Our results showed that AR stabilizes Siah2 protein by attenuating its self-ubiquitination and auto-degradation, likely by blocking its E3 ubiquitin ligase activity. Conversely, ADT decreased Siah2 protein expression but enhanced its E3 ligase activity in PCa cells. Notably, the findings that ADT decreasing Siah2 protein expression were verified in a series of paired PCa samples from the same patient. Additionally, we found that ADT-induced Siah2 activation could be abolished by Vit K3. Strikingly, ADT combined with Vit K3 treatment delayed the occurrence of CRPC and dramatically inhibited the growth of tumor xenografts compared with ADT treatment alone. AR is an inhibitor of Siah2 in PCa, and ADT leads to the continuous activation of Siah2, which may contribute to CRPC. Finally, ADT+Vit K3 may be a potential approach to delay the occurrence of CRPC.Siah2 is an E3 ubiquitin ligase that targets androgen receptor (AR) and plays an important role in the development of castration-resistant prostate cancer (CRPC). However, the regulation of Siah2 in prostate cancer (PCa) is largely unknown. In this study, we used AR-dependent and -independent cells lines to investigate the cellular roles of AR and androgen deprivation therapy (ADT) on Siah2 protein levels and E3 ligase activity using Western blotting and co-immunoprecipitation. We also validated our findings using patient samples taken before and after ADT. Finally, we used xenograft tumor models to test the effects of ADT combined with vitamin K3 (Vit K3) on tumor growth  Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer-related mortality in American men, with an estimated 174,650 new cases and 31,620 deaths expected in 2020  . Many prGiven its role in CRPC, it is important to know how Siah2 is regulated in PCa. We and others have previously reported that Siah2 is regulated by proteins, such as DHX15 and AKR1C3 in PCa , 15. HowAll plasmid constructs were created or obtained as previously described .l-glutamine at 37°C with 5% CO2. Cells were verified as mycoplasma free using PCR. Cells were cultured in phenol red-free medium supplied with 5% dextran-coated charcoal-stripped fetal bovine serum (CS-FBS) for 24 h before treatment with dihydrotestosterone . In some experiments, cells were treated with the protein synthesis inhibitor cycloheximide (Sigma-Aldrich) at 50 μg/ml and/or the proteasome inhibitor MG132 (Sigma-Aldrich) at 5 μM for various times as described in the figure legends. Cell transfection was separately performed with Lipofectamine 2000  for constructs or siRNAs according to the manufacturer’s protocols. The siRNA targeting AR was purchased from Thermo Fisher Scientific . The siRNAs targeting Siah2 were as follows: Siah2-1, 5′-UAUGACUUGCUUUCCUAGGCAAUCCAC-3′; Siah2-2, 5′-CCUCCCAUUCCUAACACACUGAUCUAU-3′.The human PCa cell lines LNCaP, 22Rv1, and PC3, and HEK293 cells were obtained from American Type Culture Collection . Cells were maintained in the appropriate medium  supplied with 10% fetal bovine serum, 5% antibiotics, and 1% Western blot and immunoprecipitation assays were performed as previously described . PrimaryHEK293 cells were transfected with Flag-AR, GFP-AR, and HA-ubiquitin as indicated for 24 h, and then were treated with 5 μM MG132 for 16 h before harvest. Cells were lysed in 100 μl RIPA buffer with 1% SDS to disrupt protein–protein interactions, and then boiled for 10 min at 95°C. The lysates were diluted 10-fold with RIPA buffer and immunoprecipitated with Flag M2 gel for 3 h followed by incubation with protein A/G Plus-Agarose for 3 h. After three washes, the immunoprecipitates were subjected to Western blot analysis.RT-PCR and real-time PCR were performed as described previously . The priFormalin-fixed paraffin-embedded (FFPE) PCa specimens were obtained from the surgical pathology archives of Shanghai General Hospital. Use of these prostate tissues was approved by the Shanghai General Hospital Review Board. Immunohistochemistry was performed as previously described  using anThe transwell migration and BrdU incorporation assays were performed as previously described .via twice weekly intra-peritoneal injections. Tumor growth was monitored twice per week for 7 weeks, at which point the mice were sacrificed and tumors were harvested for Western blot analyses. All animal studies were conducted in accordance with the Shanghai Jiao Tong University Medical School’s Animal Committee guidelines.Male athymic BALB/c nude mice (5–6 weeks old) purchased from the Animal Center of the Chinese Academy of Sciences  were subcutaneously injected in one flank with 300 μl of LNCaP cells (1 × 106) mixed 1:1 (v:v) with Matrigel (Invitrogen). Tumors were measured with calipers twice per week. Tumor volumes were calculated using the formula length × width 2 × 0.52. Mice were randomized into three groups once the tumor volume reached 0.6 mm3: the sham-operated (n=5), castration (n=5), and castration+Vit K3(Sigma-Aldrich) injection groups (n=5). For the latter, a dose of 10 mg/kg Vit K3  was administered P < 0.05 was considered statistically significant.Data are presented as mean ± standard error (SEM) or mean ± standard deviation (SD). Statistical analyses were performed with Student’s t-test or one-way ANOVA. ). To further address the role of AR in DHT-mediated Siah2 stabilization in PCa cells, siRNAs specifically targeting AR or the antagonist flutamide were used to block AR. Both siRNAs and flutamide decreased Siah2 protein expression . Consistently, DHT treatment increases Siah2 protein level in AR-positive LNCaP cells but not in AR-negative PC3 cells  for 24 h led to a significant increase in Siah2 protein expression . RING mutant Siah2 alone or co-expression with AR did not change the half-life of Spry2 , one of the classic substrates of Siah2 and a marker for Siah2 ligase activity. Overexpressing wide-type Siah2 effectively reduced Spry2 half-life from approximately 5 to 3 h, while co-expression of AR prolonged Spry2 half-life to approximately 4 h , which suggests increased Siah2 activity.Given that AR stabilized Siah2 protein and inhibited its E3 ligase activity, we hypothesized that ADT may increase Siah2 activity in PCa. To test the effect of ADT on Siah2 in PCa cells, LNCaP or 22Rv1 cells were cultured in medium supplemented with 5% CS-FBS (to mimic ADT conditions) or in complete medium. As expected, both Siah2 and Spry2 expressions were significantly decreased when the cells were cultured in CS-FBS medium compared with those in complete medium, which was consistent with AR expression , which was consistent with our in vitro findings. These data suggest that ADT decreases Siah2 protein expression and enhances its E3 ligase activity in PCa.To assess the relevance of our findings in human PCa, we evaluated Siah2 protein expression in 29 PCa patients who underwent radical prostatectomy after ADT treatment for 6–9 months. Siah2 was detected immunohistochemically both in biopsy samples (before ADT) and in radical prostatectomy samples (after ADT) from the same patient. Siah2 showed a nuclear expression pattern as described previously . NotablyGiven that Siah2 plays a key role in the development of CRPC  and that). Similarly, LNCaP cell motility was inhibited following Vit K3 treatment or Siah2 silencing, but no further changes were observed in Siah2-knockdown cells treated with Vit K3 . Collectively, these data support a model in which Vit K3 inhibits the growth and motility of PCa cells through a mechanism that involves inhibition of Siah2 activity.We next evaluated the physiologic significance of Vit K3 on PCa cells. Treatment with 20 μM Vit K3 for 24 h significantly reduced proliferation of AR-positive LNCaP and 22Rv1 cells, but not of AR-negative PC3 cells , the National Natural Science Foundation of China (81502212), and the Science and Technology Commission of Shanghai Municipality, China (18ZR1430600).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


