Recent breakthrough therapies targeting androgen receptor signalling in castration resistant prostate cancer (CRPC) involve multifunctional androgen receptor (AR) blockade and exhaustive androgen deprivation. Nevertheless, limitations to an enduring effectiveness of new drugs are anticipated in resistance mechanisms occurring under such treatments.In this study we used CRPC cell models VCaP and LNCaP as well as AR-negative PC-3- and non-neoplastic epithelial BPH-1-cells treated with 5, 10 or 25 μmol/L abiraterone hydrolyzed from abiraterone acetate (AA). The origin of CYP17A1 up-regulation under AA treatment was investigated in CRPC cell models by qRT-PCR and western-blot procedures.AA treatments of AR positive CRPC cell models led to decreased expression of androgen regulated genes such as PSA. In these cells diminished expression of androgen regulated genes was accompanied by an up-regulation of CYP17A1 expression within short-term treatments. No such effects became evident in AR-negative PC-3 cells. AR directed siRNA (siAR) used in VCaP cells significantly reduced mRNA expression and AR protein abundance. Such interference with AR signalling in the absence of abiraterone acetate also caused a marked up-regulation of CYP17A1 expression. Down-regulation of androgen regulated genes occurs in spite of an elevated expression of CYP17A1, the very target enzyme for this drug. CYP17A1 up-regulation already takes place within such short treatments with AA and does not require adaptation events over several cell cycles. CYP17A1 is also up-regulated in the absence of AA when AR signalling is physically eliminated by siAR.These results reveal an immediate counter-regulation of CYP17A1 expression whenever AR-signalling is inhibited adequately but not a persisting adaptation yielding drug resistance.The online version of this article (doi:10.1186/2193-1801-3-574) contains supplementary material, which is available to authorized users. Prostate cancer is the second leading cause of cancer death from the most frequently diagnosed malignancy in males in the USA. As well in Europe prostate cancer accounts for 9% of total cancer-related deaths. Standard treatments for localized prostate cancer are radical surgery, radiation therapy or active surveillance  and enzalutamide, effectively target the androgen pathway to arrest aberrant signalling even after multiple therapies. The mode of action of these new compounds is either inhibition of androgen synthesis at the CYP17A1 enzyme covering also adrenal and intracrine androgen sources, or a full androgen receptor (AR) blockade antagonizes the AR function by directly binding this transcription factor with high affinity. Nevertheless, clinical success of these novel drugs is not enduring in all cases. Recent phase 3 trials revealed median time to PSA progression of about eight months for both drugs and in sequential treatments of enzalutamide and AA cross resistances have to be considered . This study did not require an approval from an ethics committee and the local animal protection committee.Human prostate cancer cell line VCaP was purchased from ATCC, Wesel, Germany and kept in Phenol red-free Gibco® DMEM lot # 1089200  supplemented with 2% sodium pyruvate and 10% foetal calf serum . The other cell lines LNCaP, PC-3 and BPH-1 from permanent local stocking were certified to proof origin  was used for treatments in 5, 10 or 25 μmol/L concentration performed for 24 hours. To obtain the active drug hydroxy-abiraterone (AA), abiraterone acetate was hydrolysed in 95% ethanol prior to use . Luciferase (LUC) siRNA was used for control transfections .The VCaP cells were transfected with siRNAs against the androgen receptor . To select for best AR interference four different sequences were used Table . We chosmRNA-expression of PSA, TMPRSS2-ERG, CYP17A1, AKR1C3, AR and IGF-2 were analysed by qRT-PCR. Total cellular RNA from cultured cells was extracted with the Quick-RNA™MiniPrep . Total RNA integrity and quantity were assessed on an Agilent Bioanalyzer 2100 with a RNA 6000 Nano LabChipKit . Reverse transcription of 500 ng total cellular (tc) RNA was performed with random hexamer primers and an Omniscript RT Kit . Expression analyses were processed on an iCycler iQ real time detection system  with SsoFast EvaGreen supermix. The 20 μl reaction from the kit was supplemented with 2 μl cDNA, 0.6 μM gene-specific primers . Primers were designed by Primer 3 and PCR efficiency was assessed as previously described  was used.Protein expression was measured by western blot analysis with androgen receptor antibody Ab-2  and CYP17A1 antibody  where α-tubulin was used as loading control detected with a monoclonal anti-α-tubulin . Cells were homogenized with Pierce® RIPA buffer . Novex® precast gels  were used for electrophoresis followed by electro-transfer onto Protran® nitrocellulose membranes  from where protein-bound membrane was hybridized with antibodies. For visualization, we used Western Lightning® Plus-ECL , rabbit secondary antibodies  and ProteinSimple FluorChem E . For quantification of band intensity Image J .Statistical calculation, mean +/-SD and P values were carried out with GraphPad Prism software version 5.0 using the unpaired nonparametric CRPC cell line VCaP with overexpressed AR and LNCaP with a gain-of-function AR mutation were treated with 5, 10 or 25 μmol/L AA. In both cell lines PSA mRNA expression decreased after treatment with AA Figure A + B indA siRNA against AR transcripts was used to elucidate the relevance of AR signalling to the effects on CYP17A1 and AKR1C3 expression in the absence of the compound AA. AR directed siRNA significantly reduced AR mRNA-expression and is also seen in western blot analysis Figure A, albeitTo elucidate by which function in AR signaling, receptor status or androgen synthesis, androgen regulated genes are affected, western blot analysis for the AR and CYP17A1 were performed. In these experiments VCaP cells were treated with 5 μmol/L AA. No significant changes were detected on the AR protein level calculated from western blot analyses


Of the age-dependent protein aggregation disorders associated with neurodegeneration, diseases caused by polyglutamine tract expansions have attracted particular attention. These inherited disorders are caused by CAG microsatellite expansions in the coding regions of disease-causing genes. These alterations lead to degeneration that tends to present in mid-life, progress slowly, and exhibit toxicity that is intimately associated with misfolding of the mutant protein. This proteotoxic gain-of-function is a shared common feature that contributes to many disease manifestations.Variation in the length of CAG/glutamine tracts is also known to influence the normal function of the affected proteins. This has been well studied in the case of the androgen receptor (AR), where variations in tract length that occur within the normal population revealed that length is inversely associated with function of the receptor as a ligand activated transcription factor. Further pathological expansion of the AR's CAG repeat causes spinal and bulbar muscular atrophy (SBMA), an untreatable and progressive degenerative disorder of the neuromuscular system characterized by skeletal muscle weakness and atrophy, and loss of motor neurons in the spinal cord and brainstem. This disease is X-linked, and affected men often show signs of partial androgen insensitivity including gynecomastia, testicular atrophy and decreased fertility. These clinical features correlate with diminished intrinsic transcriptional regulatory activity of the polyglutamine expanded AR (polyQ AR).Several general principles guide our understanding of SBMA pathogenesis. Ligand binding to the polyQ AR promotes nuclear translocation and protein unfolding. Both of these steps are required for pathogenesis hence the occurrence of disease only in men. In SBMA, as in other CAG repeat disorders, the mutant protein disrupts multiple downstream pathways and toxicity likely results from the cumulative effects of altering a diverse array of cellular processes including transcription, RNA processing, axonal transport, and mitochondrial function. Notably, recent studies have established that skeletal muscle is an important site of toxicity and an attractive therapeutic target. SBMA patient muscle shows both denervation and myofiber degeneration, genetic deletion of a floxed polyQ AR allele only in skeletal muscle rescues disease in BAC transgenic mice , and trein vitro [in vitro and in gene targeted mice [While SBMA patients exhibit signs of partial androgen insensitivity, the extent to which loss of AR function contributes to pathogenesis has been poorly understood. A major challenge has been the difficulty in dissociating effects of polyQ AR proteotoxicity from impaired intrinsic transcriptional function. To accomplish this, recent work mitigated the transcriptional deficits of the polyQ AR by relieving the inhibitory effect of AR posttranslational modification by small ubiquitin-like modifier (SUMO) . SUMO prin vitro , 6 and pted mice . Moreoveted mice .This work demonstrates beneficial effects of enhancing polyQ AR transcriptional function in a mouse model of SBMA. Anabolic support of skeletal muscle by AR is well established, and promoting this function may ameliorate aspects of the disease. These findings are also notable because previous work in a cellular model indicated that acute elevations of AR SUMOylation can mitigate the ligand dependent aggregation of polyQ AR . Thus, i


