Cyp11a1-Cre to specifically ablate AR from the mouse adrenal cortex. Results show that AR-signaling is dispensable for adrenal gland development in females, and for X-zone regression during pregnancy, but is required to suppress elevation of corticosterone levels post-partum. Additionally, following disruption to adrenal AR, aberrant spindle cell development is observed in young adult females. These results demonstrate sexually dimorphic regulation of the adrenal X-zone by AR and point to dysfunctional adrenal androgen signaling as a possible mechanism in the early development of adrenal spindle cell hyperplasia.Adrenal androgens are fundamental mediators of ovarian folliculogenesis, embryonic implantation, and breast development. Although adrenal androgen function in target tissues are well characterized, there is little research covering the role of androgen-signaling within the adrenal itself. Adrenal glands express AR which is essential for the regression of the X-zone in male mice. Female mice also undergo X-zone regression during their first pregnancy, however whether this is also controlled by AR signaling is unknown. To understand the role of the androgen receptor (AR) in the female adrenal, we utilized a  The adrenal cortex has a complex multi-cellular structure comprised of specialized concentric zones that support the biosynthesis of steroids. The zones that make up the adrenal cortex consist of the zona glomerulosa (ZG), zona fasciculata (ZF), and the zona reticularis (ZR). These are responsible for the production of aldosterone, cortisol and androgens, respectively, which act both locally and systemically to support development and physiological function , 2. Adrevia AR in the male adrenal, showing that AR is essential for X-zone regression during puberty and to protect the adrenal against age-related degeneration and spindle cell hyperplasia  , 12. Stuerplasia . Howevervia AR in the adrenal cortex, controls cortex remodeling and X-zone regression in females similar to our observations in males. This study identifies AR as an essential regulator of adrenal cortex maintenance provides a significant novel entry point into advancing our understanding of X-zone regulation and sex-biased adrenal disease in women.In this study, we have used a novel knockout model targeting the AR in the adrenal gland of female mice to establish whether androgen-signaling All mice used in experiments were under a strict standard of care and experimental planning covered by licensed approval from the UK Home Office (License number 70/8804) held by L.B.S.loxP technology was used. Male C57BL/6 mice carrying a random insertion of the Cyp11a1-GC Cre  80.To specifically ablate AR from the female adrenal cortex, Cre/1-GC Cre  were mat1-GC Cre . Male ofGenotyping for the inheritance of Cre recombinase was performed as previously described . PCR ampG2 Ad-ARKO females were set up in mating pairs with wild-type (WT) C57BL/6 males. Females were allowed give birth and were culled 24 h post birth. A control group of WT pregnant females were collected under the same conditions. Females gave birth between the ages of d80 and d90.via cervical dislocation. Body weight was measured, and adrenals were removed and weighed. Reproductive organs were also examined, removed and weighed. Tissues were fixed in Bouin’s fixative  for 4 h. Bouin’s-fixed tissues were processed and embedded in paraffin wax, and 5 µm sections were used for histological analysis. Sections of adrenals were stained with hematoxylin and eosin using standard protocols and examined for histological abnormalities.Mice were culled by inhalation of carbon dioxide and death was confirmed 2O2 concentration, whereby washes were performed at 3% H202 TBS. Antibodies used are listed in Immunolocalisation was carried out under various protocols. Single antibody colorimetric (DAB) immunostaining method, as described previously , a singlvia cardiac puncture with a syringe and wide beveled needle to prevent lysis. Blood was collected in EDTA coated tubes to prevent coagulation and was spun down within 20 min of extraction. Plasma was separated by centrifugation at 13,000 revolutions per minute (rpm) for 10 min and stored at -80°C long-term.Immediately after culling, blood was collected from control and Ad-ARKO mice Post-partum corticosterone was measured using a mouse corticosterone ELISA kit  according to the manufacturer’s instructions. All samples were run as a single assay for each hormone. Females were collected at various stages throughout estrus and were not estrus synchronized.via new and sensitive isotope-dilution TurboFlow-LC-MS/MS method. Using this method we are able to quantify androstenedione, testosterone, progesterone, and corticosterone in human and mouse serum as previously described .Analysis of proliferation and apoptosis was achieved via GraphPad Prism . Statistical tests include a two-tailed unpaired t-test (if comparing two groups), a one-way ANOVA with Tukey’s post-hoc test (if comparing multiple groups), a two-way ANOVA with Tukey’s post-hoc test (if comparing multiple groups and variables), and Chi Squared test for determining proportion of a population Values are expressed as mean ± S.E.M.Groups consisted of 5 mice for morphological and presence/absence analysis. Power calculations were carried out with the software Graphpad Statmate. Power calculations based on previous stereology work determined that a sample size of 5 is appropriate for quantitative end points for cell counting and immunohistochemistry. Statistical analysis is performed We have previously shown AR to be expressed in the adrenal cortex of human and male mice . To estaCyp11a1-GC Cre line (nd Generation Ad-ARKO) completely lack AR from adrenocortical steroidogenic cells. Both generations of Ad-ARKO females are used in this study as mosaic ablation in G1 enables investigation of autocrine AR action inside cells within the adrenal cortex, in addition to establishing the effects of complete loss of adrenal AR via analysis of G2. Immunolocalization demonstrates AR is expressed in all zones of the adrenal cortex in controls, in contrast, mosaic ablation of AR from adrenocortical steroidogenic cells is observed in G1 Ad-ARKOs, with sporadic AR localization throughout the cortex and complete ablation from adrenocortical steroidogenic cells is observed in G2 Ad-ARKOs /corticosterone (rodents) that are integral in healthy pregnancy for the mother and offspring . DespiteIn the adrenal, spindle cell hyperplasia is not uncommon in ageing mice and has been noted more commonly in females; however, the development of these cells in young mice is rare , 27. Con). Immunostaining and cell counts reveal significantly fewer proliferating cells in G1 and G2 Ad-ARKOs compared to controls . Furthermore, in both G2 and G2 Ad-ARKOs, ZG cells no longer stain positive for steroid marker, 3β-HSD  held by LBS.A-LG, LO, and LBS conceived and designed the study. A-LG, LO, SS, LM, and MC. carried out the experiments. A-LG, LO, IJM, RM, and LBS analyzed the results. AJ and HF provided novel resources. A-LG and LBS wrote the paper. All authors contributed to the article and approved the submitted version.This work was funded by a Medical Research Programme Grant Award (MR/N002970/1) (to LBS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It remains unclear why chemotherapy is often ineffective in patients with bladder cancer. Meanwhile, we previously reported that male sex hormones  could considerably reduce the efficacy of cisplatin, an anti-cancer drug used as the first-line treatment against advanced bladder cancer. The present study aimed to investigate how androgen receptor signaling, which is activated by binding of androgenic hormones, modulates sensitivity to cisplatin treatment in bladder cancer, using cell line models and surgical specimens. We found that the expression levels of the androgen receptor and a molecule (BXDC2) were inversely correlated and that loss of BXDC2 was associated with cisplatin resistance. We thus provide evidence to suggest an underlying molecular mechanism responsible for androgen receptor-induced chemoresistance in bladder cancer.p = 0.083) or together with AR negativity (p = 0.047) was associated with favorable response. We identified BXDC2 as a key molecule in enhancing cisplatin sensitivity. AR-ERK activation may thus be associated with chemoresistance via downregulating BXDC2 expression in bladder cancer.Underlying mechanisms for resistance to cisplatin-based chemotherapy in bladder cancer patients are largely unknown, although androgen receptor (AR) activity, as well as extracellular signal-regulated kinase (ERK) signaling, has been indicated to correlate with chemosensitivity. We also previously showed ERK activation by androgen treatment in AR-positive bladder cancer cells. Because our DNA microarray analysis in control vs. AR-knockdown bladder cancer lines identified BXDC2 as a potential downstream target of AR, we herein assessed its functional role in cisplatin sensitivity, using bladder cancer lines and surgical specimens. BXDC2 protein expression was considerably downregulated in AR-positive or cisplatin-resistant cells. BXDC2-knockdown sublines were significantly