In a Perspective, Linda Clare proposes using a cognitive rehabilitation approach for people living with dementia. Awareness of the need to improve accessibility of services and opportunities for people with disabilities is growing, but people with “hidden” disabilities such as dementia can be excluded from these developments. Conceptualizing dementia in terms of social disability highlights the way in which symptoms such as memory problems—and the secondary effects of these, such as loss of confidence or negative reactions from others—affect the possibility of engaging in activities and participating in society . It alsoThe experience of disability confers a right to rehabilitation for people living with dementia. The United Nations Convention on the Rights of Persons with Disabilities outlines the right of people with disability to be able to attain and maintain maximum independence, with the assistance of comprehensive rehabilitation services (Article 26(1)) . We are A rehabilitation-focused service would be organized around key principles of enabling people to function optimally in the context of their intrinsic capacity and current health state. This means ensuring that people are as independent as they wish to be, have as much control as possible over daily life, have opportunities to engage in meaningful roles and activities, and are able to integrate the changes they experience into a coherent and enduring sense of identity. The rehabilitation philosophy is genuinely person centred  and reflIn cognitive rehabilitation, these principles are applied to enable people with dementia to maintain or optimize functioning. The term “cognitive” is perhaps misleading, as cognitive rehabilitation does not set out to train or improve cognition but uses a goal-oriented approach to facilitate improved management of functional disability. Potential targets include everyday functioning, activities of daily living, self-care, language and communication, social interaction, and the effects of dementia-related physical disability. Cognitive rehabilitation therapists work collaboratively with each individual to formulate meaningful goals that are realistic and potentially achievable. They evaluate the person’s strengths and the resources needed for goal attainment, identify areas of mismatch, and collaboratively develop a plan to support goal attainment or address the identified need using evidence-based methods. These might involve new learning, relearning, use of compensatory strategies, or a combination of these. The person’s intrinsic capacity may be augmented by additional resources such as assistive technology. Therapists also provide important psychological support as people confront the emotional impact of functional disability.Principles of rehabilitation can be flexibly applied to address different types of need at various stages of dementia. For example, a person in the early stages of dementia may want to learn to use email to keep in contact with friends, develop strategies to feel confident enough to go out alone, or be able to cook a meal without getting distracted, while for someone with more advanced dementia, the focus may be on maintaining the ability to dress herself, managing difficulties with swallowing, or enabling participation in an enjoyable activity. Each individual might have several episodes of rehabilitation support over time as needs and goals change or as particular circumstances arise, such as a return home after hospitalization. Cognitive rehabilitation is usually carried out in the setting where the person lives or undertakes activities, to ensure direct relevance, and carers and families, when available and willing, are fully involved and appropriately supported. There is a small but growing evidence base demonstrating that cognitive rehabilitation is effective in supporting everyday functioning, reducing disability, and delaying institutionalization –15.Many different types of nonpharmacological intervention for people with dementia have been described. Most of these are not cognitive rehabilitation; it is important that this term is understood correctly and is not applied to interventions that do not warrant it. Some approaches, however, are directly complementary to cognitive rehabilitation. These either address related aims  or address problems that negatively affect functioning and participation . Also directly relevant is support for family carers. Other intervention approaches focusing on providing pleasurable and meaningful activity or encouraging social contact can complement rehabilitation by creating opportunities to enjoy positive experiences and relationships. Within a rehabilitation-focused service, an intervention pathway would include these approaches where there is evidence that they offer benefits. Less likely to be recommended in the context of a rehabilitation model are nonpharmacological interventions that address symptoms in isolation or out of context so that gains, if any, are unlikely to transfer to daily life. People with dementia should also have full access to specialist physical rehabilitation where needed following injury or illness, as well as any other appropriate medical treatment .A rehabilitation-based model of positive support could potentially be introduced in part through a redeployment of available resources. The costs of this positive approach might be offset to some extent by preventing difficulties, limiting the costs of managing distressing symptoms, and delaying institutionalisation. There is a need to develop service systems with a clear focus on optimizing functioning and supporting relationships, identity, and engagement and a need to train staff to implement rehabilitative interventions. It is essential to fully involve people with dementia and carers to ensure a thorough understanding of their perspectives.Acknowledging the right to rehabilitation offers a tremendous opportunity to create a focused and coherent approach to positive support for people with dementia, of any age, subtype, or severity, and their families. A rehabilitation model offers a guiding framework for services and for health and social care practitioners and a practical means of providing person-centred, evidence-based interventions to maintain or enhance functioning, engagement, and participation.


Transcranial brain stimulation for post-strokeaphasia rehabilitation discusses consensus and controversies about moderntechniques for rehabilitation of language disorders arising from focal neurologicallesions.The Original Articles section, we have 11 articles, seven focused onevaluation and four on rehabilitation. Adaptation and cross-cultural validation to thePortuguese language were the aims in Semantic memory for actions and Adaptationof the CAPPA to the Portuguese. Methods of language analysis for healthyindividuals are presented in Automatic Classification of WrittenDescriptions. Oral and written language evaluations of individuals withfocal lesions were also examined in Acquired dysgraphia in adults andInferential abilities in patients with right hemisphere damage. Diffuselesions were the topic of Naming performance in bilingual speakers with dementiaand Discourse of Patients with mild to moderate Alzheimer's disease. Theissue of rehabilitation was addressed in a systematic review - Rehabilitation oflexical and semantic communicative impairments and in two interventionalstudies in dementia: Discourse intervention in Alzheimers disease andInterdisciplinary therapy in dementia.In the Case Reports different methods in therehabilitation of agrammatism were presented: in degenerative disease, Briefintervention for agrammatism in Primary Progressive Nonfluent Aphasia, andin aphasia due to vascular lesion, Sentence production and rehabilitation ofagrammatism.Finally, in the session of Logopenic aphasia andAlzheimer's.We highlight the publication of a case study awarded by ACHÉ Laboratory in aninitiative of Prof. Dr. Ricardo Nitrini and Dr. Sonia Brucki. This case study was aimedat differential diagnosis between We hope this issue will contribute to knowledge on Recent Changes in Assessment andIntervention in Neurological Disorders of Language.Leticia MansurKarin Zazo OrtizJed A. Meltzer Guest Editor


Biochemical analysis revealed that inhibiting AR resulted in decreased HER2 phosphorylation and activation of Erk and Akt, without affecting the HER2 and HER3 expression. The in vivo efficacy of Enzalutamide was further tested using the HCC1954 xenograft model. Enzalutamide impaired the growth of HCC1954 tumor at a level comparable to that by trastuzumab. Enzalutamide decreased Ki67 staining and increased activated caspase3 staining compared with vehicle control in HCC1954 tumors. Our results indicate AR plays an important role in promoting the growth of HER2 + BC by cross-talking with the HER2 signaling. AR drug may be used as an alternative second line therapy for treating HER2 + BC.Androgen receptor (AR) is widely expressed in different subtypes of breast cancer (BC). However, it is unclear how AR functions in HER2 positive (+) BC. Knockdown of AR with shRNAs and a new generation anti-androgen drug, Enzalutamide, were used to explore the involvement of AR on the growth of HER2 + BC cells (HCC1954 and SKBR3). AR shRNA or Enzalutamide inhibited the growth of SKBR3 cells at a similar extend compared to trastuzumab, an approved HER2 targeted drug. Combining Enzalutamide with trastuzumab further decreased the growth of HCC1954 and SKBR3 cells compared with single agent alone  Clinically, HER2 + breast cancer is identified by immunochemistry (IHC) of HER2 with a 3 + staining or fluorescent in situ hybridization (FISH) for HER22. HER2 mainly via heterodimerization with HER33 activates downstream signaling pathways including