This justified MFS repurposing as a potential antischistosomal drug. However, five consecutive daily 20 mg/kg doses were needed for the treatment of schistosomiasis mansoni in mice. The present study aims at enhancing MFS efficacy to allow for a single 20mg/kg oral dose therapy using a nanotechnological approach based on lipid nanocapsules (LNCs) as oral nanovectors. MFS was incorporated in LNCs both as membrane-active structural alkylphospholipid component and active antischistosomal agent. MFS-LNC formulations showed high entrapment efficiency (EE%), good colloidal properties, sustained release pattern and physical stability. Further, LNCs generally decreased MFS-induced erythrocyte hemolytic activity used as surrogate indicator of membrane activity. While MFS-free LNCs exerted no antischistosomal effect, statistically significant enhancement was observed with all MFS-LNC formulations. A maximum effect was achieved with MFS-LNCs incorporating CTAB as positive charge imparting agent or oleic acid as membrane permeabilizer. Reduction of worm load, ameliorated liver pathology and extensive damage of the worm tegument provided evidence for formulation-related efficacy enhancement. Non-compartmental analysis of pharmacokinetic data obtained in rats indicated independence of antischistosomal activity on systemic drug exposure, suggesting possible gut uptake of the stable LNCs and targeting of the fluke tegument which was verified by SEM. The study findings put forward MFS-LNCs as unique oral nanovectors combining the bioactivity of MFS and biopharmaceutical advantages of LNCs, allowing targeting via the oral route. From a clinical point of view, data suggest MFS-LNCs as a potential single dose oral nanomedicine for enhanced therapy of schistosomiasis mansoni and possibly other diseases.Miltefosine (MFS) is an alkylphosphocholine used for the local treatment of cutaneous metastases of breast cancer and oral therapy of visceral leishmaniasis. Recently, the drug was reported in  Neglected tropical diseases (NTDs) afflict more than one billion of the lowest income people in tropical and subtropical regions presenting an enormous global health and economic burden. To overSchistosoma spp., the causative agent of schistosomiasis, one of the most prevalent chronic NTDs [A drug of particular interest as repurposing candidate is miltefosine (MFS), a membrane active alkylphosphocholine with a broad pharmacological spectrum . MFS wasnic NTDs . Unlike nic NTDs , 10justiNanotechnology-based strategies offer great promise in this respect . The schMFS is a drug with relatively high bioavailability and long elimination half-life in humans and mice, 15. AltSchistosoma mansoni-infected mice by the administration of a single 20 mg/kg oral dose of MFS lipid nanocapsules using a formulation-based approach. MFS was incorporated both as a membrane active phospholipid component of the LNCs shell structure and as an active antischistosomal agent.The objective of the present study was to achieve effective treatment of in vitro hemolysis study, a 2 ml fresh blood sample was donated by one of the authors under medical supervision.All animal studies were conducted at the Animal Experimentation Facility of the Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt, in strict accordance with the Faculty of Medicine Guidelines for ethical conduct in use of animals in research. Efforts were made to minimize animal suffering. The study protocols were approved by the Ethics Committee of the Faculty of Medicine, Alexandria University . In the ® HS 15  was provided by BASF . Lipoid S100  was donated by Lipoïd GMBH. Hexadecyltrimethyl ammonium bromide (CTAB), dihexadecyl phosphate (DCP) and oleic acid (OA) were purchased from Sigma Aldrich (USA). Chitosan  was purchased from Novamatrix, Sandvika, Norway. All other chemicals were of analytical grade.Miltefosine  was obtained from Chem-Impex International Inc.. Labrafac lipophile WL 1349  was a kind gift from Gattefossé S.A. . SolutolLipid nanocapsules (LNCs) were prepared by the phase inversion method . In brie®Nano ZS Series DTS 1060, Malvern Instruments S.A., Worcestershire, UK) at a fixed angle at 25°C using a 4 mW He–Ne laser at 633 nm. LNCs dispersion was diluted 1:80 in deionized water and measurements were performed in triplicate. The stability of MFS-loaded LNCs regarding particle size, polydispersity index (PdI) and zeta potential was assessed after a 4 month-storage period at 4°C.The mean particle size and zeta potential of LNC formulations were measured by photon correlation spectroscopy (PCS ZetasizerThe morphology of LNCs was examined by transmission electron microscopy (TEM) using JEOL, JEM-100 CX Electron Microscope, Tokyo, Japan. Before analysis, the selected LNC dispersions were treated with a negative stain (2% w/v uranyl acetate solution) and sprayed onto copper grids. Shots were taken at X 7500 at 80 kV.™ Vivaspin6™, MWCO 100,000) and centrifuged for 30 min at 3663 x g. A modified spectrophotometric assay reported for quaternary ammonium compounds using bromothymol blue (BTB) [Entrapment efficiency (EE%) was calculated by determining the concentration of free MFS (un-entrapped) in the ultrafiltrate after separation of LNCs using an ultrafiltration / centrifugation technique . A 5 ml-sample of LNC dispersion was added to the ultracentrifugal concentrator (Sartoriusue (BTB)  was usedThe release of MFS was determined at 37°C at 100 rpm in phosphate buffer saline . A known volume of selected MFS-LNC dispersions was diluted to 5 ml with PBS to achieve sink conditions. At different time intervals, LNCs were separated and the whole filtrate was used to determine MFS concentration spectrophotometrically as described above. The % MFS released was calculated in triplicate relative to the theoretical initial drug content.in vitro using a reported method [The erythrocyte hemolytic activity of MFS-LNCs as a surrogate indicator of membrane activity was assessed d method . BrieflyS. mansoni, kept at the Department of Medical Parasitology, Faculty of Medicine, Alexandria University, Egypt was used. It was propagated in Biomphalaria alexandrina snails as the intermediate host. Cercariae of S. mansoni were obtained from the snails infected with miracidia from the feces of mice infected 45 days prior to the study. Forty days later, the snails were subjected to illumination at 28°C to induce cercarial shedding. The numbers and viability of cercariae were determined by light microscopy.Mice were housed under specific pathogen-free barrier conditions. The Egyptian strain of Male Swiss albino mice 4–5 weeks old and weighing 20–25 g, obtained from the animal house, Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt, were used as the definitive host. Mice were infected by the tail immersion method using a Mice of all groups were sacrificed by cervical dislocation after a heparin injection seven days post-treatment (on 49 day p.i.). The antischistosomal activity of formulations of the treatment groups compared to the appropriate control groups was assessed by determination of the % reduction in total worm burden and size of liver granulomas, histopathological changes in liver parenchyma and examination of the morphology of recovered worms by TEM and SEM.The adult worm burden was estimated by recovering schistosomes from the mice hepatic portal system by perfusion with citrated saline. The totFor histolopathological examination, specimens of the liver of mice of all study groups were fixed in 10% neutral buffered formalin. Then, 5 μm-thick histological sections were stained with haematoxylin and eosin. Pathological changes in the hepatic parenchyma were noted and the mean diameters of granulomas (μm)determined. Only granulomas containing one clearly identifiable, central-only egg were selected .Morphological examination of adult schistosomes recovered from mice of the infected untreated control group and the treatment group showing the highest % reduction in worm burden was performed by SEM and TEM after fixation in cold 1.5% glutaraldehyde.