Fibroblasts are abundantly present in the prostate tumor microenvironment (TME), including cancer‐associated fibroblasts (CAFs) which play a key role in cancer development. Androgen receptor (AR) signaling is the main driver of prostate cancer (PCa) progression, and stromal cells in the TME also express AR. High‐grade tumor and poor clinical outcome are associated with low AR expression in the TME, which suggests a protective role of AR signaling in the stroma against PCa development. However, the mechanism of this relation is not clear. In this study, we isolated AR‐expressing CAF‐like cells. Testosterone (R1881) exposure did not affect CAF‐like cell morphology, proliferation, or motility. PCa cell growth was not affected by culturing in medium from R1881‐exposed CAF‐like cells; however, migration of PCa cells was inhibited. AR chromatin immune precipitation sequencing (ChIP‐seq) was performed and motif search suggested that AR in CAF‐like cells bound the chromatin through AP‐1‐elements upon R1881 exposure, inducing enhancer‐mediated AR chromatin interactions. The vast majority of chromatin binding sites in CAF‐like cells were unique and not shared with AR sites observed in PCa cell lines or tumors. AR signaling in CAF‐like cells decreased expression of multiple cytokines; most notably CCL2 and CXCL8 and both cytokines increased migration of PCa cells. These results suggest direct paracrine regulation of PCa cell migration by CAFs through AR signaling. ARandrogen receptorCAFcancer‐associated fibroblastsEMTepithelial–mesenchymal transitionPCaprostate cancerPCDFprostate cancer‐derived fibroblastsTMEtumor microenvironment1et al., et al., et al., et al., Prostate cancer (PCa) is the second most common malignancy in men worldwide, associated with high morbidity and mortality and therefore a major health concern , a nuclear steroid hormone receptor critically involved in PCa development and progression . Two other cohorts were formed of FFPE prostatectomy specimen of patients with untreated localized PCa and patients with PCa metastasized to the locoregional lymph nodes. These cohorts were matched for Gleason score, age, initial serum PSA, and T‐stage . Core biopsies of cancer and normal prostate tissue, as identified by a pathologist, of all samples in the cohorts were included in a Tumor Micro Array and stained for AR. Pan‐cytokeratin staining was used for demarcating the border between the epithelium and stroma. ImageJ tools Color Deconvolution, Despeckle, and Watershed options were used for quantification of AR expression , 5‐μm sections of formalin‐fixed paraffin‐embedded (FFPE) prostate samples or cytospins were prepared. Hematoxylin and eosin (H&E) staining was performed according to standard protocols. IHC was performed using the BenchMark automated immunostainer and iView detection system . Antibodies used are listed in Table 2.2et al., Biopsies were taken directly after prostatectomy from locations of PCa identified by multiparametric MRI and palpation of the tumor and immediately transferred to the laboratory. Primary fibroblast cultures were established as previously described  calculated confluence.To assess cell proliferation, human PCa LNCaP cells, CWR‐R1, and short‐term cultured CAF‐like cells were seeded in a 96‐well plate and placed in an incubator outfitted with an IncuCyte Zoom microscope  with a 10× objective. Phase‐contrast pictures were taken every 4 h. The integrated analyzer within the −1 of CCL2 or CXCL8 cytokines or H2O control was added to the culture medium. Cell migration was measured at the 0‐ and 96‐h time point under a microscope . Alternatively, after scratching, medium was replaced with 1 : 1 FCS‐proficient RPMI medium and charcoal‐stripped  conditioned medium (CM) from CAF‐like cells. Migration of the cells was assessed at the 0 and 6 days after creating the scratch. Migration of cells was quantified using ImageJ and expressed as percentage of closure compared to control. LNCaP cells could not be used for scratch assays because they easily detached from the plates.For scratch assays, CWR‐R1 cells were cultured in six‐well plate in 10% FBS medium Table . Once co−7–10−9 m. Antiandrogen RD162  alone or in combination with R1881 was added to DCC medium at concentrations ranging 10−5‐10−6 m.AR signaling in CAF‐like cells was activated by adding testosterone analogue R1881  to DCC medium at concentrations ranging 10Duration of R1881/RD162 exposure was 4 or 24 h. After exposure, CAF‐like cells were washed vigorously with PBS and culturing was continued in drug‐free DCC medium for 24 h, whereafter the supernatant was removed and stored. DCC‐CM was used for IncuCyte experiments 1 : 1 with FCS‐proficient medium for cell growth assays and scratch assays.−1 to 102 pg·mL−1 in FCS‐proficient medium for growth and scratch assays with CWR‐R1 PCa cells.CCL2 and CXCL8 (Sigma) were used at concentrations ranging from 102.3Cells were scraped, spun down, and resuspended in PBS supplemented with protease cocktail inhibitor (Sigma). Subcellular fractioning was performed essentially as described  supplemented with protease cocktail inhibitor (Sigma) and used for western blot analysis using standard protocols. Antibodies used are listed in Table 2.4et al., et al., Chromatin immunoprecipitation was performed as previously described  and prostate tumors was extracted from ChIP‐seq data with CopywriteR package, which was run with default parameters .Patient‐derived CAF‐like cells were stimulated with 10m sequencing pool. The libraries were sequenced with 65 base single reads on a HiSeq2500 using V4 chemistry (Illumina Inc.).Strand‐specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit  according to the manufacturer's instructions . Libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip , diluted, and pooled equimolar into a 12‐plex, 10 net al., limma R package. BETA was run with default parameters.Integration of ChIP‐seq and RNA‐seq data was performed using BETA tool available through Galaxy Cistrome  was used to measure cytokines in CAF‐like cell medium according to the supplier's protocol. Antibody‐coated beads were specific for CXCL8, CCL2, IL‐34, CXCL5, and CXCL1 (selected based on fold change) .Human prostate‐derived CAF‐like cells were cultured in DCC medium Table  and stim2.7m pore size  were used to assess the migration and invasion ability of CWR‐R1 cells in the presence or absence of neutralizing CCL2  and CXCL8  antibodies in fibroblasts CM. CWR‐R1 cells were seeded on top of the transwell membrane. In the lower chamber CM from fibroblasts stimulated with DMSO, R1881 alone or in combination with RD162 was added 1 : 1 with FBS‐RPMI, in the absence or presence of anti‐CCL2 and anti‐CXCL8 antibodies (1 ng·mL−1). To assess invasion ability of CWR‐R1 cells, Matrigel  was added on top of the membrane before CWR‐R1 cells were seeded. After 48 h, CWR‐R1 cells that migrated on the other side of the membrane were quantified using crystal violet.Ninety‐six transwell plates with 8 μ33.1Androgen receptor is the key driver of PCa development and progression. AR staining is not only found in the epithelial compartment of human PCa specimens but also in stromal cells Fig. A. Doubleet al., et al., To assess any potential clinical implications of stromal AR levels, percentage of AR‐positive cancer‐associated stromal nuclei was compared between prostatectomy specimen with an intermediate Gleason score and a high Gleason score and between prostatectomy specimen associated with and without pelvic lymph node metastases. In line with previous reports  and two with an intermediate (3 + 4 and 4 + 3) Gleason score. Other characteristics were as follows: patient age ranging from 45 to 77 years, T‐score ranging from 2 to 3a, initial PSA ranging from 5.8 to 10 μg/L, and all without local lymph node metastases  stimulation, which suggests functionality of AR Fig. C. HoweveAs the presence of local PCa lymph node metastases was associated with a lower expression of AR in the stroma, we then speculated that AR signaling in PCDFs might affect PCa cell migration. In a scratch assay, migration of human PCa CWR‐R1 cells was inhibited by culturing in medium of PCDFs stimulated with R1881. This effect was reversed by coexposure to the AR signaling inhibitor RD162, suggesting a soluble mediator under control of AR signaling inhibits PCa cell migration Fig. D,E.Cumulatively, AR in CAF‐like cells binds the chromatin upon testosterone exposure, but does not affect CAF proliferation, morphology, or migration. However, our results suggest a direct transregulation of PCa cell migration by CAF‐like cells through AR signaling.3.4et al., et al., To determine the genomewide chromatin profiles of AR in fibroblasts, we performed chromatin immunoprecipitation followed by massive parallel sequencing (ChIP‐seq). Therefore, PCDF1 and PCDF2 were hormone‐depleted for 3 days and subsequently treated for 8 h with R1881 or DMSO control, fixed, and processed for ChIP‐seq. AR is typically considered an enhancer‐selective transcription factor  Fig. . The domCumulatively, AR in CAF‐like cells binds the chromatin at distal intergenic regions and introns upon testosterone stimulation, presumably via the AP1 complex. The vast majority of binding sites are unique to CAF‐like cells and not shared with PCa cells and tumors.3.5To identify potential direct gene targets of AR in PCDFs, we employed integration of ChIP‐seq and transcriptomic (RNA‐seq) data. Using BETA analysis, we identified 174 genes that are potentially directly upregulated Table  and 234 Using a customized Luminex kit, we then measured the protein expression level of CCL2, CXCL8, and other cytokines found to be downregulated at the RNA level upon R1881 stimulation . Expression levels of the cytokines were measured in medium of PCDFs stimulated with R1881 or vehicle for 24 h. As shown in Fig. All together, these data suggest that CCL2 and CXCL8 expressions in PCDFs were directly downregulated by AR signaling both at RNA and at protein level.3.6−1 of CCL2 or CXCL8, but only a marginal effect in reducing CWR‐R1 cell growth was seen (Fig. To explore the effect of CCL2 and CXCL8 cytokines on PCa cells growth, LNCaP and CWR‐R1 cell lines were cultured in FCS‐proficient medium with addition of 1 pg·mLeen Fig. .−1 of CCL2 or CXCL8. Pictures were taken every day. After 96 h, the percentage of repopulation of the scratch surface was compared to time point 0 and quantified. As shown in Fig. AR signaling in PCDFs inhibited migration of PCa cells via soluble mediators Fig. D. To evaThese results were validated using a transwell assay in which we assessed CWR‐R1 cell migration Fig. C and invin vitro setting.All together these data show that decreased expression of CCL2 and CXCL8 in testosterone‐stimulated PCDFs reduces migration of PCa cells in the 4et al., et al., The stromal microenvironment has emerged as a key player in the development and progression of cancer (Hanahan and Weinberg, et al., et al., et al., In agreement with others (Olapade‐Olaopa Despite the clear relationship between poor outcome and loss of stromal AR, the underlying mechanism involving AR signaling in CAFs and consequences in cancer progression and outcome remains largely unknown. Several mechanisms have been proposed, including AR‐regulated secretion of factors by CAFs, affecting PCa cell proliferation, and modification of the extracellular matrix (Leach and Buchanan, et al., et al., et al., et al., Testosterone stimulation of CAF‐like cells did not affect morphology, motility, and proliferation, which was in contrast to observations in immortalized CAFs (Leach Here, we identified CCL2 and CXCL8 as key players in CAF‐mediated PCa cell migration and invasion using an unbiased and genomewide approach. Stimulation of CAFs with testosterone resulted in AR chromatin binding at CCL2 and CXCL8 loci, subsequently leading to significant downregulation of CCL2 and CXCL8 both at the mRNA and at protein level. Importantly, we showed that blocking antibodies targeting CCL2 and CXCL8 fully abrogated migration and invasion of PCa cells cultured in CM of AR signaling inhibited fibroblasts. These results indicate a direct effect of AR‐regulated cytokines on PCa cell behavior.et al., et al., in vivo in tissue recombinant mouse models (Olumi et al., Our unbiased approach leads to the identification of secreted cytokines, suggesting that secreted factors mediate the effects on PCa cell migration. In addition, direct cell–cell contacts between fibroblasts and tumor cells may play an important role in the epithelial–stromal interaction as well and could be relevant mechanisms to explore in future work. In fact, previous studies show that CAFs are able to promote directional migration of PCa cells by aligning the fibronectin in the extracellular matrix (Erdogan et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., A relation between AR signaling and regulation of CCL2 expression was previously described in macrophages (Izumi et al. (In contrast to the effect of AR signaling inhibition on PCa proliferation, inhibiting AR signaling in CAFs might enhance PCa cell migration, as was suggested previously by Lin et al. . Therefo5In conclusion, the present study showed how inhibition of AR signaling in fibroblast by hormone therapy might lead to unwanted effects on PCa development. This would suggest that classic hormonal therapies should be combined with targeted endocrine agents to improve efficacy.BC, EN, MHMM, AMB, and WZ conceived and designed the project; BC, EN, JvB, and AMB wrote the manuscript; BC, MHMM, EH, DS, JvB carried out the experiments; EN and SS performed data analysis and interpretation; HGvdP and JdJ provided specimens material and data interpretation. All the coauthors critically reviewed the present manuscript before submission.Fig. S1. Proliferation curves of PCDFs and PCa cells.Fig. S2. Ingenuity Pathway Analysis of genes proximal to AR binding sites.Fig. S3. Upstream regulators of R1881‐stimulated genes in PCDFs.Fig. S4. CCL2 and CXCL8 effect on PCa cells proliferation.Fig. S5. Migration and invasion assay.Table S1. Antibodies list.Table S2. Culture media conditions.Table S3. R1881‐specific upregulated genes found with BETA analysis.Table S4. R1881‐specific downregulated genes found with BETA analysis.Click here for additional data file.


