The androgens/androgen receptor (AR) axis is the main therapeutic target in prostate cancer (PCa). However, while initially responsive, a subset of tumors loses AR expression through mechanisms putatively associated with epigenetic modifications. In this study, we assessed the link between the presence of CpG methylation in the 5′UTR and promoter regions of AR and loss of AR expression. Hence, we characterized and compared the methylation signature at CpG resolution of these regulatory regions in vitro, both at basal levels and following treatment with 5-aza-2-deoxycytidine (DAC) alone, or in combination with Trichostatin A (TSA). Our results showed heterogeneity in the methylation signature of AR negative cell lines and pinpointed the proximal promoter region as the most consistently methylated site in DU-145. Furthermore, this region was extremely resistant to the demethylating effects of DAC and was only significantly demethylated upon concomitant treatment with TSA. Nevertheless, no AR re-expression was detected at the mRNA or protein level. Importantly, after treatment, there was a significant increase in repressive histone marks at AR region 1 in DU-145 cells. Altogether, our data indicate that AR region 1 genomic availability is crucial for AR expression and that the inhibition of histone methyltransferases might hold promise for AR re-expression. Prostate cancer (PCa) remains a major health concern, ranking second in incidence and fifth in mortality among all cancers in men, worldwide . The andThe human AR gene is located at Xq11.2-q12, encoding a 10.6 kb transcript that comprises a 1.1 kb long 5′untranslated region (5′UTR) that contains a 24 bp upstream open reading frame (uORF), a 2.7 kb coding region and a 6.8 kb 3′ untranslated region (3′UTR) ,14, as wThe hypothesis of promoter CpG hypermethylation as a mechanism for AR gene inactivation was previously documented ,17,18. IThe observation of a methylation-related silencing mechanism prompted several attempts to reverse methylation by exposing AR negative cell lines to demethylating drugs, for relatively short or prolonged periods of time, alone or in combination with histone deacetylating drugs. Jarrard et al. showed that treatment with low doses of the demethylating drug 5-aza-2-deoxycytidine (DAC) for a short period of time was able to restore AR mRNA expression in some AR negative cell lines but not in DU-145 cells, in which AR expression was not recovered even after chronic exposure for 2 months . A similNonetheless, methylation alone does not seem to fully explain AR loss, since some AR negative cells lines and clinical specimens with heterogeneous AR staining do not exhibit this dense methylation signature . TherefoHerein, we sought to further dissect the role of promoter methylation for AR transcriptional silencing. Thus, we investigated the methylation signature of prostate cell lines with different AR transcript expression profiles at CpG resolution. CpG methylation assessment in basal conditions and following treatment with DAC alone or in combination with TSA allowed us to better understand which sites are most likely to influence AR transcriptional regulation. Importantly, a deeper analysis of chromatin architecture upon DAC/TSA treatment revealed a highly repressed AR region 1, which partially explains why DNA methyltransferases targeting alone has led to ineffective results in AR re-expression and raising the possibility of targeting histone methyltransferases as an alternative approach.w/v) heat-inactivated fetal bovine serum  and 1% (w/v) penicillin/streptomycin . Cells were maintained in an incubator at 37 °C and 5% CO2. All cell lines used in this study were routinely tested for Mycoplasma spp. contamination by using a TaKaRa PCR Mycoplasma Detection Set .In this study, AR positive (LNCaP and 22Rv1) and AR negative (DU-145 and PC-3) human PCa cell lines, as well as the non-malignant prostate cell line RWPE-1 were used . LNCaP, For DAC , a 100 mM stock solution was prepared in acetic acid and stored at −80 °C until further use. DAC working solutions of 1 mM were freshly prepared by further dilutions in DPBS along with a vehicle control (1% acetic acid in DPBS). For TSA , a 5 mM stock solution was diluted 10× to a working concentration of 0.5 mM in DMSO.5 cells were platted in T-25 cm2 culture flasks and treated daily for 3 days with 5 µM of DAC [For each experiment, 5 × 10M of DAC  or its vM of DAC  or DMSO.w/v) Tween for 1 to 2 h at room temperature. Afterwards, the membranes were incubated overnight at 4 °C with the respective primary antibodies (w/v) milk/TBS-0.1% (w/v) Tween)  for 1 h at room temperature. The signal was developed with an enhanced chemiluminescence detection kit  [Total protein extracts were prepared in RIPA Lysis Buffer  containing a protease inhibitor cocktail , sonicated and centrifuged at 13,000 rpm for 30 min at 4 °C. Protein concentration was determined using the Pierce BCA Protein Assay Kit , according to the manufacturer’s instructions. For AR detection, 20–50 μg of total protein was used for SDS-PAGE, as previously described . The protibodies . On the CA, USA) . β-actinw/v) SDS, followed by extraction using the phenol/chloroform method and precipitation with 100% (w/v) ethanol, as described by us [Genomic DNA was obtained by digestion with proteinase K (20 mg/mL) at 55 °C in the presence of 10% (ed by us . Bisulfied by us .w/v) agarose gels before proceeding to the cloning step. Two μL of the PCR product were subcloned into the TOPO TA vector  and used for transformation of One Shot® chemically competent E. coli vials  by heat-shock at 42 °C for 45 s, as adapted from [AR promoter region was divided into three subregions  that were amplified using primer pairs designed using Methyl Primer Express v1.0  and Xperted from . Followihttp://quma.cdb.riken.jp/, last accessed on 12 November 2022) software was used to analyze bisulfite sequencing data [QUMA  following normalization to β-actin. For each gene, both an NTC and a bisulfite modified HCT116 DKO non-methylated DNA standard  were used as negative controls.Bisulfite modified DNA was amplified by qMSP using Xpert Fast SYBER Mastermix Blue  and specific primers , as prev2 culture flasks containing 90% confluent DU-145 cells from each experimental condition were used for ChiP-PCR experiments, based on a protocol previously established by us [w/v) formaldehyde final concentration). The flasks were then incubated at room temperature (RT) for 10 min with constant agitation, after which the reaction was quenched by the addition of glycine , followed by incubation at RT for 5 additional minutes with agitation. Crosslinked cells were then washed twice with ice cold DPBS before 10 mL of ice cold DPBS containing a protease inhibitor cocktail  was added to each flask and a rubber cell scraper was used to collect cells. A centrifugation step (4 °C) at 800× g allowed for cell collection. A cell lysis buffer  containing a protease inhibitor cocktail  was added to the cell pellet and lysis was performed for 1 h 30 min on ice, with intermittent vortexing. A centrifugation step (4 °C) at 800× g that allowed nuclei collection was followed by incubation on ice for 15 min with an ice-cold nuclei lysis buffer  SDS) containing a protease inhibitor cocktail . The supernatant was diluted with 2× volume of IP dilution buffer  SDS) before sonication for 2 × 10 min cycles of 30 s ON and 30 s OFF, replacing warm with cold water and ice between each cycle. Sonicated samples were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatant (containing chromatin) was collected in a new 1.5 mL tube. Thirty µg of chromatin was set aside to assess shearing efficiency. An input fraction was set aside, and the sheared chromatin was diluted 10x in ChIP dilution buffer  SDS, 1.1% (w/v) TritonX-100). The final volume was divided in 3 parts, to which 20 µL of (Dyna)Beads A + G  plus either (mono)clonal anti-acetyl histone 3 antibody or (mono)clonal anti-RNA polymerase II (positive control) or mouse/rabbit IgGs (negative control) were