PI3K/Akt and Ras/Erk to drive the growth of the breast cancer cells5. Various HER2 targeted therapies have been developed over the years7. Trastuzumab (Herceptin), a humanized monoclonal antibody against HER2, when combined with chemotherapy, is very effective in treating HER2 + breast cancer, with a 50–80% response rate8. However, 20–50% of patients with HER2 + tumors do not respond to trastuzumab initially or develop acquired resistance after one year of treatment9. Thus, it is important to explore alternative second line therapies.HER2 positive (+) breast cancer accounts for ~25% of breast cancer and has a poor prognosis10 and recent studies reveal that AR is more widely expressed than estrogen receptor (ER) or progesterone receptor (PR) in breast cancer12. According to the Nurses’ Health Study in 2011, AR expression can be detected in 77% of the breast cancer with the breakdown of 88% of ER+, 59% of HER2+, and 32% of triple negative breast (TNBC) expressing AR respectively12. Although as with ER, AR expression in breast has been largely associated with a better prognosis than AR negative disease,16, recent studies indicate that AR promotes the growth of ER + breast cancer17 and TNBC19 via distinct mechanisms. In ER + BC, studies from the use of a new generation of anti-androgen drug, Enzalutamide 20, indicate that AR signaling is required for androgen and estrogen-induced tumor cell growth in vitro and in vivo17. In the luminal AR21 subset of TNBC, AR activity is required for the growth of LAR-TNBC through the upregulation of HER319. In other subset of TNBC, AR activity promotes the growth of TNBC at least in part via upregulating the expression of amphiregulin22. Various clinical trials using androgen antagonists bicalutamide and Enzalutamide in treating AR + TNBC are ongoing .Androgen receptor (AR) is a ligand-activated transcription factor that belongs to the steroid hormone receptor family. AR signaling is well known to drive the growth of prostate cancer cells as reviewed23 or worse14 patient survival of HER2 + breast cancer (BC). Previous studies suggested a role of AR in the molecular apocrine BC (maBC), which is a subtype of estrogen receptor negative (ER-) BC that overexpresses AR and frequently HER2 as well24. Treating MDA-MB-453 cells, a maBC line, with the first generation anti-androgen drug flutamide reduced cell proliferation and induced apoptosis of maBC in vitro25. Furthermore, AR promoted Erk activation via upregulation of HER2 gene transcription26. Considering the role of AR and its interaction with HER2 in maBC, we hypothesize that AR expression may contribute to the growth of HER2 + BC.In contrast, the role of AR in HER2 + breast cancer is less well understood. The prognostic value of AR expression have been reported to be associated with either better27, our study should help dissect the role of AR in HER2 + BC independent of ER. Our data revealed that inhibiting AR impairs the growth of HER2 + breast cancer cells in vitro and vivo, in comparable to the effect of trastuzumab. AR inhibition also reduced HER2 phosphorylation and activation of Akt and Erk without affecting HER2 and HER3 protein expression. Our results suggest that AR plays a novel role in HER2 signaling and AR targeting therapy may be useful in treating HER2 + breast cancer.In the current study, we examined the functional role of AR in HER2 + BC by blocking AR expression with shRNA and AR activity with androgen antagonist Enzalutamide in HER2 + BC cell lines, SKBR3 and HCC1954. These two cell lines are well known models of HER2 + BC that are estrogen receptor negative. Considering the reported interaction between AR and estrogen receptor in breast cancerTo test whether AR can drive the growth of HER2 + breast cancer, AR shRNAs were used to knockdown AR expression in HCC1954 and SKBR3. Both of the AR shRNAs significantly inhibited AR protein expression in HCC1954 and SKBR3 cells as indicated from the western blot analysis , an FDA approved AR targeting drug, was used to treat HCC1954 and SKBR3 cells sed Fig. . Similarsed Fig. . Althougsed Fig. , treatmesed Fig. . Again, 18. Western blot analysis was used to examine the phosphorylation status of HER2, HER3, and total HER2, HER3 protein expression in HER2 + breast cancer cells treated with Enza nza Fig.  and 3a,cnza Fig.  and 3d,enza Fig.  and 3a. nza Fig. . These din vitro, the effect of Enza on HER2 + tumor growth was examined in HCC1954 xenograft model. HCC1954 cells mixed with matrigel were injected into both flanks of the Balbc/nude mice. Mice were monitored for tumor growth by palpation and caliper measurement, and randomized into four groups for treatment with Vehicle, Enza, Trastuzumab, or Enza + Trastuzumab two weeks after cell injection.Because of its efficacy in reducing the growth of HER2 + breast cancer cells While HCC1954 tumors treated with vehicle control grew gradually, Enza treatment significantly decreased the tumor growth. Likewise Trastuzumab treatment dramatically reduced HCC1954 tumor growth Fig. . AdditioImmunohistochemistry (IHC) using antibodies against Ki67 and activated caspase 3 (Casp3) was used to examine the effects of Enza on cell proliferation and apoptosis respectively in xenograft tumors Fig. . While vin vitro  in the host mice33. In any event, because the efficacy of Enza in inhibiting HCC1954 cell growth in vitro and in vivo, our data suggest that Enza may be used to overcome trastuzumab resistance.Two well-known HER2 + breast cancer cell lines, SKBR3 and HCC1954, were used in our study. Our data showed that SKBR3 cells were sensitive Fig.  whereas del Fig. , which iin vitro  (Hyclone) supplemented with 5% fetal bovine serum (FBS) (Sigma), 1 mM sodium pyruvate, 100 units/ml penicillin and 100 μg/ml streptomycin (Hyclone). Breast cancer cell line HCC1954 cells were grown in RPMI-1640 medium (Hyclone) supplemented with 5% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. All cell lines were cultured in 37 °C humidified tissue incubator with 5% CO2 and 95% air. Enzalutamide was purchased from Selleck and dissolved in DMSO. Trastuzumab (Herceptin) was purchased from Roche.6 cells 24 hours before were cotransfected with 1.88 μg pHR8.9deltaR, 1.25 μg CMV-VSVG, 2.5 μg pLKO1 shRNA using 16.8 μg PEI. Virus supernatants were harvested between 48–72 hours post-transfection, filtered through 0.45 μm low protein-binding sterile filter, and used to infect HCC1954 and SKBR3 cells in the presence of 8 μg of polybrene (Sigma). Pools of HCC1954 and SKBR3 cells expressing shRNAs were selected in the presence of 1.5 μg/ml puromycin (TaKaRa) for 3 days before using for further experiments. Sequences for AR shRNAs are:Lentiviral vector PLKO.1 expressing control-shRNA and two different AR-shRNAs (1 and 2), and packaging plasmids pHR8.9deltaR and CMV-VSVG were purchased from Sigma. 293T17 cells plated in 6-cm dish at 2.5 × 10ShRNA1: 5′-GAGCGTGGACTTTCCGGAAAT-3′;ShRNA2: 5′-CACCAATGTCAACTCCAGGAT-3′.Cells were seeded in 24 well plates at 5000/well in quadruplicate and treated with vehicle control, 20 μM enzalutamide, 20 μg/ml trastuzumab, and combined enzalutamide with trastuzumab. Fresh medium containing the drugs were replaced every two days. Six days later, cells were fixed and stained with 0.5% crystal violet for 20 minutes at room temperature. Crystal violet dye was extracted with 10% acetic acid and quantitated at OD 540 nm using Varioskan flash plate reader (Thermo scientific).Breast cancer cell lines were lysed in 1X SDS sample . Tumors from xenografts were homogenized in modified RIPA lysis buffer  using Brinkmann Polytron PT2100. Tumor lysates were clarified by centrifugation (>15 K rpm) at 4 °C for 20 minutes. Protein concentrations in clarified lysates were quantified using BCA Protein Assay Kit (Beyotime Institute of Biotechnology) according to manufacturer’s protocol. Equal amounts of cell lysateswere resolved by 8% SDS-PAGE and transferred to Immobilon-P membrane (Millipore Inc.), immunoblotted using the appropriate primary and the HRP-conjugated secondary antibodies (Beyotime Institute of Biotechnology), and developed using enhanced chemiluminescence (ECL) reagent (Thermo Scientific). ECL signals were captured by the ChemiDoc MP imaging system (Bio-Rad) and were analyzed using the Image Lab 4.0 software (Bio-Rad). Alpha -Tubulin (Abcam) was used as the loading control. Monoclonal antibody (mAb) against ErbB2 was purchased from Santa Cruz Biotechnology. Antibodies recognizing phospho-HER2 (Tyr1221/1222) (P-HER2), HER3, phospho-HER3(Tyr1197) (P-HER3), cleaved Caspase-3(9G10), phospho-Akt (P-Akt) (Ser473) and phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (P-Erk) were from Cell Signaling Technology. Antibodies recognizing AR (06–680) was from Merck Millipore.in vivo experiments. HCC1954 cells (2 × 106/100 μl) were mixed with 100 ul growth factor reduced Matrigel (BD Bioscience) and injected subcutaneously at the flanks of the mice. Tumor growth was measured by caliper measurement every 3 days. The modified ellipsoid formula was used to calculate the tumor volume: (width)2 × (length)/2. Once all the tumors reached the tumor volume of 300–500 mm3 (~15 days after injection), four group of mice were randomized and treated with the following four regiments: 1) vehicle control: 2.7% DMSO + 30% PEG300 plus PBS; 2) 25 mg Enzalutamide/kg plus PBS; 3) 2.7% DMSO + 30% PEG300 plus 10 mg trastuzumab/kg; 4) 25 mg Enzalutamide/kg plus 10  mg trastuzumab/kg. Enzalutamide was dissolved in 2.7% DMSO, formulated in 30% PEG300 and administrated daily to mice by oral gavage. Trastuzumab was dissolved in PBS and injected intraperitoneally twice a week. Twenty one days after treatments, the animals were sacrificed after the last measurement of tumor size. Tumor growth was presented as fold change in tumor