+ and MFS-LNCs-OA-CTAB+  was set at 20 psi and the collision gas (nitrogen grade 5.0) at 8 psi. The ion spray voltage was set at 5500V, while the source temperature was 600°C. Quantification was achieved using a calibration curve for peak area ratios of MFS in spiked plasma obtained under the same conditions.Mass spectrometric detection was performed on SCIEX Triple QuadThe plasma concentrations versus time data were analyzed based on a non-compartmental pharmacokinetic model using Excel pharmacokinetic solver add-in . The maiSPSS software package version 20.0. Quantitative data were described using the parameters: range (minimum and maximum), mean, standard deviation and median. Comparison between different groups was analyzed using F-test (ANOVA) and Post Hoc test (LSD) for pair-wise comparison. Significance test results were quoted as two-tailed probabilities. In the pharmacokinetic study, data were analyzed using a paired t-test. In both studies, p values ≤0.05indicated significance.Data of the preclinical study in mice were analyzed using IBM + which showed a slightly but significantly (p<0.05) larger mean diameter , LNCs remained monodisperse with PdI< 0.3. Formulations incorporating oleic acid (MFS-LNC-OA and MFS-LNC-OA-CTAB+) showed no significant increase in particle size (56.21±0.3 and 39.64±0.19 nm respectively). Zeta potential did not significantly change upon storage.Storage of LNCs dispersions for 4 months at 4°C did not significantly change colloidal properties (results not shown). With the exception of MFS-LNC-DCP¯ and MFS-LNC-CTAB+, MFS-LNC-OA and MFS-LNC-OA-CTAB+, respectively. With the exception of MFS-LNC-OA, LNCs were almost spherical, homogenously distributed and not aggregated. MFS-LNC-OA (S-LNC-OA  showed v+ (EE % was 96.5±1.31and 95.6±0.95 respectively). However, a limited but significant (p<0.05) reduction in EE% was observed for standard MFS-LNC, MFS-LNC-CTAB+ and MFS-LNC-DCP- to 93.4±2.05, 94.1±0.98 and 93.8±1.25%, respectively.Entrapment efficiency values (EE%) exceeded 97.5% in all LNC formulations with an average drug payload of ~25.6 mg/g. Reassessment of MFS EE% following 4 month-storage at 4°C indicated no significant (p>0.05) leakage from MFS-LNC-OA and MFS-LNC-OA-CTABIn vitro MFS release in PBS pH 7.4 at 37°C under sink conditions (+) and increased by oleic acid (MFS-LNC-OA) to a limited extent, nonetheless, differences at 2, 4, 8 and 24 h were statistically significant (p<0.05).nditions  for 24h +, MFS-LNC-OA, and MFS-LNC-OA-CTAB+) reduced MFS hemolytic activity, the effect being dependent on both MFS concentration and LNCs composition . LNCs  or MFS solution (MFS-sol) did not significantly change the mean worm burden compared to un-treated mice.However, administration of the six MFS-LNC formulations reduced the mean worm burden to different extents (42.31 to 88.46%). While cationic MFS-LNCs (MFS-LNC-CTAB+) and those containing oleic acid (MFS-LNC-OA) induced the greatest reduction, the least reduction was induced by the negatively charged MFS-LNC-DCP-. In all cases, the mean worm burden for MFS-LNC formulations was significantly lower compared to that for MFS-sol or the untreated control groups. The difference between% reduction among MFS-LNCs treatment groups was significant while the difference between MFS-LNC-CTAB+ and MFS-LNC-OA did not reach significance.Data for worm burden and their statistical analysis are shown in +(31.36%) and MFS-LNC-OA (32.99%) although the difference between both formulations was not statistically significant. In all cases, the difference in granuloma diameters was statistically significant when MFS-sol or the infected untreated group was used for comparison. None of the corresponding blank formulations had an effect on granuloma size (data not shown).Regarding granuloma size, MFS-LNC formulations brought about a reduction in granulomas diameter (μm) ranging from 17.1 to 31.4% . The larHistopathological changes in liver sections of infected untreated mice are shown in S. mansoni male worm  showed erosion of the tegument in some areas  whereas,ale worm  are show2 ≥0.998). The limits for detection and quantification for MFS in plasma were less than 1 ng/ml. The coefficient of variation for intra-day and inter-day precision for three quality control MFS solutions  was 14 and 12% respectively. The MFS retention time was 3.4 min.The LC-MS/MS method used for the quantification of MFS in plasma was linear within the concentration range 1–100 ng/mL(rmax), time to reach Cmax (tmax), half-life (t½) and mean residence time (MRT) are shown in max values for all MFS-LNCs compared to MFS solution was observed (p <0.05). However, the differences between Cmax, t½, AUC and MRT for MFS solution and each of the test MFS-LNC formulations did not reach statistical significance.In the treatment of schistosomiasis, oral administration provides for mass chemotherapy and patient compliance. Efficacy of oral treatment depends on drug bioavailability and interaction with therapeutic targets including mainly the fluke tegument, a prime site of vital and immunological functions , 12, 13.Incorporation of MFS, a membrane active phospholipid with intestinal barrier modulating effect , 30, repLNCs could be prepared using MFS as the only phospholipid component. Blank and MFS-LNCs with good colloidal properties  were mod+ did not show the oleic acid-induced vesicular structures, formation of more stable CTAB/oleic vesicles of the LNCs size range cannot be ruled out [TEM images revealed generally spherical and uniform LNCs except for MFS-LNC-OA which showed apparently composite vesicular structures . Unsaturuled out .High EE% values  despite water solubility and relatively small molecular size of MFS, implied integration of MFS within the stable LNC structure, most probably at the core/pegylated surfactant shell interface with the hydrophobic alkyl chain extending into the lipid core, a property of hexadecylphosphocholines. This wa+ compared to MFS-LNCs(p<0.05) can be explained by the dense MFS/CTAB interfacial packing [in vitro release data presumably indicated integrity of the MFS-LNCs structure during the 6 h gastrointestinal transit time in mice [Sustained release of MFS-LNCs at physiological pH for a 24 h study period with a limited burst effect  can be a packing . Convers packing –36. More in mice  with limin vitro hemolytic activity study was conducted to provide information on the effect of LNCs on MFS membrane activity and cytotoxicity.MFS is known to exert extensive hemolysis of erythrocytes, precluding intravenous administration [+ exerting 100% inhibition. Hemolysis data combined with high MFS entrapment efficiency (EE %) and slow release data , administered in a single 20mg/kg, significantly enhanced antischistosomal activity of MFS in s in mice. Enhancein vitro ,crosses in vitro , 30. Furcted mice, an effected mice, 48 posscted mice. On the cted mice, vesiclected mice, 50 and/cted mice might ex+was in agreement with their hemolytic activity and pointed to preservation of structural integrity of MFS-LNC-CTAB+ post absorption. The role played by cationic properties was verified by the enhanced activity of another cationic formulation with post-inserted chitosan (MFS-LNC-CS+) and conversely, the reduced activity of anionic MFS-LNC-DCP¯. Chitosan nanoparticles were shown recently to cross the intestinal mucosa [Inclusion of CTAB and chitosan (CS) as positive charge imparting agents or oleic acid (OA) as membrane permeabilizer significantly enhanced the effect of MFS-LNCs on adult worm load  and granl mucosa . However+. It is worth noting that the MFS-LNC-OA formulation was less hemolytic than MFS-LNC-CTAB+. Thus, this formulation was used for histopathological examination , exerted the greatest antischistosomal effect expressed as reduction in worm load  and granmination . Mild hiy Tables  and 4 vey Tables . Combinemax) for MFS solution  was adjusted to account for species differences . The timon 10 h,  and the  1/262h,  were con for t1/2. Lack ofsolution  indicatesolution  and 4. Trom LNCs  and possLNCs were demonstrated to enhance the pharmacological activity of orally administered drugs mainly by modifying pharmacokinetics. Enhanced bioavailability accounted for increased activity of various poorly absorbed drugs such as paclitaxel and fondCombined data of the preclinical and the pharmacokinetic studies tend to indicate that the antischistosomal activity enhancement of MFS by LNCs most probably involved the relatively stable MFS-LNCs and accordingly was not solely dependent on systemic exposure to molecular MFS. Such findings corroborated those of previous studies in pointing out that classical drug PK/PD relationships should be considered prudently for oral nanomedicines , 63. In in vitro structural stability of MFS-LNCs, poor release characteristics and formulation-dependent antischistosomal activity suggested dependence of efficacy enhancement on the structural integrity of MFS-LNCs and their potential gut uptake and translocation to the target fluke tegument. Remarkably, this was verified by scanning electron microscopic visualization of nano-objects on the surface of the tegument of blood dwelling flukes in Schistosoma mansoni-infected mice . MFS-LNCs integrating the membrane activity of MFS, the biopharmaceutical advantages of LNCs and the biological effects of CTAB and oleic acid offered unique attributes that significantly enhanced antischistosomal activity and liver protection in mice. Poor pharmacokinetic / pharmacodynamic relationship indicated that the efficacy of orally administered nanomedicines may not necessarily relate to systemic drug exposure, particularly for stable nanostructures incorporating drugs with good bioavailability that are taken up by the gut and translocated to a therapeutic target(s). Data obtained implied possible targeting via the oral route by selected nanocarriers. From a clinical standpoint, effective single oral dose therapy of schistosomiasis mansoni would promote repurposing of MFS as an antischistosomal drug for mass chemotherapy with lower gastrointestinal toxicity and put forward single dose oral MFS-LNCs as a potential alternative to praziquantel chemotherapy.


