Work-related injuries of the shoulder complex represent a challenge for clinicians because of the large variety of clinical entities involved and the broad anatomic structures that can be affected. Furthermore, commonly performed orthopedic tests have demonstrated limited accuracy for diagnosing the injury despite considerable research efforts. The aim of this study protocol is therefore to describe a comprehensive approach integrating both a clinical- and functional status-based pathology and an adapted rehabilitation prescription.A longitudinal cohort study will be performed at the Department of Rehabilitation and Medical Assistance of a mutual insurance society for work-related injury management in Spain . Patients will be attended by an occupational physician who specializes in work-related injuries and is part of the project team that will systematically visit all the participants. After the medical diagnosis and any requested supplementary evaluations , the patients will be referred to the rehabilitation service. Before the physiotherapeutic rehabilitation program is initiated, the patients will undergo a comprehensive functional screening at the biomechanics laboratory. Using a decision-making scheme, the identified functional deficits will be used to customize the individual rehabilitation plan.The proposed objective criteria-based shoulder diagnosis and rehabilitation model could be a new effective strategy for minimizing the time required to regain functional capacity and recover from symptoms among patients with work-related shoulder injuries.NCT02732002 (April 10th 2016).The study protocol has been registered on Clinical Trials.gov as The online version of this article (doi:10.1186/s12891-017-1435-2) contains supplementary material, which is available to authorized users. Persistent shoulder pain and functional impairments are common among patients with shoulder injuries, accounting for 5% of all medical practice visits . As repoShoulder pain can arise from different anatomical structures or a combination of several structures. Thus, subacromial or external impingement syndrome (SIS), rotator cuff pathology, superior labrum anterior posterior (SLAP) injury and glenohumeral (GH) ligament insufficiency are the most frequently cited anatomical pain-eliciting structures . Other aSeveral orthopedic tests are commonly performed to identify the clinical entity causing the pain. In short, these tests involve moving the affected arm into different positions, which are believed to irritate different shoulder structures , 6. DepeSeveral biomechanical models of shoulder function in the presence or absence of shoulder injury –16 have However, in the authors’ opinion, a comprehensive rehabilitation model for shoulder injuries that integrates the clinical entity and functional status and provides an individualized exercise rehabilitation program based on all the information obtained from both clinical and functional examinations is lacking. The purpose of the present study protocol is therefore to establish a comprehensive clinical model for shoulder injury management that integrates both clinical and biomechanical assessments and targets individual deficits in the posterior rehabilitation program. The present study protocol will be implemented by a non-lucrative public medical insurance corporation that functions as a satellite of the national public health system in Spain.If our hypothesis is correct, an objective criteria-based rehabilitation algorithm (OCBRA) for rehabilitation could become a new effective strategy for improving functional capacity and symptom recovery among patients with shoulder injuries. This intervention is designed to improve the rate of complete return to previous activity levels, for which a concise clinical diagnosis and functional status picture seems imperative.This longitudinal cohort study with a historical clinical cohort serving as control group will be conducted at the Department of Rehabilitation and Medical Assistance of a mutual insurance society for work-related injury management in Spain . The patients will be attended by a physician trained in the protocol implemented during this research. Following medical diagnosis and supplementary evaluations when requested , the patients will be referred to the rehabilitation service. Before initiating the physiotherapeutic rehabilitation program, the patients will undergo comprehensive functional screening at the biomechanics laboratory. Using a decision-making scheme, the identified functional deficits will be used to customize an individual rehabilitation plan. The rehabilitation plan will be conducted at the physical rehabilitation facilities of the institution by any of the 7 physical therapists working in the area . All the participants will be informed of the objectives and risks of the intervention. All the participants will sign an informed consent. The subjects’ names will be coded to preserve their anonymity.The study decision-making scheme, i.e., the Objective Criteria Based Rehabilitation Algorithm (OCRBA), is presented in Table Full recovery will be determined by negative results on the last medical examination  and positive results on the biomechanical evaluation . As the clinical cohorts will not undergo functional evaluations, the time to full recovery and the number of physician and physiotherapy visits will be analyzed to compared the cost-effectiveness of the two injury management models .Every Spanish-speaking patient over 18 years of age seeking treatment from a physician at our institution due to work-related shoulder complaints of a mechanical origin  lasting more than 6 weeks will be eligible for our rehabilitation model. This preliminary time-based criterion for exclusion from the program is designed to prevent the inclusion of transient shoulder ailments that could adequately resolve with rest and NSAIDs .The clinical reasoning for patient investigation using the rehabilitation model is adapted from previously published data by Cools et al. . These aThus, in the present study, we aim to complement the model in two different ways. First, we will record the quantitative variables of ROM (°) and force output (N) for each of the orthopedic tests performed (see the primary outcome measures section for more details). This protocol supplementation will reinforce the posterior rehabilitation program by addressing the specific muscle force deficits observed during the complete evaluation, thus complementing the clinical diagnosis made during the evaluation itself. Thus, a functional deficits-based rehabilitation program that addresses both the clinical entity and the functional status of the shoulder will be prescribed.The patient will complete the initial clinical and functional evaluation in the medical room and the laboratory. Afterwards, all the gathered information will be assembled to generate the patient-specific functional  and clinical  status-based rehabilitation program. This program will be generated in conjunction with the physical therapy staff Table . For eacPain levels during the complete clinical and functional examinations will be recorded using the previously validated visual analogue scale (VAS)  at the tThe shoulder range of motion (ROM) will be measured using 3 iso-inertial unit STT-IBS -based technology.© is a 9-degrees-of-freedom inertial measurement unit that integrates an accelerometer, a gyroscope and a magnetometer in each of its axes. The system measures the relative orientation, acceleration and position  of the STT-IBS© sensors and sends this information to a computer with a Bluetooth-enabled host. The raw signals are processed online using iSens software , which provides the angular velocity, the acceleration and the angular position of each STT-IBS. Furthermore, after the preferred model is selected  and the sensor units are placed accordingly, the software provides the angular measurement of the selected movement in each plane.The STT-IBSAfter determining a reference position or zero position, the software measures this angular position as the projection of the vector position of each sensor in the corresponding plane determined by the reference sensor .The 3 STT-IBS units will be placed on the arm and forearm of the upper limb with straps (moving sensors) and at the patient’s inter-scapular level (reference sensor) with double-sided tape according to the specifications of the models provided by the manufacturer . Three consecutive FLX and EXT movements starting from a neutral position will be performed. Next, the ABD movement will be measured. Similar to the previous movement, the patient will be asked to perform 3 consecutive ABD maneuvers, returning to a neutral position between repetitions.Finally, the IR/ER task will be analyzed. The patients will be positioned with the arm parallel to the ground (90°ABD), with the forearm perpendicular to the arm and the ground and parallel to the trunk Fig. . When reData analysis will be performed offline. The raw angular data of each pair of movements  will be exported to an Excel file , and the maximum and minimum values will be obtained. The maximum values correspond to the FLX, ABD and IR movements. Conversely, the minimum values correspond to the EXT, ADD and ER. The mean of 3 repetitions for each movement will be obtained for further statistical analysis.The shoulder range of motion (ROM) will be measured using 3 iso-inertial unit STT-IBS -based technology.© is a 9-degrees-of-freedom inertial measurement unit that integrates an accelerometer, a gyroscope and a magnetometer in each of its axes. The system measures the relative orientation, acceleration and position  of the STT-IBS© sensors and sends this information to a computer with a Bluetooth-enabled host. The raw signals are processed online using iSens software , which provides the angular velocity, the acceleration and the angular position of each STT-IBS. Furthermore, after the preferred model is selected  and the sensor units are placed accordingly, the software provides the angular measurement of the selected movement in each plane.The STT-IBSAfter determining a reference position or zero position, the software measures this angular position as the projection of the vector position of each sensor in the corresponding plane determined by the reference sensor .The 3 STT-IBS units will be placed on the arm and forearm of the upper limb with straps (moving sensors) and at the patient’s inter-scapular level (reference sensor) with double-sided tape according to the specifications of the models provided by the manufacturer . Three consecutive FLX and EXT movements starting from a neutral position will be performed. Next, the ABD movement will be measured. Similar to the previous movement, the patient will be asked to perform 3 consecutive ABD maneuvers, returning to a neutral position between repetitions.Finally, the IR/ER task will be analyzed. The patients will be positioned with the arm parallel to the ground (90°ABD), with the forearm perpendicular to the arm and the ground and parallel to the trunk Fig. . When reData analysis will be performed offline. The raw angular data of each pair of movements  will be exported to an Excel file , and the maximum and minimum values will be obtained. The maximum values correspond to the FLX, ABD and IR movements. Conversely, the minimum values correspond to the EXT, ADD and ER. The mean of 3 repetitions for each movement will be obtained for further statistical analysis.Throughout the screening examination, each of the orthopedic tests will be performed using a hand-held dynamometer  to register the peak force (N) exerted during each task in addition to the standard clinical interpretation of the pain elicited during the maneuver. Three repetitions will be performed for each limb at each testing position. The first repetition will serve as familiarization, whereas the subsequent two repetitions will be recorded for further analysis.Self-reported shoulder function will be registered using the Simple Shoulder Test Questionnaire (SST). The questionnaire consists of 12 items answered with dichotomous responses (yes/no). Two questions are related to pain, 7 are related to function and strength, and 3 are related to range of motion perceptions. The minimum clinically important difference between pre- and post-rehabilitation evaluations was set at 2 to 2.33 points . The queWhen the study concludes, the number of rehabilitation and medical visits administered and the number of working days lost among the participants will be analyzed. Thus, the economic burden of the rehabilitative process  will be determined for comparison with historical cohorts from the same medical institution (60 patients recruited the year before the OCBRA model is implemented). The number of overall rehabilitation and medical visits made will by multiplied by their typical economic costs at the hosting medical institution. The number of sessions administered and the number of work days missed will be provided by the mutual insurance society for work-related injury management. The economic burden of both medical costs and missed work days will be provided by the same institution following the recommendations of the National Social Health Agency of the Spanish Heath Service Ministry. The cost effectiveness ratio will be calculated as the number of visits + missed work days divided by the sum of the economic burdens.Considering the multi-factorial nature of the present study’s design, statistical power should be adjusted (and sufficient) for each of the primary outcomes analyzed. A minimum of 80% statistical power will be guaranteed for all of the primary outcome variables . Thus, an is the number of patients required in each group, d is the standardized difference and cp,power is a constant defined by the values chosen for the P (0.05) value and power (0.8) for this study, resulting on a constant value of 7.9.Where X2 analysis. Following logarithmic transformation if required, mean changes in pain intensity, functional limitation (Simple Shoulder Test questionnaire), ROM and force application capability will be compared using a repeated measures paired T-Test. All analysis will be performed with SPSS© . The level of statistical significance will be set at p < 0.05. Simultaneously, the magnitude of the treatment effect will be tested by the determination of the effect size (Cohen’s d). This analysis will be performed as the standardized difference between two means (pre and post rehabilitation program). Cohen’s standard attributes and effect size for the intervention based on the magnitude of the observed size will be described as a large effect size for 0.8 or greater, medium for 0.5 to 0.8 and small for 0.2 or less [In relation to statistical analysis of the collected variables, descriptive statistics and normality (Shapiro Wilk test) and variance homogeneity (Levene test) tests will be performed for intra-group analysis (patients recruited in the proposed injury management model). Perceived changes will be compared using cross tabulations with  or less .The present study protocol aims to complement previously published shoulder injury management algorithms. The proposed objective criteria-based rehabilitation algorithm (OCBRA) focuses on the clinical entity of the examined shoulder and its functional status with respect to the joint range of motion, the force exertion capability and the pain elicited during the evaluation. Thus, the prescribed rehabilitation program is designed to treat the clinically observed affectation and the actual and quantitative deficits identified through a standardized functional evaluation.Hegedus et al.  recentlyIn relation to exercise protocols for shoulder injuries, a wide source of scientific investigations has addressed EMG activity , 27, 28,However, in the authors’ opinion, this is the first study aiming to fuse the clinical entity with an objective and standardized functional evaluation and a coordinated  rehabilitation program to help patients fully recover from multi-etiological shoulder complaints. If this new rehabilitation model proves to be useful, it could be instituted by several health companies to improve shoulder injury management routines, thereby hastening patients’ return to previous activity levels and reducing the economic burden associated with these pathological conditions.The trial will start recruitment on January 2017. The trial will remain undecidedly open given that the present intervention program is the standard for shoulder injury care at the friendly society for work-related injuries management wherein the study is being performed. Once 100 patients are enrolled, a case- series retrospective study will be performed to compare the cost efficiency in terms of time duration, number of physical therapy and physician visits perceived and economical burden of the present shoulder injury management program vs. historical cohorts of patients receiving care at our institution.