volume, which was calculated by dividing the tumor volume on a given day with the tumor volume on day 3 after injection of cells into mouse flank. The tumor tissues were dissected, fixed in 10% formalin for 24 hours and processed for immunohistochemistry or flash-frozen in liquid nitrogen and stored at −80 °C for western blot analysis later.Protocols for the animal studies were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. All animal experiments were performed in accordance with the relevant guidelines and regulations. BALB/c female nude mice (6 to 8 week old) purchased from Shanghai SLAC laboratory animal center were used for the  Sections (5 μm thickness) of the formalin-fixed, paraffin-embedded (FFPE) tumor tissues were deparaffinized in xylene, rehydrated with a series of graded ethanols, then stained in Hematoxylin solution for 15 min and counterstained in eosin solution using the Hematoxylin and Eosin Staining Kit (Solarbio).2O2 for 8 min at room temperature (RT), then blocked with 10% normal goat serum (Solarbio) diluted in PBS for 20 min at RT, and incubated with Ki-67 mouse mAb (1:1200)  and cleaved caspase 3 rabbit mAb (1:400)  overnight at 4 °C. Normal mouse IgG (Beyotime#A7028) (1:1200) and rabbit IgG (Beyotime#A7016) (1:200) were used as negative controls. For detection of Ki-67, polyperoxidase conjugated anti-mouse/rabbit IgG Fab (ZSGB-BIO) was used followed by incubation with 3, 3′-diaminobenzidine chromogen DAB detection Kit (ZSGB-BIO). For detection of cleaved caspase 3, biotin labeled anti-rabbit secondary antibodies was used followed by incubation with streptavidin-horseradish peroxidase (SABC kit SA1052) (Boster) and 3, 3′-diaminobenzidine chromogen DAB detection Kit. Sections were counterstained with diluted hematoxylin for 2 min before mounted. For quantification of Ki-67 and cleaved caspase 3 IHC, images from ten random representative 400 × fields per mouse tumor were captured by Nikon Eclipse 80i Microscope. Ki-67 and cleaved caspase 3 staining positive cells in each field were counted and the average of positive staining cells per field was presented. The IHC data shown was representative from three independent experiments.For immunohistochemistry, sections were deparaffinized, rehydrated as above, and subjected to heat induced epitope retrieval in 10 mM citrate buffer, pH 6.0 using Supor Pressure Cooker (at 125 °C for 5 min). Slides were immersed in 3% HStatistical analyses were performed using SPSS, version 19.0. Quantitative data from at least three independent experiments were expressed as mean ± S.E.M. Student’s t-test was used to compare data between two groups. One-way ANOVA with Bonferroni’s multiple comparison test correction was used to analyze data among multiple groups. All statistical tests were two-sided and P < 0.05 was considered statistically significant.Supplementary Information


Currently, two pathogenic pathways describe the role of obesity in osteoarthritis (OA); one through biomechanical stress, and the other by the contribution of systemic inflammation. The aim of this study was to evaluate the effect of free fatty acids (FFA) in human chondrocytes (HC) expression of proinflammatory factors and reactive oxygen species (ROS).HC were exposed to two different concentrations of FFA in order to evaluate the secretion of adipokines through cytokines immunoassays panel, quantify the protein secretion of FFA-treated chondrocytes, and fluorescent cytometry assays were performed to evaluate the reactive oxygen species (ROS) production., and the reactive nitrogen species increased significantly in a at the two-dose tested (250 and 500 μM). In addition, we found an increase in the cytokine secretion of IL-6 and chemokine IL-8 in FFA-treated HC in comparison to the untreated HC.HC injury was observed at 48 h of treatment with FFA. In the FFA-treated HC the production of reactive oxygen species such as superoxide radical, hydrogen peroxideIn our in vitro model of HC, a hyperlipidemia microenvironment induces an oxidative stress state that enhances the inflammatory process mediated by adipokines secretion in HC. OA is one of the most common musculoskeletal disorders of the aging population –3, and ae (H2O2) –13. The e (H2O2) –16. An ie (H2O2) .The deposition of free fatty acids (FFA) within the joint stimulates both a deregulation of the metabolic process and an imbalance of the oxygen available. To address this issue, we used an in vitro HC model where we used FFA intra-cartilage as inductors of oxidative and inflammatory state.2. All participants were appropriately informed about the study aims and signed an informed consent form. This study meets all the criteria contained in the Declaration of Helsinki and was approved by the Ethics and Research Committee of the Instituto Nacional de Rehabilitación (INR) (Ref. INR-08/11).Hyaline cartilage biopsies, macroscopically healthy and  4 mm in diameter, were harvested by arthroscopy from a non-weight bearing area of the knees , from four consecutive patients who underwent surgery for Anterior Cruciate Ligament (ACL) repair. Three of these patients were female whereas one was male, with an average age of 44.25 ± 24.81 years, and a mean body mass index of 26.97 ± 5.33 kg/m2, at saturation humidity. Chondrocytes from the fourth passage were used for all experiments, the HC were characterized using Western Blot analysis for SOX9 transcription factor to ensure the phenotype of the cells were maintained all along the study . HC were treated with a FFA mixture composed of palmitic acid (C16), and oleic acid  , in a ratio 1:2 respectively at concentrations of 250 and 500 μM for 48 h, respectively. As a positive control for oxidative stress, 100 μM of H2O2 was used, and cells maintained under the same condition, while HC not exposed to FFA or H2O2 were used as a negative control. The lipid overload within the HC was confirmed by Oil red staining [To assure statistical significance each experiment was performed in triplicate for all patients . O2.- and H2O2 were quantified, by oxidation of the dihydroethidium (DHE) and the 5-, 6- carboxy-2′, 2′, 7′-dichlorofluorescein diacetate (carboxy- H2DCFDA) respectively (kit Image-iT LIVE Green Reactive Oxygen Species Detection). NO was detected using the commercial kit DAF-FM . DHE was quantified in the 530 ± 20 nm emission filter, and the carboxy-H2DCFDA and DAF-FM fluorescence in the 458 ± 20 nm emission filters, according to the manufacturer’s instructions.ROS and RNS production was evaluated through intracellular determination of O2O2 treated HC cells by Milliplex Human Adipocyte Magnetic Panel . The pro-inflammatory cytokine quantification was performed by Magpix Merck-Millipore and the results were expressed in ng/ml. The samples were analyzed by triplicate according to manufacturer’s instructions.Pro-inflammatory cytokines were evaluated in the supernatant from FFA-untreated, FFA-treated, and HP-values adjusted by Bonferroni test combining data from both FFA concentrations. All P-values lower than 0.05 were considered as statistically significant. Prism v6.01  and STATA v12.1  were used to perform the analysis and graphs.Data are presented as mean ± standard deviation (SD). Statistical differences between experimental groups were assessed by one-way ANOVA test adjusted by Bonferroni multiple-comparison test. The mean of the estimated difference in ROS intracellular production relative units, as well as in secreted proteins concentration between groups was assessed by linear regression models. To assess the association between ROS intracellular production and secreted protein concentrations, Spearman correlation coefficients were calculated with their respective Treatment of HC with 250 or 500 μM of FFA for 48 h lead to increase HC lipid overload by Oil red staining. Figure 2.- increased to 14% in HC exposed to 250 μM of FFA  and 35% when exposed to 500 μM of FFA  compared to untreated HC , and 30% when treated with 500 μM , compared to untreated HC  and 24%  in HC exposed to 250 and 500 μM of FFA, respectively, compared to untreated HC.  in the O2.- concentration, an increase of 34%  in the H2O2 production and an increase of 35%  in the NO production.  and 26.3 ng/ml (P < 0.01) with 250 and 500 μM FFA, respectively in comparison to 18.5 ng/ml in the untreated HC. In a similar fashion, the secretion of IL-8 increased from 6.1 ng/ml (untreated HC) to a concentration of 7.29 ng/ml (P < 0.01) and 8.11 ng/ml (P < 0.01) in the two concentrations of FFA treated HC , for IL-8, the concentration was 8.6 ng/ml P < 0.01).In order to address the secretion of pro-inflammatory mediators elicited by lipid overload in HC, we determined the concentration of IL-6, and IL-8 in the supernatant of HC exposed to 250 and 500 μM of FFA for 48 h. IL-6 increased in a dose-dependent manner, having an average concentration of 21.7 ng/ml . Although, there was a positive correlation between NO and H2O2 production and IL-8 concentration, the magnitude was not statistically significant . None of the IL-6 concentration and ROS production correlations were statistically significant, however, this data showed similar trend as IL-8 (Fig. A statistically significant positive correlation was identified between Oion Fig.  (rho = 0L-8 Fig. .Fig. 5St2.-, H2O2, and NO, respectively, as well as the production of the cytokine IL-6 and chemokine IL-8 at the two FFA doses tested.In this study, we observed that a lipidic microenvironment in human chondrocytes induces oxidative stress and elicits a proinflammatory response, that could be reflected in joint diseases. Our results show that FFA stimulates the production of ROS and RNS such as O2.