheal (ET) tube. After confirmation of successful intubation by capnometry, the ET tube was connected to a breathing circuit and anaesthesia was maintained with halothane in air. Piglets were mechanically ventilated  to allow a virtually instantaneous switch from room air to test gas, mimicking study one in which piglets were placed in a pre-filled chamber.2 tensions, body temperature and blood flow were monitored throughout.Continuous monitoring of heart rate, pupil reactivity and lacrimation was carried out to ensure maintenance of anaesthesia throughout the procedure. End tidal halothane and COEEG and ECG data were recorded continuously until death (as determined by isoelectric EEG and the cessation of cardiac contractile activity), at which time gas exposure ceased. EEG was analysed off-line at the conclusion of the experiment.Raw EEG data were visually inspected and classified into active, transitional or isoelectric waveforms, as described for Study one. EEG data were band pass filtered between 0.1 and 100 Hz during recording. Frequency spectra were generated for sequential non-overlapping 1 s epochs of recorded data. Briefly, data were low-pass filtered at 30 Hz and Fast Fourier Transformation applied to each epoch, yielding sequential frequency spectra with a resolution of 1 Hz. From these, the summary variables median frequency (F50), 95% spectral edge frequency (F95) and total power (PTOT) were derived. For each animal, baseline values for F50, F95 and PTOT were obtained by calculating the mean value for each variable over the final 5 min of baseline immediately preceding test gas exposure. Mean values for consecutive 5 s blocks from the start of gas exposure were then calculated, and transformed to a percentage of baseline mean.ECG data were band-pass filtered between 1.0 and 500 Hz during recording. Raw ECG recordings were used to generate heart rate for sequential 1 s epochs using the ratemeter function in LabChart 8.1 .All statistical analyses were carried out in SAS 9.1.3 . Plots of standardized residuals versus predicted values were evaluated to test the assumption of normally distributed within-group errors, centred at zero with constant variance. Where the residuals were found to approximate a normal distribution, parametric analyses were carried out, whilst non-parametric tests were used to analyse data with non-normally distributed residuals. All values are reported as non-transformed mean ± standard error of the mean (SEM).For study one, all data were subjected to non-parametric one-way analysis of variance , followed by the post-hoc Bonferroni’s test for multiple comparisons when a significant main effect was found. For study two, latency to the appearance of transitional and isoelectric EEG, along with maximum heart rate and time to cessation of blood flow, was subjected to non-parametric analysis of variance , with Bonferroni’s post-hoc tests for multiple comparisons carried out where a significant treatment effect was identified.p-values were manually corrected for multiple comparisons by dividing by the number of comparisons of interest. Differences were considered significant at p < 0.05.Fourier-transformed EEG data and heart rate data from study two were analysed using the mixed procedure in SAS with age, weight and treatment as fixed effects, piglet as a random effect and time as a repeated measure. Where a significant treatment effect was identified, 2: 7.6 ± 21.86, Ar: 18.7 ± 21.86, Ar/CO2: 24.0 ± 21.86; p = 0.811). Plasma epinephrine concentrations increased in response to treatment and there was a tendency for a smaller increase following exposure to Ar/CO2 than Ar (change in epinephrine concentrations (ng/mL): CO2: 24.7 ± 2.30, Ar: 26.1 ± 2.30, Ar/CO2: 16.9 ± 2.30; p = 0.075).Plasma cortisol concentrations increased in response to treatment, irrespective of gas treatment (change in cortisol concentrations (ng/mL): COEEG data were unavailable for four individuals, due to the displacement of recording electrodes as a result of vigorous escape behaviour during the experiment. Of the remaining 11 datasets, EEG data were incomplete due to contamination of the recordings caused by voluntary (escape behaviour) and involuntary (convulsions) skeletal muscle activity. The precise time to appearance of a transitional EEG waveform could not be determined for any individual. The time to appearance of isoelectric EEG could only be precisely determined for three individuals; therefore