House dust mite (HDM)‐induced allergic rhinitis (AR) is a highly prevalent disease with bothersome symptoms. Genetic variants of the Hippo pathway genes play a critical role in the respiratory disease. However, no study has reported associations between variants of the Hippo pathway genes and HDM‐induced AR risk.Forty‐three key genes in the Hippo pathway were selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome pathway database, and previous reported studies. A case‐control study of 222 cases and 237 controls was performed to assess the associations between 121 genetic variants in these genes and HDM‐induced AR risk. DNeasy Blood & Tissues Kits were used for extracting genomic DNA from the venous blood and Infinium Asian Screening Array BeadChips for performing genotyping. A logistic regression model was applied to evaluate the effects of variants on HDM‐induced AR risk. The false discovery rate (FDR) method was utilized to correct for multiple testing. The receiver operating characteristic (ROC) curve was plotted to obtain the cut‐off value of total IgE for the diagnosis of HDM‐induced AR. Histone modification and transcription factor binding sites were visualized by UCSC genome browser. Moreover, expression qualitative trait loci (eQTL) analysis was obtained from Genotype‐Tissue Expression (GTEx) database.DLG5 was significantly associated with a decreased HDM‐induced AR risk after FDR correction . The rs754466 A allele reduced the risk of HDM‐induced AR in the subgroup of moderate/severe total nasal symptom score (TNSS). Furthermore, rs754466 was associated with a high mRNA expression of DLG5. Additionally, histone modification and transcription factor binding sites were rich in the region containing rs754466.We found that rs754466 in DLG5 decreased the susceptibility to HDM‐induced AR.Our findings indicated that rs754466 in  Cases with asthma, atopic dermatitis, sinusitis, other nasal disorders, and malignant diseases were excluded. The healthy controls were randomly selected from physical examination centers in the same geographical region from April 2015 to October 2019 and with matched age (±5 years) and sex. The healthy controls met all the following criteria: (1) no symptoms of nasal disorders; (2) no symptoms and family history of atopic diseases; (3) specific allergen‐IgE < 0.35 kUA/L measured by the Phadiatop test . All the individuals were Han Chinese and signed informed consent. Approximately 5 ml of peripheral venous blood was donated by each individual. The research approval was obtained from the ethics committee at Nanjing Medical University.A total of 222 cases and 237 controls were included in this case‐control study. The cases were recruited from the First Affiliated Hospital of Nanjing Medical University between May 2008 and October 2017. The diagnosis of HDM‐induced AR was established according to Allergic Rhinitis and its Impact on Asthma criteria.2.2Der p, Der f, cat dander, dog dander, Blatella germanica, Alternaria alternate, Ambrosia elatior, and Artemisia vulgaris were also detected in cases by ImmunoCAP system. Phadiatop tests were conducted in controls.A total nasal symptom score (TNSS) on a scale of 0–12 evaluating sneezing, rhinorrhea, nasal itching and nasal obstruction was used to assess the disease severity. The score of each symptom is: 0 = no symptoms; 1 = mild symptoms ; 2 = moderate symptoms ; 3 = severe symptoms .2.3p value of Hardy–Weinberg equilibrium (HWE) > 10−6. After that, pairwise linkage disequilibrium (LD) analysis (r2 ≥ 0.8) was performed to obtain tagging SNPs by Haploview 4.2 software. Finally, web‐based tools (SNPinfo Web Server (https://snpinfo.niehs.nih.gov/), HaploReg , and RegulomeDB (https://www.regulomedb.org/regulome‐search/)) were used to predict putative functions of genetic variants. In addition, genetic variants with RegulomeDB Score ≥6 were removed.Forty‐three key genes in the Hippo pathway were selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome Pathway database, and reported studies.2.4Genomic DNA was successfully extracted from the venous blood of all individuals using DNeasy Blood & Tissue Kits . Genotyping was performed by Infinium Asian Screening Array BeadChips . The genetic variants and samples were selected according to a uniformed quality control protocol.2.5https://www.gtexportal.org/home/) for genetic variants.Histone modification and transcription factor binding sites were visualized by UCSC genome browser from chromatin immunoprecipitation sequencing (ChIP‐Seq) data stored in ENCODE. The expression qualitative trait loci (eQTL) analysis was obtained from Genotype‐Tissue Expression (GTEx) database  curve was plotted to obtain the cut‐off value of total IgE for the diagnosis of HDM‐induced AR. The levels of total IgE and specific IgEs to both Der p and Der f were analyzed after logarithmic transformation. The logistic regression model was performed to calculate adjusted and crude odds ratios (ORs) and their 95% confidence intervals (CIs). The false discovery rate (FDR) method was utilized to restrict the probability of false‐positive findings, due to the large number of genetic variants selected. The heterogeneity was tested with Cochran Q‐test and I2 statistic. p < 0.05 was considered as a statistically significant level. All statistical analyses were computed by R 3.6.2 and PLINK v1.09.Student's 33.1p = 0.739) and sex (p = 0.974) were found between cases and controls. The level of total IgE was significantly higher in cases than in controls (p < 0.001). According to the ROC curve  (Table DLG5 was found to be associated with a decreased risk of HDM‐induced AR (PFDR = 3.93 × 10−2).Of 49 candidate genes from KEGG and Reactome Pathway database, we teased out 43 genes having vital functions in diseases from reported studies Figure . The flo5) Table . After Fhet = 0.57, 95% CI = 0.37–0.87, p = 9.16 × 10−3; ORhom = 0.18, 95% CI = 0.05–0.65, and p = 8.88 × 10−3). In the additive model, we observed that individuals with the A allele had a reduced risk of HDM‐induced AR, compared with those with the T allele . Compared with the TT genotype, the TA/AA genotypes were associated with a lower risk of HDM‐induced AR . Furthermore, the AA genotype was also associated with a decreased risk of HDM‐induced AR compared with TT/TA genotypes .To comprehensively investigate the association between rs754466 and HDM‐induced AR risk, we used four genetic models  to analyze the correlation of rs754466 with HDM‐induced AR risk Table . The fre3.3p < 0.05). Additionally, the rs754466 A allele decreased the risk of HDM‐induced AR in all subgroups of total IgE, specific IgE to Der p, and specific IgE to Der f . However, no significant heterogeneity was observed in all these subgroup analyses .Subgroup analyses based on different demographic characteristics were performed Figure . The rs7p = 9.70 × 10−4). A stronger association was also observed between the rs754466 A allele and a decreased HDM‐induced AR risk in moderate/severe rhinorrhea, moderate/severe nasal obstruction, mild sneezing and mild nasal itching subgroups , Salvador family WW domain‐containing protein 1 (SAV1), large tumor suppressor half (LATS1/2), MOB kinase activator 1 A/B (MOB1A/B), Yes‐associated protein (YAP), and transcriptional co‐activator with PDZ‐binding motif (TAZ).DLG5, discs large MAGUK scaffold protein 5, encodes a membrane‐associated guanylate kinase (MAGUK) containing a coiled‐coil domain, a GUK domain, a SH3 domain, a CARD domain, a Duff domain, and four PDZ domains.DLG5 can increase the phosphorylation of YAP and TAZ to inhibit the nuclear translocation of YAP and TAZ.DLG5 lowly expressed in Crohn's disease,DLG5 downregulation leads to emphysema‐like lesions,DLG5 might function in respiratory diseases. Notably, HDM may initiate HDM‐induced AR through impairing nasal epithelial barrier.DLG5 might participate in the pathogenesis of HDM‐induced AR. However, the expression of DLG5 is required to be detected for further validating our hypothesis. And further biological function experiments are necessary to fully illuminate the detailed molecular mechanisms underlying the role of DLG5 in HDM‐induced AR.DLG5 are significantly associated with Crohn's disease susceptibility.DLG5 and HDM‐induced AR have not been reported. In the stratification analyses, we found that the rs754466 A allele decreased the risk of HDM‐induced AR in all subgroups of age and sex, indicating that this variant might be of predictive value for HDM‐induced AR population. Furthermore, rs754466 in DLG5 had a significantly reduced risk of HDM‐induced AR in subgroups of serum indexes  and nasal symptoms. The nasal epithelial barrier dysfunction results in an increased allergen passage and then B cells produce IgE after allergens uptake.DLG5, thereby restraining the allergens intrusion and thus decreasing IgE production and the occurrence of nasal symptoms. This hypothesis may be supported by our findings in GTEx database. Although nasal tissues were unavailable in the GTEx database, we observed that the rs754466 A allele came with a high expression of DLG5 in lung tissues. Given the anatomic relationship between nose and lung , we dared to speculate that the rs754466 A allele might increase the expression of DLG5 in nasal tissues. As a consequence, the nuclear translocation of TAZ was inhibited and barrier function was strengthened, all contributing to the lower risk of HDM‐induced AR. These findings may refresh our understanding of the pathogenesis, provide a novel target for early prevention and precision treatment of HDM‐induced AR.Many genetic variants in Our study has some limitations. First of all, AR is a complex disease induced by gene‐environment interactions. Many environmental factors such as nitric oxides, sulfur pollutants, organic chemical agents, particulate matter and tobacco smoke have been shown associated with AR risk.5DLG5 had a significant decreased risk of HDM‐induced AR. Our findings might provide new insight into the pathogenesis of HDM‐induced AR and a new therapeutic target.We performed the first analysis for associations between genetic variations in the Hippo pathway genes and the risk of HDM‐induced AR in a Chinese population. And we found that rs754466 in All participants signed their written consent for publication.The authors declare that they have no competing interests.Supporting Information S1Click here for additional data file.