- by a donor compound to form peroxynitrite, creating a deleterious state in the cartilage as described by Scher et al. [The role of ROS in the development of OA has been well documented. Henrotin et al.  showed tr et al. .In our HC in-vitro model, we observed that at 48 h, the internalization of FFA by chondrocytes induces the production of ROS and RNS, suggesting that a hyperlipidemic microenvironment plays a key role in situ oxidative stress-state related to cartilage injury, characteristic in the OA development. In addition to the ROS production, we show evidence that HC stimulated with FFA leads to an increase on the cytokine IL-6 and chemokine IL-8 in  an autocrine fashion. IL-6 has been strongly associated with the development of OA as it acts as a mediator in the degeneration of the cartilage , 26; furOn the other hand, IL-8 chemokine has also been shown to have a deleterious effect in OA . IL-8 fuWe provide evidence that the increase oxidative stress caused by HC exposed to FFA leads to ROS and RNS production, autocrine activation of IL-6 and IL-8 by chondrocytes leading to cartilage damage. Our data suggest that the oxidative stress along with an inflammatory response triggers an imbalance in the cartilage homeostasis as seen in OA patients with obesity . Taken tIn conclusion, this work suggests an alternative mechanism in which an increase in the lipid composition in the cartilage also contributes to the development of OA and may suggest why non-weight-bearing joints such as the small joints of the hand are affected in OA patients with obesity and dyslipidemia Fig. . Althoug


Here, we characterized a novel splice variant which has never been investigated, NRP1-Δ7, differing from the NRP1-FS by a deletion of 7 amino acids occurring two residues downstream of the O-glycosylation site. This short sequence contains two aspartic residues critical for efficient glycosylation. As expected, the high molecular weight products appearing as a smear in SDS-PAGE and reflecting the presence of GAG in NRP1-FS were undetectable in the NRP1-Δ7 protein. NRP1-Δ7 mRNA was found expressed at an appreciable level, between 10 and 30% of the total NRP1, by various cells lines and tissues from human and murine origin. To investigate the biological properties of this isoform, we generated prostatic (PC3) and breast (MDA-MB-231) cancer cells able to express recombinant NRP1-FS or NRP1-Δ7 in a doxycycline-inducible manner. Cells with increased expression of NRP1-Δ7 were characterized in vitro by a significant reduction of proliferation, migration and anchorage-independent growth, while NRP1-FS had the expected opposite “pro-tumoral” effects. Upon VEGF-A165 treatment, a lower internalization rate was observed for NRP1-Δ7 than for NRP1-FS. Finally, we showed that NRP1-Δ7 inhibited growth of prostatic tumors and their vascularization in vivo. This report identifies NRP1-Δ7 as a splice variant displaying anti-tumorigenic properties in vitro and in vivo, emphasizing the need to consider this isoform in future studies.Neuropilin-1 (NRP1) is a transmembrane protein acting as a co-receptor for several growth factors and interacting with other proteins such as integrins and plexins/semaphorins. It is involved in axonal development, angiogenesis and cancer progression. Its primary mRNA is subjected to alternative splicing mechanisms generating different isoforms, some of which lack the transmembrane domain and display antagonist properties to NRP1 full size (FS). NRP1 is further post-translationally modified by the addition of glycosaminoglycans (GAG) side chains through an O-glycosylation site at serine The neuropilin-1 (NRP1) gene is located on chromosome 10p12 and composed of 17 exons –7, and i612 has been identified as the site of O-glycosylation where long chains of oligosaccharides assemble on the protein. The nature of the chains attached on NRP1 depends on the cell type. In smooth muscle cells, NRP1 is predominantly glycosylated with chondroitin sulfate while it is derivatized with heparan sulfate in endothelial cells [612 into Ala prevents O-linked glycosylation and markedly affects the biological functions of NRP1 [NRP1 can be considered as a proteoglycan since it can be modified by the addition of glycosaminoglycan side chains –12. Seri of NRP1 , 13. The of NRP1 .165 but exert anti-angiogenic and anti-tumorigenic effects by sequestration of NRP1 binding growth factors and competition with their signaling pathways [The full size NRP1  is known as a pro-angiogenic and pro-tumorigenic factor , 14. Altpathways , 16. NRPpathways .612 and contains two aspartic residues that are critical for efficient glycosylation [612 affected the glycosylation status and the functions of this new NRP1 variant.In the present work, we have identified and studied a so far uncharacterized splice variant of NRP1. Although this variant is quite abundant , it was never specifically reported before, most probably because it differs from the NRP1-FS variant by the absence of only 7 amino acids, which prevents its identification on SDS-PAGE. This deletion is located two residues downstream of the O-glycosylation site on serinesylation . We haveDoxycyline hyclate (D9891) was from Sigma-Aldrich (USA). Rabbit monoclonal anti-neuropilin-1 (ab81321) was from Abcam (UK). Rabbit polyclonal anti–extracellular signal-regulated kinase (ERK1/2) (M-5670) and mouse monoclonal anti-FLAG (F1804) were from Sigma-Aldrich. Rabbit polyclonal anti-Tet-repressor antibody (TET01) was from MoBiTec (Germany). The secondary horseradish peroxidase–conjugated rabbit anti-mouse IgG (P0260) and swine anti–rabbit IgG (P0217) were from DAKO (Belgium). PC3 human prostate adenocarcinoma cells were cultured in F-12 Kaighn's medium  supplemented with 7% fetal bovine serum (FBS) . MDA-MB-231 human breast adenocarcinoma cells were cultured in DMEM (Lonza) supplemented with 10% fetal bovine serum. Human skin fibroblasts, HSMC (human smooth muscle cells), HUVEC , HT1080 (human fibrosarcoma cells) and MCF7 (human breast adenocarcinoma cells) were cultured in DMEM supplemented with 10% FBS, MO59J  were cultured in DMEM/F12 (Lonza) supplemented with 10% FBS and OCI-LY-19 (human lymphoma cells) were cultured in RPMI 1640 (Lonza) supplemented with 10% FBS. Murine 4T1, LLC, NIH3T3 and B16F10 were cultured in DMEM supplemented with 10% FBS. RNA from human normal breast, placenta and skin were obtained from the Surgery, Obstetrics and Gynecology Department and from the Dermatology Department. Mouse RNA was purified from 8-weeks old C57Bl6 mice.Cells were lysed in Laemmli buffer and proteins were separated by polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF Transfer Membrane  which was then blocked for 1 hour with 3% dry milk in PBS-0.05% Tween 20 and incubated for 4h with the primary antibody. After 3 washings, membranes were incubated with the secondary horseradish peroxydase-conjugated antibody for 1h, and revealed by chemoluminescence using home-made ECL acquired with the ImageQuant LAS4000 device . The membranes were re-probed with anti-ERK1/2 antibodies used as a control for protein loading.in vivo experiments.Total RNA was purified from cells with the High Pure RNA Isolation Kit  and from tissues with Ambion kit using TRIzol reagent , following the instructions of the manufacturer. Reverse Transcription-Polymerase Chain Reaction was performed using Tth DNA Polymerase kit (Roche). The primers 5’-GCTGTGAAGTGGAAGCCCCTACA-3’ (forward) and 5’-GGTCTTGTGAGAGCCCCAGCCA-3’ (reverse) were used to amplify NRP1-FS and NRP1-Δ7 in human samples. The primers 5’-GTGGATGAGTGTGACGACGACCA-3’ (forward) and 5’-GAGAGCCCCAGCCAAACTCGCA-3’ (reverse) were used to amplify NRP1-FS and NRP1-Δ7 in murine samples. The amplification products of the two isoforms were separated on a polyacrylamide gel and quantified. Primers 5’-CTTGGTGGGATTGCTGTGGATGAC-3’ (forward) and 5’-GGGCACTCATGGCTATGATGGTGA-3’ (reverse) were used to amplify NRP1-ΔE16. Primers 5’-GGTGGATGAATGTGATGACGACCA-3’ (forward) and 5’-GTCACATTTCGTATTTTATTTGATACCTGA-3’ (reverse) were used to amplify NRP1-s12. Primers 5′-GTTCACCCACTAATAGGGAACGTGA-3′ (forward) and 5′-GATTCTGACTTAGAGGCGTTCAGT-3′ (reverse) were used to amplify human and mouse 28S rRNA. Primers 5’-TCCAGCTCTCTCCACGCGATTCA-3’ (forward) and 5’-ATGACCGTGGGCTTTTCTGTGGC-3’ (reverse), specific of human NRP1, were used in the (-ΔΔCt). The specificity of the amplification products was assessed by gel electrophoresis.RT-qPCR was performed as previously described . One μg www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5104.MCF7 were isolated and prepared for next-generation sequencing analysis, as described in a previous publication . The aveMDA-MB-231 cells were transfected with pcDNA6/TR (Invitrogen) and selected in medium supplemented with blasticidin (4 μg/ml). One clone expressing a high level of tetracycline repressor (MDA-MB-231/TR) was isolated. PC3 cells expressing a high level of tetracycline repressor (PC3/TR) were described previously . The entCells were chilled on ice and lysed in buffer containing 1% Igepal, 150 mM NaCl, 20 mM Tris/HCl pH 7.4, 0.1mM AEBSF and 4 μg/ml aprotinin. Lysates were centrifuged for 10 min at 16000g at 4°C. Supernatants were incubated with 1 μg/ml rabbit anti-NRP1 or anti-FLAG antibodies. After 2h of incubation at 4°C, 30 μl of protein A–Sepharose beads  were added for 1h to capture immune complexes. Beads were washed four times in ice-cold lysis buffer and boiled in 60 μl of SDS–PAGE lysis buffer. The samples were subjected to western blotting as described above.A suspension of 50 000 PC3 or 35 000 MDA-MB-231, each inducible for NRP1-FS or -Δ7, was seeded into culture-inserts  with or without 200 ng/ml doxycycline in the medium. This type of insert allows the creation of a homogeneous 500 μm width cell-free space in the middle of a confluent cell monolayer. Twenty-four hours after seeding, the inserts were removed and pictures by phase-contrast