no further data analysis was conducted. In addition, the lack of clean ECG or respiration rate data following transfer to the chamber precluded further analysis of these variables.The mean time to onset of specific behaviours identified in the ethogram , along wp = 0.007), frequency (p = 0.01) and duration (p < 0.001) of escape attempts occurred sooner, more often and for a longer duration in piglets exposed to 100% CO2 compared to those exposed to 100% Ar. Latency, frequency and duration of escape attempts did not differ between Ar/CO2 and CO2 treated piglets.Escape attempts were observed in all individuals. The latency to onset (p < 0.05) grunts, for a longer (p < 0.05) duration, than piglets exposed to either CO2 or Ar/CO2 treatments.There was no effect of gas treatment on frequency or duration of squealing; however, piglets exposed to the 100% Ar treatment performed more  and Ar/CO2  treated piglets compared with Ar treated piglets. In addition, laboured breathing was rated as being more (p < 0.05) intense when piglets were exposed to 100% CO2 than 100% Ar, but did not differ between Ar and Ar/CO2 treatments. Prior to placement in the chamber, all piglets exhibited normal quiet breathing.All animals in the CO2 and Ar/CO2  than Ar treated piglets. Loss of posture immediately preceded the onset of convulsions in all animals and convulsions were observed in all individuals. No piglets regained postural control after cessation of initial convulsions.Loss of posture occurred sooner in CO2 (p = 0.045) and Ar/CO2 (p = 0.004) than the Ar treatment. The number and total duration of convulsions was greater in the Ar than the CO2  or Ar/CO2  treatments.The latency to onset of convulsions was shorter in the CO2 or Ar/CO2 treatments compared with Ar .Gasping was observed in all individuals, following apparent loss of consciousness. In 10 of 15 piglets (67%), gasping commenced prior to the cessation of convulsions. Gasping was observed sooner in piglets exposed to the CO2 treatment compared with those exposed to Ar (p < 0.001) or Ar/CO2 (p = 0.006).Respiratory arrest occurred sooner in piglets exposed to the COHead shaking, coughing or sneezing were not observed in any individuals. Only three of 15 piglets urinated or defecated during exposure to the gas, with this occurring after loss of posture on all occasions. Therefore, these behaviours are not presented in 2 than in the Ar treatment and intermediate in the Ar/CO2 treatment  and tended to occur sooner in CO2 treated than Ar/CO2 treated piglets , whereby F50 was lower than baseline from 25–40 s after gas exposure (p = 0.014), with mean F95 tending to be lower than baseline after 35 s of gas exposure  and time (p < 0.001), along with a treatment × time interaction (p < 0.001), on the change in PTOT of the piglet EEG. Total power decreased more rapidly following exposure to CO2 than to either Ar or Ar/CO2 (2 treatment was lower than the Ar treatment after 25 (p = 0.033), 30, 35 and 40 s (p < 0.001), and lower in the Ar/CO2 treatment after 25 (p = 0.032), 30 (p < 0.001), 35 and 40 s (p < 0.001). After 40 s of gas exposure, PTOT was also lower in the Ar/CO2 than the Ar treatment (p = 0.003). There was an effect of time on the change in F50 of the EEG . In previous studies evaluating the suitability of hypoxic and hypercapnic gas atmospheres for stunning pigs, squealing prior to loss of consciousness has been interpreted as a sign of stress or aversion [2 may have affected their ability to vocalise. Moreover, grunting prior to loss of posture was only observed in the 100% Ar group. Grunting generally occurred soon after the piglet was placed in the chamber, while it appeared to investigate the novel environment. No grunting was recorded during the performance of escape behaviours. Given that grunting appeared to be associated with exploratory behaviour, it may indicate that Ar is less aversive to piglets, at least initially, than the CO2 or Ar/CO2 treatments tested in this study.Interestingly, vocalisations were only performed by piglets exposed to Ar  and prior to loss of conscious whist in the chamber . Exposure to these stressors could potentially have affected the piglets’ subsequent responses to gas exposure in study one. However, given that piglets in each of the gas treatments were exposed to the same stressors, these are unlikely to account for the behavioural differences observed among the gas treatments tested. It would be of interest to differentiate