added. The immunoprecipitation was performed overnight at 4 °C. The next day, the (Dyna)Beads were successively washed for 5 min at 4 °C, with agitation with 1 mL of (i) low salt wash buffer  SDS, 0.1% (w/v) Triton X-100); (ii) high salt wash buffer  SDS, 0.1% (w/v) Triton X-100); (iii) LiCl wash buffer  NP-40, 1% (w/v) deoxycholate); and (iv) TE buffer . The DNA was eluted by adding 100 µL of elution buffer (1% (w/v) SDS, 0.1 M NaHCO3) to the beads in the presence of 0.5 µL of RNase A and incubated for 30 min at 37 °C with agitation. The input fraction was subjected to the same conditions. Afterwards, treatment with proteinase K was performed for 2 h at 62 °C, with agitation. The crosslinking was reversed by placing the samples at 95 °C for 10 min.T-175 cmed by us . For thaThe supernatant was then collected, and the DNA was purified using the Qiaquick extraction kit  according to the manufacturer’s instructions, followed by RT-qPCR with four pairs of primers . Data sh® gene expression assays  for AR (assay ID Hs00171172_m1) and housekeeping gene β-GUS (assay ID Hs99999908_m1). LNCaP and 22Rv1 cDNAs were used as positive controls. Three technical replicates were made for each biological replicate and results were normalized to β-GUS. Fold-changes to mRNA expression were calculated using the cycle threshold (ΔΔCt) method [RNA extraction was performed using TripleXtractor  according to the manufacturer’s instructions. Afterwards, 1 µg of RNA was converted to cDNA using a RevertAid RT kit  . Quantit) method .U test was used to compare two groups. For comparisons between three or more groups, non-parametric Kruskal–Wallis test, with Dunn’s correction, was used. All in vitro experiments were performed at least 3 times.Statistical analysis was performed using the GraphPad Prim 7.0 software . Non-parametric Mann–Whitney U-test was used for the evaluation of an entire set of CpG sites.Moreover, QUMA software Fisher’s exact test was used to determine the statistical significance of methylation levels between two bisulfite sequence groups at each CpG, whereas the Mann–Whitney p-values were considered statistically significant when inferior to 0.05. Significance is shown vs. the respective control and depicted as follows: * p < 0.05, ** p < 0.01, *** p <0.001; **** p < 0.0001. All data are represented as mean ± standard deviation of the mean (SEM), unless otherwise specified.AR gene of both AR negative (DU-145 and PC-3) and positive (RWPE-1) cell lines, was analyzed by bisulfite sequencing. Based on prior reports [The methylation status of individual CpGs, present in regulatory regions of the  reports ,17,19, t reports  and it e reports . Region We observed methylation heterogeneity of CpG sites across regions and clones . In DU-1Conversely, in DU-145 clones, the least frequently methylated CpG site in region 1 was that embedded in the Sp1-1 binding site (−42) . Furthermore, the CpGs located near −42 were also more heavily methylated in DU-145 than in the other cell lines. Of note, CpG −51, which was seldom methylated in PC-3 (6.7%) and RWPE-1 (0%), was the CpG with the highest difference in percentage of methylation between DU-145 and the other cell lines B.For region 2, DU-145 exhibited, again, the highest overall methylation of all cell lines 66.3%, C. HoweveImportantly, whereas DU-145 cells presented high levels of methylation in both region 1 and 2 for almost all of the clones (13/15 clones with methylation levels ~75%), for both regions the clones obtained from PC-3 cells could be separated into two opposite groups: those with slight (~30%) or no methylation across the entire region and those with high methylation levels (above 75%). The same was observed for region 2 in RWPE-1 cells, in which 6/15 clones had methylation levels above 75% . This suGlobally, for all the cell lines, regions 1 and 2 were more densely methylated than region 3 B,C. The We next focused on characterizing the methylation status of individual CpG sites in regions 1 and 2 following exposure to the demethylating drug DAC. Bisulfite sequencing was performed in DNA obtained from treated cell lines . OverallIn DU-145, treatment with DAC only accomplished a reduction in overall methylation of region 2, whereas region 1 was particularly resistant to DAC-mediated demethylation A,B. SimimiR-130a [CCND2 [RASSF1A [To further demonstrate that the lack of an obvious demethylation effect following DAC treatment was not due to the absence of drug action, methylation levels of miR-130a , CCND2 [a [CCND2  and RASS[RASSF1A , previouFinally, we checked whether AR transcript and protein expression could be rescued by DAC treatment alone. However, no AR expression, either at mRNA or protein levels D, was obSince synergy is frequently observed in epigenetic mechanisms, concomitant treatment with DNA methyltransferase inhibitor (DNMTi) and histone deacetylase inhibitor (HDACi) was performed to investigate whether the HDACi TSA would enhance the DAC demethylating effect. Since DU-145 was more packed with methylation throughout regions 1 and 2, we focused on this cell line for the assay. To our knowledge, this is the first study analyzing the CpG site specific methylation following this combined approach.AR expression was found upon exposure to this combined treatment, both at transcript and protein levels  and 5′UTR (+44 to +54 bp from the TSS) were reported as critical for AR expression, mostly based on comparison to methylation patterns of AR positive cells and on recovery of AR transcript expression following exposure to DAC, alone or in combination with TSA or sodium butyrate (NaB) [AR regulatory regions in DU-145. Surprisingly, DAC treatment did not significantly impact CpG methylation at the proximal promoter region, and only minor effects were observed for 5′UTR hotspots. Importantly, significant demethylation of the promoter region of AR was only observed upon DAC and TSA concomitant treatment, but the CpG hotspots retained high methylation levels, especially in CpGs at −123 and −121 bp from the TSS.Promoter CpG island hypermethylation has been classically associated with transcriptional silencing . Initialte (NaB) ,19,20,40AR expression. Indeed, blocked translation, and not transcription, has been suggested for AR-negative cell line PC-3, which bears much lower methylation levels across those regions than DU-145 [AR gene methylation heterogeneity, especially in region 1, with some of the sequenced alleles displaying extensive methylation in almost all CpGs. This may indicate that, within the same tumor, cells may use different mechanisms to inactivate AR expression, some being DNA methylation-dependent while others not. Indeed, other epigenetic mechanisms, particularly histone methylation, might further explain the lack of AR expression in PC-3 cells [Our results further confirm that not all AR negative cells exhibit dense methylation in these same regions, indicating that methylation-independent mechanisms also influence n DU-145 . In linen DU-145 . However-3 cells . Advance-3 cells  may help-3 cells . Additio-3 cells ,45,46.AR transcription, is a methylation indifferent transcription factor, and methylation in CG boxes is not expected to prevent DNA binding [Traditionally, methylation is perceived as a chemical repellent for transcription factor (TF) binding. However, recent studies challenge this observation by showing that TFs respond differently to the presence of DNA methylation . In this binding . Indeed, binding . For ins binding , also fu binding .AR region 1 was found, possibly impairing the binding of TF and transcription itself, which might explain the differences observed between our study and the results of Nakayama et al. [Importantly, our ChIP-PCR data corroborate this argument, since a heavily packed chromatin architecture around a et al. . Moreovea et al. , which ia et al. . With ana et al. , our data et al. , alone oOverall, our findings pave the way for the use of histone methyltransferase inhibitors (HMTi) and/or DNMTi, in combination with ADT, for the management