The objective of the present study was to study CD45RC expression in a large cohort of KT recipients exposed to modern immunosuppressive regimens. CD45RC expression on T cells was analyzed in 128 KT patients, where 31 patients developed AR, of which 24 were found to be T-cell mediated (TCMR). Pre-transplant CD4+ and CD8+ CR45RChigh T cell proportions were significantly higher in patients with AR. The frequency of CD45RChigh T cells was significantly associated with age at transplantation but was not significantly different according to gender, history of transplantation, pre-transplant immunization, and de novo donor specific anti-Human Leucocyte Antigen (HLA) antibody. Survival-free AR was significantly better in patients with CD8+ CD45RChigh T cells below 58.4% (p = 0.0005), but not different according to CD4+ T cells (p = 0.073). According to multivariate analysis, CD8+ CD45RChigh T cells above 58.4% increased the risk of AR 4-fold . Thus, pre-transplant CD45RC expression on CD8+ T cells predicted AR, mainly TCMR, in KT patients under modern immunosuppressive therapies. We suggest that CD45RC expression should be evaluated in a prospective study to validate its usefulness to quantify the pre-transplant risk of AR.Predictive biomarkers of acute rejection (AR) are lacking. Pre-transplant expression of CD45RC on blood CD8 Significant progress has been made over the past few years in the immunological and histological fields, allowing for better differentiation and to refine the diagnosis and prognosis of T cell-mediated rejection (TCMR) and of antibody-mediated rejection (ABMR) in kidney transplant patients . While bSeveral risk factors of AR have been identified in previous studies including young age, female gender, black race, and immunological characteristics  . HoweverThe risk of allograft rejection relies on graft-, environmental-, and host-related factors . However+ and CD8+ T cell subsets have an essential role in the development of alloimmune response, defining T cell subpopulations with higher and lower alloimmune properties may constitute an interesting approach.The identification of biomarkers reflecting the level of tolerance emerges as a major goal in solid organ transplantation. This would allow one to tailor the immunosuppressive regimens, especially in low immunological risk kidney transplant candidates. Given that CD4+ T cells (high and low expression) and CD8+ T cells  [CD45 is a transmembrane protein tyrosine phosphatase heavily expressed on T cells and critical for signal transduction by regulating kinases of the Src-family ,9. Four nd high) ,11. Thesnd high) ,11,12.+ T cells before kidney transplantation was associated with the risk of AR after transplantation [+ CD45RChigh T cells above 54.7% conferred a 6-fold increased risk of developing AR after 4.8 years of follow-up [We demonstrated in a previous work that the level of CD45RC expression at the surface of blood CD8antation ,13. Thisollow-up . The aimThis is a monocentric cohort study that included patients transplanted in the University Hospital of Angers between 2007 and 2015. During the period of the study, after giving their written consent, patients were offered the chance to participate to a biocollection (“Collection Néphrologie et voies urinaires”). Samples were collected before kidney transplantation and stored at the dedicated department (“Centre de Ressources Biologiques BB-0033-00038”). All patients that gave their written consent to the study were included. The primary aim of the study was to analyze the value of CD45RC expression on T cells for AR prediction. The study was approved by the Medical Ethics Committee of Angers University Hospital (2009/10).The immunosuppressive treatment was not imposed by the study and was based on the assessment of immunological risk according to clinical practice in our department as detailed hereafter. Low immunological risk patients, defined as first time kidney transplant recipients and with PRA < 20%, received two injections of Basiliximab , while higher immunological risk recipients  were more likely to receive antithymocyte globulines  during the first 3 to 7 days post-transplant. ATG was also used for induction in donors with cardiac arrest before brain death, in non-heart-beating donors, and when delayed graft function was anticipated by clinician. Moreover, between 2010 and 2013, no induction therapy was performed in patients aged >70 years old. All patients received a single methylprednisolone bolus of 500 mg followed by prednisone (1 mg/kg/day) with a progressive tapering and discontinuation at the end of month 5 post-transplant, unless there was an occurrence of AR. A maintenance immunosuppressive regimen relied mainly on mycophenolate mofetil or mycophenolic acid and tacrolimus.Characteristics of the study population were collected prospectively via the systematic screening of patients’ medical records. All clinical events and biological data were retained until last follow-up: anthropometric data, nature of original kidney disease, and graft donor characteristics. Diagnosis of acute rejection (AR) episodes was based on conventional clinical and laboratory criteria and confirmed using a histological examination of a graft biopsy (according to the last Banff Classification) . AR diagPeripheral blood mononuclear cells (PBMC) of kidney transplant candidates were prospectively harvested before transplantation and stored in liquid nitrogen. Patients with samples showing PBMC viability below 80% were excluded from the analysis.After giving their written consent, fresh samples of end-stage renal disease (ESRD) patients and heathy individuals (HD) were used to monitor the proliferation capacities of CD45RC T cells.6 cells were incubated with the viability dye according to the manufacturer’s recommendations before incubation with the antibodies. Data were collected using a FACS-Canto II (BD Biosciences) cytometer and analyzed using the FlowJo software, Ashland, OR, USA. The expression of CD45RC is bimodal on CD4+ T cells, some cells expressing low levels of CD45RC (CD45RClow), and others expressing high levels (CD45RChigh). On CD8+ T cells, expression of CD45RC is trimodal, the first fraction of cells expressing low levels (CD45RClow), the second fraction expressing intermediate levels (CD45RCInt), and the last fraction expressing high levels of CD45RC (CD45RChigh). The following conjugated antibodies were used to characterize CD45RC T cell subpopulations: CD3-VioGreen (REA613), CD4-PerCP-Vio700 (REA623), CD8-PE-Vio770 (REA734), from Milteny Biotec, Bergisch-Gladbach, Germany; CD45RA-APC (HI100) from BD Biosciences, San Jose, CA, USA; and CD45RC-FITC (MT2) from IQ Product, Houston, TX, USA. Cell viability was systematically assessed . Briefly, 107 PBMCs were stained using a Cell Trace Violet proliferation kit  for proliferation assessment and then stained using CD4-BV421 , CD8-PE-Vio770 , and CD45RC-FITC . CD45RChigh and CD45RClow subpopulations were sorted among CD4+ and CD8+ T cells. Purity was always routinely above 95%. Then, 5 × 104 T cells were cultured at 37 °C in RPMI 1640 medium  in 96-well round-bottomed microplates , with or without a 1 µg/mL plate-bound anti-CD3  and 0.5 µg/mL soluble anti-CD28 (Beckman-Coulter). After 72 h of culture, cells were harvested and proliferation was assessed using flow cytometry .CD45RC T cells were sorted from freshly isolated PBMC of end-stage renal disease (ESRD) patients and age/sex matched healthy donors (HD) using a FACS-Aria cytometer, BD Bioscience, San Jose, CA, USA. Briefly, after a gradient centrifugation, 2 × 102 or Fischer’s exact test and Mann–Whitney U tests, respectively. The Wilcoxon matched-pairs rank test was used to compare the proliferative capacities of T cells. The predictive values of the CD45RC subset frequency for the first AR episode were analyzed using receiver operating characteristics (ROC) curves. Subsequently, cut-off values were determined by using the Youden index. The Kaplan–Meyer method was used to analyze AR-free survivals according to predetermined cut-off values of CD45RC subset frequencies. A log-rank test was used to compare survival curves. Correlations were analyzed using Spearman’s rank correlation test. Multivariate cox models were used to analyze the association between CD45RC subset frequencies and AR. Results are reported as hazard ratio (HR) with 95% CIs. All p-values were two-sided and a p-value lower than 0.05 was considered statistically significant. Statistical analysis was performed using Graphpad Prism® version 7  and SPSS® software version 22.0 .Data were expressed as a median with minimum to maximum values for continuous variables and absolute count with percentage for categorical variables. Categorical and continuous data were analyzed with χn = 6) or poor blood cell viability (n = 6). Thus, 128 patients were included and finally analyzed  during follow-up. These data are reported in The mean follow-up of the cohort was 3.82 ± 2.22 years. During the follow-up, AR occurred in 31 patients (24.2%) at a mean delay of 0.73 ± 1.24 years post-transplant. When considering only the first AR episode, 28 were histologically-proven and 3 were diagnosed based on clinical and biological criteria. Among the histologically-proven AR cases, 24 were TCMR, and 6 being borderlines. The four other AR episodes were ABMR in one case and mixed AR (TCMR and ABMR) in the three other cases. At one-year post-transplant, mean serum creatinine was 141.4 ± 75.2 µmol/L and mean glomerular filtration rate (GFR) was 53.2 ± 21.8 mL/min/1.73 mp = 0.035). Baseline characteristics, including age and pre-transplant immunization, were not significantly different between groups. These data are reported in Patients that experienced AR received more frequently Basiliximab as induction therapy as compared to patients that did not experienced AR, who received more-frequent ATG .We first analyzed the association of CD45RC expression on T cells with patient’s characteristics. As shown in subjects ,11, CD45+ and CD8+ CD45RChigh compared to patients that did not develop AR. The difference between groups remained significant regardless of whether all AR episodes were considered, when analysis was restricted to biopsy-proven ARs, or when borderline AR episodes were excluded. Moreover, the absolute number of both CD4+ and CD8+ CD45RChigh cells was significantly greater in patients that experienced AR , while AR-free survival was not significantly different according to CD4+ CD45RChigh T cell frequencies (p = 0.073)  and biopsy-proven AR. When borderline ARs were excluded, ATG was no longer significantly associated with AR occurrence.We finally analyzed the risk factors of AR in a cox model analysis . We succ+ CD45RChigh T cell subset was associated