To compare the quantitative angiographic aortic regurgitation (AR) of six self-expanding valves after transcatheter aortic valve replacement (TAVR).Quantitative videodensitometric aortography (LVOT-AR) is an accurate and reproducible tool for assessment of AR following TAVR.This is a retrospective central core-lab analysis of 1,257 consecutive cine aortograms performed post-TAVR. The study included 107 final aortograms of consecutive patients who underwent TAVR with first-generation VitaFlow in four Chinese centers and 1,150 aortograms with five other transcatheter aortic valves . LVOT-AR analyses of these five valves were retrieved from a previously published pooled database.p < 0.001). Post hoc 2-by-2 testing showed that CoreValve had significantly higher LVOT-AR compared with each of the other five THVs. No statistical difference in LVOT-AR was observed between VitaFlow, Evolut Pro, Evolut R, Acurate Neo, and Venus A-valves. The VitaFlow system had the lowest proportion of patients with LVOT-AR >17% (4.7%) , followed by Evolut Pro (5.3%), Evolut R (8.8%), Acurate Neo (11.3%), Venus A-valve (14.2%), and CoreValve (30.1%) (chi-square p < 0.001).Among 172 aortograms of patients treated with VitaFlow, 107 final aortograms (62.2%) were analyzable by LVOT-AR. In this first in man eight cases necessitated a procedural valve in valve due to inappropriate TAVR positioning and severe aortic paravalvular regurgitation. In the VitaFlow group, the mean LVOT-AR of the intermediate aortograms was 7.3 ± 7.8% and the incidence of LVOT-AR >17% was 8.6%. The mean LVOT-AR of the final aortogram was 6.1 ± 6.4% in the VitaFlow group, followed by Evolut Pro (7.3 ± 6.5%), Evolut R (7.9 ± 7.4%), Venus A-valve (8.9 ± 10.0%), Acurate Neo (9.6 ± 9.2%), and lastly CoreValve (13.7 ± 10.7%) (analysis of variance Compared to other commercially available self-expanding valves, VitaFlow seems to have a low degree of AR and a low proportion of patients with ≥moderate/severe AR as assessed by quantitative videodensitometric angiography. Once the learning phase is completed, comparisons of AR between different transcatheter heart valves should be attempted in a prospective randomized trial. Modifications in the design of THVs, such as radial force, sealing skirt, frame composition or size of struts, may, among others, influence the THV's sealing capacity. A wide variation in AR severity among different THVs has been reported (2–30% AR in the left ventricular outflow tract [LVOT-AR] >17%), with lower degree of LVOT-AR in THVs that feature an anti-leak skirt .In the current study, first data on quantitative videodensitometric assessment of AR in TAVR patients with the VitaFlow system  are presented, and compared to five other self-expanding THVs.This is a retrospective, multi-center analysis of consecutive aortograms in 172 consecutive TAVR patients treated with the VitaFlow THV at four Chinese centers. The study complied with the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by each center's ethics committees.in vivo, in animal models as well as in clinical correlations with magnetic resonance imaging, transthoracic and transesophageal echocardiography have been extensively reported in the literature  and remotely supervised by an independent core laboratory  using the CAAS A-valve 2.0.2 software , not financially subsidized by industry. The quantitative assessment of the AR from the aorta into the LVOT is reported as the LVOT-AR parameter. The results are expressed in percentages and quantify the fraction of AR defined as the ratio between the area under the time-density curves assessed by videodensitometry in the LVOT  and in the aortic root (reference area) during a conventional aortography . Technicterature , 18, 20.terature . Importaterature , 18. Theterature , 12.The first-generation VitaFlow system consists of a self-expanding nitinol frame with robust radial strength, a tri-leaflet bovine pericardial valve, and a delivery caheter that does not afford the option for valve repositioning. The double-layer (inner and outer) polyethylene terephthalate (PET) skirt at the LVOT is designed to reduce post TAVR PVL. We aimed to compare, in the six SEVs, the quantitative AR in the left ventricular outflow tract (LVOT-AR). Comparison among the THVs was performed with regard to the mean LVOT-AR (continuous variable assessment of LVOT-AR) and with regard to the proportion of patients  with that particular THV presenting with moderate or severe regurgitation, with a pre-determined threshold criterion of LVOTAR >17%, previously identified as moderate or severe regurgitation with respect to echocardiography , 17.post-hoc Bonferroni test. Continuous variable regurgitation was stratified into categorical variables according to the following pre-determined threshold criteria: (1) none or trace (LVOT-AR <6%); (2) mild (6% to ≤ 17%); and (3) moderate or severe (>17%) .Continuous variables were reported as mean ± standard deviations. Comparison of LVOT-AR was performed using 1-way analysis of variance and 2-by-2 comparisons using the e (>17%) . The pron = 33, 50.8%) overlapping of the descending aorta on ascending aorta , duration of the acquisition is too short , deep breathing or table motion , insufficient contrast , radiopaque structure in LOVT , others .Out of 172 aortograms of patients treated with VitaFlow THV, 107 final aortograms (62.2%) were analyzable by quantitative assessment of AR. The study flow chart is shown in In this first in man experience 8 cases nessessitate a valve in valve treatment due to inappropriate positioning of the valve resulting in severe aortic paravalvular regurgitation. Among the eight valve in valve cases, only six were analyzable by videodensitometric assessment . The twoThe mean LVOT-AR of the intermediate aortograms prior to the valve in valve treatment in 6 cases was 23.8 ± 8.5%, after the second valve implantation, the mean LVOT-AR of the final aortogram in these six cases was 2.5 ± 2.4%.n = 107) for VitaFlow. The mean LVOT-AR of the final aortogram was 6.1 ± 6.4% (n = 105) in the VitaFlow group followed by Evolut Pro , Evolut R , Venus A-valve , Acurate Neo , and lastly CoreValve   (The mean LVOT-AR of the intermediate aortograms was 7.3 ± 7.8% (p <0.05) . Post hop <0.05) .p < 0.001) (The proportion of patients with LVOT-AR >17% (AR after the intermediate aortograms) was 8.6% in the VitaFlow group. If the final aortograms was analyzed VitaFlow THV had the lowest proportion of patients with LVOT-AR >17% (4.7%) followed by Evolut Pro (5.3%), Evolut R (8.8%), Acurate Neo (11.3%), Venus A-valve (14.2%), and CoreValve (30.1%) (chi-square < 0.001) .The cumulative frequency curves of LVOT-AR after TAVR for the six THVs are shown in This is the first in man study comparing by quantitative aortography the degree of acute regurgitation following TAVR with VitaFlow in comparison with multiple cohorts of consecutive “real world” patients treated with other SEVs. The main findings of the present study are as follows:(1) The VitaFlow THV, in comparison with other self-expanding percutaneous valve analyzed in our database, showed numerically low mean degree of LVOT-AR. The first-generation CoreValve had the highest mean LVOT-AR among the evaluated valves.(2) The VitaFlow THV had a low proportion of patients with LVOT-AR >17% compared with other SEVs analyzed in our database.(3) The four contributing centers were in their learning phase, which could explain the high incidence of procedural valve in valve implantation in order to correct a severe aortic paravalvular regurgitation due to malpositioned implantation in the initial attempt.Paravalvular regurgitation, even in those with mild PVL, following TAVR is associated with mortality , 21, 22.The Seller's visual grading  of aortiFurthermore, echocardiographic assessment of regurgitation is semiquantitative, operator dependent, and not reproducible, even when performed by core laboratories . PreviouThe first-generation VitaFlow THV is a novel Chinese transcatheter valve system with a unique combination of characteristics such as a self-expanding nitinol frame with high-radial force, a double-layered PET skirt, and features the only commercially available motorized delivery system worldwide ; Table 2It was granted China's National Medical Products Administration approval in July 2019.In the current study, the VitaFlow THV, in comparison with the other commercially available SEVs analyzed in our database, showed a low degree of mean LVOT-AR and had a low (4.7%) occurrence of moderate or severe PVL (>17 % LVOT-AR). This is in line with the previous study reporting that only 2% of patients had moderate PVL (assessed by echocardiography) at discharge and at 30 days . Of notePost-TAVR AR has been associated with mortality and adverse events , 21, 22;ClinicalTrials.gov NCT04275726)  .Compared to other commercially available self-expanding THVs, the VitaFlow THV seems to have a low degree of AR and a low proportion of patients with ≥moderate/severe AR as assessed by quantitative videodensitometric angiography. These results should be confirmed in prospective randomized comparisons of AR of different THV types.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Xijing Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.PS and LT had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis and including and especially any adverse effects. RW, CG, and HK contributed to the data collection, analysis and interpretation, and the writing of the manuscript. FM, JZ, PL, JY, JL, and LT contributed to the data collection and analysis and interpretation. DM, WW, YO, and OS contributed substantially to the data analysis and interpretation and the critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This work RW, CG, and WW were supported by Science Foundation Ireland Research Professorship Award (15/RP/2765).PS reports consultant fees from Merillife, Novartis, Philips/Volcano, SMT, Sinomedical Sciences Technology, Xeltis outside the submitted work. DM is a proctor and consultant for Microport, Boston Scientific, and Medtronic. WW is co-founder of Argonauts, medical advisor Rede Optimus Research and reports honoraria from MicroPort. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


