Prenatal androgen exposure has been suggested to be one of the factors influencing handedness, making the androgen receptor gene (AR) a likely candidate gene for individual differences in handedness. Here, we examined the relationship between the length of the CAG-repeat in AR and different handedness phenotypes in a sample of healthy adults of both sexes (n = 1057). Since AR is located on the X chromosome, statistical analyses in women heterozygous for CAG-repeat lengths are complicated by X chromosome inactivation. We thus analyzed a sample of women that were homozygous for the CAG-repeat length (n = 77). Mixed-handedness in men was significantly associated with longer CAG-repeat blocks and women homozygous for longer CAG-repeats showed a tendency for stronger left-handedness. These results suggest that handedness in both sexes is associated with the AR CAG-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. Since longer CAG-repeat blocks have been linked to less efficient AR function, these results implicate that differences in AR signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization. LRRTM1, PCSK6 and AR) as influencing handedness8910111213141516AR) gene and different aspects of handedness, since the interrelationships constitute a conceptual bridge between the theories that invoke testosterone as a factor in the development of cerebral asymmetries1116181921232526With more than 90% of all humans preferring to use their right hand, handedness is the most noticeable functional expression of cerebral lateralization in humans456Since prenatal testosterone (pT) has organizing effects on brain and behavior, and male fetuses temporarily produce more than 2.5 times the levels observed in females19AR gene is located on chromosome Xq11.2–q12 and codes for a nuclear transcription factor that mediates the actions of testosterone and dihydrotestosteronen repeat in exon 1 encoding a glutamine tract in the N-terminal transactivation domain of the protein31The human AR CAG-repeat length and handedness has been suggested by two previous studies. Medland et al. (2005) examined the role of the AR CAG-repeat length in association with handedness for writing in 783 twin pairsAR CAG-repeat is positively correlated with testosterone effects in males, and negatively correlated in females, they conclude, consistent with the callosal hypothesis, that the likelihood of left handedness is increased in those individuals with variants of the AR associated with lower testosterone levels. Recently, the association with the AR CAG-repeat and handedness was confirmed in an independent sample of 180 males by Hampson and Sankar (2012)AR CAG-repeat lengths and different handedness phenotypes. Overall, we tested in a sample of 1057 unrelated healthy Caucasian adults and specifically analyzed handedness in a subsample of women that were homozygous for CAG-repeat length (n = 77).Interestingly, a relationship of vs. right-handedness  or consistent vs. inconsistent handedness . Although also not significant, the laterality quotient (LQ) was slightly higher in men as compared to women  = 0.61; p = 0.55). Because the AR gene is X-linked, males are hemizygous for the CAG-repeat polymorphism . In contrast, females carry two alleles, of which only one is expressed in any cell because of the phenomenon of X chromosome inactivation. Therefore, in accordance with previous studiesAR CAG-repeats in males ranged from 6 to 35, with a mean repeat length of 22.20 (+/−3.00), a finding that is in line with previous data on Caucasian populations(428) = 1.15; p = 0.25), the results for Direction 2  = 4.13; p = 0.017; MSError = 8.83; partial η2 = 0.02) indicated that mixed-handed males (24.00+/−0.82) had a significantly higher CAG-repeat length than left-handed (21.22+/−0.53) or right-handed males (22.21+/−0.15) ((428) = 1.53; p = 0.13).The +/−0.15) . For con(428) = 0.71; p = 0.48).To investigate, whether the relation between mixed handedness (as reflected by low absolute LQ numbers) and CAG-repeat length was not only qualitative but also quantitative, we calculated a two-sided Neyman-Pearson correlation coefficient between absolute LQ and CAG-repeat length. Interestingly, men with lower absolute LQ had significantly higher CAG repeat lengths  for the short allele and 23.48 (+/−2.63) for the long allele, a finding that is in line with previous data on Caucasian populations(624) = −0.55; p = 0.58; Direction 2: F = 0.40; p = 0.65; MSError = 5.48; Consistency: t(624) = −0.44; p = 0.66). For the long allele, Direction 1 (t(624) = −1.16; p = 0.25) and Direction 2  = 1.26; p = 0.29; MSError = 6.92) also failed to reach significance, while for Consistency a trend (t(624) = 1.87; p = 0.06) towards a higher repeat length in inconsistently handed females (23.68+/+2.66) as compared to consistently handed females (23.28+/+2.48) was obtained. For females, the correlation between absolute LQ and CAG-repeat length failed to reach significance for both the short  and the long alleles .The number of (75) = −1.76; p = 0.08), Direction 2  = 1.17; p = 0.31; MSError = 4.01) and Consistency (t(75) = 0.79; p = 0.43) the effect failed to reach significance. While no significant correlation between CAG-repeat length and absolute LQ was observed , a strong trend was recognizable for a correlation between CAG-repeat length and LQ , indicating that in homozygous women longer CAG-repeats had a tendency to be related with stronger left-handedness AR polymorphism firm conclusions cannot be drawn on the active AR allele. Yet, in homozygous genotypes the allelic expression is unambiguously the same, since inactivation of either allele will yield identical active alleles. In our study, 77 female participants were homozygous for the CAG-repeat expansion, and analyzing this subgroup separately revealed that longer CAG-repeats showed a tendency to be related with stronger left-handedness. These data support previous findings by Medland et al. (2005) who showed that left-handedness was more common among females with a greater number of CAG-repeats when considering the longer of the two alleles or the mean allele length of both allelesAR CAG-repeat length, with longer repeats being related to a higher incidence of non-right-handedness.Handedness is a sex-dependent phenotype, with males showing a higher frequency of left-handedness than females2734AR CAG-repeats have been demonstrated to be inversely related to the capacity of the receptor to activate target genes in vitro373839AR CAG-repeats could paradoxically play a more important role than the androgen-AR effectInterestingly, the lengths of the vs. asymmetric callosal axon pruning) the early theories developed by Geschwind and Witelson, both posit fetal testosterone as one common biological substrate that may be responsible for influencing sexually dimorphic patterns of brain development1841Despite mechanistic differences  known to underlie visuospatial tasks, whereas shorter repeats in females were associated with a more masculine pattern of cortical maturation in regions of the left inferior frontal gyrus (IFG), implicated in language and impulse-control domains, which favor femalesThe influence of fetal levels of testosterone was tested indirectly by comparing brain sizes of 9-year old dizygotic twins; children with a male AR CAG-repeats decrease AR activity in the hypothalamus resulting in decreased negative feedback and increased serum androgen levels in menAR and ER beta genesAR CAG-repeats are associated with higher androgen levels in women. These data thus support the notion that differences in androgen levels as well as the AR CAG genotype in males as compared with females could contribute to between-sex differences in sexual dimorphisms in hemispheric asymmetry like handedness.Also the present results support a different mode of AR action in men and women, which may be explained by sex-specific feedback influence of AR on serum levels of androgens. Krithivas et al. suggest that longer AR CAG-repeat. The observation that homozygous women with longer CAG-repeats were more often left-handed supports previous findings by Medland et al.1920AR repeat length was observed as compared to right-handersAltogether, our findings suggest that handedness in both males and females is associated with the length of the AR CAG-repeat may be in linkage disequilibrium with another functional variation.The current study has some limitations. The classification system of handedness is controversial and tests available for assessing handedness are numerous and diverse in the way handedness is measured. Preference tests like the here used Edinburgh handedness inventory allow for handedness to be subjectively reported based on unimanual tasks. Yet, based on their high level of subjectivity and lack of consistent reliability the validity of the handedness report may be prone to errorsAR CAG-repeat could be one of the factors that lead to individual differences in hand preference perhaps through its influence on early developmental events that promote sexually dimorphic brain specialization. Providing insight into mechanisms that may give rise to the genetic and developmental basis of brain asymmetry shed light on cerebral specialization and also disorders that alter brain laterality.In conclusion, our findings suggest that the length of the In total, 1057 genetically unrelated, healthy adult participants of Caucasian descent for at least two generations participated in the present study. One male participant had to be excluded from further analysis, because he did not fill out the handedness questionnaire, therefore 626 females and 430 males were included in the analysis. The participants had a mean age of 31.88 , and none of them had a history of any neurological or psychiatric diseases. No participants are included in the cohort that would have been forced to write with the right hand in school, although they have actually preferred the left. The study was approved by the ethics committee, Ruhr-University Bochum, Germany. All participants gave written informed consent and were treated in accordance with the declaration of Helsinki.Handedness was assessed using the Edinburgh handedness inventoryFor non-invasive sampling, exfoliated cells were brushed from the oral mucosa of the participants. DNA isolation was performed with QIAamp DNA mini Kit . Genotyping of the AR exon 1 CAG repeat was performed by fragment analysis of the PCR product. The PCR products were electrophoresed on an ABI PRISM 3500xL Genetic Analyzer and the fragment lengths were determined using GeneMapper v4.1 Software. Oligonucleotides were designed using Primer Express 2.0 Software (Applied Biosystems). Further details of methodology and primer sequences are available upon request.All authors contributed significantly to this work. L.A., S.O., S.S., V.N., W.M.G., J.T.E., O.G. and C.B. designed the research study, performed the research study and collected the data; J.G.H. and M.F. have provided a subset of samples, L.A. and S.O. analyzed the data and wrote the paper. All authors reviewed the manuscript. In addition, all authors approved the final draft.Supplementary Figures