Value in health care represents the relationship between health outcomes achieved and resources used. In a highly prevalent condition it is important to ensure that the highest value interventions are employed effectively In COPD these are influenza vaccination, smoking cessation and pulmonary rehabilitation . Muscle fatigue is an important symptom limiting exercise. The main driver is physical inactivity and this occurs in early in the course of the disease in particular this may be before it has been diagnosed. Physical inactivity may itself drive lung disease progression.As well as a loss of muscle bulk there is a shift away from a Type I fibre, oxidative endurance muscle phenotype. The underlying biology is complex and in addition to inactivity, inflammation, corticosteroids, reduced anabolic hormones, corticosteroid treatment, hypoxia, poor nutrition and increased resting energy expenditure may all play a role. There is a recent ATS/ERS statement on limb muscle dysfunction in COPD.Exercise has a wide range of beneficial effects. It can improve exercise capacity, lipid profile, reduce falls, reduce cardiac risk, improve depression, insulin sensitivity, and systemic inflammation as well as protecting against cognitive decline and osteoporosis.Pulmonary rehabilitation has been defined as “an evidence-based, multidisciplinary, and comprehensive intervention for patients with chronic respiratory diseases who are symptomatic and often have decreased daily life activities. Integrated into the individualized treatment of the patient, pulmonary rehabilitation is designed to reduce symptoms, optimize functional status, increase participation, and reduce health care costs through stabilizing or reversing systemic manifestations of the disease.”A standard PR course might be ∼8 weeks of twice weekly sessions as well as home exercise being encouraged. A mixture of aerobic and strength training is used and it is supervised by health professionals. The venue can be a hospital gym or a community gym or other space. It should be integrated into clinical management and include education about self-management and other issues.There is a compelling evidence base for benefit of pulmonary rehabilitation in COPD, reversing the effects of deconditioning improving exercise capacity and quality of life. The cost per QALY of pulmonary rehabilitation is significantly better than for pharmacotherapy in COPD.Pulmonary rehabilitation in the early post-exacerbation phase reduces readmission and improves survival with a number needed to treat of 4 to prevent one readmission.This recent review on PR for non-COPD lung disease is useful.- Smoking cessation- Inhaler technique- Written self-management plan- Assessment for pulmonary rehabilitation- Follow up arrangement made before dischargeOne way to improve pulmonary rehabilitation uptake is the use of a COPD discharge care bundle has been developed with input from guidelines, clinicians and patients which includes the following items:Patients also complete a safe discharge checklist with the nurse responsible for their care. Evidence suggests that it may reduce readmission rates and improve quality of care. The documents are all available in the online supplement (open access).


Integrin αVβ3 is a malignant driver of anchorage-independence and tumor angiogenesis, but its dysregulation in hepatocellular carcinoma (HCC) remains unclear. In this study, we observed that sulfatide significantly promoted integrin αV(ITGAV) expression and wound closure in HCC. We also noted that elevated sulfatide profoundly stimulated integrin αVβ3 clustering and signaling. In the cells with integrin αVβ3 clustering induced by sulfatide, integrin β3 subunit was phosphorylated. Simultaneously, focal adhesion kinase (FAK), Src and paxillin were also phosphorylated. Treatment with FAK inhibitor resulted in robust suppression of FAK-Y397 and Src-Y416 phosphorylation stimulated by sulfatide, but not suppression of integrin β3 phosphorylation. Src inhibitors repressed Src-Y416 and FAK Y861 and Y925 phosphorylation, but not FAK-Y397 and integrin β3 phosphorylation. After mutation of integrin β3 (Y773F and Y785F), FAK or Src phosphorylation failed to be stimulated by sulfatide. Moreover, β3 Y773 and Y785 phosphorylation was suppressed by insulin-like growth factor receptor knockdown even in cells stimulated by sulfatide. In assays of immunoprecipitation and immunostaining with integrin αV or β3 antibody, labeled sulfatide was found in the complex and co-localized with integrin αVβ3. Taken together, this study demonstrated that elevated sulfatide bound to integrin αVβ3 and induced clustering and phosphorylation of αVβ3 instead of matrix ligand binding, triggering outside-in signaling. Hepatocellular carcinoma (HCC) is one of the most malignant diseases worldwide, especially in Asia, where there is a high prevalence of chronic viral hepatitis infection. Tumor metastasis is the major cause of HCC-related mortality. Invasive primary HCC breaches the basement membrane and invades the lymphatic or blood microvessels, from where the tumor cells are transported to distant sites to form dormant micrometastases which may eventually acquire the ability of colonization and angiogenesis, enabling them to form a macroscopic metastasis . HCC comThe noncatalytic cytoplasmic tail (CT) domain of integrin β3 subunit is laden with various phosphorylation sites, including two tyrosine residues, one serine, and multiple threonine residues . The twoSulfatide is a cell membrane component that is modified from cerebroside by adding a sulfate group by cerebroside sulfotransferase(CST). Sulfatide is significantly elevated in some cancers such as HCC. Nevertheless, the biological and pathological significance of elevated sulfatide in HCC is largely unknown . Our preAs compared to DMSO control, galactocerebroside, lactocerebroside and ganglioside, sulfatide interestingly enhanced the expression of integrin αV subunit Figure  in both To uncover the initial regulation event in the HCC cells, we investigated and observed integrin clustering activity after treatment. Compared to control groups, cells treated with sulfatide showed extensive clustering of integrin αV, which was similar to the clustering induced by specific antibody against integrin αV Figure . In ordeLateral association and clustering of integrins require integrin activation which is accompanied by tyrosine phosphorylation of the β3 subunit. Although the level of phosphorylated integrin β3 at Y773 and Y785 seemed not significantly different 24 h after sulfatide treatment Figure , a signiNow in the cells treated with sulfatide, integrin αVβ3 was activated. FAK and Src were also observed that they were phosphorylated and activated. To study further their role in the integrin activation and signaling, we examined both FAK and Src inhibitors, and observed that they were phosphorylated and activated sequentially. FAK inhibitor 14 significantly suppressed the sulfatide-stimulated phosphorylation of FAK on Y397 and attenuated the activation of Src (p-Src-Y416), but not phosphorylated integrin β3 (β3-Y773) Figure . HoweverThe integrin clustering correlates with integrin activation, which needs ligand binding in the process since the activated integrins require ligands to form integrin clusters and to induce tyrosine phosphorylation of the β subunit. OPN is a small integrin-binding protein and can bind to integrin αV via its RGD motif . To undeIntegrin αVβ3 binding to ligand molecules is through RGD-binding sites, and upon ligand binding, the cytoplasmic domain of the integrin β subunit associates with signaling and cytoskeletal proteins. To investigate the role of RGD-binding site in αVβ3 and sulfatide interaction, we used the linear synthetic RGD peptide (GRGDSP) to block the RGD binding site of integrin αVβ3 with the negative control GRGESP. RGD peptide inhibited sulfatide-stimulated phosphorylation of integrin β3-Y773, β3-Y785, FAK-Y397, FAK-Y861, FAK-Y925 and Src-Y416 in both SMMC-7721 and BEL-7404 cells Figure . To deteIn sulfatide-induced activation of integrin β3/FAK/Src signaling, integrin β3 was phosphorylated. However, both integrin β3 and sulfatide lack kinase activity. The phosphorylation of integrin β3 subunit needs an additional kinase molecule. In this process of sulfatide-stimulated activation of integrin αVβ3, the inhibitors of Src kinase failed to prevent the phosphorylation of β3 subunit. We silenced IGF1R and IGF2R to investigate their roles in the phosphorylation of integrin αVβ3 and the results indicated that the sulfatide-stimulated phosphorylation of integrin β3 was suppressed in the cells with silenced IGF1R or IGF2R expression (siIGF1R and siIGF2R) Figure . TherefoIntegrin αV abnormality is associated with malignancy in many cancers. The tissues of non-small cell lung cancer were observed abnormal elevation of integrin αV expression that was believed to promote tumor proliferation and progression . OverexpTyrosine phosphorylation of integrin β1 subunit inhibits talin and kindlin binding to NPXY sequence leaving room for other proteins to bind, for example, filamin and tensin . Loss ofHow sulfatide induces integrin αVβ3 phosphorylation by IGFR after stimulation should be the next question. We then investigated the interaction between sulfatide and integrin αVβ3, and observed that sulfatide bound to integrin αVβ3 via the RGD binding domain. Blocking this domain by RGD peptide suppressed sulfatide binding and integrin αVβ3 clustering or phosphorylation. Therefore, it was the sulfatide binding that induced clustering and phosphorylation of integrin αVβ3. Integrin clustering classically requires the interaction with and binding of ECM ligands to induce activation of integrin. As integrin binds to the ECM ligand, it forms the first contact with the ECM in the lamellipodia, and the integrin activator, talin or kindlin associates with the cytoplasmic domain of the β subunit to induce conformational change and integrin activation. This process is followed by integrin lateral movement on the membrane, clustering, and phosphorylation of β subunit. Sulfatide, as an important component of the cell membrane, is often elevated in HCC . The eleRabbit antibodies against phosphorylated FAK , phospho-paxillin-Y31, phospho-caveolin-1-Y14, phosphorylated integrin β3 (Tyr773/Tyr785) and non-phosphorylated FAK, paxillin and caveolin-1 were from Bioworld Technology . Phosphorylated FAK Tyr407 was from Abcam . Phospho-Src-Y416 was from Cell Signaling Technology . Antibodies against integrin αV, β3, Src and FAK inhibitor 14 were from Santa Cruz Biotechnology . Integrin αVβ3 antibody LM609 was from Millipore . Integrin β1 and β5 antibodies, PP2-1--) and SU6656((Z)-N, N-dimethyl-2-oxo-3-methylene)indoline-5-sulfonamide) were from Merck . Sulfatide, galactocerebroside, lactocerebroside and ganglioside (GM3) were from Sigma–Aldrich . Peptides GRGDSP and GRGESP with 99% purity were provided by China Peptides Co. . Bodipy FL-C5 was from Molecular Probes .HCC cell lines SMMC-7721 and BEL-7404 were obtained from Shanghai Institute of Biochemistry and Cellular Biology, Chinese Academy of Science, and authenticated. Cells were