L0 penalties. In this paper we introduce an adaptive ridge procedure (AR), where iteratively weighted ridge problems are solved whose weights are updated in such a way that the procedure converges towards selection with L0 penalties. After introducing AR its specific shrinkage properties are studied in the particular case of orthogonal linear regression. Based on extensive simulations for the non-orthogonal case as well as for Poisson regression the performance of AR is studied and compared with SCAD and adaptive LASSO. Furthermore an efficient implementation of AR in the context of least-squares segmentation is presented. The paper ends with an illustrative example of applying AR to analyze GWAS data.Penalized selection criteria like AIC or BIC are among the most popular methods for variable selection. Their theoretical properties have been studied intensively and are well understood, but making use of them in case of high-dimensional data is difficult due to the non-convex optimization problem induced by  L0 penalty, and include the classical information criteria AIC         grad = crossprod-pen*w*beta        hess=-crossprod-pen*diag(w)        beta = beta-solve        }      We want to point out that the resulting code for adaptive ridge is compact and extremely easy to understand and to implement. This is in stark contrast to the available LASSO implementation of the same problem  which usp = 50 and p = 500, where now count data are simulated from Poisson regression models of size k = 10 and k = 25, respectively. All other aspects of this simulation study are identical with the last simulation study on linear regression, where we compare again AR with stepwise regression, SCAD and ALASSO. The results are summarized in To evaluate the performance of the AR procedure for Poisson regression we consider again two simulation scenarios with The biggest difference to the results from linear regression is concerned with runtime, where AR has now lost its big advantage compared with stepwise regression and ALASSO. On the contrary, AR now actually tends to be slower than ALASSO. However, this comparison is not entirely fair because ALASSO uses the lqa R-package which is based on a highly efficient implementation in C, whereas AR uses only the extremely simple R code sketched above to solve the weighted Poisson regression problem. It is evident that an efficient C implementation of this solver could speed up the procedure substantially, but this is not the main focus of this article. On the contrary, our emphasis is that even such a simple implementation using only a few lines of R-code remains competitive and provides very good results in terms of classification. The biggest advantage is that AR can be easily adapted to more complicated models as we will see in the next section.The AR procedure can be applied in contexts which go beyond regression models, like for example for least squares segmentation of a one-dimensional signal which was recently applied in the context of analyzing pathological patterns of DNA in tumor tissues . We wantn measurements which are spatially  ordered. Then the problem of segmentation can be formalized by introducing L0 penalties for changing the estimated mean between neighboring measurements,w(0) ≃ 1 and for k ≥ 1 perform the iterationsSL. Minimization of the loss function then corresponds to solving the following set of linear equations:i = 1, …, n − 1, let us write μi = ai + biμi+1 where Di = 1 + λwi + λwi−1(1 − bi−1), and finallykmax ≥ 1 segments in Segmentor3IsBack R package [Let 0(20)In  it was sained in  and impl package .n = 500 consecutive measurements and three breakpoints at positions 100, 250 and 375. Based on a Gaussian model 200 data sets were generated with mean values −0.3, 0.7, 1.5, 0.5 in the four different segments, and a common standard deviation of σ2 = 1.0. After performing some calibration of the parameter λ = 2log(n) is the penalty of the original criterion. This rescaling factor appeared to be quite stable for various scenarios, though perhaps increasing slightly with growing n (data not shown).In order to validate the adaptive ridge approach in the context of least squares segmentation we will compare its performance with the exact approach in a small simulation study. We consider a simple Gaussian design with od of AR  we decidWe can see in n = 4843 for LDL) compared with the data set used in [n ≤ 4518 for LDL). On the other hand the covariates of smoking status and alcohol intake, which play a crucial role for some of the metabolic traits, are not provided by dbGaP. One thus has to be a little bit careful when comparing our results with those of [To illustrate the applicability of AR in practice we want to reanalyze a GWAS on metabolic traits from a Finnish cohort . Our ana used in  . For the AR approach one needs complete genotype data for all SNPs. To achieve this we performed imputation of missing values using Beagle 4.1 [Sabatti et al.  performeagle 4.1 .−7 which corresponds approximately to a Bonferroni correction at nominal level 0.16. The mBIC criterion we are going to adopt is of the formc can be interpreted from a Bayesian point of view as the a priori expected number of causal SNPs. From a frequentist perspective the mBIC criterion is closely connected with the Bonferroni multiple testing rule, where in [c) for m independent regressors and n individuals. In our case the typically recommended choice of c = 4 yields FWER = 0.008, which appears to be extremely conservative compared with the multiple testing correction used by Sabatti et al. Using mBIC with c = 10 gives a FWER of 0.02 which is still rather conservative, whereas c = 23 yields FWER = 0.05.Concerning multiple testing correction Sabatti et al. applied the Benjamini Hochberg procedure at a nominal level of 0.05 and then argued that equivalently they might use a significance threshold of 5 × 10rs693 also reported in [rs207150 lying on chromosome 1. Note that this SNP has a marginal p-value of 5.6E-07 and would therefore not have been selected applying the threshold 5 × 10−7 suggested by Sabatti et al. This goes along with the fact that model based analysis will typically have larger power to detect SNPs than single marker tests, even when the selection criterion guarantees that the type I error rate is controlled at a stricter level Click here for additional data file.


AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of  Autophagy is a conserved process responsible for the turnover of long-lived proteins or removal of damaged organelles in eukaryotic cells. It is regulated by series of autophagy related genes (ATGs) that are involved in the initiation, autophagosome formation and maturation . Autophain vitro and in vivo [ATG4D, RAB5A and STMN1 [miR-101 gene [miR-101 gene by luciferase reporter and ChIP assays. The expression of miR-101 was found to correlate with AR status in prostate cancer cell lines. Despite AR depletion by celastrol, transfection of miR-101 mimic in LNCaP cells led to inhibition of autophagy. When miR-101 was blocked, AR inhibition on autophagy was relieved. Furthermore, mutagenesis of the AR binding site in the upstream region of the miR-101 gene led to decreased inhibition of AR-mediated autophagy.Celastrol, a triterpenoid isolated from the traditional Chinese medicine ‘Thunder of God Vine’ has shown effectiveness against human prostate cancer  in vivo , 4. It p in vivo , 6. AR i in vivo . Blockin in vivo –11. Indu in vivo , 9, 11.  in vivo . Several in vivo , 9. Howend STMN1 . In addi101 gene . These fCelastrol was purchased from Cayman chemical company . All the oligonucleotides were synthesized by Ribio  with the following sequences: miR-101 mimic, 5'-UACAGUACUGUGAUAACUGAA-3'; miRNA mimic Negative control (Ncontrol) #22: 5'-UUUGUACUACACAAAAGU ACUG-3', miR-101 inhibitor: 5'-UUCAGUUAUCACAGUACUGUA-3'; miRNA inhibitor Ncontrol #22: 5'-UCACAACCUCCUAGAAAGAGUAGA-3'. siRNA for AR: 5'-GGTGATCACAGGATAGGTATT-3', siRNA for control: 5'-GGGCCATGGCA CGTACGGCAAG-3'.2/95% air.Human prostate cancer cell lines LNCaP, 22Rv1, DU145 and PC-3 were obtained from Shanghai Institute of Biochemistry and Cell Biology , and cultured in RPMI 1640  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin in a 37°C humidified atmosphere containing 5% COFor androgen starvation, LNCaP cells were cultured in phenol red-free RPMI 1640 media  containing 1% charcoal-stripped FBS  for 24 h before experiments.Transfection was performed by using Lipofectamine 2000 . Transient transfection of pEGFP-C1-AR or empty vector (Addgene) was performed in LNCaP or PC-3 cells. After transfection, medium were replaced with fresh medium containing 1 nM of R1881. Stable transfection of pEGFP-C1-AR or empty vector was performed in DU145 cells. Cells were pretreated with R1881 (1 nM) for 24 h before experiment. LNCaP cells were transfected with pEGFP-LC3 (Addgene) and selected with 0.8 mg/ml G418  to obtain stable transfectants.miR-101 gene that contains the predicted AR binding site and its shorter fragment with deletion of the predicted AR binding site were amplified using primers 5'-CGCACGCGTAATGGATTTATTTCCTACCCT ACAT-3'; 5'-CCGCTCGAGTATTCCCTGCCACCCAGCTCACC-3', and 5'-CGCAC GCGTAATGGATTTATTTCCTACCCTACAT-3'; 5'-CCGCTCGAGTATTCCCTGCC ACCCAGCTCACC-3', respectively, and cloned into pGL3-Basic vector. The reporter construct that contains a 300 bp fragment with the predicted wild-type AR binding site, pGL3-B-miR-101-WBS, was amplified with PCR using primers 5'-CGCACGCGTAATGGATTTATTTCCTACCCTACAT-3', and 5'-CCGCTCGAG CTTTCTTCTTTTGTTTTTCATTTTC-3'. To mutagenize the predicted AR binding site in pGL3-B-miR-101-WBS (designated pGL3-B-miR-101-MBS), two-step PCR was performed. For the first step PCR, the following primers (mutated nucleotides were indicated by lower cases): 5'-CGCACGCGTAATGGATTTATTTCCTACCC TACAT-3'; 5'-TAACTTTAtAgaATATTtaAgATAG-3', 5'-CTATcTtaAATATtcTaTAA AGTTA-3'; and 5'-CCGCTCGAGCTTTCTTCTTTTGTTTTTCATTTTC-3' were used. The PCR products were used as the template for the second step PCR using the outer pair of primers.pGL3-Basic was a generous gift from Dr. Li Yu . To generate reporter constructs, pGL3-B-miR-101-L and pGL3-B-miR-101-S, a 1793 bp DNA fragment in the upstream of PGL3-B-miR-101W construct was generated by PCR amplification of a 2166 bp DNA fragment containing both AR binding site and miR-101 gene sequence using primers 5'-CGCACGCGTGCCTTGGTCAGACTGGAT-3', 5'-CCGCTCGAGAT GTTACCACGCCATTTA-3', and cloning into the pGL3-Basic vector. Its mutant form of construct, PGL3-miR-101M, was generated with two-step PCR as described above using two pairs of primers containing a mutant AR binding site 5'-CGCACGCGTGCCTTGGTCAGACTGGAT-3'; 5'-TAACTTTAtAgaATATTtaAgATAG-3', and 5'-CTATcTtaAATATtcTaTAAAGTTA-3'; 5'-CCGCTCGAGATGTT ACCACGCCATTTA-3'. Letters in