M-ITC-LNC showed the greatest enhancement of the ITC-induced tumor growth inhibition, proliferation, and necrosis. At the molecular level, the tumor content of Gli 1, caspase-3, and vascular endothelial growth factor verified superiority of M-ITC-LNC in enhancing the ITC antiangiogenic, apoptotic, and Hedgehog pathway inhibitory effects. Finally, histopathological and biochemical analysis indicated greater reduction of ITC systemic toxicity by M-ITC-LNC. Superior performance of M-ITC-LNC was attributed to the effect of MFS on the structural and release properties of LNC coupled with its distinct bioactivities. In conclusion, MFS-modified LNC provides a simple nanoplatform integrating the potentials of LNC and MFS for enhancing the chemotherapeutic efficacy of ITC and possibly other oncology drugs.Itraconazole (ITC), a well-tolerated antifungal drug, exerts multiple anticancer effects which justified its preclinical and clinical investigation as potential anti-cancer agent with reduced side effects. Enhancement of ITC anti-cancer efficacy would bring valuable benefits to patients. We propose herein lipid nanocapsules (LNCs) modified with a subtherapeutic dose of miltefosine (MFS) as a membrane bioactive amphiphilic additive (M-ITC-LNC) for the development of an ITC nanoformulation with enhanced anticancer activity compared with ITC solution (ITC-sol) and unmodified ITC-LNC. Both LNC formulations showed a relatively small size (43–46 nm) and high entrapment efficiency (>97%), though ITC release was more sustained by M-ITC-LNC. Cytotoxicity studies revealed significantly greater anticancer activity and selectivity of M-ITC-LNC for MCF-7 breast cancer cells compared with ITC-sol and ITC-LNC. This trend was substantiated by  Itraconazole (ITC) is a well-tolerated fungicidal triazole compound  and an oily core surrounded by a pegylated surfactant tensioactive shell  incorporating MFS (0.06%) as a new bioactive nanocarrier to enhance the anticancer activity of ITC. Such a system would provide multiple advantages including MFS-induced enhancement of the structural integrity of ITC-LNC, sustainment of ITC release and improvement of the anticancer activity of ITC via MFS membrane activity and an MFs/ITC combinational effect. The efficacy of the proposed M-ITC-LNC relative to ITC solution (ITC-sol) and unmodified ITC-LNC was assessed against breast cancer. The cytotoxicity and selectivity of the test formulations were appraised using human adenocarcinoma MCF-7 breast cancer cells and normal human fibroblasts. Their antitumor efficacy and systemic toxicity were determined utilizing a murine Ehrlich ascites breast cancer model and doxorubicin (DOX) for comparison. Assessments included % change in tumor weight and volume alongside tumor proliferation and necrosis determined by histopathological and immunohistopathological examination. The tumor content of the biomarkers, Gli 1, caspase-3, and vascular endothelial growth factor (VEGF), was determined to assess the effect of test LNC formulations on the anticancer effects of ITC. Systemic toxicity was assessed biochemically and by histopathological examination of the liver and kidney of mice post treatment.® M1944 CS (oleoyl polyoxyl-6 glycerides), and Transcutol HP® (diethylene glycol monoethyl ether), were gift of Gattefossé S.A. . Lipoid® S75-3  was purchased from GMBH . Kolliphor® HS 15 (Solutol® HS-15) was purchased from BASF . Sodium lauryl sulfate (SLS), ethylene diamine-tetraacetic acid (EDTA), hematoxylin and eosin (H&E) solutions, 3--2,5-diphenyltetrazolium bromide (MTT), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) were purchased from Sigma-Aldrich . Fetal bovine serum (FBS) was purchased from GIBCO® Life Technologies . Methanol, acetonitrile, and dimethyl sulfoxide were purchased from Fisher Scientific .Itraconazole was purchased from Neuland Laboratories Limited , >99% and MFS (1-hexadecylphosphocholine) was purchased from Chem-Impex International , 98–100%. Labrafac™ lipophile WL 1349 , Labrafil® HS 15 (Kolliphor) (10%), Transcutol (7%), sodium chloride (1%), Lipoid (1.5%), and demineralized water (20.5%). The mixture was subjected to three cycles of progressive heating and cooling between 60 and 90 °C at a rate of 4 °C/min with the formation of a w/o emulsion and a o/w emulsion, above and below the phase inversion temperature, respectively. An irreversible shock was induced by a twofold dilution with deionized cold water (0–2 °C) added at a temperature 1–3 °C from the beginning of the phase inversion zone. The LNC dispersion was stirred slowly using a magnetic stirrer for 5 min and kept at 4 °C. For the preparation of ITC-LNC, the same procedure was adopted following addition of ITC to the oil phase in a 0.3% w/w concentration. For M-ITC-LNC, ITC (0.3% w/w) and MFS (0.06% w/w), at a ratio of 5:1 by weight, were included in the oil phase. Blank fluorescent LNC labeled with the DiI dye (DiI-LNC) were prepared as reported  at 25 °C and a 4 mW He–Ne laser at 633 nm . Zeta potential was determined at 25 °C in water  using a cell voltage of 150 V and 5 mA current. The morphology of ITC-LNC and M-ITC-LNC was examined by TEM using JEOL, JEM-100 CX Electron Microscope . Before analysis, LNC dispersions were sprayed onto copper grids and stained with 2% w/v uranyl acetate solution. Shots were taken at ×50k at 80 kV. Entrapment efficiency was calculated based on the difference between the amounts of entrapped and un-entrapped ITC. LNC were separated using an ultrafiltration/centrifugation technique and ITC was assayed in the ultrafiltrate by an isocratic reverse phase HPLC method with UV detection at 225 nm  of LNC were determined by dynamic light scattering (DLS) using Malvern Zetasizer−1 using FT-IR Spectrometer  after compression of the samples with IR grade KBr into discs.The thermal behavior of ITC and MFS alone and in freeze-dried LNC formulations was examined by differential scanning calorimetry (DSC) using a DSC-6 differential Scanning Calorimeter . DCS traces were recorded between 40 and 400 °C at a constant 20 °C/min rate under an atmosphere of nitrogen purged at a flow rate of 20 mL/min. An empty pan was used as reference. Fourier transform infrared (FT-IR) spectra of the same samples were recorded from 4000 to 500 cm®6 centrifugal filters. The filtrate containing the released ITC was injected into the HPLC column. Results expressed as cumulative ITC release (%) are the average of three determinations. The release kinetics of ITC-LNC were determined by fitting release data to different mathematical models and choosing the best fit by regression analysis using an Excel add-in.ITC release from M-ITC-LNC in comparison with ITC-LNC was investigated at 37 ± 0.5 °C in a thermostatically controlled shaking water bath at 100 rpm. A developed release medium allowing sink conditions was based on a solubility study of ITC in phosphate buffer saline  containing SLS in increasing concentrations. A known volume of the LNC dispersion was added to 5 mL of the selected release medium (5% SLS in PBS pH 7.4) in closed flasks. At different time intervals, LNC were separated by ultracentrifugation at 12,000 rpm for 10 min at 4 °C using Vivaspinin vitro erythrocyte hemolytic activity using fresh human blood  and were then seeded (5 × 103/well) in a 96-well plate containing 100 μL of DMEM and allowed to adhere to the plate for 24 h. The medium was replaced with a fresh medium containing ITC-sol, ITC-LNC or M-ITC-LNC and incubated for another 24 h. The cells were washed twice with PBS pH 7.4 after removal of the treatments and were incubated with 100 μL MTT solution (0.5 mg/mL in DMEM) for further 4 h at 37 °C in the dark. After removal of the supernatant by centrifugation at 2000 rpm for 10 min, 100 μL of DMSO was added to the wells to dissolve the MTT-formazan crystals by agitation for 15 min. Absorbance was measured at 570 nm using a microplate reader . Values for IC50 were determined using Origin 8.0 software . Dose–response curves were plotted after correction by subtracting the background absorbance from the controls. The relative cell viability (%) was calculated relative to the untreated control cells as follows:A is the absorbance of the treated wells and Ac is the absorbance of control wells. Values for IC50 were extrapolated graphically from the plotted data using a polynomial regression equation.The cytotoxicity of M-ITC-LNC in comparison with ITC-sol and ITC-LNC at different ITC concentrations on human breast adenocarcinoma MCF-7 cell line (American Type Culture Collection (ATCC), Manassas, VA) was evaluated in triplicate using the MTT assay. DMSO and blank LNC were used as controls. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS in a CO3 cells/well) that were cultured in DMEM containing 5% FBS. Following 24 h incubation, the % cell viability was calculated using the MTT assay and the IC50 values for normal cells estimated. Selectivity for MCF-7 cancer cells was assessed by calculating the selectivity index (SI) using the following equation . The study protocol was approved by the Institutional Animal Care and Ethics Committee of the Faculty of Pharmacy, Alexandria University, Egypt . A total of 30 female BALB/c mice 7–8 weeks old and weighing 20–25 g were randomly housed at 22 ± 5 °C in a 12 h light/dark cycle at 6 mice/cage. Mice were fed rodent chow and water at libitum. In vivo studies were conducted for visualizing the biodistribution of fluorescent blank DiI-LNC and assessment of the antitumor efficacy and systemic toxicity of M-ITC-LNC relative to ITC-sol and ITC-LNC using DOX for comparison. Mice were weighed at baseline and 7- and 14-days post treatment initiation.TM Optima, Biospace Lab, Nesles-la-Vallée, France).Biodistribution of LNC was assessed by fluorescence imaging of DiI-LNC  14 days post cell inoculation and continued for 14 days. Tumor-bearing mice were randomly divided into five groups, six mice each, as follows: group 1: control (untreated), group 2: DOX injection , group 3: ITC-sol in DMSO (10 mg/kg once daily), group 4: ITC-LNC (10 mg/kg once daily), and group 5: M-ITC LNC providing the dose of 10 mg/kg ITC and 2 mg/kg MFS, once daily. At the end of the 14 day-study, animals were sacrificed with a large dose of thiopental (50 mg/kg. i.p.) and the excised tumors were washed with ice-cold phosphate buffer and their weights determined. Tumors were then divided into portions for different assessments. Antitumor efficacy was assessed by determining the % change in tumor volume and tumor weight, histopathological and immunohistopathological examination of the tumor for necrotic and antiproliferative effects, respectively, alongside quantitative determination of three tumor biomarkers.Mammary tumors were induced in female balb/c mice using Ehrlich ascites tumor (EAT) cells obtained from the National Cancer Institute, Cairo, Egypt. In brief, approximately 10The % change in tumor volume in all mice was determined at days 7 and 14 from the start of treatment compared to baseline tumor volume. Tumor volume was calculated by measuring the tumor length (major axis) and width (minor axis) with a Vernier caliper as follows:Tumor necrosis was examined histopathologically. Sections of the fixed excised mammary tumors, 5 μm thick, were stained with H&E, dehydrated in alcohol, mounted in Canada balsam, and examined blindly by optical microscopy. Necrosis was assessed semi-quantitatively as the ratio of necrosis area to the total area in 10 random sections of each tumor using a scoring scale based on % necrosis in sections in poorly differentiated tumor , caspase-3, and VEGF were determined quantitatively in samples of excised tumors using ELISA Kits  according to the respective manufacturer’s protocols.in vivo study and before animal sacrifice, blood was collected by aortic vein puncture in microcentrifuge tubes and centrifuged at 2500 rpm for 5 min at 4 °C. The supernatant serum was assayed for alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine levels using commercial kits (Accurex®). In parallel, biopsies of the livers and kidneys of sacrificed mice were collected, fixed in 10% neutral buffered formalin, and embedded in paraffin for histopathological examination. Sections were stained with H&E and digital shots were taken.Systemic toxicity of the test ITC-based formulations was evaluated by monitoring animal weight at day 7 and day 14, histopathological examination of the mice livers and kidneys and biochemical analysis of the liver and kidney functions. At the end of the p < .05 was indicative of significance.Data were analyzed using Minitab ver. 17 software. Data expressed as means ± SD are representative of at least three measurements. The differences between multiple groups were assessed by one-way analysis of variance (ANOVA) and Dunnett’s test. A value of ® HS 15 as the pegylated surfactant forming the tensioactive shell  and PDI (0.18–0.24) which were not affected by ITC loading . For lip−1 attributed to the aromatic CH and NH2 groups, peaks at 1613 cm−1 and 1425 cm−1 due to the C=N and C–N bonds, respectively, and a sharp peak at 1699 cm−1 ascribed to the stretching of the C=O bond  sustained (22.5%) by M-ITC-LNC. As lipophilic drugs are released from LNC via diffusion into the aqueous medium across the tensioactive interfacial barrier  was less than 1, denoting a case 3 parabolic release profile  reduced the hemolytic activity of ITC to ≈6% due to the dense protective Lipoid/Kolliphor® stealth shell of LNC. Stealth nanocarriers hinder interaction with the erythrocyte membrane, enhancing hemocompatibility of the entrapped drug  by MFS modification. Free MFS is known to induce hemolysis by destabilization of the erythrocyte membrane  reduced hemolysis to less than 5%, implying hemocompatibility of both formulations.p < .05) reduced the IC50 of ITC-sol (1.5-fold) (p < .05) increased the selectivity of ITC for MCF-7 cells (1.4-fold). M-ITC-LNC achieved a further significant (p < .05) selectivity enhancement (3.5-fold). Results implied substantial reduction of indiscriminative cytotoxicity and greater selectivity of ITC treatment by MFS-modified LNC.The cytotoxicity of M-ITC-LNC in comparison with ITC-sol and ITC-LNC over an ITC concentration range of 25–800 µg/mL was assessed using MCF-7 cell line following 24 h contact with the cells at 37 °C. Results for cell viability and IC50 are shown in .5-fold) . Such a .5-fold) . Notably.5-fold)  or conju.5-fold) . Selecti.5-fold) . Lipid nBiodistribution of blank DiI-LNC in comparison with Dil-sol was examined to verify the structural stability and accumulation of LNC in vital organs following i.p. injection. Dil is a lipophilic indocarbocyanine dye proven to be retained in LNC  reduced by Dox and ITC test formulations to less than ±45% (p<.05) decreased by Dox and ITC formulations. The tumor growth inhibitory effect of these formulations was in the order M-ITC-LNC > ITC-LNC > ITC-sol. Results were consistent with digital images of the tumors (Results for tumor growth inhibition are shown in han ±45% . At day e tumors  as well e tumors . The weie tumors .p < .05). Regarding ITC formulations, ITC-sol significantly inhibited cell proliferation , an effect induced by Hh pathway inhibition  and further boosted by MFS-LNC . The difference in % proliferation reduction by M-ITC LNC and Dox treatments was not significant (p> .05).Tumor cell proliferation and treatment efficacy can be assessed by the Ki-67 proliferative protein expression determined by immunohistochemical staining of the tumor  increased by ITC-LNC and to a significantly greater extent (p<.05) by M-ITC-LNC. Although the difference between the necrotic score of M-ITC-LNC and Dox was relatively small, yet it reached statistical significance. Results were generally consistent with those of antiproliferative activity  reduced by Dox. A significant reduction in Gli 1 expression was also induced by ITC-sol . ITC is a potent inhibitor of the Hh pathway via a complex signaling cascade culminating into suppression of the Gli family of transcription factors leading to multiple anticancer effects including apoptosis and tumor growth inhibition . Increased caspase-3 expression, an apoptotic mechanism of ITC  by LNC and further potentiated by M-ITC-LNC . Possible MFS-induced bioactivity of LNC, sustained ITC delivery and/or caspase-3 dependent MFS-induced apoptosis of tumor cells  lowered by Dox (56%). Significant VEGF suppression (40%) was also induced by ITC-sol known to exert specific and dose-dependent inhibition of endothelial cell proliferation, migration, and tube formation in response to VEGF angiogenic stimulation . Inhibition of VEGF expression in cancer cells by MFS could be a contributing factor , suggesting lack of severe systemic toxicity. As indicated by liver histopathology  by Dox to normal but was elevated two-fold relative to untreated control by ITC-sol, an effect attributed to ITC hepatotoxicity  reduced 55% and 59% by ITC-LNC and M-ITC-LNC, respectively. A generally similar trend was observed with AST levels, indicating LNC-induced hepatoprotective effect. Regarding kidney function, BUN, and serum creatinine levels were within the reference ranges of both markers, 0.2–0.8 mg/dL and 30–40 mg/dL, respectively  integrating the distinct biopharmaceutical properties of LNC and the structural properties and bioactivity of MFS significantly enhanced the efficacy of ITC against breast cancer. This was verified at both the cellular and molecular levels compared with ITC-sol and ITC-LNC. Apart from increasing ITC anticancer efficacy, M-ITC-LNC enhanced the safety of ITC treatment in a murine mammary pad Ehrlich tumor model. Accordingly, M-ITC-LNC offer promise as a bioactive nano-platform for the delivery of ITC and possibly other chemotherapeutic agents. The challenge of encapsulating drugs having different physicochemical properties into LNC can be overcome by modifying the lipid/surfactant composition of LNC.