cultured in Dulbecco's Modified Eagle's Medium  complete medium containing 10% newborn calf serum. For treatment, cells were cultured in DMEM containing 2 μM sulfatide, galactocerebroside, or lactocerebroside from stock solution in DMSO. An equal amount of DMSO (0.1% v/v) was added to the control group.http://rnaidesigner.lifetechnologies.com/rnaiexpress/design.do). Synthetic 55-bp oligonucleotides containing the siRNA sequences were annealed and ligated to pSilencer 4.1 vector. Tyrosine to phenylalanine mutations of integrin β3 at 773 and 785 were introduced into the integrin β3 cDNA by PCR mutagenesis using primers consisting of 5′-AAAGAATTCATGCGAGCGCGGCC-3′, 5′-TGGCT TCTTTAAACAGTGGGT-3′, 5′-CAACAACCCACTGT TTAAAGAGG-3′, and 5′-GGGTCTAGATTAAGTGCCCCGGAACGTGAT-3′.The short hairpin sequences, 5′-GATCCGCTGAGCTCATCGTTTCCATTCAAGAGATGGAAACGATGAGCTCAGCAGA-3′ and 5′-AGCTTCTGCTGAGCTCATCGTTTCCATCTCTTGAATGGAAACGATGAGCTCAGCG-3′, which specifically targeted the mRNAs of integrin αV subunit, 5′-GATCCCCAAGACTCATATAGCATTTTCAAGAGAAATGCTATATGAGTCTTGGAGA-3′ and 5′-AGCTTCTCCAAGACTCATATAGCATTTCTCTTGAAAATGCTATATGAGTCTTGGG-3′ for targeting integrin β3 subunit, 5′-GATCCGCGGCTGGAAACTCTTCTATTCAAGAGATAGAAGAGTTTCCAGCCGCAGA-3′ and 5′-AGCTTCTGCGGCTGGAAACTCTTCTATCTCTTG AATAGAAGAGTTTCCAGCCGCG-3′ for insulin-like growth factor 1 receptor (IGF1R), 5′-GATCCCCTGGGAACTCCTGAATTTTTCAAGAGAAAATTCAGGAGT TCCCAGGAGA-3′ and 5′-AGCTTCTCCTGGGAACTC CTGAATTTTCTCTTGAAAAATTCAGGAGTTCCCAG GG-3′ for IGF2 receptor (IGF2R) were designed through the website of Life Technologies . The protein was resolved on 10% SDS-polyacrylamide gels and electrophoretically transferred to a polyvinylidene fluoride membrane. Blocked with 8% milk for 2 h at room temperature, membranes were incubated overnight with primary antibodies. After washing, the blots were incubated with horseradish-peroxidase-conjugated secondary antibody, and protein bands were visualized using enhanced chemiluminescence detection kit and captured by an E/M camera. The density of protein bands was analyzed with Band Analyzer software.After cells were harvested and lysed with 1% SDS containing 50 mM NaF, 1.5 mM NaThe cells were incubated with 2 μM Gal-Cer, sulfatide or 10 μg/ml anti-integrin αVβ3 antibody for 1 h at 10°C in DMEM. Cells were fixed with freshly prepared 4% paraformaldehyde in PBS for 20 min at room temperature, and blocked with 1% bovine serum albumin in PBS for 2 h at room temperature without permeabilization. Cells were incubated with primary antibodies against integrin αV or αVβ3 overnight at 4°C, followed by FITC-conjugated secondary antibody and DAPI  at room temperature in the dark for 2 h. The coverslips were mounted on glass slides. Images were captured using an Olympus fluorescent microscope .Before fixing, cells in a Lab-Tek chamber  were treated with 2 μM Gal-Cer or sulfatide for 48 h at 37°C. After washing with PBS four times and permeabilization with 0.1% Triton-X-100 in PBS in a shaker for 5 min, the cells on slides were incubated with rhodamine–phalloidin (1 μg/mL) and DAPI for 20 min at room temperature. Then slides were rinsed in PBS and mounted for microscopy. Images were captured using an Olympus fluorescent microscope.5 cells into each well of six-well plates. The cell monolayers were scratched with a sterile micropipette tip, washed with PBS to remove the detached cells, and incubated with 2 μM sulfatide and Gal-Cer. The scratches were monitored every 24 h until they closed. The rate of wound closure was determined by calculating the percentage of the area covered by migrating cells in the initial wound. All experiments were repeated three times independently.The cells were seeded at a density of 5.0×105/mL in serum-free DMEM in triplicate, and medium containing 10% fetal bovine serum was added to the lower chamber. Cells were allowed to migrate across polyporous membrane for 12 h at 37°C. The migrated cells on the downside of the membrane were fixed with methanol, stained with 0.5% crystal violet, and counted. The average number of migrated cells was counted from three independent experiments. Each set of experiments was performed in triplicate separately.Cell suspensions (100 μL) with 2 μM Gal-Cer or sulfatide were seeded in Millicell inserts  with polyethylene terephthalate microporous membrane (8 μm pore size) at a density of 2×103VO4, 10 μg/mL aprotinin, 2 mM PMSF and 10 mM NaF for 1 h at 37°C. The lysates were immunoprecipitated with anti-integrin αV or β3 antibody along with protein A/G-agarose beads. Immunocomplex-captured beads were washed with lysis buffer. Bodipy fluorescence was determined . Afterwards, immunocomplexes captured by beads were boiled in Laemmli sample buffer. The proteins were separated by 10% SDS-PAGE and western blotted using anti-integrin αV or β3 antibody.Bodipy labeling assay was performed as described previously . Sulfatit test. Statistical significance was set at P < 0.05 or P < 0.01.Data are presented as means ± SD. Statistical differences were measured using one-way ANOVA and Student's


Previous studies show that androgens are involved in hypertrophy and excitability of cardiomyocytes and that their effects are mediated through their receptor. The aim of this study was to evaluate the presence of androgen receptor (AR) in mouse heart during prenatal and early postnatal stages.The expression of AR and related genes, alpha myosin heavy chain -Myh6-, beta myosin heavy chain -Myh7- and atrial natriuretic factor –Nppa- was simultaneously evaluated by semiquantitative RT-PCR. AR was also detected by immunohistochemistry. Androgen receptor mRNA was detected in hearts from 10.5 days post coitum to 16 postnatal days. A higher expression of AR mRNA in atria compared to ventricles was observed in neonatal mouse. A positive correlation between mRNA levels of AR and Nppa was observed in mouse heart at early postnatal development. Androgen receptor expression is similar in males and females during cardiac development. Finally, androgen receptor protein was observed by immunohistochemistry in myocardial cells of atria and ventricles from 12.5 days onwards and restricted after 16.5 days post-coitum to nuclei of cardiomyocytes.Present results provide evidence that androgen receptor is expressed from prenatal stages in mouse heart, supporting the proposition that androgens could be involved in mammalian heart development. The involvement of androgens in gender-related cardiovascular diseases  explainsThe presence of androgen receptor in embryonic heart would be important to indicate a role of androgens in prenatal cardiac development. The aim of this work was to determine the expression of androgen receptor simultaneously with the expression of alpha and beta myosin heavy chain genes (Myh6 and Myh7) and atrial natriuretic peptide gene (Nppa) at prenatal and early postnatal stages of mouse heart development. The presence of mRNA and the protein of the androgen receptor was observed in the nuclei of cardiac myocytes from embryonic stages and a positive correlation between AR and Nppa mRNA’s was registered at 2 and 9 postnatal days.emouseatlas.org. Mouse pups from 2, 9 and 16 postnatal days (pnd) were euthanized by decapitation. The sex of embryos and pups was determined after 12.5 dpc by gonad examination. All the procedures were approved by the Ethics and Research Committee of the Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM) and according to the National Institutes Health guidelines.CD1 mice were caged with food and water ad libitum under a 12-h light/12-h dark cycle in a room with a constant temperature of 23 ± 2 °C. Female mice were mated with males overnight, when vaginal plug was found, it was determined as 0.5 days post coitum (dpc). Pregnant females from 8.5, 10.5, 12.5, 14.5, 16.5 and 18.5 dpc were euthanized by cervical dislocation to obtain the embryos. The developmental stage was corroborated according to characteristics described at Atria and ventricles were obtained at pre-and postnatal stages and directly stored in RNA later solution . Samples of males and females were pooled separately to obtain enough tissue; at least three hearts were included in each pool. Total RNA was isolated using Trizol  according to manufacturer’s instructions. Total RNA was measured using NanoDrop . Total RNA (1.0 μg) was reversely transcribed to cDNA by Transcriptor Reverse Transcriptase (Roche) in 20 μL reaction mixture. For semi-quantitative PCR analysis 1 μL of cDNA as template was amplified with Platinum Taq DNA polymerase  in 20 μL PCR reactions. The number of cycles was selected to be in the linear portion of the exponential curve. The sequences of primers used were: for mouse beta-actin (NM_007393) sense primer sequence: 5′-gtatgcctctggtcgtacca-3′ and antisense primer sequence: 5′-ttgctgacaggatgcagaag-3′; mouse glyceraldehyde-3-phosphate dehydrogenase (NM_008084) sense primer sequence: 5′-atggtgaaggtcggtgtgaa −3′ and antisense primer sequence: 5′-gattgtcagcaatgcatcctgc-3′; mouse androgen receptor (NM_013476) sense primer sequence: 5′-gagtgactactctgcctccgaag-3′ and antisense primer sequence: 5′-gttatgaagcagggatgactctggg-3′; mouse myosin, heavy polypeptide 6, cardiac muscle, alpha (NM_001164171)sense primer sequence: 5′-atctctgacaacgcctatc-3′ and antisense primer sequence: 5′-gataggcgttgtcagagat-3′; mouse myosin, heavy polypeptide 7, cardiac muscle, beta (NM_080728) sense primer sequence: 5′-tgtgctgtacaacctcaagg-3′ and antisense primer sequence: 5′-ccttgaggttgtacagcaca-3′; mouse natriuretic peptide type A (NM_008725) sense primer sequence: 5′-aataaacttcagcaccaaggac-3′ and antisense primer sequence: 5′-gtccttggtgctgaagtttatt-3′. The PCR products were size-fractionated by 1% agarose gel electrophoresis and visualized with ethidium bromide using an EpiChemi II Darkroom . PCR bands were subjected to densitometry analysis with Quantity One software (Bio-Rad). Measurements were done by triplicates and the average was registered as the value of the sample. Data were normalized to beta-actin values, which were obtained simultaneously in each sample, before statistical analysis. The sequence determination was carried out using the Automated DNA sequencer model 373 (PE- Applied Biosystems Foster CA).Complete embryos before 14.5 dpc and hearts from older time points were fixed by immersion overnight in saline phosphate buffer containing 4% paraformaldehyde and paraffin embedded. Tissue sections 3 μm thick, were deparaffinized and rehydrated. Antigen retrieval was carried out in a pressure chamber for 5 min in Diva decloaker citrate buffer . Non-specific binding sites were blocked with 10% goat serum for 1 h at room temperature. Tissue slices were incubated overnight at 4 °C with anti-AR polyclonal antibody diluted 1:50 . AR antibody evaluated by Western blot technique binds to a 110 kDa protein, the expected size of androgen receptor. Slides were further incubated with Mach2 rabbit HRP polymer (Biocare) for 1 h at room temperature. Signal detection was achieved with diaminobencidin chromogen kit (Biocare). Color development was stopped by PBS rinsing and counterstained with Gill’s hematoxylin. Samples without first AR antibody were used as negative controls. Histological sections