Low androgen levels have been linked with an increased risk of cardiovascular disease in men. Previous studies have suggested that androgens directly inhibit atherosclerotic lesion formation although the underlying mechanisms for this remain unclear. This study addressed the hypothesis that endogenous androgens inhibit arterial remodelling by a direct action on the androgen receptor (AR) in the vascular wall.7 ± 4.5 × 106 vs. 3.9 × 107 ± 4.9 × 106 µm3, P = 0.04, n = 9–10).We studied a series of novel mouse lines with cell-specific deletion of the AR in either the endothelium or in smooth muscle cells or both cell types. Findings were compared with a model of global androgen deficiency in wild-type mice (castrated). We characterized the cardiovascular phenotype, vascular pharmacology and histology, and assessed neointimal lesion formation following vascular injury to the femoral artery. Cell-specific AR deletion did not alter body weight, circulating testosterone levels or seminal vesicle weight, but caused limited alterations in arterial contractility and blood pressure. Neointimal lesion formation was unaltered by selective deletion of AR from the vascular endothelium, smooth muscle, or both cell types. Castration in wild-type mice increased neointimal lesion volume (Sham vs. Castration: 2.4 × 10Vascular cell-specific AR deletion had no effect on neointimal lesion formation, while low systemic androgen levels adversely affect neointimal lesion size. These findings suggest that the cardio-protective effects of androgens are mediated either by AR outside the vasculature or by AR-independent mechanisms. Cross-sectional studies have associated low testosterone levels with increased cardiovascular risk factors  and increased cardiovascular risk in men.5 This is particularly important given the progressive population-level decline in serum testosterone concentrations in men from developed countries8 which has resulted in a dramatic increase in the use of androgen replacement therapy (ART). Indeed, there has been a 10-fold increase in prescribed ART in the USA9 and nearly a 3-fold increase in the UK10 in the past decade. ART improves the muscle/fat mass ratio, bone mineral density, and blood lipid profile12 in hypogonadal men. It has also been suggested that ART could provide a novel strategy to reduce cardiovascular risk. More recently, however, concerns have been raised about safety and the Food and Drug Administration in the USA has announced an investigation into the risk of stroke, heart attack, and death in men taking testosterone products.13 This follows recent reports demonstrating an excess of cardiovascular events in apparently hypogonadal men using ART.16 Given the inconsistent findings from clinical studies, there is a clear need for additional pre-clinical studies to improve our understanding of how endogenous androgens and pharmacological androgen supplements influence CVD. Previous pre-clinical studies have focused on pharmacological testosterone supplementation and/or deficiency (by castration) and have largely supported a cardio-protective role for androgens, with pharmacological testosterone replacement in castrated animals reducing atherosclerotic plaque formation.19 However, the mechanism of this effect is not clear. It may be indirect, following modification of conventional cardiovascular risk factors, and/or due to direct modulation of vascular remodelling. Furthermore, it has not been established whether androgens alter vascular remodelling by direct stimulation of the androgen receptor (AR), by testosterone-mediated AR-independent actions, or, indirectly, via aromatase-mediated conversion of testosterone to oestrogens. In models of arterial injury that lack elevated systemic cardiovascular risk factors, the findings are contradictory, with studies showing that androgens either reduce20 ,21 or have no effect on22 neointimal lesion formation.Male sex hormones have traditionally been linked to the greater risk of cardiovascular disease (CVD) in men.Tfm) mouse, which lacks a functional AR.19 However, interpretation of results from this animal is confounded by the fact that it lacks AR in all tissues, has low (∼10%) circulating testosterone, and, consequently, has sub-physiological concentrations of oestradiol. Generation of a similar total AR knockout mouse on an atherosclerosis-prone (apoE−/−) background suggested that androgens reduce total serum cholesterol via an AR-dependent mechanism but implicated both AR-dependent and AR-independent mechanisms in the observed anti-atherosclerotic effects.23 Recognizing the limitations of these models and the complex role of androgens in influencing a number of aspects of cardiovascular risk, we generated mice with vascular cell-specific deletions of AR in order to address the hypothesis that endogenous testosterone inhibits neointimal proliferation by stimulation of AR in the vascular wall.Endogenous androgens play a complex role in determining cardiovascular risk and thus investigation of their mechanism of action is challenging. The influence of AR stimulation on vascular lesion formation has been investigated previously using the testicular feminised  Act 1986.24 or smooth muscle cells (SM-ARKO)25 were established in our laboratory as previously described. In this study, these two lines were mated to generate stud males hemizygous for both Tie2-Cre and SM22-Cre, which were then mated with female ARfl/fl mice. The mouse line was maintained by breeding male SM22-Cre+/−:Tie2-Cre+/−:ARfl/y mice with female ARfl/fl mice. Four genotypes were identified in the resultant offspring at expected Mendelian ratios :WT: SM22-Cre−/−:Tie2-Cre−/−:ARfl/y. Used as controls.SM-ARKO: SM22-Cre+/−:Tie2-Cre−/−:ARfl/y. Smooth muscle cell (SMC) ARKO.VE-ARKO: SM22-Cre−/−:Tie2-Cre+/−:ARfl/y. Endothelial cell (EC) ARKO.SM/VE-ARKO: SM22-Cre+/−:Tie2-Cre+/−:ARfl/y. Smooth muscle and EC double ARKO.In this study, only male mice were used for onward analysis.C57Bl/6J mice were supplied by the University of Edinburgh Biomedical Research Facility. Mice with selective ablation of AR from vascular endothelial (VE-ARKO)2.224To verify AR ablation in target cells, genomic DNA was extracted immediately from freshly isolated aortic EC and SMC cells (without any culturing) and subjected to PCR amplification using primers GCTGATCATAGGCCTCTCTC and TGCCCTGAAAGCAGTCCTCT. An amplicon of 1142 bp indicated the presence of a floxed AR, while an amplicon of 612 bp indicated recombination between loxP sites and deletion of AR exon 2.26 and CGCATAACCAGTGAAACAGCATTGC and CAGACACCGAAGCTACTCTCCTTCC for SM22-cre.27 An amplicon of 608 bp indicated the inheritance of the Cre Recombinase transgene in EC under control of the Tie2 promoter, while an amplicon of 575 bp for the Cre Recombinase transgene in SMC under control of SM22 promoter.Inheritance of Cre Recombinase was used to determine genotype. Genomic DNA from ear clips was amplified using primers CGCATAACCAGTGAAACAGCATTGC and CCCTGTGCTCAGACAGAAATGAGA for Tie2-cre;2.32 and aortic EC and SMC isolated, by collagenase digestion, and cultured, as described.28 Isolated cells were either used directly for DNA extraction, or cultured  for investigation of AR expression. Testosterone (1 × 10−7 M), DHT (1 × 10−8 M), or vehicle  was added from the 3rd day of culture and media were replenished twice weekly.Mice were euthanized by CO2.42.4.1Systolic blood pressure was assessed in conscious, restrained mice using tail-cuff plethysmography .2.4.2Plasma testosterone , total plasma cholesterol , and plasma triglyceride  were measured using commercially available kits, in accordance with manufacturer's instructions.2.4.32. Femoral and mesenteric arteries were isolated for functional analysis, as described previously.29 A linear relationship between the increment of cyclic force and the increment of diameter was used to describe arterial compliance.30 Arteries were then exposed to high (125 mM) potassium physiological saline solution (KPSS), phenylephrine , acetylcholine , and sodium nitroprusside . A further set of arteries from the same animals were exposed to testosterone (10−9–10−4 M) and endothelin-1 .Mice (12–16 weeks) were euthanized by CO2.5Surgical procedures were performed in mice under isoflurane-induced anaesthesia with analgesic cover.2.6In C57Bl/6J mice, a small incision was made in the mid-line of the scrotum and both testes externalized and removed (castration) or returned to the scrotum (Sham). The mice were allowed to recover for 1 week prior to induction of femoral artery injury.2.7et al.31 Ligation injury was performed on the common femoral artery immediately proximal to the femoropopliteal bifurcation. Wounds were sutured and mice were allowed to recover (21 days) to allow lesion development.Wire injury was performed by inserting a guidewire using the method of Sata 2.832 Longitudinal lesion distribution and total neointimal volume in the first 1.2 mm segment of the artery were used to describe the overall neointima formation . The maximum cross-sectional neointimal area was determined from serial histological sections indicated the level of stenosis .Mice were killed  and plasma harvested and stored (−20°C). Mice were then perfusion-fixed, femoral arteries excised from the femoropopliteal branch to the bifurcation with the iliac artery, and then processed for optical projection tomography (OPT), as described.2.9After OPT scanning, tissues were processed for histology, sectioned (5 µm) and stained with Masson's trichrome. Intimal and luminal area were measured using Image Pro Plus 7.0 rom images obtained using a CoolSNAP camera . Immuno-florescent staining was with Tyramide Signal Amplification  was applied using primary antibodies against: AR , CD31 , von Willebrand factor , and smooth muscle alpha-actin . Fluorescent images were analysed using confocal microscopy.For cultured cells, samples were fixed, stained without antigen retrieval, and images were captured using a Zeiss Axiovert 200M epi-fluorescent microscope .2.10n mice, unless indicated otherwise. Analysis was performed (GraphPad Prism v5.0) using Student's t-test, one-way or two-way ANOVA with a Bonferroni post-hoc test, as appropriate; P < 0.05 indicated statistical significance.Data are mean ± standard error of the mean (SEM) for 3.3.1Figure 1A) and in cultured aortic EC  and SMC . AR expression in mouse aortic EC and SMC was increased following incubation with testosterone (1 × 10−7 M) .Immunohistochemistry confirmed AR expression in intact mouse aorta , VE-ARKO , and SM/VE-ARKO , and floxed-AR mice (WT), which were used as controls.Three vascular cell-specific AR-ablated mice were generated using the Figure ). A recombined 612 bp band representing the recombined non-functional AR allele was only observed in SM-ARKO SMC, VE-ARKO EC, and SM/VE-ARKO EC/SMC. Genomic DNA samples isolated from ear biopsies were used for genotyping. The SM22-cre amplicon consistently correlated with AR ablation in SMC and the Tie2-cre amplicon with AR ablation in EC .In