with an increased risk of AR. We could determine that patients with a proportion of CD8+ CD45RChigh T cell frequency above 58.4% had a 4-fold increased risk of AR. Thus, these results are in line with our previous work [In the present work, we confirmed that the CD8ous work ,13 and c+ T cell subset [As compared to our previous work ,13, in tl subset ,11. A gel subset ,15; howehigh T cell subset frequency according to subsequent DSA development, which suggests that CD45RC status does not allow one to assess the risk of ABMR. TCMR still represents a significant cause of morbidity in the era of ABMR, the latter being the main cause of late graft lost [AR incidence, including borderline AR, in our study was 24.2% after a mean follow-up of almost 4 years. As expected, most AR episodes were TCMR, with ABMR being implicated in only four AR episodes. This observation is in line with recent reports ,16 showiaft lost . Moreoveaft lost  and probaft lost ,21,22. Thigh CD4+ and especially CD8+ T cells. In vitro, CD45RChigh CD8+ T cells from HD mainly produced interferon gamma a key cytokine in TCMR, and poor levels of regulatory cytokines [Interestingly, not only CD45RC proportion was associated with AR occurrence, but also the absolute count of CD45RCytokines . Whether+ CD45RChigh T cells for AR prediction. In our previous work, the best threshold was determined at 54.7% [+ CD45RChigh T cells in the present study. The very closed cut-off values as determined in the two works, conducted in two different populations with different immunosuppressive regimens, reinforces the strength of this biomarker.We could determine in the present study that 58.4% was the best threshold of CD8at 54.7% . FinallyThe present study has several limitations. First, only 32% of patients transplanted during the period were included and we could not exclude a selection bias. Next, AR diagnosis was based on for-cause biopsies and not protocol biopsies. Thus, the value of CD45RC expression for subclinical rejection remains to be investigated. Moreover, given the relatively short follow-up, we were not able to analyze the relationship between pre-transplant CD45RC expression and the risk of ABMR, which occurred in only four patients in the present study.+ CD45RChigh T cells is associated with a 4-fold increased risk of AR, mainly TCMR. Thus, CD45RC could be used to help define the immunological risk and the level of immunosuppressive regimen before kidney transplantation. However, the value of CD45RC expression on T cells should be evaluated in a multicenter prospective cohort study with protocol biopsies to confirm its usefulness and to study its predictive value for subclinical AR.In conclusion, the present study confirmed and extended the data on CD45RC that has appeared as a promising biomarker to assess the risk of AR before transplantation. This work confirmed that the pre-transplant proportion of CD8


Robot-assisted therapy has become a promising technology in the field of rehabilitation for poststroke patients with motor disorders. Motivation during the rehabilitation process is a top priority for most stroke survivors. With current advancements in technology there has been the introduction of virtual reality (VR), augmented reality (AR), customizable games, or a combination thereof, that aid robotic therapy in retaining, or increasing the interests of, patients so they keep performing their exercises. However, there are gaps in the evidence regarding the transition from clinical rehabilitation to home-based therapy which calls for an updated synthesis of the literature that showcases this trend. The present review proposes a categorization of these studies according to technologies used, and details research in both upper limb and lower limb applications.The goal of this work was to review the practices and technologies implemented in the rehabilitation of poststroke patients. It aims to assess the effectiveness of exoskeleton robotics in conjunction with any of the three technologies  in improving activity and participation in poststroke survivors.A systematic search of the literature on exoskeleton robotics applied with any of the three technologies of interest  was performed in the following databases: MEDLINE, EMBASE, Science Direct & The Cochrane Library. Exoskeleton-based studies that did not include any VR, AR or gamification elements were excluded, but publications from the years 2010 to 2017 were included. Results in the form of improvements in the patients’ condition were also recorded and taken into consideration in determining the effectiveness of any of the therapies on the patients.Thirty studies were identified based on the inclusion criteria, and this included randomized controlled trials as well as exploratory research pieces. There were a total of about 385 participants across the various studies. The use of technologies such as VR-, AR-, or gamification-based exoskeletons could fill the transition from the clinic to a home-based setting. Our analysis showed that there were general improvements in the motor function of patients using the novel interfacing techniques with exoskeletons. This categorization of studies helps with understanding the scope of rehabilitation therapies that can be successfully arranged for home-based rehabilitation.Future studies are necessary to explore various types of customizable games required to retain or increase the motivation of patients going through the individual therapies. Stroke refers to a sudden, often catastrophic neurological event that can lead to long-term adult disability. The American Heart Association (AHA) is responsible for providing up-to-date statistics related to heart disease and stroke. According to Benjamin et al , the AHAThere are several reasons for stroke occurrence, which could be related to an increased risk of a collection of symptoms caused by disorders affecting the brain  . VariousOne of the most effective techniques is robot-aided therapy, which has been gradually increasing in use primarily because patients may consider traditional rehabilitation therapy to be tiring and exhaustive. This may decrease their motivation and cohesion to the treatment, thus resulting in only minor improvement in the health of poststroke patients -6. VarioApart from these studies, Housman et al  showed uVR is a virtual form of a real entity, object or environment. According to Schultheis et al , VR can As mentioned above, traditional methods of rehabilitation might lead to a patient’s loss of interest in their therapy, as it often involves daily repetitive tasks. VR encourages patients to participate in their therapy by either incorporating games in the form of exercises or through other interactive means. With the current state of VR in rehabilitation services, a new form of therapy has gradually emerged that is known as Virtual Rehabilitation . VirtualWe must also consider the advancements in VR technology in recent years by the inclusion of sixth and seventh generation gaming systems, which include various popular systems such as the Xbox 360 Kinect and the Nintendo Wii. Yates et al  discusseSlowly, AR is also gaining traction in the field of rehabilitation. According to Khademi et al , when a In addition to these two technologies (VR and AR), video games are often used in rehabilitation services these days as they play an essential part in encouraging patients to participate in therapeutic exercises. It should be noted that VR- or AR-interaction can be nongamified or nonplayful, which is why we prefer to delineate them. Games are used with specific hardware depending on the physical condition of the patients, and various game attributes are considered while developing games for rehabilitation purposes. There are several types of games used in rehabilitation services, such as two-dimensional (2D), 3D, VR and AR games, and other natural user interfaces such as Wii, PlayStation, Wii Balance, Xbox, and Kinect. Alankus et al  developeRabbit Chase, Bubble Trouble and Arrow Attack), and identified game design principles which were significant for upper limb stroke rehabilitation. However, the aim of our paper is to investigate and review the coupling of such gaming elements, or virtual reality, with an exoskeleton or robotic device.It should be noted that most of the games used in rehabilitation are commercial, off-the-shelf games. In a study conducted by Acosta et al , the feaExoskeleton means an extension to the (human) skeleton, but in simplistic terms, some researchers have defined an exoskeleton as any transparent device that a user or patient may wear or attach upon themselves and that extends their natural motor capabilities by determining their intent . They arTo summarize, in our review we intended to ascertain the possible interactions of rehabilitation robotics or exoskeletons with AR or VR , that is, to use the intermediate interfaces employed as a means of supplementing the rehabilitation process using exoskeleton-based hardware. Therefore, we set out to perform an exploratory review of the field of rehabilitation robotics with an additive aspect of technical scope, focusing on solutions and prototypes in the area of exoskeletons that interfaced with software mediums. Our methodology focused on the common approach carried out when doing systematic reviews; however, our analysis and reflections were mainly based on qualitative grouping and meta-synthesis, due to the preliminary nature of our work and the heterogeneous and multidisciplinary character of our considered papers.To further motivate our approach, work and research objectives, we scanned the literature to extract review articles like ours, and two recent studies emerged. The first focused primarily on the prospect of exoskeletons for stroke rehabilitation  and the Therefore, to conclude, the aim of this review was to examine the potential and latest trends in the area of exoskeleton- or robotic-aided therapy in combination with VR, AR, or gamification for the improvement of motor function for poststroke patients. Specifically, we aimed to determine: (1) if such a coupled approach or setting could provide positive outcomes for patients; (2) trends and popular configurations across both types of exoskeletons and software mediums; and (3) future challenges in the field of exoskeleton-based HCI therapy.We conducted this review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The folInclusion criteria were as follows: (1) experimental, explorative or RCT studies on poststroke rehabilitation, (2) VR, AR, or gamification visual feedback, (3) stroke-affected patients; and (4) use of an exoskeleton or robotic device. As per our interpretation for this review, a VR or AR environment in the field of robot-aided rehabilitation is a replica of the real-world environment that is achieved after using hardware devices and a wearable exoskeleton device in liaison with each other.The exclusion criteria for our study were: (1) studies done without the use of any robotic device or exoskeleton; (2) studies with nonvirtual or nonaugmented environments, or an absence of games; and (3) publications or articles in languages other than English. As mentioned before, due to the heterogenous nature of the collated studies, data was synthesized qualitatively.A total of 504 articles were identified from electronic searches and a total of 56 were identified through reference searches or other sources. We excluded 129 citations which were only at title and abstract stage, resulting in 204 full-text articles. Of these, 132 citations were excluded at the full-text stage, with reasons mentioned in All articles published in English and in the years 2010 to 2017 were included. Four studies were published in the year 2010 and one in the year 2017, with size of the samples involved in the studies ranging from 1-47 participants per study; however, 13 studies had less than 10 participants. In two of the studies, healthy users of the devices were used as controls to compare the improvements of the patients ,33. AparThe average time poststroke for the participants involved in these studies, as mentioned in the articles, varied from less than six months to several years. Four studies did not mention the poststroke period at all ,33,35-37The studies were categorized into different fields, and of all 30 studies half of them used VR therapy while the other half used some gaming concepts. The studies that involved the use of an exoskeleton or robotic device along with VR, AR, or gamification were within the