Background: Vitamin E is an effective antioxidant, protecting cells against oxidative stress.Objective: In this investigation the protective effect of vitamin E on the testis during development and spermatogenesis in rats exposed to sodium arsenite was evaluated.Materials and Methods: Pregnant Wistar rats were divided into 4 groups (n=8) control, sodium arsenite (8 mg/kg/day), sodium arsenite+vitamin E (100 mg/kg/day) and vitamin E. Treatment was carried out from day seven of pregnancy till 90 days. Finally the right testis was stereologically studied. The obtained data was analyzed using one way ANOVA and Tukey's test and the means difference was considered significant at p<0.05.Results: The weight and volume of testis, volume of seminiferous tubules and its diameter, volume of interstitial tissue, height of germinal epithelium and the total number of types A and B spermatogonia, spermatocyte, spermatid and sertoli cells reduced significantly in sodium arsenite group compared to the control. Co-administration of vitamin E and sodium arsenite compensated the adverse effects of sodium arsenite on the above parameters.Conclusion: We concluded co-treatment of rats with sodium arsenite and vitamin E could prevent the adverse effects of sodium arsenite exposure on the testicular tissue during the prenatal stage till sex maturity. Arsenic as an environmental toxicant is used in the production of agricultural pesticides, herbicides, insecticides, rodenticides, food and wood preservatives, metallurgic applications, glass production, as a catalyst in several manufacturing process and in medicine. Human are exposed to arsenic mainly through water, food and drugs. The main source of environmental arsenic exposure in most populations is the drinking water -3. Arsenic exposure causes both acute and chronic disorders in human, such as diabetes, gastrointestinal tract disorders, degenerative, inflammatory, and neoplastic changes of the respiratory, hematopoietic, cardiovascular, and nervous systems -6. InvesIn addition, the effects of postnatal sodium arsenite exposure on Leydig cells of Wistar rats testes has been studied, revealing a significant decrease in testicular weight and the total number of Leydig cells at postnatal day 21 in the treated rats . Since LArsenic can also cross the placenta. This experimental study investigate the reproductive and developmental toxicity of prenatal arsenic exposure in rats , 13. OraVitamin E (α-tocopherol) is a naturally occurring antioxidant that plays an important role by inactivating harmful free radicals produced through normal cellular activity and from various stressors thus terminating lipid peroxidation and stabilizing the molecular composition of cellular membranes, preventing the harmful effects of reactive oxygen species (ROS). Therefore vitamin E is used to ameliorate the toxic effects of arsenic -20. The present study was designed to investigate the toxic effect of sodium arsenite treatment on the testicular tissue of rats from prenatal to maturation for a period of 90 days. Since vitamin E is vital for normal reproduction and testicular function, therefore, the preventing role of vitamin E, as a strong antioxidant, on the undesired effects of sodium arsenite on the testis was also evaluated in rats co-treated with sodium arsenite and vitamin E using stereological techniques , 22.Animals and treatmentsoC, 12h light - 12h dark and enough food and water. Male and female Wistar rats with average weight of 200±25g were purchased from Pasteur institute, Iran and kept in the animal house of Arak University under standard condition of 21±2-1 per day) , vitamin E (100mg kg-1 per day)  and sodium arsenite + vitamin E. After mating, based on vaginal plug observation, female pregnant rats were divided into 4 groups (n=8), including control, sodium arsenite  were divided into the same groups (n=8) as their parents, and the same treatments were continued orally till maturation (90 days) . The expChemicals and dosageAll the chemicals in this study were purchased from Merck Company, Hohenbrunn, Germany unless it is mentioned elsewhere. The sodium arsenite dose was selected on the basis of the doses that have been used in the previous investigations with pathological consequences on male reproductive systems of rats and the same logic was also considered for vitamin E dose , 24-26. Tissue preparation's fluid fixative .At the end of the treatment, the rats were weighed and anesthetized using diethyl ether, then they killed were, and their right testis were taken out and weighed. The volume of the testis was estimated using the immersion method and then fixed in the modified Davidsonfixative , 28.Stereological studyThe orientator method was used to obtain isotropic uniform random (IUR) sections , 30. ForThe other piece that resulted from the cut made on the φ clock was placed on the θ clock vertically so that its cut surface overlapped the 0-0 axis. This piece was also cut parallel along a randomly selected number. After tissue processing, sections of 5 and 20μm thickness were cut using a microtome and stained using Heidenhain Azan method.Estimating the shrinkage and the total volume of testisbefore). To estimate the shrinkage, three random segments were prepared using trocar from IUR sections. For each segment the two vertical diameters were measured and their mean radius was estimated and considered as pre-fixing radius (rafter). Using the following equitation, the amount of shrinkage in each testis was estimated  by the total volume of the testis.Estimating the length, diameter, height of the germinal epithelium and the basement membrane thickness of seminiferous tubuleTo estimate the length of seminiferous tubules, 5-7 random fields from 5 μm thick sections, using the objective of ×10, were selected and the number of the selected seminiferous tubules profiles was counted using an unbiased counting frame -31, thuswhere ΣQi is the total number of the selected tubules, a/f for frame area in tissue scale and ΣPi is the total points superimposed on the testis tissue. To obtain the absolute length of the seminiferous tubules, the lengths density (Lv) was multiplied by the total volume of the testis.To estimate the mean diameter of seminiferous tubule, we used the Olympus DP12 microscope equipped with camera. The diameter of the tubules was measured on the sampled tubules in the counting frame used for estimating the length of the seminiferous tubules. The diameter was measured perpendicularly to the long axis of the tubules where the tubules are widest . Approxiin which Vv is the volume density of the germinal epithelium and Sv is the surface density of the germinal epithelium. For this purpose, an average of 8 to 10 fields, using the objective of ×10, from all of the 5μm thick sections of rat testis were studied using the systematic random sampling method.To obtain the volume density of the germinal epithelium, the total number of points of the point probe superimposed on each image of the testis was counted and then the total number of points superimposed on the germinal epithelium was also counted and divided into the total number of points counted for the testis.To estimate the surface density (Sv) of the germinal epithelium, the total number of points superimposed on the germinal epithelium of the seminiferous tubule (ΣPi), the length of the linear test probe in actual tissue scale (L/P), along with the total number of intersections of linear test probe with the inner surface of the germinal epithelium (ΣIi) were counted. The surface density (Sv) was then estimated using the following equation:To estimate the mean basement membrane thickness of seminiferous tubules, the harmonic mean of basement membrane thickness was estimated . A numbeThe distance of the drawn line was considered as the thickness of basement membrane. An average of 110-120 intercepts was estimated, and the mean basement membrane thickness was calculated using the following equation:Harmonic mean layer thickness= 8/3π × Harmonic mean of orthogonal intercepts where harmonic mean= number of measurements/ sum of the reciprocal of orthogonal intercepts (oi) lengths =Estimating the number of spermatogonia (A and B), spermatocyte, spermatid and sertoli cellsTo estimate the number of cells, the optical dissector method and the unbiased counting frame were used , 21. A nWhere ΣQi is the total number of the counted cells, h is the tissue thickness considered for counting, a/f is the frame area in true tissue scale and ΣPi is the total number of points superimposed on the selected fields. The result of the equation was then multiplied by the total volume of testis to obtain the total number of cells .Statistical analysisThe results were analyzed by one–way analysis of variance (ANOVA) and Tukey’s test using the SPSS V11/0 software. The means were considered significantly different at p<0.05. Histopathological findingsAn irregular vacuolated germinal epithelium with a reduction in height was observed in the sodium arsenite treated group. Giant cells were seen following germinal epithelium degeneration in the mentioned group. The amount of sperm also seemed to be less in this group compare to the other groups .The total volume of testis, seminiferous tubules and interstitial tissueA highly significant reduction in the mean total volume of testis and seminiferous tubules was observed in the sodium arsenite group compared to the control ones (p<0.01). While the sodium arsenite + vitamin E group showed no significant reduction in the mentioned parameters compared to the control group (p>0.05). Comparing the mean volume of the interstitial tissue showed a significant reduction in the sodium arsenite group relative to the control group (p<0.01). The same result was also obtained for the sodium arsenite + vitamin E group .The length, diameter, basement membrane thickness and the germinal epithelium height of seminiferous tubules The sodium arsenite group showed a highly significant reduction in the mean diameter and the height of the germinal epithelium of the seminiferous tubules compare to the control group (p<0.001). While the sodium arsenite+ vitamin E group did not show a significant difference in the above parameters when compared to the control group (p>0.05). Sole treatment with vitamin E lead to a significant increase (p<0.01) in the mean diameter, basement membrane thickness and the height of the germinal epithelium of seminiferous tubules compared to the other groups. Meanwhile the length of the seminiferous tubules showed no significant difference (p>0.05) in all four groups .Number of the spermatogonia cells (A and B), spermatocyte, spermatid and sertoli cellsA significant reduction in the mean number of spermatocyte, spermatid and sertoli cells was seen in the sodium arsenite group compared to the control rats (p<0.05). While the sodium arsenite + vitamin E group did not show a significant difference in the above parameters when compared to the control group (p>0.05), except for the mean number of spermatids which didn't increase to the normal level in this group (p<0.05). Sole treatment with vitamin E lead to a significant (p<0.01) increase in the mean number of spermatogonia cells (A and B) and spermatocytes compared to the other groups .Body and testis weightAt the end of weaning, no significant difference was found in the mean body weight of the four groups. While, the mean body weight in the sodium arsenite group reduced significantly (p<0.001) compared to other groups at the end of the treatment. Co-treatment of vitamin E + sodium arsenite could compensate the body weight reduction observed in the sodium arsenite group (p>0.05). Rats treated with sodium arsenite showed a significant reduction in the testis weight when compared to the control group. However, the application of sodium arsenite + vitamin E was not able to ameliorate the reduction in the testis weight compared with the sodium arsenite group .Our results showed that treatment with sodium arsenite caused a significant reduction in the mean total volume of testis and the volume, diameter and the germinal epithelium height of the seminiferous tubules. Other studies have also indicated that treatment with sodium arsenite causes shrinkage, atrophy and diameter reduction in the seminiferous tubules and a significant reduction in the volume of testis , 32-35. Since the seminiferous tubules are the main component of the testis, it can be deduced that testis volume reduction may be due to the reduction in the volume of seminiferous tubules. Treatment with sodium arsenite also lead to a significant decrease in the number of spermatogonia type A and B, spermatids and sertoli cells compared to the control group. Other studies have also indicated that sodium arsenite can disturb spermatogenesis. et al reported a reduction in the number of spermatid and spermatozoa cells and also damage to the Leydig and sertoli cells following administration of 6mg/kg of sodium arsenite in rats , ABP-heat shock protein 27, N-cadherin and desmoglein, therefore damage to sertoli cells can alter the structural and functional activity of these proteins which may be another reason in the reduction of the number of germ cells . et al (2004) reported the alterations in the activity of testicular enzymes including sorbitol dehydrogenase, acid phosphatase and lactate dehydrogenase in mice treated with 53.39mmol/L of sodium arsenite, which can damage the germ cells. Furthermore, changes in the enzymes of testis following exposure to sodium arsenite is also involved in the damage of testicular tissue. In this case Pant γ-GT activity, a sertoli cells marker, which may be as a result of arsenite interference with the normal physiology of sertoli cells, influencing the development of spermatocytes and spermatids  showed that treatment of rats with a single dose of 100mg/kg of alpha-tocopherol could protect testis mitochondria from lipid peroxidation  concentration and decrease in lipid peroxidation . In thisThe obtained desirable changes in the testicular tissue following treatment with vitamin E could be due to the fact that vitamin E  can interfere with the reactions that lead to lipid peroxidation, compensating for the sodium arsenite induced toxicity .In conclusion, according to our results, vitamin E, as a strong antioxidant, could compensate for the majority of the undesirable changes due to sodium arsenite exposure in the rat testis structure. In addition, sole treatment with vitamin E could significantly increase the seminiferous tubules diameter and basement membrane thickness, height of germinal epithelium as well as the number of types A and B spermatogonia and spermatocytes. Therefore, consumption of vitamin E in the case of sodium arsenite toxicity is recommended.