lower case indicate the mutated nucleotides.Total RNA was extracted from cells using TRIzol reagent . A total of 1.0 μg of total RNA was used to synthesize complementary DNA using EasyScript First-Strand cDNA Synthesis SuperMix . qPCR was performed to determine the expression levels of each gene using the following primers: ATG5: 5'-GCAAGCCAGACAGGAAAAAG-3', 5'-GACCTTC AGTTGGTCCGGTAA-3'; ATG7: 5'-CAGGAGATTCAACCAGAGAC-3', 5'-AGAT ACCATCAATTCCACG G-3'; AR: 5'-CAGCAACAGCAGCAGGAAGC-3'; 5'-CTTT TGCATTCGGCCAATGG-3'; GAPDH: 5'-TGCACCACCAACTGCTTAGC-3'; 5'-G GCATGGACTGTGGTCATGAG-3'. Pri-miR-101: 5'-GTATTTCGTAGGACAGG-3'; 5'-TCTACAGGAAGCGAGT-3'. Data was normalized to GAPDH expression levels.et al [MiRNA expression was analyzed as reported by Shi et al . BrieflyWhole cell lysates were prepared using RIPA buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.8% Triton X-100 and protease inhibitor cocktail . Western blotting was performed as described in [5 per well) for 24 h and co-transfected with 300 ng of reporter constructs , 300 ng of pEGFP-C1-AR or pEGFP-C1 plasmids and 10 ng of pRLSV40  using Lipofectamine 2000. After transfection, the medium was replaced with fresh medium containing R1881 (1 nM) and harvested 24 h later. Luciferase activities were assayed using the dual-luciferase reporter assay system  in a Turner TD20/20 luminometer and normalized to Renilla luciferase activity.DU145 prostate cancer cells were plated in 24-well plates (1×102 and 10 mM KCl), followed by homogenization. Collected nuclei were resuspended in hypotonic buffer and sonicated to an average DNA length of 200 to 1000 bp. Aliquots (1%) of sheared DNA were removed as input, and the remainder was used for individual ChIP reaction equally. The chromatin solutions were precleared by addition of protein A-sepharose  for 2 h and incubated with 10 μg of AR antibody or IgG as a negative control overnight at 4°C. The protein/DNA immune complexes were collected by centrifugation at 4°C, washed with dilution buffer , and elution buffer (0.1M NaHCO3 and 1% SDS). Protein/DNA cross-links were reversed by addition of NaCl to a final concentration of 200 mM followed by incubation at 65°C for 4 hr. After DNA purification, PCR was performed to detect miR-101 expression using the following primers: 5'-CGCACGCGTAATGGATTTATTTCCTACCCTA CAT-3', and 5'-CCGCTCGAGCTTTCTTCTTTTGTTTTTCATTTTC-3'. The PCR products were analyzed on agarose gels (2%) with ethidium bromide.ChIP was performed as previously reported . BrieflyLNCaP cells with GFP-LC3 expression were seeded in 6-well plates. After celastrol treatment, the cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS three times. Cells were then permeabilized with 0.05% (v/v) of Triton X-100, stained with DAPI  and analyzed using confocal microscope . The number of GFP-LC3 punctate was quantified. Positive cells which contained five or more puncta were selected. Fifty cells per condition per experiment were analyzed.LNCaP cells were plated in a 96-well plate (5000/well). After treatments, MTT (5 mg/ml) was added and incubated at 37°C for 4 h to allow for complete cleavage of the tetrazolium salt by metabolically active cells. Cell lysis buffer  was added to each well, followed by colorimetric analysis using an iMark Microplate Absorbance Reader  at 595 nm.LNCaP cells were treated with celastrol in the presence or absence of miR-101 mimic or negative control or bafilomycin A1  for 24 h. After treatments, LNCaP cells (500/well) were seeded in six-well plate and cultured for 15 days without disturbance. Cells were then fixed with 4% formaldehyde in PBS and stained with crystal violet. Colonies with over 50 cells were counted.P<0.05; **, P<0.01.Statistical analysis was performed using SPSS statistical software. All values were expressed as the mean ± SD, two group comparisons were carried out using paired Student’s t-test. Data from multiple groups was analyzed by one-way ANOVA, followed by Dunnett's test. For all the tests, the level of significance was defined as * or #, ATG5 and ATG7 was observed at early time points after celastrol treatment in LNCaP prostate cancer cells . Suppression of miR-101 expression by celastrol was confirmed by RT-PCR, which revealed a ~3-fold decrease of pri-miR-101 and mature miR-101 in LNCaP cells post celastrol treatment , right, 101 gene . Thus, isfection  bottom, reatment .miR-101 transcription, but not maturation.AR status could affect miR-101 expression in human prostate cancer cells. Real-Time PCR revealed that the expression levels of pri-miR-101 as well as mature miR-101 were significantly higher in AR-positive LNCaP and 22Rv1 cells than that in AR-negative DU145 and PC3 cells . AR was Next, we determined if AR negatively regulates celastrol-induced autophagy through inhibition of miR-101 expression in prostate cancer cells. MiR-101 mimic was used to increase the miR-101 level in LNCaP cells. With miR-101 upregulation by addition of miR-101 mimic , upper, Since AR reduction also results in reduced cell viability, whether the observed miR-101 suppression and autophagy induction were due to cell death was determined. LNCaP cells were treated with AR antagonist MDV3100  for different times. As expected, AR protein was decreased . Along wAutophagy plays dual roles in cancer cell survival under stresses, either protects cancer cells from apoptosis or itself leads to cell death, termed as type II programmed cell death. To evaluate the effect of autophagy on cell growth upon celastrol treatment, miR-101 mimic was used to inhibit autophagy and cell proliferation was determined post celastrol treatment. As shown in AR plays a critical role in the growth of prostate cancer cells, thus targeting AR signaling pathway is an effective strategy for prostate cancer treatment. Celastrol has been reported targeting AR signaling by promoting AR degradation through inhibition of Hsp90 or activation of calpain , 6. AR rmiR-101gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. Celastrol-induced autophagy was suppressed by AR, but compromised by using miR-101 inhibitor. Transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.The exact mechanism underlying AR inhibition and autophagy induction has not been fully understood. AMPK activation or Grp78 downregulation has been proposed as the underlying mechanisms for autophagy induction upon androgen deprivation . ProlongmiR-101 transactivation depends on other transcriptional factors than AR, as EZH is reported to be one such factor in these cells [Our work demonstrates the negative regulation of AR/miR-101 on celastrol-induced autophagy in AR positive prostate cancer cells. We found that celastrol could also induce autophagy in AR negative cells, which could be suppressed by exogenous AR. These results support the negative regulation of AR on celastrol-induced autophagy that was found in AR positive cells. It has been reported that celastrol induces autophagy in AR negative prostate cancer cell line such as PC-3 as well as other type of cancers such as pancreatic cancer and hepatocarcinoma , 22. Actse cells .Abiraterone and Enzalutamide are representatives of the second generation of androgen deprivation therapy (ADT) that have been moved into clinical trials for the treatment of metastatic prostate cancer –25. AndrmiR-101 in prostate cancer cells. Targeting AR with celastrol in the presence of miR-101 would be in favor of the inhibition on cell proliferation in prostate cancer cells.In summary, our results demonstrate that AR plays a negative role of in the regulation of celastrol-induced autophagy; it suppresses autophagy via transactivation of


Sprouting blood vessels are led by filopodia-studded endothelial tip cells that respond to angiogenic signals. Mosaic lineage tracing previously revealed that NRP1 is essential for tip cell function, although its mechanistic role in tip cells remains poorly defined. Here, we show that NRP1 is dispensable for genetic tip cell identity. Instead, we find that NRP1 is essential to form the filopodial bursts that distinguish tip cells morphologically from neighboring stalk cells, because it enables the extracellular matrix (ECM)-induced activation of CDC42, a key regulator of filopodia formation. Accordingly, NRP1 knockdown and pharmacological CDC42 inhibition similarly impaired filopodia formation in vitro and in developing zebrafish in vivo. During mouse retinal angiogenesis, CDC42 inhibition impaired tip cell and vascular network formation, causing defects that resembled those due to loss of ECM-induced, but not VEGF-induced, NRP1 signaling. We conclude that NRP1 enables ECM-induced filopodia formation for tip cell function during sprouting angiogenesis.  •NRP1 enables morphological, but not genetic, specialization of endothelial tip cells•NRP1 is essential for matrix-induced CDC42 activation in endothelial cells•NRP1 promotes filopodia extension and actin remodeling via CDC42•Endothelial NRP1 loss and CDC42 inhibition similarly impair angiogenesis During angiogenesis, new blood vessel sprouts are led by filopodia-studded tip cells to sense environmental signals and enable directional migration. NRP1 is a tip cell protein that senses angiogenic cues. Fantin et al. now show that NRP1 promotes tip cell function by enabling CDC42 activation for filopodia formation and actin remodeling. Developing organs, ischemic tissues, and growing tumors produce the vascular endothelial growth factor VEGF-A to signal to its receptors on the endothelial cells (ECs) that line all blood vessels, and the resulting angiogenic expansion of local vasculature ensures the delivery of oxygen and nutrients to sustain fundamental metabolic processes . VEGF-A The main regulators of filopodia formation are members of the RHO-GTPase family, in particular CDC42, which is activated by VEGF-A signaling in cultured ECs . AgreeinNeuropilin 1 (NRP1) is a non-tyrosine kinase transmembrane protein that regulates vascular development through dual roles in endothelial VEGF-A and ECM signaling . Using tHere, we have combined the analysis of vascular development in the mouse hindbrain with functional studies in primary human ECs, zebrafish embryos, and mouse retina to demonstrate that NRP1 is dispensable for the genetic specification of tip cells but essential for CDC42 activation. Unexpectedly, NRP1 enabled CDC42-dependent actin remodeling and filopodia formation in endothelial tip cells independently of VEGF-A stimulation, and loss of CDC42 activation did not phenocopy the vascular defects of mice with impaired VEGF-A signaling through NRP1. Instead, NRP1-mediated CDC42 activation was induced by stimulation with ECM, and loss of this pathway caused defective vessel sprouting and branching similar to loss of the ECM-induced, NRP1-dependent activation of ABL kinases. In addition to demonstrating