of CRPC. Indeed, in AR-negative tumors, epigenetics might explain the lack of the receptor’s expression. Even thought we did not manage to re-express AR in DU-145 CRPC cells, in primary tumors, due to intra-tumor and inter-patient heterogeneity, the possibility of reverting AR promoter repressive chromatin state may induce AR expression. Therefore, combining epigenetic drugs with the standard of care might improve PCa patients’ management .However, before clinical implementation, the rationale of this study would need to be performed with additional AR-negative cell lines to enlarge the spectrum of in vitro models to account for tumor heterogeneity and strengthen the results. Moreover, our current work lacks validation in patient-derived materials, as well as validation of AR methylation by qMSP, since we were not able to optimize specific primers. Additionally, a ChiP-seq approach might give a more in-depth knowledge of the chromatin architecture nearby the AR promoter, and the usage of AR specific antibodies would allow to further support our hypothesis. Importantly, studies involving HMTi and DNMTi are still required to fully address this question and validate the relevance of the epigenetic mechanisms for AR expression, which could ultimately provide clinical benefit for patient management.


Allergic rhinitis (AR) has a significant impact on the community as a whole with regard to quality of life and its relationship to allergic multi-morbidities. Appropriate diagnosis, treatment and review of the efficacy of interventions can ameliorate these effects. Yet, the importance of AR is often overlooked, and appropriate therapy is neglected. The availability of effective medications and knowledge as to management are often lacking in both public and private health systems.This review is based on a comprehensive literature search and detailed discussions by the South African Allergic Rhinitis Working Group (SAARWG).The working group provided up-to-date recommendations on the epidemiology, pathology, diagnosis and management of AR, appropriate to the South African setting.Allergic rhinitis causes significant, often unappreciated, morbidity. It is a complex disease related to an inflammatory response to environmental allergens. Therapy involves education, evaluation of allergen sensitisation, pharmacological treatment, allergen immunotherapy (AIT) and evaluation of the success of interventions. Regular use of saline; the important role of intranasal corticosteroids, including those combined with topical antihistamines and reduction in the use of systemic steroids are key. Practitioners should have a thorough knowledge of associated morbidities and the need for specialist referral.This review summarises the latest developments in the diagnosis and management of AR such that it is a resource that allows easy access for family practitioners and specialists alike. Funds for medications for AR are frequently not a priority in the public health sector. However, untreated or poorly treated AR may have a greater economic cost as a result of absenteeism or reduced productivity.22Practical issues: Distance to hospital or clinic, single-parent households and inability to take time off work make clinic visits difficult.Understanding of health-related issues: This may be affected by poor health literacy and dominant traditional beliefs, negatively affecting compliance.The aim of this consensus document, produced by the SAARWG, is to review up-to-date recommendations for AR applicable to SA.8 approximately 80% developing before the age of 20 years, with a peak at 20–40 years and then a gradual decline.8 The burden in low- and middle-income countries is similarly substantial and has been increasing since the 1990s.6 In the International Study of Asthma and Allergies in Childhood (ISAAC), the SA cohort of 13- to 14-year olds showed substantial and increasing prevalence from 30.4% in 1995 to 38.5% in 2002.2 Urbanisation and increasing levels of pollutants, as well as changes in pollen concentrations, allergenic potential and composition because of climate change, have been implicated in the increase in the prevalence of AR.24Allergic rhinitis affects between 10% and 40% of children and adults worldwide,25 The late phase of the allergic response, which precipitates a cycle of chronic allergic symptoms, manifests 4 h – 6 h after allergen exposure, with nasal mucosal inflammation from activation and influx of inflammatory cells, including T-cells, eosinophils, basophils and neutrophils.25Allergic rhinitis is a result of a Type 1 hypersensitivity reaction of the nasal mucosa. Allergens deposited onto the nasal mucosa of sensitised individuals bind to allergen-specific immunoglobulin E (IgE) on the surface of mast cells, resulting in the release of preformed mediators such as histamine. This causes the early phase of the allergic response and leads to acute symptoms such as itching, sneezing and rhinorrhoea.8 Air pollutants can also contribute to priming. Treatment with intranasal corticosteroids (INCS) can suppress the priming response.25Priming  occurs as a result of increased numbers of mast cells in the epithelium, increased permeability of the epithelium and easier allergen penetration to IgE-bearing cells and exaggerated responses of the nasal end organs.7 Nasal and non-nasal symptoms can occur.26The diagnosis relies chiefly on clinical assessment and laboratory tests indicating allergic sensitisation. Clinical assessment should include a thorough history recording duration, seasonality and severity of symptoms, and examination.Symptoms of AR  include the following:Nasal symptoms:Rhinorrhoea (anterior and posterior), sneezing, nasal blockage and itching and hyperreactivity of the mucosa to other allergens and non-allergic stimuli Non-nasal associations:Allergic conjunctivitis, palatal itching, cough from postnasal drip, asthma exacerbations, sinusitis or otitis media.An impact on QoL, specifically cognitive dysfunction and sleep disturbance.The examination should assess for signs of atopy such as the ‘allergic facies’ . The inferior turbinate should be examined for swelling and pallor. The patient should be evaluated for concomitant allergic diseases such as eczema and asthma. Comorbidities such as chronic rhinosinusitis (CRS), otitis media and hearing loss should be quantified. Other factors that can also cause nasal obstruction such as nasal polyposis, septal deviation, nasal deformities or mid-facial hypoplasia should be excluded.Plain film sinus X-rays have no place in the diagnosis. Computed tomography scanning should be reserved for suspected chronic sinus disease, particularly where surgery is contemplated.17 despite the recognition of some weaknesses.27 These guidelines divide symptoms into intermittent or persistent and severity into mild or moderate-severe  guidelines are widely appliede-severe .7,928The differential diagnoses of AR must be considered to plan for appropriate testing .6,7,8,2529 However, indiscriminate and extensive specific IgE testing is not indicated where history and examination suggest AR.Objective laboratory testing can identify patients at increased risk for severe disease, direct preventative steps to minimise allergen exposure(s) and tailor treatment and allergen immunotherapy (AIT).31 The 2015 iteration included the pooled specific IgE aeroallergen screen (Phadiatop®) for which there is an extensive evidence base to support its use as a single rule-out test.32Various recommendations regarding prevailing aero-allergens in South Africa have been made and subsequently modified.Dermatophagoides pteronissinus [Der p] and farinae [Der f]), cat, dog, moulds , and outdoor allergens . Since then, specific IgE testing over 3 years (November 2019 – October 2022) from two private laboratories revealed that 36.6% of Phadiatop® tests were negative, confirming its usefulness as a screen-out tool and that testing analysis showed low positivity to the outdoor mould Cladosporium (m2).A panel of specific IgEs to aeroallergens deemed most relevant