between the stress response caused by gas inhalation alone and that associated with the other stressors experienced by the piglets as part of the euthanasia process, however this was beyond the scope of this study. The measurements of EEG does allow the assessment of nociception without the confounding effects of external stressors, hence assessing welfare using behaviour in conscious animals and electrophysiological changes in unconscious animals may provide a good model for assessing the welfare implications of different gaseous methods of euthanasia in piglets.2 than the Ar treated piglets, based on the development of transitional and isoelectric EEG waveforms. In conscious animals, transitional EEG is thought to be indicative of loss of awareness, whilst isoelectric EEG is deemed incompatible with consciousness [2. However, despite the likely faster onset of unconsciousness with CO2, conscious piglets exposed to 100% CO2 still performed escape behaviours and laboured breathing for longer prior to loss of consciousness than piglets exposed to 100% Ar.Loss of consciousness may be inferred behaviourally, based on loss of posture  or by exiousness . The app2 concentrations of 30 to 40% [2 reportedly causes pain in the eyes, nose and throat of humans, through stimulation of nociceptors in the mucosa of the corneal, nasal and upper respiratory regions [2, in this study was unexpected. Whilst the absence of a clear nociceptive response in the EEG of piglets exposed to either 40% or 100% CO2 in this study may suggest that CO2 does not induce a nociceptive response in young pigs, it is more likely that methodological limitations prevented the detection of any such response. The method employed to measure EEG in the present study required the animals to be intubated, preventing exposure of the corneal, nasal or upper respiratory tract mucosa to the test gas, which may have affected our results. Further study is required to rule out a nociceptive response to different gasses in pre-weaned piglets.Spectral analysis of EEG revealed no clear evidence of nociception prior to onset of transitional EEG waveform for any of the gas treatments tested in the present study. Given that transitional EEG likely indicates loss of awareness, analysis of the EEG beyond this point was not carried out. The typical pattern of mammalian cortical response to noxious stimulation is a transient increase in the F50 along with a transient decrease in the PTOT of the EEG in the period immediately following application of the noxious stimulus ,42,43,440 to 40% ,28,29, a regions ,22. Ther2 or a mixture of 60% Ar/40% CO2 for on-farm euthanasia of pre-weaned piglets. Furthermore, there appeared to be no advantage of using a mixture of 60% Ar/40% CO2 to euthanise piglets. However, piglets euthanised with 100% Ar still experienced some degree of stress or aversion in the period prior to loss of consciousness, as evidenced by performance of escape attempts and a brief period of laboured breathing before loss of posture and convulsions. Since an ‘ideal’ euthanasia method should induce no  pain, stress or anxiety prior to loss of consciousness, there is still a need to continue to evaluate alternative on-farm methods of euthanasia that meet these criteria. This study has identified that 100% Ar is preferable in welfare terms to 100% CO


MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer’s disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (The online version of this article (doi:10.1007/s10522-017-9718-4) contains supplementary material, which is available to authorized users. Human induced pluripotent stem cells (hiPSC) can generate neural stem cells, as well as neural and glial progenitors , which attenuated disease phenotype ; early neural progenitors (eNP) and neural progenitors (NP). We have investigated cell viability, ROS level, mitochondrial membrane potential and mitochondrial biogenesis , changes in total cell number as well as the expression of ® Human Episomal iPSC Line, Thermo Fisher Scientific), as described in Augustyniak et al. 5 cells/cm2 on 6-well, 24-well or 96-well (Nunc) plates covered with the solution of Matrigel:DMEM/F12 (1:30) in medium dedicated to NSC , eNP (differentiation medium type I), NP (differentiation medium type II). The next day the media were replaced by the fresh ones supplemented with idebenone at concentrations of control; 0.125; 0.25; 0.5 µM. The cells were incubated with idebenone for 5 