order to confirm deletion of AR in targeted cell types, genomic DNA from freshly isolated aortic EC and SMC were subjected to PCR analysis. An 1142 bp band representing the wild-type AR allele was only observed in WT EC and SMC, SM-ARKO EC and VE-ARKO SMC  and seminal vesicle weights . Total plasma cholesterol and triglyceride were not affected by vascular ARKO . Tail-cuff plethysmography revealed a small but significant increase in blood pressure in VE-ARKO mice .Mice of all four genotypes were healthy. In contrast to Tfm or global ARKO mice,Ex vivo myography was used to determine whether vascular AR deletion was associated with functional changes in smooth muscle contraction or endothelium-dependent relaxation. Vascular AR deletion did not alter femoral or mesenteric arterial compliance . PhE-induced contraction, however, was reduced in femoral arteries that lack smooth muscle cell AR from both SM-ARKO and SM/VE-ARKO mice , while there was a small reduction in ET-1 mediated contraction in all vascular ARKOs . Vascular AR ablation did not alter PhE-  or ET-induced  constriction in mesenteric arteries. KPSS-induced receptor-independent constriction  and B(i)), ACh-induced endothelium-mediated dilation , and SNP-induced endothelium-independent dilation  and B(ii)) were not affected by vascular ARKO. Testosterone-induced dilation, which occurred at supra-physiological concentrations (1 × 10−4 M), showed no dramatic alterations following deletion of AR from vascular EC and/or SMC, despite some small differences in response at specific concentrations .3.4Figure ) and seminal vesicle weight  and decreased AR expression in femoral arteries . Body weights following surgery were lower in castrated mice than in controls . Castration also increased neointimal lesion formation following wire injury , resulting in increased lesion volume but without increasing the maximal cross-sectional area. In contrast, castration had no effect on the neointimal lesion formation following arterial ligation .Castration reduced circulating testosterone concentrations . Vascular ARKO did not alter the profile , volume , or cross-sectional area  of lesions induced following wire injury. Similarly, SM-ARKO and SM/VE-ARKO had no effect on lesion size following arterial ligation . In contrast, deletion of AR from vascular ECs alone (VE-ARKO) resulted in an altered lesion profile  and a small increase in lesion volume , but not cross-sectional area , following ligation.Body-weight changes after arterial injury were similar among the four genotypes of mice  activation of AR-independent pathways, (ii) secondary to aromatization of androgen to oestrogen, (iii) stimulation of AR in other cell types.Tfm19 mice. Interpretation of data from these animals is complicated, however, by low endogenous levels of testosterone and oestradiol . In contrast, consistent with previous demonstrations,23 vascular cell-specific deletion of AR did not reduce circulating testosterone levels and, therefore, no exogenous androgen administration was required. This is important since pharmacological administration of androgens does not satisfactorily ‘replace’ endogenous hormone. The pharmacokinetics and tissue accumulation of pharmacologically administered androgen are not fully understood . This may explain (i) the unexpected increase in adverse cardiovascular events associated with androgen treated in a clinical trial involving elderly (>65 years old) hypogonadal men,16 and (ii) the increased death and incidence of stroke and myocardial infarction (regardless of the pre-existing CVD) associated with clinical androgen replacement in hypogonadal men, as demonstrated in a large scale observational study.14The generation of vascular-specific ARKO mice was central to this investigation. The functional role of AR has been investigated previously using Tfm rats is reduced by castration and restored by administration of testosterone.33 This contrasts with vascular selective ARKO which showed either no change or, in the case of VE-ARKO, a small elevation in blood pressure. The implication, therefore, is that testosterone-mediated elevation of blood pressure is not mediated by vascular AR. The increased blood pressure in VE-ARKO mice suggests slight EC dysfunction, but this was not supported by our findings in isolated arteries.Androgens contribute to elevation of blood pressure by acting on catecholamines in the brain, independent of classical AR; blood pressure in wild-type and 34 In Tfm mice, contraction in response to high potassium  was reduced in femoral arteries, suggesting altered smooth muscle cell function.34 Relaxation in response to ACh was also impaired, suggesting EC dysfunction.34 Vascular AR ablation had no effect on passive arterial compliance and did not alter endothelium-dependent or -independent relaxation, confirming normal activity of the endothelium-derived nitric oxide system. It was notable that EC function was maintained in VE-ARKO mice, despite the  increase in blood pressure in these animals. Unlike the Tfm, smooth muscle AR deletion impaired agonist-mediated contraction without altering the response to potassium, suggesting a change in receptor-dependent signal transduction pathways. Reduced contraction was agonist dependent  and tissue-specific . An androgen-mediated alteration of adrenoceptor activation would be consistent with androgen-induced production and release of noradrenaline in rodents.35 The differences between the current results and those reported for Tfm suggest that AR expressed outside the vasculature contributes to regulation of vascular function. The low testosterone in Tfm could also lead to a loss of rapid, non-genomic androgen signalling. Alternatively, since endogenous oestrogen regulates EC function in males,37 the impaired endothelium-mediated dilation in the Tfm mice could be a result of its low oestrogen levels.Disruption of systemic androgen/AR signalling impairs normal vascular function.38 testosterone caused relaxation of arteries, although only at supra-physiological concentrations. The maintenance of this response in the vascular-specific ARKO mice is consistent with data from Tfm mice.34 This indicates, therefore, that vascular AR does not mediate this response. Similar arterial relaxation has been observed with high concentrations (≥1 × 10−4 M) of corticosteroids, oestrogen, and cholesterol40 and may be due to direct alteration of the cell membrane. Given the very high concentrations required to produce this response, testosterone-induced vasodilation is very unlikely to have any physiological relevance.As shown previously,22 This investigation addressed the influence of androgens/AR on neointimal lesion formation using models of denuding (wire) and non-denuding (ligation)41 injury. Wire injury denudes the endothelium and produces lesions formed predominantly from circulating bone marrow-derived progenitor cells. On the other hand, femoral artery ligation does not introduce direct endothelial damage. The disturbed blood flow and altered shear stress may induce endothelial dysfunction and stimulate neointima formation by migration and proliferation of media-derived mural smooth muscle cells.42 Increased lesion volume in castrated mice following wire-injury, but not following ligation, is consistent with evidence that the influence of androgens is dependent on the type of lesion.20 Endothelial denudation is one of the major differences between the wire injury and ligation injury models. Androgens promote endothelial proliferation and migration in vitro43 and improve angiogenesis in vivo following ischaemic injury.45 It is possible, therefore, that endothelial regeneration following wire injury was impaired by systemic androgen deprivation, thus favouring formation of lesions. In addition, blocking AR signalling improves self-renewal and migration of bone marrow-derived stem cells46 which may promote lesion formation following wire injury. The well-recognized androgen/AR mediated immune-suppression47 may also modulate adventitial inflammation and subsequent neointima formation and could explain the increased lesion volume following castration.Evidence for androgen-mediated inhibition of vascular neointimal lesion formation under normolipidemic conditions is less conclusive than for inhibition of atherosclerosis.44 Our VE-ARKO mice will be a useful tool to address the question whether endothelial AR truly regulates endothelial function or behaviour using endothelial-specific models.Following arterial injury, vascular AR ablation had very little impact in neointima formation. Deletion of AR from the endothelium increased lesion volume following arterial ligation, but it was notable that no similar increase was detected in mice with double knockout of AR in EC and SMC. This may suggest that deletion of AR in the smooth muscle opposes the effect of deletion from the endothelium. However, given the variability of the data in the VE-ARKO following ligation, it seems more likely that this result is an anomaly, and that, as proposed, AR in the vascular EC and SMC does not contribute to regulation of neointimal lesion formation. Excluding a role for the EC and SMC AR suggests that any influence of androgens on neointimal lesion formation may be mediated by AR-independent mechanisms, or by conversion to oestrogen by aromatization either systemically or locally in the vascular wall, or by AR expressed in other cell types in the vascular wall. It should be noted, however, that AR expression in the vasculature is not restricted to EC and SMC. In healthy arteries, cells bearing strong AR expression were shown to be present in the adventitia, while the population of AR-positive adventitial cells was increased in injured arteries. It was notable that expression of AR in the adventitia of injured arteries was dramatically reduced following castration. However, the identity of these AR positive cells and their pathophysiological function require further investigation. Interestingly, androgens were recently reported to increase ischaemia-induced angiogenesis, indicating a direct association between androgen and ECs.The use of vascular selective ARKO mice has shown that AR in the arterial wall have little role in regulating androgen-dependent neointimal lesion formation. These results suggest that any protective effects of androgens in atherosclerosis are likely to be mediated by conversion of testosterone to oestrogens, by effects on classical cardiovascular risk factors such as cholesterol, or by AR outside the vascular wall. Future investigations should try to determine whether androgens inhibit atherosclerosis through direct modulation of non-vascular AR or following conversion to oestrogens.Cardiovascular Research onlineSupplementary material is available at .British Heart Foundation Project Grant award to L.B.S., P.W.F.H., and M.A.D. (PG/11/72/29334), an MRC Programme Grant award to L.B.S. (G1100354), and the BHF Centre for Research Excellence. W.G.L. received a BHF CoRE Summer Studentship. I.M. received a Study-abroad Fellowship of the German National Academic Foundation. Funding to pay the Open Access publication charges for this article was provided by The British Heart Foundation.Funded by a