of testis were used as positive controls.post-hoc Tukey test, Student’s t test and Pearson correlation coefficient as indicated in figures. Results were considered significant when P values were less than 0.05.Data were analyzed by ANOVA and Androgen receptor mRNA was evaluated by semi quantitative PCR in the heart of mouse embryo; the presence of AR mRNA was observed from 10.5 dpc until birth. The AR expression increases gradually with the highest levels registered at 16.5–18.5 dpc ; at 9 pnd r = 0.75 (n = 20). Moreover, AR correlates with Nppa in atria  and ventricles  indicating that correlation is observed in both chambers despite the highest level of Nppa observed in atria. There was no correlation of AR with Nppa, Myh6 or Myh7 in prenatal stages.Androgen receptor mRNA displayed a significant correlation with the expression of Nppa at 2 and 9 dpn heart development Fig.  but not Androgen receptor mRNA was similarly expressed in male and female hearts, independently of the age and the chamber studied. Androgen receptor mRNA values determined in atria and ventricles at 14.5 dpc, and 16 pnd separated by gender is shown in Fig. Immunoreactivity for androgen receptor was evaluated in heart samples obtained at 12.5, 14.5, 16.5, 18.5 dpc, and 2, 9 and 16 pnd. The presence of the androgen receptor was detected in the cell nuclei of myocytes and endocardial cells at 12.5 dpc Fig. . At moreAndrogen receptor expression is herein described through prenatal and early postnatal heart development. We demonstrated that AR mRNA is present during the major stages of heart morphogenesis: looping heart (10.5 dpc) and chamber formation ; AR is also detected during heart functional maturation . Expression of AR was similar in males and females in the evaluated developmental stages. The presence of AR is higher in atria than in ventricles at 2, 9 and 16 pnd and mRNA levels for AR correlates with Nppa levels at 2 and 9 pnd of postnatal heart development.The role of androgens in heart development has been previously analyzed; testosterone and dihydrotestosterone, acting through androgen receptor, induce the differentiation of stem cells into beating cardiac myocytes , 9. More. Similarly, in adult rat atria, calcium-handling proteins from sarco-endoplasmic reticulum were altered after orchiectomy and prevented by testosterone replacement [Moreover, present results show that AR displays a higher expression in atria than in ventricles at 2, 9 and 16 days after birth. In neonatal mouse, cardiac myocytes perform terminal differentiation and display ion channel expression profiles distinct from that of the adult mouse . AR knoclacement . These rDuring heart development, there is a dynamic expression of several genes, included the alpha myosin heavy chain, Myh6, beta myosin heavy chain, Myh7, and atrial natriuretic factor A, Nppa , 17. ThiNo variations in the expression of AR between males and females are herein detected, either at prenatal or postnatal stages. Similarly, it has been reported that AR did not differ between male and female in the cytosolic and nuclear fractions of adult mouse ventricles and atria . There aThe immunoreactivity for AR protein is displayed in nuclei of atrial and ventricular myocardium from 12.5 dpc to 16 pnd. The presence in the nuclei of the heart cells suggests that AR is an active transcription factor. Myocardial and endocardial cells are positive for AR at 12.5 dpc. Meanwhile, at postnatal stages, it is clearly identified that cardiac myocytes are positive while cardiac fibroblasts are negative. Previous studies have described the presence of androgen receptors in nuclear subcellular fraction of mouse cardiac myocytes . CardiacThe androgen receptor is expressed during the morphogenesis and maturation of mouse heart, primarily restricted to cardiac myocytes. A high expression of AR in atrial tissue is observed at early postnatal heart development, together with a positive correlation between AR and Nppa expression in atria and ventricles. Present data support that androgen receptor action would be relevant in mammalian heart development.


Patients with metastatic angiosarcoma undergoing chemotherapy, radiation, and/or surgery experience a median progression free survival of less than 6 months and a median overall survival of less than 12 months. Given the aggressive nature of this cancer, angiosarcoma clinical responses to chemotherapy or targeted therapeutics are generally very poor. Inhibition of beta adrenergic receptor (β-AR) signaling has recently been shown to decrease angiosarcoma tumor cell viability, abrogate tumor growth in mouse models, and decrease proliferation rates in preclinical and clinical settings. In the current study we used cell and animal tumor models to show that β-AR antagonism abrogates mitogenic signaling and reduces angiosarcoma tumor cell viability, and these molecular alterations translated into patient tumors. We demonstrated that non-selective β-AR antagonists are superior to selective β-AR antagonists at inhibiting angiosarcoma cell viability. A prospective analysis of non- selective β-AR antagonists in a single arm clinical study of metastatic angiosarcoma patients revealed that incorporation of either propranolol or carvedilol into patients' treatment regimens leads to a median progression free and overall survival of 9 and 36 months, respectively. These data suggest that incorporation of non-selective β-AR antagonists into existing therapies against metastatic angiosarcoma can enhance clinical outcomes. Angiosarcomas are rare vascular tumors of aberrant endothelial histology that exhibit an aggressive and often highly lethal natural course. These tumors can manifest at various sites on the body, but are most frequent in the skin, soft tissue, liver, breast, spleen, bone, or heart , 2. MetaSeveral recent studies have reported beta adrenergic receptor (β-AR) expression across diverse tumor types -23, and In addition to vascular tumors, β-AR antagonists have shown preclinical efficacy against a variety of malignant tumors including breast cancer , 39-41, In the current study, we evaluated the efficacy of selective (targeting only one β-AR) and non-selective (targeting multiple β-ARs) beta blockers using in vitro angiosarcoma models. We then quantified clinical outcomes of patients with metastatic disease prescribed non-selective beta blockers in combination with standard treatment regimens.β-AR antagonism disrupts sarcoma cell viability. We have previously shown that β-AR antagonists inhibit the viability of angiosarcoma cells and limit tumor progression [gression , 32, 36.Non-selective β-AR antagonists are superior to selective β-AR antagonists for inhibiting angiosarcoma viability. Propranolol is a non-selective β-AR antagonist that classically inhibits both β1-AR and β2-AR. Our lab and others have previously shown that non-selective β-AR antagonists are superior to selective β-AR antagonists with regard to decreasing tumor cell proliferation and enhancing clinical outcome in breast, liver, and ovarian cancer [n cancer , 57, 58.Non-selective β-AR antagonists extend progression free and overall survival in patients with metastatic angiosarcoma. Patients with an initial diagnosis of metastatic angiosarcoma were prescribed the non-selective beta blockers propranolol or carvedilol in addition to their anti-cancer treatment regimen. Six women and three men were included in this study, with a median age of 53.7 (range=34-75). Each patient was given propranolol  or carvedilol  in combination with treatment protocols, which varied by treating oncologist . Membranes were blocked in tris buffered saline plus 3% bovine serum albumin and 0.05% Tween-20, and incubated with the following antibodies as indicated for each experiment: anti-phospho-p38 (T180/Y182) , anti-phospho-SAPK/JNK (T183/Y185) , anti-phospho-p44/42 (T202/Y204) , anti-phospho-p53 (S46) , anti-cyclin D1 , anti-cyclin D3 , anti-CDK4 , anti-CDK6 , anti-p21 , anti-p27 , and anti-β actin (Santa Cruz Biotech #sc8432). Each primary antibody was detected with an appropriate 1:1000 HRP-conjugated secondary antibody, subjected to Supersignal West Dura Extended Duration Substrate (ThermoFisher Scientific #34075), and digitally captured using a GE Image Quant Las4000 imaging system.Antibody array. The Phospho-Mitogen-Activated Protein Kinase (MAPK) Antibody Array (R&D Systems #ARY002B) was performed according to the manufacturer's instructions using SVR cell lysates. Densitometry of each antibody array signal was performed using ImageJ software. Reference spots on each array were used to normalize the pixel densities. The numerical protein expression data was normalized, and centroid linkage based on an uncentered correlation similarity metric was performed using Gene Cluster 3.0 software. Heatmaps were generated using JavaTreeView software.Animal models. Animal experiments were performed in accordance to Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee regulations for the care and use of animals in experimental procedures, and all efforts were made to minimize suffering. Mice were housed 4 per cage in a temperature-controlled animal facility on a 12h-12h light-dark cycle. Animals had free access to mouse chow and water. Xenograft angiosarcoma tumor models were generated via subcutaneous injection of 1x105 SVR cells into the dorsolateral flank of 6 week old male J/Nu mice . Tumors were allowed to grow until ~1cm3, and injected with saline sham or 15 mg/kg propranolol for 15 minutes. Tumor tissue was collected and immediately flash frozen in liquid nitrogen. Tissue lysates were pooled prior to use in immunoblotting.Immunohistochemistry (IHC). IHC was performed on tumor tissue samples collected from an angiosarcoma patient before and after one week of treatment with propranolol [pranolol . The folClinical study. This clinical study was approved by the Texas Tech University Health Sciences Center Institutional Review Board (IRB# E17109). As angiosarcoma is a very rare tumor and most individual clinical sites do not see enough patients with this tumor type to accommodate a clinical trial, we recruited nine angiosarcoma patients (six women and three men) with a diagnosis of metastatic disease through the Angiosarcoma Awareness patient support group on Facebook. The median age for this cohort was 55 years with a range from 34 to 75 years. Similar recruitment strategies using social media have been effective for a number of rare diseases where any single treatment center would lack the patient volume necessary to conduct a clinical study [al study -73. PFS Due to the poor prognosis of metastatic angiosarcoma patients, many are faced with the decision to undergo chemotherapy, radiation, and/or surgery or simply accept supportive care—choices that generally lead to similar outcomes. The data presented in this study suggest that incorporation of non-selective β-AR antagonists into a variety of standard treatment regimens enhances PFS and OS in patients with metastatic angiosarcoma. Use of inexpensive and relatively safe non-selective β-AR antagonists along with established treatments against angiosarcoma could improve prognoses for metastatic patients.