In silico analysis of gene expression data revealed PABPC1 up-regulation in prostate cancer tissue specimens and this up-regulation correlates to increased disease recurrence. Co-immunoprecipitation assays confirmed the association of PABPC1 with a.a. 50-250 of the NTD of the AR. Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells. Additionally, knockdown of PABPC1 inhibited transactivation of the PSA promoter by NAR (AR lacking the LBD) and attenuated proliferation of AR-positive prostate cancer cells. These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC.The androgen receptor (AR) is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. We recently reported that amino acids (a.a.) 50-250 of the NTD are capable of modulating AR nucleocytoplasmic trafficking. To further explore the mechanism associated with a.a. 50-250, GFP pull-down assays were performed in C4-2 CRPC cells transfected with GFP tagged a.a. 50-250 of the AR. Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR.  Prostate cancer is the second leading cause of cancer death in the United States killing almost 30,000 men annually . LocalizThe AR is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. It plays a vital role in all stages of prostate cancer development and progression, including CRPC. The N-terminal domain (NTD) of the AR is the least conserved of the four domains of the receptor and does not have an ordered structure ,10. It cIn the present study, we explored potential mechanisms involved in the modulation of AR subcellular localization and function by the a.a. 50–250 region of the NTD using immunoprecipitation coupled with proteomics. This led to the identification of poly (A) binding protein cytoplasmic 1 (PABPC1) as a binding partner of the region in the NTD capable of promoting AR cytoplasmic localization. PABPC1 is known to bind to the 3’ poly (A) tail of eukaryotic mRNA via RNA recognition motifs . It is i2 in a humidified incubator. For androgen induction, cells were grown in 5% charcoal stripped FBS RPMI for 24 hours followed by addition of R1881  for an additional 24 hours.LNCaP, 22Rv1, and PC3 cells were purchased from American Type Culture Collection . C4-2 cells were obtained from Dr. Leland Chung . All cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin . Cells were grown at 37°C in the presence of 5% COThe expression vector pEGFP-C1  was used to generate fusion protein constructs with GFP at the N terminus of AR and various AR mutants as described . The PSA50-250) were excised and sent for analysis by mass spectrometry at the Cancer Biomarkers Facility (CBF) of University of Pittsburgh Cancer Institute (UPCI) .C4-2 cells were transiently transfected with indicated AR deletion constructs tagged with GFP using Polyjet transfection reagent  in 10% FBS RPMI media. Cells were lysed with NP-40 lysis buffer 48 hours after transfection. GFP pull-down assays were performed using anti-GFP tagged agarose beads (20μl)  followed by SDS-PAGE. SDS-PAGE gels were stained with coomassie blue to visualize banding patterns in immunoprecipitation compared with GFP control immunoprecipitation. Gel bands that were uniquely pulled down by a.a. 50–250 of the NTD of the AR (GFP-AR°C. Peptides were extracted with 1:2 (vol/vol) 5% formic acid/acetonitrile for mass spectrometric analysis. MALDI TOF/TOF analysis was done on a 4800 MALDI-TOF/TOF analyzer . MALDI TOF/TOF data was analyzed for protein identification with ABSciex Protein Pilot 3.0 using Mascot (version 2.1.0). Searches used the IPI_human_3.68 human  protein database. The search parameters used trypsin as the proteolytic enzyme with one missed cleavage permitted, carbamidomethylation of cysteines and oxidation of methionines as variable modifications, and mass tolerance of 50ppm and 0.4 Da for precursor ions and fragment ions, respectively. Protein score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores were derived from ion scores as a non-probabilistic basis for ranking protein hits. Protein scores greater than 62 were considered to be significant (p<0.05).Gel bands were excised and destained, disulfides were reduced with Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) , and sulfhydryls were alkylated with iodoacetamide  as previously described . Gel pluC4-2 cells were transiently transfected with AR deletion constructs tagged with GFP using Polyjet transfection reagent . C4-2 cells were also stably transfected with GFP or GFP-tagged AR deletion constructs in the presence of G418. GFP pull-down assays were performed using anti-GFP tagged agarose beads  followed by Western blot analysis using antibodies specific for PABPC1 (F-2)  and GFP .2 dishes followed by knockdown of PABPC1. Nuclear and cytoplasmic fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction kit from Thermo Fisher Scientific following the manufacturer’s recommendations . GAPDH and Lamin A/C  were used as loading controls. For mRNA expression following knockdown, cells were assayed as described below. For luciferase assay, cells were transfected with appropriate reporter plasmids and assayed as described below.Control siRNA (sc-37007) and siRNA oligos specific for PABPC1 (sc-108012) were purchased from Santa Cruz Biotechnology  and transfected for knock down of PABPC1. Cells were transfected in OPTI-MEM media  using Lipofectamine 2000  according to the manufacturer’s instructions and harvested 72 hours after transfection in RIPA buffer for analysis by Western blot using antibodies specific for PABPC1 (F-2), PSA (C-19) and GAPDH (FL-335) . GAPDH was included as a loading control. For localization of the AR, C4-2 cells were seeded into 10cmC4-2 and LNCaP cells were cultured in 5% charcoal stripped FBS RPMI for 24 hours and then transfected with either siControl (40 pmol/mL) or siPABPC1 (40 pmol/mL) for 48 hours followed by treatment with DMSO or 0.1nM R1881 for an additional 24 hours. RNA was isolated from subconfluent cells using Trizol  followed by phenol chloroform extraction. Following quantitation, 1μg of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit . qRT-PCR was performed using Taqman Universal Master Mix  with either Taqman primers specific for PABPC1 (Hs01598422_mH) or primers/probes spanning exon/exon junctions designed using Primer 3 software : GAPDH For: 5’-CAT GTT CGT CAT GGG TGT GA-3’; Rev: 5’-GGT GCT AAG CAG TTG GTG GT-3’; Probe: 5’-6FAM ACA GCC TCA AGA TCA TCA GCA ATG CCT C TAMRA-3’ and PSA For: 5’-CAT CAG GAA CAA AAG CGT GA-3’; Rev: 5’-AGC TGT GGC TGA CCT GAA AT-3’; Probe: 5’-6FAM CAC AGC CTG TTT CAT CCT GAA GAC ACA TAMRA-3’. The comparative Ct method  was used202. After blocking with 10% goat serum, cells were incubated with anti-BrdU antibody  overnight, followed by addition of Cy3 secondary antibody . Cells were visualized with a Nikon Eclipse TE 2000-U fluorescence microscope . Images were taken at 100x total magnification and all BrdU positive cells were counted in each image. The mean average of three experiments from cells transfected with control siRNA was compared to cells transfected with siPABPC1.C4-2, LNCaP, PC3, and 22Rv1 cells were transfected with siPABPC1 (40 pmol/mL) or control siRNA (40 pmol/mL) for 72 hours followed by addition of BrdU  for 1 hour at 37°C. Cells were then fixed with Carnoy’s fixative , incubated with 2M HCl, washed with 0.1M boric acid, followed by incubation with 3% HTo determine the effect of PABPC1 knockdown on PSA promoter activity, siControl oligos or oligos specific for PABPC1 were transfected with PSA6.1 luc and pRL-CMV renilla plasmids into C4-2 cells using Lipofectamine 2000. After 24–48 hours cells were lysed and luciferase activity was measured on an LmaxII384 luminometer  using a Promega luciferase assay kit  following the manufacturer’s recommendations. The PSA6.1 firefly luciferase activity was normalized to pRL-CMV renilla luciferase activity. For hormone induction, cells were cultured in 5% charcoal stripped FBS RPMI for 24 hours followed by treatment with 0.1nM R1881 for an additional 24 hours. All experiments were done in triplicate and repeated at least three times. Significance was determined by Student’s t-test using GraphPad Prism software .50-250) of the NTD can promote cytoplasmic localization of the AR [50-250, GFP pull-down was performed followed by MALDI TOF/TOF mass spectrometry analysis. MALDI TOF/TOF data was analyzed for protein identification with ABSciex Protein Pilot 3.0 using Mascot (version 2.1.0). Searches used the IPI_human_3.68 human  protein database. This led to the identification of proteins as potential interacting partners with the region of a.a. 50–250 of the NTD of the AR (The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. Thus, identification of binding partners of the NTD is crucial to the design of specific therapies targeting this region. Recently, our group found that the region of amino acids 50–250  and normal prostate stromal fibroblasts (WPMY-1) were examined to determine the relative expression levels of PABPC1 . Western50-250 detected by mass spectrometry, co-immunoprecipitation was performed. C4-2 cells were transiently transfected with AR deletion mutants tagged with GFP  and activate transcription of androgen responsive genes such as PSA , Nkx3.1, and VEGRecently, splice variants of the androgen receptor lacking the LBD have been shown to activate transcription of androgen response genes ,30. HereTo determine the effect of inhibition of PABPC1 expression on AR nucleocytoplasmic trafficking, C4-2 cells were transfected with siPABPC1 for 48 hours followed by treatment with 0.1nM R1881 or vehicle for an additional 24 hours. Western blot analysis of nuclear and cytoplasmic fractions revealed that nuclear AR protein levels were reduced with the knockdown of PABPC1 in C4-2 cells . AdditioTo determine the effect of PABPC1 on prostate cancer cell proliferation, siRNA specific for PABPC1 knockdown was transfected into C4-2, LNCaP, PC3, and 22Rv1 cells. Knockdown efficiency was determined by Western blot . AR-posi50-250) that can promote AR cytoplasmic localization. Knockdown of PABPC1 decreased AR transcriptional