microscopy of the resulting scratches were taken at T0 . PC3 and MDA-MB-231 were cultured for a further 24h and 8h, respectively. Cell migration was evaluated by measuring the cell-free surface at the beginning of the experiment (T0) and the surface covered by the cells at the end of the experiment by using ImageJ Software . Results are expressed as the percentage of wound closure in each condition.Cells were seeded in 24 wells costar plates (7500 cells/well) and allowed to attach overnight (PC3) or for 2h (MDA-MB-231). Half of the cultures were supplemented with doxycycline (day 0) and cells were collected after 1, 3 and 5 days (PC3) or after 1, 2 and 3 days (MDA-MB-231). DNA content was assayed by a fluorimetric technique using bisbenzimide and a microplate Gemini EM spectrofluorimeter .PC3/NRP1-FS and PC3/NRP1-Δ7 cells were cultured in absence and presence of doxycycline and apoptosis was evaluated by FACS after annexin V–FITC and propidium iodide staining, as previously described  using a 3 PC3 cells or of 5 × 103 MDA-MB-231 cells overexpressing NRP1-FS or NRP1-Δ7 in a doxycycline-dependent way were plated in 60-mm dishes in growth medium containing 0.3% agar, on top of a 0.6% agar gel. The overexpression of the transgene was induced by addition of 1 μg/ml doxycycline. After 21 days, colonies were counted in the whole dishes using an inverted phase-contrast microscope. Results are expressed as the mean number ± SD of two independent experiments performed in quadruplicate.Anchorage-independent growth was determined by soft agar assay . A totalCells were serum starved and treated with doxycycline during 24h. Recombinant VEGF-A (isoform 165 at 37 ng/ml) [6 PC3 conditionally expressing NRP1-Δ7. Control mice had access to regular water while doxycycline (2 mg/ml) was added in drinking water of induced mice. The general condition of mice was monitored daily by visual examination during the entire experiment and tumor volume was evaluated ((length x width x thickness) x π/6, as determined using a caliper). Signs of pain were never observed and the calculated tumor volumes remained always largely below 1 cm3 which is considered as the upper acceptable limit for subcutaneous tumors. After 7 weeks, mice were sacrificed by cervical dislocation followed by decapitation and the collected tumors were photographed, dissected, measured and weighed. Fragments of tumors were then used for immunohistological analyses and RNA extraction.Experimental protocol was approved by the local ethical committee . Nude mice  received an intraperitoneal injection of 60 mg/kg Nembutal before to be subcutaneously injected in each flank with 2 x 10Antigen retrieval was performed on 5 μm sections of PC3 tumors by heating at 126°C for 11 minutes in Target Retrieval Citrate buffer . After blocking endogenous peroxydases and non-specific antigenic sites , sections were incubated with primary anti-CD31 antibodies (diluted 1:150 in BSA/PBS 1%) for 1h, followed by biotinylated secondary antibodies (diluted 1:300) for 30 minutes and with HRP-conjugated streptavidin (diluted 1:500) for 30 minutes. Signal was amplified by an incubation in Biotinylated Tyramide  for 10 minutes, followed by additional 30 minutes in HRP-conjugated streptavidin. Staining was performed with AEC+ High Sensitivity Chromogen  followed by a counterstaining with hematoxylin. Stained sections were scanned with a NanoZoomer slide scanner  and quantification was performed using ImageJ software . Tumor vascularization was quantified both as the number of blood vessels per unit surface and as the % of surface covered by blood vessels.Statistical analyses were performed using U Mann-Whiney test or by an Anova test  with p ≤ 0.05 considered as significant.http://www.ncbi.nlm.nih.gov/nuccore/BX648250.1) by entries in Genbank under the accession numbers NM_001244973 (NRP1 variant 5) and NP_001231902 (NRP1 isoform E). Based on the exon content of the previously described variants of NRP1, NRP1-s12 and NRP1-ΔE16 should be also affected by the 21 bases deletion. It was confirmed for NRP1-s12 (NRP1-s12-Δ7), while it was not further investigated for NRP1-ΔE16 which is barely detectable in our cell models  and exon12/exon13 (reverse) junctions . In addil models .612 affects its glycosylation because it changes the consensus sequence required for optimal O-glycosylation. These results also show that our cell models are adequate for investigating the biological functions of NRP1-Δ7. To improve the significance of the results of our biological models and avoid potential off-target effects linked to the use of different clones, we then generated inducible clones overexpressing NRP1-FS and NRP1-Δ7 upon treatment by doxycycline.Recombinant proteins were produced to identify potential functional consequences of the 7 aa deletion. HEK293 cells were transiently transfected with an expression vector for NRP1-FS or NRP1-Δ7, while PC3 cells were transfected with a vector expressing FLAG-tagged NRP1-FS or NRP1-Δ7 to allow discriminating exogenous and endogenous NRP1. They were immune-precipitated, respectively, with anti-NRP1 and anti-FLAG antibodies and subjected to western blot analysis revealed by anti-NRP1. As illustrated in PC3 and MDA-MB-231 cancer cells are known to constitutively express a significant level of NRP1 and to respond to NRP1-mediated signaling –24. In oin vitro tumorigenic properties of PC3 and MDA-MB-231 cells were further evaluated in an anchorage-independent growth model. Cells were seeded in soft agar in absence and presence of doxycycline and the total number of colonies formed after 3 weeks was counted. The overexpression of NRP1-FS in both cell types significantly increased the number of colonies while overexpression of NRP1-Δ7 had the opposite effect  act in several settings as antagonists of neuropilin-1 signaling, mainly by competitive binding to the ligands [612, the main O-glycosylation site on NRP1. We have shown that NRP1-Δ7 is significantly and differently expressed in various cell types of human and mouse origin, varying between 10 and 30% of total NRP1, which suggests a specific physiological function. The ability of cells to add glycosaminoglycan chains strongly depends upon the amino acids sequence around the glycosylation site [612 (S612GTGDDF) contains two “D” acidic residues, while the “GDDFQLT” deletion in NRP1-Δ7 results in a more hydrophobic consensus sequence (S612GTGGTT) which is likely responsible for the defective O-glycosylation of NRP1-Δ7 reported here. Alterations of NRP1 glycosylation, induced enzymatically or by mutations of Ser612, profoundly alter cancer cells phenotype [The neuropilin-1 pre-mRNA is alternatively spliced in several mature mRNA encoding proteins with various, sometimes opposite, properties. The full size NRP1 is anchored in the cell membrane, functions as a co-receptor for multiple ligands such as VEGF-Aion site . For insion site . In NRP1henotype . The rolhenotype –24. Theyin vitro [in vivo tumor growth and vascularization [in vivo by using inducible PC3/TR/NRP1-Δ7 in a xenograft model. As observed in vitro, the addition of doxycycline in drinking water allowed continuous expression of NRP1-Δ7 in the xenografts. Using this model, we clearly demonstrated that increased expression of NRP1-Δ7 inhibits tumor growth and intratumoral angiogenesis.NRP1-FS is known to regulate cell functions involved in cancer progression such as proliferation, migration or anchorage-independent growth , 13, 27.in vitro , 13, 27 rization . The potin vitro and in vivo. Although the precise mechanisms of regulation depending on NRP1-Δ7 were not identified, our observations emphasize the critical importance of considering specifically the different NRP1 variants, especially NRP1-Δ7, instead of analyzing the whole NRP1 expression.This report is the first study characterizing the specific functions of NRP1-Δ7, a glycosaminoglycan-defective neuropilin-1 splice variant that displays anti-tumorigenic properties S1 FigSpecific primers were designed to co-amplify the NRP1-FS and the NRP1-ΔE16 variant in PC3 and MDA-MB-231. NRP1-ΔE16 variant corresponds to 1% and 3% of NRP1-FS in, respectively, MDA-MB-231 and PC3 cells.(TIF)Click here for additional data file.S2 FigRT-qPCR measurements were performed on total RNA extracted from cells and tissues from human (A) and mouse (B) origin as detailed in Materials and Methods. The ΔCt and the ratio Δ7/FS were calculated as described in Materials and Methods. (C) At the end of the run, the amplification products of NRP1-FS and NRP1-Δ7 were separated by gel electrophoresis and displayed the expected size.(TIF)Click here for additional data file.S3 FigPC3 or MDA-MB-231 (MDA) clones with inducible expression of recombinant full size NRP1 (FS) or recombinant NRP1-Δ7 (Δ7) were cultured for 24h in absence (-) or presence (+) of doxycycline (dox) at 200 ng/ml. For each clone, cells were processed for total RNA extraction to perform RT-qPCR analyses. The ΔCt and the ratio Δ7/FS were calculated as described in Materials and Methods.(TIF)Click here for additional data file.S4 FigMigration, proliferation and growth in soft agar assays were performed in absence and presence of doxycycline on cells transfected with an empty vector to assess the absence of influence of doxycycline by itself on these processes.(TIF)Click here for additional data file.S5 FigAdditional clones of PC3 and MDA-MB-231 were used for the evaluation of the NRP1 isoform-related effects on the cell migration.(TIF)Click here for additional data file.S6 FigApoptosis was evaluated by flow cytometry analysis of doxycycline-inducible clones of PC3 overexpressing NRP1-FS and NRP1-Δ7 after annexin V-FITC and Propidium Iodide staining as detailed in Materials and Methods.(TIF)Click here for additional data file.