The progress in information and communication technology (ICT) led to the development of a new rehabilitation technique called “serious game for functional rehabilitation.” Previous works have shown that serious games can be used for general health and specific disease management. However, there is still lack of consensus on development and evaluation guidelines. It is important to note that the game performance depends on the designed scenario.The objective of this work was to develop specific game scenarios and evaluate them with a panel of musculoskeletal patients to propose game development and evaluation guidelines.A two-stage workflow was proposed using determinant framework. The development guideline includes the selection of three-dimensional (3D) computer graphics technologies and tools, the modeling of physical aspects, the design of rehabilitation scenarios, and the implementation of the proposed scenarios. The evaluation guideline consists of the definition of evaluation metrics, the execution of the evaluation campaign, the analysis of user results and feedbacks, and the improvement of the designed game.The case study for musculoskeletal disorders on the healthy control and patient groups showed the usefulness of these guidelines and associated games. All participants enjoyed the 2 developed games , and found them challenging and amusing. In particular, some healthy subjects increased their score when enhancing the level of difficulty. Furthermore, there were no risks and accidents associated with the execution of these games.It is expected that with the proven effectiveness of the proposed guidelines and associated games, this new rehabilitation game may be translated into clinical routine practice for the benefit of patients with musculoskeletal disorders. Musculoskeletal disorders lead to high medical costs all over the world. These disorders affect the working performance and well-being of the involved people ,2. Age-rSome research studies focused on games that could improve general health for adults and elders. Chen et al 2012) developed a lower limb power rehabilitation system. Each user needs to execute a squat motion, with sufficient power, to correctly build a virtual tower made of blocks . The sys developeParkinson disease has been a subject of interest among serious game projects. Yu et al (2011)  developeAnother rehabilitation field that has been studied is stroke rehabilitation. Cho et al (2014) developed a proprioception rehabilitation system for stroke patients . The useLiterature showed that serious games have been intensively developed for general health and specific disease management. One of the most important aspects of serious games is the game playing scenario to motivate the patient. Moreover, user acceptability also plays an important role in promoting this new technology to clinical practice. Finally, the user security aspect needs particular attention to avoid new clinical complications for patients. Different game systems have been developed and tested. There is still lack of development and evaluation consensus guidelines to achieve these important aspects. It is important to note that the game performance depends on the designed scenario. Some authors have attempted to propose specific guidelines for game development-based learning  or for PThe development of serious games for functional rehabilitation of musculoskeletal disorders is a complex engineering task. To deal with such complexity, a two-stage workflow was proposed. The first workflow relates to the development guideline , whereasThis subsection describes the work done using a proposed development guideline for creating specific serious games for functional rehabilitation of musculoskeletal disorders.The selection of available computer graphics technologies and tools plays a crucial role in the success of the rehabilitation game. To ensure a user-friendly, human-system interaction, cutting-edge technologies benefiting the most recent progress of ICT solutions need to be used. In this study, open source Blender design software (Neo Geo) was selected for human body modeling. XNA Game Studio (Microsoft) was selected as game engine. Microsoft Kinect camera was selected as human motion capture tool. Computer screen was used as human-system interface. The pertinence of these technological choices has been proven in our previous studies -8,21.A 3D avatar model was developed using Blender design software to represent the human body. This is a 3D surface mesh model including a collection of vertices, edges, and faces that defines the external shape of the human body. Moreover, an internal skeleton structure was also created to define body segments  and their interaction  during the motion . Game enGames with 3D interactive objects need to establish interaction rules between them. An algorithm was designed and implemented to detect collisions between objects within the scene. The challenge was to find a way to differentiate between the detection between different avatar bones and 3D objects; therefore, we created spheres around each bone of the body .1 be a sphere with a 3D center C1 and a radius r1, and S2 another sphere with center C2 and radius r2. The distance between the 2 centers of the 2 spheres is d drawn in Let S1 and r2, a collision is detected, and the game reacts to it by a certain preprogramed reaction.This distance is computed between every 2 objects at each updated iteration during the game. If d is found to be less than the sum of the 2 radiuses rTwo task-oriented game scenarios  were designed and implemented. The football game aims at practicing body orientation and lower limb motions, allowing the rehabilitation of spinal and lower limb systems. The object manipulation aims to practice the upper limb and lower limb motions with a focus on the detailed hand skill. The description of each game is given in the following paragraphs.This game requires the player to execute many consecutive gestures. First, players have to stand in front of the Kinect and the computer screen. Then, they need to target the left or right cones by pivoting their body . Once thIn this scene, the user needs to take a flower from the given vase and put it in the other one . They reVisual Studio.Net, with C# programming language, was adopted for image acquisition and processing, body tracking, object manipulation, as well as for the development of graphical user interfaces (GUIs).This part presents the work done, as well as outcomes issued from the application of the proposed evaluation guideline for assessing the developed games.The game-playing performance was evaluated by the points acquired at the end of each scenario. For the usage acceptability aspect of the designed games, a questionnaire was defined. At the end of each game scene, players were required to fill out a questionnaire. The questionnaire consists of 13 questions for each specific game scenario. The feedback focuses on the game, exercise, and user aspect. For the game, the objective, the level of difficulty, the ignorance of achievement, the attractiveness of the 3D environment and GUI, and the game management  were investigates. For the exercise, the game instructions, the variation of scenarios, the suitability of the game to the goal, and the clearness of the feedback were examined. For the user, the motivating challenge, the possibility to make mistakes, and the security feeling were investigated.The developed game scenarios were evaluated by a normal healthy group , to ensure the security condition, and then evaluated by a population of 20 pathological subjects  at the “Centre Hospitalier Universitaire de Limoges” (France). The patient group included different musculoskeletal disorders . Each participant signed an informed consent agreement before playing the rehabilitation games. It is important to note that the execution of rehabilitation serious game was monitored by clinicians, to ensure the ability and the security of the patients when using this new rehabilitation tool. Each healthy subject was asked to play every level of difficulty of each game, which means a total of 6 trials per subject. Some patients were not able to try all levels or even one of the two games due to the severity of their state . Medical experts were given the decision to accept or decline the participation of their patient in a game or a level of a certain game. Therapists accompanied their patients by standing behind them and supporting them, to ensure their security. The duration of each game level was around 60 seconds. A rest time of around 2 min was also allowed for each participant when necessary  after each game execution. The total time of the test for one subject was equal to 20 min approximately.t-test, implemented in Matlab R2010b software [The MathWorks Inc.]) showed no significant difference. In particular, some subjects (ID4 or ID6) increased their score when enhancing the level of difficulty. According to the healthy control group, the performance of the pathological population was significantly  lower for all levels of difficulty  lower than that of the normal population. The number of the patients able to perform harder levels was also reduced from 17 patients for the easy level to 5 patients for the hard level.Regarding the object manipulation game, the same results were noted -10. The For the responses to the questionnaires, 29 users  rated the football game, and 27 rated the object manipulation game.Regarding the user acceptability of the evaluated games, all healthy subjects found the 2 developed games motivational, attractive, and challenging. A synthesis of the patients’ responses to the football game questionnaire is depicted in Finally, players were asked to give some specific comments on this project and the developed games. Comments and suggestions from the patient groups are summarized as follows:Interesting game and this game needs to be developed in bigger scales.The games are amusing, motivational and not bad at all. It made me really move my legs.The football scene is excellent. I am a football fan and I watch all the games.I recommend you to force the player to hit the left cone at first and then rotate towards the right cone. This improves the efficacy of spine rehabilitation.In my opinion this can really help patients. Even if I am not a florist!The exercises are adapted to rehabilitation at the final stages.The project is suitable for younger players.The project is very fun, helps in performing rehabilitation while enjoying it. It should please young and old people.Very attractive games.Very interesting project for movement coordination.The avatar's movements should be improved.Difficult but interesting. More games need to be developed.Based on these