to SA was established in 2014 by the Allergic Rhinitis Diagnostic Working Group (ARDWG). This included indoor allergens (house dust mites (HDM)  may be relevant in certain areas33 (updated pollen data can be found at https://pollencount.co.za/).ss trees  includedhttps://pollencount.co.za/).Specific tree panels should be considered based on clinical history, and where available, local sensitisation and pollen data .Pharmacological treatment:Patient evaluation for AIT.Measuring response to therapy.Patient evaluation for referral to a specialist.Education is the cornerstone of effective management. Key points include explaining that AR is a chronic disease and that, apart from AIT, there is no curative treatment. Treatment options should be discussed with regard to cost, efficacy, ease of use and side-effect profile.35 which is required for the successful treatment of AR.36 This is achieved through discussing the available therapeutic options and agreeing on a treatment plan that best serves the needs of the patient and which ensures compliance with and persistence with the plan.35Shared decision-making is of utmost importance. One of the main goals of shared decision-making is long-term adherence to treatment,Daily medication for persistent symptoms or intermittent use for seasonal symptoms should be discussed, as should the correct dosage, frequency and time of dosing.28Poor treatment adherence is an important barrier to treatment success, and questionnaires, such as the Medication Adherence Report Scale, a validated 5-item tool that assesses adherence, can be used in routine clinical practice.The correct method of using an INCS, by means of a physical demonstration, should be emphasised at the initial visit:After shaking and removing the lid, the nasal spray should be aimed towards the turbinates, which are on the lateral wall of the nasal passage.Ideally, the head should be tilted slightly forwards and, while closing the opposite nostril, one puff should be administered towards the outer side wall of the nose, aiming towards the ear and the back of the head, avoiding the nasal septum .Repeat the process in the other nostril.After each puff, the patient should try not to sniff, but rather pinch the nose between the thumb and index finger, holding the head neutrally forwards for a count of 10.Afterwards, the nose may be wiped, without blowing.Written educational material containing this information may be helpful to cement the key points.40 The major outdoor allergens are pollens and fungal spores, while the major indoor allergens include HDM, pets, moulds and cockroaches. Avoidance measures can be cumbersome, expensive and not always practical, and hence, allergen sensitisation needs to be proven before advising on allergen reduction strategies.Allergen avoidance and environmental control measures aim to decrease exposure to aeroallergens and irritants to reduce the severity of symptoms .37,38,3941Nasal irrigation with hypertonic or isotonic saline is a simple, inexpensive and effective adjunct to therapy, by squirt, pump, gravity  or spray bottle systems. Nasal rinses remove allergens, irritants and inflammatory mediators and clear accumulated mucus, optimising mucociliary clearance. Saline rinsing is safe in children and adults and reduces disease severity and symptom scores.Whenever possible, saline irrigation should precede the administration of INCS to remove debris for better delivery of INCS.42:In a resource-constrained environment, the following recipe can be used in place of commercially available products for nasal irrigationIn a clean container, mix 3 teaspoons iodide-free salt with 1 teaspoon bicarbonate of soda.Add 1 teaspoon of this mixture to 250 mL of lukewarm distilled or boiled water.Using a soft ear bulb or a commercial device, draw up the solution, lean over a sink with the head held sideways and insert the mixture into the top nostril till it comes out of the bottom nostril.Then repeat on the opposite side.44 and 19The efficacy of the various classes of drugs for the treatment of AR is listed in Table 329 They are used intermittently for seasonal disease and continuously for perennial disease.30 Efficacy should be reviewed after 2–4 weeks in treatment-naïve patients. If still symptomatic, a combination of INCS and antihistamine is advised.Intranasal corticosteroids are the pharmacological treatment of choice for all forms of AR as they are effective against a wide range of symptoms.21 Hydrating the nose with saline may reduce these side effects. Erroneously aiming the spray towards the nasal septum is an important contributor to local side effects.Side effects of INCS are mostly because of local irritation and include nasal dryness, a burning sensation inside the nose, blood-tinged nasal secretions and epistaxis.46 Molecules such as fluticasone propionate, fluticasone furoate, ciclesonide and mometasone furoate have increased glucocorticoid receptor selectivity and binding affinity and greater uptake and retention in the nasal tissue and have negligible systemic bioavailability (< 1%) compared to molecules such as budesonide, beclomethasone dipropionate and triamcinolone.47All newer INCS are safe and effective. However, these molecules have structural differences that influence glucocorticoid receptor-binding affinity and topical anti-inflammatory potency. These differences also alter the physicochemical properties such as solubility, lipophilicity and permeability, which in turn influence the pharmacokinetic properties and the systemic activity and therapeutic index.50Intranasal corticosteroids decrease the release of inflammatory mediators and cytokines from inflammatory cells and provide effective symptomatic relief when used continuously or as needed. They are most effective when used regularly, or at least in prolonged ‘blocks’ of treatment, as the onset of action is 7 h to 12 h, with maximum benefit after 2 weeks of regular use. Intranasal corticosteroids with increased topical potency do not necessarily offer a therapeutic advantage relative to those with less potency.48 Care should still be taken when multiple different steroid formulations  are used.Intranasal corticosteroids are less likely to cause systemic side effects  compared to oral and inhaled corticosteroids because of the lower dose and lower bioavailability.Short-term use of INCS drops can be considered for severe congestion (1–2 weeks), but long-term use of INCS drops, as opposed to nasal sprays, is strongly discouraged, as these have higher systemic bioavailability and are significantly more likely to cause systemic side effects.51 Complications associated with their use include hypothalamic–pituitary–adrenal‑axis (HPA) suppression, hyperglycaemia, osteoporosis, avascular necrosis of the femoral head and gluteal subcutaneous atrophy.51Depot intramuscular steroid injections should not be used for the treatment of AR.H1-AH dampen the effects of histamine during the early and late phase of allergic reactions. They are effective against itching, sneezing and rhinorrhoea but have little efficacy against congestion.1-AH are functionally classified as first- or newer- (second and third) generation AH. Third-generation formulations  are metabolites or enantiomers of second-generation AH and are theoretically safer and more efficacious than second-generation types.52H53 and high lipid solubility, causing significant blood–brain barrier transgression. Because of the non-selectivity of receptor binding and propensity to side effects ,53 SAARWG strongly discourages the use of first-generation AH in the routine management of AR.30 Second- and third-generation AH are less sedating than the first-generation AH because of reduced brain H1 receptor occupancy.54 Fexofenadine does not cross the blood–brain barrier. Rupatidine is a platelet-activating factor antagonist in addition to its antihistaminic properties.55 In a systematic review of 45 randomised controlled trials, second-generation AH use in children was generally safe; however, some may cause sedation in