AR) gene status measurement in plasma DNA using multiplex droplet digital PCR (ddPCR) in pre- and post-chemotherapy CRPC.There is an urgent need to identify biomarkers to guide personalized therapy in castration-resistant prostate cancer (CRPC). We aimed to clinically qualify androgen receptor  and progression-free survival (PFS) in 73 chemotherapy-naïve and 98 post-docetaxel CRPC patients treated with enzalutamide or abiraterone (Primary cohort) and 94 chemotherapy-naïve patients treated with enzalutamide .We optimized ddPCR assays for AR gain was observed in 10 (14%) chemotherapy-naïve and 33 (34%) post-docetaxel patients and associated with worse OS , PFS  and rate of PSA decline ≥50% . AR mutations [2105T>A (p.L702H) and 2632A>G (p.T878A)] were observed in eight (11%) post-docetaxel but no chemotherapy-naïve abiraterone-treated patients and were also associated with worse OS . There was no interaction between AR and docetaxel status . In the PREMIERE trial, 11 patients (12%) with AR gain had worse PSA-PFS (sPFS) , radiographic-PFS (rPFS)  and OS . Plasma AR was an independent predictor of outcome on multivariable analyses in both cohorts.In the primary cohort, AR status assessment using ddPCR identifies CRPC with worse outcome to enzalutamide or abiraterone. Prospective evaluation of treatment decisions based on plasma AR is now required.Plasma NCT02288936 . Testing for plasma epidermal growth factor receptor (EGFR) mutations has FDA clearance for selection of mutant lung cancer patients for EGFR tyrosine kinase inhibitors and studies of plasma DNA in multiple indications have suggested clinical utility for monitoring of mutations or copy number (CN) gain  as associating with resistance to abiraterone, shown previously to be activated by prednisone or progesterone, respectively  as aectively , 15. Forectively , 17 althectively , we aimeAR and an orthogonal approach, NGS, in samples collected before starting treatment and after disease progression. Our second objective was to evaluate associations between pre-treatment plasma AR and clinical outcome in a primary cohort, representative of both pre- and post-docetaxel patients, and test for interactions with prior chemotherapy exposure. As no trial to date has randomized patients between first-line enzalutamide or abiraterone and taxanes, we combined data from four cohorts of men recruited to two biomarker protocols and defined by treatment with enzalutamide or abiraterone and prior chemotherapy status. Our third objective was to test our ddPCR assay in a second cohort of chemotherapy-naïve men treated with enzalutamide in the PREMIERE trial.This was a multi-institution analysis of plasma samples collected prospectively in studies with the primary aim of biomarker evaluation. The objectives were defined after sample collection but before plasma analysis. Our first objective was to determine the correlation between ddPCR testing for plasma TMPRSS2-ETS in response to enzalutamide in patients with prostate cancer. Exploratory endpoints included circulating cell-free DNA and circulating tumor cell (CTC) analysis. Data emerging after the trial was designed and initiated  for splitting patients into two distinct prognostic groups . Use of this cut-off was also supported by 95.5% concordance between NGS and ddPCR for classifying AR CN status . Overall, 10 (14%) chemotherapy-naïve and 33 (34%) docetaxel-treated patients had AR gain  abiraterone patients were nt Table . We planAR gain and OS in both chemotherapy-naïve        (P = 0.41 for OS and P = 0.11 for PFS) or chemotherapy status  examined in the Cox models were not significant. We also evaluated the association of AR status with the rate of PSA decline in the chemotherapy-naïve and post-docetaxel groups. Chemotherapy-naïve patients with AR gain were 4.7 times less likely to have a ≥50% decline in PSA   , 6.3; 95% CI 0.72–54.59; P = 0.12) . We then carried out multivariable analysis with stepwise backwards elimination and the sole variables that remained significant were plasma AR status , total plasma DNA concentration and ALP levels for PFS  patients. CTCs were detected in 35 patients (37%). AR gain was detected in seven (20%) CTC-positive and four (7%) CTC-negative patients    . Patients with AR gain were less likely to have a ≥50% decline in PSA     in chemotherapy-naïve patients. Our assay detects point mutations present in at least 2% of plasma DNA. Greater sensitivity is obtained with higher input DNA [AR CN cut-off that best predicts associations with outcome was built with 171 patients. We plan to perform a meta-analysis of multiple trials when the data on AR CN acquired from different institutions and trials exceeds 1000 patients.We did not detect nput DNA  althoughAR CN is higher in the population with detectable CTCs although AR gain can also be observed in CTC-negative patients, accounting for one third of AR gained in the PREMIERE cohort. The overlap between AR-V7 positive and plasma AR gained patients and a comparison of the two tests in prospective trials is warranted to develop the best biomarker strategy for identifying resistant patients. Testing plasma AR status by ddPCR is affordable and can be widely implemented in clinical laboratories but does not control for plasma DNA tumor content [AR by ddPCR provides information on the outcome of men starting enzalutamide or abiraterone that is independent of other factors previously reported to be prognostic [AR aberrations is 10%–15% in this setting compared with 30%–40% post-docetaxel. As our study is single arm, the associations we report are prognostic although the association with PSA decline rate suggests plasma AR CN could identify patients resistant to enzalutamide or abiraterone. The aims of our study were defined after sample collection and therefore larger studies with a pre-specified primary objective of defining the association with outcome by plasma AR status could provide further supportive evidence for the role of AR CN as a biomarker in CRPC. For level one evidence to change clinical practice, our findings require confirmation in prospective trials where plasma AR CN defines treatment selection.Detection of AR splice variants in CTCs is also associated with shorter PFS and OS with enzalutamide or abiraterone , 29. AR  content  that mayognostic , 26, 30.Supplementary DataClick here for additional data file.


CREB3L4 resulted in decreased androgen-dependent PC cell growth. LNCaP cells transfected with siCREB3L4 underwent G2/M arrest, with upregulation of the proteins cyclin B1, phospho-CDK1, p21Waf1/Cip1, and INCA1, and downregulation of cyclin D1. Moreover, depletion of CREB3L4 resulted in significantly decreased expression of a subset of androgen-receptor (AR) target genes, including PSA, FKBP5, HPGD, KLK2, and KLK4. We also demonstrated that CREB3L4 directly interacts with the AR, and increases the binding of AR to androgen response elements (AREs). We also identified a role for the unfolded protein response , in activating CREB3L4. Cumulatively, we postulate that CREB3L4 expression is mediated by an AR-IRE1α axis, but is also directly regulated by AR-to-ARE binding. Thus, our study demonstrates that CREB3L4 plays a key role in PC cell proliferation, which is promoted by both AR and IRE1α.The incidence of prostate cancer (PC) is growing rapidly throughout the world, in probable association with the adoption of western style diets. Thus, understanding the molecular pathways triggering the development of PC is crucial for both its prevention and treatment. Here, we investigated the role of the metabolism-associated protein, CREB3L4, in the proliferation of PC cells. CREB3L4 was upregulated by the synthetic androgen, R1881, in LNCaP PC cells (an androgen-dependent cell line). Knockdown of  Prostate cancer is one of the most commonly diagnosed cancers among men in industrialized nations, and is a leading cause of cancer-related deaths326prostate-specific antigen (PSA) gene is the best-characterized androgen-responsive gene in the prostate. Biochemical and genetic studies revealed that both the enhancer and the promoter of the PSA gene are androgen-responsive (independent of each other), but for the maximum expression of PSA, both its promoter and enhancer activities are necessaryThe inositol-requiring enzyme 1 (IRE1α), as well as x-box binding protein 1 (XBP1). The latter two proteins target the ribosome-associated membrane protein 4 (RAMP4) and the ER degradation-enhancing alpha-mannosidase-like 1 (EDEM1) genes, in prostate cancer cellsIt has been well shown that the endoplasmic reticulum (ER) plays a key role in normal prostate function, and various stimuli that cause ER stress could result in prostate pathologies, including benign prostate hyperplasia (BPH) and prostate cancerCreb3l4 is expressed in preadipocytes and functions as a gatekeeper, inhibiting adipogenesisCreb3l4 disruption causes ER stress, formation of abnormal epididymal sperm nuclei, and caspase activation15CREB3L4  is an ER membrane-bound bZIP domain-containing transcription factorTo this end, we explored a role for CREB3L4 in the androgen-dependent prostate cancer cell line, LNCaP. Our overall results demonstrate a role for CREB3L4 in modulating AR action, suggesting an interrelationship, and an AR-ER stress-CREB3L4 signaling axis, in prostate cancer cell proliferation.CREB3L4 in prostate cancer cell lines, including the androgen-dependent prostate cancer (ADPC) cell line LNCaP, the androgen-insensitive metastatic subline C4-2B , the androgen-independent prostate cancer (AIPC) cell line PC-3, and the nontumorigenic prostatic epithelial cell line RWPE-1. As shown in CREB3L4, fatty acid synthase (FASN), and PSA, as compared to the other cell lines. Along with its mRNA, CREB3L4 protein expression was higher in LNCaP than other cell lines  in the presence or absence