activity and also reduced nuclear AR in C4-2 prostate cancer cells. Furthermore, PABPC1 knockdown inhibited proliferation of all the tested AR-positive but not the AR-negative PC3 prostate cancer cells. In silico analysis of several publicly available datasets including the MSKCC cBioPortal for Cancer Genomics database revealed PABPC1 up-regulation in prostate cancer specimens and this up-regulation was associated with rapid disease recurrence. These findings suggest a significant role for PABPC1 in modulating AR activity and prostate carcinogenesis.The AR plays a major role in all stages of prostate cancer development and progression, including CRPC, thus it is important to determine the mechanisms involved including the impact of AR co-regulators. This study identified PABPC1 as a novel AR co-regulator capable of modulating AR function and subcellular localization in prostate cancer cells. PABPC1 is associated with the AR through the NTD region  because PABPC1 is associated with this region in co-immunoprecipitation assays. Since AR50-250 promotes AR cytoplasmic localization using the CRM-1-dependent nuclear export pathway [50-250 in the context of the full-length AR can modulate intracellular trafficking via PABPC1 because PABPC1 association with AR is specifically mediated through AR50-250.PABPC1 appears to inhibit AR nuclear export and/or enhance AR nuclear import, because PABPC1 knockdown decreased endogenous nuclear AR protein levels in C4-2 cells. The modulation of AR subcellular localization by PABPC1 is likely mediated through AR pathway , the modPABPC1 modulation of the AR is likely to have a significant impact on prostate cancer progression because the AR is a key factor regulating prostate cancer cell growth and factors capable of modulating AR function could have profound effects in modulating prostate cancer cell growth. We recognize that PABPC1 has the potential to modulate multiple signaling pathways. As a poly A binding protein, it could influence translation of multiple signaling molecules. However, its influence on prostate cancer cell growth is likely mediated through the AR because PABPC1 knockdown inhibited proliferation of AR-positive prostate cancer cells, but not AR-negative PC3 cells. The up-regulated expression of PABPC1 in prostate cancer specimens and the association of high-level PABPC1 expression with recurrent prostate cancer further suggest that PABPC1 plays an important role in promoting prostate cancer progression.Identification of PABPC1 as a co-factor of the AR provides a potential link between AR activity and active translation in prostate cancer cells. PABPC1 is thought to stimulate efficient mRNA cap-dependent translation. Thus, elevated PABPC1 expression levels in prostate cancer cells can simultaneously enhance translation and androgen action, which may lead to more efficient cell growth. As a CRM-1 dependent shuttling protein between nuclear and cytoplasmic regions ,32, PABPIn summary, the results of our present study suggest that PABPC1 is an important AR co-regulator capable of promoting AR nuclear localization and function via interaction with the NTD region. Elevated expression of PABPC1 in prostate cancer specimens is likely to play an important role in prostate carcinogenesis by enhancing AR signaling pathways. Future studies aimed at defining the precise mechanism of PABPC1 regulation of AR signaling may lead to new approaches to prevent and/or treat prostate cancer patients.S1 FigcDNA lysates used in (TIF)Click here for additional data file.S2 FigC4-2, LNCaP, 22Rv1, and PC3 cells were transfected with siC or siPABPC1 for 72 hours followed by Western blot analysis. Antibodies specific for PABPC1 and GAPDH were used to probe the blots as previously described.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.


Hypertrophy (H) is an adaptive response of the heart to a hemodynamic overload. Severe left ventricular (LV) volume overload (VO) from valve regurgitations (aortic (AR) or mitral regurgitation) leads to eccentric LVH. Increased protein turnover is a major event during development of LVH and the mechanistic target of rapamycin (mTOR) is a key molecule for its control. The role of mTOR inhibition in the development of LVH using rapamycin for relatively short periods of time (days to a few weeks) has been studied in the past in pressure overload models but not in VO models. We investigated if mTOR pathway was activated during LVH development in a model of severe VO (AR) in rats and if a rapamycin treatment can slow heart remodeling in this situation.Male rats with severe AR were studied acutely at 2 days, at 8 weeks (compensated phase) and 6 months (late phase) after VO induction. mTOR complex (mTORC) 1  was activated early after AR induction but not later in the disease whereas mTORC2 activity levels (Akt phosphorylation at Ser473) remained stable. We observed that a moderate dose of rapamycin  for 8 weeks prevented severe LVH caused by AR (−46 %: p < 0.001). Rapamycin treatment specifically inhibited LV mTORC1 without altering mTORC2 activity at 8 weeks. Rapamycin also prevented cardiac myocyte hypertrophy caused by AR.Rapamycin slows hypertrophy in LV VO by inhibiting early activation of mTORC1 without modulating mTORC2. Chronic aortic valve regurgitation (AR) will slowly alter left ventricular LV morphology and function. The remodeling associated with AR is characterized by dilation, eccentric hypertrophy and eventually diastolic and systolic heart failure (HF). The management of patients with heart valve regurgitation is currently limited to surgical valve replacement  activation by a phosphoinositide-3-kinase (PI3K)-dependent and/or independent mechanism is often present  models causing eccentric hypertrophy. This is also true for the effects of rapamycin inhibition of mTOR in the context of eccentric LVH. We therefore designed a study to evaluate mTORC1 and mTORC2 activity in LV VO hypertrophy acutely (48 h post-AR), during the compensated phase of the disease (8 weeks) and at a later stage (6 months). We hypothesized that blocking protein translation from mTORC1 activation using rapamycin would slow LVH development in a model of severe VO from chronic AR. Severe AR is associated with significant diastolic LV stretch from increased preload caused by the regurgitating blood. This mechanical stress on the myocardium is an important trigger of LVH. We therefore also studied the state of mTOR activation during a mechanical stress (cyclic stretching) imposed to cardiac myocytes in culture in order to investigate the role of this pathway in initiating eccentric LVH.Forty male Wistar rats  were divided in four groups. Severe AR was induced by retrograde puncture of the aortic valve leaflets as previously described  immediately before, during surgery and at the end of protocol , 100 IU/ml penicillin and 100 μg/ml streptomycin  and maintained at 37 °C under 5 % CO2.HL-1 cells  were cultured in fibronectin /gelatin  (Sigma)-coated flasks as described /gelatin . The day after, cells were serum-starved for 4 h then subjected to cyclic stretch to produce an 18-20 % elongation at 1 Hz. Cells not subjected to mechanical stretch were used as controls.HL-1 cells were subjected to equi-biaxial mechanical stretch using a Flexcell FX 5000 Tension System . Cells were seeded at 10The analysis of LV mRNA levels by quantitative RT-PCR has been described in details elsewhere  or from Santa Cruz Biotechnology  as described elsewhere (6).Results are presented as mean ± SEM. Intergroup comparisons were done using 2-way ANOVA followed by Bonferroni post-test if interaction between disease (AR) and treatment (Rapa) was significant. One-way ANOVA followed by Tukey’s post-test was used for culture cells studies. Student t test was used when two groups were compared directly. Statistical significance was set at p values <0.05. Data and statistical analysis were performed using Graph Pad Prism version 6.02 for Windows, Graph Pad Software (San Diego CA).AR caused severe heart hypertrophy. Rapamycin treatment significantly reduced LVH in AR rats Table . RapamycChronic rapamycin treatment has been shown to inhibit not only the activity of the mTORC1 but also of mTORC2 by binding free mTOR and blocking complex formation  as an index of mTORC2 activity at other stages of the disease  is implicated in the signal transduction of mechanical stress. Two days post-AR, both FAK and pFAK were increased in the LV Fig. a while iAutophagy can help eliminate damaged proteins and help survival in cardiac hypertrophy but excessive autophagy may lead to heart failure. mTORC1 is a known regulator of autophagy and we were interested to see if the acute mTORC1 activation in AR LVs could influence this process. We studied the levels of protein markers associated with autophagy namely LC3 and p62/sequestrin. As illustrated in Fig. We report that rapamycin can reduce LV dilation and cardiac myocyte hypertrophy caused by severe LV VO from AR. We observed that mTORC1 and mTORC2 activities were not increased in the myocardium during the compensated phase of the disease (8 weeks) but that mTORC1 activity was increased acutely 2 days after post-AR induction. The rapamycin dosage used in our study led to the inhibition of mTORC1 without altering LV mTORC2 activity. These findings are somewhat different from those of previous studies in PO LVH where mTORC1 activation was shown to be sustained over time   after induction of severe LV VO. Our results at 8 weeks are in line with those we obtained in a recent study where we observed that S6K (mTORC1) and Akt S473 (mTORC2) levels were unchanged at 8 weeks in this model  resulting in a decrease in adipose tissue mass  at the dose of 2 mg/kg/day orally was not associated with mTORC2 inhibition. Our results also suggest that mTORC1 becomes activated following a mechanical stress in cardiac myocytes and that activation probably decreases when wall stress is normalized after LV dilation Our findings open the door to potential new therapeutic molecular targets in LV VO that deserves further investigation.