inclusion criteria of this review, so thirty exoskeletons or robotic devices were included. Of these devices, three exoskeletons emerged as slightly more popular in use: ARMin, Bi-Manu Track and ArmeoSpring. These devices each had repeated use in two studies while 24 studies made use of entirely different exoskeletons or robotic devices.Klamroth-Marganska  made useAnother exoskeleton device, Armeo Spring, was used in two other papers selected for analysis ,43. In tIn the year 2011, several studies were published that made use of exoskeletons with other technologies, and here we summarize a few as case studies. Lambercy et al  had a stThe studies mentioned so far mostly involved one part of the human body (arm), but Connelly et al  discusseWe noticed that there was less academic literature for lower limb rehabilitation compared to upper limb rehabilitation, but our review still included two studies that involved lower limb rehabilitation. Forrester et al and Mirelman et al ,63 descrAlthough positive results and improvements in motor function were observed in most of the studies, the results from this systematic review also depict that most rehab services are carried out in groups in clinics while home-based rehab is rarely attempted using the current configuration of interactive technologies. Group therapies in a clinical or lab setting are done so that patients feel motivated by collaborating with, or competing against, each other. In home-based rehabilitation, it is possible that patients might feel overwhelmed or isolated with the advanced forms of technology necessary for their therapy. In this case, the technology and the therapy sessions need to be designed in a way such that patients feel motivated and confident during home-based rehabilitation sessions as well (such as through online-tailored gaming). Thus, game-based rehabilitation can play a key role and provide a suitable interfacing medium for VR or AR, with 10 of our sample of 30 papers associating gaming with virtual reality. Use of customized games should be encouraged so that games are designed to keep in mind a particular target user, which could drive motivation in those people who play these specialized games at home as a part of their rehabilitation process. Articles in our sample indicated the key considerations that researchers must focus on while designing games for rehabilitation , with key elements of discussion including: whether the play is meaningful, if engagement or motivation is retained, the difficulty level of the game, the role of customization, the range and type of feedback acquired, and the overall usability of the gameplay. Lastly, the interaction technique used is also a key consideration in game-based rehabilitation, with what gestures result in what game event easily being dictated by the motor movements required .However, there are some limitations to this review. For example, different types of assessment scale and quality of collected data were used, which makes it difficult to compare the outcomes and results accurately or quantitatively against each other. In addition, some articles could have been missed in the review due to very specific search criteria.This review was carried out to collect data from different clinical trials and then to categorize and explore them to find the effectiveness of VR, AR, or gamification when used in combination with an exoskeleton or robotic device for the rehabilitation of poststroke patients. It was found that very little work is done to make use of these technologies for rehabilitation of lower limbs when compared to upper limbs, and that there are a wide variety of exoskeleton-based devices currently in use. Apart from this, the review also states that these exoskeleton-based devices are rarely available for home-based trials. This shows that there is a considerable gap in the transition of rehabilitation services from a clinical environment to a home-based setting. Future work should focus on the successful application of VR, AR, or gamification technology to engage poststroke patients in rehabilitation therapies done at their homes. In addition, commercial, off-the-shelf games may be deployed easily, but efforts must be dedicated to designing games for rehabilitation to keep in mind the user and allow for customization to facilitate their motivation.


The androgen receptor (AR) plays key roles in the development of prostate tissue and the development and progression of prostate cancer (PC). AR guides cytodifferentiation and homeostasis in benign luminal epithelial cells; however, in PC, AR instead drives the uncontrolled proliferation of these cells. This ‘AR malignancy shift’ (AMS) is a central event in tumorigenesis. Using a ChIP‐seq approach in primary human tissues, cell lines, and mouse models, we demonstrate that the AMS occurs in every sample analyzed, suggesting that it is necessary for PC development. Using molecular and genetic techniques, we demonstrate that forkhead box (FOX)A1, HOXB13, GATA2, and c‐JUN are involved in the regulation of the AMS. AR‐binding sites (ARBS) are enriched for FOX, HOX, and GATA motifs in PC cells but not for c‐JUN motifs in benign cells. We show that the SPOP mutation commonly found in localized PCs can cause the AMS but is not transformative on its own and must be coupled to another mutation to transform cells. We show that the AMS occurs in mouse models of PC as well and that chronic low T, which is associated with increased PC risk and aggressiveness in humans, also causes the AMS in mice. We have discovered a previously unrecognized, fundamental tenet of PC, one which explains how and why AR signaling is different in cancer and benign cells. Our work has the potential to be used to stratify patients with localized PC for specific treatments. Furthermore, our work suggests that the AMS is a novel target for the treatment and/or prevention of PC. The androgen receptor plays key roles in the development of prostate tissue; however, an ‘AR malignancy shift’ (AMS) can occur which allows AR to also drive prostate cancer (PC). We have characterized this shift, and our work has the potential to be used to stratify patients with localized PC for specific treatments and suggests that the AMS is a novel target for the treatment and/or prevention of PC. AMSandrogen malignancy shiftARandrogen receptorIPAingenuity pathway analysisN‐ARBsnormal androgen receptor‐binding sitesPCprostate cancerTtestosteroneT‐ARBstumor androgen receptor‐binding sitesTFtranscription factorWTwild‐type1et al., et al., et al., et al., et al., et al., Despite advances in detection and treatment, prostate cancer (PC) remains the second leading cause of cancer death in American men  can cause the AMS in benign mouse prostate tissue.22.1De‐identified patient samples were received from City of Hopes’ umbrella discarded tissue protocol for pilot studies as approved by the City of Hope ethics committee. Eleven normal and three tumor (two matched) fresh tissue samples were taken at time of radical prostatectomy and immediately snap‐frozen in OCT and stored at −80° C. A 5‐µm section was used for H&E staining by the City of Hope pathology core and areas of tumor and benign identified by a pathologist. A precooled scalpel was used to isolate the tumor and normal cells on a bed of dry ice to keep samples frozen and then stored at −80 °C until used in subsequent experiments.2.2et al., et al., et al., et al., n = 5 for each cohort) and another after chronic low T study consisted of a normal cohort (n = 9) and a low T cohort (n = 7) had prostates collected at 60–80 weeks and immediately frozen down and stored at −80 °C. Mouse prostate tissue from the +/−; Pten+/−Nkx3.1 model, and appropriate control tissue from mice of the same background was a kind gift from the Abate‐Shen Laboratory .All animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the City of Hope IACUC #13023. Models of low T were created as previously described .The LHSAR cells were a kind gift from the Freedman laboratory  as per standard protocol with various plasmids and siRNAs. Specifically, the plasmids were vector , FOXA1 , FOXA1 L455M was created by site‐directed mutagenesis, GATA2 , PIK3CA , and SPOP . For transient GATA2 and c‐JUN siRNA transfections, the Flexitube™ siRNA kits were used  as per standard protocol.2.5t‐test in prism V 7.02 . A P‐value of > 0.05 was considered significant.After 48 h, the cells were harvested and 20 000 cells were seeded in 96‐well plates in triplicate for each treatment for subsequent CytoSelect™ soft agar cell colony‐forming assays  to analyze cell transformation via attachment independent growth rates as per standard protocol. Statistical analysis was by a two‐way 2.6et al., m glycine for 5 min. Cells in 150 mm dishes were harvested at ~ 80% confluency and fixed in formalin for 10 min and quenched for 5 min at room temp before washing 2× in cold PBS and then kept on ice. 0.1% and 0.5% SDS buffers were used to isolate and then disrupt nuclei, respectively. Chromatin was sheared in the Bioruptor™ pico  to 100–500 bp in length with six cycles (30 s on/30 s off). Lysates were precleared with Pierce A/G magnetic beads (Thermo Fisher), and 10% of lysate was stored at −80 °C as input. IP was overnight at 4 °C with anti‐AR antibody  or 10 µg rabbit IgG. Antibody/chromatin conjugates were isolated with A/G magnetic beads. Chromatin was uncross‐linked overnight at 65° C with proteinase K and DNA extracted with phenol–chloroform. For quality control, 5 µL was run out on an agarose gel for chromatin size determination and DNA quantified and quality checked on a NanoDrop  or q‐bit dsDNA HS assay kit .ChIP was performed as previously described . While there were some differences between the two, including more promiscuous binding by the Abcam antibody, the two antibodies performed similarly in our hands  according to the manufacturer's protocol. Briefly, 5–10 ng of immunoprecipitated DNA underwent end‐repaired, A tailing, and adaptor ligation. Ten cycles of PCR were performed to produce the final sequencing library. The libraries were validated with the Agilent Bioanalyzer DNA High‐Sensitivity DNA Kit  and quantified with Qubit (Invitrogen). ChIP‐seq libraries were sequenced on Illumina HiSeq 2500 with V4 SBS reagent in the single‐read mode of 51 cycles of read 1 and 7 cycles of index read. Real‐time analysis 2.2.38 software was used to process the image analysis and base calling.2.8http://www.novocraft.com/products/novoalign/). Mapped reads were filtered to exclude PCR duplicates using Picard (https://broadinstitute.github.io/picard/). Data quality measurements and fragment length estimation were performed using Phantompeakqualtools  or mouse (mm9) reference genome using NovoAlign (2.9n = 9179) or normal tissue  . A P‐value of > 0.05 was considered significant.The Galaxy/Cistrome ‘seqPos Motif tool’ was used to discover candidate TF motifs both in our AR ChIP‐seq data and that of publicly available datasets  to identify genes canonical pathways, disease networks associated with the peaks.2.13et al., t‐test in prism V 7.02 (GraphPad). A P‐value of > 0.05 was considered significant.ChIP‐qPCR was used to quantify the enrichment of IP compared to input as previously described , HOXB13 , and GATA2  motifs were significantly enriched in tumor compared with normal tissue, and conversely, c‐JUN was enriched in normal tissue  . Conversely, knockdown of FOXA1 in LNCaP PC cells caused them to adopt AR‐binding patterns more similar to benign cells. We repeated these experiments in LHSAR cells by overexpressing FOXA1 or GATA2, or knocking down c‐JUN  and also a decrease in c‐JUN motifs (P = 0.001) in AR peaks from FOXA1‐transfected cells compared to AR peaks from control cells  with several specific members from each family listed as potential associated factors. FOXA1 and HOXB13 were chosen as specific family members to investigate because of their strong literature connections to AR signaling in PC, which is nicely described in Pomerantz et al.  or mutations ’  and N20× (Santa Cruz) anti‐AR antibodies.Click here for additional data file.Fig. S2. Recapitulation of previous heatmaps.Click here for additional data file.Fig. S3. Validation and analysis of transfections.Click here for additional data file.Table S1. Alignment summary.Table S2. MACS2 peak calling summary.Table S3. Genes associated with unique AR peaks in normal tissue.Table S4. Genes associated with unique AR peaks in tumor tissue.Click here for additional data file. Click here for additional data file.