of R1881 (CREB3L4 with or without R1881 inhibited proliferation at D3 by 20% (p < 0.05), D6 by 50% (p < 0.05), and D8 by 50% (p < 0.05), respectively. Specifically, from D3 to D8 after transfection, androgen-dependent proliferation of LNCaP cells was suppressed by siCREB3L4 transfection  interacting with cyclin A1), in complex with CDK2, respectivelyCREB3L4  activated the PSA promoter activity by 50-fold, in the absence of androgen and AR , showing that the two proteins interact with each other  LNCaP cells  (inverted repeated (IR) sequence-IR3; AGAACANNNTGTTCT; ARE1~ARE4) within the 3 kb region of the human CREB3L4 promoter . We also showed that CREB3L4 directly associates with AR, to enhance AR-induced transactivation of its downstream genes, in prostate cancer cells. Furthermore, we observed that AR-induced CREB3L4 expression is also mediated by IRE1α, ER stress response signaling. Our data thus suggest that the novel link between AR-CREB3L4 and the IRE1α pathway is an axis critical for driving prostate cancer progression , and in part mediated indirectly, via an IRE1α pathway. Thus, we posit that CREB3L4 is an essential mediator of AR-IRE1α-induced prostate cancer progression.The unfolded protein response (UPR) has critical roles in development and normal physiology, as well as in pathological states such as cancerIRE1α . In addi pathway . These rCREB3L4 knockdown also increased the number of cells in G2/M, and upregulated p21Waf1/Cip1 and INCA1, both inhibitors CDK, regardless of the presence of androgen , in contrast to another studyPSA and other AR-target genes. We assume that independently bound CREB3L4 may interact with AR (bound to its ARE) on the PSA promoter, presumably by bending, “looping,” or some other unknown mechanism. Hence, more study is required to understand CREB3L4 activation of PSA, or other downstream genes, in a manner that is synergistic with AR.How does CREB3L4 regulate androgen-dependent cancer cell proliferation? We showed here that pression . Besidespromoter . This inFASN was increased in siCREB3L4 knockdown cells. The mechanism of downregulation of FASN by the AR-CREB3L4 axis may be different from that of other AR target genes. Distinct cofactor complexes with CREB3L4/AR may occur in its regulation of lipogenesis-related genes. Thus, the molecular mechanism of this distinct regulation needs further study.Previous reports have demonstrated that the enzymes involved in fatty acid synthesis and cholesterol synthesis are upregulated in prostate cancer cells by androgens27PSA)  . AdditioHEK293 (embryonic kidney) and PC3 (androgen-independent prostate cancer cells) were obtained from ATCC  and grown in Dulbecco’s modified Eagle’s medium , supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100-μg/ml streptomycin. LNCaP  and RWPE-1  cells were grown at the Roswell Park Memorial Institute  in medium 1640, supplemented with 10% FBS, and penicillin/streptomycin. C4-2B cell lines were supplied as a kind gift from Dr. H.G YoonsiRNA-transfected LNCaP cells were harvested by trypsinization, and fixed in ice-cold 70% ethanol overnight. The cells were then washed twice with cold PBS, and incubated for 30 min at room temperature in 1-mL PBS containing 50 μg/ml propidium iodide (Invitrogen) and 50 μg/ml RNaseA . The stained samples were then analyzed with a LSR-II flow cytometer .3 cells/well). Media containing 5% CT-FBS, with 10 nM R1881 or DMSO/EtOH (control), was changed every 2 days. At D3/D6/D8 after transfection, cell viability was measured, using the Cell Counting Kit-8 (Sigma) according to the manufacturer’s instructions. Briefly, 10-μl of CCK-8 solution was added to each well, and plates incubated for 4 hr, followed by measurement of absorbance at 450 nm, using a microplate reader.siRNA-transfected LNCaP cells were trypsinized and seeded in 5% CT-FBS-containing media, in the presence/absence of 10 nM R1881, in 96-well plates , according to the manufacturer’s instructions. This RNA was used to generate cDNA using the GoScript Reverse Transcription System (Promega). Quantitative real-time PCR (qPCR) was performed using the Step One Real-Time PCR Systems instrumentation and software , according to the manufacturer’s protocol. The relative amount of mRNA in each sample was normalized to that of the gene Waf1/Cip1 (#2947) , anti-HMGCS1 , and anti-Flag . Antibodies for FAS, ACCα, ACLY, and SREBP1 were kindly provided by Dr. K.S. Kim2HPO4, 2 mM KH2PO4, 0.1% Tween-20), for 1 h at room temperature. Antibody-bound proteins were visualized using an enhanced chemiluminescence detection system . The protein bands were also detected using a Fujifilm LAS-3000 Imager .Proteins from LNCaP/PC3/RWPE1/C42B cells were isolated using RIPA solution  containing appropriate protease inhibitors. Protein concentration was determined using the BCA protein assay (Thermo Scientific), and 40 μg were subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes . Membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies: anti-CREB3L4 , anti-INCA1 (sc-243077) , anti-AR (#5153s), anti-PSA (#2475s), anti-GAPDH (#2118), anti-IRE1α (#3294s), anti-Bip (#3711p), anti-eIF2α-total (#2103s), anti-eIF2α-phospho (#3597s), anti-cyclin B1 (#12231), anti-phospho CDK1 (#4539), anti-cyclin D1 (#2922), anti-CDK2 (#2546), anti-p21CREB3L4 siRNA, combined with 10 nM R1881 treatment. Cell lysates were then incubated for 15 hr at 4 °C with 5-μg anti-AR  or normal rabbit IgG  antibodies. After reversal of crosslinking, immunoprecipitated DNA was quantified by qPCR. All reactions were normalized relative to total input to account for chromatin sample preparation differences (ΔCt), determined as the difference between the α-AR IP sample (ΔCt [AR]) and α-IgG IP sample (ΔCt [IgG]) for fold enrichment. Relative expression was determined by the method ΔΔCt [AR - IgG] = ΔCt [AR] - ΔCt [IgG], and fold-change in occupancy = 2(−ΔΔCt[AR-IgG])PSA gene is described in ref. ChIP experiments were carried out according to the standard protocol . LNCaP cells were plated in 15-cm tissue culture plates and transfected with sihCREB3L4N and GFP-tagged AR were cotransfected into HEK293 cells using FuGENE HP transfection reagent (Promega). To confirm the endogenous interaction between CREB3L4 and AR, LNCaP cells were treated with 10-nM R1881 for 48 hr, harvested, and lysed in cold cell lysis buffer , containing appropriate protease inhibitors. Whole cell lysates (1000 μg) were precleared, with protein G-agarose , followed by incubation with anti-AR, anti-CREB3L4, or anti-Flag antibodies, for 16 h with protein G-agarose, for 2 h at 4 °C. After centrifugation, the protein G-agarose pellets were washed several times with washing buffer (1/3 dilution of cell lysis buffer in PBS) and resuspended in sample buffer, before being subjected to SDS-PAGE.Constructs expressing Flag-tagged CREB3L4  (Invitrogen) were synthesized. For silencing IRE1α, siRNA against IRE1α  was used. Medium GC Duplex was used as a negative control . Each siRNA (40 nM) was transfected into appropriate experimental sets of LNCaP cells, using Lipofectamine RNA iMAX (Invitrogen) for at least 48 h, followed by cell lysis for RNA and protein preparation.RNA oligonucleotides for human hCREB3L4 and hXBPs were PCR-amplified and cloned directly into the pcDNA3.1-Flag2 vector. The construct pEGFP-C1-AR was purchased from Addgene . The human PSA promoter, covering about 4.7 kb, was a generous gift from Dr. K.S. Kim . The mouse Creb3l4 gene promoter covering a −2500/+209 region was PCR-amplified and cloned directly into the pGL4b luciferase reporter vector.Expression constructs encoding the nuclear form of How to cite this article: Kim, T.-H. et al. The role of CREB3L4 in the proliferation of prostate cancer cells. Sci. Rep.7, 45300; doi: 10.1038/srep45300 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Robot-aided rehabilitation has become an important technology to restore and reinforce motor functions of patients with extremity impairment, whereas it can be extremely challenging to achieve satisfactory tracking performance due to uncertainties and disturbances during rehabilitation training. In this paper, a wire-driven rehabilitation robot that can work over a three-dimensional space is designed for upper-limb rehabilitation, and sliding mode control with nonlinear disturbance observer is designed for the robot to deal with the problem of unpredictable disturbances during robot-assisted training. Then, simulation and experiments of trajectory tracking are carried out to evaluate the performance of the system, the position errors, and the output forces of the designed control scheme are compared with those of the traditional sliding mode control (SMC) scheme. The results show that the designed control scheme can effectively reduce the tracking errors and chattering of the output forces as compared with the traditional SMC scheme, which indicates that the nonlinear disturbance observer can reduce the effect of unpredictable disturbances. The designed control scheme for the wire-driven rehabilitation robot has potential to assist patients with stroke in performing repetitive rehabilitation training. Stroke, an acute cerebrovascular disease typically caused by hemorrhage or blockage in brain blood vessels, is a major cause of motor dysfunction or even permanent disability . RepetitMechanical structure is one of the most important factors that can affect the effectiveness of the robot-assisted rehabilitation. In the past two decades, many robotic systems have been designed and applied in rehabilitation training. Conventional rehabilitation robots usually consist of several rigid links. MIT-MANUS has two degrees-of-freedom (DOF) and can guide the upper-limb over a horizontal plane . Mirror-Control scheme is another important factor affects the effectiveness of robot-assisted rehabilitation. When