Demographic changes are putting the healthcare industry under pressure. However, while other industries have been able to automate their operation through robotic and autonomous systems, the healthcare sector is still reluctant to change. What makes robotic innovation in healthcare so difficult? Despite offering more efficient, and consumer-friendly care, the assistive robotics market has lacked penetration. To answer this question, we have broken down the development process, taking a market transformation perspective. By interviewing assistive robotics companies at different business stages from France and the UK, this paper identifies new insight into the main barriers of the assistive robotics market that are inhibiting the sector. Their impact is analysed during the different stages of the development, exploring how these barriers affect the planning, conceptualisation and adoption of these solutions. This research presents a foundation for understanding innovation barriers that high-tech ventures face in the healthcare industry, and the need for public policy measures to support these technology-based firms. Technology has always been a vital ally for healthcare , from thThis disparity is generally attributed to the market barriers present in the sector that are impeding the adoption of innovations and therefore their spread. Innovations in healthcare do not follow a linear pathway even though there is still an inclination to see these as such . Indeed,Innovations today are desperately needed. Healthcare systems worldwide are under increasing pressure. Life expectancy has increased, from 52.6 in 1960 to 72.0 in 2016 , it is eFor the healthcare sector, the ageing of the population translates into many more individuals with one or more long-term conditions, and more complex patterns of diseases . While sBased on these previously introduced challenges, the assistive robotics (AR) market is surging, providing support to patients and care workers, in the globalisation of healthcare. Assistive robots are shifting the model of healthcare from one focused on hospital treatment to one that supports independent life . HoweverIdentifying obstacles to innovation has clear policy relevance, since addressing these barriers could increase the population of innovators and boost the performance of current ones ,17,18. PBy offering insights into the current barriers of the AR market, we seek to assist this process for a market we have not seen before. The development of assistive robots for the healthcare sector is not only an issue of technology or societal acceptance. We need to work on the broader market barriers and ways to overcome them. Only then, entrepreneurs and firms will be aware of the venture challenges, and governments could take measures to support these inventions that are highly needed .Consequently, this paper starts by giving an overview of the current state of this market in The AR market relates to robotics and autonomous systems with the primary role of providing assistive help to carers or directly to patients in hospital, specialist care facility or domestic healthcare settings . This maThe AR market features robots that organise and deliver medication , that suThe assistive robotic market is different from any software or hardware application, or even other AR technologies. Manufacturing or agricultural robots have required a focus on the design of the mechatronic structure. Aerial, marine and transportation robots have focused on autonomous navigation and object detection. On the other hand, assistive robots build around the nascent human–robot interaction field . They arThe size of the global AR market in 2015 was over USD 200 million, with an 18.9% compound annual growth rate estimation from 2016 to 2024 . It is cHowever, despite the potential for the technology, there is limited evidence of AR projects that transform into new products and services for the user . The IntFurthermore, from February 2019 to March 2019, we contacted 11 robotic companies in the field, inquiring their trending activities . Only onInnovation research may be divided into two general areas of analysis: an economic-oriented tradition and an organization-oriented tradition . The firMarket barriers to new technologies can be perceived in different ways . For insNone of these studies consider the development of assistive robots as a market-orientated process; the obstacles of producing the technology from concept design to the commercialisation of the AR. We argue, therefore, that since the application of robotics in healthcare is not only an issue of technology or societal acceptance, but special attention has also to be paid to broader market barriers and ways to overcome them. As a result, this paper aimed to address the following question: what are the market barriers that are inhibiting the development of AR?To thoroughly understand the barriers, we have explored them from a market transformation perspective. Here, we do not focus on the nature of the technology or typical operating characteristics of conventional markets, but rather on the development of technology as part of a market process and what needs to be carried out in practical terms to create markets for new technologies . To dateThere is, therefore, a predominant focus only on software products. A distinction exists between the development of a hardware product and its commercialisation. Consequently, market barriers may also be different ,56.This study seeks to apply the Painuly (2001) framework, which builds upon the barriers approach to innovation, focusing on a market transformation perspective . PainulyDifferent models describe product development processes for different industries. Ulrich and Eppinger’s (2016) six phases of product development balance hardware and software development in the product development process  58]. Th. Th58]. To summarise, using a market transformation perspective, this paper explores and identifies market barriers as perceived by companies engaged or trying to engage in the AR market. We argue the study is particularly pertinent at a time of growing care needs, coupled with a minimal understanding of the AR market and the implications for health innovation policy. The current literature focuses on soft elements of AR innovation and not on the implications of the market deployment of these technologies.Moreover, the current literature around market barriers for healthcare focuses only on digital technologies. The AR market is fundamentally different from the digital or any other robotics sector. Therefore, only by exploring market barriers will policymakers and entrepreneurs be aware of the venture challenges and take measures to support these inventions that are highly needed for the healthcare sector.Semi-structured interviews were conducted with 17 people from different assistive robotic companies. The companies were based either in the UK or France . The intThe study explored their perspectives relating to the challenges they perceived and were facing during the product development and commercialisation process. Apart from having to be involved in AR, the other criterion used to select businesses was the age of the business. We aimed to obtain companies covering different business stages: seed and development, start-up, expansion, and maturity.We interviewed start-ups and companies working from at least six months to up to 11 years. This was intended in order to map the different obstacles assistive robotic companies face during the market process/product development and life cycle. Sampling comprised a mixture of purposive and convenience, given that companies had to meet our selection criteria . ParticiTo design and structure the questions for the interview, we drew on Ulrich and Eppinger’s (2015) six phases of product development. The process includes the tasks and responsibilities of the critical functions of the company for each phase, including marketing, design, and manufacture. The linearity of the process allowed us to structure the interview, but it did not unduly restrain or influence the conversation. In this sense, interviews were semi-structured and akin to Kvale’s notion of conversations with a purpose . For eacThe questions were designed following the study objectives: explore AR market barriers from the perspective of the companies, the critical functions of the development process and an initial literature review from AR projects . ExampleInterviews lasted between 20 and 50 min and were audio recorded. They started with an introduction of the objectives of the study and by the participant’s overview of their business.Then, interviews were transcribed using IBM Watson Speech to text. The transcripts were cleaned and put into a standardised format. The general inductive approach was used to analyse the transcripts to identify themes in the text that were related to the study evaluation objectives . The anaThe present paper follows  for clasThese tables present a useful heuristic device as a means of understanding the nature and scope of critical barriers in the AR market, as evidenced in the interviews. However, we recognise that the classification of barriers shown in This refers to the lack of conditions needed for perfect competition in the market, most notably access to information. The impact of hereof can be seen through the whole development process.The most frequent barrier identified was access to the healthcare industry. Specifically, for innovation to take place, entrepreneurs need access to patients, families and carers’ needs, to know and understand their problems. Only with this knowledge is it possible to generate and implement ideas for new improvements. User-centred design is seen as a vital tool when it comes to AR innovation for healthcare, and its input could overcome further barriers of adaptability and implementation. The interdisciplinary field of the innovation process demands specialised knowledge in methods of care, presenting new challenges for innovators who ask themselves whom they should involve in the process. Being able to define and then access an ideally representative sample of early adopters for the market is the initial challenge that AR companies face.