certain patients.56First-generation AH have poor receptor selectivity exclusive use of newer generation AH for AR treatment, with careful selection based on each patient’s unique profile. If side effects occur, a different, non-sedating AH may be tried; however, the EUFOREA guidelines on AR in children discourage AH switching, and an intranasal AH or INCS is preferred.57The SAARWG recommends the 58 They are effective and safe in children with AR.58 The major side effect is a bitter taste in the mouth, which is less with olopatadine than azelastine.57 However, INAH  are costly and not readily available in SA. In 2021, a combination intranasal spray, mometasone/olopatadine became available in SA, with approval for use in teenagers and adults.Topical INAH act rapidly (within 15 min) and have proven to be more effective than oral AH in the control of AR.59Nasal decongestants contain phenylephrine, oxymetazoline and xylometazoline that cause vasoconstriction of the nasal mucosa when applied topically, increasing airflow and relieving congestion. However, they have no effects on the other symptoms of AR and may worsen rhinorrhoea. Use for more than 5 to 10 consecutive days can cause rhinitis medicamentosa (rebound congestion). They should be used only for a short period when nasal congestion is dominant and always with an INCS.59 They are less effective than INCS in improving overall symptoms and QoL and should not be used as first-line treatment.25 Combinations of AH and LTRA are discouraged because of cost, unless concomitant asthma  is present.17 In this case, an LTRA  should be considered as an add-on to INCS, together with guideline-directed asthma treatment.60 A summary of South African Allergic Rhinitis Working Group – recommended practice points for the pharmacological treatment of allergic rhinitis is available in 60Leukotriene receptor antagonists are similar in efficacy to oral AH but are more effective in improving night-time than daytime nasal symptoms than AH.17Allergen immunotherapy is a desensitisation process for IgE-mediated hypersensitivity to common allergens such as pollens, HDM and insect venoms. It is the only disease-modifying treatment available for AR.63The administration of high-dose allergen, using sublingual (SLIT) or subcutaneous (SCIT) immunotherapy, suppresses the pro-allergic dendritic cell phenotype by inducing T-cell differentiation to regulatory phenotypes (Tregs). Induction of B regulatory cells to stimulate blocking antibodies further reduces mast cell degranulation.66 Allergen immunotherapy studies have further demonstrated a reduced need for asthma medications67 and a reduction in new aeroallergen sensitisations.65Allergen immunotherapy improves short- and long-term symptom severity, decreasing the need for medication for AR and protecting against the progression from AR to asthma.68Subcutaneous and SLIT are both effective once the causative allergen has been accurately identified by history and allergen sensitisation tests. Allergen provocation tests might be necessary in cases of high suspicion and inconclusive allergy test results.68According to the ARIA-EAACI care pathway, both monosensitised (single dominant antigen) and polysensitised (multiple antigens) patients can benefit.Patients who should be considered for AIT include the following:Those in whom symptom control is not achieved with pharmacotherapy and allergen avoidance.Those in whom high medication doses with potential side effects are required, particularly corticosteroids.Those in whom adverse events have occurred on normal doses of pharmacotherapy.Those who would prefer not to have to take pharmacotherapy for prolonged periods.Children in whom AIT would potentially be a modifying intervention to prevent further sensitisations and to reduce the chance of developing asthma.Potentially adolescents or adults with pollen-food syndrome.69The duration of treatment should be for at least 3 years but needs to be individualised and might need to be continued for up to 5 years according to symptom severity and control.71Asthma should be well controlled, and practitioners should be well versed in the management of adverse events, including rare cases of anaphylaxis.Currently, acquisition of AIT is made difficult by the fact that it is an expensive, unlicensed product with a single distributor in South Africa. For greater access, it would be preferable for products to be registered with the SA Health Products Regulatory Authority (SAHPRA). The acquisition cost of AIT is high, but the reduction in morbidity and medication costs makes it cost-effective.72 Tests of control should be validated and quick and easy to perform in routine clinical practice.73 Validated control tests include the ‘Control of Allergic Rhinitis and Asthma Test’ (CARAT), ‘Rhinitis Control Assessment Test’ (RCAT), ‘Allergic Rhinitis Control Test’ (ARCT) and ‘Sinonasal Outcome Test’ (SNOT) for CRS.28 The RQLQ questionnaire has been translated into Afrikaans, isiXhosa and isiZulu.74 The clinician should use the same control test consistently and regularly to monitor the AR.Visual analogue scales (VAS)  or AR control tests are increasingly used to evaluate control and treatment response, to detect adverse effects and to gauge the need for treatment adjustment in a reproducible manner.Patients with AR can be successfully initiated on AR treatment by general practitioners. Treatment success should be evaluated 2–4 weeks after the initiation of therapy.The following are indications for referral to a specialist :Poor or no response to treatment (based on VAS assessment).Need for initiation of AIT.Assessment of aeroallergen sensitisation if not available at the primary health care level.Atypical nasal symptoms and signs, including unilateral involvement, epistaxis and anosmia.The presence of nasal polyps, septal perforation, facial deformities and significant cervical lymphadenopathy.Severe co-morbid allergic diseases .Warning symptoms and signs of a possible underlying immune deficiency .The presence of severe ocular involvement .76; to improve access to topical medications and to decrease disease burden before other procedures such as AIT, aspirin desensitisation or initiation of biological therapies.79Surgery for ‘pure’ AR is rarely needed but may be needed in severe cases to improve airflow 18 Such patients should be referred for appropriate procedures by a surgeon skilled in rhinology and base of skull surgery.Surgery may be needed to manage complications of AR such as chronic or recurrent otitis media or for overlapping chronic conditions involving the nose and sinuses such as CRS with and without nasal polyps, non-steroidal (aspirin) exacerbated respiratory disease, cystic fibrosis, eosinophilic granulomatous polyangiitis (Churg-Strauss syndrome) and allergic fungal rhinosinusitis.Multi-morbidity is defined as the presence of one or more additional disorders co-occurring with a primary disorder. Multi-morbidities associated with AR include the following:Allergic disorder spectrum: asthma, atopic dermatitis, food allergy, eosinophilic oesophagitis, allergic conjunctivitis and anaphylaxis.Disorders of the upper airway, middle ear and Eustachian tube disease, sinusitis, turbinate and adenoid hypertrophy and pharyngeal and laryngeal disorders.Sleep disorders with secondary effects on concentration, behaviour and mood.80Treatment of AR will often result in an improvement of these associated multi-morbidities.Allergic rhinitis causes significant, often unappreciated, morbidity in the community. It is a complex disease related to an inflammatory response to environmental allergens. Therapy involves education, evaluation of allergen sensitisation, pharmacological treatment, AIT and evaluation of the success of interventions. Regular use of saline, the important role of INCS, including those combined with topical antihistamines and the reduction in the use of systemic steroids are key. Practitioners should have a thorough knowledge of associated morbidities and the need for specialist referral.