suggestions, our game scenarios were updated to take them into consideration. Note that only technical improvement feedbacks were considered in the updated version. In particular, the order of the football game, as suggested in the third comment above, was redefined to adapt to the rehabilitation of spinal patients. Moreover, avatar’s movement has been improved by using multi-sensor fusion approach . Some paSerious gaming technologies target audience ranging from young to adults to the elderly population. The objective of this work was to propose development and evaluation guidelines of serious games for musculoskeletal disorders. The simplicity and challenging aspect are the main advantages of this new technology. Research studies have proposed some interesting solutions over the past decade for the “gamification” approach -26. SeabRegarding our case study, patients’ scores were lower than those of the healthy group. Some of them were not able to play the football scene because of their amputation. Others could not try the object manipulation scene because they cannot move their hands at all. In general, all of them accepted the challenge and wanted to participate in this study. Hemiplegic patients were the top testers among all patients. Medical doctors and physiotherapists thought that these task-oriented games were more adapted to this particular disorder. Previous study came to the same conclusion about the use of the task-oriented games for these patients ,17. We cThe design of rehabilitation game scenarios plays a crucial role in the success of the serious game for health. The game scenario must not only be attractive but also needs to be clinically useful. In this study, a task-oriented rehabilitation game scenario was proposed. The football and object manipulation games respond to the challenging objective: patient practices rehabilitation exercises without recognizing that it is a rehabilitation exercise when playing the game. Thus, the football game allows the player to practice the two motor tasks (body rotation motion and the leg motion) and two decision-making actions (observation of time and identification of right moment). The object manipulation game allows the player to practice two motor tasks (leg and arm motion) and two decision-making actions . This study suggests that rehabilitation game scenarios should be designed, implemented, and evaluated with similar strategies. The outcome of the case study confirmed the robustness and effectiveness of this strategy.The design of a motivating, challenging, and safe serious games for functional rehabilitation requires particular attention on the development and evaluation processes. This study proposes useful guidelines to achieve this objective. Thus, the development and evaluation of the 2 developed games  followed the proposed guidelines. In general, a guideline is defined as a principle to determine a set of actions in a standard way. This study aimed to propose a coherent set of development and evaluation steps for rehabilitation-oriented serious games for musculoskeletal disorders. It is expected that this proposition may help define a development and evaluation consensus in the health-oriented serious games community. It is important to note that some published works already followed these guidelines ,10,12 buOur developed system used the Kinect camera as motion capture sensor. Currently, the virtual avatar imitates player movements correctly. However, clinical experts require more precision in analyzing the joint behavior during the exercise. It is well known that the accuracy of this device is limited for joint angle estimation. A deviation range of 11° to 14° was noted for the knee joint motion ,33. To oThe main limitation of the 2 developed games is the lack of evident cognitive actions, which could maximize the effect of game outcomes to better manage the functional rehabilitation of musculoskeletal disorders with cognitive impairment ,35. ThusIn summary, this study is an explanatory work aiming to show the usefulness and applicability of the proposed guidelines and associated serious games for functional rehabilitation of musculoskeletal disorders. However, more investigations such as a long-term evaluation campaign for effectiveness analysis and a more quantitative analysis on the user‘s engagement in the games are needed to fully validate these guidelines .Development and evaluation guidelines dedicated to serious games for health were established in this study. The case study showed the effectiveness and usefulness of these guidelines and associated games. The developed serious game system used the Kinect camera to allow users to interact with two 3D environment scenes . Healthy subjects and patients enjoyed the games and found them challenging and amusing. In this work, we concentrated on the assessment data of the developed games. In perspective, the effectiveness and clinical relevance of these games will be studied through a long-term evaluation campaign. And, in the case of positive outcomes, this new rehabilitation game may be translated into clinical routine practice in the near future for the benefit of patients with musculoskeletal disorders.


In allergic disease, mast cell activation is conventionally triggered by allergen-mediated cross-linking of receptor-bound IgE on the cell surface. In addition to its diverse range of intracellular roles in apoptosis, cell proliferation and cancer, Histamine-Releasing Factor (HRF) also activates mast cells and basophils. A subset of IgE antibodies bind HRF through their Fab regions, and two IgE binding sites on HRF have been mapped. HRF can form dimers, and a disulphide-linked dimer is critical for activity. The current model for the activity of HRF in mast cell activation involves cross-linking of receptor-bound IgE by dimeric HRF, mediated by HRF/Fab interactions. HRF crystal and solution structures have provided little insight into either the formation of disulphide-linked HRF dimers or the ability of HRF to activate mast cells. We report the first crystal structure of murine HRF (mHRF) to 4.0 Å resolution, revealing a conserved fold. We also solved the structure of human HRF (hHRF) in two new crystal forms, one at the highest resolution (1.4 Å) yet reported. The high resolution hHRF structure reveals a disulphide-linked dimer, in which the two molecules are closely associated, and provides a model for the role of both human and murine HRF in mast cell activation. In addiPlasmodium knowlesi , solved at 4.0 Å resolution, revealing the conserved HRF fold. We also report two structures of human HRF (hHRF) in new crystal forms, one of which was solved at the highest resolution yet reported (1.4 Å) for HRF. One hHRF structure, and the mHRF structure, contain non-covalent HRF interactions, but reveal different packing arrangements. However, the high resolution hHRF structure reveals a disulphide-linked HRF dimer, with substantial contact between the two monomers, finally providing a model for the activity of dimeric HRF in allergic disease.22.1mHRF and hHRF were prepared according to a previously described protocol . Both pr2.2xia2 program (Coot (Data were collected at beamlines I04 (mHRF), I03 (hHRF-1) and I04-1 (hHRF-2) at the Diamond Light Source . Data were integrated with XDS  or DIALS program , and sca program  from the program . The mHR program . mHRF an program  using PD program  as a sea program  and manuam  in new crystal forms, the latter revealing a disulphide-linked dimer.3.1The structure of mHRF was solved at 4.0 Å resolution, and contains two molecules in the asymmetric unit A. ResiduH. sapiens, P. falciparum and P. knowlesi)    with RMS. pombe)  with RMS2 at their interface, in which Glu80-Thr84 from one chain pack against equivalent residues from the other, with a hydrogen bond between Ser82 from each chain .Like hHRF, mHRF contains two cysteine residues at positions 28 and 172, and thus has the potential to form a covalently-linked dimer; while Cys28 is buried, Cys172 is surface exposed. However, the two molecules of the mHRF asymmetric unit, which are related by non-crystallographic two-fold symmetry, form only non-covalent interactions A. The twch chain C. A simich chain  and PDB ch chain ]; howevech chain D, and th3.2The structure of hHRF-1, solved at 1.75 Å resolution, contains two molecules in the asymmetric unit A. Residu2  pack against one another; Met1 from one chain contacts Met1, Ile3 and Glu12 from the other; Ile17 packs against Asn75 and His76, while Met74 packs against the mobile loop; furthermore, a single water molecule forms two hydrogen bonds, one with Asn75 in each chain. The disulphide bond between Cys172 in each chain is accommodated in a shallow depression created by the Met1 and Ile3 side chains B. Due toOnly one other HRF structure [human HRF containing a Glu12Val mutation (PDB 3EBM)], contains a dimer covalently linked by a disulphide bond . In thisWild-type hHRF described here, and the Glu12Val hHRF mutant, both contain a C-terminal purification tag (Leu173-His180), and in each dimer the tag in one chain contacts the other monomer; however, this interaction differs between the two structures. In the structure of the Glu12Val hHRF mutant , the Leu2, of which 40% is contributed by the C-terminal His-tag. By contrast, the interface in hHRF-2 buries a substantially larger surface area, ∼954 Å2, of which a smaller proportion, ∼25%, is contributed by the tag. Thus, the two molecules in the wild-type hHRF-2 dimer are much more closely associated with one another, compared with the mutant (The interface between the two molecules of the Glu12Val hHRF mutant buries a surface area of ∼652 Åe mutant A-D, and 3.5The current model for the activity of HRF in mast cell activation is that dimeric HRF cross-links FcεRI-bound IgE on the mast cell surface, mediated by interactions between HRF and the Fab regions of a subset of IgE molecules . IgE bin2 if the C-terminal His-tag is omitted from the structure. Crucially, the two binding sites necessary for the Fab interactions are surface exposed in each molecule (The hHRF-2 structure comprises a wild-type hHRF dimer, with a disulphide bond between Cys172 from each monomer. In this dimer, the two monomers are closely associated, burying a surface area of ∼666 Åmolecule , and wou4We report the first crystal structure of murine HRF and the structure of human HRF in two new crystal forms, one at the highest resolution yet reported. The high resolution human HRF structure contains a disulphide linked dimer, with closely packed monomers, which provides a model for the activity of human and murine HRF in allergic disease.Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: mHRF, PDB: 5O9K; hHRF-1 PDB 5O9L; hHRF-2, PDB 5O9M.