more resistant to cisplatin, compared with respective controls. Without cisplatin treatment, BXDC2-knockdown resulted in significant increases/decreases in cell proliferation/apoptosis, respectively. An ERK activator was also found to reduce BXDC2 expression. Immunohistochemistry showed downregulation of BXDC2 expression in tumor (vs. non-neoplastic urothelium), higher grade/stage tumor (vs. lower grade/stage), and AR-positive tumor (vs. AR-negative). Patients with BXDC2-positive/AR-negative muscle-invasive bladder cancer had a significantly lower risk of disease-specific mortality, compared to those with a BXDC2-negative/AR-positive tumor. Additionally, in those undergoing cisplatin-based chemotherapy, BXDC2 positivity alone ( Urinary bladder cancer, mostly urothelial carcinoma, has been one of the most commonly diagnosed malignancies, especially in men ,2. In adWhile several immune checkpoint inhibitors have recently been approved for clinical use, cisplatin (CDDP)-based combination chemotherapy, such as “MVAC” (methotrexate (MTX)/vinblastine (VBL)/doxorubicin /CDDP), “GC” (gemcitabine (GEM)/CDDP), and “CMV” (CDDP/MTX/VBL), remains the mainstay of the treatment of locally advanced or metastatic urothelial carcinoma in a neoadjuvant or adjuvant setting ,6. NonetThe mechanisms for CDDP resistance remain poorly understood, although multiple molecular pathways, including extracellular signal-regulated kinases (ERKs) and PTEN, have been suggested to involve the modulation of the cytotoxic and anti-proliferative potential of CDDP ,8. We haBXDC2 whose expression was significantly upregulated in an AR-knockdown bladder cancer subline, compared with AR-positive control cells [Our recent DNA microarray analysis identified candidate genes, including ol cells . To furtWe had recently employed DNA microarray analysis in control AR-positive UMUC3 versus a UMUC3 subline stably expressing AR-short hairpin RNA (shRNA) . Of thosWe first examined the expression of BXDC2 in 4 human bladder cancer lines that were known to be AR-negative  or AR-positive  . WesternWe next assessed the impact of BXDC2 expression on sensitivity to CDDP treatment in bladder cancer cells. Western blotting confirmed significant downregulation of BXDC2 expression in CDDP-resistant cells, compared with control cells A. To comTo see if BXDC2 could modulate the cytotoxic effects of other chemotherapeutic drugs used for the treatment of bladder cancer, the cell viability in the presence of various concentrations of MTX, VBL, DOX, or GEN was compared in the control-shRNA vs. BXDC2-shRNA sublines. However, there were no significant differences in sensitivity to these anti-cancer agents between the control and knockdown sublines derived from 5637 A or UMUCUsing control-shRNA vs. BXDC2-shRNA sublines, we assessed the role of BXDC2 in the cell proliferation (via MTT assay A), apoptAs aforementioned, AR activation has been implicated in CDDP resistance in bladder cancer cells . AndrogeWe stained immunohistochemically for BXDC2 in two separate sets of bladder tissue microarray (TMA). Positive signals were detected predominantly in the cytoplasm of non-neoplastic and neoplastic epithelial cells .p = 0.007)  of non-neoplastic and 99 (77%) of neoplastic specimens . Thus, t007) see .p = 0.074) or progression (p = 0.056)  or progression-free survival (p = 0.406) in patients with non-muscle-invasive disease between BXDC2-negative/AR-positive versus BXDC2-positive/AR-negative tumors. However, patients with BXDC2-negative/AR-positive muscle-invasive tumor had an insignificantly (p = 0.070) or significantly (p = 0.041) higher risk of disease progression  . In patigression D or cancgression E, respecp = 0.083). Additionally, when the status of AR expression stained in our previous study [p = 0.047; In another set of TMA consisting of muscle-invasive bladder cancer specimens from those who had subsequently undergone CDDP-based neoadjuvant chemotherapy, BXDC2 was positive in 26 (60%) of 43 cases. These BXDC2-positive cases included 13 (76%) responders and 13 (50%) non-responders to the neoadjuvant chemotherapy . Thus, lus study  was consAlthough resistance to CDDP-based chemotherapy is commonly seen in patients with urothelial cancer, its underlying mechanisms remain to be established. Meanwhile, AR activation in bladder cancer cells has been suggested to induce chemoresistance ,17,18. IBRIX1 gene in myocardial cells [BXDC2 gene in immortalized lung fibroblasts, lung cancer cell lines, and non-small cell lung cancer tissues [BXDC2, also named BRIX1, belongs to the Imp4 superfamily and is known to play an important role in ribosome biogenesis via involving, for instance, RNA processing, subunit assembly, and export to the cytoplasm ,20,21,22al cells . Moreoveal cells . By cont tissues , suggestAgain, both AR and ERK signals have been associated with CDDP resistance ,8,9. OurIn addition to chemoresistance, AR signaling in urothelial cancer cells has been implicated in urothelial tumorigenesis and tumor progression , the for® shRNA Bacterial Glycerol Stock, TRCN0000122361, Millipore Sigma) was stably expressed in 5637 and UMUC3 lines. In addition, a 647V subline resistant to CDDP (or a control cultured for the same period without CDDP) was established by ≥12-week culture with low/increasing doses of CDDP in our previous study [Human urothelial carcinoma cell lines, 5637, 647V, UMUC3, and TCCSUP, were originally obtained from the American Type Culture Collection and recently authenticated by the institutional core facility. Sublines stably expressing human wild-type AR (or vector only) or AR-shRNA (or control-shRNA) were established in our previous studies , anti-BXDC2 (C-1), and anti-GAPDH (6c5) antibodies, and anti-p-ATF2 (Thr71), anti-p-p44/42 MAPK (ERK) (Thr202/Tyr204), anti-PTEN (D4.3), and anti-cleaved caspase-3 (Asp175) antibodies from Santa Cruz Biotechnology and Cell Signaling Technology, respectively. CDDP, DHT, and HF were from Sigma-Aldrich, while MTX, VBL, DOX, GEM, C6 ceramide, and SCH 772984 were from Cayman Chemical.Equal amounts of proteins (30 µg) obtained from cell extracts were subjected to electrophoresis with 10% sodium dodecyl sulfate-polyacrylamide gel, which was transferred to polyvinylidene difluoride membrane electronically. After blocking with 0.03–0.3% Blotting-Grade Blocker (BioRad), the membrane was incubated with a primary antibody at 4 °C overnight, followed by 1-h incubation with an HRP-conjugated secondary antibody  at room temperature. Chemiluminescent signals were generated by Clarity western ECL substrate and detected by ChemiDOC™ MP (Bio-Rad).3/well) seeded in 96-well tissue culture plates were cultured for up to 144 h and then incubated with 0.5 mg/mL of MTT (Sigma-Aldrich) for 3 h at 37 °C. MTT was dissolved by dimethyl sulfoxide, and the absorbance at 570 nm was measured.We used the MTT assay to assess cell viability. Cells , followed by counterstaining for DNA with 4′,6-diamidino-2-phenylindole (DAPI). Apoptotic index was determined in the cells visualized by the fluorescence microscopy. For cell cycle phase quantification, the Cell Cycle Assay Cell-Clock™ (Biocolor) was used according to the manufacturer’s protocol. Data were analyzed, using ImageJ software .Scratch wound-healing assay was adapted to evaluate the ability of cell migration. Cells at a density of ≥90% confluence in 12-well tissue culture plates were scratched manually with a sterile 200 μL plastic pipette tip. The wounded monolayers of the cells were allowed to heal in serum-free medium for 24 h, and the width of the wound area was monitored with an inverted microscope. The normalized cell-free area in photographed pictures (24 h/0 h) was quantitated, using the ImageJ.Two sets of TMA consisting of retrieved bladder tissue specimens obtained by transurethral surgery performed at the Johns Hopkins Hospital were constructed previously ,16. NoneImmunohistochemical staining was performed on the 5-µm sections, using a primary antibody to BXDC2, as we described previously ,9,15. Alt-test was used to compare numerical data. Survival rates in patients were calculated by the Kaplan–Meier method and comparison was made by log-rank test. p < 0.05 was considered to be statistically significant.Fisher’s exact test or the Chi-square test was used to evaluate the associations between categorized variables. Student’s We identified BXDC2 as a key downstream effector of AR in modulating CDDP sensitivity in bladder cancer. Specifically, AR-ERK activation was suggested to induce chemoresistance via downregulating BXDC2 expression. While BXDC2 activators are not currently available, our data further support that concurrent anti-androgen therapy has the potential of being a means of chemosensitization, especially in male patients with AR-positive bladder tumor. In addition, because BXDC2-negative/AR-positive tumors were strongly associated with resistance to CDDP-based chemotherapy, the expression status of BXDC2, along with that of AR, might serve as a predictor of chemosensitivity in patients with bladder cancer. Meanwhile, further investigation of BXDC2 activity in animal models for bladder cancer is required to address the feasibility of future therapeutic intervention.