a physiological role for ECM-induced CDC42 activation in sprouting angiogenesis, our work has therefore identified a mechanism that ensures the integration of growth factor signals with ECM cues via NRP1 to enable tip cell function in sprouting angiogenesis.Nrp1-deficient mouse embryo hindbrains in greater detail than previously done. Immunohistochemistry for the vascular endothelial marker platelet endothelial cell adhesion molecule (PECAM) showed that vessel ingression was mildly delayed in hindbrains carrying one, and severely delayed in hindbrains carrying two, Nrp1 null alleles . As expected, Nrp1 expression was not detectable in homozygotes due to the null mutation (Nrp1 levels and the NRP1 dose-dependent delay in brain vascularization (see above), Pecam levels were significantly decreased in both types of mutants, with a milder defect in heterozygotes . Similar results were obtained for another endothelial gene, VE-cadherin .To investigate whether defects in endothelial tip cell and filopodia formation were a consequence of reduced tip cell molecular specification, we performed qPCR of genes known to be upregulated in tip cells in E11.5 hindbrains. For this analysis, we first validated the labeling , Nrp1 mRmutation A. CorrelApln and Ang2 . When normalized to Pecam as a measure of overall vascular volume, the mRNA expression of all tip cell markers examined, including that of the notch ligand DLL4, was significantly increased in homozygous mutants  or NRP1 siRNA (si-NRP1), stimulated them with FN, and compared levels of total and activated CDC42 with this method. While NRP1 knockdown did not affect the overall level of CDC42 B, it effPhalloidin staining for visualization of F-actin showed that many control cells after 2 hr on FN had assumed an elongated appearance with irregular edges and numerous stress fibers, typical of motile cells H. In conAdditional VEGF-A stimulation further increased the number of microspikes in cells plated on FN compared to cells plated on FN without VEGF-A stimulation M. SurpriTaken together, our observations establish that NRP1 enables CDC42 activation and CDC42-dependent actin dynamics and filopodia extension in ECM-stimulated ECs, independently of VEGF-A signaling.The phenotype of HDMECs after CDC42 inhibition or NRP1 knockdown resembled the cellular phenotype caused by ABL1 knockdown, including a rounded cell shape, increased cortical actin, reduced stress fibers, and impaired microspike formation . Moreovenrp1 homologs, nrp1a and nrp1b, and both are expressed in vascular endothelium and reported to regulate vascular development  that had originally been designed to target Nrp1 at a time when Nrp1a, but not Nrp1b, had been identified  sprout from the dorsal aorta and then fuse into the dorsal longitudinal anastomosing vessels (DLAV) . Zebrafielopment . Howeverogenesis . This obentified  and is rd y5 embryos expressing a fluorescent endothelial reporter y5 zebrafish embryos further allowed us to compare the process of ISV sprouting over time y5 embryos by adding ML141 to the aquarium water at late gastrula stage (8 hpf) and imaged the trunk vasculature at 32 hpf y5 zebrafish embryos relative to controls  alleles (Nrp1fl/fl) expressing a tamoxifen-inducible Cre transgene under the control of the endothelial Pdgfb promoter (Pdgfb-iCreER-Egfp) or littermate controls lacking the Cre transgene  because it is accessible to small-molecule inhibitors and suited to the tamoxifen-inducible, endothelial specific deletion of floxed target genes and (2) because this model was recently used to establish distinct roles for ECM-induced and VEGF-A-induced NRP1 pathways in angiogenesis . To obtaransgene . Tamoxifransgene A. As preransgene , NRP1 knSimilar to NRP1 knockdown, CDC42 inhibition with ML141 from P2 to P5 reduced vascular network density at P6, with sprouts at the vascular front appearing longer and larger and fewer lateral connections between neighboring vessels C. The nuNrp1Y297A/Y297A mice  and cDNA prepared using Superscript III reverse transcriptase (Invitrogen) for qRT-PCR using SYBR Green (Applied Biosystems). For more information, see the Tg(fli1a:EGFP)y5 (Tg(kdrl:HsHRAS-mCherry)s896  was used to align nucleotide and amino acid sequences. For more information, see the MOs were injected into :EGFP)y5  or Tg(kdrry)s896  zebrafisSilencer negative control siRNA (Applied Biosystems) using Lipofectamine RNAIMAX (Life Technologies). For more information, see the HDMECs were cultured in MV2 media with supplements (Promocell) and transfected with SMARTpool siRNA targeting NRP1 or ABL1 (Dharmacon) or GTP-bound CDC42 was isolated with glutathione agarose beads bound to the p21-binding domain of PAK1 (Millipore) or the CDC42-binding domain of WASP (Cytoskeleton) via a GST tag and identified by immunoblotting of eluted proteins with an antibody for CDC42 (Millipore). For more information, see the We calculated p values with a two-tailed unpaired Student’s t test or one-way ANOVA followed by a Tukey post hoc test with Prism 5 (GraphPad Software) or Excel 12.2.6 (Microsoft Office); p < 0.05 was considered significant.A.F. performed and analyzed the retina and zebrafish experiments, A.L. and C. Raimondi performed and analyzed the cell culture experiments, G.G. performed zebrafish experiments, V.S. performed mouse genotyping and husbandry, I.Z. provided a vital reagent, and A.F. and C. Ruhrberg designed the study, performed hindbrain experiments, and prepared the manuscript. All authors have approved the manuscript.


Different delivery modes may affect the susceptibility to allergic diseases. It is still unknown whether early intervention with probiotics would counteract this effect.Bifidobacterium breve(B. breve) were examined.The effect of different delivery modes on immune status and nasal symptoms was investigated on established allergic rhinitis (AR) mouse model. In addition, the immunoregulatory effects and mechanisms of different feeding manners with B. breve was orally administered to BALB/c mice born via vaginal delivery(VD) or cesarean delivery (CD) for 8 consecutive weeks, after which they were sensitized by ovalbumin(OVA) to establish experimental AR. Nasal symptoms, serum immunoglobulins, cytokines, splenic percentages of CD4+CD25+Foxp3+ regulatory T(Treg) cells and nasal eosinophil infiltration were evaluated.Live lyophilized +CD25+Foxp3+Treg cells after establishing experimental AR. These parameters were reversed by administering B. breves hortly after birth. However, the effect of B. breve did not differ between different delivery modes.Compared with VD mice, mice delivered via CD demonstrated more serious nasal symptoms, higher concentrations of OVA-specific immunoglobulin (Ig) E, more nasal eosinophils and lower percentages of splenic CD4B. breve shortly after birth can significantly alleviate the symptoms of AR mice born via both deliveries, probably via activation of the regulatory capacity of CD4+CD25+Foxp3+Treg cells.CD aggravates the nasal symptoms of AR mice compared to VD. This is the first report that oral administration of  Allergic rhinitis (AR) is a chronic inflammatory condition of the upper airways, characterized by symptoms such as nasal itching, sneezing, rhinorrhea and elevated serum immunoglobulin E (IgE) levels. In recent decades, the incidence of AR has increased significantly worldwide, which may be related to diet structure changes, popular use of antibiotics and other factors involving microbes. According to the “hygiene hypothesis”, the changes in microbial burden early in life would lead to increased susceptibility to allergy in later life.Bifidobacterium (B.), the susceptibility of T helper type 2 (Th2) responses to oral tolerance induction is restored to a level comparable with specific pathogen-free mice[The homeostasis of early gut microbiotais considered essential for the maturation and maintenance of the host immune system. Germ-frfree mice. Furtherfree mice. Multiplfree mice.Lactobacillus, Prevotella or Sneathia species in the intestine, resembling their mother’s vaginal microbiota; infants born via cesarean delivery(CD) acquire bacterial communities from the maternal skin surface, dominated by Staphylococcus, Corynebacterium and Propionibacterium species[The first exposure to environmental microbiota occurs during the initial hours of delivery. The mode of delivery influences the establishment of microbiota and its subsequent roles in immune regulation. Infants born via vaginal delivery (VD)are predominantly colonized with m species. In latem species. HoweverB. breve, which is one strain of Bifidobacteria originally isolated from healthy infant feces, has been reported to successfully prevent or modify some immune disturbance caused by atopy[B.breve, Lactobacillus GG and mixed probiotics (B.breve and Lactobacillus) were compared, by measuring the concentration of IgE in serum and the splenic percentage of CD4+CD25+regulatory T (Treg) cells in BALB/c mice with established AR (AR) mice. This study showed that B.breve was one of the most potent anti-allergic strains (unpublished data). However, the exact regulatory mechanisms by which B. breve regulates the immunological status disturbed by CD in infants with AR still need to be studied.Probiotics, defined as “live microorganisms that confer a health benefit on the host when consumed in adequate numbers”, are major components of the gut flora. Current by atopy,11. In a+CD25+Foxp3+Treg cell percentages. Furthermore, by administration of B. breve via different feeding manners to the pups shortly after CD, it was analyzed whether B. breve has inconsistent immunomodulating capacities in experimental AR mice.In the present study, the effects of CD on nasal symptoms and immune status of ovalbumin (OVA)-sensitized BALB/c AR mice were examined. As AR is generally associated with an imbalance of Th1 and Th2 cells and with a dysfunction of Treg cells, the levels of Th1- and Th2-associated cytokines, total IgE and OVA-specific IgE were measured in serum as well as splenic CD4Female and male BALB/c mice in specific pathogen-free conditions, aged 6-8weeks, were obtained from the Dashuo Laboratory Animal Co., Ltd . All animals were kept in plastic cages and allowed free access to food and water in an environment-controlled room. Generally, 3–5 female mice were kept with 1 male mouse within one cage for mating. When a sperm plug was observed in the vagina, the female mouse was considered pregnant and reared in a separate cage until VD or CD. After birth, only female pups were used for further research. The female mice were randomly assigned to 7 groups (n = 12–15 per group) and received different experimental operations:VD (VD)CD (CD)experimental AR establishment after VD (VD+AR)experimental AR establishment after CD (CD+AR)B. breve feeding and experimental AR establishment after VD (VD+B.