A range of new user interfaces and systems have been developed as novel and relevant tools in neurorehabilitation, allowing the implementation and exploration of new rehabilitation approaches and protocols to be complemented and to improve upon traditional methods.In this special issue both medical and engineering aspects of these tools as well as state-of-the-art research trends have been addressed in order to explore these types of solutions and how they can support and/or extend current clinical or home-based rehabilitation practices. The issue provides six original research articles by some of the leading experts in the field, covering development studies and studies investigating evidence of the effectiveness of new technologies, devices, specific applications, and treatment methodologies.The paper of F. Trincado-Alonso et al. presents a study on the development of new strategies based on virtual reality (VR) that can provide additional information to clinicians regarding rehabilitation assessment. It includes the definition of set of metrics combining kinematic data in order to obtain parameters of reaching amplitude, joint amplitude, agility, accuracy, and repeatability during the evaluation sessions.W. Chinthammit et al. report a pilot study that employs a prototype telerehabilitation system called “Ghostman”. It is a visual augmentation system designed to allow a physical therapist and patient to inhabit each other's viewpoint in an augmented real-world environment. The presented initial results look promising and suitable to be used in the field of stroke rehabilitation.J. R. Octavia and K. Coninx focus on integrating adaptivity and personalization in rehabilitation training for multiple sclerosis patients. Their findings showed that adaptive personalized training trajectories have been successfully provided. Furthermore, they report on the development of positive social interaction during the collaborative rehabilitation training.A. M. Fenuta and A. L. Hicks present a detailed comparison of two commercial rehabilitation treadmills in terms of oxygen demand and muscle activation during therapy. Important and clear indications on the use of both devices have been stated in the paper's conclusions.C. Cortes et al. provide a mathematical formulation and implementation of a method to estimate the limb posture in VR and robotic assisted rehabilitation systems. Additionally, they present the details of a VR game platform for stroke rehabilitation and the quantitative assessment of the method during the analytic training of the elbow and wrist.Finally, the paper of D. A. G. Galeano et al. proposes a novel balance training platform which combines postural analysis with synergistic electrical muscle stimulation and is based on low-cost gaming interfaces. Results include the technical validation of the platform using mediolateral and anteroposterior sways as basic balance training therapies.As the editors of this special issue, we hope that readers will find these articles representative of the state-of-the-art in novel user interfaces for neurorehabilitation. This special issue highlights the latest research and developments in this important research field and the contributions it has made to improve treatment outcomes for patients, clinical research methodology, and the development of many practical applications.


Virtual rehabilitation approaches for promoting motor recovery has attracted considerable attention in recent years. It appears to be a useful tool to provide beneficial and motivational rehabilitation conditions. Following a stroke, hemiparesis is one of the most disabling impairments and, therefore, many affected people often show substantial deficits in walking abilities. Hence, one of the major goals of stroke rehabilitation is to improve patients' gait characteristics and hence to regain their highest possible level of walking ability. Because previous studies indicate a relationship between walking and balance ability, this article proposes a stroke rehabilitation program that targets balance impairments to improve walking in stroke survivors. Most currently, available stroke rehabilitation programs lack a theory-driven, feasible template consistent with widely accepted motor learning principles and theories in rehabilitation. To address this hiatus, we explore the potential of a set of virtual reality games specifically developed for stroke rehabilitation and ordered according to an established two-dimensional motor skill classification taxonomy. We argue that the ensuing “exergame”-based rehabilitation program warrants individually tailored balance progression in a learning environment that allows variable practice and hence optimizes the recovery of walking ability. Virtual reality technique can be combined with targeted exergames development with the aim to promote motor rehabilitation. Recently, a number of researchers have pointed to the potential of virtual reality applications in health care , 48, 50 For best results in rehabilitation, video games specifically designed for therapy should be used . Becausehttp://ec.europa.eu/research/fp7/index_en.cfm?pg=understanding]. The research project “Rehabilitative Wayout In Responsive home Environments” (REWIRE) [FP7 is the short name for the Seventh Framework Programme for Research and Technological Development, the European Union's main instrument for funding research in Europe running from 2007 to 2013. FP7 is also designed to respond to Europe's employment needs, competitiveness, and quality of life [c.f. (REWIRE) , which i(REWIRE) , the for(REWIRE) ) in a ro(REWIRE) .When creating a virtual reality-based stroke treatment approach, the development of suitable exergames is undoubtedly an important requirement. There have been, to date, some studies that deal with the development of video games adequate for therapy purposes. Based on specific game design principles, they intend to provide engaging and challenging treatment conditions to achieve a high level of motivation for rehabilitation practicing , 43, 59.Worldwide, there are about 4.8 million survivors of stroke of whom about 1.1 million suffer lasting functional disabilities . The spevariable practice and progression. These principles are known to positively affect gait rehabilitation and, hence, should be considered in developing a rehabilitation program.Good balance skills are an important determinant of walking performance and impaired balance ability is assumed to be related to a decreased locomotor function , 62. ConCurrent Opinion in Neurology—stated that variable practice is a fundamental principle in terms of retaining learning over time and that a consistent agreement in the literature exists indicating that varied practice is superior to repetitive identical tasks when it comes to motor learning. The principle of progression holds that motor learning and rehabilitation programs benefit from a continuous adaptation of task difficulty to increasing skill level [The literature to date has found that variable practice leads to better transfer and retention of motor skills than if a constant practice structure is used , 38, 52.ll level . For sucll level . Furtherll level , 53, 62.From the above, it follows that rehabilitation programs for stroke survivors are well-advised to train balance and walking skills by means of a variety of balance- and walking-specific exercises, the difficulty of which is progressively adapted to patients' skill level, in combination with direct visual feedback on performance.Although previous studies have emphasized the importance of theory-based practice and rehabilitation , 37, 57,Four prevalent classification systems exist for identifying common characteristics of motor skills. Three of these categorize motor skills according to one common characteristic of the skill and lead to one-dimension classification systems. In contrast, Gentile's taxonomy considers two general skill characteristics and offers, therefore, a broader concept leading to a two-dimensional classification system of motor skills. To highlight the high potential of Gentile's two-dimensional approach, it is worthwhile to focus at first on the one-dimensional classification systems and discus some of their limitations:A description of these classification systems is given by Magill   regulatory conditions and (b) intertrial variability. The regulatory conditions indicate relevant environmental features that constrain movement execution and may either be stationary (stationary regulatory conditions) or moving (in-motion regulatory conditions). With the indicator intertrial variability, Gentile's taxonomy differentiates between regulatory conditions that change between trials  and those that do not . The second dimension, action function, is also characterized by two indicators: (a) body orientation and (b) object manipulation. Body orientation indicates whether an action requires the performer to move from one location to another (body transport) or not (body stability). Object manipulation indicates whether an object has to be controlled during the action performance (object manipulation) or not (no object manipulation).Gentile presented a systematic classification system to categorize motor skills and movement according to two general dimensions of physical actions . The firThrough the interaction of the resulting four environmental context characteristics and four action function characteristics, Gentile defines 16 different motor skill categories that provide a comprehensive template to classify motor skills . Moving either rightward or downward in the table renders the skill category more difficult, so that the most difficult skill category can be found at the bottom right of the table (4D). For the action function dimension, this implies that body transport is more difficult than body stability and object manipulation more difficult than no object manipulation. Importantly, it also assumes a hierarchy in the action function characteristics: skills involving body transport but no object manipulation are more difficult than those involving object manipulations but not body transport. For the environmental context dimension, the same pattern of progression is assumed.progression. Moreover, each of the 16 categories is associated with unique features based on the two-dimensional approach and, consequently, the taxonomy involves task variations. Obviously, Gentile's classification system is consistent with the motor learning principle variable practice.Thus, Gentile's taxonomy allows a systematic progression in difficulty of motor tasks and meets the demand of the motor learning principle progression and variable practice. The taxonomy provides a well-structured framework and the 4 × 4 table can serve as a useful guide for preparing a systematically coherent motor learning concept in a simple manner. For implementing Gentile's taxonomy-based approach, exergames might demonstrate an optimal opportunity for providing beneficial treatment conditions. On the one hand, as we referred above, there is high potential for creating a motivating rehabilitation environment using virtual reality. On the other hand, the taxonomy seems suited for virtual reality applications where the environment, task complexity, and other contextual factors related to exercise performance can be manipulated. Exergame parameters can be easily modified and, thereby, adjusted to the demands of the taxonomy-included skill categories.There are several reasons for assuming that this taxonomy provides a valuable tool for developing a theory-based rehabilitation program using virtual reality. Considering the explanations above, it is evident that Gentile's taxonomy is in accordance with the generally accepted motor learning principles To provide a post-stroke rehabilitation program focused on improved balance skills in standing and walking in accordance with Gentile's framework, we designed six basic exergames. Based on these six basic exergames, we created 16 virtual reality scenarios, either by making game parameter modifications or by substituting another basic game. Each scenario corresponds to one of the 16 skill categories included in the taxonomy Table .Table 2EIn accordance with a phased iterative approach suggested by Campbell et al. , this paThe first phase of the rehabilitation program focuses on the basic physical skill of standing quietly. In this exergame, the patient is represented as a scarecrow avatar and is required to maintain a cursor indicating the body's center of pressure (COP) within a predefined marked circle on a computer screen . The exeScarecrow described for skill category 1A