The crystalline and multilayer structures of the multilayers were determined by a glancing angle X-ray diffractometer (XRD). A nanoindenter was used to evaluate the hardness, the elastic modulus and scratch scan of the multilayers. The chemical bonding was investigated by a X-ray Photoelectron Spectroscopy (XPS). The maximum hardness (36.3 GPa) and elastic modulus (466 GPa) of the multilayers was obtained when Ar/N2 ratio was 18:1. The TiAlN/Al2O3 multilayers were crystallized with orientation in the (111) and (311) crystallographic planes. The multilayers displayed stably plastic recovery in different Ar/N2 ratios. The scratch scan and post scan surface profiles of TiAlN/Al2O3 multilayers showed the highest critical fracture load (Lc) of 53 mN for the multilayer of Ar/N2 = 18:1. It indicated that the multilayer had better practical adhesion strength and fracture resistance.TiAlN/Al The multi-element systems, in recent years, have received more attention in terms of further improving performance ,2,3. Whe2O3 and SiO2, are much more stable at high temperature than nitrides. Unfortunately, oxides lack the strength that nitrides have to withstand the mechanical abuse of cutting. In practice, oxides have to be combined with nitride or carbide material, such as TiN or TiC, in the form of multilayered structure when being used in tool coatings. The nitride or carbide layer provides strength, while the oxide layer provides chemical resistance. Pronounced strength enhancement, optimal hardness/toughness ratios and excellent wear resistance can be obtained through a proper critical bilayer thickness design for nanoscale multilayers [As is well-known, TiAlN coating is relatively excellent coating material due to its high hardness, wear, chemical inertness and superior oxidation resistance ,10. In atilayers ,12,13. Ttilayers . Films stilayers . 2O3 multilayers with constant Λ (10 nm) and tTiAlN: tAl2O3 (10:1) were deposited on Si substrate at a fixed substrate bias of −140 V in different N2 partial pressure by magnetron sputtering. High purity Ti3Al (99.99%) target and Al2O3 (99.99%) target were respectively connected to RF source sputter guns, which were installed with the horizontal plane. First of all, Si (100) substrates were cleaned by an ultrasonic agitator with acetone and alcohol for at least 15 min and dried using compressed air after each cleaning cycle. Subsequently, all substrates were sputter-cleaned by −500 V bias for 10 min. The deposition of the multilayers started with the deposition of 80-nm-thick Ti buffer layer to improve coating adhesion. Then, the TiAlN/Al2O3 multilayers were fabricated in a flowing Ar and N2 gas mixture with different N2 partial pressure and the working pressure was kept at 0.4 Pa. The ratios of Ar/N2 were from 10:1 to 22:1. In the process of deposition, all the depositions were conducted using a power of 100 W for the Ti3Al target and Al2O3 target. The thickness of the monolayer was controlled by open time of baffle and calculated by multiplying the deposition rate by the sputtering time. All multilayers were deposited 30 periods. Total thicknesses of the multilayers were around 400~500 nm.TiAlN/AlThe element compositions, their depth profiles and chemical states of the coatings were investigated by a PHI5000 Versa Probe X-ray Photoelectron Spectroscopy  using an Al Kα X-ray source (1486.6 eV). A D/MAX 2500 diffractometer was used for low-angle X-ray reflectivity (XRR) and wide angle X-ray diffraction (XRD) of layered structure and crystalline analysis, operating with Cu Kα radiation at 1.54056 Å. The nanohardness, the elastic modulus and loading-unloading curves of the multilayers were investigated by the continuous stiffness measurement technique using a Nano Indenter XP system. This system was also used to perform scratch test. Residual stress generated during the coating growth process was calculated according to the curvature measured using an XP-2 profiler.2O3 multilayers. Clear reflection peaks are observed in XRR pattern of TiAlN/Al2O3 multilayer, indicating that the multilayer has distinct chemical modulation structure and sharp interfaces. Sharp interfaces between the two layers throughout whole multilayer can cause an enhancement in hardness. The modulation period (Λ = 10 nm) is calculated according to the periodic position of the reflection peaks using the modified Bragg’s law [λ = 1.54056 Å is the X-ray wavelength, and−5) which is related to the average reflective index. Using equation above, the Λ is calculated by the slope of linear least-squares fitted straight line of sin2n versusθn2 which is shown in the inserted figure.gg’s law :sin2θn=2O3 multilayers deposited at Si substrates with different Ar/N2 from 10:1 to 18:1. All the investigated multiayers were highly textured with a preferential orientation in the (111) direction, but the intensities of (311) orientation was very low. With increasing Ar/N2, TiAlN(111) and AlN(111) peaks began to appear, the intensity of three different phases (111) have reached maximum and become sharper, when Ar/N2 ratio is 18:1. It indicates that multilayer (Ar/N2 = 18:1) have larger grains and higher degree of crystallinity [2O3 multilayers. The peak broadening behavior was usually originated from the diminution of grain size [2O3 phase is observed, meanwhile, there has also been the presence of Al2O3 in XPS. It indicates that Al2O3 layer is amorphous phases in all multilayers. This structure change is ascribed to grains growth induced by amorphous Al2O3 periodic insertion. The combined nanocrystallities and amorphous phases can produce a positive effect on mechanical properties.allinity . The radain size . No refl2p, N1s, Al2p and O1s for TiAlN/Al2O3 multilayer are shown in 2p3/2 stoichiometric TiN and TiOx, respectively [2p1/2–N and Ti2p1/2–O. The N1s peaks at 396 eV and 398 eV are related to Ti–N bonding and Al-N bonding in 2p spectrum, 2O3 (BE = 72 eV). The rich aluminum oxide in multilayer undoubtedly provides better protection against the environment attack. x (530 eV) and Al2O3 (531.5 eV). The existing of TiOx in deep layer might because Ti is preferred to combine with O more than N for its higher activity. This result is in agreement with that concluded from XRD tests. From XPS measurement and combined with our XRD results with no Al2O3 diffraction peaks, we concluded that Al2O3 was amorphous phase and the multilayers existed as nc-TiAlN/a-Al2O3. It can be conceived that the amorphous nature of Al2O3 enables it to imbed among TiAlN grains cohesively, which would contribute to the enhancement of the hardness of the TiAlN/Al2O3 multilayers.The high-resolution XPS core-level spectra of Tiectively . The bin2O3 multilayers with Λ ≈ 10 nm and different Ar/N2. With the increasing of Ar/N2 ratio, the hardness and elastic modulus of the multilayers all increase before decrease, the maximum hardness and elastic modulus of the multilayers are found to be 36 GPa and 466 GPa, respectively, when Ar/N2 ratio is 18:1. Excellent mechanical properties have come from TiAlN layer. Appropriate N2 is beneficial for TiAlN crystallinity, but an excess of N2 is to the disadvantage of Al2O3 layer formation. The increase in hardness and elastic modulus can be understood by studying the XRD patterns mentioned above. We believe that a strong mixture of TiAlN(111), AlN(111), TiN(111) and TiAlN(311) textures with larger grain sizes is responsible for hardness and elastic modulus increase. In addition, Hardness enhancement has been attributed to the periodic insertion of thinner amorphous Al2O3 into TiAlN related to suppressed dislocation generation and impeding dislocation motion. Interfaces act as barriers to the motion of dislocations and provide sites for dislocation pile-up [ pile-up .2 ratios as is shown in σ) is the main reason for coating delamination and plastic deformation. Therefore, the reduced residual stress in coatings is a key factor for these coatings to explore more applications. The residual stress calculated by the Stoney formula [σ = −Es, ts and vs are elastic modulus (131 GPa), thickness (0.0005 m) and Poisson’s ratio (0.28) of the substrate, separately; tc is the coating thickness; and R is the radius of curvature of the multilayer coated on substrate. R can be determined by the film curvature using surface profilometry. The compressive stresses decrease with the increase of Ar/N2. When the Ar/N2 flow rate is 18:1, the compressive stress gets to the highest (−4.3 GPa) connected with highest hardness (36.3 GPa). Multilayers with Ar/N2 ratio more than 18:1 have lower compressive stress than others. It is due to periodically introduction of Al2O3 layers into TiAlN layers that helps to relax the stress.The compressive stress of the multilayer coatings determined by XP-2 profiler is influenced by Ar/N formula : σ = −Es2 are shown in Lmax), followed by stepwise unloading. Usually, the elastic recovery (ER) is defined as ER = (dmax − dres)/dmax, where dmax and dres are the displacement at the maximum load and residual displacement after unloading, respectively [2O3 multilayers are parallel. The value of (dmax − dres)/dmax is about 0.4. This indicates that plastic recovery (ER) has nothing to do with depth of penetration into surface. The stably plastic recovery (ER) is observed. With the change of Ar/N2, the plastic recovery displays invariable due to stably value of (dmax − dres)/dmax.Typical observations of the loading-unloading curves on the different Ar/Nectively . Obvious2O3 multilayers with different Ar/N2 ratios. The scratch scan profiles of all multilayers indicate an abrupt increase point in scratch depth. The normal load corresponding to an abrupt increase point in the scratch scan profile is the critical fracture load of coating (Lc). The Lc can characterize the adhesion strength of the coating. The Lc of Ar/N2 = 10:1 and 12:1 are 11 and 40 mN, respectively. However, no abrupt change in scratch depth is observed until Lc = 53 mN for the multilayer of Ar/N2 = 18:1, which has better practical adhesion strength and fracture resistance. In terms of the above results, the multilayer’s fracture resistance may be interrelated with inherent internal stress, hardness, and plastic recovery of the coatings with multilayered structures.2O3 multilayers were deposited on Si substrate by magnetron sputtering. The effect of Ar/N2 ratios on the microstructure and properties of TiAlN/Al2O3 multilayers was investigated. With increasing Ar/N2, the intensity of (111) and (311) peaks becomes stronger. Meanwhile, the hardness and elastic modulus of the multilayers have all increased before decrease. When Ar/N2 ratio was 18:1, the maximum hardness and elastic modulus of the multilayers were found to be 36 and 466 GPa, respectively. The TiAlN/Al2O3 multilayer with Ar/N2= 18:1 has displayed better practical adhesion strength and fracture resistance. Therefore, the TiAlN/Al2O3 multilayers could be an effective and suitable candidate for hard coating system such as coated cutting tools. Such optimal processing parameters (Ar/N2= 18:1) would be quite favorable for industrial application in the future.TiAlN/Al


Erectile dysfunction (ED) following treatment for clinically localized prostate cancer, particularly radical prostatectomy (RP), is a major quality of life issue that remains unsatisfactorily addressed. With the introduction and use of cavernous nerve–sparing procedures over the past 25 years, many men recover erections postoperatively that enable sexual intercourse unlike in the prior surgical era, when permanent ED postoperatively was certain. Despite this advance, 26–100% of these patients may never recover normal erectile function (EF). Recent advances in the understanding of ED after RP have stimulated great attention to develop penile rehabilitation programs and neuromodulation. The purpose of penile rehabilitation is to prevent adverse corpus cavernosal tissue structural alterations and thereby maximize the chances of recovering functional erections. Rehabilitation programs are common in clinical practice, but there is no definitive evidence to support their efficacy. Neuromodulation represents another strategy for promoting erection recovery postoperatively. This therapy involves the application of neuroprotective interventions, conceivably targeting biological elements involved in the erection response that are affected by neuropathic injury. Well-conducted, controlled trials with adequate follow-up are required in order to determine the erection preservative benefits of these therapeutic strategies. The purpose of this essay is to describe the mechanisms related to post-RP ED, assess the need for penile rehabilitation and neuromodulation following surgery, and analyze the basic science and clinical trial evidence associated with these applications for preserving EF following prostate cancer treatment.