‘Without contacts, there is not a really a way into it […] you are not exactly going to be able to walk into any care home ask them; do you want a robot? Can we now work with you?’The fragmented structure of healthcare systems was seen as the primary reason for this barrier since there is not a clear go-to point. Take, for instance, the National Health Service (NHS) in the UK. Commonly seen as one entity, it is, in fact, a group of many individual organisations: NHS England, NHS Scotland, NHS Wales, and the affiliated Health and Social Care in Northern Ireland, Public Health England, 195 Clinical Commissioning Groups (CCGs), 245 hospital or acute trusts, around 7454 general practices only in England, and thousands of community providers. Each often includes clinicians, financial managers, commissioners and Information Technology managers.‘GBP 1500 for a 3 h consultation’. Besides being an elevated price for new ventures, 3 h is unrealistic about the actual time needed to gain sufficient understanding of consumer needs. Every decision taken during the innovation process should count with the feedback of lead-users and stakeholders involved. Some companies mentioned that for conceptualisation, development and testing of their prototype product, consultation time ranged between 10 and 15 h.Respondents mentioned that some health organisations charge for preliminary assistance, even up to look in the streets’ for potential lead-users.The alternative is to look to family and friends to become involved in the process. This is a reason why so many entrepreneurs innovate around the needs of their relatives. Participants also mentioned the extreme option of having to ‘‘doors are not open to entrepreneurs with good ideas’.Distrust towards entrepreneurs was also mentioned as a barrier. Participants mentioned there was an expectation for eHealth entrepreneurs to have an extended portfolio of developing robots as if they were app developers. Therefore, many mentioned that ‘We don’t have a reputation, or much more, products to our names, so that we can go and say, this is a current problem, look what we have done, we got the solution … no one listens’.In particular, this last quotation illustrates the credibility problem these new entrepreneurs have in gaining acceptance in the eHealth marketplace.What makes the mentioned lack of access different for AR companies compared to digital companies is its combination with social, cultural and behavioural barriers. Where digital technologies also suffer from negative attitudes and beliefs from the healthcare members, individuals have been widely exposed to apps and programs in their daily lives, greatly improving the user perception of the innovation and adoption of these solutions ,63. RoboThe complex structure of the healthcare system has a direct impact on our target market infrastructure. According to interviewers, multiple people influence the procurement process, making it challenging to identify and locate who is, ultimately, responsible for making the final decision on a purchase or commission. Interviewers also mentioned that the spread of AR technologies within healthcare systems is slow and fails to achieve widespread use.The number of individuals involved in the adoption process not only defines the demand of the market but also slows down the diffusion of a product. It also raises a fundamental problem: transparency regarding the structures and purchasing processes within each organisation. This barrier has further impacts that discourage private funding from supporting AR ventures.‘[regarding the purchasing processes] for someone that is new to the market it is exceptionally difficult to get to the right people, to go to the people that make the choices’.The lack of a technological appraisal tool or method for AR that could benefit companies as a route to widespread use was also mentioned. There is currently a market search friction problem; purchasing and supply agencies do not know what technologies are currently available. There is no standard platform for AR to prove their benefits to healthcare stakeholders and look for potential buyers. Moreover, there are no distribution channels for AR technologies.‘it is quite hard to reach the client, and distributors ask for a lot of money, raising prices’.Currently, initiatives such as Innovation, Health and Wealth, introducing a legal obligation on all UK’s CCGs to offer National Institute for Health and Care Excellence (NICE) approved technologies to patients, have a positive impact on new medicine use, but it does not cover non-medicines . TherefoThis category describes those barriers which had an impact on the access to finance and the conditions attached to obtaining financial backing. From the interviews, it was clear that the main barriers focused on the lack of seed funding with implications for scalability of the market. These issues have a significant impact during the first stages of the development process .According to interviewees, on average, three to four prototypes are required before obtaining a minimum viable product. This could translate into four years of work and at least three rounds of funding. This demonstrates that it is not only the technical challenges that discourage AR innovation but also economic viability considerations. The fear of financial failure appears to be thwarting most projects before they properly have the chance to flourish.As in other markets, robotics companies frequently start with entrepreneurs working part-time on their projects. However, while app developers can find solutions to avoid incurring initial costs, for robotics start-ups, the scope here is more limited. A high initial investment is needed for buying off-the-shelf devices, as well as fast prototyping tools.‘Finance, is so difficult, is not cheap, is an expensive journey, and this stops people from doing it’.Several participants explained that there is an underdeveloped capital market, scarcity of capital, restricted entry, unavowed regulations and lack of access to affordable capital for AR ventures. Others mentioned that there were few investors and venture capital providers who understood hardware development and therefore, the seed capital needed. Those who are willing to invest perceive this as a high-risk involvement, demanding high-interest rates to offset the risks they take.There is poor creditworthiness for AR and inadequate recovery regulations. Without a repayment history, credit score or available assets, financial institutes perceive early-stage AR companies as a high-risk investment. This translates into high interest rates and low credit limits. This has a direct impact in particular on indebted businesses. AR companies are often forced to recover through the sale of the collaterals, which in this market is the manufacturing equipment or the technology inside the robot, not allowing a real recovery for start-ups.The lack of openness from the healthcare sector makes it difficult for start-ups to prove cost-savings of their products, how they affect the right treatment and the downstream healthcare system. Besides, assistive robots have an impact on the quality of life of patients , making it difficult to define the real value of innovation without standard technological appraisal tools. Additionally, participants’ views were that health commissioning bodies take their investment decisions motivated more on cost and risk concerns than the healthcare outcomes for patients.However, it was also noticed that companies interviewed were not aware of funding opportunities provided by the European Union through their various initiatives . FurtheThe cost of acquisition and use of assistive robots was reported as a significant barrier. Business to consumer schemes are not viable for most companies since final AR products are commonly too expensive for their end-users due to the costs associated with manufacturing and the technology behind the product. Even if consumers want to purchase AR products, because of under-developed credit facilities, this could present a barrier. Thus, currently, there are no government policies, strategies and incentives for encouraging the adoption of AR technologies that extend to offering either credit facilities or other elements of financial support.On the other hand, due to the complex structure of healthcare systems, the end-user generally has no input on pricing considerations, only purchasing and supply agencies. Therefore, if the product has not been prescribed, the final user will have to pay the full price for a product, irrespective of affordability. All of this has an impact on the current market size for AR companies.Healthcare systems around the world not only vary in their structures and entry channels but also in terms of their regulations and economic context, different leadership styles and environments. Product or service development strategies may need to be tailored to each unique system. This makes access to new markets, i.e., ones with which the eHealth entrepreneur is not familiar, difficult. Interview responses indicated that companies from France and the UK did not have any knowledge about the other’s healthcare market, and both mentioned that they could not enter a new market without support. Some companies mentioned difficulties expanding to other regions within the same country due to the fragmented structure