delivering task-oriented rehabilitation training, the control scheme is required to assist the robot in guiding the paretic limb to finish predefined movements or trajectory accurately and compliantly. Linear control techniques such as PID , 15 and In this paper, a wire-driven rehabilitation robot is designed to deliver task-oriented training exercises for upper-limb, which can work over a three-dimensional space. First, sliding mode control with nonlinear disturbance observer (SMCNDO) is designed for the wire-driven rehabilitation robot against unpredictable disturbances. Then, simulation of tracking a predefined trajectory is conducted to investigate the performance of the designed control scheme. Moreover, a square-shaped and a circle-shaped trajectory are designed, and the forearm of the subject is controlled by the robot to follow the predefined trajectories. Both simulation and experimental results of the designed SMCNDO are compared with that of a traditional SMC.Mechanical architecture of the designed wire-driven rehabilitation robot is shown in Figure x, y, z) represents the position of the end-effector, i varying from 1 to the number of wires, and  represents the position of the associated ith winch pulley.The geometry of the designed wire-driven rehabilitation robot is shown in Figure i, yi, zix) can be expressed specifically as follows:a represents the length of |OA|, b represents the length of |AB|, and c represents the length of |AC|. Therefore, from Eqs According to Table ows:2)x,y1,z1= is the vector of gravity terms, and d denotes the vector of all the lumped uncertainties . In this study, the designed wire-driven rehabilitation robot is considered to perform only translational movements, low velocities, and accelerations are involved in rehabilitation training, therefore Coriolis forces can be neglected. Hence, Eq. x, dyd, and zd represent the disturbances in X, Y, and Z directions. Therefore, referring to Eqs p generalized coordinates can be obtained as:Moreover, in this study, we assume that the motion is within the workspace and the gravity of the end-effector is able to keep all the wires under tension. Therefore, the relation between externally applied forces and the wire tensile forces can be expressed as follows :6)uxuy follows :7)M(p¨M(p¨(6)uxuxuy follnce, Eq. M(p)p¨+G is a discontinuous function, it can be given as follows:s) = [sgn(xs) sgn(ys) sgn(zs)]T, k  = diag(x ky kzk) and η = diag(ηx ηy ηz) are symmetric diagonal positive matrices need to be designed in practice. In this study, a switching sliding surface is considered by defining control gain as follows:Note that According to Eqs d is estimated as q is an auxiliary vector, γ = diag(γx γy γz) is a symmetric diagonal positive matrix. In this study, we consider that the disturbances are slow time-varying signal, which means A nonlinear disturbance observer has been introduced in many studies –28. In t^=q+γMp˙q˙=γ function is a discontinuous function, the control law is discontinuous across the sliding surface, which may cause chattering. Chattering is undesirable since it can cause damage to the actuators being controlled. Chattering reduction can be achieved by using a nonlinear disturbance observer to estimate and compensate disturbances; furthermore, chattering can be reduced by replacing the discontinuous sgn(s) function with a continuous sat(s) function. Using sat(s) function, the discontinuity of the controller is smoothed in a thin boundary layer neighboring the sliding surface. sat(s) function is defined as follows:As mentioned previously that via SMCNDO are compared with that of the traditional SMC. Parameters for the wire-driven rehabilitation robot are shown in Table pd(t) once and twice, respectively. The vector of disturbances along three coordinate directions is defined as follows:d1(t) = [5sin(t) 5sin(t) 5sin(t)]T, d2(t) is a vector of pulse-like disturbances which is defined as follows:To investigate the effectiveness of the presented SMCNDO for the wire-driven rehabilitation robot, first, simulation is carried out in Matlab/Simulink. Both SMCNDO and the traditional SMC are considered in the simulation, and the results of tracking a predefined trajectory λ = diag, k = diag, η1 = 0.1, η2 = 80, β = 0.5, and proper control parameters for SMCNDO are given as: λ = diag, k  = diag, γ = diag, η1 = 0.1, η2 = 80, β = 0.5.Both the traditional SMC and SMCNDO can be designed according to Eq. λ = diag, k  = diag, η1 = 2,000, η2 = 500, β = 0.1, and proper control parameters for SMCNDO is chosen as: λ = diag, k  = diag, η1 = 2,000, η2 = 500, β = 0.1, γ = diag. The square-shaped trajectory which is predefined as the reference trajectory for the experiment is shown in Figure Experiments are carried out based on a real-time system to verify the performance of the presented controller for practical applications. It has been approved by the ethics committee of the Injury Rehabilitation Hospital of Guangdong Province. During the experiments, a healthy subject  is seated beside the wire-driven rehabilitation robot with his forearm placed on the splint. The subject is instructed to keep relaxed during the experiments. Then, the forearm of the subject is controlled by the robot to follow predefined trajectories, and two different types of trajectories are considered in this section, including a square-shaped and a circle-shaped trajectory. The results of SMCNDO are compared with that of the traditional SMC. The traditional SMC and SMCNDO are designed according to Eq. The desired velocity and acceleration can be obtained by differentiating the desired position once and twice, respectively, each experiment of tracking the circle-shaped trajectory lasts 62.8 s. The predefined circle-shaped trajectory is visually shown in Figure X, Y, and Z directions are shown in Figures via the traditional SMC and SMCNDO are drawn in blue dashed line and red dashed line, respectively, and the predefined trajectory is drawn in black-solid line. The output forces for the three wires in simulation via SMC and SMCNDO are shown in Figure via SMC for wire 1, wire 2, and wire 3, respectively. Figures via SMCNDO for wire 1, wire 2, and wire 3, respectively. From Figure via SMC is relatively severe, whereas chattering is reduced effectively by implementation of SMCNDO. These simulation results indicate that when control the wire-driven robot follow a predefined trajectory in the presence of unknown disturbances, SMCNDO has better tracking performance and lower chattering compared with the traditional SMC.Tracking performance of the traditional SMC and SMCNDO in simulation are shown in Figure The tracking errors of the two applied control schemes in the three coordinate directions when a healthy subject follows the predefined square-shaped trajectory are shown in Figure The tracking errors along the three coordinate directions when tracking the predefined circle-shaped trajectory by implementation of SMC and SMCNDO are shown in Figure The designed wire-driven rehabilitation in this paper has three DOF and its architecture makes it possible to assist the subject in performing predefined movements in a three-dimensional space. Coordinate system that applied in the control scheme design procedure is considered in this paper, which can be classified into the wire length coordinate system and the task space coordinate system. In the wire length coordinate system, the length of each wire is measured by scaling the associated motor encoder count; however, due to the unavailable flexibility of wires, using the wire length coordinate system is not reliable when the implementation may require high accuracy. In this paper, this problem can be effectively addressed by using the task space coordinates , where tvia SMCNDO. The results of experiments are consistent with those of simulation. In literature, many studies have found that the traditional SMC is not able to achieve satisfactory tracking performance and can cause high chattering (From the results of both simulation and experiments, the traditional SMC has degraded tracking performance and can cause high chattering in all the three coordinate directions, while tracking performance can be obviously improved and chattering can be effectively reduced attering –31. Mohaattering , Deshpanattering , the resattering , human-aattering , and uncattering , whereasThere are a few limitations of this study that should be addressed in the future. The positions of the wires should be well arranged to ensure enough workspace and the safety for upper-limb rehabilitation in clinical application. In this pilot study, we mainly focus on the investigation of the feasibility of the wire-driven rehabilitation robot using SMCNDO, in the future, we will recruit enough patients after stroke to investigate its clinical effectiveness in rehabilitation training.In this paper, a wire-driven rehabilitation robot is designed for upper-limb rehabilitation training, and SMCNDO is designed for this robot. Simulation and experimental results of trajectory tracking show that the wire-driven rehabilitation robot with the designed control scheme has exhibited two superiorities including tracking performance improvement and chattering reduction as compared with the traditional sliding mode control scheme. The wire-driven rehabilitation robot with the designed control scheme may have great potential in robot-aided rehabilitation training.This study was approved by the ethics committee of the Injury Rehabilitation Hospital of Guangdong Province.JN and RS conceived and designed the study. JN and QY performed the experiments, wrote the paper, contributed to the work equally, and should be regarded as cofirst authors. RS reviewed and edited the manuscript. XW made a contribution to experiments. All authors had read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.