Humans are uniquely cooperative and form crucial short- and long-term social bonds between individuals that ultimately shape human societies. The need for such intense cooperation may have provided a particularly powerful selection pressure on the emotional and communicative behaviours regulating cooperative processes, such as guilt. Guilt is a social, other-oriented moral emotion that promotes relationship repair and pro-sociality. For example, people can be more lenient towards wrongdoers who display guilt than towards those who do not. Here, we examined the social consequences of guilt in a novel experimental setting with pairs of friends differing in relationship quality. Pairs of participants took part in a cooperative game with a mutual goal. We then induced guilt in one of the participants and informed the other participant of their partner's wrongdoing. We examined the outcome using a dictator game to see how they split a joint reward. We found that guilty people were motivated to repair wrongdoing regardless of friendship. Observing guilt in others led to a punishment effect and a victim of wrongdoing punished close friends who appeared guilty more so than acquaintances. We suggest, therefore, that guilt has a stronger function between close friends as the costs of relationship breakdown are greater. Relationship context, therefore, is crucial to the functional relevance of moral emotions. EvidCooperation is a widespread strategy in humans, but some cultural differences have previously been identified –29. In c1.2.In an experimental study, pairs of friends and pairs of strangers took part in a cooperative task. We then artificially induced guilt in one participant by attributing failure in the task to this person, and informed the other participant of their partner's wrongdoing. We examined the influence of friendship, facial expression and culture on the participants' responses using a dictator game . We deci2.2.1.The sample size for this study was based on previous research looking at the induction of guilt. Moreover, due to financial limitation, and to keep the design as simple as possible, we did not include a ‘no guilt’ condition for Player 1. This allowed us to minimize the number of variables, already extensive, and reduce the number of participants needed. Therefore, in order to capture as many facial expressions as we could in a culturally varied sample, we aimed to have 50–60 participants posing as Player 1 in each condition (two conditions for Player 1: ‘blame’ versus ‘no blame’). As a result, we aimed to collect data from 110 pairs of participants.Two hundred and eighteen participants were recruited as pairs . Participants were given the opportunity to either sign up with one of their friends or sign up alone and be randomly paired up with a stranger . Participants had various ethnicities and nationalities, constituting a sample made of individuals with various places of origin (PoO) . The or2.2.Stage 1, participants were separated to complete questionnaires . Due to the nature of the task, the attribution of responsibility remained largely ambiguous throughout and participants were not able to determine whose fault it was as the game went on. On completion of the task, the participants were separated again for Stage 3, where they completed more questionnaires  ; t106 = −5.62, p < 0.001) and shame feelings . Players reported a significantly higher level of guilt than shame after the induction task , suggesting that guilt was the primary induced emotion .After receiving the feedback in Stage 4, Player 1 experienced an increase in guilty feelings  based on the control condition and comparing those with the observed distribution of action units in the guilt condition, revealed that participants in the guilt condition exhibited facial muscle activation that was significantly different from the control condition. Electronic supplementary material, table S5 presents the summary of the comparison for the entire guilt dataset. In the upper face, AU4 was more active in the guilt condition more than in the control videos. In the lower face, AU10, AU12 and AU20 were active more often than would have been predicted based on the control videos. Most striking was the difference in the likelihoods of participants to touch their face, hair, ear, scratch or laugh in the guilt videos. Participants in the guilt videos were significantly less likely to show activation of AU24.3.1.1.2.In the direct comparison between the two groups , individuals who reported strong feelings of guilt were more likely than expected to touch their ear and laugh (corroborating the results of comparing guilt videos with the control videos). AU1, AU2, AU4, AU14 and AU24 were significantly less likely to occur in participants who reported strong guilt.3.1.2.M = 5.97, s.d. = 1.88) and giving nine coins to their partner . Player 1 gave more money to their partner the guiltier they felt . There was no effect of friendship on reward split  and whether Player 1 was blamed or not for his/her performance did not influence reward split .On the whole Player 1 gave a bigger share to Player 2, keeping an average of six coins (out of 15) for themselves . They also showed less activity of AU1, AU2, AU7 and AU20.Guilt videos. The guilt videos that were judged as presenting guilt differ significantly from other guilt videos by showing more activity in AU18, AU24 and ear touching . They also show less activity of AU10, AU12, AU14 and face touching.β = −0.13, s.e. = 0.083, p = 0.123; PoO β = −0.062, s.e. = 0.079, p = 0.435; friendship index β = 0.032, s.e. = 0.076, p = 0.673).We investigated which other factors influenced the judgement of guilt by Player 2: none of the investigated variables affected judged guilt  and giving eight coins to their partner . However, this does mean that Player 2 changed the original split presented to them, from 10 coins for their partner to 8 on average . In relationships with a strong friendship index , Player 2 was more likely to change the original split  when they judged their friends as seeming guilty . This interaction did not hold with the removal of these data points; either potentially due to extreme values driving the model, or, due to the reduction in sample size. We, therefore, want to highlight that this finding should be approached with caution.On the whole, Player 2 did not favour themselves over their partner in the reward split, keeping seven coins (out of 15) on average for themselves . Similarly, players punished partners more when they looked guilty, and this effect was modulated by friendship: players punished their close friends more when they were perceived as feeling guilty.When told they performed poorly, participants gave more money to their partners the guiltier they felt, regardless of their friendship, which is consistent with previous findings highlighting the positive social consequences associated with feelings of guilt –19,21. TWhen presented with an unfair reward split, the victim changed this unequal split to a fairer split  and did so more when the wrongdoer was both a close friend and guilty. This resulted in an almost 50–50 split of the shared reward, which is atypical of the dictator game. However, we need to acknowledge the various differences between a ‘typical’ dictator game and the version used here. In most studies using a dictator game, one participant needs to divide a set reward between two people (either themselves and another player or two distinct people ). In ourOverall, this is in contrast with previous studies showing people being more lenient towards people expressing guilt ,50,51. OThe punishment function of guilt was further enhanced when victims had a strong friendship with the wrongdoer suggesting that the victim punished their guilty friends more. This result could be due to at least two reasons. First, Player 2 might have acted differently when facing someone they perceived as guilty when that someone was a close friend compared with when it was an acquaintance or a stranger. Second, Player 2 might have perceived their close friends as expressing more guilt than an acquaintance or a stranger, so the nature of their relationship might have impacted Player 2's perception of Player 1's emotional state. People sharing a strong friendship bond have invested a lot of time and resources into the relationship; any disruption to the relationship ought to be repaired in order to save and restore the relationship , regardlWe used a bottom-up coding scheme to identify facial patterns associated with the experience (production and perception) of guilt. We found that players informed of their poor performance and experiencing guilt (Player 1) displayed more frown (AU4 brow lowerer), upper lip movement (AU10 upper lip raiser), smile (AU12 lip corner puller) and lip stretch . They aIt is important to remain cautious when interpreting these new findings. First, even though both participants reported variation in the amount of guilt they felt throughout the experiment, the reported values remained under the scale midpoint . This experiment was designed to induce ethically appropriate levels of guilt and mimic real-life situations. Our goal was, therefore, to induce mild levels of guilt and mostly to observe variations in the levels of guilt throughout the experiment. Second, caution should always be used when interpreting self-report measures; it is possible participants down-played how much guilt they were feelings or were not able to identify the feelings they experienced as the study went on. Third, the experimental induction of guilt might have had different outcomes based on participants' propensity to believe what they were told. If both players were aware of the number of times the marble fell off the platform, some Player 1s might not have believed that they were more to blame for those falls. Fourth, another confounding factor that could explain Player 2's decision is having just taken part in a cooperative task with a partner; the face might not have influenced Player 2's final decision even when Player 2 reported that Player 1 ‘looked’ more guilty (Stage 4—Player 2). It would be interesting to take this into account in a follow-up study, designing a task where participants are introduced but do not need to cooperate, as well as introducing a solitary task in order to disentangle possible confound factors present in our experiment.5.This is the first study examining the impact of friendship on the consequences of guilt in a social interaction between participants. We examined genuine social interactions between dyads differing in relationship quality and tested the impact of guilt within the interaction. In line with previous research, we demonstrated the pro-social role of guilt: guilty players gave more money to their partners. We might have also revealed a new finding demonstrating a punishment effect of guilt, and a modulating effect of friendship on victims' behaviours: victims of a wrongdoing punished their close guilty friends more than less close guilty friends. It could, therefore, seem costly for the wrongdoer to communicate guilt; however, as punishment after wrongdoing has been demonstrated to benefit the relationship in the long term ,13, the