The purpose of this study was to investigate the expression of autophagy-related proteins in relation to androgen receptor (AR) status in estrogen receptor (ER)-negative breast cancers.We extracted 334 ER-negative breast cancer samples to construct tissue microarrays (TMAs), which were immunohistochemically stained for autophagy-related proteins  and for AR and HER-2.P<0.001), while the AR-positive group was associated with tumoral BNIP3 expression (P<0.001). Tumoral LC3A was most highly expressed in the AR-negative and HER-2 negative group, while stromal LC3A showed the highest expression in the AR-negative and HER-2-positive group. Tumoral BNIP3 and stromal BNIP3 were highest in the AR-positive and HER-2-negative group. In the AR-positive and HER-2-negative group, stromal p62 positivity was an independent factor that was statistically significant in its association with shorter disease-free survival (DFS) . Shorter DFS was associated with tumoral LC3A positivity  in the AR-negative and HER-2-positive group.There were 127 AR-positive cases and 207 AR-negative cases, and 140 HER-2-positive cases and 194 HER-2 negative cases. The AR-negative group was associated with tumoral LC3A expression (In ER-negative breast cancers, AR status was associated with expression of different types of autophagy-related proteins. Tumoral LC3A was most highly expressed in AR-negative breast cancers, while tumor BNIP3 was highest in AR-positive breast cancers. Autophagy is defined as the lysosomal degradation of cellular components within cells. There are three types of autophagy: microautophagy, chaperone-mediated autophagy and macroautophagy, which is the most commonly employed type. Autophagy removes dysfunctional or damaged cellular components while recycling cellular components that can be re-used, thereby playing an important homeostatic role within the cell The development and natural history of breast cancer is significantly influenced by the status of steroidal hormones, such as those of estrogen. It is common practice to evaluate the status of estrogen receptor (ER) and progesterone receptor (PR) in order to treat and prognosticate breast cancer. In addition to ER/PR, androgen receptor (AR) is another steroidal hormone that influences and is associated with breast cancers, but the relationship is still not clearly understood. In general, androgen receptors are expressed in 70% of all breast cancers The purpose of this study was to investigate how autophagy-related proteins are expressed in relation to AR status in ER-negative breast cancers and to determine the corresponding clinical implications.+) membranous staining was observed, whereas cases with 0 to 1+were regarded as negative. The cases showing 2+ HER-2 expression were evaluated for HER-2 amplification by fluorescent in situ hybridization (FISH).Formalin-fixed paraffin-embedded tissue samples of patients diagnosed with invasive ductal carcinoma, no specific type, from January 2005 to December 2012 at Severance Hospital were included in this study. The study was approved by the Institutional Review Board (IRB) of Severance Hospital. IRB board waived the need for written informed consent. Those cases that had undergone pre-operative chemotherapy were excluded. Information on ER, AR and HER-2 status was collected from pathology reports. A cut-off value of 1% or more positively stained nuclei was used to define ER and AR positivity All cases were retrospectively reviewed by a breast pathologist (Koo JS), in which histological evaluation was based on hematoxylin and eosin (H&E)–stained slides. The histological grade was assessed using the Nottingham grading system After reviewing H&E–stained slides, the most suitable formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples were retrospectively selected. The most representative tumor region on the FFPE sample was then marked and a 3-mm tissue core sample was extracted using a punch machine and planted onto a 6×5 recipient block. A total of 2 tissue cores were taken for all samples in this TMA construction.The antibodies used for immunohistochemistry in this study are shown in Interpretations of IHC stains were standardized as the proportion of stained cells multiplied by the intensity of the immunohistochemical staining. The proportion of stained cells was scored with a system ranging from 0 to 2, defined as follows: 0 represented a negative result, 1 represented a section in which less than 30% of cells were positively stained, and 2 represented a section in which more than 30% of cells were positively stained. Immunostaining intensity was scored with a system ranging from 0 to 3, defined as follows: 0 represented a negative result, 1 represented weak, 2 represented moderate, and 3 represented strong. The number obtained after the multiplication of stained cell proportion by immunostaining intensity resulted in the overall interpretation score: 0–1 was defined as negative, 2–6 as positive t test and Fisher’s exact test were used for continuous and categorical variables, respectively. To analyze data with multiple comparisons, a corrected P-value with application of the Bonferroni method for multiple comparisons was used. Statistical significance was assumed when P<0.05. Kaplan-Meier survival curves and log-rank statistics were employed to evaluate time to tumor metastasis and time to survival. Multivariate regression analysis was performed using a Cox proportional hazards model.Data were statistically processed using SPSS for Windows version 12.0 . Student’s P = 0.003, and P<0.001, respectively). While within the AR negative group, HER-2positive group was shown to be associated with older age at diagnosis and HER-2 negative group with higher Ki-67 LI/HER-2 (–) group, 74 cases in the AR (+)/HER-2 (+) group, 66 cases in the AR (–)/HER-2 (+) group and 141 cases in the AR (–)/HER-2 (–) group. When ER negative cancer was divided into groups according to AR and HER-2 status, these groups exhibited a noticeable difference in age at diagnosis and Ki-67 expression levels. AR negative group was associated with older age and higher Ki-67 LI. Stromal LC3A was highest in the AR (–)/HER-2 (+) group, and lowest in the AR (–)/HER-2 (–) group (P<0.001). Tumoral BNIP3 and stromal BNIP3 had the highest expression in the AR (+)/HER-2 (–) group and lowest in the AR (–)/HER-2 (–) group /HER-2 (–) group, and lowest in the AR (+)/HER-2 (+) group (P<0.001), while tumoral BNIP3 was most highly expressed in the AR-positive group , higher Ki-67 LI (P<0.001), AR negativity (P<0.001), and HER-2 negativity (P<0.001), while stromal LC3A positivity showed a significant relationship with HER-2 positivity (P<0.001). Tumoral LC3B positivity was associated with higher histologic grade (P = 0.016), while tumoral BNIP3 positivity was significantly associated with AR positivity (P<0.001) and HER-2 positivity (P<0.001).Autophagy-related protein expression according to clinicopathologic parameters was analyzed in ER-negative breast cancer . TumoralP = 0.032) as well as higher T stage /HER-2 (+) group was the only group that showed a significant association between tumoral p62 positive and higher histologic grade (P = 0.016), higher Ki-67 LI (P = 0.001), AR negativity (P<0.001) and HER-2 negativity (P<0.001). Stromal LC3A positivity was associated with HER-2 positivity (P<0.001) and tumoral BNIP3 positivity was associated with HER-2 positivity (P<0.001).In each of the groups, autophagy-related protein expression with clinicopathologic parameters was assessed according to AR status . Within P = 0.006), while in the AR (+)/HER-2 (+) group, shorter DFS was associated with tumoral beclin-1 negativity (P = 0.029). In the AR (–)/HER-2 (+) group, shorter DFS and shorter overall survival were significantly associated with tumoral LC3A positivity /HER-2 (–) group, stromal p62 positivity was an independent factor significantly associated with shorter DFS /AR (–)/HER-2 (+) group can be presumed to be mostly the HER-2 type, and the ER (–)/AR (–)/HER-2 (–) group is thought to be the basal-like/triple negative types. Because HER-2 type and basal-like/triple negative type are both distinct clinicopathologic entities Our study observed cytoplasmic expression of LC3A, LC3B and BNIP3 and nuclear expression in beclin-1 and p62. Previous reports noted that different expression patterns of LC3A had resulted in different biologic behaviors of tumors; in tumors with diffuse cytoplasmic or perinuclear LC3A expression had an association with ER and PR positivity, whereas those tumors with stone-like pattern of LC3A expression was associated with ER and PR negativity and a worse prognosis Although there was no association between autophagy-related protein expression and breast cancer prognosis in ER-negative breast cancer, in the AR (+)/HER-2 (–) group, stromal p62 positivity was an independent factor associated with shorter DFS, while in the AR (–)/HER-2 (+) group, tumoral LC3A positivity was an independent factor for shorter DFS. Previous studies have revealed that LC3A expression in ovarian clear cell carcinoma In conclusion, there was a significant difference in autophagy-related protein expression according to AR status in ER-negative breast cancer. Tumoral LC3A expression was higher in AR-negative breast cancer, while tumoral BNIP3 was higher in AR-positive breast cancer.