N-acyliminium ion intermediates, obtained from activation of the quinoline and isoquinoline heterocycles or from the appropriate δ-lactam with benzyl chloroformate. Finally, the hydrolysis of phosphonate moiety with simultaneous cleavage of the carbamate afforded the target compounds.We report here a practical and efficient synthesis of α-aminophosphonic acid incorporated into 1,2,3,4-tetrahydroquinoline and 1,2,3,4-tetrahydroisoquinoline heterocycles, which could be considered to be conformationally constrained analogues of pipecolic acid. The principal contribution of this synthesis is the introduction of the phosphonate group in the  After stirring at this temperature for 2 h, the reaction was treated with saturated aqueous sodium acetate (5 mL) and warmed to room temperature. A 3:1 mixture of diethyl ether and saturated aqueous ammonium chloride (16 mL) was then added and the resulting mixture was stirred at room temperature until a suspension was formed. The solid was filtered off under reduced pressure and washed with diethyl ether (2 × 10 mL). The organic layer was separated and the aqueous phase was extracted with diethyl ether (2 × 20 mL). The combined organic extracts were washed with brine (20 mL), dried, filtered, and evaporated to provide the hemiaminal as an oil. It was dissolved in methanol (6 mL) and treated with pyridinium p-toluenesulfonate . After stirring at room temperature for 2 h, triethylamine  was added. The solvent was evaporated and the crude methoxyaminal 9 thus obtained was dissolved in anhydrous dichloromethane (7 mL) and kept under argon. Trimethyl phosphite  was added and the resulting solution was cooled to −20 °C. Boron trifluoride-diethyl ether  was added dropwise and the reaction mixture was slowly warmed to room temperature and stirred for 12 h. After quenching with water (2 mL), the two layers were separated and the aqueous phase was extracted with dichloromethane (2 × 10 mL). The combined organic extracts were dried, filtered, and concentrated. Purification by column chromatography (eluent:ethyl acetate/hexanes 4:1) afforded 11 as a colourless oil . IR (neat) νmax 1702, 1279, 1029 cm−1. 1H-NMR : δ = 7.53–7.46 , 7.37–7.28 , 7.22–7.16 , 7.14–7.06 , 5.31 , 5.16 , 5.04 , 3.63 , 3.51 , 2.85–2.75 , 2.67–2.57 , 2.54–2.40 , 2.17–2.03  ppm. 13C-NMR : δ = 154.68 (CO), 136.84 (Ar), 136.08 (Ar), 132.78 (Ar), 128.54 (Ar), 128.16 (Ar), 127.88 (Ar), 127.56 (Ar), 126.36 (Ar), 125.97 (Ar), 125.22 (Ar), 68.11 (CH2Ph), 53.07 , 52.82 , 49.33 , 25.75 (C-3), 25.66 (C-4) ppm. 31P-NMR : δ = 26.39 ppm. HRMS (ESI): calcd. for C19H22NNaO5P [M + Na]+ 398.1128; found 398.1145.A 1 M solution of diisobutylaluminium hydride in hexanes  was slowly added to a solution of 13 as a colourless oil . IR (neat) νmax 1706, 1653, 1603, 1263, 1125, 1026 cm−1. 1H-NMR : δ = 7.58–7.52 , 7.44–7.30 , 7.25–7.07 , 6.71–6.65 , 6.07 , 5.61 , 5.27 , 3.47  ppm. 13C-NMR : δ = 152.90 , 135.68 (Ar), 134.66 (Ar), 128.05 (Ar), 127.70 (Ar), 127.40 (Ar), 127.35 (Ar), 127.01 , 126.77 , 126.06 , 124.35 (Ar), 123.66 (Ar), 122.55 , 67.44 (CH2Ph), 52.70 , 52.39 , 50.84  ppm. 31P-NMR : δ = 20.41 ppm. HRMS (ESI): calcd. for C19H20NNaO5P [M + Na]+ 396.0971; found 396.0991.Benzyl chloroformate  was added dropwise to a solution of quinoline  in anhydrous acetonitrile (6 mL) kept at 0 °C under argon. After stirring at this temperature for 10 min, trimethyl phosphite  was slowly added followed by sodium iodide . The mixture was heated at 50 °C for 10 min. The solvent was evaporated and the crude product was partitioned between dichloromethane (15 mL) and saturated aqueous sodium bicarbonate (15 mL). The organic phase was separated and the aqueous layer was further extracted with dichloromethane (3 × 15 mL). The combined organic extracts were washed with brine (10 mL), dried, filtered, and concentrated. Purification by column chromatography (eluent:ethyl acetate/hexanes 4:1) afforded 13  and 10% Pd/C (20 mg) in ethyl acetate (10 mL) was stirred at room temperature under an atmospheric pressure of hydrogen gas for 12 h. Filtration of the catalyst and evaporation of the solvent provided pure 14 as a colourless oil . IR (neat) νmax 3316, 1231, 1057, 1029 cm−1. 1H-NMR : δ = 7.02–6.93 , 6.70–6.63 , 6.58–6.53 , 4.16 , 3.82 , 3.81 , 3.71 , 2.88–2.75 , 2.29–2.19 , 2.12–1.98  ppm. 13C-NMR : δ = 143.08 , 129.40 (Ar), 127.11 (Ar), 121.05 (Ar), 118.26 (Ar), 114.97 (Ar), 53.83 , 53.08 , 49.05 , 26.09 , 22.39  ppm. 31P-NMR : δ = 27.39 ppm. HRMS (ESI): calcd. for C11H16NNaO3P [M + Na]+ 264.0760; found 264.0752.A mixture of 11: A 33% solution of hydrogen bromide in acetic acid (2 mL) was added to compound 11  and the reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated and the residue was taken up in water and lyophilised to afford 4 as a white solid . M.p. 94–96 °C (dec.). IR (nujol) νmax 3381, 1171, 1077, 1057 cm−1. 1H-NMR : δ = 7.40–7.28 , 3.78 , 3.08–3.01 , 2.52–2.43 , 2.19–2.06  ppm. 13C-NMR : δ = 132.21 , 131.87 (Ar), 131.78 (Ar), 130.25 (Ar), 128.64 (Ar), 124.49 (Ar), 52.52 , 25.94 , 22.37  ppm. 31P-NMR : δ = 14.61 ppm. HRMS (ESI): calcd. for C9H13NO3P [M − Br]+ 214.0628; found 214.0632.From 14: A 33% solution of hydrogen bromide in acetic acid (2 mL) was added to compound 14  and the reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated and the residue was taken up in water and lyophilised to afford 4 as a white solid . Spectroscopic data were identical to those described above.From 16 as a colourless oil . IR (neat) νmax 1715, 1342, 1295, 1238, 1121, 1028 cm−1. 1H-NMR : δ = 7.51–7.11 , 6.91 , 6.08–5.94 , 5.83 , 5.27 , 3.57–3.38  ppm. 13C-NMR : δ =  152.37* (CO), 151.88 (CO), 136.01 (Ar), 135.91* (Ar), 131.03* , 130.84 , 128.75* , 128.68 , 128.54 (Ar), 128.47* (Ar), 128.26 (Ar), 128.11* (Ar), 127.92 (Ar), 127.55 , 127.30 , 127.18 , 125.43 (Ar), 125.35 (Ar), 124.86 , 124.79* (C-3), 109.86 (C-4), 67.86* (CH2Ph), 67.77 (CH2Ph), 53.40* , 53.16* , 53.12 , 52.64  ppm. 31P-NMR : δ = 21.34 ppm. HRMS (ESI): calcd. for C19H20NNaO5P [M + Na]+ 396.0971; found 396.0982.Benzyl chloroformate  was added dropwise to a solution of isoquinoline  and trimethyl phosphite  in anhydrous acetonitrile (6 mL) kept at 0 °C under argon. Sodium iodide  was slowly added and the mixture was heated at 50 °C for 10 min. The solvent was evaporated and the crude product was partitioned between dichloromethane (15 mL) and saturated aqueous sodium bicarbonate (15 mL). The organic phase was separated and the aqueous layer was further extracted with dichloromethane (3 × 15 mL). The combined organic extracts were washed with brine (10 mL), dried, filtered, and concentrated. Purification by column chromatography (eluent: ethyl acetate/hexanes 4:1) afforded 19 as a colourless oil . IR (neat) νmax 1715, 1294, 1253, 1120, 1023 cm−1. 1H-NMR : δ = 7.50–7.10 , 6.91 , 5.98 , 5.77 , 5.26 , 3.95–3.69 , 1.08 , 1.07  ppm. 13C-NMR : δ =  152.48* (CO), 151.91 (CO), 136.05 (Ar), 135.88* (Ar), 131.15* , 130.98 , 128.69* , 128.61 , 128.56 (Ar), 128.47* (Ar), 128.28 (Ar), 128.25* (Ar), 128.13* (Ar), 127.97 (Ar), 127.53 , 127.22 , 127.11* , 125.59 , 125.56* , 125.42 (Ar), 124.84* (C-3), 124.81 (C-3), 109.95 (C-4), 67.84* (CH2Ph), 67.71 (CH2Ph), 62.54 , 62.42* , 62.38* , 53.97* , 53.15 , 16.17 , 16.15*  ppm. 31P-NMR : δ = 18.77 ppm. HRMS (ESI): calcd. for C21H25NO5P [M + H]+ 402.1465; found 402.1466.Benzyl chloroformate  was added dropwise to a solution of isoquinoline  and triethyl phosphite  in anhydrous acetonitrile (6 mL) kept at 0 °C under argon. Sodium iodide  was slowly added and the mixture was heated at 50 °C for 10 min. The solvent was evaporated and the crude product was partitioned between dichloromethane (15 mL) and saturated aqueous sodium bicarbonate (15 mL). The organic phase was separated and the aqueous layer was further extracted with dichloromethane (3 × 15 mL). The combined organic extracts were washed with brine (10 mL), dried, filtered, and concentrated. Purification by column chromatography (eluent:ethyl acetate/hexanes 3:2) afforded 19  in anhydrous dichloromethane (15 mL) kept at 0 °C under argon. The solution was allowed to warm to room temperature and stirred for 18 h. Evaporation of the solvent followed by column chromatography (eluent:hexanes/ethyl acetate 1:1) afforded 20 as a colourless oil . IR (neat) νmax 1701, 1294, 1249, 1230, 1051, 1022 cm−1. 