days.Before the exposition to idebenone (Sigma-Aldrich) at concentrations of 0.5; 0.25; 0.125 μM; control NSC, eNP and NP were generated from human induced pluripotent stem cells (hiPSC) (The Gibco5 cells/cm2) in the medium dedicated to the stage of development. At 80% confluency cells were fixed with 4% of PFA (15 min). At the next steps, cells were washed with PBS and 0.1% Triton X-100 was used for cells permeabilization. Before primary antibodies (Supplementary Table 1) were added, blocking solution of 10% goat serum was applied for 1 h and cells were incubated with primary antibodies for 24 h. After this time secondary antibodies (Supplementary Table 1) were added and incubated for 1 h in the dark. Nuclei were contrast gained with Hoechst 33258 (Sigma-Aldrich).NSC, eNP, and NP were characterized by immunofluorescence staining. Images were prepared in Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Centre Polish Academy of Sciences using Confocal Laser Microscope LSM 510 (Zeiss). hiPSC- derived neural stem cells, early neural progenitors and late neural progenitors were seeded on coverslips covered with solution of Matrigel: DMEM/F12, (1:30 ratio) in a 24-well plate  was performed. Fluorescence of resorufin was read at wavelengths: 544 nm (excitation) and 590 nm (emission) 3 h after adding reagent to the culture medium (1:10). The results are shown as the ratio (%) of the fluorescence intensity of test samples to the control (untreated) samples measured by Fluoroscan Ascent  plate reader. Data presented on the graphs are normalized to cell number which was obtained by Janus Green (Abcam) staining performed according to the manufacturer’s protocol.After 5 days of exposition of NSC, eNP, and NP to idebenone, ROS level was measured by DCFH-DA  assay. Cells were incubated with DCFH-DA reagent (1 µM) for 3 h. After this time fluorescent DCF was detected by a plate reader Fluoroscan Ascent  at wavelengths: 485 nm (excitation)—538 nm (emission). The results are shown as the ratio (%) of the fluorescence intensity of test samples to the untreated control. Normalization of ROS level results to cell number was obtained using Janus Green (Abcam) staining.® Red CMXRos (Thermo Fisher Scientific) detected at the wavelength: 544 nm (excitation) 590 nm (emission) on a plate reader. The measurements were performed 4 h after the 50 nM solution of MitoTracker Red CMXRos was added to the culture medium. The results are shown as the ratio (%) of the fluorescence intensity of the test sample to the untreated control, after normalization of data to the total cell number with Janus Green staining according to manufacturer’s protocol.After 5 days of exposition to idebenone, NSC, eNP and NP mitochondrial membrane potential was measured using fluorochrome MitotrackerCells at the three stages of neural differentiation were seeded separately on a 96-well plate covered with Matrigel solution (1:30). Idebenone was added to cells for 5 days. After this time the levels of two mitochondrial proteins was measured with MitoBiogenesis In-Cell ELISA Colorimetric kit (Abcam), according to the manufacturer’s instructions, on cells fixed with 4% PFA. SDHA, mt-COX-1 proteins level was obtained on a Fluostar plate reader OMEGA (BMG Labtech). After washing with PBS, cells were incubated for 30 min in 1X Permeabilization Buffer. Prior to the addition of primary antibodies (anti-SDHA and anti-COX-1), the cells were incubated in 2X Blocking Buffer for 2 h followed by a mix of secondary antibodies conjugated with enzymes: (1) alkaline phosphatase and (2) horseradish peroxidase for 60 min. After this time substrate for alkaline phosphatase was added and absorbance was measured at OD 405 nm wavelength, then the substrate for horseradish peroxidase was added, and absorbance was measured at OD 600 nm wavelength. Changes in SDHA and COX-1 levels were normalized to total cell number measured according to manufacturer’s protocol using Janus Green (Abcam) staining method. SDHA and COX-1proteins levels were shown independently as the absorbance ratio (%) of cell samples treated by idebenone to the untreated control.After 5 days of incubation with idebenone, cells were fixed with 4% PFA (RT). 