+AR)B. breve feeding and experimental AR establishment after CD (CD+B.+AR)B. breve feeding immediately after CDand thereafter experimental AR establishment (CD+B.-immediate +AR)All procedures were performed according to the guide for the care and use of laboratory animals and approved by the Laboratory Animal Ethics Committee of Sichuan University.The procedures of cesarean section were followed as previously described by Murphy. BrieflyB. breve was provided by Jiao Da Onlly Lmt., Co.. Starting at the first day afterVD or CD, pups were givenB. breve by gavage. In the CD+B.-1stbite+AR group, pups were orally given B. breve before breastfeeding or any other food shortly after CD. The concentration of B. brevewas109 colony-forming units(CFU)/ml distilled in phosphate buffered saline (PBS), and the volume of daily administration increased according to the age of the offspring:25μl at 0-1weeks, 50μl at 2–3 weeks, 75μl at 4–5 weeks and 100μl at 6–7 weeks after birth. Pups in control groups were given the same volume of PBS. The oral administration was carried out once a day for 8 consecutive weeks  to establish experimental AR. Briefly, mice were primarily sensitized twice by intraperitoneal injection of 200μl PBS containing 20μg OVA at week 8 and 9 after birth. At week10, they were intranasally instilled with200μg OVA distilled in 20μl PBS once daily for 6 consecutive weeks as well as OVA-specific IgE levels in serum were measured by enzyme-linked immunosorbent assay(ELISA) according to the manufacturer’s instructions. For the measurement of total IgE, samples were diluted 1:100 in deionized water, while 1:1–1:6 dilutions were used for OVA-specific IgE. Absorbance values were read at 450nm on a Bio-Rad 680 microplate reader. Total immunoglobulin levels were calculated with reference to a standard. The minimal detection concentration was 4ng/ml for total IgE and 20.7pg/ml for OVA-specific IgE.Interleukin (IL)-4, IL-5and interferon (IFN)-γ levels in serum were measured by using Luminex kits according to the manufacturer’s instructions and analyzed using a Luminex100 instrument. Samples were diluted1:2 in deionized water.6splenocytes suspended in 1 ml PBS were stained with FITC-labelled anti-mouse CD4 and PE-labelled anti-mouse CD25 for 30 minutes at 4°C. After permeabilization and fixation, the splenocytes were intracellularly stained with APC-labelled anti-mouse Foxp3 following the manufacturer’s instructions. Treg cells, defined as CD4+CD25+Foxp3+ cells, were detected in the splenocytes by flow cytometry  and data were analyzed by FlowJo 7.6 software.After removal of the spleen, half of the spleen was gently grinded and filtered through a 40μm filter to obtain cell suspensions. Approximately 1x10When mice were sacrificed at week 16, their noses were collected for histological observation. The head was removed and the lower jaw, skin and soft tissue were discarded. The noses were decalcified in 10% nitric acid solution for a total of 5 days before regular paraffin embedding. Serial sections were cut at the level of middle turbinate and inferior turbinate and stained with hematoxylin and eosin for microscopic analysis of the morphology of the nasal mucosa. Investigator was blinded for the treatment groups and calculated the average numbers of eosinophils in 10 high-magnification (×400) fields in each group.Statistical analysis was performed using SPSS version 17.0. Data with normal distribution and homogeneity of variance were analyzed using the Student—Newman—Keuls and Dunnett-t methods for pairwise comparison; data without normal distribution and homogeneity of variance were analyzed using a Kruskal-Wallis H rank sum test. A p-value<0.05 was considered statistically significant.B. breve administration. As shown in Experimental AR of mice was established by sensitization with OVA following8 weeks of B. breve significantly decreased the frequency of nasal rubbing in both VD- and CD-born AR mice, especially at week5 of nasal challenge. However, B. breve did not cause any differences in inhibition of nasal rubbing between mice born by VD and mice born by CD with established AR. Similarly, there were also no obvious differences in nasal rubbing between the CD+B.+AR group and the CD+B.-immediate+AR group.B. breve shortly after birth significantly suppressed the sneezing frequency in both VD- and CD-born AR mice at week5 of nasal challenge. However, B. breve did not show any different degree of inhibition between VD- and CD-born AR mice, and there was also no significant difference in sneezing suppression when B. breve was given first immediately or not to CD-born AR mice.As shown in B. breve did not affect total IgE levels of mice with AR born by VD or CD.Compared with the corresponding negative control groups, the concentration of total IgE was significantly higher in VD+AR and CD+AR groups . AdminisB. breve orally for 8 weeks in the VD+AR group and the CD+AR group, the concentration of serum OVA-specific IgE was significantly decreased. After treatment with the B.breve, the serum OVA-specific IgE levels in the VD+B+AR group and CD+B+AR/ CD+B.-immediate+AR group decreased by 5.7 times and 5.3 times respectively, compared to untreated rhinitis groups. No significant differences were observed between the three groups fed with B. breve.The serum OVA-specific IgE concentration in both the VD+AR group and the CD+AR group was significantly higher compared to the concentration in the corresponding negative group . FurtherNo differences in serum levels of IL–4, IL–5 and IFN-γ were observed between the 7 groups (data not shown).+CD25+Foxp3+Treg cells was significantly decreased in mice with established AR compared with the corresponding negative groups of B. breve to CD mice after establishing AR. These results may indicate that B. breve affects the immune status of mice with experimental AR via a mechanism involvingCD4+CD25+Foxp3+Treg cells.In the present study, the effects of Interestingly, some studies focused on using bispecific antibodies and antib+CD25+Treg cell development and function, and a mutation in Foxp3 leads to hyper-IgE, eosinophilia and dysregulated Th1 and Th2 cytokine production[Bifidobacteria and lactic acid bacteria have been reported to induce immunoregulatory responses and attenuate both Th1 and Th2 responses by Treg cells, which in turn ameliorated the severity of some allergic diseases[+Foxp3+Treg cells could affect atopic patients by unknown factors, which were unrelated to Th1/Th2 markers[Enterococcus faecalisFK–23, a probiotic product of Enterococcus faecalis, to BALB/c mice alleviated nasal symptoms, reduced nasal eosinophil infiltration and increased the percentage of CD4+CD25+Treg cells in experimental AR, without regulating serum and splenocyte culture supernatant levels of IL–4, IFN-γ and OVA-specific IgE. Therefore, Ozdemir et al concluded that, except for inhibition of Th1 and Th2-type cytokines, Treg cells could potently suppress IgE production and directly or indirectly suppress the activity of effector cells of allergic inflammation[+CD25+Foxp3+Treg cells by B. breve were also observed. However, no effects were seen on systemic IL–4, IL–5 and IFN-γ concentrations. It may be hypothesized that B. breve increased the percentage of CD4+CD25+Foxp3+Treg cells in the spleen, which might decrease serum allergen-specific IgE and local eosinophils infiltration. However, further investigations are needed to demonstrate whether the mechanism of reducing OVA-specific IgE by B. breve occurs via CD4+CD25+Foxp3+Treg cells, independent of regulating the Th1/Th2 balance.AR is a respiratory disease of the upper airways characterized by high concentrations of serum allergen-specific IgE, infiltration of inflammatory cells in the nasal mucosa and release of several inflammatory cytokines. Previous studies have suggested that the pathogenesis of allergic diseases is mainly associated with the imbalance of Th1/Th2 cells. In 1995, Sakaguchireported roduction. Further2 markers. Luping 2 markersalso obselammation. The aboB. breve with non-digestible oligosaccharides in a chronic asthma murine model, Sagar et al[+CD25+Foxp3+Treg cell response. Hougee et al[B. breve M-16V was identified as the most potent anti-allergic strain. Furthermore, several other studies combined B. breve with probiotics or prebiotics to evaluate the efficacy on a given kind of cell or allergic disease[B. breve may play an important role in the pathogenesis and severity of allergic diseases. This study therefore investigated how B. breve individually could affect a murine model with established AR.By combining the administration of gar et al found thgee et al comparedc disease,24,25,26B.breve on a murine allergic rhinitis model, presenting results of decreased IL–4 and OVA-specific IgE levels and increased CD4+CD25+ Tregs in the spleen at a dose of 109CFU.B. breve installed a Th1/Th2 equilibrium mainly through the inhibition of the Th2 reponse, without promoting a Th1 response. Thus, we concluded that B.breve exerted its anti-allergic effects by inhibiting Th2 immune responses and enhancing CD4+CD25+ Treg activity in that study. However, in the present study, no evidence was found that B.breve had the capacity to regulate the Th1/Th2 balance, which was partly contradictory to the previous findings and our unpublished study. We hypothesize that the conflicting conclusions might be due to different combinations used, different kinds of allergic diseases, or selections of different study populations with possibly underlying discrepant immune status, which deserves more in-depth research.Ashour et al,28 estab+CD25+Foxp3+Treg cells. Moreover, we investigated how B. breve regulated the immune status of CD mice after being established with AR, since Mikael Kuitunen et al[B.breve shortly after CD significantly alleviated nasal symptoms in mice with AR, and the strength was almost equal to that in VD mice. Most current literature has suggested that the environment of a normal fetus is sterile, and microbiota begin to colonize rapidly during birth, which emphasizes the powerful effects of delivery modes on the composition of the microflora in the gastrointestinal tract of newborns. Nevertheless, some recent studies demonstrated that the microbial community has already dwelled in the fetus before birth, as bacteria or bacterial ribosomal DNA was detected in meconium or amniotic fluid[B. breve was orally administered to cesarean-delivered mice immediately after birth or later. However, there was limited information concerning the intestinal microbiome in this study, which restricted the possibility of an in-depth exploration of the effects of B.breve in the gut.In recent years, with the increasing prevalence of CD in many areas of the world, an epidemic of allergic diseases has been postulated. A numbenen et alobserved tic fluid. In the B. breve shortly after birth for 8 consecutive weeks, whether immediately fed after birth or not, could significantly alleviate the nasal symptoms of CD mice with AR. The regulatory capacity did not vary between the delivery modes; B. breve increased the number of CD4+CD25+Foxp3+Treg cells, which might suppress allergen-specific IgE production and local mucosal infiltration of eosinophils.This study showed that the nasal symptoms  of cesarean delivered mice with AR were more severe than vaginal delivered mice. The oral administration of