demands a centered and stable COP position, this game  uses controlled COP displacements for successful performance. This exergame targets stroke patients' dynamic stability, because subjects with hemiparesis often suffer from reduced limits of stability [stationary regulatory conditions are given and no intertrial variability. To meet the requirement of object manipulation, a virtual basked filled with blueberries is placed on the farmer's head. In order to prevent berries from falling down, the patient must adopt an ideal upright body posture.Whereas the game tability . The virFrog jumping) requires the control of the COP displacements during stepping movements. The virtual environment represents a pond with several stones placed in it. By doing steps in a specific direction, the patient can control a frog jumping from one stone to another. A virtual arrow indicates which particular stone has to be targeted by the patient. In accordance with the taxonomy conditions, the position of each stone within the pond is fixed (stationary regulatory conditions) and the order of the targeted stones unchangeable from one exergame trial to the next .Previous exercise interventions have shown that stepping exercises indicate an effective treatment method to improve postural stability and balance , 29, 41,Frog jumping described for 1C can be extended by integrating an object manipulation task. Specifically, in this game, the virtual frog is wearing a crown and it is the patient's task to perform stepping tasks with an upright body position in order to keep the virtual crown correctly positioned.According to Gentile's taxonomy, the skill category 1D can be seen as a progression of 1C. Thus, the game Blueberry collector described above is not only adequate for skill category 1B, but also for category 2A by making small adaptations. On the one hand, the virtual basket on the farmer's head has to be removed to get the condition no object manipulation. On the other hand, because intertrial variability is required, the position of the berries varies within exergame trials.The game Blueberry collector for category 2A can be extended with an object manipulation task. Thus, the virtual farmer is balancing a basket on his head such as in the exergame described for skill category 1B.For skill category 2B, the exergame Frog jumping described for category 1C with the only modification of providing a random order of targeted stones—marked by the arrow—to realize intertrial variability. Consequently, each time when the game has been played, a unique step performance pattern is required by the patient.We use the game Frog jumping of category 2C can easily be adapted. The only modification that has to be done concerns the need for object manipulation. Hence, in this game, the frog is wearing a crown for which the patient has to control his/her position while playing the game.To meet the taxonomy requirements in skill category 2D, the game Tractor driver. The exergame presents a moving tractor for which the driving speed is defaulted by the video game. The direction of driving is controlled by the patient's weight shifting movements. When playing this exergame, the ability to move the COP in a standing posture without loss of balance (dynamic stability) is being trained [in-motion regulatory conditions are given. There is a fixed position of each hay bale unchangeable from one exergame trial to the next to ensure no intertrial variability.For skill category 3A, we created a game called  trained . SpecifiFruit catcher), the patient is represented as an avatar balancing a fruit basket on the head. Virtual apples are falling down from a tree (in-motion regulatory conditions) that should be caught by the basket. The patient has to perform target-oriented COP shifts to get an optimal avatar position and the apples are falling into the basket. This exergame focuses on dynamic stability due to controlled weight shifting movements to selected targets. For presenting no intertrial variability, the game parameters are always the same, even when the game is repeatedly played.In this exergame  requires stepping tasks. Specifically, the patient represented as an avatar has to overstep a crawling worm coming closer alternately from the right and the left side (in-motion regulatory conditions). The parameters of the game are unchangeable and, thus, no intertrial variability is given.According to Gentile's taxonomy, this exergame .For integrating an Below, we will briefly describe the content of each of the 16 skill categories.When implementing a therapeutic program to improve stroke victims motor skills, therapists must select exercises that are tailored to the demands of the individual patient. To achieve optimal outcomes in rehabilitation, the activities should be matched to a patient's functional abilities and limitations. In a well-elaborated training plan, the (exercise) tasks which have to be performed should maintain an optimal challenge for the patient , 19. ObvConsidering the allocation of the skill categories to one of the seven difficulty levels, Gentile's taxonomy offers a simple and easy-to-follow way for incorporating an ongoing progression into a rehabilitation process. Specifically, for selecting appropriate activities that provide rehabilitation at the optimal level of challenge, we propose the following procedure: A patient may only move up one level of difficulty when all skill categories within the current level are completed. For clarification, beginning with skill category 1A in difficulty level 1, the patient is required to move to the skill categories in difficulty level 2  as soon as category 1A is successfully performed. Within level 2, patients and treating therapists are free to choose the order of execution of the belonging skill categories. Once a patient is capable to perform both skill categories 1B and 2A successfully, the next difficulty level 3 has to be incorporated into rehabilitation. This procedure can be continued until the patient finally achieves skill category 4D and, consequently, the highest level of difficulty . In this way, challenging situations are ensured at any point in time during rehabilitation and the patients are encouraged to exercise towards their functional limits. Following the procedure suggested to guide someone's therapy plan, one ensures that the selection of skill categories is constrained. Due to this constraint, a variation of both general dimensions defined by Gentile's taxonomy will be considered during the rehabilitation process. Accordingly, the procedure prevents a skill category-based progression only in one direction and includes the modification of both the environmental context and the action function characteristics.Apart from these constraints, this approach also enables a certain degree of self-determination by therapy-involved actors. Within a specific level of difficulty, the performance order of the skill categories can be freely determined. This autonomy in decision-making increases the potential of tailoring the rehabilitation process to the demands of each individual patient and leaves freedom of choice to the treating therapists for the most adequate exercises that should currently be trained.body stability that represents less complex tasks than motor skills that require body transport . In our view, maintaining a one-leg stance position needs good balance skills. Therefore, this activity might be perceived by some stroke patients as an advanced motor task which challenges them more than a body transport activity. Furthermore, where this theoretical approach focused on the “general stroke patient population”, we are aware of the fact that different accompanying diseases and impairments; e.g., dizziness or muscle weakness due to stroke, may cause differences in how patients perceive tasks as being more or less difficult. It can be hypothesized that some patients have more difficulty maintaining a given posture without loss of balance whereas others experience performing a body transport task as more difficult. We think, however, that these potential differences may be resolved by clinicians that treat and monitor patients and decide, together with the patients and according their skill levels, how and when to progress through the taxonomy. It is clear to us that several pilot studies assessing feasibility and usability in several reference and subpopulations are needed as necessary next step in our program development process. Feasibility studies are comparative randomized trials designed to provide preliminary evidence on the clinical efficacy of a drug or intervention [Using Gentile's taxonomy for designing a rehabilitation program that targets balance and walking deficits of stroke patients, an aspect must be considered critically. According to Gentile's framework, a horizontal shift to the right or a vertical shift downward within the taxonomy provides an increase of task complexity. However, when we focus on a motor task such as standing still on one leg, a potential inconsistency in Gentile's approach emerges. Standing still on one leg can be categorized by the taxonomy dimension rvention . Usabilirvention .In this paper, we elucidate the theoretical basis for a theory-driven rehabilitation program to improve stroke sufferers' balance skills and walking capability using exergames. According to the framework proposed by Campbell et al. , investiWith stroke, the changes in the central nervous system and lifestyle may impair an individual's ability to regain and/or maintain certain levels of motor functioning. By using virtual reality, we might be able to offer continued rehabilitation in an attractive and meaningful way. Specific targeted exergames have been designed that aim to minimize stroke-induced walking impairments. Considering the aim of presenting a well-elaborated training concept with a strong theoretical rationale, in this paper we demonstrate that Gentile's taxonomy presents a feasible template providing assistance for developing and implementing a theory-driven rehabilitation program. The motor skill taxonomy suggested by Gentile defines two general dimensions and structures a table that consists of 16 different skill categories (Table


The fusiform face area (FFA) is known to play a pivotal role in face processing. The FFA is located in the ventral region, at the base of the brain, through which large blood vessels run. The location of the FFA via functional MRI (fMRI) may be influenced by these large blood vessels. Responses of large blood vessels may not exactly correspond to neuronal activity in a target area, because they may be diluted and influenced by inflow effects. In this study, we investigated the effects of large blood vessels in the FFA, that is, whether the FFA includes large blood vessels and/or whether inflow signals contribute to fMRI signals of the FFA. For this purpose, we used susceptibility-weighted imaging (SWI) sequences to visualize large blood vessels and dual-echo gradient-echo echo-planar imaging (GE-EPI) to measure inflow effects. These results showed that the location and response signals of the FFA were not influenced by large blood vessels or inflow effects, although large blood vessels were located near the FFA. Therefore, the data from the FFA obtained by individual analysis were robust to large blood vessels but leaving a warning that the data obtained by group analysis may be prone to large blood vessels. Brain imaging via functional MRI is used to identify the functional sites that play specific roles in information processing in the brain. For an identified functional site, we expect that the site is as specific as possible to a function without exhibiting an overlap with other functional sites.In BOLD imaging, on which most fMRI studies are based, the paramagnetic property of blood produces a bulk susceptibility difference between a blood vessel and the surrounding brain tissue, thus producing resonance frequency shifts in extravessel molecules. Thus, signals from oxygenation modulation can be expected in the tissues and for some distance into the venous side of the capillary bed . This isHowever, two factors make an undesirable contribution to BOLD-based fMRI signals, leading to an exaggeration of the spatial specificity and signal amplitude compared with those expected. One factor is the propagation of the bolus of oxygen-enriched blood by neuronal activation to the larger draining venous structures, which leads to the appearance of BOLD contrast in remote regions downstream of the actual site of neuronal activation , 2. AnotFace recognition is important for social communication. The fusiform face area (FFA) is known to play a pivotal role in face processing , 6. The In this study, we investigated the effects of large blood vessels in the FFA, that is, whether the FFA includes large blood vessels and/or whether inflow signals contribute to fMRI signals of the FFA. For this purpose, we used susceptibility-weighted imaging (SWI) sequences to visualize large blood vessels , 8 and dTen healthy volunteers participated in this study. After subjects were given a complete description of the study, written informed consent was obtained. This study was approved by the Institutional Review Board of Tohoku Fukushi University.All MRI experiments were performed using a Verio system  with a standard 12-channel head matrix coil operating at 3 Tesla.The dual-echo EPI sequence was modified from the default single-shot GE-EPI of the Siemens apparatus to acquire images at the two echo times of 13 and 38 ms, which were determined as the shortest echo times possible. For functional imaging, the dual-echo GE-EPI sequence was used with a 2000 ms repetition time (TR), 90° flip angle (FA), 220 mm field of view (FOV), 64  × 64 mm matrix size, and 3.4 mm slice thickness with 0.5 mm gaps. Twenty slices parallel to the AC-PC were acquired for each volume.An SWI 3D sequence was used with the parameters of TR of 28 ms, TE of 20 ms, FA of 15°, 294 × 320 matrix size, 220 mm FOV, and 1.13 mm thickness with 0.23 mm gaps. The orientation and center of slice positions were the same as those used in EPI. SWI image processing was performed using the Siemens default program.T1 of a voxel and is responsible for inflow effects; TE is the effective echo time; and T2* is the apparent transverse relaxation time. As reported in a previous study S0 is calculated directly using the MRI signals S(TE1) and S(TE2) corresponding to TE1 and TE2. The equation that yields S0 for the corresponding voxel is as follows ., 8.7, 8]http://dx.doi.org/10.1155/2014/234345).S0 images were acquired from images of two TEs of a dual-echo sequence. The activation map of S0 was acquired by contrasting the face conditions (SF and PF) to the control condition (this contrast included the contrast of the face versus scene conditions). Five of 10 subjects exhibited activation maps of S0 in the visual ventral region, which reflects inflow effects, while another five subjects did not. Among the data from the five subjects that showed inflow effects, variation was found in the location of activation sites  of the dual-echo sequence by contrasting the face conditions (SF and PF) with the scene conditions (SS and PS). The data from some subjects showed that the FFA was located near the sites that reflected inflow signals. Other data showed that the FFA was located a little bit more remotely from those sites  but PF was larger than SF .To assess the effect of large surrounding blood vessels in the FFA, we processed the FFA data at individual and group levels. The results of the comparison of the SF and PF conditions were different between these two analyses . SF and Our aim was to examine the contribution of large blood vessels to the fMRI response of the FFA. The data demonstrated, based on the presence of separate locations of activation sites of BOLD and S0, that fMRI responses of the FFA were not affected by inflow signals. The data also showed that no large blood vessels were present in the FFA. This means that the FFA was not affected directly by inflow signals and that its location was not biased by large blood vessel signals.However, the presence of large blood vessels and the activation by inflow effects around the FFA represent a warning that the evaluation of FFA responses via intersubject averaging may lead to a wrong direction, because a representative location of the FFA acquired by averaging activated sites across subjects can pick up signals that originate from large blood vessels that surround the FFA. In fact, the comparison between an averaged signal from the locations of individual subjects and a signal from a common location based on the group-averaged data yielded different results; that is, the difference between the SF and PF conditions was larger in the group analysis than in the individual subjects' analysis . It is cThe contribution of signals originating from large blood vessels to BOLD activation has been reported, and several previous studies attempted to remove this contribution or identify the location of large blood vessels –15. One In the present study, we used SWI sequences to acquire blood vasculature information and dual-echo EPI sequences to measure inflow effects. Imaging using SWI sequences represents anatomical scanning, whereas imaging using dual-echo sequences represents functional scanning. Therefore, there was no penalty regarding scanning time for function or regarding signal intensity, because the second echo signal can be used for typical BOLD.Signals originating from large blood vessels may lead to misinterpretation of the functional characteristics of brain areas examined using typical fMRI, particularly in high spatial resolution studies or high functional resolution studies . Therefore, the consideration of the contribution of large blood vessels in parallel with the typical functional analysis would be needed for ensuring reliability. The present study revealed that large blood vessels do not run through the FFA and that inflow effects did not affect fMRI signals of the FFA. This assures that the fMRI data from the FFA do not include functional artifacts resulting from large blood vessels, such as the variation in the location of the FFA between subjects or the modulation of signal intensity. Conversely, information on the large vessels that surrounded the FFA provided a warning regarding group analyses. The results obtained for the FFA can also be applied to other functional areas. Therefore, it may be advisable to use SWI and dual-echo EPI sequences for high spatial resolution studies or for perception or cognitive studies that deal with subtle differences in signals , 17.Our study has some limitations. Although SWI images provided a strong contrast to large blood vessels, cerebral spinal fluid (CSF) also provided a contrast of a similar level. Some additional efforts may be needed to delineate vessels if greater detail regarding the blood vasculature is required. The method used in the present study consisted of the observation of the effects of large blood vessels rather than the removal of the effects. Therefore, other methods may be used in combination with the present approach if the proposed method shows that some target areas are influenced by large blood vessels.Taken together, the results of the present study demonstrated that the location or response signals of the FFA were not influenced by large blood vessels or inflow effects, despite large blood vessels being located near the FFA and the variation in the location of the FFA across subjects. However, a group analysis of multiple subjects may be affected by large blood vessels. This suggests that the data from the FFA obtained by individual analysis would be robust to large blood vessels, whereas the data obtained by group analysis may be prone to large blood vessels.The data from the FFA obtained by individual analysis were robust to large blood vessels but leaving a warning that the data obtained by group analysis may be prone to large blood vessels. This shows that SWI sequences and dual-echo GE-EPI sequences can be used to probe functional characteristics of brain areas.Common description for Supplementary Figures 1-3: SWI images and activation maps for one subject in whom activations of BOLD and inflow signals appeared in different image slices. The crossing of the two white lines indicates the center of the FFA for (a) and the center of the S0 activation map for (d). (a) SWI-noMIP (left) and SWI-MIP (right) images. (b) Activation maps on the SWI image of the FFA were obtained by BOLD signals using the contrast of the face and scene conditions (F > S) (left), but not by inflow (S0) signals using the contrast of the face and control conditions (F > 0) (right). (c) Activation maps on the SWI image using SF > 0 (left) and PF > 0 (right). (d) Activation maps by S0 signals in a different image slice obtained by BOLD signals. Common description for Supplementary Figures 4-8: SWI images and activation maps for one subject in whom activation of BOLD and inflow signals appeared in the same image slice. The crossing of the two white lines indicates the center of the FFA. (a) SWI-noMIP (left) and SWI-MIP (right) images. (b) Activation maps on the SWI-noMIP of the FFA by BOLD signals using the contrast of the face and scene conditions (F > S) (left), and an activation site caused by inflow (S0) signals using the contrast of the face and control conditions (F > 0) (right). (c) Activation maps on the SWI image using SF > 0 (left) and PF > 0 (right).


The androgen receptor (AR) is required for prostate development and is also a major driver of prostate cancer pathogenesis. Thus androgen deprivation therapy (ADT) is the mainstay of treatment for advanced prostate cancer. However, castration resistance due to expression of constitutively active AR splice variants is a significant challenge to prostate cancer therapy; little is known why effectiveness of ADT can only last for a relatively short time. In the present study, we show that PCGEM1 interacts with splicing factors heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and U2AF65, as determined by RNA precipitation and Western blot, suggesting a role for PCGEM1 in alternative splicing. In support of this possibility, PCGEM1 is correlated with AR3, a predominant and clinically important form of AR splice variants in prostate cancer. Moreover, androgen deprivation (AD) induces PCGEM1 and causes its accumulation in nuclear speckles. Finally, we show that the AD-induced PCGEM1 regulates the competition between hnRNP A1 and U2AF65 for AR pre-mRNA. AD promotes PCGEM1 to interact with both hnRNP A1 and U2AF65 with different consequences. While the interaction of PCGEM1 with hnRNP A1 suppresses AR3 by exon skipping, its interaction with U2AF65 promotes AR3 by exonization. Together, we demonstrate an AD-mediated AR3 expression involving PCGEM1 and splicing factors. Prostate cancer is the most common malignancy among elderly men in Western countries. The androgen receptor (AR) is a nuclear receptor transcription factor required for normal prostate development and prostate cancer pathogenesis. Furthermore, AR serves as an important therapeutic target. For instance, androgen deprivation therapy (ADT) has been the frontline therapy for treatment of advanced prostate cancer. Although initial response of prostate cancer to ADT is effective, these patients inevitably develop the resistance, i.e., castration-resistant prostate cancer (CRPC) , 2. ThisAlternative splicing represents an important mechanism of genetic diversity in eukaryotes. In this regard, the vast majority of eukaryotic genes including protein-coding genes and long non-coding RNAs (lncRNAs) can be expressed as various alternative splice variants  and theyLncRNAs are a large group of poorly characterized non-coding RNAs with >200 nucleotides in length . AccumulIn the present study, we show that androgen deprivation (AD) induces PCGEM1 expression and causes its subcellular re-distribution. Moreover, PCGEM1 functionally interacts with splicing factors including hnRNP A1 and U2AF65. Of considerable interest, the PCGEM1-hnRNP A1 interaction suppresses binding of hnRNP A1 to AR pre-mRNA whereas the interaction of PCGEM1 with U2AF65 enhances its binding to AR pre-mRNA, leading to expression of AR3.PCGEM1 is prostate cancer specific lncRNA . A recenin vitro and in vivo possibly through regulation of AR3 expression.It is well known that AR splice variants play a significant role in castration resistance , 6. NextGiven the positive relationship between PCGEM1 and AR3, we decided to determine whether PCGEM1 specifically impacts AR3 expression, leading to castration resistance. We found that PCGEM1 was significantly induced in LNCaP cells by AD Fig. . This ADNuclear speckles are important to RNA splicing and gene expression . To bettSince hnRNP A1 is well-known for its role in mRNA processing or alternative splicing , 23, andAlthough other members of hnRNP family such as hnRNP C have been implicated in suppression of alternative splicing of Alu elements , RNA preNuclear speckles have been shown to be critical sites for RNA processing . AD causTo further determine the role of PCGEM1 in AR3 expression, we generated a mini-gene cassette reporter carrying mCherry . As expein vitro and