NPR1-dependent salicylic acid signalling controls sodium entry into the roots while preventing potassium loss through depolarization-activated outward-rectifying potassium and ROS-activated non-selective cation channels during salt and oxidative stresses. Arabidopsis SA signalling mutants, namely npr1-5 (non-expresser of pathogenesis related gene1), which lacks NPR1-dependent SA signalling, and nudt7 (nudix hydrolase7), which has both constitutively expressed NPR1-dependent and NPR1-independent SA signalling pathways, were compared with the wild type (Col-0) during salt or oxidative stresses. Growth and viability staining showed that, compared with wild type, the npr1-5 mutant was sensitive to either salt or oxidative stress, whereas the nudt7 mutant was tolerant. Acute salt stress caused the strongest membrane potential depolarization, highest sodium and proton influx, and potassium loss from npr1-5 roots in comparison with the wild type and nudt7 mutant. Though salt stress-induced hydrogen peroxide production was lowest in the npr1-5 mutant, the reactive oxygen species (ROS) stress  caused a higher potassium loss from the roots of the npr1-5 mutant than the wild type and nudt7 mutant. Long-term salt exposure resulted in the highest sodium and the lowest potassium concentration in the shoots of npr1-5 mutant in comparison with the wild type and nudt7 mutant. The above results demonstrate that NPR1-dependent SA signalling is pivotal to (i) controlling Na+ entry into the root tissue and its subsequent long-distance transport into the shoot, and (ii) preventing a potassium loss through depolarization-activated outward-rectifying potassium and ROS-activated non-selective cation channels. In conclusion, NPR1-dependent SA signalling is central to the salt and oxidative stress tolerance in Arabidopsis.The role of endogenous salicylic acid (SA) signalling cascades in plant responses to salt and oxidative stresses is unclear.  Soil salinity is one of the major abiotic stresses that threaten sustainable food production worldwide. About 831 million ha of land is affected by natural salinization worldwide . In addiArabidopsis sid2 (SA induction deficient 2) mutant defective in the expression of the isochorismate synthase (ICS1) gene is hypersensitive to salt stress  activity, allowing for the possibility that SA signalling might be turned on before NahG converts SA into catechol  hydrolases (AtNUDTs) that hydrolyse nucleoside derivatives  was found to have three- to four-fold higher concentration of SA than the wild type under control growth conditions  protein was identified as one of these (Arabidopsis NPR1 knockout mutant (npr1-1) accumulated SA upon salt stress and showed enhanced salt tolerance (Arabidopsis double mutant (npr3npr4) failed to undergo programmed cell death (nudt7 mutant (which has both constitutively expressed NPR1-independent and NPR1-dependent SA-mediated pathways) with a NPR1 knockout mutant (without SA-mediated NPR1-dependent pathway) will pave the way for characterizing a SA-mediated defence response against salt stress.Recently, an 2−), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hydroxyl radical (.OH) in plants  can induce K+ loss via ROS-activated channels and trigger programmed cell death during salt stress  namely superoxide , is a NPR1-knockout mutant without the SA-mediated NPR1-dependent defence response  retain K+ both in the roots and shoots by controlling K+ loss through depolarization-activated outward-rectifying K+ channels (KOR) and ROS-activated non-selective cation channels (NSCC).The present study hypothesized that the elevated SA concentration may mediate adaptive responses against salt and oxidative stresses through both NPR1-independent and NPR1-dependent pathways. This hypothesis was tested by characterizing roots of Arabidopsis thaliana L. wild type (Col-0) and mutant seeds of loss-of-function of NPR1 gene npr1-5  and NUDT7 gene nudt7  were obtained from the Arabidopsis Biological Resource Centre (http://www.Arabidopsis.org/abrc/). Arabidopsis seeds were surface sterilized with 1 % v/v sodium hypochlorite  plus 0.01 % v/v Triton (wetting agent) for 10min followed by at least three rinses with sterile deionized water.Seeds of nudt7, and npr1-5) were sown on the surface of 90-mm Petri dishes containing solid 0.35 % w/v phytogel, full strength Murashige and Skoog medium , 1% w/v sucrose, and various concentrations of NaCl . Media pH was adjusted to 5.7 by adding either KOH or HCl. The Petri dishes were divided into three equal parts to accommodate three genotypes per dish , and the germination percentage was assessed after 7 d in the growth chamber. These experiments were repeated at least twice, with four replicates each time.For genotype comparison, 15 surface-sterilized seeds of each genotype  and weighed. Shoot water content (%) was calculated as the difference between fresh and dry weight.At the end of the experiment, plants were harvested and thoroughly rinsed with ice-cold 0.5mM CaSO2 at pH 5.7  technique was used to measure net fluxes of H+, K+, and Na+. The principles and methods of this MIFETM technique can be found in The Microelectrode Ion Flux Estimation  for at least 30min before commencing MIFE measurements  for 30min at 30 °C. Following DCF-DA incubation, the amount of H2O2 produced in roots was assessed by visualizing fluorescence intensity using a confocal microscope . The Argon, visible laser power was set at 20%. Given that the H2O2 fluorescence intensity at 4h was stronger than at 24h time point, two different settings  were used to resolve the signal. The acousto-optic tuneable filter (AOTF-488) was set at 10 % and 40 %, and the hybrid detector (HyD) gain was set at 19 and 120 for 4-h and 24-h time points, respectively. The software Leica Application Suite Advanced Fluorescence  used to acquire images, and ImageJ  was used to calculate the mean fluorescence intensity.The HP≤0.05 level.Data are reported as means±SEM. Statistical significance of mean values was determined using the standard LSD test at Arabidopsis genotypes tested . This decline was smallest in nudt7 plants, followed by the wild type, and then by npr1-5 . Furthermore, under control conditions , the fresh  were slightly lower in npr1-5 plants than the wild type and nudt7, but the difference was not statistically significant. At 150mM NaCl, salt-sensitive npr1-5 had fewer radicles emerging than nudt7 and the wild type  showed green fluorescence even after 12h, suggesting the control roots were viable and healthy in our experimental solutions  and imply that roots of npr1-5 were sensitive to salt stress, whereas nudt7 was salt-tolerant.An hour of salt stress severely affected the viability of ion zone . However7 mutant . ProlongArabidopsis roots , whereas nudt7 showed about a 3-fold smaller peak K+ efflux  and 6-fold (mature zone) higher in salt-sensitive npr1-5 than salt-tolerant nudt7 mutant  and Col-0  at both the elongation and mature root zones was highest in the npr1-5 mutant followed by Col-0 and was least in nudt7  throughout the measurement period in npr1-5 and the wild type, while hovering around zero in nudt7  membrane depolarization in all three Arabidopsis genotypes tested. The time-course of membrane potential changes (+ (+ flux (+ influx and K+ efflux (the magnitude of the former being greater than that of the latter) , whereas salt-tolerant nudt7 showed the least membrane depolarization (to –30±1 mV)   and 5. Ilication . Among t30±1 mV) . A ≈25mVt period .In vivo imaging of H2O2 production in root tissue was done 4h and 24h after 100mM NaCl addition  to acquire images for each time point to avoid oversaturation and photobleaching. Among the genotypes, mutant npr1-5 with SA signalling blockage had lower capacity to increase H2O2 production under salt stress, whereas nudt7 mutant showed sustained elevation in H2O2 production under salt stress at both time points.addition . The sal+ concentration and a decrease in the shoot K+ concentration in all three Arabidopsis genotypes tested  for 1h  for 1h . Fluorespe Col-0 . No dama7 mutant . Furthersymptoms . With reo npr1-5 . The dam5 . The K+ flux gradually recovered after reaching the peak, although it remained negative for the treatment duration in all three Arabidopsis genotypes  is similar for different genotypes exposed to either concentration of H2O2 (npr1-5 mutant showed significantly higher (4- to 5-fold) H+ influx compared with the other two genotypes (nudt7 and Col-0) in either 1 or 10mM H2O2  restrict the initial entry of Na+ ions into the root tissue, (ii) excrete Na+ from root tissue back into the rhizosphere, (iii) sequester Na+ inside the root vacuoles, and (iv) reduce the long-distance transport of Na+ into the shoots  and viability of root cells  activity in the plasma membrane , and it was shown that exogenous H2O2 application increased the stability of SOS1 in a rapid (within 30min) concentration-dependent manner   exacerbate Kll death . Hence, s tested . However5 mutant , suggestArabidopsis, comparison of the depolarization-activated KOR knock-out mutant gork1-1 with rbhoD (a mutant lacking ROS production via NADPH oxidase) during acute 100mM NaCl stress revealed that 3/4 of K+ loss were mediated by depolarization-activated KOR and the remaining 1/4 through H2O2-activated channels  throughout the measurement period  compared with up to 10mM H2O2. Among the genotypes, the npr1-5 mutant showed a higher K+ loss than the wild type and nudt7 mutant under hydroxyl radical and hydrogen peroxide treatments , the K+ efflux of nudt7 mutant did not differ from the wild type, and was lower than in the npr1-5 mutant  NPR1-mediated SA signalling is pivotal for H2O2 production during salt stress, and also for decreasing K+ loss through the NSCC and KOR channels activated by hydrogen peroxide and hydroxyl radicals, and (ii) the nudt7 mutant shows no response to hydrogen peroxide and is tolerant to hydroxyl radicals.An increase in the production of superoxide (channels  showed teatments , suggest7 mutant . The nud was not  suggesti5 mutant . This sunpr1-5 mutant lacking the NPR1-dependent SA signalling was unable to control both the entry of Na+ into roots and its long-distance transport into the shoot, and to prevent K+ loss via depolarization-activated KOR and the ROS-activated NSCC channels during salt stress. As a result, the npr1-5 mutant was sensitive to salt stress. On the other hand, the constitutive expression of NPR1-dependent SA signalling enhanced the salt tolerance of a nudt7 mutant by controlling Na+ entry into the root tissue and subsequent transport to the shoot, as well as minimizing K+ loss during salt stress. In conclusion, NPR1-dependent SA signalling is a crucial component of salt and oxidative stress tolerance in Arabidopsis.In summary, an JXB onlineSupplementary data are available at Figure S1. Effect of salt stress on dry weight and water content of Arabidopsis thaliana seedlings grown in the full-strength MS medium with 2% w/v phytogel for two weeks.