Thus, AR’s function as a novel ETS-1 co-activator or potentially therapeutic target of HCC has been demonstrated.The expression of androgen receptor (AR) has been detected in hepatocellular cancer (HCC). However, there is no universal model detailing AR’s function and mechanism in HCC. This study’s results show that treatment with dihydrotestosterone (DHT), an endogenous androgen, promoted HCC cells’ proliferation and up-regulated the transcription factor activity of ETS-1 (E26 transformation specific sequence 1), which mediates the migration and invasion of cancer cells via protein-protein interaction between AR and ETS-1. Results from luciferase assays showed that ETS-1’s activity was significantly up-regulated following androgen treatment. AR mediated ETS-1’s DHT-induced transcription factor activity. A potential protein-protein interaction between ETS-1 and AR was identified via glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. The mechanisms’ data indicated that enhancing AR activity increases ETS-1’s activity by modulating its cytoplasmic/nuclear translocation and recruiting ETS-1 to its target genes’ promoter. Moreover, while overexpression of AR significantly increased the proliferation or  In mammalian cells, androgen responds to androgen receptor (AR), which plays a central role in male health and the maintenance or progress of prostatic carcinoma . AR, a mThe transcription factor ETS-1, which belongs to the ETS protein family, contains the ETS (transcription activation domain) and helix DNA-binding domains . In the in vitro and in vivo.HCC is the most common type of human cancer . DespiteEC50 value was 28.61 ± 4.75 nM. At the same time, the antagonist of AR, mifepristone, down-regulated the activity of EBS-Luc induced by DHT  and antagonist tivantinib (ARQ-197) of the ETS-1 signaling pathway were used. As shown in Figure EC50 value = 7.55 ± 1.02 ng/ml), ARQ-197 inhibited this activity . Together, these results indicate that enhancing AR activity increased ETS-1transcriptional activity.To discover whether androgen modulates the transcription factor activity of ETS-1, luciferase assays were performed in HepG2 cells, which were co-transfected with ETS-1 binding site EBS-Luc reporters. As shown in Figure T Figure ; the IC5Then, the potential cross-talk of AR and ETS-1 signaling pathways was detected. HepG2 cells, which were co-transfected with EBS-Luc, or ETS-1 targeted genes MMP1/9’s luciferase reporters MMP1-Luc or MMP9-Luc, were harvested and analyzed by luciferase assays. As shown in Figure Next, AR’s effect on the expression of ETS-1 response genes was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting assays. As shown in Figure To determine the specificity of DHT’s effect on ETS-1, HepG2 cells were used in co-transfection experiments. To investigate the role of endogenous AR in ETS-1 mediated transcription, HepG2 cells Figure  were staTo further determine whether the observed effects of androgen on ETS-1 transactivation were specific to endogenous AR in HepG2 cells, an AR-negative cell line was used. Human prostate cancer PC-3 cells, which are AR negative and ETS-1 positive, were co-transfected with EBS-Luc, AR vector, or empty vector. As shown in Figure Next, ETS-1 signaling’s involvement in AR-mediated transcription was examined. HepG2 cells, which were co-transfected with EBS-Luc, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.5% charcoal-stripped fetal bovine serum (FBS) added with or without HGF and analyzed by luciferase assays. As shown in Figure Next, the possible interaction between ETS-1 and AR was investigated. HepG2 cells were transfected with FLAG-AR or FLAG empty vector. Then the co-immunoprecipitation (co-IP) and immunoblotting (IB) assays were performed. The results shown in Figure in vitro is valuable. The purified GST-AR or GST-ETS1 was incubated with purified FLAG-ETS1 or FLAG-AR for GST pull-down. A protein-protein interaction between GST-AR with FLAG-ETS1  assays were performed. The recruitment of ETS-1 and its co-factors to the MMP1 Figure  promoterTo decipher whether AR activity modulated the proliferation of HepG2 cells, 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) and soft agar assays were performed. HepG2 cells were stably transfected with empty vectors, AR vectors, control siRNA, or AR siRNA and cultured in phenol red-free DMEM supplemented with 2% charcoal-stripped FBS added with 100 nM DHT or not. As shown in Next, the effect of AR on HepG2 cell’s anchor-independent growth was examined. Enhancing AR’s activity promoted the anchor-independent growth of HepG2 cells Figure  and 6C cin vitro invasion or migration.Then, the effect of AR on HepG2 cell’s invasion and migration was also examined. DHT treatment enhanced HepG2 cells’ invasion and migration Figures  and 7B, in vivo growth in male mice but not female mice. These results indicate that AR may function via a ligand-dependent manner in HCC.Additionally, the effect of AR deletion on HepG2 cells was tested in nude mice. As shown in Figure To further confirm the effect of androgen-AR in HCC cells’ proliferation and invasion or migration, other HCC cells were tested. First, the expression of ETS-1 and AR was detected in HCC cells. As shown in Figure in vitro invasion or migration -dependent transcription factor, has been implicated as targeted effector of human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR) signaling pathway . In the At the same time, the transcription factor activity of ETS-1 is regulated by multiple factors. Myers et al. showed that ETS-1 should function by interacting with transcriptional co-regulators such as SRC-1or AIB-1 . SequencThe ETS family also includes many transcription factors or regulatory proteins. All-ETS family members share DNA-binding domains named as ETS domains in the C-terminal part of the protein and similar DNA-binding sites  and playProliferation and invasion or migration are the main features of metastatic malignancies, which explore critical points in cancer progression and are a major cause of mortality. AR contributes to several kinds of human cancer, including prostate cancer, urothelial carcinoma, and especially HCC . AR partRecently, nuclear receptors have been suggested as targets for anti-tumor therapy , 31. WanThis study’s results provide important details about AR’s function and mechanism in HCC’s cell proliferation ability. By establishing AR’s roles and mechanisms, this study identified AR as a useful molecular target for HCC therapy.8 sequences were synthesized by using chemical synthesis methods  and cloned into a pGL4.26 plasmid. All vectors were confirmed by DNA sequencing. The siRNA of ETS-1 was synthesized by Shanghai GenePharm Company, China. All siRNA were transfected into the cells according to the manufacturer’s protocol.The expression vectors of ETS-1, FLAG-ETS1, AR, FLAG-AR, AR siRNA, ARE-Luc, MMP1-Luc, and MMP9-Luc were gifts from Dr. Jiajun Cui and Dr. Fan Feng as described in references  and 3]..3]. The 2. The human PC-3 prostate cancer cell line was a gift from Dr. Fan Feng and described in reference [The ARQ-197 was obtained from Active Biochemicals, Selleck Company, USA. The dihydrotestosterone  and mifepristone (AR’s antagonist) were obtained from Sigma . Compounds were configured to a 10 mM DMSO solution and stored at 4°C. Recombinant human HGF was obtained from Pepro-Tech . The human HCC cell line HepG2, Hu7, MHCC-97H, or MHCC-97L was obtained from the Cell Resources Center of the Chinese Academy of Medical Sciences in China. HepG2 cells were cultured in complete Dulbecco’s modified Eagle’s medium  and incubated at 37°C with 5% COHepG2 or MHCC-97L cells were seeded in 24-well plates  containing phenol red-free DMEM supplemented with 0.5% charcoal-stripped FBS  in the presence or absence of DHT or HGF. PC-3 cells were cultured in an RPMI1640 medium containing 0.5% charcoal-stripped FBS ; in the presence or absence of DHT or HGF. Transfection was performed using Lipofectamine 2000 . Cells which were co-transfected with luciferase reporters  were harvested for luciferase analysis according to the methods described in . The lucThe total RNA of the HepG2 cells was extracted using a PARISTM Kit  according to the manufacturer’s instructions. A Multiscribe ™ Reverse Transcriptase  was used to synthesize the complementary DNA templates. Real-time reverse transcription-polymerase chain reactions were performed by an Applied Biosystems 7500 detection system using Maxima SYBR Green/ROX qPCR Master Mix Assays  and following the protocols provided in reference . The expTotal protein samples were performed by SDS-PAGE and incubated with antibodies following the methods provided in reference . AntibodHepG2 or MHCC-97L cells were transfected with plasmids using Lipofectamine 2000. Then the cells were harvested and lysed in an immunoprecipitation buffer culture for 18–24 h at 4°C. The Co-IP analysis was performed with an anti-FLAG monoclonal antibody  and then detected by immunoblotting assays treated without or with 100 nM DHT following the protocol described in reference .GST-AR or GST-ETS1 was expressed in E. coli strain DH5α and bound to glutathione-Sepharose beads purified as described by the manufacturer . FLAG-AR or FLAG-ETS1 was expressed in HEK293T cells and purified by FLAG-beads. FLAG-AR or FLAG-ETS1 was incubated with GST alone, GST-ETS1, or GST-AR bound to glutathione-Sepharose beads from reference  at 4°C. The subcellular localization of AR and ETS-1 was determined by subcellular fractionation assays following the protocol described in reference . Cells wThe chromatin immunoprecipitation (ChIP) was performed following the protocol provided by the ChIP kit  and as described in reference . HepG2 c® nonradioactive cell proliferation assay kit , per the manufacturer’s instructions. Cells, which were transfected with plasmids or treated with agents, were seeded onto 96-well plates and incubated at 37°C with 5% CO2. After incubating for 24, 48, 72, 96, 120, and 144 h, cells were harvested and analyzed. Finally, growth curves for each cell group were drawn according to the volume of O.D.490 nm values from the 96-well plate reader. The MTT cell growth assays were performed three times independently.Cell proliferation was analyzed by MTT-assay as described previously in reference . HepG2 cHepG2 cells were treated with compounds. Cells were plated on six-well plates (500 per well), with a bottom layer of 0.7% low-melting-temperature agar in phenol red-free DMEM supplemented with 2% charcoal-stripped FBS and a top layer of 0.25% agar in phenol red-free DMEM supplemented with 2% charcoal-stripped FBS. The colony number was the mean ± SD of triplicate independent experiments scored after 3–4 weeks of growth .5 cells/ml) in serum-free medium, and the bottom chambers were filled with 0.25 ml of an RPMI 1640 medium containing 10% FBS. The cells were incubated in the transwells at 37°C in 5% CO2 for 4 h or 24 h. The relative invading/migrating cells were measured following the methods described previously in [The invasion and migration assays were performed in 24-well plates using a transwell chamber  fitted with a polyethylene terephthalate filter membrane with 8-μm pores following the protocol described in . For theously in .P < 0.05 was considered statistically significant. The EC50/IC50 values were calculated by Origin 6.0 software.The results of the RT-PCR and WB assays were analyzed by ALPHA INNOTECH software. The relative expression level  = (indic