Evidence indicates that post − stroke rehabilitation improves function, independence and quality of life. A key aspect of rehabilitation is the provision of appropriate information and feedback to the learner.Advances in information and communications technology (ICT) have allowed for the development of various systems to complement stroke rehabilitation that could be used in the home setting. These systems may increase the provision of rehabilitation a stroke survivor receives and carries out, as well as providing a learning platform that facilitates long-term self-managed rehabilitation and behaviour change. This paper describes the application of an innovative evaluative methodology to explore the utilisation of feedback for post-stroke upper-limb rehabilitation in the home.‘what works for whom and in what circumstances and respects?’Using the principles of realistic evaluation, this study aimed to test and refine intervention theories by exploring the complex interactions of contexts, mechanisms and outcomes that arise from technology deployment in the home. Methods included focus groups followed by multi-method case studies (n = 5) before, during and after the use of computer-based equipment. Data were analysed in relation to the context-mechanism-outcome hypotheses case by case. This was followed by a synthesis of the findings to answer the question, Data analysis reveals that to achieve desired outcomes through the use of ICT, key elements of computer feedback, such as accuracy, measurability, rewarding feedback, adaptability, and knowledge of results feedback, are required to trigger the theory-driven mechanisms underpinning the intervention. In addition, the pre-existing context and the personal and environmental contexts, such as previous experience of service delivery, personal goals, trust in the technology, and social circumstances may also enable or constrain the underpinning theory-driven mechanisms.Findings suggest that the theory-driven mechanisms underpinning the utilisation of feedback from computer-based technology for home-based upper-limb post-stroke rehabilitation are dependent on key elements of computer feedback and the personal and environmental context. The identification of these elements may therefore inform the development of technology; therapy education and the subsequent adoption of technology and a self-management paradigm; long-term self-managed rehabilitation; and importantly, improvements in the physical and psychosocial aspects of recovery. Stroke is a global problem and the worldwide incidence of stroke is set to escalate from 15.3 million to 23 million by 2030  says I haven’t… It’s disappointing; it must be the sensors” Mr Gray.All of the users were uninterested in watching their replays using the avatar (knowledge of performance feedback). One participant explained how the qualitative knowledge of results chart was easier to understand and enabled her to interpret how well she had performed,“I didn’t really grasp the two men on the screen. Those dots were much easier but it still took a few go’s to get used to it. I knew if I got in the green I‘d done well” Mrs. Green.Explicit knowledge of results such as, the provision of scores with the ‘catch the ball’ game (Mr Redmond) also increased the determination of the user to improve,“It’s a good idea; you can see a target to aim for! I can see the dots getting higher and my score going up on that game” Mr. Redmond.Mr Redmond’s carer suggested that providing a score also enabled him to tell others about his progress, leading to him receiving praise from others, which reinforced his engagement with rehabilitation.“It is easier for him to tell other people that ‘I did catch the ball and I got 3100’ or… ‘I’m better this week I got 3600’ and even though it might not make much sense to other people, they can tell it is going up and it always seems to appeal to him and he wants to do better on it each time” Mr Redmond’s carer.Contextual factors also influenced the activation of mechanisms. These included; the pre-existing context, the personal and environmental context, and the influence of the carer and researcher. These are described below.Pre-existing context - Previous experience of rehabilitation (i.e. therapist led rehabilitation to patient led rehabilitation) could influence expectations of service delivery. One participant did not believe it was his responsibility to lead his rehabilitation,“It’s not up to me to sit here and do it all myself is it… You’re the experts you should be doing it” Mr Brown.Personal and Environmental context - This included personal preferences, stroke severity, acceptance of technology and motivation to improve and carry out rehabilitation independently which the research confirmed all impacted on the utilisation of feedback from the technology. In addition, the environmental restrictions of space and ferromagnetic interference also detrimentally influenced the use of the SMART system. For example, not having somewhere to store the SMART system when not in use and the sensitivity of the inertial sensors to large metal objects (i.e. radiators) resulted in the SMART system being stowed away upstairs. In other words, if the sensors were kept near a large metal object, this would affect the accuracy of the sensors and distort the on-screen image. Because of this moving the system from its place of use to its place of storage increased the amount of setting up time and effort required prior to use.Carer and researcher influence - For the carer, issues such as personal skills, feeling empowered to be involved, accepting technology in the home and as part of a therapeutic package were also key to the utilisation of the SMART system. For the researcher, the provision of adequate training and support was essential.Analysis of the NEADL scores -44 for eThis research aimed to test and refine intervention theories by exploring the complex interactions of contexts, mechanisms and outcomes. This involves refining CMOC propositions which can then be retested Figure .The combination of determining what works/does not work for whom in what circumstance and respects has identified elements of feedback that are essential for mechanisms to work. This has enabled the refinement of theories underpinning the use of computer-based feedback for post-stroke upper-limb rehabilitation in the home. Indeed, this research has made significant refinements to the proposed CMOC’s. Two examples of these refinements are set out in Table Existing literature suggests that concurrent feedback, knowledge of performance (KP), knowledge of results (KR) and explicit feedback may be key components in the promotion of improved performance. However, existing literature also highlighted the heterogeneity in studies that have explored the use of feedback in post-stroke rehabilitation . This suThe findings have revealed that the mechanisms underpinning the utilisation of feedback from computer technology for upper-limb rehabilitation in the home are influenced by the pre-existing, personal and environmental contextual factors surrounding the user. The identification of the interaction between feedback, mechanisms and outcome(s) has also revealed how underpinning theories  may work in combination with other theories to produce an outcome within a given context. For example, knowledge of results (feedback theory)  providedThis research has highlighted the need for computer feedback to be both accurate and reliable. Indeed, the accuracy (i.e. it would show the arm moving backwards when it is moving forwards) and reliability (i.e. the system would crash and/or not boot up correctly) of the equipment used in this study influenced the utilisation of feedback which in turn, hindered the mechanisms. Because the on-screen display was sometimes inaccurate, the participants (and in some instances the carers as well) became frustrated, lost patience, and trust in the feedback provided. This resulted in the participants being less willing to use the equipment and/or dismissing the feedback as an inaccurate evaluation of their performance. Therefore, if similar systems are to be used in the future for mainstream rehabilitation, accuracy and reliability is essential.However, despite the encouraging qualitative findings for the promotion of self-managed rehabilitation, the quantitative findings suggest that this did not result in functional improvement. Therefore, further work is required to explore how overcoming technological and contextual challenges may lead to the increased intensity and longer periods of use (months not weeks) which will drive neuroplastic adaptation .Developing technology needs to account for the clinical needs of the practitioners as well as the end users themselves. If technology is to be used in mainstream therapy, service providers are required to consider the impact and variance of the context, the reliability and accuracy of the technology and importantly, what forms of feedback are provided to facilitate the users’ understanding of performance.http://www.physiotools.com) [To have clinical utility, technology systems would need to incorporate greater clinical adaptability, such as, including greater variety of exercises into the programme(s). For example, the PhysioTools (ols.com)  exerciseWith the growing interest in gaming technology such as the Nintendo® Wii™; future users may be more receptive to computer interaction; however, they may also have higher expectations in terms of the interface and motivational components of rehabilitative devices. Therefore, if future systems are to be embraced they will have to meet the rising expectations of system users and therapists for both usability and clinical utility.Research also suggests that therapists currently provide a model of service delivery that is led by their empirical knowledge, which may not be conducive to motor learning and self-management. This may therefore limit the stroke survivor’s ability to utilise computer-based feedback to facilitate self-managed rehabilitation. Work is required to educate therapists in the provision of therapy that includes newer innovative methods of delivering