Most acute stroke patients with disabilities do not receive recommended rehabilitation following discharge to the community. Functional and social barriers are common reasons for non-adherence to post-discharge rehabilitation. Home rehabilitation is an alternative to centre-based rehabilitation but is costlier. Tele-rehabilitation is a possible solution, allowing for remote supervision of rehabilitation and eliminating access barriers. The objective of the Singapore Tele-technology Aided Rehabilitation in Stroke (STARS) trial is to determine if a novel tele-rehabilitation intervention for the first three months after stroke admission improves functional recovery compared to usual care.This is a single blind , parallel, two-arm randomised controlled trial study design involving 100 recent stroke patients. The inclusion criteria are age ≥40 years, having caregiver support and recent stroke defined as stroke diagnosis within 4 weeks. Consenting participants will be randomized with varying block size of 4 or 6 assuming a 1:1 treatment allocation with the participating centre as the stratification factor. The baseline assessment will be done within 4 weeks of stroke onset, followed by follow-up assessments at 3 and 6 months. The tele-rehabilitation intervention lasts for 3 months and includes exercise 5-days-a-week using an iPad-based system that allows recording of daily exercise with video and sensor data and weekly video-conferencing with tele-therapists after data review. Those allocated to the control group will receive usual care. The primary outcome measure is improvement in life task’s social activity participation at three months as measured by the disability component of the Jette Late Life Functional and Disability Instrument (LLFDI). Secondary outcome variables consist of gait speed  and endurance , performance of basic activities of daily living (Shah-modified Barthel Index), balance confidence , patient self-reported health-related quality-of-life [Euro-QOL (EQ-5D)], health service utilization  and caregiver reported stress (Zarit Caregiver Burden Inventory).The goal of this trial is to provide evidence on the potential benefit and cost-effectiveness of this novel tele-rehabilitation programme which will guide health care decision-making and potentially improve performance of post-stroke community-based rehabilitation.NCT01905917).This trial protocol was registered under ClinicalTrials.gov on 18 July 2013 as study title “The Singapore Tele-technology Aided Rehabilitation in Stroke (STARS) Study” (ID: The STARS Study, ClinicalTrials.gov Identifier:  Supervised therapy is an important factor in determining post-stroke functional recovery. In a 1-year cohort study on local post-stroke patients in the community, only a third of the subjects undergo supervised rehabilitation one month after discharge into the community –5.In a mixed methods study on non-adherence with recommendations for continued rehabilitation, although 86 % acknowledged that rehabilitation was beneficial, only 40 % intended to continue with rehabilitation after discharge, citing functional and social (e.g. unwillingness to inconvenience their caregiver) barriers . Based oSolutions to addressing non-adherence to rehabilitation include the use of transportation and mobility systems such as stair-lifts for stroke survivors living in apartments on floors without access to an elevator. Paid handicap-fitted transport services to ferry stroke survivors to and back from outpatient rehabilitation centers can also overcome physical barriers. Providing paid caregivers for social support and subsidies for outpatient rehabilitation may reduce financial and social barriers. Nevertheless, all such services and/or equipment are expensive; moreover, caregivers are in short supply around the world .Given the heavy cost and inconvenience incurred in transporting stroke survivors to outpatient rehabilitation centers, home rehabilitation seems to be a viable option in the delivery of post-stroke recovery. Even through home rehabilitation was shown to be equally effective as inpatient rehabilitation , 7, it iRecent studies focus on the use of home-based tele-health technologies to provide support and guidance by rehabilitation therapists at a distance, representing a novel potential approach for addressing the access to post-acute rehabilitation care problem for stroke survivors . Tele-reGiven the current heterogeneity and room for improvements in solutions related to post-stroke rehabilitation, there is a need for well-designed intervention and rigorous trial to close the gap. The Singapore Tele-technology Aided Rehabilitation in Stroke (STARS) Study is a parallel, two-arm (intervention-control), single blind  randomised controlled trial to determine if a novel tele-rehabilitation intervention can improve functional recovery of post-stroke patients. For the tele-rehabilitation intervention arm of this study, the team developed small affordable lightweight wireless wearable sensors which can be strapped onto patients to allow real-time biofeedback and collection of sensor data from the sensors for subsequent transmission to patient’s rehabilitation team wirelessly via the internet. The sensor data is automatically summarized in chart form for quick review by therapist to allow them to prescribe further rehabilitation exercises remotely. The system also automatically allows the patient to self-record videos of them exercising which will also be transmitted to the therapist (termed ‘tele-therapist’) for review where needed, and allow patients and their therapists to communicate with each other using video-conferencing. The study’s primary hypothesis is that this novel tele-rehabilitation intervention during the first three months after stroke admission will result in greater functional recovery compared to usual care three months after stroke admission, as determined by the Jette Late Life Functional and Disability Instrument (LLFDI). Secondary hypotheses are that tele-rehabilitation intervention during the first three months after stroke admission will result in greater functional recovery as determined by gait speed, 2-min walk test and Shah modified Barthel Index, less health service utilization, greater contact time with therapist, better balance, better quality of life, less caregiver burden and be more cost-effective compared to usual care three months after stroke admission. Process measures in the form of a diary will be used to assess improved adherence in rehabilitation. Outcomes will also be assessed again at six months post-stroke to determine whether any improvement in outcomes is sustained in both groups.All procedures conducted during this trial with human participants were carried out in compliance with institutional ethical standards. All research procedures were approved by an Institutional Review Board (IRB) at each participating site: Singapore General Hospital (SGH) from Centralised Institutional Review Board (CIRB Reference No. 2011/779/D), and Ang Mo Kio Thye Hua Kwan Hospital (AMKTHKH) from National University of Singapore (NUS) IRB (NUS IRB Reference Code 11–013) and accepted by the National Healthcare Group Domain Specific Review Board (DSRB). SGH is an acute hospital with an acute stroke unit and an inpatient rehabilitation unit, whereas AMKTHKH is an inpatient rehabilitation hospital.This is a parallel, two-arm (intervention-control), and single blind  randomised controlled trial of a novel home-based tele-rehabilitation intervention provided for 3 months after discharge for stroke admission compared with usual care. Prior to the study, a pilot study was done to test the procedures, interventions and assessments. Eligible participants are randomized to the intervention and control groups at baseline before discharge from the hospital. With regards to mean length of stay in hospital, Chen. et al. reported that patients admitted in local inpatient rehabilitation hospitals (e.g. AMKTHKH) for stroke stayed for 33 days . In anotParticipants are individuals with recent onset of ischemic or hemorrhagic stroke. For purposes of inclusion in this study, a stroke is defined, according to the World Health Organization definition, as “a rapid onset event of vascular origin reflecting a focal disturbance of cerebral function, excluding isolated impairments of higher function and persisting longer than 24 h ”. StrokeThe inclusion criteria for the trial are as follows: (1) age ≥ 40 years, (2) recent stroke , (3) able to sit unsupported for 30 s, (4) able to stand on the non-paretic leg for >4 s, (5) able to walk at least 2 m with maximum of 1 person assisting, (6) able to follow a 2-step command, (7) living in the community before stroke and expected to be discharged home, (8) has a caregiver when the patient is doing the exercises for safety reasons, as the tele-therapist is not present when the patient is doing the exercise and a caregiver is needed to catch the patient if he/she should fall.The exclusion criteria for the trial are as follows: (1) has a pacemaker in-situ , (2) unable to ambulate at least 45 meters (150 feet) prior to stroke, or intermittent claudication while walking less than 200 meters (656 feet), (3) serious cardiac conditions , (4) history of serious chronic obstructive pulmonary disease or oxygen dependence, (5) severe weight bearing pain, (6) pre-existing neurological disorders such as Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis or severe dementia, (7) history of major head trauma with severe residual deficits, (8) lower extremity amputation, (9) legal blindness or severe visual impairment, (10) severe uncontrolled psychiatric illness such as psychosis, schizophrenia or medication refractory depression (11) life expectancy less than three months, (12) severe arthritis or orthopedic problems that limit passive ranges of motion of lower extremity , (13) history of sustained alcoholism or drug abuse in the last six months, (14) hypertension with systolic blood pressure greater than 200 mmHg and diastolic blood pressure greater than 110 mmHg at rest, that cannot be medically controlled into the resting range of 180/100 mmHg.Participants will be recruited from the inpatient rehabilitation unit of an acute hospital (i.e. SGH) and an inpatient rehabilitation hospital (i.e. AMKTHKH). Each site is expected to recruit approximately 50 participants (25 intervention and 25 controls).Recruitment will be initiated while the stroke patient is still in the hospital. The research assistant will first screen the medical records for current admissions and medical histories including the list of diseases and conditions at the hospital sites. Once potential patients are identified, the research assistant will approach them and provide information about the study, both in written and verbal form, only after they have indicated their interests. The research assistant has to review the inclusion and exclusion criteria of patients that cannot be determined through medical records.After eligibility is confirmed, details of study will be explained to potential participant. Once participant agrees to participate, Informed consent is obtained, patient is enrolled and randomization only occurs after enrollment.The data collected from all participants at baseline are detailed in Table Informal caregivers will rate each item/symptom on the 11-item Centre for Epidemiologic Studies Depression (CES-D) scale as occurring none/rarely (score = 0), sometimes (score = 1) or often (score = 2) during the past week. Items scores will be summed to obtain a CES-D score (range: 0–18). A previous study from Singapore has reported that the 11-item CES-D has good internal consistency  and its factor structure matched with that reported in the original validation of the scale. In sensitivity analysis, the CES-D score, a continuous variable, is considered as the outcome.The Mini-Mental State Examination (MMSE) is the most commonly used screening tool for cognitive impairment and dementia worldwide. A number of studies have examined the accuracy of the MMSE in the detection of dementia. In Singapore, the localised MMSE version discriminated well between elderly with and without dementia  .Research assistants and tele-therapists will receive training provided by the study coordinator. The research assistants will be trained in assessing the eligibility of potential participants, obtaining informed consent from participants, randomization process, STARS tele-rehabilitation intervention protocol, the use of tele-rehabilitation system from the patient user interface, patient education on the use of the tele-rehabilitation system (including provision of written instructions to the participant), obtaining outcome measures for both intervention and control groups and documenting the measures. On the other hand, the tele-therapists will be trained in the use of tele-rehabilitation system from the tele-therapist user interface, progression protocols and documenting patients’ progress.Competency of the tele-therapists and research assistants in their respective responsibilities will be assessed by the study coordinator at the end of the training programme. 