1X Janus green reagent (Abcam) was added for the 5 min. After this time cells were washed five times with PBS, then 0.5 M HCl (10 min in RT) was added. Absorbance used to determine total cell number was measured on Fluostar plate reader OMEGA (BMG Labtech) at a wavelength: OD 595 nm. Total cell number was calculated from the standard curve and presented as the % of control cells untreated with idebenone.Duet™ DNA/RNA Mini-Prep Kit (Zymo Research) according to the manufacturers’ protocols. Before reverse transcription reaction, total RNA was purified with Clean-Up RNA Concentrator kit , and RNA integrity on 2% agarose gels was validated. RNA and DNA concentration was measured using NanoDrop ND-1000 . The purity of nucleic acids was assessed by calculating the 260/280 absorbance ratio.Total RNA and total DNA was extracted from NSC, eNP, NP samples with ZR-® Green supermix (Bio-rad) and 0.25 μM/μl each primer (Supplementary Table 2). RT-qPCR was performed in the following steps: initial denaturation step at 95 °C for 3 min, 45 cycles of denaturation at 95 °C for 10 s, and annealing/extension at 60 °C for 1 min. Samples were tested in four replicates. For each stage of development  reference gene was selected in NormFinder software as shown in Fig. ® 96 Software .cDNAs to RT-qPCR reaction were obtained by High-Capacity RNA-to-cDNA™ Kit, (Thermo Fisher Scientific) on CivivCycler Thermocycler (Biotech INC). Gene expression analysis was evaluated with 1 μl (10 ng) cDNA template in 25 μl reaction mixture containing 12.5 μl iTaq™ Universal SYBRmt-ND1/SCLO2B1 ratio and (2) mt-ND5/SERPINA1 ratio . The qPCR was performed with 1 μl (10 ng) DNA template in 25 μl reaction mixture containing 12.5 μl iTaq™ Universal SYBR® Green Supermix (Bio-rad) and 0.25 μM/μl each primer (Supplementary Table 3). PCR conditions were as folows: hot start at 95 °C for 3 min followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s.The mtDNA copy numbers were calculated as (1) GraphPad Prism5.0. was used to perform statistical analysis. Kolmogorov-Smirnov was used as a normality test. After checking whether the distribution is normal test groups were compared with (1) the t-student test, (2) One-way ANOVA followed by Tukey’s Multiple Comparison Test (3) two-way ANOVA followed by Bonferroni Multiple Comparison Test; (*) p < 0.05 ; (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001; for comparison inside the group; 2) (#) p < 0.05; (##) p < 0.01; (###) p < 0.001; (####) p < 0.0001 for comparison between group. Results represent three independent experiments, each in at least four replicates. Results presented at the graphs were shown as mean with standard error of measurement (SEM). Data in the text were present as mean with standard deviation (SD).NSC, eNP and NP were obtained from hiPSC as described before ], but at 0.5 µM small, no significant increase was observed. The largest increase of viability was detected at eNP stage [138.19% (±10.78)] at the highest dose 0.5 µM of idebenone. The increase in cells viability was also high and significant for NSC for doses 0.25 µM [128% (±6.31) and 0.5 µM (130% (±4.40)].When the tested populations were compared at the highest dose of idebenone (0.5 µM), the differences between NSC [130% (±4.40)] versus NP [103.81% ±2.45)] and eNP [138.19% (±10.78)] versus NP [103.81% (±2.35)], were highly significant . The results are shown in Fig. .45] and Antioxidant properties of idebenone, revealed by reactive oxygen species (ROS) detection as the decrease in ROS level, have been observed at eNP and NP stages of neural differentiation. The most effective antioxidant capacity was recorded at the highest tested dose 0.25 µM for NSC [79.77%(±14.47)] and 0.5 μM for eNP [85.45% (±4.81)]; NP [88.44% (±7.09)]. A significant difference between compared populations (NSC vs. eNP vs. NP) at the dose 0.5 μM was not notified, as presented in Fig. There were no significant changes in the accumulation of fluorescent dye between stages of differentiation at any tested dose of idebenone Fig. c. MitochThe SDHA (Flavoprotein (FP) subunit A of succinate dehydrogenase) protein level shown a significant effect for the highest dose of idebenone 0.5 µM: [123.12% (±20.94)] in the eNP and [117.04% (±10.79)] in the NSC stage. We did not observe any statistically significant difference of SDHA protein level between: NSC versus eNP contrary to eNP versus NP   protein level in the early neural progenitors at dose 0.25 µM [144.13% (± 16.26)];  and 0.5 µM [137.24% (±11.83)]; . At the highest dose significant enhancement of COX-1 protein level was detected in NSC stage of development [113.90% (±3.01)]; (* p < 0.05). No significant changes between treated and untreated control were detected