of the healthcare sector.Since AR in the healthcare sector represents an emerging market, policies and governments are still playing a catch-up game concerning regulation and support. We refer here to the notion of institutional burdens, that is underdeveloped or absent institutional structures which can hinder aspiring entrepreneurs from exploiting opportunities fully .The AR market lacks specialised agencies at a planning level that can develop and ensure a safety adoption framework for AR innovations. As mentioned in In the same way, there is no education for health stakeholders of the technology available and opportunities this new market presents. Besides, liability concerns interrupt the development process and AR adoption. All of this is compounded by a missing regulatory body that supports early-stage companies, regulates the development process, and promotes the adoption of assistive robots.A lack of planning and the absence of policies to foster the development of innovations in the eHealth sector was also mentioned. Barriers such as access to health experts and lead users could be overcome with the right regulatory body. It could also address the absence of legislation surrounding liability while testing or adopting new AR products at the organisational and health professional levels. This includes critical appraisal tools for testing AR, approved by the relevant bodies.These barriers are the result of the reduced involvement that the supply side has in decision making. Different from traditional markets, the healthcare stakeholders’ counterparts do not involve engineers, developers, and entrepreneurs in the development of policies for innovations. There is currently a consultation culture missing, driven by a tension surrounding change alongside social, cultural and behavioural barriers, described in Another essential subject mentioned was the lack of support for entrepreneurs to protect the intellectual property (IP) of their products. The current process takes around five years to complete and requires a substantial investment in applications for patents and design rights. In the UK, only one in twenty applications get a patent without professional support. Subsequently, this might need to be renewed on an annual basis. This problem prevents companies from accessing quality manufacturers recognised by the healthcare industry, which often requires IP before getting involved in the process.Most companies that took part in this study mentioned the vital role that universities played in this market, from access to technical and medical knowledge to the recruitment of new talent. Nevertheless, it was acknowledged that doors are not open to entrepreneurs and businesses.‘You can’t put a price [university support] but, unfortunately, they are not interested in product development’.Moreover, current policies from some of the universities mentioned during the interviews highlighted the regulations against university spinout. Companies believe that some universities currently retain intellectual property over any technological development. Therefore, if an entrepreneur wants to apply the research carried out, they will have to address the corresponding payback to the university.Universities’ investment and support for AR companies is visible on launchpads and incubators. Besides, previous studies have evidenced that IP-based spin-offs are an ideal mechanism for technology transfer . HoweverNavigating the tricky channels to obtain medical certification was regarded by the businesses interviewed as burdensome and costly.A device must receive a Conformitè Europëenne (CE) mark as a medical device before being used in the healthcare sector of the European Economic Area . In the For all classes of device, the manufacturer is required to provide a technical file . TherefoNone of the companies younger than five years considered it worth the effort. Most companies mentioned that understanding, meeting and measuring the regulation requirements translates into a cost AR companies cannot afford. For the companies, there is no transparent process to get their products certificated.Therefore, instead of looking for medical certification, most companies only seek and achieve conformity with the CE marking standards that rule the use of products sold within the European Economic Area. To put this in perspective, this means most of these AR companies follow the same procedure as toy manufacturers. The practical implication of this choice is that doctors cannot prescribe toys and medical insurance do not recognise toys either. Therefore, the patient should have to cover the full cost of the product.We mentioned in previous sections the lack of an appraisal tool. Existing legal and regulatory systems, initially designed for medicine, have not been adapted to the characteristics of AR. However, most importantly, they do not allow entrepreneurs and start-ups to enter and meet requirements without creating significant expenses.This category discusses technological viability for AR in the healthcare market, not state of the art challenges. It includes barriers for the adoption due to the nature of these technologies.There appears to be a gap in the provision of education and training of healthcare professionals regarding AR, as all the companies interviewed currently trading said they had to spend time and money training healthcare professionals and carers on how to use their products. It seemed that this could be addressed by improving the curriculum of healthcare staff.This acts as one constraint for the adoption of new technologies. Integration problems, such as obsolete data records, or other current software being used as per regulatory requirements in health settings also slows down the development process of RAS. The lack of infrastructure was also a concern; the availability of resources such as connectivity to the internet in remote healthcare organisations or directly with the final user, limit the market further.This category describes the opposition from the healthcare professionals to AR, the willingness of the healthcare stakeholders to incorporate AR into their work environment and care of patients.Attitudes and beliefs were seen as a crucial barrier for the market, slowing the introduction of assistive robots in healthy environments. There is currently a distrust in AR companies, enhanced by a lack of understanding of how the final product works. This impacts on stakeholders’ understanding of the benefits and opportunities from AR.The current distrust is also driven by concerns over patient safety, but also strong resistance from staff to change their current practices, considering AR will disorder the delivery of care. Some concerns were also raised about the belief among healthcare professionals that robots will take their jobs.Furthermore, ethical concerns were mentioned concerning AR and its underpinning technology, to include robots’ levels of autonomy, and more general concerns around automation. This fear of automation and AR is leading to an industry that is technology lagging and consequently, the sector and crucially patients are missing out on these advances. The consequence for eHealth start-ups is that they have to commit their resources, to face the market misconceptions of AR.Education is a critical aspect. Like many other implementation evaluation studies , better Given the potential of assistive robotics (AR) to improve lives, and upon a backdrop of concerns around growing costs in health services, research on the adoption of AR in a health care setting is still minimal. There is a clear need for research into improving the efficiency of the AR marketplace . The neeThe presented work presented new insight into and addressed the source and nature of the barriers that are inhibiting a more rapid and widespread adoption of advances in AR in healthcare.Identifying market barriers has clear policy relevance. Addressing these barriers, policymakers could increase the entrepreneurship and boost the performance of the sector. Our goal is to raise awareness of these barriers, that further research may be conducted and that policymakers may develop a sustainable framework that ensures AR and associated technologies are realised in the healthcare sector.Some of the main findings are in agreement with previous research that identifies barriers in the adoption of digital technologies in eHealth and the co-evolutionary and path-dependent nature of the innovation process more generally . HoweverThere is a need to strengthen bonds to sources of specific and codified knowledge in more traditional industries . FosteriAll of these show that to address the significant barrier of access to the healthcare sector, governments have to take a more proactive role. To overcome market failure or inefficiencies that result from these barriers government intervention is vital . From thHowever, this governmental effort should be a joint effort among different countries. Supportive legislation to ease the adoption of innovation among healthcare systems from different countries should be encouraged. Only then can the real potential of AR be realised, providing the companies with new opportunities, expanding the size of their market. As a result, venture capital firms, angel investors, crowdfunding providers and other financial backers will start reducing their risk perception.Although we presented a comprehensive evaluation of market barriers, further research is needed, in particular in other types of regions, including less developed economies.