90 % competency prior to participating in the study must be attained. To avoid drift, maintenance of competency is important in a multi-site clinical trial. Re-assessment will be done by the study coordinator every 6 months during the study at both sites.ralloc package of Stata 11 .Randomization will be conducted after baseline assessment. The participants will be randomized into one of the two groups: tele-rehabilitation intervention and usual care based on equal allocation with randomly varying block sizes of four and six. As the study is conducted in two sites, stratified randomisation will be carried out assuming study site as the stratification factor. The randomisation list is generated using the The tele-rehabilitation intervention has three components: (a) the hardware, (b) the software and (c) the progressive rehabilitative exercises. Prior to the tele-rehabilitation intervention, the research assistant, who previously recruited the participant, will train the participant and their caregiver on the usage of the devices and sensors. The participant and their caregiver will learn how to wear the sensors, operate the hardware and perform all the rehabilitation exercises with the hardware. The training is usually done within 1–3 sessions, with each session being an hour long, until the participant and caregiver are competent in its use. These sessions are done before the discharge of participant and, if necessary, in their homes after discharge. To ensure that the participant/caregiver is able to use the devices and sensors, the participant/caregiver must pass a competency check list, such as switching on the iPad and applying the sensors. Additionally, a baseline visit is scheduled for set-up of tele-rehabilitation system in the participant’s house.The hardware component comprises of an iPad, two limb sensors, a heart rate and blood pressure monitoring set and chargers for these devices. A stable movable floor-mounted flexible iPad stand will be provided to allow easy positioning of iPad to video-record exercises performed by the patient. Engineering details on the tele-rehabilitation system infrastructure and the accuracy of the sensors have been published , 19. OnlThe limb sensor node is used in the system to determine the orientation of the limb being exercised, which is in turn sent to iPad to determine the number of repetitions and range of motion. Each sensor node consists of an ATMEGA328 microprocessor embedded on a printed circuit Board with three motion sensors and attached to a Bluetooth module. The three motion sensors attached are: ITG3200 (gyroscope) and LSM303DLHC (accelerometer and magnetometer). The Bluetooth module, which is a BLE112-A low energy single mode module produced by Bluegiga tech, is required for wireless communication with iPad.2C connection. The microprocessor will perform sensor fusion which involves the fusion of data to a 4-dimensional quaternion orientation vector using the algorithm devised by Madgwick [The data collected from these motion sensors are polled into ATMEGA328 microprocessor through a daisy-chained IMadgwick  to denotThe MySQL relational database management system will be used to store data in the system. The database is used to contain data regarding the patients and therapists such as log-in information. In addition, exercises selected and remotely prescribed by the therapist, and the sensor and video data on the exercises performed by the patients are also stored in the database.The user interface for the patient was designed to be simple and intuitive. A patient guide is provided within the application if the patient needs additional help on its usage. Three languages are provided: English, Chinese and Malay, to cater to the diverse ethnic groups in Singapore. Before the exercises, the iPad needs to be connected to the internet. A log-in is not required as the iPad’s unique identifier is registered to the patient before the initial set-up at the participant’s house Fig. . After sAdditionally, another application is created for the patients to practise and familiarize themselves with the exercises prescribed prior to the actual exercise regime. Videos of the prescribed exercises are provided in the application Fig. . When thThe user interface for the tele-therapist was designed for efficient display of information on exercises performed by patients. The application allows the tele-therapist to initiate a Facetime video call during a scheduled tele-consultation. On selecting the patient’s name, the tele-therapist will be directed to a page to remotely prescribe exercises and adjust the difficulty (progression) level and the minimum range of motion for each exercise. A summary of the rehabilitative exercises completed by the patient will be presented in graphs for easy interpretation of adherence to prescribed exercise and quality of exercises by the tele-therapist. This is in addition to the videos of patients performing the exercises. The tele-therapist can enter also clinical notes and parameters in the application to record a tele-consultation.A website was devised for the usage by both the research assistants and the study coordinator. The website allows research assistants to enter baseline data, primary and secondary outcome data, and tele-therapist to document patient’s clinical information during the assessment. Only the study coordinator is allowed to view all patients’ and therapists’ information and update the information in the event of data entry error. Any amendment of data entered will be recorded and date/time-stamped. In addition, the study coordinator can designate therapists and research assistants to the patients. In the occasion when an adverse event happens, the study coordinator will enter the details in the adverse events log in the application.The tele-rehabilitation intervention group will receive a standardized rehabilitation programme for three months, which was based on the rehabilitation programme for the control group in a randomized controlled trial on weight-supported locomotion by Duncan et al. . The typStroke patients in Singapore typically receive acute stroke care in an acute hospital, which usually has a dedicated stroke unit. Those who are totally dependent or fully independent in activities of daily living (ADL) usually do not receive post-stroke rehabilitation. Those who are mild to moderately dependent in ADL are usually referred for centre-based rehabilitation after discharge from acute hospital. For those who are moderately to severely dependent in ADL, they usually receive inpatient rehabilitation for 4–6 weeks at inpatient rehabilitation hospitals after discharge from the acute stroke unit. These moderately to severely ADL dependent patients usually would have recovered to become mild to moderately ADL dependent during their inpatient rehabilitation hospitals stay, and hence would be referred for centre-based rehabilitation after being discharged home from inpatient rehabilitation hospitals. In acute hospitals, rehabilitation is usually started soon after stabilization of stroke and would be provided once a day for a total of 1–2 h a day by a physiotherapist and occupational therapist. In inpatient rehabilitation hospitals, rehabilitation is continued upon admission and would be provided twice a day for a total of 2–3 h a day by a physiotherapist and occupational therapist. After discharge, it usually takes 1–2 weeks for centre-based rehabilitation to be initiated and this is usually provided 1–2 times a week for an hour each time.Home rehabilitation in Singapore is relatively under-developed and generally unavailable and unaffordable for the majority of stroke patients. Government subsidies for acute hospital and inpatient rehabilitation hospital stays are available, and government subsidies for centre-based rehabilitation are also available. However, government subsidies for home rehabilitation, previously unavailable, were only made available mid-way through this trial in July 2014.It should be noted that for ethical reasons, the intervention group is allowed to supplement their tele-rehabilitation sessions with centre-based rehabilitation if they desire. To control for this at the data analysis stage, we will capture data on the total number of hours of use of centre-based and home rehabilitation for both intervention and control groups throughout the six months from study recruitment.In this study, exercise done by the participant refers to those that are prescribed by the therapist. Both unsupervised  and supervised exercise  that participants have performed are tracked during the trial for both intervention and control subjects. Adherence to supervised and unsupervised therapy will be recorded by the subject in a diary to record the number of minutes subject spent each day in therapy with a therapist (excluding time with the tele-therapist) and the number of minutes subject spent each day doing their prescribed rehabilitation exercises. The tele-therapist will check that the intervention subject is entering these data during tele-consultations. For the control  subjects, the Study Coordinator will call the subject every 4 weeks to check that the intervention subject is entering these data as well.For intervention participants, feedback about their rehabilitation progress is given via real-time and Facetime. For real-time biofeedback, visual and audio feedback is given to the participant through the iPad for every repetition that exceed the target angle when the participant is performing the rehabilitation exercises. For Facetime, the tele-therapist will have a face-to-face consultation with the participant via video call once a week. They will evaluate the participant based on the data collected from the sensors (which capture the progress of the participant when he/she performs the rehabilitative exercises), and the exercises which the participant may be asked to perform during the video call.The primary outcome variable and the secondary outcome variables chosen for the study are based on the notion that patients undergoing rehabilitation are more concerned with their improvement in activities and participation instead of reducing their impairment . AlthougFunctional ability may be quantified using performance based measures or self-report. Self-report measures remain the most commonly used instruments in many studies involving older adults because of their low cost and practicality . DisabilThere will be 7 secondary outcomes for this trial: (1) gait speed, (2) two-minute walk distance, (3) functional status, (4) balance confidence, (5) patient quality of life, (6) health service utilization and (7) caregiver burden.Gait speed is related to the severity of impairment in the home and the community and has been used in many studies . Gait spIn addition to the gait speed, the distance a