The latter were followed longitudinally six and 12 months after rejection. We found higher levels of circulating LNC-EPHA6 during rejection, compared with renal recipients with a stable kidney function (p = 0.017), that normalized one year after AR. In addition, LNC-RPS24, LNC-EPHA6, and LIPCAR levels correlated significantly with the vascular injury marker soluble thrombomodulin. We conclude that AR and microvascular injury are associated with higher levels of circulating LNC-EPHA6, which emphasizes the potential role of lncRNAs as biomarker in the context of AR.Acute rejection (AR) of a kidney graft in renal transplant recipients is associated with microvascular injury in graft dysfunction and, ultimately, graft failure. Circulating long noncoding RNAs (lncRNAs) may be suitable markers for vascular injury in the context of AR. Here, we first investigated the effect of AR after kidney transplantation on local vascular integrity and demonstrated that the capillary density markedly decreased in AR kidney biopsies compared to pre-transplant biopsies. Subsequently, we assessed the circulating levels of four lncRNAs , that were previously demonstrated to associate with vascular injury in a cohort of kidney recipients with a stable kidney transplant function ( Acute rejection (AR) is considered to be a prominent cause of graft failure in the first year after transplantation in kidney transplant recipients ,2, althon = 102 and n = 29). Subsequently, we compared these parameters to the available pre-transplant biopsies (resp. n = 78 and n = 66) of these patients [p < 0.0001) as well as pericytes  in AR, indicating loss of the peritubular capillary network in AR.To assess the impact of AR on the local capillary density in the kidney, we quantified the number of endothelial cells (EC) and pericytes in archival acute rejection biopsies by immunohistochemical staining of the EC for CD34 antigen and the pericytes for the CD73 marker (resp. patients . Patientp < 0.001 and p = 0.003). Factors that can influence the amount of vascular injury next to rejection, such as donor age, dialysis before transplantation, and months since transplantation, did not differ significantly. Incidence of active smokers was 7% in AR patients and 13% in stable patients. Panel reactive antibodies (PRA), mismatch, immunosuppressive regimen and the presence of previous transplantations did not differ between stable patients and patients with AR. Patients with AR had interstitial rejection, with or without involvement of the vasculature, and were treated with methylprednisolone (67%), ATG alone (13%), or a combination of methylprednisolone and ATG (13%) or alemtuzumab (13%).Next, we sought to investigate the relation of circulating lncRNAs with AR. To that end, we included plasma samples of a different cross-sectional study cohort that included patients with acute T cell mediated rejection and a control group of patients with stable kidney transplant function after transplantation (hereafter mentioned as ‘stable’). In addition, AR patients were studied longitudinally at 6 and 12 months after rejection to determine the dynamics after AR. The baseline characteristics of the transplant recipients in this cohort are described in p = 0.017; p = 0.11 and p = 0.16).In order to assess the relationship between AR and vascular injury related lncRNAs LNC-RPS24, MALAT1, LNC-EPHA6, and LIPCAR, circulating levels of these lncRNAs were measured in stable patients and AR patients. In this cohort, MALAT1 levels were only detectable in less than 30% of patients and therefore not included in further analyses. Relative expression of circulating LNC-EPHA6 was significantly higher in patients with AR, compared with stable patients (p < 0.001) and decreased significantly one year after rejection, although LNC-EPHA6 levels at one year after rejection remained slightly higher levels than in stable patients , while LNC-RPS24 increased one year after transplantation vascular damage in the context of rejection and emphasizes the potential role of lncRNAs as biomarkers to monitor vascular injury in kidney transplant rejection.Renal biopsies were selected from patients that had a biopsy proven acute renal allograft rejection, as previously described . Patientn = 15) and a control group consisting of renal recipients 12 months after transplantation without rejection and with a stable kidney transplant function (n = 32). In addition, recipients from the rejection group were followed longitudinally. Plasma samples were obtained at 6 and 12 months after rejection.A total of 47 patients were enrolled in a cross-sectional, observational, single center study. All patients were transplanted between 2006 and 2012 in the Leiden University Medical Center (LUMC) in Leiden, The Netherlands. The cohort consisted of 2 groups, namely renal transplant recipients with AR .All patients received immunosuppressive drug therapy according to the standard of care at the time of transplantation. IL-2 receptor inhibitor as induction therapy was the standard of care and alemtuzumab was administered in case the treating physician expected a higher risk of rejection. The presence and type of rejection was assessed using the Banff classification. The choice for a specific rejection treatment was made according to the standard of care at the time of rejection .The RNeasy Micro Kit  was used with an adapted protocol, to isolate total RNA from 200 μL plasma. In summary, using 800 trizol μL reagent , the plasma/Trizol sample was centrifuged for 15 min  after the addition of 160 μL chloroform. Then, 100% ethanol (1.5 volume) was added to the aqueous phase and transferred to a MinElute Spin column (Qiagen) followed by centrifugation for 15 s . Subsequently, 700 μL RWT buffer and twice 500 μL RPE buffer was used to wash the column. The column was centrifuged  for 15 s after the first two washing steps and 2 min  after the third washing step. 15 μL RNase-free water was added for elution of the RNA.To quantify circulating lncRNA levels we performed RT-qPCR. Isolated RNA was reverse transcribed using Iscript (Biorad) according to the protocol of the manufacturer. RT-qPCR of target genes was done using SYBR Green Master Mix . The primer sequences of target lncRNAs are given in t test and Mann–Whitney U test for parametric and non-parametric data.Categorical data are described as total count and percentages, parametric data as mean ±standard deviation (SD), and non-parametric data as median and interquartile range (IQR). Testing for differences of baseline characteristics was performed by using Fishers exact test for categorical data and the unpaired Circulating lncRNA levels were normalized by the double delta CT method to miR-16 and subsequently transformed logarithmically (with base 10). The logarithmic relative expression of all three lncRNAs was normally distributed. In the longitudinal study the data was analyzed by using a linear mixed model analysis. Analysis of correlations between the lncRNAs and vascular markers was performed using Spearman rank correlation.p-value < 0.05 was considered to be statistically significant. SPSS version 23.0  was used for the data analysis and Graphpad Prism version 8.0 .A


Abnormal activity of oncogenic and tumor-suppressor signaling pathways contributes to cancer and cancer risk in humans. Transcriptional dysregulation of these pathways is commonly associated with tumorigenesis and the development of cancer. Genetic and epigenetic alterations may mediate dysregulated transcriptional activity. One of the most important epigenetic alternations is the non-coding regulatory element, which includes both enhancers and super-enhancers (SEs). SEs, characterized as large clusters of enhancers with aberrant high levels of transcription factor binding, have been considered as key drivers of gene expression in controlling and maintaining cancer cell identity. In cancer cells, oncogenes acquire SEs and the cancer phenotype relies on these abnormal transcription programs driven by SEs, which leads to cancer cells often becoming addicted to the SEs-related transcription programs, including prostate cancer. Here, we summarize recent findings of SEs and SEs-related gene regulation in prostate cancer and review the potential pharmacological inhibitors in basic research and clinical trials. Prostate cancer (PCa) is one of the leading causes of cancer-related deaths in men worldwide3. Although clinically beneficial treatment options for localized PCa include surgery, radiotherapy, and androgen ablation therapy, there is basically no cure for metastatic castration-resistant PCa (CRPC)6. Therefore, it is most necessary than ever to further understand the crucial regulators within the PCa and develop new therapies7.The hallmarks of cancer, including sustaining proliferation, activating invasion, metastasis, and aberrant replicative immortality, are closely connected to cancer-specific regulatory mechanisms of gene expression8. It increases the promoter activity to activate target genes through specific transcription factors (TF)9. For cancer-type-specific gene expression, enhancer closely interact with the promoter, over either short or long distances, independent of the corresponding orientation and position concerning the transcription start sites (TSS)11. Enhancers often contain conserved recognition DNA sites for RNA polymerase, TFs, and co-activators and function as binding platforms12. Accumulated evidence reveals that enhancers are bound by epigenetic modifications, such as mono-methylation at H3 lysine 4 (H3K4me1) and acetylation at H3 lysine 27(H3K27ac), generally14. In addition, the three-dimensional structure of enhancers to TSS affects the interaction activity and expression output, with the distance varying from less than 10 Kb to more than 1 Mb16.The enhancer is a class of regulatory DNA sequence that defines the genetic regulatory circuitry4. Similar to the typic enhancer, SE incorporates modelized regulation mechanisms. Specific TFs bind to SE to trigger promoter-enhancer interaction, mediated by chromatin looping, to load the SE to the cognate promoter. Then the basal machinery is recruited to initiate the downstream transcription activity  algorithm is designed to search SEs by locating genomic proximity for grouping elements to a putative target gene18. Generally accepted models of SEs are considered to be large clusters of regulatory elements (after over 20 Kb) binding with dense transcriptional coactivators, such as BRD4 and CDK7, and with high potential to activate target gene expression output19  is defined as large clusters of enhancers spanning across a long-range region of genomic DNA that drives stronger transcriptional activation ability than individual enhancers25. In essence, the SE can be described as a co-assembly of high-density transcription factors, co-factors, chromatin regulators, non-coding RNA, and RNA polymerases27. Computer simulations and experimental validations indicate that, in the context of the number and valence of interacting components and the affinity of the interaction between transcription factors and nucleic acids, phase separations play essential roles in the SE assembly and function28. Inhibition of the activity of SE function highlights sensitivity and vulnerability in cancers along with cancer-specific oncogene downregulation, which could be a possible therapeutic target for cancer treatment29. SE complexes are dynamic, highly enriched condensates, particularly sensitive to SE-related inhibition28. The essential components of SE are dysregulated in PCa, such as increased MED1 phosphorylation at T1457 in a CDK7-dependent manner in metastatic CRPC and enzalutamide-resistant cells. Bromodomain-containing proteins (BRDs) are overexpressed in CRPC and show particular prognostic value in PCa.The most cutting-edge research has revealed that membrane-less organelles, such as the nucleolus, stress granule, processing body, and nuclear speckles form subcellular compartments to facilitate signaling transduction and transcriptional regulation by liquid–liquid phase separationGiven the cancer-specific SE complexes in PCa, we summarize the critical transcriptional regulation and therapeutic targets of the SEs and SE-associated regulator proteins and provide promising future directions for SEs in the prostate cancer community . BRD4, as a chromatin reader, recognizes