xenograft assays with castrated male mice support the role of PCGEM1 in castration resistance. Together, these findings highlight the importance of PCGEM1 in AR signaling and castration resistance.The present study demonstrates that AD induces PCGEM1 and causes its localization of nuclear speckles. Through interaction with splicing factors, such as hnRNP A1 and U2AF65, PCGEM1 promotes AR3 expression. As a major AR splice variant, AR3 has been shown to play a significant role in castration resistance  and thusAs components of the basic machinery for RNA splicing, hnRNP A1 and U2AF65 are important to AR3 expression although AD has no effect on their expression levels. The hnRNP proteins belong to the RNA binding protein family that play multiple functions. Along with other RNP proteins, they participate in pre-mRNA processing such as splicing, and are important determinants of mRNA export, localization, translation, and stability . SeveralRegulation of alternative splicing is a complex process. It is known that splicing factors select splice sites often in a concentration-dependent manner and thus, the relative expression of these factors may decide a particular splice pattern . The preHowever, the detail mechanism still remains to be determined as to why the PCGEM1-hnRNP A1 interaction causes the loss of its suppressive function whereas the PCGEM1-U2AF65 interaction promotes the binding of U2AF65 to AR pre-mRNA, leading to expression of AR3. We speculate at least two possibilities for this opposing role of PCGEM1 once bound by these splicing factors. Although hnRNP A1 is a RNA binding protein, interacting with many types of RNAs, several reports suggest that hnRNP A1 prefer the UAG motif , 30. ForPCGEM1 is an interesting molecule, but the role of PCGEM1 in prostate cancer is still controversial. For example, it has been reported that PCGEM1 along with PRNCR1 can impact AR signaling through interaction with AR to promote castration resistance , howeverIn summary, our study suggests that the interaction of PCGEM1 with splicing factors such as hnRNP A1 and U2AF65 determines the fate of AR3 . One fun2 in the humidified chamber.Prostate cancer LNCaP and CWR22Rv1 cells were purchased from ATCC; LNCaP95 was a generous gift from Dr. Alan K. Meeker, Johns Hopkins University School of Medicine. Cells were grown in phenol free RPMI 1640  supplemented with charcoal stripped 5% FBS (Sigma-Aldrich). HECK293T cells were grown in DMEM supplemented with charcoal stripped 10% FBS. All media were supplemented with 2 mM glutamine, 100 units of penicillin/ml, and 100 μg of streptomycin/ml (Lonza). Cells were incubated at 37°C and supplemented with 5% COLentiviral packaging was carried out in HEK-293T cells using a packaging system from SBI per the manufacturer's protocol, as described previously . For infTo determine which proteins are associated with PCGEM1, we performed RNA precipitation assay using synthesized PCGEM1 as a probe. In brief, the DNA fragment covering the entire PCGEM1 sequence was amplified by PCR using a T7 containing primers (T7-PCGEM1-5.1 and T7-PCGEM1-Not1-3.1), and then cloned into pCR8 (Life Technology). In addition, two lncRNAs, ncR-UPAR and HULC, were also cloned in the same way as negative controls. The resultant plasmid DNA was linearized with restriction enzyme Not I which was introduced from the reverse PCR primer, and then used to synthesize RNA by T7 polymerase. A 20 μl reaction contained 400 ng linearized plasmid DNA, 20 U ribonuclease inhibitor, 2.5 mM NTP mixture supplemented with 10% biotin labeled UTP (Perkin Elmer) and 20 U T7 RNA polymerase (New England BioLabs); and then it was incubated at 37°C for 60 min, followed by 25 U RNase-free DNase I (New England BioLabs) at 37°C for 30 min. The labeled RNA was purified by a column-based kit (Zymo Research). Cellular extract was prepared from a 10 cm dish culture (∼80% confluence) with cell lysis buffer . For preTo determine the interaction of hnRNP A1 or U2AF65 with PCGEM1 and AR pre-mRNA, we used hnRNP A1 or U2AF65 antibody for pulldown assays and then detected PCGEM1 or AR pre-mRNA by qRT-PCR using specific primers listed in FISH was used to detect PCGEM1 levels in prostate cancer cell lines. Biotin-labeled antisense LNA probes derived from PCGEM1 were listed in Immunofluoresence staining was used to detect AR, hnRNP A1 and U2AF65, as described previously . In brieMTT assay was performed to determine the effect of PCGEM1 knockdown on cell growth as described previously . InfecteThe animal studies were conducted in accordance with NIH animal use guidelines and the experimental protocol was approved by the UMMC's Animal Care and Use Committee. Male SCID mice at 5∼6 week old were first castrated and one week later infected CWR22Rv1 cells (control shRNA or PCGEM1-shRNAs) were then injected subcutaneously into these mice with 1 million cells containing 50% matrigel per spot, two spots per animal and 6 animals per group. Tumor growth was monitored every other day and harvested at day 31 after injection. The two-group t test was used to compare two means at each time point. All animals were included for analysis.To facilitate the selection of positive clones resulted from CRISPR/Cas9, we generated a donor vector in such a way that targeting sequence is replaced by marker genes  once it is integrated into the genomic DNA by homologous recombination. Donor vector carried ∼800 bp targeting sequence at each side and EF1-GFP-T2A-PU in the middle, flanked by a LoxP site. The dual gRNA construct carrying Cas9 and donor vector were introduced into CWR22Rv1 cells by transient transfection. One week later, the transfected cells were subject to puromycin selection; and surviving cells were sorted by FACS based on GFP signal into individual wells of 96-well plates and incubated for 2∼3 weeks, as described previously . The sorThe two-sample t test was used to compare two mean expressions. Bonferroni correction was used to adjust the p values of pairwise comparisons among three mean expressions. Relationship between PCGEM-1 and AR expression was studied by depicting scatter plot and calculating Pearson correlation coefficient. All p values were two sided and p values less than 0.05 were considered as significant.


Despite the increased interest about tele-rehabilitation, virtual reality and outcome measures for stroke rehabilitation, surprisingly little research has been done to map and summarize the most common outcome measures used in tele-rehabilitation. For this review, we propose to conduct a systematic search of the literature that reports outcome measures used in tele-rehabilitation or virtual reality for stroke rehabilitation. Specific objectives include: 1) to identify the outcome measures used in tele-rehabilitation studies; 2) to describe the psychometric properties of the outcome measures in the included studies; 3) to describe which parts of the International Classification of Functioning are measured in the studies. Methods: we will conduct a comprehensive search of relevant electronic databases . The scoping review will include all study designs. Two reviewers will pilot-test the data extraction forms and will independently screen all the studies and extract the data. Disagreements about inclusion or exclusion will be resolved by consensus or by consulting a third reviewer. The results will be synthesized and reported considering the implications of the findings within the clinical practice and policy context. Dissemination: we anticipate that this scoping review will contribute to inform researchers and end-users , regarding the most appropriate outcome measures for tele-rehabilitation or virtual reality as well as help to identify gaps in current measures. Results will be disseminated through reports and open access journals, conference presentations, as well as newsletters, podcasts and meetings targeting all the relevant stakeholders. Tele-rehabilitation (TR) and virtual reality (VR) are alternative and innovative approaches of delivering rehabilitation services for stroke survivors . Tele-reSeveral tools have been developed to evaluate the consequences of stroke and the effectiveness of virtual realityand telerehabilitation interventions on stroke outcomes . EvidencFormer systematic reviews on tele-rehabilitation and virtual reality addressed the difficulties of finding evidence coming from the small number of eligible studies. Scoping reviews may overcome this problem since their approach may address other questions beyond those related to intervention effectiveness, and generate findings that can complement the findings of clinical trials.The scoping review will use a methodologically rigorous approach, guided by the Arksey and O’Malley framework from the University of York . The YorWhich tele-rehabilitation and virtual reality outcome measures are used and when are they administrated  following a stroke?Which measures are psychometrically sound and what are the author(s) trying to measure? .Which parts of the International Classification of Functioning are measured in the outcome measures?  and structures , activities and participation  and environmental factors .A possible limitation of scoping reviews is the quality of the included studies since generally it’s not assessed; to overcome this limitation a quality assessment will be conducted in parallel to the stages of the scoping review, which will be based on the CASP tools . Results of this activity will be qualitatively summarized and added to the main findings of the studyThe scoping review will include studies: 1) involving stroke patients; 2) describing a rehabilitation intervention using tele-rehabilitation and/or virtual reality; 3) written in English, French, Spanish, Italian or Portuguese. The exclusion criteria are: articles published in other languages; articles not reporting the outcome measures or only reporting laboratory measures or only reporting the development of the technology. The search will not be limited by study design. Two authors will be in charge for the assessment of the retrieved papers; disagreement about inclusion or exclusion of a specific paper based on the review of its abstract will be resolved by reaching a motivated consensus or consulting a third reviewer.The literature search will be performed by a librarian expert in the field of rehabilitation. The search will include the major databases in which rehabilitation articles are found: PubMed (until December 2014), EMBASE (until December 2014), Cochrane Library-The Cochrane Central Register of Controlled Trials (until January 2015), Cumulative Index to Nursing and Allied Health Literature (CINAHL) (until December 2014) and PEDRO (until January 2015) to identify potentially relevant records. A combination of medical sub-headings (MeSH) and or keywords will be adapted as needed for each database (the details of the search strategy are available upon request).Data collection process and charting the data within the selected studiesData-exstraction forms will be developed based on the current literature in the field and on the research questions. A pilot test will be conducted using a subset of articles covering the research questions; changes may be necessary in the data extraction form during this validation phase in order to reach a final version. We expect to collect data on: 1) articles’ authors, 2) year of publication, 3) objective(s) of the study, 5) study design, 6) country 7) outcome measures reported; 8) psychometric properties of measures reported, and 9) participants’ characteristics ; 10) relevant International Classification of Functioning domains.; 11) period of time at which the measure is taken ; 12) technology used ; 13) details on tele-rehabilitation and/or virtual reality intervention and 14) outcome improvement.The outcome measures will be organized according to the domains of the international classification of functioning.Based on the recommendations by The results of the proposed scoping review are addressed primarily to clinicians and researchers in rehabilitation. In addition, the results will be disseminated to a larger audience including clinicians, policy makers, researchers and stakeholders and those interested in standardizing outcome measures for stroke tele-rehabilitation and virtual reality. Our research team will ensure broad dissemination of our findings through numerous knowledge translation strategies including podcasts, publications in open-access and peer-reviewed journals, conference presentations, web seminars and meetings including diverse audiences such as clinicians, stakeholders, researchers, healthcare managers, policy-makers and citizens, among others.