feedback that facilitates independent rehabilitation. This will place less demand on services whilst empowering stroke survivors and close family members to carry out and continue their recovery beyond the acute and sub-acute period.Finally, the adoption of a realistic evaluation methodological approach used in this research gave an important innovative dimension to the study since this methodology has not been used in the development of technology for rehabilitation. In line with the MRC framework for complex interventions , this apThe SMART 2 project is currently exploring the implementation and adoption of the key elements of feedback highlighted in this research through the development and user testing of the Personalised Self-Management Rehabilitation System for stroke (SMART 2) .This research has exposed the interaction between the feedback delivered through computer technology, the mechanisms underpinning the intervention, the context, and how this leads to variable outcome(s).The identification of these interactions may therefore inform the development of technology; therapy education and the subsequent adoption of technology and a self-management paradigm; long-term self-managed rehabilitation; and importantly, improvements in the physical and psychosocial aspects of recovery. Further work is required to; develop technology so that it incorporates the elements of feedback highlighted by this research; ensure the technology is robust, reliable and accurate; investigate the clinical utility of technology for home-based stroke rehabilitation, and the extent to which it might encourage utilisation by the end user.ICT: Information and communications technology; EPSRC: Engineering and Physical Sciences Research Council; MR: Medical Research Council; RE: Realistic evaluation; SMART: Self-Management supported by Assistive, Rehabilitation and Telecare Technologies; FAST: Frenchay aphasia screening test; MMSE: Mini Mental State Examination; NEADL: The Nottingham Extended Activities of Daily Living Scale; CMOC: Context-mechanism-outcome configuration.The authors declare that they have no competing interests.JP carried out the research, participated in its design and coordinated the manuscript. SM assisted in the study design and coordination and helped to draft the manuscript. GM assisted in the study design and helped to draft the manuscript. NN helped to conceptualise the methodological application and draft the manuscript. HZ provided the technical expertise and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/14/46/prepubAdditional Material: Refined CMOCs.Click here for file


Triple-negative breast cancer (TNBC) patients testing positive for androgen receptor (AR) expression are thought to be chemotherapy resistant, similar to other hormone receptor-positive breast cancers; however, this has not been substantially validated in the clinic. In this study, we investigated the association between chemotherapy sensitivity and AR expression in patients treated with neoadjuvant chemotherapy (NAC) using standardised chemotherapy criteria and regimens.A total of 177 patients with resectable early-stage breast cancer were treated with NAC. Oestrogen receptor, progesterone receptor, HER2, Ki67 and AR status were assessed immunohistochemically.P<0.001). The rate of pathological complete response after NAC was significantly lower (P=0.001), and disease recurrence was more common (P=0.008) in patients with AR-positive compared with those with AR-negative TNBC. In TNBC cases, as expected, the non-recurrence period in cases that were negative for AR expression was significantly extended .Sixty-one patients were diagnosed with TNBC; AR expression was identified in 23 (37.7%), which was significantly less common than that found in non-TNBC patients (103 of 116; 88.8% Androgen receptor expressions may be useful as biomarkers to predict treatment responses to NAC in TNBC. Moreover, induction of a change in subtype to the AR-negative phenotype was observed after NAC. Breast cancer is a typical hormone-dependent malignant tumour. Expression of the oestrogen receptor (ER) is frequently observed in breast cancer and plays a central role in disease development and progression. In addition, expression of the androgen receptor (AR) has also been frequently noted, suggesting that androgens may also play a role in breast cancer biodynamics  2 enriched, claudin-low, basal like) and a normal breast-like group . Among tGiven the lack of treatment choices and the biological attributes of TNBC, this type of breast cancer generally has a poor prognosis, and new therapeutic targets are being investigated . SimilarIn the present study, we retrospectively investigated the outcomes of NAC in patients with TNBC, using standardised criteria and regimens. The aim of this study was to clarify the differences in chemosensitivity, clinically and pathologically, based on AR expression in patients with breast cancer.A total of 177 patients with resectable early-stage breast cancer diagnosed as stage IIA , IIB  or IIIA  were treated with NAC between 2007 and 2013. Tumour stage and T and N factors were stratified based on the TNM Classification of Malignant Tumours, UICC Seventh Edition  every 3 weeks, followed by 12 courses of paclitaxel (80 mg m−2), administered weekly (−1) or tri-weekly (6 mg kg−1) basis, during paclitaxel treatment  in an autoclave . Specimens were then incubated with 3% hydrogen peroxide in methanol for 15 min to block the endogenous peroxidase activity, and then incubated in 10% normal goat or rabbit serum to block non-specific reactions.All patients had a core needle biopsy prior to receiving NAC, and following treatment underwent either a mastectomy or breast-conserving surgery with axillary lymph node dissection at Osaka City University. Immunohistochemical studies on core needle biopsy specimens were performed as previously described , PgR , HER2 , Ki67  and AR  were used. Tissue sections were incubated with each antibody for 70 min at room temperature or overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (HISTOFINE (PO) kit; Nichirei, Tokyo, Japan). Slides were subsequently treated with a streptavidin-peroxidase reagent and incubated in phosphate-buffered saline-diaminobenzidine and 1% hydrogen peroxide (v/v), followed by counterstaining with Mayer's haematoxylin. Positive and negative controls for each marker were used according to the supplier's data sheet.in situ hybridisation (FISH). For FISH analysis, each copy of the HER2 gene and its centromere 17 (CEP17) reference were counted. Interpretation of the results followed the criteria of the ASCO/CAP guidelines for HER2 IHC classification for breast cancer: positive if the HER2/CEP17 ratio was >2.0 . Categorical data are reported with numbers and percentages, and continuous data as a median and range. The association between AR and other clinicopathologic variables, and the significance of different prognostic markers were analysed using the chi-squared test (or Fisher's exact test when necessary). The association with survival was analysed using the Kaplan–Meier plot and log-rank test. The Cox proportional hazards model was used to compute univariable and multivariable hazard ratios (HR) for the study parameters with 95% confidence intervals, and used in a backward stepwise method for variable selection in multivariate analysis. In all of the tests, a P<0.001) and HRBC (P<0.001), but was not more commonly observed in patients with HER2BC (P=0.071). There was no significant difference in relation to other factors including pCR (P=0.108) for all breast cancer subtypes  and HRBC (P=0.056) patients.The breast cancer subtypes of the 177 patients who received NAC were as follows: 61 (34.5%) were TNBC, 36 (20.3%) were HER2BC and 80 (45.2%) were HRBC . Androgen receptor expression was observed in 126 cases (71.2%). Among the 61 cases of TNBC, AR expression was positive in 23 cases (37.7%) and negative in 38 cases (62.3%). Among the 116 cases of non-TNBC, AR expression was positive in 103 cases (88.8%) and negative in 13 cases (11.2%) . Androgen receptor expression was more commonly observed in patients with non-TNBC . However, multivariate analysis revealed that negative AR expression was not an independent factor (P=0.080, HR=3.78; Analysis of all 177 patients receiving NAC revealed that no significant difference in DFS was associated with AR expression (Overall, 11 patients experienced recurrence events among the 23 patients with AR-positive TNBC . The disTriple-negative breast cancer is typically associated with poor prognosis, based on the limited treatment options and the biological attributes of TNBC itself. At present, the NCCN guidelines and St Gallen Consensus conference also consider chemotherapy to be the only alternative for treatment. Despite this, a significantly higher rate of pCR is observed in TNBC patients following treatment with NAC compared with HRBC patients, with reports also suggesting that patients achieving pCR also demonstrated an extended period of non-recurrence (We observed frequent disease recurrence in patients with AR-positive TNBC. The disease-free intervals in these cases were very short, showing the typical pattern of recurrence in TNBC. We also observed frequent loss of AR expression in postoperative specimens after NAC, or in the recurrent foci of AR-positive TNBC in these patients, which might be due to the genetically unstable nature of TNBC during disease progression (In our previous study, we demonstrated favourable prognosis in patients with AR-positive TNBC compared with those with AR-negative TNBC, following standard therapeutic strategies. Similar observations have been demonstrated in other studies (Recent research indicates that TNBC can be categorised into seven specific subcategories based on genetic expression profiles . Furtherin vitro. In hormone-dependent breast cancer cells, induction of 7βHSD2 mRNA by active androgen dihydrotestosterone was inhibited by oestradiol (Given the results of this study, which indicate that AR-positive TNBC has low sensitivity to chemotherapy, AR-targeted-endocrine therapy may be one such promising therapeutic strategy. It is also known that AR and ER interact. The aim of this study was to clarify, based on AR expression in patients with breast cancer, the differences in chemosensitivity, clinically and pathologically. The pCR rate was significantly higher in AR-negative than in AR-positive patients with TNBC. Our study demonstrates that AR-negative TNBC patients receiving NAC under standardised protocols consisting of FEC, followed by weekly paclitaxel, do not show any survival benefit compared with patients with AR-positive TNBC. Moreover, induction of a change in subtype to the AR-negative phenotype was observed after NAC.