at stage of NP at the dose 0.25 μM and 0.5 μM after 5 days of exposition to idebenone. We noticed significant difference in response to idebenone between: NSC versus eNP ; eNP versus NP , and NSC versus NP  in the highest dose ]; 0.25 µM [119.75% (±18.59)]; 0.5 µM [138.57% (±12.69)], but only for the highest dose it was statistic significant. The statistically significant differences between treated cells and untreated control were shown at highest dose of 0.5 µM for NSC [138.57% (±12.69)] and eNP [118.29% (±0.11)]. At stage of late neural progenitor (NP) idebenone inhibited total cell number, about ~7–12% versus control samples at all tested doses, however this effect was found to be statistically significant only for 0.25 µM dose of idebenone [88.10% (±3.87)]  from 42.60 (±2.73) to 55.33 (±4.03) only at early neural progenitors stage . Significant difference between tested groups was observed at untreated cells populations: NSC versus NP  and eNP versus NP . In cells treated by idebenone significant difference in response to stimulations was noted between NSC and NP .Idebenone significantly increased ratio of ND5/SERPINA1 ratio analysis has shown significant increase at eNP stage  [128.00(±7.00)] as compared to untreated control [99.50(±2.50)]. At NSC and NP stages ND5/SERPINA1 ratio was not changed at significant level  increased about ~sevenfold [6.86 (±0.416)]. The increase in expression has also been observed in NSC and NP stages but it was much lower and ranged [0.311 (±0.156)] and [1.092 (±0.113)], respectively. We have also observed statistically significant differences in NRF1 mRNA expression level between every tested developmental stage  ]. Idebenone stimulated about ~5 times increase of PPARGC1A expression in NSC [5.304 (±0.164)] and NP [4.903 (±1.098)] stage of development. Relative gene expression in NSC versus eNP and eNP versus NP differed significantly  while NSC versus NP population was not significantly different ], in early neural progenitors. At the developmental stages NSC and NP expression of TFAM was reduced about three times [−2.578 (±0.252)] and one time [−0.612(±0.131)], respectively .Idebenone raises the expression of MAP2 relative gene expression only at the stage of early neural progenitors ~threefold increase [3.313 (±0.537) of expression]. At the stage of neural stem cells and neural progenitors (NP) the drug has the opposite effect: in NSC expression of MAP2 decreased about ~onefold [−1.088 (±0.168)] and for NP about ~twofold [−2.391 (±0.347)]. The observed changes were significantly different between all stage of differentiation  . Up regulation has been observed also at NSC ~fivefold [4.765 (±0.124)] and NP ~threefold [2.805 (±0.591)] stages of differentiation. Significant differences were recorded in all tested populations: NSC versus eNP versus NP  ] the ] the GFAIn this paper, we demonstrate the cellular and molecular response of hiPSC-derived cell populations to the synthetic analog of coenzyme Q10, idebenone during early stages of neural development. To the best of our knowledge, the effect of idebenone has not been studies in neural stem cells so far. We assumed that due to the positive effect of idebenone on electron transport in mitochondria , mtDNA copy number , important marker of mitochondrial biogenesis and the level of neural differentiation related markers (MAP2 and GFAP) were evaluated upon the treatment with idebenone. We have observed that most effective was the highest concentration of idebenone tested by us (0.5 μM) therefore, the further comparison between NSC, eNP and NP cells was performed at the highest tested concentration of idebenone. The summary of the influence of idebenone (0.5 µM) on three different stages of neural differentiation is presented in Table To elucidate whether idebenone can influence the neural differentiation process and content of mitochondria, and gene expression of key regulators of mitochondrial biogenesis have been investigated in the three above-mentioned cell populations. Thus, viability, total cell number, ROS level and mitochondrial membrane potential, expression of proteins  and genes  a complex of the mitochondrial respiratory chain . Idebenone induced significant increase of mtDNA copy number only in the stage of eNP, while NSC and NP populations did not respond significantly Supplementary material 2 (DOCX 17 kb)Supplementary material 3 (DOCX 14 kb)Below is the link to the electronic supplementary material.