acetylated histones and facilitates the transcription activation by propelling the recruitment of the positive transcription elongation factor P-TEFB31. In particular, BRD4 shows a density binding activity in SE and drives the cell-identical gene expression32. In PCa, AR-positive or AR-signaling-component cell lines  are selectively sensitive to BRD4 inhibition, but not in AR-negative cell lines (PC3 and DU145)34. BRD4 has been found that sequence-specific DNA-binding TFs may physically interact with it in a gene-specific manner, such as AR35. Mechanically, BRD4 physically interacts with the N-terminal domain of AR, and BRD4 colocalizes with AR at AR target loci to drive AR-mediated gene transcription. For the BMPR1B gene, AR-and BRD4-associated binding signals in the enhancers and SEs are significantly induced by dihydrotestosterone (DHT) treatment. The expression levels of BMPR1B are corroborating with the ChIP-seq data. Moreover, almost half of the known BRD-containing proteins are related to PCa, which also contribute the main chromatin-related processes and changes in PCa, beyond BRD436. These BRD-containing proteins  perform a wide range of downstream functions by recognizing acetylated histones, also present to be TFs, AR co-activators or methyltransferases39. Therefore, BRD-containing proteins are important transcriptional regulators that initiate chromatin restructuring beyond the SE function.The bromodomain and extra-terminal domain (BET) family proteins act as “readers” of acetylated histones, and are important transcriptional regulators. BRD2, BRD3, BRD4, and BRDT (bromodomain testis-specific protein) are part of the BET family40. On one hand, BRD4, by functioning downstream of AR signaling, appears to be effectively blocking the oncogenic drivers of PCa and less likely to be bypassed by acquired treatment resistance of AR therapy. BRD4 inhibition preferentially blocks both BRD4 and AR recruitment (the BRD4 and AR cistrome) to target loci on a genome-wide scale and leads to defects in transcriptional elongation. The acquisition of BRD4-associated SEs leads to prompt expression of key oncogenic genes, including TMPRSS2-ETS, KLK3, and BMPR1B in PCa, especially CRPC. Urbanucci et al. demonstrated that the transcription of certain BET proteins (BRD2 and BRD4), and ATAD2, a recently reported common coactivator of AR, may be induced by androgen41. The upregulated AR leads to a positive loop that boosts the expression of BRDs to expand the AR cistrome by increasing chromatin accessibility. On the other hand, BET inhibitors can also operate via mechanisms other than AR signaling. Shah et al. demonstrated that BET inhibitors resensitized drug-resistant tumors to enzalutamide by inhibiting the glucocorticoid receptor42. Cai et al. showed that the BRD4 inhibitor, JQ1, significantly inhibited androgen-independent growth of CRPC cells in vitro and in vivo by blocking AR-V7 chromatin binding and transcriptional programs co-activated by AR-V7 and ZFX in addition to the canonical AR signaling43.AR signaling remains the most common resistance mechanism in most CRPC patients45. CDK7 is a ubiquitously expressed kinase that activates the cell cycle controlling through phosphorylation of CDKs and plays a key role in transcription regulation46. The mRNA and protein expression levels of CDK7 alone showed no significant change in benign cells and PCa cells47. However, CDK7 activity affects chromatin modification by promoting the recruitment of histone methyltransferases SETD1A/B and SETD2, which are phosphorylated by the C-terminal domain49. CDK7 also phosphorylates a range of TFs, including p53 and nuclear hormone receptors , to promote the transcription activation activity and subsequent protein-degradation53. Studies show that CDK7 inhibition can transiently block the functional activity and expression of the oncogenic drivers  in prostate cancer, and all these changes require the involvement of MED1 as a cofactor56. CDK7 activates MED1 via ligand-specific phosphorylation, and the IDRs  of MED1 form phase-separation properties to be recruited to the AR-bound SEs, resulting in the high-density assembly of the SE-related transcription apparatus. CDK7 inhibition has been effectively tested in several aggressive cancer types, including MYCN-amplified neuroblastoma, T-cell acute lymphoblastic leukemia, triple-negative breast cancer, and small-cell lung cancer60. The CDK7-related SEs mediate the recruitment of AR and RNA polymerase II to boost the expression of a host of target genes, known as the “Achilles cluster” genes, thus becoming transcriptional addictive and sensitive to CDK7 inhibition50.The mammalian cyclin-dependent kinases (CDKs) contain subfamilies with specific functions related to cell cycle  and transcriptional regulation 61. ERG protein is the master transcription factor for endothelial, hematopoietic, and luminal cell differentiation65. It interacts with other transcription factors by forming complexes to establish the cell-type-specific patterns. In PCa, ERG expression and rearrangement is per se not a strong prognostic biomarker and only relevant in the context of a specific molecular subtype68. Gerke et al. found that it was crucial to determine the prognostic value with other biomarkers, such as RRM2 and TYMS, when applying the ERG status to predict the outcomes69. ChIP-seq data show ERG binds to the vast majority of SEs in VCaP (a TMPRSS2-EGR fusion-positive cell line) cell65. The SE-associated lineage-specific machinery of ERG is linked with the TFs like FOXA1 and HOXB13, which play important roles in prostate-cancer-specific gene expression. Mechanically, ERG increases the SEs activity partially through the BRG1-associated chromatin remodeling complex to establish accessible chromatin of SEs and transcriptional regulation. In summary, ERG drives the prostate-cancer-specific lineage genes by regulating SEs. TMPRSS2-ERG structural rearrangements occur in close to 50% of PCa patients and contribute to the ERG overexpression70. The proposed model of ERG overexpression was previously thought to be driven by TMPRSS2 promoter hijacking. Ken et al. showed that TMPRSS2, along with the rearranged ERG allele, formed an expanded SE71. The expanded SE still contains cis-regulatory elements and extends into the ERG locus, which promotes ERG overexpression. This work firstly confirms that the expansion of the SE region after chromosomal rearrangements could positively drive the target gene expression. As for ERG, it synergistically regulates by physically interacting with prostate-cancer-specific regulators AR, HOXB13, and FOXA1. The regulatory landscape difference between TMPRSS2-ERG fusion and non-fusion PCa types may depend on the SE-related ERG-specific transcriptional profile, including activated NOTCH pathway. In light of the present findings, we conclude that ERG contributes to the SE-driven oncogenic transcriptional addiction, and that SEs lead to the overexpression of the ERG gene, leading to subsequent overexpression of ERG-target genes that drive the development of PCa.The erythroblast transformation-specific (ETS) family proteins are the essential transcription factors necessary for cell-type-specific lineage differentiation and expression patterns74. Chen et al. demonstrated that high enrichment of PCa-specific risk variants in SE regions, particularly in the disease-specific regulatory regions and the DNA regulatory elements, may lead to prostate carcinogenesis75. O-GlcNAc transferase (OGT), a glycosyltransferase, catalyzes the addition of a single O-GlcNAc sugar to serine and threonine residues76. OGT, as a major metabolic integration point in human cells, is also involved in the hexosamine biosynthetic pathway (HBP) to increase O-GlcNAcylation-modified nuclear proteins77. RNA polymerase II is reported to be the most prominent O-GlcNAcylation-modified protein that regulates the formation of the transcriptional pre-initiation complex formation. Also, increased OGT expression has been found in many cancers, including prostate cancer, where high O-GlcNAc protein levels are associated with poor clinical prognosis. Harri et al. showed that OGT regulated SE-dependent transcription through chromatin compaction78. Over 70% of the SE-related genes are related to the OGT chromatin mark, and the OGT inhibition significantly decreases the SE-related mRNA expression. The activity of OGT is required for the expression of MYC-target mitotic proteins. Thus, OGT could be an effective target for MYC-addicted PCa.Abundant evidence shows that the transcriptional regulatory regions of SEs are practically enriched with cancer-related single nucleotide polymorphisms (SNPs), resulting in dysregulation of target genes and contribution to cancer development79. The androgen-regulated SE-target genes are associated with cell-proliferation-associated gene sets, stem cell properties, and hallmark AR signaling, such as KLK2, KLK3, TMPRRSS2, and FKBP5. In addition, abundant evidence shows that AR also tightly cross-talks with other factors80. In low androgen environments or AR-overexpression status, AR promotes the expression of some SE-complex-associated proteins, such as BRD441. AR closely interacts with the BRDs in the N-terminal domain (NTD) instead of the ligand-binding domain (LBD), since the NTD was essential for the transcriptional activity of AR81. Alternative splicing variants of the AR that lack LBD are overexpressed in patients who are resistant to enzalutamide or the androgen synthesis inhibitor abiraterone acetate82. For AR mutants (full-length AR with point-mutated forms and AR with lack of LBD splice variants and nonsense mutants), such as AR-V7, AR T878A, and AR H875Y, some BET inhibitors (PFI-1 and BETi) could reduce these mutants’ expression by regulating RNA processing and reducing alternative splicing84. In this regard, we suspect that the formation of AR-bound-SE may be partly affected and the cistrome of AR mutants could redistribute while retaining some binding sites. The current inhibitors may still be effective for AR mutants, but the extent of the effect still needs to be specifically evaluated.AR is a predominant target for PCa, owing to the functional AR signaling in the early-and late-stage PCa. Simon et al. demonstrated that R1881-activated AR bound at a comparable number of SE sites in VCaP cells, and indicated that AR attributed to the SE-associated action85. FOXA1 directly binds to and de-compacts condensed chromatin to increase accessibility of the binding sites for partnering nuclear hormone receptors, including estrogen receptor and AR86. Lupien et el. showed that FOXA1 functionally collaborated with AR and was predominantly recruited to the AR-regulated enhancers and SEs to establish a lineage-specific program in PCa87. FOXA1 plays a key role in prostate tumorigenesis by reprogramming the AR cistrome to new binding sites and driving the transformation of normal prostate epithelial cells88. Besides, FOXA1 also interacts directly with AR89. Considering the AR-bound-SE complexes, the alterations in FOXA1 may impact subsequent effects on the AR cistrome and lineage-specific programs in PCa.The forkhead box A1 (FOXA1) transcription factor plays a pivotal role for the development and differentiation of several endoderm-derived organs, including prostateSEs drive PCa cells into becoming addicted to dysregulated transcriptional programs mediated by BRD4, CDK7, ERG, and other factors, but also become a powerful rationale for therapeutic interventions. Targeting SE may disrupt the dysregulated networks of the oncogenic functions, and some small molecule inhibitors and blockers have been tested to selectively target PCa cells. We summarize these inhibitors and their mechanisms as below and an overview description in Table 90. JQ1 exhibits a high binding