The androgen receptor (AR) is a critical target in all the clinical stages of prostate cancer. To identify a new AR inhibitor, we constructed a new screening system using the androgen-dependent growth of prostate cancer cell lines as a screening indicator. We screened 50,000 culture broths of microorganisms using this screening system and found that the fermentation broth produced by a fungus inhibited androgen-dependent growth of human prostate cancer LNCaP cells without cytotoxicity. Purification of this culture medium was performed, and this resulted in deoxynortryptoquivaline (DNT) being identified as a novel inhibitor of AR function. DNT showed potent inhibition of androgen-dependent growth of human prostate cancer LNCaP cells. The AR competitor assay was performed, and DNT did not act as an AR antagonist. However, DNT inhibited AR-dependent transcriptional activity and AR nuclear translocation, it suggested that the suppression of AR function leads to inhibition activity against androgen-dependent growth. The estimated cancer incidence in Japan in 2021 was approximately 1,009,800 of which 95,400 was prostate cancer and this was the most common male cancer site  forms a complex with its fluorescence-tagged ligand, resulting in a high polarization value. Competitors displace the fluorescently tagged ligand from the AR/ligand complex, resulting in reduced polarization values. DNT did not displace the fluorescent ligand from the complex and showed a high polarization value. By contrast, dihydrotestosterone  and bicalutamide showed low fluorescence polarization with IC50 values of 1.1 and 490 nM, respectively . This reNext, we researched the effect of DNT on AR transcriptional activity using a dual luciferase reporter system. In this assay, all three human prostate cancer cell lines LNCaP, VCaP, and 22Rv1 cells showed an induction of luciferase activity with R1881. DNT inhibited the AR transcriptional activity in a dose-dependent manner in LNCaP, VCaP, and 22Rv1 cells, but the inhibitory effect on VCaP was slight .Next, we investigated the effect of DNT on the expression of PSA mRNA expression in LNCaP cells. DNT repressed R1881-induced expression of PSA mRNA at 24 h of treatment .Moreover, we investigated the effect of DNT on the expression of AR and PSA using western blotting in LNCaP cells. DNT did not affect the expression of AR protein and slightly reduced the expression of PSA protein. We determined the change of cell-cycle proteins and found that DNT repressed the expression of the cyclin D1 protein .Finally, we investigated the effect of DNT on the AR nuclear translocation in LNCaP cells. Western blotting showed that R1881 induced and DNT repressed AR nuclear translocation in LNCaP cells  and 3E. AR is a principal driver of prostate cancer progression and an important therapeutic target for all clinical stages of prostate cancer. AR is activated by binding to androgens, dissociating from heat shock proteins and chaperones, dimerizing, and translocating into the nucleus. It then binds to the androgen response elements in the regulatory region of AR-dependent genes and activates the androgen signaling pathway .Although second-generation AR antagonists have improved the overall survival of patients with prostate cancer, both androgen-dependent and CRPC, but potential side effects have been reported. Enzalutamide can cross the blood–brain barrier and antagonize the GABAα receptor in the central nervous system (CNS), and thus it is associated with CNS-related events such as seizure and falls –32. The We selected the androgen-dependent growth of a prostate cancer cell line as a screening indicator because androgen-dependent growth reflects the androgen activity and can detect multiple AR signaling steps. Human androgen-sensitive cells, LNCaP and VCaP, can grow with R1881 stimulation, and androgen-dependent growth was detected using LNCaP and VCaP cells. Because the inhibition of androgen-independent growth is an indication of cytotoxicity, a compound which inhibits only androgen-dependent growth is a candidate. Consequently, androgen-dependent and androgen-independent growth of LNCaP and VCaP cells needed to be differentiated. However, the common medium FBS contains androgens or AR ligands and activates the androgen signaling pathway. Because of this, 18 different sera were screened using a reporter assay to identify an FBS that does not activate the androgen pathway and it was found that LNCaP and VCaP cells grew in serum 18, which slightly activated the androgen pathway . MoreoveWe screened 50,000 culture broths of microorganisms and found two broths that showed specific androgen-dependent growth inhibition in LNCaP prostate cancer cells. One of them contains androprostamines that have been previously reported by us ,37 and tAndroprostamine A repressed R1881-induced androgen-regulated gene expression, and dramatically inhibited R1881-induced prostate-specific antigen (PSA) levels. However, APA did not act as an AR antagonist, and did not inhibit AR transcriptional activity. Androprostamine A significantly inhibited the growth of VCaP cells in severe combined immunodeficient mice upon oral administration , howeverIn conclusion, through this screening study, we identified that DNT specifically inhibited the androgen-dependent growth of human prostate cancer cells. DNT inhibited the AR-dependent transcriptional activity and AR nuclear translocation. Unfortunately, although DNT did not show an anti-tumor effect in mice due to the short half-life in blood, derivative research of DNT may overcome this challenge.Prostate cancer is a long-term disease and progresses from androgen-dependent to CRPC and NEPC with various treatments. While the prognosis is good for patients diagnosed with localized disease, the prognosis remains poor for patients with advanced or metastatic prostate cancer.This study proposed a new screening method for the detection of AR function inhibitors and identified DNT as a possible anti-prostate cancer agent.


Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for several ligands, is highly expressed in many kinds of mesenchymal stem cells (MSCs), but its function is poorly understood. In this study, we explored the roles of full-length NRP1 and glycosaminoglycan (GAG)-modifiable NRP1 in adipogenesis in C3H10T1/2 cells. The expression of full-length NRP1 and GAG-modifiable NRP1 increased during adipogenic differentiation in C3H10T1/2 cells. NRP1 knockdown repressed adipogenesis while decreasing the levels of Akt and ERK1/2 phosphorylation. Moreover, the scaffold protein JIP4 was involved in adipogenesis in C3H10T1/2 cells by interacting with NRP1. Furthermore, overexpression of non-GAG-modifiable NRP1 mutant (S612A) greatly promoted adipogenic differentiation, accompanied by upregulation of the phosphorylated Akt and ERK1/2. Taken together, these results indicate that NRP1 is a key regulator that promotes adipogenesis in C3H10T1/2 cells by interacting with JIP4 and activating the Akt and ERK1/2 pathway. Non-GAG-modifiable NRP1 mutant (S612A) accelerates the process of adipogenic differentiation, suggesting that GAG glycosylation is a negative post-translational modification of NRP1 in adipogenic differentiation. Adipogenesis is a complicated process that involves multipotent stromal cell commitment toward preadipocytes, followed by proliferation and differentiation of preadipocytes into mature adipocytes . The imbNeuropilin 1 (NRP1) is a highly conserved transmembrane glycoprotein and plays a co-receptor role for multiple ligands, such as class 3 semaphorins (Sema3A) , vasculaC-Jun N-terminal kinase (JNK)–interacting protein 4  was first identified as a scaffold for mitogen-activated protein kinase (MAPK) signaling modules . SubsequThe specific purpose of this study was to investigate the role of NRP1 in the response to the adipogenic induction in C3H10T1/2 cells, and to reveal the underlying molecular mechanisms of full-length NRP1 and its GAG-modifiable form in adipogenic differentiation. We demonstrated that the full-length NRP1 and its GAG-modifiable form were involved in adipogenesis in C3H10T1/2 cells. NRP1 binds to JIP4 and promotes adipogenic differentiation by activating the Akt and ERK1/2 signaling pathways. Interestingly, there is a novel observation that overexpression of non-GAG-modifiable NRP1 mutant (S612A) promotes adipogenic differentiation in C3H10T1/2 cells. These findings elucidated a new mechanism of NRP1-mediated adipogenesis of C3H10T1/2 cells.C3H10T1/2 cells can differentiate into adipocytes A. After C3H10T1/2 cells were infected with a control lentivirus (sh-cont) or with lentiviruses containing shRNA for NRP1 (sh1 and sh2). The stable single-cell clones  were selected. Sh2-1 showed the highest decrease in NRP1 mRNA A and proAfter the induction of adipogenic differentiation, numerous neutral lipid droplets stained with oil red O were observed in C3H10T1/2 cells. NRP1 silencing resulted in a decreased number of neutral lipid droplets as determined by direct observation or quantification C,D. Sh2-In our study, JIP4 remained highly expressed before and after adipogenic differentiation in C3H10T1/2 cells A. To anaWe next examined in greater biochemical detail whether NRP1 may promote adipogenic differentiation by binding JIP4. First, NRP1 WT overexpression was achieved through transduction of lentivirus containing the NRP1 gene. The expression levels of NRP1 in these cellular models were determined using qPCR and Western blotting A,B. SecoIt is well established that NRP1 can be post-translationally modified by the addition of GAGs at Ser612 , phosphorylated Akt (CST), Akt (CST), phosphorylated ERK (CST), ERK (CST), and GAPDH (CST). Signal was detected with horseradish peroxidase–conjugated secondary antibodies (diluted at 1:2000) and Immobilon Forte Western HRP substrate detection substrate (Millipore) using a Bio-Rad Chemidoc imaging system. The proteins were normalized to GAPDH, and the intensity of each band was quantified using ImageJ software .C3H10T1/2 cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 3 min on ice. After blocking with 5% bovine serum albumin (BSA) in PBS for 2 h, the cells were incubated with corresponding antibodies. For localization of NRP1 and β-actin, the cells were incubated with NRP1 antibody  overnight at 4 °C, and then incubated with Alexa Fluor 594–conjugated secondary antibodies  (1:300), followed by staining with ActinGreen™ 488 ReadyProbes™ Reagent (Invitrogen). The stained cells were counterstained with DAPI and were observed using a confocal laser scanning microscope LSM780  at Central Research Laboratory, Okayama University Medical School. The procedure of colocalization of NRP1 and JIP4 with immunocytochemistry was shown in ® Universal qPCR Master Mix  in a LightCycler System . The relative number of mRNAs was determined through the comparative threshold cycle (ΔΔCT) method and was normalized to the levels of β-actin mRNA. Primer sequences are listed in RNA was isolated from C3H10T1/2 cells using Trizol reagent  in accordance with the instructions of the manufacturer. cDNA was generated from 1 μg of mRNA using PrimeScript™ RT Master Mix . Analysis of gene expression was performed using LunaE. coli DH5α competent cells (Takara). The plasmid was extracted and sequenced, and the lentivirus was packed with HEK 293T cells.Based on the results of bioinformatics, we selected two short-hairpin RNA (shRNA) molecules targeting mouse NRP1 sequence . The lentivirus vector pLKO.1-puro used in this experiment was produced and kindly provided by Dr. Takarada (Okayama University). Briefly, two single-stranded DNA molecules were designed and synthesized. Then, double-stranded DNA, formed by annealing the single-strands, was connected to the linearized vector, and was transformed into C3H10T1/2 cells were transduced with lenti-sh1, lenti-sh2, or empty vector (referred to as lenti-sh-cont). Following transduction, the cells were selected with 5 μg/mL puromycin. Then, stable single-cell clones were selected and verified by qPCR and Western blotting. The clones sh-cont, sh1-1, sh1-2, sh2-1, and sh2-2 were finally selected for subsequent experiments.C3H10T1/2 cells were transfected with Ambion Silencer Select siRNA targeted to mouse JIP4 , or negative control siRNA (Invitrogen) at a final concentration of 50 nM, using 2 μg/mL polyethylenimine (PEI) (Invitrogen) in an antibiotic-free medium. All experiments were performed 48 h after transfection, and the efficiency of transfection was confirmed using qPCR and Western blotting.The mouse NRP1 wild type with Flag (NRP1 WT), NRP1 S612A mutant with Flag (NRP1 S612A), and control plasmid (cont) were designed, synthesized, and cloned into the GV218 vector by the GeneChem Corporation . Pseudotyped lentiviral vectors were produced in HEK293T cells transiently co-transfected with 10 μg of the corresponding lentiviral vector plasmid, 6 μg of the packaging plasmid pSPAX2 , and 6 μg of the VSV g envelope protein plasmid pMD2G (Addgene) using Lipofectamine Plus reagent, in accordance with the manufacturer’s instructions (Invitrogen). The cells were infected in the presence of 4 μg/mL polybrene (Sigma-Aldrich) and selected with 10 μg/mL puromycin (Sigma-Aldrich).g, and resuspended in 1 mL of lysis buffer . The lysate was incubated overnight at 4 °C in rotation with 40 μL anti-Flag M2 affinity gel (Sigma). On the next day, the complex was washed five times with lysis buffer and incubated for 30 min with 100 μg/mL of Flag Peptide (Sigma) at 4 °C in rotation. The supernatant was separated in a fresh tube and 20 μL of electrophoresis sample buffer was added, boiled for 5 min, and examined via Western blotting.C3H10T1/2 cells cultured in 100 mm plastic dishes were washed twice with PBS, scraped into PBS, pelleted at 3000× t-test for comparison of two groups. Asterisks indicate statistically significant differences . All of the statistical analyses were performed using GraphPad Prism 8 for Windows . The experiments were repeated at least three times, and representative images are shown in the figures.All data are presented as the mean ± standard deviation (SD). Data were analyzed using either a one-way analysis of variance (ANOVA), followed by a Tukey’s post hoc test for comparison of multiple groups, or an independent Student’s