PITX1 and the adjacent long intergenic non-coding RNA (lincRNA) C5orf66-AS1 in publicly available datasets.Patients with squamous cell cancer of the head and neck region (HNSCC) are at risk for disease recurrence and metastases, even after initial successful therapy. A tissue-based biomarker could be beneficial to guide treatment as well as post-treatment surveillance. Gene methylation status has been recently identified as powerful prognostic biomarker in HNSCC. We therefore evaluated the methylation status of the homeobox gene PITX1 and lincRNA C5orf66-AS1 was correlated with overall survival and other parameters. Thus, ten beads targeting PITX1 exon 3 and three beads targeting lincRNA C5orf66-AS1 were identified as significant candidates. The mean methylation of these beads was used for further correlation and the median was employed for dichotomization.Gene methylation and expression data from 528 patients with HNSCC included in The Cancer Genome Atlas  were evaluated and methylation and expression levels of PITX1 exon 3 and lincRNA C5orf66-AS1 were significantly higher methylated in tumor tissue than in normal adjacent tissue (NAT) . In a univariate analysis, hypermethylation of both loci was significantly associated with the risk of death (univariate: exon 3: Hazard ratio (HR): 4.97 [1.78–16.71], p = 0.010, lincRNA C5orf66-AS1: Hazard ratio (HR): 12.23 [3.01–49.74], p<0.001). PITX1 exon 3 and lincRNA C5orf66-AS1 methylation was also significantly correlated with tumor localization, T category, human papilloma virus (HPV)-negative and p16-negative tumors and tumor grade. Kaplan-Meier analysis showed, that lincRNA C5orf66-AS1 hypomethylation was significantly associated with overall survival (p = 0.001) in the entire cohort as well in a subgroup of HPV-negative tumors (p = 0.003) and in patients with laryngeal tumors (p = 0.022).Both PITX1 and even more so of lincRNA C5orf66-AS1 is a promising prognostic biomarker in HNSCC, in particular for HPV-negative patients. Further prospective evaluation is warranted.Methylation status of  Squamous cell carcinoma of the head and neck region (HNSCC) is a common malignancy with 5-year survival rates ranging from 61% for laryngeal cancers to 63% for cancers of the oral cavity and pharynx .th edition of the TNM-classification , p = 0.011; HRC5orf66-AS1lncRNA = 1.47, 95%CI [1.16–1.86], p = 0.002). T category, nodal status and age were also associated with a risk of death in univariate analyses  of the methylation as continuous variable. The analysis as continuous variable without the introduction of a cut-off for result dichotomization allows for an accurate assessment of the biomarker performance without the risk of statistical artifacts resulting from overfitted cut-offs. In univariate analysis, both Parameters that tested significant in univariate analyses were further analyzed in multivariate analyses. Here, T category remained as the only significant independent prognostic marker in multivariate Cox proportional hazard analysis.PITX1 exon 3 methylation: 58.0%, median C5orf66-AS1 methylation: 27.4%). The median was also used to dichotomize RNA levels . lincRNA C5orf66-AS1 hypomethylation was significantly associated with an improved overall survival . In addition, lincRNA C5orf66-AS1 RNA levels were not associated with survival (p = 0.22). An optimized cut-off might have resulted in a significant result for all parameters, but was not applied in order to avoid overfitting.Methylation status was dichotomized in hypo- and hypermethylation using the median as cut-off for both gene loci in the entire cohort  revealed a survival benefit for patients, whose laryngeal tumors were hypomethylated at the lincRNA C5orf66-AS1 hypo- and hypermethylation with p16 expression, a trend towards significance was seen in survival  identified spectrin, a cytoskleton protein, as a promising new biomarker [C5orf66-AS1 could emerge as a useful prognostic biomarker to identify HPV-negative patients at risk for disease recurrence and metastases, who might benefit from additional therapy like immune checkpoint inhibitors.The present study evaluates for the first time the clinical utility of nfection –47. As yiomarker . Expressiomarker . Expressiomarker . MethylaPITX1 and lincRNA C5orf66-AS1 is strong, since both are lost in some tumor tissues. Moreover, genome-wide association studies have identified single nucleotide polymorphism associated with susceptibility of colorectal cancer in East Asian patients, notably rs647161 (A/C) on 5q31.1, which matches the position of PITX1 [PITX1 dysregulation in cancer development than previously thought. The present study indicates, that lincRNA C5orf66-AS1 might also be involved in tumorigenesis, likely by acting as (post)-transcriptional regulator of PITX1. LincRNA C5orf66-AS1 is down-regulated in esophageal squamous cell cancer and re-expression inhibits migration and invasion in vitro [PITX1 mRNA in the TCGA HNSCC dataset cannot be explained by genomic events like chromosomal perturbances or loss-of-function mutation. Only two out of 279 patients in the TCGA HNSCC dataset with HPV-status determined by RNAseq had a genomic alteration, one deletion and one -likely passenger- missense mutation, . HoweveoPortal, . Since oPITX1 is an upstream inducer of RASAL1 and thus an important mediator of the Ras signaling pathway[PITX1 and lincRNA C5orf66-AS1 methylation status should be investigated ideally in a prospective randomized clinical study.g pathway. Loss ofg pathway. The prePITX1 and lincRNA C5orf66-AS1 in patients with HNSCC. lincRNA C5orf66-AS1 methylation is emerging as promising new prognostic biomarker to guide clinical treatment.This is the first study that investigates methylation status of S1 Table(XLSX)Click here for additional data file.