1H-NMR : δ = 7.44–7.16 , 5.53 , 5.17 , 4.14–3.77 , 3.72–3.52 , 2.98–2.78 , 1.17 , 1.08  ppm. 13C-NMR : δ =  154.60 , 154.18* , 136.66 (Ar), 136.46* (Ar), 134.73 , 134.63* , 129.22 (Ar), 129.13* , 128.95 , 128.85 (Ar), 128.38 (Ar), 128.33* (Ar), 127.94* (Ar), 127.91 (Ar), 127.86* (Ar), 127.67 , 127.58 (Ar), 127.38 (Ar), 125.88 , 125.83* , 66.92* (CH2Ph), 66.82 (CH2Ph), 62.58 , 62.19 , 52.76* , 52.34 , 39.05* (C-3), 38.84 (C-3), 27.43 (C-4), 27.15* (C-4), 16.08* , 16.05 . 31P-NMR : δ = 20.75. HRMS (ESI): calcd. for C21H27NO5P [M + H]+ 404.1621; found 404.1611.Triethylsilane  and trifluoroacetic acid  were added to a solution of 20  and the reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated and the residue was taken up in water and lyophilised to afford 5 as a white solid . M.p. 85–87 °C (dec.). IR (nujol) νmax 3421, 1212, 1118, 1019 cm−1. 1H-NMR : δ = 7.45–7.41 , 7.37–7.27 , 4.70 , 3.78 , 3.58–3.51 , 3.18–3.12  ppm. 13C-NMR : δ = 132.88 , 130.40 , 129.47 , 128.94 , 127.92 , 127.40 , 54.00 , 41.15 , 25.88 (C-4) ppm. 31P-NMR : δ = 10.02 ppm. HRMS (ESI): calcd. for C9H13NO3P [M − Br]+ 214.0628; found 214.0633.A 33% solution of hydrogen bromide in acetic acid (2 mL) was added to H)-isoquinolinone 21  in anhydrous tetrahydrofuran (5 mL) kept at −78 °C under argon. After 30 min, benzyl chloroformate  was added dropwise and stirring was continued for additional 3 h. The reaction was then treated with saturated aqueous ammonium chloride (10 mL) and allowed to warm to room temperature. The two layers were separated and the aqueous phase was extracted with dichloromethane (2 × 10 mL). The combined organic extracts were dried, filtered, and concentrated. Purification by column chromatography (eluent:hexanes/ethyl acetate 7:3) afforded 22 as a white solid . M.p. 63–65 °C. IR (nujol) νmax 1692 cm−1. 1H-NMR : δ = 7.49–7.44 , 7.40–7.25 , 7.23–7.19 , 5.34 , 4.92 , 3.73  ppm. 13C-NMR : δ = 169.43 (CO), 153.62 (COO), 135.38 (Ar), 132.25 (Ar), 132.13 (Ar), 128.74 (Ar), 128.50 (Ar), 128.46 (Ar), 128.22 (Ar), 127.35 (Ar), 126.99 (Ar), 125.81 (Ar), 68.97 (CH2Ph), 48.75 (C-1), 41.53 (C-4) ppm. HRMS (ESI): calcd. for C17H15NNaO3 [M + Na]+ 304.0944; found 304.0943.A 1 M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran  was slowly added to a solution of 1,4-dihydro-3 in anhydrous tetrahydrofuran (5 mL) kept at −78 °C under argon. After stirring at this temperature for 2 h, the reaction was treated with saturated aqueous sodium acetate (10 mL) and allowed to warm to room temperature. A 3:1 mixture of diethyl ether and saturated aqueous ammonium chloride (16 mL) was then added and the resulting mixture was stirred at room temperature until a suspension was formed. The solid was filtered off under reduced pressure and washed with diethyl ether (2 × 10 mL). The organic layer was separated and the aqueous phase was extracted with diethyl ether (2 × 10 mL). The combined organic extracts were washed with brine (10 mL), dried, filtered, and evaporated to provide the hemiaminal as an oil. It was dissolved in methanol (5 mL) and treated with pyridinium p-toluenesulfonate . After stirring at room temperature for 2 h, triethylamine  was added. The solvent was evaporated and the crude methoxyaminal obtained 23 was dissolved in anhydrous dichloromethane (5 mL) and kept under argon. Trimethyl phosphite  was added and the resulting solution was cooled to −20 °C. Boron trifluoride-diethyl ether  was added and the reaction mixture was slowly warmed to room temperature and stirred for 12 h. After quenching with water (10 mL), the two layers were separated and the aqueous phase was extracted with dichloromethane (2 × 10 mL). The combined organic extracts were dried, filtered, and concentrated. Purification by column chromatography (eluent:ethyl acetate/hexanes 9:1) afforded 25 as a colourless oil . IR (neat) νmax 1703, 1410, 1246, 1055, 1031 cm−1. 1H-NMR : δ =  7.42–7.30 , 7.23–7.04 , 5.31–5.12 , 5.11–5.03 , 5.00–4.86  overlapped with 4.97*–4.89* , 4.52 , 4.45* , 3.65 , 3.51* , 3.35 , 3.33–3.16  overlapped with 3.24*  ppm. 13C-NMR : δ =  155.72 (CO), 155.05* (CO), 136.37 (Ar), 136.18* (Ar), 132.61* (Ar), 132.46 (Ar), 131.41 (Ar), 131.00* (Ar), 128.64 (Ar), 128.53 (Ar), 128.45* (Ar), 128.24 (Ar), 128.17* (Ar), 127.95 (Ar), 126.70 (Ar), 126.64* (Ar), 126.60 (Ar), 126.09* (Ar), 125.86 (Ar), 67.79 (CH2Ph), 52.74 , 52.58 , 52.34* , 47.07* , 46.29 , 44.37 (C-1), 28.56* (C-4), 28.32 (C-4) ppm. 31P-NMR : δ =  27.67, 27.07* ppm. HRMS (ESI): calcd. for C19H22NNaO5P [M + Na]+ 398.1128; found 398.1144.A 1 M solution of diisobutylaluminium hydride in hexanes  was slowly added to a solution of 25  and the reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated and the residue was taken up in water and lyophilised to afford 6 as a white solid . M.p. 102–103 °C (dec.). IR (nujol) νmax 3404, 1232, 1010 cm−1. 1H-NMR : δ = 7.35–7.22 , 4.52 , 4.42 , 3.94–3.83 , 3.34–3.25  ppm. 13C-NMR : δ = 131.71 , 129.97 (Ar), 129.27 (Ar), 128.69 (Ar), 128.32 (Ar), 127.77 (Ar), 51.34 , 47.01 , 27.44 (C-4) ppm. 31P-NMR : δ = 14.06 ppm. HRMS (ESI): calcd. for C9H13NO3P [M − Br]+ 214.0628; found 214.0631.A 33% solution of hydrogen bromide in acetic acid (2 mL) was added to 4, 1,2,3,4-tetrahydroisoquinoline-1-phosphonic acid 5, and 1,2,3,4-tetrahydro-isoquinoline-3-phosphonic acid 6 have been prepared in high overall yields using efficient methods that make use of easily available substrates. The compounds obtained can be viewed as higher homologues of phosphopipecolic acid that bear a benzene ring fused at different positions of the six-membered piperidine cycle. In particular, the latter is the phosphonic surrogate of Tic, an amino acid of extraordinary value in the design of pharmacologically useful peptides. The synthetic routes developed rely on the addition of trialkyl phosphites to iminium ions generated from quinoline/isoquinoline or from the appropriate δ-lactam bearing a fused benzene ring. Thus, lactams have proven to be versatile starting materials for the efficient and selective synthesis of α-aminophosphonic acids containing an azacyclic skeleton. The presence of the carbonyl group in the starting lactam determines, with full regiocontrol, the position at which the phosphonate substituent is incorporated. Formation of the intermediate N-acyliminium ion with the adequate regiochemistry has allowed the synthesis of the phosphonic counterpart of Tic (TicP). Importantly, this α-aminophosphonic acid is not accessible through other methodologies traditionally used to generate iminium ions.The synthesis of α-aminophosphonic acids characterized by a tetrahydroquinoline or tetrahydroisoquinoline heterocycles is described. Specifically, 1,2,3,4-tetrahydroquinoline-2-phosphonic acid


Grain yield was lower under N90 than under N150 in DSR by 11–18%, whereas the difference was not significant in SSR. Grain yield under N0 was 36–63% lower in DSR than in SSR. There was a significant relationship between percentage change in grain yield comparing N90 to N150 with grain yield under N0. Furthermore, it was observed that grain yield under N0 was not significantly associated with growth duration but was closely associated with daily grain yield under N0, and the relationships of daily grain yield under N0 with seasonal average daily mean temperature and solar radiation were not significant. These results indicate that response of grain yield to reducing N rate was more sensitive in DSR compared to SSR due to lower soil N productivity. Growth duration and climatic conditions did not explain the difference in soil N productivity between DSR and SSR. The findings of this study should encourage more research into comparing the inherent traits of plants, especially the morphology and physiology of the root system, between DSR and SSR.Reducing N rate without sacrificing grain yield is crucial for sustainable rice production in China. In this study, field experiments were conducted in 2016 and 2017 to determine whether the response of grain yield to reducing N rate varied between double-season rice (DSR) and single-season rice (SSR). Three N rates were applied for each rice crop, i.e. 150 kg ha In the past several decades, rice yield has more than tripled in China as a result of the development of new cultivars and improved crop management practices2. This increase in rice yield has been important for ensuring food security in China. However, at the same time, fertilizer