affinity to the bromodomain pocket and displaces BRD4 from the active chromatin in most SE sites and is believed to act predominantly on BRD4, BRD2, BRD3, and, BRDT. In acute myeloid leukemia (AML) and myeloma, pre-clinical models and clinical trials already show the BRD4 inhibition induces strong suppression of tumor progression91. In CRPC xenograft mouse models, BRD4 inhibition (JQ1) is more effective than direct AR antagonism 84. This novel approach can be used to synergistically block the oncogenic drivers in advanced PCa for better treatments. BRD4 contains two conserved bromodomains, BD1 and BD2. Dual-bromodomain BET inhibitors are designed to competitively inhibit the binding of the BD1 and BD2, such as OTX015, CPI-0610, and ABBV-07596. However, in some monotherapy clinical trials, dose-limiting adverse events, such as reduced numbers of thrombocytes in the blood and some gastrointestinal toxicity, are limited the clinical activity. Fairre et al. proposed a highly potent and selective inhibitor of the BD2 domain by a medicinal chemistry campaign named ABBV-74497. ABBV-744 selectively maintains high activity in PCa cell lines and xenografts and has a lower toxicity than ABBV-075. Further analyses demonstrate that ABBV-744 displaces BRD4 from AR-bound SE sites and disrupts the AR-target transcriptional programs. The successful development of the preclinical compound JQ1 for BET inhibition has also enabled several compounds of BET inhibitors  to successfully enter the clinical trials.First, BET proteins have been targeted by JQ1 in preclinical models in vitro and in vivo58. The inhibition of CDK7 by THZ1 is related to the global transcriptional downregulation at high dose levels, but studies have found that cancer cell lines are sufficiently sensitive to lower doses of THZ1. Further reports indicate that THZ1 may selectively target SE-driven transcription programs, including MYC-dependent transcription amplification and the expression of other cancer-specific oncogenic TFs and signaling molecules99. Rasool et al. demonstrated that THZ1 attenuated the AR-signaling and maintained efficacy in CRPC and enzalutamide-resistant PCa cells47. Also, CDK7 selective inhibitors have been developed, such as SY1365 and CT7001, and evaluated in clinical trials in other advanced solid tumors101.THZ1, a covalent inhibitor of CDK7, shows the ability to suppress the CDK7-dependent phosphorylation activity to achieve clinical activity102. Another small-molecule inhibitor, YK-4-279, reduces the ERG-positive PCa patient-derived xenograft growth103.ERG overexpression is observed in a large group of primary PCa and CRPC. A highly selective small-molecule inhibitor of ERG, NSC139021, inhibits the growth of ERG-positive cancer cells105. Also, the relevant information will feed to the pharmacologic industry for therapeutic interventions.Advances in SE profiling and chromatin landscape profiling make it possible for identifying essential SE regulators and SE-target genes in cancersSuper-enhancers are a large cluster of active transcriptional enhancers and are rich in enhancer-related chromatin features. Compared to typical enhancers, SEs are larger, exhibit a higher density of TFs, and are often associated with critical lineage-specific genes.107.In PCa, fundamental components of the SE complexes, such as BDR4 and ERG, densely bind to the enhancer and SE elements to promote tumorigenesis and tumor growth. In advanced CRPC patients, the SE activity created by these transcription factors remains stable and evolves to be more specific, which makes it possible that SEs may be promising therapeutic targets21. BRD4 inhibition has been validated and investigated in leukemia, lymphoma, myeloma, neuroblastoma, breast cancer, prostate cancer, and other cancer types. Compared to castration-sensitive status, advanced CRPC patients exhibit an AR deregulation status41. Evidence shows that BRDs  are prognostic markers that are overexpressed in CRPC. AR-deregulation-mediated BRDs upregulation intensifies the SE activity and feeds back a positive loop for AR chromatin binding. In other words, AR deregulation forms a transcription addiction status by enhancing the SE activities. To define the BET inhibition response in CRPC, a ten-gene signature, BROMO-10, was designed and used to guide patient selection for combinatorial trials of the BET inhibition against other agents. Remarkably, the BROMO-10 signature still needs to be refined and evaluated for AR signaling status and BRDs expression as well. Using this method, we can design specific gene signatures to evaluate the efficacy of other inhibitors and screen patients for potential therapeutic benefits. CDK7 inhibition has shown surprising therapeutic effects in preclinical models without evident systemic toxicity, which gives us high hopes for further human clinical trials108. Besides, it has been reported that SEs tend to undergo double-strand breaks and are therefore susceptible to deficiencies in cellular DNA-repair mechanisms. This shows that the combination of endocrine therapy and drugs targeting DNA damage repair will improve the anti-tumor efficiency, and clinical studies have already been conducted. Strikingly, Ma et al. found that SEs reorganization was tightly linked to drug resistance109. In repetitive cisplatin-treated cancer cells, the developmental transcription factor ISL1 invokes an unconventional trans-differentiation identity via SE reorganization to escape the drug-induced near-to-death status and facilitate tumor colonization. This may raise the question of how to effectively target cancer cells by avoiding the acquisition resistance in a SE-reorganization manner. The inhibition of transcription factors, such as SOX10 and ISL1, might potentially blockade the SE reorganization beyond BRDs or CDK7 inhibitors.Several pre-clinical and clinically relevant studies targeting SEs have been successfully carried out in various tumors, including PCa111. PCa cell lines and organoids from individuals with SPOP mutations show therapeutic resistance to cell growth arrest and apoptosis induced by BRD4 inhibitors113. The resistance to BRD4 inhibition in SPOP-mutant PCa can be overcome by combining with AKT inhibitors, and SPOP mutations may be used as biomarkers to guide the choice of treatment options for PCa patients, including those with urgent needs seeking precision medicine, and to determine whether the treatment is valid or not115. Moreover, it is worth noting that targeting SEs for cancer can cause side effects that cannot be ignored, because blocking SEs may also suppress specific SE-dependent tumor suppressor genes that are associated with cancer risk, such as cancer cell death117. Therefore, before using SEs as a therapeutic approach for the treatment of specific cancers, we urgently need to do more detailed research and make effective decisions.However, researchers have discovered that SE-related inhibitors might not have therapeutic effects in certain PCa types


The third isotype of beta-adrenoreceptors (β3-AR) has recently come (back) into focus after the observation of its expression in white and beige human adipocytes and its implication in metabolic regulation. This coincides with the recent development and marketing of agonists at the human receptor with superior specificity. Twenty years ago, however, we and others described the expression of β3-AR in human myocardium and its regulation of contractility and cardiac remodeling. Subsequent work from many laboratories has since expanded the characterization of β3-AR involvement in many aspects of cardiovascular physio(patho)logy, justifying the present effort to update current paradigms under the light of the most recent evidence. Beta3-adrenergic receptors (β3-AR) have traditionally been considered as metabolic receptors in the adipose tissue. After a long period of relative disinterest due to disappointing performance of early agonist drugs, this is now being actively studied again, with new exciting findings in human white or beige adipocytes. In the meantime, the demonstration of β3-AR expression in cardiac myocytes and endothelial cells sparked international efforts from many research groups to decipher the role of this receptor in cardiovascular physiology and pathology. The present review recapitulates current knowledge on the structure, coupling and function of β3-AR in these tissues, with a view to evaluate translational applications in human disease based on the availability of more selective agonists.361/363, shared by the all β-ARs, involved in G-protein coupling and AC activation in β1/β2AR. A recent study further observed that Cys361/363 is involved in β3 receptor-effector coupling (readout by ligand-induced cAMP production) but also in β3-AR abundance and stability, and described new specific human β3-AR S-palmitoylated sites on Cys153 and Cys292, within the second and third intracellular loops, respectively, which may also regulate membrane receptor abundance (assessed with FLAG-β3-AR immunostaining) [The third isoform of human β-AR was identified by cloning in 1989 ,3 and botaining) . Severaltaining) , but nottaining) , reportetaining) . Howevertaining) ,14 (see taining) ,17, suppPrevious reports on expression and function of β3-AR, especially in cardiac tissue, have been controversial owing to the dubious specificity of radioligands and antibodies for protein immunohistological detection (versus β1/β2AR isoforms), and limitations regarding affinity and potency for selective agonists to dissect specific cellular signaling . MoreoveThe human β3-AR was first identified in human cardiac biopsies in 1996 . Under p−/− mice [Functionally, β3-AR were initially shown to have inotropic effects antipathetic to β1/β2AR by decreasing cardiac myocyte contractility through eNOS/cGMP signaling in human myocardial biopsies , as conf−/− mice . Another−/− mice . Convers−/− mice .Such discrepancies point out that precautions should be taken for the comparison and interpretation of results obtained with acute β3-AR stimulation of human tissues, depending on (i) the origin of the tissue sample (ventricle vs. atrium) and its physio-pathological phenotype , (ii) the pharmacology applied and (iii) the experimental conditions .2+ channel current (Ica) in isolated human cardiac ventricular myocytes (at odds with the absence of any effect on contractility). Contractile and electrophysiological effects of β3-AR may also differ in atrial myocytes. Although few data are available on this specific tissue, β3-AR stimulation was reported by Skeberdis et al. [2+ channel current, through cAMP/PKA signaling [In human endomyocardial biopsies from the right interventricular septum of cardiac transplanted patients , Gauthier et al.  demonstrs et al.  to slighignaling . These cignaling  later reignaling , but thiignaling  observedignaling  to firml2+ transient induced by β3-AR stimulation are abolished in cardiac myocytes isolated from knockout mice lacking either eNOS or nNOS ; the same was observed in cardiac myocytes treated with a specific nNOS inhibitor (SMTC). This regulation of the β3-AR response was explained by a nNOS-eNOS crosstalk in which nNOS is required to inhibit xanthine oxidoreductase (XOR)-dependent superoxide anion production, thereby protecting eNOS function from oxidative uncoupling.The β3-AR/cGMP downstream signaling involves not only the eNOS pathway, but also nNOS, with the two enzymes cooperating to maintain signaling in the face of catecholaminergic stress . In this2+ handling and sarcomere function in cardiac myocytes (reviewed in [2+ transient may operate both through the NOS-dependent inhibition of L-type Ca2+ channels (LTCC)-mediated Ica current [2+ re-uptake during cardiac myocyte relaxation through PKG-mediated phosphorylation of phospholamban. cGMP/PKG signaling