person can walk and the amount of daily walking that a person is able and willing to do are strong indicators of his or her health and condition . Hence, Shah-modified BI will be used to assess and compare the improvements in the performance of ADL between the intervention and control groups. The Shah-modified BI is considered to be a valid measurement of ADL, as it is an empirically derived scale with proven inter-observer and test-retest reliability and validity which measures the patient’s functional ability . It invoThe 16-items Activities-Specific Balance (ABC) Scale will be used to assess self-perceived efficacy in maintaining balance while performing a number of activities common in community-dwelling older adults such as bending, reaching, and walking both inside and outside the home . This meWe will use the Euro-QOL (EQ-5D) to measure patients’ quality of life. The EQ-5D is a standardized health-related quality of life questionnaire applicable to a wide range of health conditions and treatments, and it provides a simple descriptive profile and a single index value for health status which can be used in the clinical and economic evaluation of health care . It is dSingapore Stroke Study Health Service Utilization Form will be used to perform cost-effectiveness analysis in both tele-rehabilitation and usual care. This form is chosen because it has been contextualized to the local Singaporean context. It comprises of 11-items and for each item, utilisation of a particular service is asked. If the answer is “Yes”, the duration and the expenditure of its usage will be asked. Quality-of-life data based on EQ-5D instrument will be combined with resource utilization and cost data to develop incremental cost-effectiveness ratios that will allow investigators to evaluate the cost-effectiveness of tele-rehabilitation compared to usual care .The Zarit Burden Interview (ZBI), which provides a comprehensive assessment of caregiver burden, has been validated in many culturally and ethnically different populations. In this study, the 12-item version of the ZBI will be used with response options ranging from never =1 to nearly always = 5 . The totAll patient information to and from the server will be encrypted via Hypertext Transfer Protocol Secure (HTTPS) which ensures no external parties are able to view it during transfer, a protocol similar to those used by online banking systems. Data access from server is also authenticated by a passphrase which is supplied from the therapist application, ensuring only the patient’s therapist will be able to request and review patient data. Regarding security on the server side, we will be using the Apache server running on a Unix-based operating system and have taken recommended measures to minimise vulnerabilities from hacking.Only an administrator  will be able to view all patients (both groups) and therapists (intervention group only) data, and update the system in an event of data entry error. The administrator will have privileges to add patients & therapists to the database, pair patients to therapists as well as to update and view all patient data. The independent assessor  will be able to input outcome data into the system for both groups at 3 month and 6 month. However, independent assessor will not have access to any participant information other than the latter’s initials and unique study number. The biostatistician will only have read-only access to all the patients’ data for research purposes. However, the biostatistician will not be able to manipulate or distort the data. The biostatistician can also download data for research purposes in the form of an Excel file. All these measures have been put in place to ensure that there is no leakage of patients’ information and only authorized individuals have full access to the secured data.Chumbler et al’s paper had similar tele-rehabilitation intervention at 0, 3 and 6 months, and the same primary outcome measure, the Difficulty dimension of the Disability component of the Jette LLFDI . Even thData entered will be checked for accuracy at least twice by at least two independent assessors. To understand the structure of missing data and to reduce bias from uncertainty of missing data, multiple imputations will be performed using the Markov Chain Monte Carlo method. All scale-based measures used in this study will be screened via an internal consistency test  before creating a summative score. Furthermore, rigorous validity tests will be performed in two ways: person-centered tests  and variable-centered tests .t-test and Analysis of Covariance (ANCOVA) will be performed on all continuous outcome measures  between tele-rehabilitation vs. usual care group if outcomes are normally distributed. Mann–Whitney U test will be performed on data with skewed distribution to test differences between treatment groups. Chi-square test will be performed on categorical variables. Confounders are age, gender, ethnicity and marital status. Multiple linear regressions will be used to adjust for differences in confounders between intervention and control groups for continuous outcome variables. Ordered logistic regressions will be used to adjust for differences between intervention and control groups for continuous categorical variables. For these multivariate linear and logistic regressions, we will use survey analysis processes with robust standard errors which accounts for nesting effects within patient data as a result of clustering by different recruitment centers. For all statistical analyses, the level of significance (α) will be taken at 0.05. Analyses will be based on the intention-to-treat approach. We will use STATA version 12 for statistical analysis and significance level is set at P < 0.05.For descriptive statistics, independent two-sample Cost effectiveness analyses from both the health system perspective will be performed using the standard quality of life measure EQ-5D and measures of health service utilization and cost over the study period. Quality adjusted life years (QALYs) will be computed for each individual using the EQ-5D score. Incremental cost-effectiveness ratio (ICER) will subsequently be computed to assess incremental costs per QALY gained between treatment and control group. To determine confidence intervals for ICER, a non-parametric bootstrapping method with 1000 replications of the data will be performed. When dominance is observed, full cost analyses will be conducted to determine mean savings per patient.Adverse events will be carefully monitored during the STARS trial. The teams at the clinical intervention sites will monitor and report all minor or serious adverse events that occur in any participant from the point of enrollment through the 6-month follow-up assessment. Investigators will report to IRB of any adverse events or unanticipated problems, serious or continuing noncompliance, and suspensions or termination.An adverse event is defined as any untoward or unfavorable medical occurrence in a human subject, including any abnormal signs, symptoms, or diseases, temporally associated with the subject’s participation in the research, whether or not considered related to the subject’s participation in the research. All adverse events regardless of the seriousness and its relation to the study will be reported to the principal investigator. The research member who observed the adverse event will report the following: the date of onset, action taken with respect to study procedures, corrective treatment/therapy given, outcome and his/her opinion as to whether the adverse event is related to the intervention exercises.In general, an unanticipated problem includes any incident, experience, or outcome that meet all the following criteria: (1) unexpected in terms of nature, severity, or frequency given the research procedures that are described in the protocol-related documents and the characteristics of the subject population being studied, (2) related or possibly related to participation in the research and (3) suggestions that the research places subjects or others at a greater risk of harm  than previously known or recognized and requires changes to the research protocol or informed consent document or other corrective actions in order to protect the safety, welfare, or rights of subjects or others. Even though the IRB does not require unanticipated problems to be reported, the principal investigator would be informed of them.Adverse events which will be monitored include unplanned re-hospitalization, emergency department visit, death, falls and fractures, recurrent stroke and myocardial infarction. The principal investigator will determine if the adverse event or unanticipated problem has to be reported to IRB such that any corrective changes to the research protocol, informed consent document or other corrective actions are needed. Reporting of any adverse event or unanticipated problem would be done within five working days from the day the principal investigator is aware of the issue.The STARS Study is a multi-site randomized controlled trial designed to determine the impact of video-conferencing and the use of wearable monitoring devices compared to usual care on the functional recovery of stroke survivors. Stroke results in disabling limitations for patients and supervised rehabilitation can improve functional recovery, but only a minority is undergoing supervised rehabilitation one month after discharge into the community . This tr


What makes a protein a good immune regulator? We can make some intuitive guesses based on the characteristics of an immune response. Because immunity is not only dangerous to the pathogen but also potentially harmful to self, its regulators should only be active when the threat of infection is imminent, and their activities should be spatially and temporally appropriate to the infection pressure. In-depth studies of the plant immune regulator non-expressor of pathogenesis-related (PR) genes 1 (NPR1) have revealed the molecular mechanisms contributing to these characteristics and also discovered some surprising connections that the immune system has to normal plant physiological processes to minimize the fitness costs. Because NPR1 transcription is not dramatically affected by pathogen challenge . Bo. Bo55/SeS55/59A mutant in which sumoylation is no longer inhibited by phosphorylation at this site, defense response is constitutively elevated . Th. Th35]. 3a/cul3b . TherefoBecause NPR1 is not only a positive regulator of SAR but also an inhibitor of ETI, it is not difficult to imagine that NPR1 degradation plays a role in ETI induction. Because this PCD-associated immune response is tightly restricted to the site of infection, there is likely a threshold at which NPR1 is degraded to relieve its repression on cell death. Fu et al. proposed that this is achieved by the SA concentration gradient radiating from the site of pathogen challenge , combine-elements in the PR-1 promoter and their activities [Posttranslational modifications of the master immune regulator, NPR1, play a major role in the signaling decision for cell death or cell survival during pathogen challenge. However, there are still some very basic questions that remain to be answered: How is the transcription cofactor activity of NPR1 orchestrated, and what genes does it control during specific immune responses? Modification of NPR1 affects its ability to interact with TFs which may affect their associations with both positive and negative cistivities . Anothertivities –41. SincAlthough phosphorylation has been shown to regulate multiple NPR1 functions, the specific kinases responsible for some key phosphorylations remain elusive. To date, two members of the SnRK family of kinases have been shown to interact with and phosphorylate NPR1 ,42, both521 and Cys529) in the C-terminus and that this activity is crucial for the SA-induced transactivation activity of NPR1 [Several methods have been used to demonstrate that NPR1 can directly bind SA ,44. One