consumption in China has had an almost linear increase, especially for nitrogen (N) fertilizer3. The average N application rate for rice production in China at present is 180 kg ha−1, which is about 75% higher than the world average5. The high N rate has caused substantial environmental costs including enhanced N deposition, soil acidification, and surface water eutrophication8. Therefore, reducing the N rate without sacrificing grain yield is necessary for sustainable rice production in China.Rice is the staple food crop for about 65% of the population in China9. Improving soil fertility is an effective way to achieve high inherent soil productivity and consequently reduce the dependence on external fertilizer inputs in rice production10. In addition to the soil conditions, the inherent soil productivity also can be affected by the crop. For example, Huang et al.11 observed that inherent soil N productivity (i.e. grain yield without N application) was increased with the development of new rice cultivars in China.The capacity of productivity in a soil is referred to as inherent soil productivity and can be expressed by the actual crop yield without fertilizer inputshttp://ricestat.irri.org:8080/wrs). There are two rice cropping systems in this province: (1) double-season rice (DSR) cropping with early-season rice (ESR) grown from March (sowing) to July (harvesting) and late-season rice (LSR) grown from June to October; and (2) single-season rice (SSR) cropping from April to September. Growth duration is shorter in DSR compared to SSR. This difference in growth duration can result in shorter duration of soil N uptake and consequently may cause lower soil N productivity in DSR than in SSR. Moreover, climatic conditions during growing-seasons are also different between DSR and SSR. In general, seasonal average temperature and solar radiation are lower in DSR than in SSR, because ESR and LSR have lower temperature and solar radiation than SSR during the early and late growth periods, respectively. These differences in temperature and solar radiation may lead to slower crop growth and hence lower soil N productivity in DSR compared to SSR. The potentially different soil N productivity resulted from differences in growth duration, and climatic conditions may further lead to different N responses between DSR and SSR, but no studies have been conducted to confirm and clarify these differences.Hunan province is one of the major rice-producing provinces in China and contributes more than 10% of total rice production  in two years. Our objectives were (1) to determine whether the response of grain yield to reducing N rate is different between DSR and SSR, and if so, (2) to identify the factors that contribute to the difference in N response between DSR and SSR.Average daily mean temperatures during early and late rice-growing seasons were lower than that during the single rice-growing season by 1.8–4.1 °C in 2016 and by 1.1–3.2 °C in 2017 Fig. . In 201612. High temperatures shorten the growth duration and vice versa. However, in this study, the shorter growth duration in ESR and LSR than in SSR was not related to temperature because, as mentioned above, the temperature was lower during early and late rice-growing seasons than during the single rice-growing season. Therefore, the difference in growth duration between DSR and SSR should be mainly attributed to different genotypes.Growth duration from transplanting to maturity was shorter in ESR and LSR compared to SSR by 18–24 d in 2016 and by 23–26 d in 2017 Fig. . It is wGrain yield was significantly affected by rice crop, N rate, and their interaction Table . In gene−1 increase in grain yield under N0. These findings support results of previous studies suggesting that improving soil N productivity is an effective way to reduce the dependence of external N inputs in rice production11. More importantly, the results of this study suggest that the magnitude of the potential of reducing N rate may be smaller in DSR than in SSR due to lower soil N productivity.These results indicate that response of grain yield to reducing N rate was more sensitive in DSR (ESR and LSR) compared to SSR. This different N response could be related to the difference in soil N productivity (grain yield under N0) between DSR and SSR, which was significantly lower in ESR and LSR than in SSR by 38–45% in 2016 and by 36–63% in 2017 Fig. . Consist14. The findings of this study should encourage more research into comparing the morphology and physiology of the root system between DSR and SSR. Such research would provide useful information to improve soil N productivity and consequently reduce the dependence of external N inputs in rice, especially for DSR.Similar to the grain yield under N0, daily grain yield under N0 was significantly lower in ESR and LSR than in SSR by 25–29% in 2016 and by 19–53% in 2017 Fig. , and the16, but further studies are required to confirm this hypothesis. In addition, our study has potential limitations that need to be acknowledged. First, the experiment was conducted on only one soil type. Second, only one cultivar was used for each rice crop. Therefore, more experimentation should be done on different soil types with various rice cultivars to obtain broader results.Perhaps also interestingly, in contrast to the grain yield under N0, increase in grain yield with N fertilization was generally higher in ESR and LSR than in SSR Fig. . This meDSR had lower soil N productivity than SSR. As a result, response of grain yield to reducing N rate was more sensitive in DSR compared to SSR. Growth duration and climatic conditions did not explain the difference in soil N productivity between DSR and SSR.−1, total N = 1.50 g kg−1, total P = 0.61 g kg−1, total K = 7.92 g kg−1, NaOH hydrolysable N = 179 mg kg−1, NaHCO3 extractable P = 16.2 mg kg−1, and NH4OAc extractable K = 102 mg kg−1. The soil characteristics were determined based on samples taken from the 0–20 cm layer, with the methods described by Bao17.Field experiments were conducted in a farmer’s field in Yongan Town , Hunan Province, China, in 2016 and 2017. The soil of the field was a clay type with the following properties: pH = 6.26, organic matter = 44.3 g kg−1 , 90 kg ha−1 (N90), and 0 kg ha−1 (N0). Treatments were arranged in a split-plot design with rice crops (DSR and SSR) as main plots and N rates as subplots. The experiment was replicated six times and subplot size was 20 m2.Two rice cultivars, Zhongzao 39 and Y-liangyou 1, were used in this study. Zhongzao 39 was grown in early and late rice-growing seasons, while Y-liangyou 1 was grown in the single rice-growing season. These two cultivars were selected because they have been widely grown by rice farmers in the study region. Three N rates were applied to each rice crop , i.e. 150 kg haRice crops were established according to locally recommended practices. Pre-germinated seeds were sown in a seedbed. For DSR, 25- and 15-day-old seedlings were transplanted on 22 April and 21 July in the early and late seasons, respectively. Transplanting was done at a hill spacing of 20 cm × 16.7 cm with three seedlings per hill. For SSR, 25-day-old seedlings were transplanted on 4 June. Transplanting was done at a hill spacing of 20 cm × 20 cm with two seedlings per hill.2O5 ha−1 as basal) and potassium  were applied in all subplots. All fertilizers were manually broadcasted and incorporation was made only for basal fertilization. The experimental field was kept flooded (5–10 cm) from transplanting until 7 days before maturity, when the field was drained. Insects and diseases were intensively controlled by chemicals to avoid yield loss.N was applied in three splits: 50% as basal (1 day before transplanting), 30% at early-tillering (7 days after transplanting), and 20% at panicle initiation. Phosphorus  were calculated.Daily mean temperature and solar radiation during rice-growing seasons were measured using a Vantage Pro2 automatic weather station . Growth duration was recorded as number of days from transplanting to maturity. Grain yield was determined from a 5-mStatistical analysis was performed in Statistix 8.0 . Data were analyzed using analysis of variance and linear regression analysis. Means of treatments were compared based on the least significant difference (LSD) test at the 0.05 probability level.