downstream β3-AR may also regulate cardiac diastole by decreasing myofilament Ca2+ sensitivity and modulating myocardial stiffness through troponin I (TnI) [cGMP-activated PDE) and PDE3 (a cGMP-inhibited PDE) are able to hydrolyze both cAMP and cGMP, but with higher affinity and/or Vmax activity for cAMP, while PDE5 is a selective cGMP hydrolase. Interestingly, in cardiac myocytes expressing a FRET-cGMP biosensor, the authors also observed that cGMP production induced by β3-AR stimulation is itself under the control of PDE5 and PDE2.The β3-AR-mediated attenuation of contractility and positive lusitropic effects can be attributed to the well-described NOS/NO/cGMP/PKG signaling regulation of Caiewed in ). β3-AR  current  (thereby I (TnI)  and titi I (TnI)  phosphor I (TnI) . This re I (TnI) . PDE2  [Ks), but the functional implications on contractility (or rhythm control) remain to be investigated [Other targets of NOS/sCG may participate in β3-AR regulation of myocyte contractility. In ventricular myocytes from sheep with HF, activation of β3-AR improved contractility by increasing Nactively) . In guinstigated  , during a first adaptive period, sustained catecholamine  stimulation is recruited, and required, to maintain cardiac contractile function for appropriate blood supply to peripheral organs (compensated stage). However, chronic (over)stimulation of the receptors progressively leads to β1/β2AR desensitization and subsequent loss of contractile function (decompensated stage), in an interdependent manner with morphological hypertrophic-to-dilated cardiac remodeling and fibrosis ,47. In t2+ transient of isolated ventricular myocytes from failed hearts [Accordingly, in a dog model of rapid pacing-induced HF associated with an increase in cardiac β3-AR expression, acute β3-AR stimulation further decreased contractility and Cad hearts . A simild hearts , leadingd hearts . A simild hearts . Howeverd hearts . β3-AR ad hearts , and coud hearts .In contrast to the above, the majority of studies report cardioprotective effects of β3-AR stimulation on heart function and remodeling, which mainly involve (i) preservation of contractile function mediated by NOS-signaling and antioxidant effects, (ii) antihypertrophic and antifibrotic effects and (iii) metabolic effects. As β3-AR signaling regulates cardiac relaxation—notably through PKG-downstream signaling—and prevents hypertrophic remodeling, it recently appeared as a promising therapeutic target for HF with preserved ejection fraction (HFpEF). These points are discussed in the following section.−/− mice submitted to pressure overload-induced HF (by transverse aortic constriction—TAC) displayed exacerbated left ventricular (LV) contractile dysfunction (reduced EF) and cardiac remodeling compared to wild type (WT) mice. These deleterious effects of β3-AR genetic deletion were associated with decreased eNOS phosphorylation on its activation site (Ser1177) and increased uncoupling, responsible for higher NOS-dependent superoxide production. On the contrary, β3-AR stimulation  conferred cardioprotection against haemodynamic stress [1412 and mostly dephosphorylation at Ser847, associated with increased nNOS activity, higher cGMP synthesis and decrease in ROS production. Such protective effects were lost in mouse cells expressing phosphomimetic nNOS mutants on Ser847. Moreover, inhibition of Gαi protein (PTX incubation) blocked BRL37344-induced nNOS post-translational modifications and ROS reduction. Similar cardiac nNOS post-translational modifications were reproduced upon BRL37344 treatment of mice submitted to 3 weeks of TAC [1412 [Cardioprotective effects of β3-AR signaling against HF were initially inferred from the phenotype of knockout mice lacking the receptor . β3-AR−/c stress . In the c stress . Furthers of TAC . Interessite Ser17 and incAC [1412 ), and thAC [1412 , the ant+/K+ pump function upon β3-AR stimulation also contributes to the attenuation of congestive cardiac remodeling induced by coronary ligation in rabbits [+/K+ pump induced by NADPH oxidase-dependent oxidation of its β1-subunit [+/K+ pump function would oppose the Na+ overload commonly observed in failing myocytes and restore EC coupling and systolic function . Diastolic dysfunction in HFpEF patients mainly results from the combination of increased cardiomyocyte stiffness with LV hypertrophic remodeling and interstitial fibrosis ,64,65. Cpatients  and subspatients ,68,69. TOur group and others showed that activation of cardiac myocyte β3-AR protects against the deleterious effects of chronic adrenergic stimulation, particularly against hypertrophic remodeling and myocardial fibrosis.An antihypertrophic effect of β3-AR was initially observed by pharmacological stimulation of primary myocytes and in vivo infusion of β3-AR agonists in rodents ,54. The Strikingly, the β3-AR-mediated protection extended to myocardial interstitial fibrosis in the above models. A similar antifibrotic effect was also observed following myocardial infarction upon treatment of mice with β3-AR agonists  along wiAs extensively described in the previous section, β3-AR signaling in cardiac myocytes is linked to the PKG/cGMP/NOS pathway. The cardioprotective effects of β3-AR against hypertrophic remodeling and fibrosis following neurohormonal and haemodynamic stress were also shown to be mediated by nNOS ,26,52,541412) [Mechanistically, the reduced ROS levels following β3-AR activation attenuate fibrosis through reduced release of paracrine profibrotic agents in β3-AR expressing myocytes; in superfusion experiments using conditioned media and secretome analysis, connective tissue growth factor (CTGF) was identified as one of the major contributors, as its silencing in cardiac myocytes significantly attenuated the pro-fibrotic effect upon their stimulation with phenylephrine  Figure . In addi1412)  and eNOS1412) . HoweverAMPK was found to colocalize with β3-AR at the caveolae. Transgenic human β3-AR expression sustained the activation of AMPK and its downstream effectors in the face of neurohormonal and haemodynamic stress. Conversely, silencing of crucial AMPK α1/α2 catalytic subunits in cardiac myocytes significantly blunted the antihypertrophic effect of β3-AR . While Aβ3-AR have traditionally been linked to metabolic effects in adipose tissue—e.g., through activation of brown fat in rodents and beiging of white adipocytes in rodents and humans. The transcription factor, Prdm16, is a main regulator of the beiging process in white adipose tissue (WAT) ,78. In aIn addition to oxidative stress handling, β3-AR was strongly associated with metabolism in a proteomic study analyzing differentially expressed proteins from hearts of β3-AR systemic KO mice compared to WT . This stSimilarly, the interpretation of cardiac effects of β3-AR agonists administered systemically must take into account potential effects mediated by β3-AR in adipose tissue. The latter deserve special consideration, as improvements in the overall metabolic status—i.e., decreased triglyceridemia, and decreased obesity, with increased insulin sensitivity and/or β3-AR-mediated endocrine/paracrine signaling from adipose tissue—can greatly affect the cardiovascular system.In brown adipose tissue, non-shivering thermogenesis in rodents has long been shown to be regulated by the activation of the β3-AR coupled to Gαs leading to activation of AC and intracellular of cAMP/PKA signaling ; this le2+ cycling by the sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) when Ca2+ transport is uncoupled from ATP hydrolysis [While the argument for β2-AR mediated thermogenesis in human BAT is solid, it probably does not extend to human beige adipose tissue where the evidence points to a β3-AR mediated signaling. Beige adipocytes with thermogenic ability interspersed within white adipose tissue (WAT) were recently identified in mouse and human ; in addidrolysis . In beigdrolysis . Whetherdrolysis ,103. In drolysis ,98. Humadrolysis . While adrolysis , recent drolysis ,94,98. AAt this stage, experiments performed with systemic β3-AR stimulation do not allow to discriminate beneficial effects linked to WAT lipolysis from those due to beige adipocytes stimulation and thermogenesis. Nevertheless, stimulation of adipocytes by mirabegron was associated with a significant improvement in glucose tolerance and insulin sensitivity in both healthy and obese subjects ,94 despiIn a manner similar to this observation in skeletal muscle, a single β3-AR agonist injection in a WT mouse was found to strongly upregulate Perlipin 2 (PLIN2) and Perlipin 5 (PLIN5) expression in cardiac myocytes . Again, Systemic administration of β3-AR agonists is likely to affect the adipose tissue in different locations—i.e., visceral, epicardial or perivascular—with various effects depending on their content in beige adipocytes and lipolytic capacity. From the perspective of clinical applications, the resulting cross-talk between adipocytes and cardiac myocytes is of particular importance as recent studies highlight a higher content of beige adipocytes in human visceral (VAT) than in subcutaneous adipose tissue  contraryApart from VAT, stimulation by β3-AR of the perivascular adipose tissue (PVAT) was shown to generate a cAMP-dependent release of NO that attenuates the contractile response of rodent mesenteric arteries . In anotBased on the evidence reviewed in the previous sections, β3-AR represent a promising therapeutic target for the treatment or prevention of cardiovascular diseases (CVD), be it through their systemic or direct activation in cardiac myocytes, in particular given their increased expression in the pathological myocardium . This caEvaluation of the cardiovascular safety of mirabegron is an important prerequisite given the recent report of mirabegron-mediated increase in human atrial force, albeit at high concentration in vitro (see discussion above). This side-effect of mirabegron was attributed to an accrued norepinephrine release from sympathetic nerves leading to stimulation of the β1-AR response in isolated atrial trabeculae . Regardl+ levels—the effect involved protection of the Na+-K+ -ATPase activity from oxidative inactivation, as developed in The first randomized trial on CVD examined the effect of mirabegron on 70 patients suffering from HFrEF. The rationale for this trial was based on preclinical data in failing sheep in vivo and rabbit cardiac myocytes where β3-AR stimulation was found to reduce the detrimental increase in intracellular NaWhile the BEAT HF trials test the beneficial effects of a chronic β3-AR activation in advanced HF, no trial has yet tested the reverse hypothesis—i.e., that a β3-AR antagonist, administered acutely, may improve LV function by antagonizing the negative inotropic effect of β3-AR at the early phase of acute HF see  above. TIn a porcine model of pulmonary hypertension, β3-AR agonists also improved right ventricular (RV) performance and pulmonary hemodynamics, particularly pulmonary vascular resistance (PVR) . Accordi2+ sensitivity and lower titin stiffness; and it mediates NOS-dependent vasorelaxation in the human coronary microvasculature [As mentioned in culature . β3-AR aculature . This isculature ; a sub-sculature .The substantial body of evidence reviewed above clearly indicates pleiotropic roles of the human β3-AR beyond metabolic regulation in adipose tissue. Direct and systemic, indirect influences on the contractility, remodeling and, possibly metabolism of the cardiac muscle justify past and current efforts to harness the human β3-AR for the treatment of specific forms of HF. Careful determination of the dose and timing of administration of the newly available agonists  at the human β3-AR should enable the accumulated knowledge to be translated into new avenues of treatment for this deadly disease.


Wearing smart glasses may be distracting and thus annihilate the beneficial effects of cues on freezing of gait in Parkinson's disease. Furthermore, augmented reality cues might be effective in reducing FOG specifically in cueing-responsive patients. We present a single-patient study in which a patient with Parkinson's disease traversed a doorway under different cueing conditions. Wearing augmented reality (AR) glasses did not deteriorate FOG nor affect the beneficial effects of cues. The AR visual cues did not improve FOG. This single-patient study implies that the current design of AR glasses does not stand in the way of the development of augmented reality visual cues. However, the effectivity of augmented reality visual cues remains to be proven. External cues, such as transverse bars on the floor or the beat of a metronome, can alleviate freezing of gait (FOG) in people with Parkinson's disease (PD) –3. We prWe hypothesized that wearing AR glasses could have two negative effects: (1) eliciting or worsening FOG; and (2) annihilating the effects of conventional cues. Additionally, we hypothesized that AR cues could alleviate FOG in a cueing-responsive patient.To test our hypotheses, we tested one person with PD with an established clear response to conventional cues under different cueing conditions. This person was examined with and without wearing AR glasses. The patient was a 63-year-old man who was diagnosed with PD 17 years earlier and who had experienced FOG for 16 years. He had no cognitive impairments and no relevant comorbidities. His FOG partially improved with dopaminergic medication  but remained bothersome nevertheless. He reported no beneficial or negative effects of bilateral deep brain stimulation of the subthalamic nucleus on his FOG. He successfully used a wide variety of cueing strategies, such as auditory and haptic metronomes, bars and lines on the floor, stepping over a broom, and kicking against a small box on the floor.t-tests.The experiment was conducted in a medication OFF state, >12 hours after the last intake of dopaminergic medication. The deep brain stimulator was left switched on. The patient traversed a doorway four times under each of the following conditions: (1) no AR glasses worn, no cues applied (“Control”); (2). The AR glasses worn but switched off (“SG OFF”); (3) AR three-dimensional bars displayed through the AR glasses (“SG AR”); (4) real transverse bars on the floor, no AR glasses worn ; (5) real transverse bars on the floor while wearing AR glasses switched off ; (6) stepping over a hand-held broom, no AR glasses worn (“Broom”); and (7) kicking against a small box on the floor, no AR glasses worn (“Box”). The AR glasses used were the Microsoft HoloLens . FOG was annotated from video recordings by two independent experienced raters. The percent time of the trial spent on freezing was compared amongst the different cueing conditions with paired The real bars, broom, and box caused a remarkable and significant improvement in percent time frozen compared to the control condition, confirming that this person was particularly responsive to visual cues . WearingFrom this single-patient study, we conclude that wearing AR glasses does not worsen FOG nor affect the beneficial effects of conventional cues. This implicates that the current design of AR glasses does not stand in the way of the development of AR visual cues. However, we found no evidence that AR visual cues could improve FOG even in a cueing-responsive patient. Whether these conclusions can be extended to other patients requires further study in a larger cohort. We recommend future studies involving cueing through AR glasses to include control conditions without AR glasses.


Pharmacists have a valuable role in the management of allergic rhinitis (AR) at the community pharmacy level. This role has been reported extensively in numerous papers. However, a systematic review of the available literature and a comprehensive analysis of the outcomes has not been published. This systematic review aimed to evaluate the impact of interventions developed by pharmacists on clinical AR outcomes. A thorough search was performed in three electronic databases, including studies published between January 2000 and June 2019. After the selection process, only three articles met the inclusion criteria and were further analysed. Despite the scarcity of the available studies, in all of them was clear that the pharmacist plays a pivotal role in the management of AR, significantly improving the patients’ quality of life and symptom control. This systematic review also stresses the utmost importance to investigate and report practices and interventions developed by pharmacists using measurable outcomes. Allergic rhinitis (AR), commonly referred to as hayfever, is a chronic upper respiratory condition resulting from inflammation of the nasal mucosa. The common symptoms of AR include sneezing, rhinorrhoea and nasal congestion, induced by an immunological response following exposure to allergens such as pollen, house dust mites, moulds and animal dander in sensitised individuals . While AThe socioeconomic and health burden of poorly controlled AR on individuals and society can be minimised with optimal AR management strategies, that encompass patients education, including allergen minimisation strategies, pharmacotherapy and the addition of allergen-specific immunotherapy in severe cases of AR . AR can The non-governmental organization Allergic Rhinitis and its Impact on Asthma (ARIA) issued specific guidelines for the management of AR in the community pharmacy, first in 2004  with a rPharmacists can interview patients and determine whether they require a referral and further medical investigation . They plWhile there is a large body of literature available regarding the management of AR within the community pharmacy setting, to date, no systematic review has evaluated community pharmacists’ intervention in AR management. This systematic review aims to evaluate the impact of community pharmacists’ interventions on clinical AR outcomes among adult patients.This systematic review was performed according to the methodology recommended by the PRISMA guidelines  from JanTo be included in the systematic review, the studies had to meet the following inclusion criteria: (1) published between January 2000 and June 2019; (2) written in Portuguese, English, Spanish or French; (3) original article/investigation (primary literature); and (4) report the results of community pharmacists interventions in AR. All articles that did not meet the mentioned inclusion criteria were not considered. No limitation regarding the age of the participants was considered. References obtained from different databases were compared to identify and remove duplicates.The titles and abstracts of all articles obtained from the database search were screened to identify potentially relevant articles (which fulfilled the inclusion criteria). Non-relevant articles were deleted while potentially relevant articles identified were obtained. The full text of the eligible studies was read and evaluated, considering the objectives of this review. All non-original and non-experimental studies that did not report the clinical results from the implemented intervention or interventions that were not exclusively implemented by community pharmacists were also excluded. Additionally, potentially relevant studies in the bibliographic references of the selected studies of interest were searched. The studies identified were reviewed by two investigators (J.J. and O.L.).The relevant information that was extracted from each eligible article, was (1) the source (author and year of publication), (2) the country where the intervention was performed, (3) the objective of the intervention, (4) the type of study (5) the outcomes assessed, (6) the tools used for outcome assessment, (7) the number of participants , (8) the intervention results, and (9) study limitations.https://www.strobe-statement.org).For assessment of study quality and risk of bias, we applied the ‘Risk of Bias’ tool described in the Cochrane Review Group Handbook and the STROBE statement checklists (available on Electronic searching from the different databases resulted in a total of 478 citations, of which 78 duplicates were removed. Their titles and abstracts were then read to assess their agreement with the initially defined inclusion criteria, which resulted in 31 studies. Of these, 28 were excluded, which resulted in three studies included in the systematic review. This process is illustrated in The three intervention studies ,28,29 thThe intervention study conducted by Arsoy et al. focused on AR education, including avoidance of allergen exposure, counselling on medication and training on the administration technique of nasal medication . The stuThe study conducted by O’Connor et al.  comparedBoth studies used the Mini RQLQ© to assess the QoL of the patients before and after the interventions.The Mini RQLQ© scores were significantly reduced between the pharmacy visits resulting in statistically significant QoL improvements ,28. HoweThe study by Todorova et al.  exploredRegarding the severity of symptoms, both the Arsoy G. et al.  and O’CoThis systematic review highlights the paucity of research exploring pharmacist interventions in AR management and draws to attention the urgency of the requirement for further reseach in this field, especially given the burden caused by sub-optimal management of AR internationally ,30. AlthA major limitation of this review was the scarcity of intervention studies in AR in the community pharmacy setting, as well as the limited quality of existing studies. The most profound limitation across all the three studies was the limited number of participants, both pharmacists and patients. Similarly, the studies were conducted were confined to specific geographical areas and not necessarily applicable to wider health care settings international. O’Connor et al., also has limitations of not having a control group and focussing on OAH use, whose overuse is currently associated with poorer AR outcomes and is only recommended in the most mildest of AR cases ,33.However, despite the limited number of pharmacy interventions evaluated, this review has shown that pharmacists play a crucial role in helping patients minimise their AR symptoms, providing information and advice on treatment and monitoring outcomes. From these studies it can be concluded that interventions by community pharmacists in the management of AR can significantly improve patients’ QoL while also improving disease control; however, further research in this field is needed. Further interventions are imperative to fully understand the pharmacist’s role in the management of AR since the community pharmacy is the venue of choice for patients to receive counselling regarding AR and often the only place where patients seek healthcare for their AR.Patients with AR have needs that are not being met by current practices, e.g., diagnosis is absent in many cases, and there is inadequate counselling and treatment selection by pharmacists. These facts may be related to pharmacists’ lack of knowledge about ARIA guidelines ,34 and u