The evidence from post-mortem biochemical studies conducted on cortisol and catecholamines suggest that analysis of the adrenal gland could provide useful information about its role in human pathophysiology and the stress response. Authors designed an immunohistochemical study on the expression of the adrenal β2-adrenergic receptor (β2-AR), a receptor with high-affinity for catecholamines, with the aim to show which zones it is expressed in and how its expression differs in relation to the cause of death. The immunohistochemical study was performed on adrenal glands obtained from 48 forensic autopsies of subjects that died as a result of different pathogenic mechanisms using a mouse monoclonal β2-AR antibody. The results show that immunoreactivity for β2-AR was observed in all adrenal zones. Furthermore, immunoreactivity for β2-AR has shown variation in the localization and intensity of different patterns in relation to the original cause of death. To the best of our knowledge, this is the first study that demonstrates β2-AR expression in the human cortex and provides suggestions on the possible involvement of β2-AR in human cortex hormonal stimulation. In conclusion, the authors provide a possible explanation for the observed differences in expression in relation to the cause of death. The adrenal response to stress involves the activation of the sympathoadrenal (SA) system and the hypothalamic–pituitary–adrenocortical (HPA) axis; in this way, homeostasis in emergencies such as “fight or flight” situations is maintained . SeveralIn the forensic field, many researchers analyzed the individual differences in the stress response during the death process, mostly investigating both catecholamine and glucocorticosteroid levels, using a biochemical approach ,6,7,8 anARs are cell membrane receptors included in the seven-transmembrane-spanning G-protein coupled receptor (GPCR) superfamily, formed by membrane proteins triggered by extracellular ligands such as small organic molecules, peptides, and neurotransmitters . StructuAccording to their pharmacological response, ARs were subdivided into α and β types: these latter types were further divided into three families called β1, β2 and β3 on the basis of molecular classification ,20. β2-AThe purpose of this study was to analyze the adrenal glands obtained from forensic autopsies in order to evaluate the expression of β2-AR in the adrenal glands and to investigate its expression in subjects with different causes of death.The study revealed an evident immunoreactivity for β2-AR expressed in the human adrenal gland  .p < 0.05 versus the fasciculata zone)  e;p < 0.05) between the glomerulosa and the medulla   o;road accident: if compared to the other adrenal zones  a,b,d, osudden cardiac death: the glomerulosa and the medulla showed mild β2-AR immunostaining f,i, whilsepsis: the highest β2-AR immunostaining was observed in the glomerulosa k; the poMoreover, the positive staining varied in localization and intensity in the specimens obtained from subjects included in the six groups examined, as shown in As a consequence of the above results, the comparison between β2-AR expression in the single zones showed that: (a) the glomerulosa zone has a higher expression in hanging, drowning and sepsis deaths; (b) the fasciculata zone was significantly positive in sudden cardiac death and fire fatalities; (c) the reticularis zone shows a higher β2-AR expression in hanging, fire fatalities, and road accidents; (d) medulla expression was greater in cases of hanging and drowning .The aim of this study was to analyze the structural and/or molecular changes in adrenal glands obtained from forensic autopsies of different causes of death, in order to evaluate the presence of β2-AR in the four zones of the glands and to investigate its expression in subjects that died during different stressful conditions. In particular, the study was directed to analyze an adrenal tissue marker involved in the stress response process to activated/stimulated receptors by the activation of the SA system and HPA axis, focalizing the attention exclusively on the adrenal gland and its specific functions.(i)to histologically demonstrate β2-AR expression in the human cortex;(ii)to provide suggestions on the possible involvement of β2-AR in human cortex hormonal stimulation, and(iii)to study β2-AR expression in the adrenal gland in different causes of death.The study showed that β2-ARs were expressed in the human medulla and cortex and that cortical expression was observed in all zones, suggesting that in the stress response, the adrenergic system could contribute, as a complementary pathway, to stimulate steroid production and release involving the glomerulosa, the fasciculata and the reticularis, respectively secreting mineralocorticoids, glucocorticoids and androgens . While tThe presence of β2-AR in the cortical zone of the human adrenal gland might indicate that the cortex and medulla, even if embryologically different, could share common mechanisms in the coordination of the stress response.As to the possible involvement of β2-AR in human cortex hormonal stimulation, the results have demonstrated that an in vivo cross-talk between cortical and medullary cells was present according to previous evidence based on vascular and nervous adrenal features. Adrenomedullary cells receive blood from the cortex in which high concentrations of adrenocortical steroids are present  and cortMoreover, the study showed differing β2-AR expression, for both localization and quantity, in the adrenal glands of subjects who died from different causes, suggesting that the activation/stimulation of adrenergic receptors might vary on the basis of different stressful conditions related to the death process.Although many studies have highlighted the increase of catecholamines and glucocorticoids in relation to stress ,6, the fIn the sudden cardiac death group, the greater expression in the fasciculata suggested that the adrenal gland response could be mainly associated with the synthesis and secretion of glucocorticoids, possibly related to their effect on blood pressure regulation and on evidence suggesting the involvement in heart rate control by the arterial baroreceptor reflex ,32,33.In the hanging group, the asphyxia condition could induce a greater stimulation of medullary β2-ARs to promote the synthesis and secretion of catecholamines as supported by evidence on adrenalin and noradrenaline increase due to both blood pH alteration induced by hypoxia and activation of a sympathetic–adrenal mechanism regulating the cardiovascular response to asphyxia (redistribution of blood flow toward the brain and muscle beds). In previous experimental studies on the analysis of the effects of asphyxia, an increase of systemic arterial pressure, sympathetic vertebral nerve activity, nitric oxide formation and the release of plasma catecholamine components of norepinephrine and epinephrine was observed ,35; moreIn the road accident group, including subjects with brain injuries, the effects of DHEA and DHEA sulfate (DHEAS) could explain the higher β2-AR expression observed in the reticularis zone, as these hormones have positive effects in brain lesions as documented by experimental studies ,41.In the fire fatality group, the significant β2-AR expression in the reticularis could be related to the modulation of the nociceptive function of DHEA and DHEAS ,43, in pIn the sepsis group, the higher β2-AR expression in the glomerulosa suggested a role for mineralocorticoid synthesis and secretion, related to compensatory mechanisms activated to increase volemia and arterial blood pressure through the renin–angiotensin–aldosterone system (RAAS) ,45. It iIn the drowning group, the higher expression of β2-AR in the glomerulosa is possibly related to the systemic effects of drowning fluid. Studies on pathophysiology highlighted that drowning determines different systemic responses and outcomes, in relation to immersion (upper airway above water) and/or submersion (upper airway under water), depending also on the characteristics of the drowning liquid . These systemic effects affect the cardiocirculatory, respiratory and neural systems by several mechanisms, mediated by hypothermia, hypovolemia, diving response, autonomic conflict, upper airway reflexes, water aspiration and swallowing, and electrolyte disorders ,49. In lTherefore, the present study highlights the post-mortem profile of β2-AR expression in the human adrenal gland, showing topographic and quantitative differences, possibly related to different causes of death. These results suggest that other studies aiming to evaluate the correlation between adrenal β2-AR expression and the activation of SA system and HPA axis (not investigated in the present study) could be very useful to improve the knowledge of stress response processes. Furthermore, in a medicolegal approach to the causes and mechanisms of death, adrenal gland analysis could prove to be, as well as other second level investigations ,54,55,56The major limits of our study are represented by the relatively small number of samples and by the possible influence on β2-ARs expression caused by the subject’s previous pharmacological therapy (data not known). Furthermore, post-mortem changes cannot be evaluated in this study, thus no comments can be offered on the effects of post-mortem phenomena on receptor expression. The authors did not analyze the relationship between the obtained results and other factors , which would also possibly affect the expression of the receptors.n = 8; 3 females and 5 males; mean age 51.4 ± 17 years); (ii) road accident ; (iii) fire fatality ; (iv) sudden cardiac death ; (v) sepsis ; and (vi) drowning . All autopsies were performed 24–48 h after death.Forty-eight forensic autopsies, carried out between March 2017 and December 2017, were included in this study . The folThis is a retrospective study carried out on filed judiciary cases in which the specimens were taken during autopsy as ordered by a judicial authority. The authors asked for ethical approval  from the Local Ethics Committee  that declared no-competence because the study used judiciary specimens. The authors got the permission to use the autopsy samples for research purposes by the General Public Prosecutor.2O2 in PBS. The primary antibody  was incubated overnight at 4 °C in a moisturized chamber. The following day, peroxidase-conjugated secondary antibody  was added and the reaction was visualized with 3,3′-diaminobenzidine (DAB) . Counterstaining was performed in Mayer’s hematoxylin . Negative controls from all the regions of the adrenal gland  were obtained using PBS instead of the primary antibody , dehydrated in graded ethanol, cleared in xylene and embedded in paraffin . The histological sections (5 μm) were deparaffinized in xylene and rehydrated in ethanol. Antigen retrieval was performed with pH 6.0 citrate buffer and endogenous peroxidase blocked with 0.3 % Hantibody . For theThe sections were photographed with a Nikon Ci-L  light microscope. All images were taken with a digital camera (Nikon Ds-Ri2) and saved as Tagged Image Format Files (TIFF) with the Adobe Photoshop CS software. Five randomly chosen microscopic fields (MFs) from five non-serial sections of each group were considered. All micrographs were captured at the same magnification and were blindly assessed by two trained observers who ignored which of the groups the examined specimens belonged to. Immunopositivity of β2-AR in each zone of the adrenal gland  was graded according to the following semi-quantitative score: 0, negative; 1, slight and/or limited (to a few groups of cells) positivity; 2, patchy and scattered positivity; 3, diffuse and intense positivity.p-value of ≤0.05 was considered statistically significant.For each cause of death, data referring to the semi-quantitative grading of β2-AR expression assigned to the different zones of all glands were expressed as mean ± standard deviation (SD). A non-parametric approach was used since the examined variables were not normally distributed, as verified by the Kolmogorov–Smirnov test. For each cause of death, a non-parametric ANOVA was applied in order to assess the existence of possible differences among the four adrenal gland zones . The same analysis was performed in order to assess, for each zone, possible differences among the six causes of death . Statistical analysis was performed using the SPSS 17.0 for Window package. A This study revealed the expression of β2-ARs in all layers of the human adrenal gland and supports the presence of a cross-talk between cortical and medullary cells.The obtained results suggest, also, that the adrenal gland could respond to stress with a kind of “specificity”, managing homeostatic changes determined by different stressors and stressful conditions, possibly providing a specific response in different death processes.The authors state that, considering the poor literature about adrenal gland β2-ARs and the absence of previous studies focused on adrenal gland functional changes during death processes, the obtained results must be considered as preliminary evidence supporting the opportunity to perform further research aimed to improve the knowledge of the adrenal gland’s functionalities and to discover its role in human pathophysiology during stress conditions.