Augmented reality (AR) and virtual reality (VR) have increasingly appeared in the medical literature in the past decade, with AR recently being studied for its potential role in remote health care delivery and communication. Recent literature describes AR’s implementation in real-time telemedicine contexts across multiple specialties and settings, with remote emergency services in particular using AR to enhance disaster support and simulation education. Despite the introduction of AR in the medical literature and its potential to shape the future of remote medical services, studies have yet to investigate the perspectives of telemedicine providers regarding this novel technology.This study aimed to understand the applications and challenges of AR in telemedicine anticipated by emergency medicine providers with a range of experiences in using telemedicine and AR or VR technology.Across 10 academic medical institutions, 21 emergency medicine providers with variable exposures to telemedicine and AR or VR technology were recruited for semistructured interviews via snowball sampling. The interview questions focused on various potential uses of AR, anticipated obstacles that prevent its implementation in the telemedicine area, and how providers and patients might respond to its introduction. We included video demonstrations of a prototype using AR during the interviews to elicit more informed and complete insights regarding AR’s potential in remote health care. Interviews were transcribed and analyzed via thematic coding.Our study identified 2 major areas of use for AR in telemedicine. First, AR is perceived to facilitate information gathering by enhancing observational tasks such as visual examination and granting simultaneous access to data and remote experts. Second, AR is anticipated to supplement distance learning of both minor and major procedures and nonprocedural skills such as cue recognition and empathy for patients and trainees. AR may also supplement long-distance education programs and thereby support less specialized medical facilities. However, the addition of AR may exacerbate the preexisting financial, structural, and literacy barriers to telemedicine. Providers seek value demonstrated by extensive research on the clinical outcome, satisfaction, and financial benefits of AR. They also seek institutional support and early training before adopting novel tools such as AR. Although an overall mixed reception is anticipated, consumer adoption and awareness are key components in AR’s adoption.AR has the potential to enhance the ability to gather observational and medical information, which would serve a diverse set of applications in remote health care delivery and education. However, AR faces obstacles similar to those faced by the current telemedicine technology, such as lack of access, infrastructure, and familiarity. This paper discusses the potential areas of investigation that would inform future studies and approaches to implementing AR in telemedicine. Telemedicine, telehealth, and virtual health are terms commonly used interchangeably. Telemedicine focuses on clinical services, whereas telehealth entails remote health care services that can be clinical or nonclinical in nature, including public health and health care administration purposes  Interviewees also described the potential of AR to allow concurrent access to reference images or guidelines that they would normally access through the internet, such as specific physical examination findings, anatomy, or expected ranges for laboratory test values:There’s the ability to, rather than me running to go and try to look at my computer or look at my phone, to say “okay, what are some images of that again?” or “is this what Bell’s palsy looks like? Well, let’s pull up a picture of what Bell’s palsy facial weakness looks like.” And it’s right there, right at the eyeball. Assistance for clinical decision-making and for education.Participant 5The seamless integration of AR with internet-connected tools that collect clinical data was also of interest. Examples of information that could be displayed in the AR-enhanced field of view include real-time vitals from monitors and results from electrocardiograms, sonography, and stethoscopes at the patient’s location. An interviewee stated the following:We call them peripherals. Can I hear their heartbeat? Do I have a stethoscope? Can I listen to their lungs? These are features that would be great. So I could hear it myself rather than just going by secondary signs...If I could get the Apple Watch technology, to place that on and just see what’s going on for them, I think that’s where we’re at with this.Participant 13A few respondents commented about the role of artificial intelligence, in conjunction with AR, in identifying the appropriate data to display.Respondents mentioned the use of AR in facilitating inpatient consultations and commented about the potential for patients to access hard-to-reach specialists as outpatients. Interactions with more experienced experts could yield advice that supplements the information used for medical decision-making. Remote specialty consultants could obtain footage of the patient and provide clinical input or instructions for a specific process such as resuscitation or a stroke code. Respondents also commented about the potential for interpreters and tool specialists  to provide support remotely through the use of AR:Think smaller institutions that don’t have all the consults available to them. A lot of city hospitals don’t have ophthalmology hanging out in the middle of the night. That would be interesting, if you could do dilated eye exams...having people not come in the middle of the night, but still having the ability to get to the patient’s bedside and having a good point of view to do physical exams.Participant 11Respondents also described remote health delivery services that could integrate AR to allow remote experts to better support local staff, including emergency medical and disaster response services, teleparamedicine, telerehabilitation, and telewound programs. Few respondents mentioned uses in psychiatric care and less conventional settings including military, in-flight aircraft, and space.In the context of remote learning, interviewees felt that the addition of AR could enrich education beyond the teaching of subject matter, such as procedural and nonverbal skills including cue recognition and empathy. Furthermore, the integration of AR tools could enable long-distance training programs, including international training.Respondents commented about the use of AR for providing active feedback, step-by-step guidance, and planning, with a participant describing how AR’s ability to display hand gestures provided additional feedback alongside existing communication modalities:One of the opportunities that AR provides is that you can augment your verbal instructions with gestures, and those gestures are in the field of view of the local person...You have to think about how you add verbal, nonverbal and gestures together to enhance communication and jointly complete a task. They may be individually powerful but together, the most powerful tools.Participant 16Most of the interviewed participants  described the use of AR in teaching unfamiliar processes to the less experienced individuals, whether through interactive simulations and supervision of a trainee or allowing the trainee to shadow an experienced provider from a different location. Approximately half of the respondents  considered AR to particularly enhance simulations of high-risk case scenarios that are of low frequency. A respondent described how AR would allow instructors to remotely guide learners in real-time and gauge where the learner’s attention is focused without interrupting the simulation experience:One of the things that AR can help with is teaching thought process. Let’s say we do a simulation, and I have the learners going through a case. The only way I can really get into their thought process is by stopping the simulation and incorporating a “talking about it after the fact” kind of thing. Or pausing the simulation and talking about it. With AR, I can see what they’re looking at, and I can perhaps annotate in real-time. I can introduce labs in real-time. I can gesture and point them to look somewhere else. I can coach through the encounter, which allows them to continue to work through the case, and I can see what they’re seeing.Participant 1Among the most cited contexts for AR in remote procedural learning are both general and specialized telesurgery and minor interventions including those nonspecific to emergency medicine, such as intravenous access. An interviewee stated the following:You may have a PA and a surgeon remote. You may have a hospitalist and then an intensivist remote...That was the classic example of just telemedicine in general. So AR with telemedicine, you could screw that on. Interventionally then, you could do anything better essentially than what you’re already doing via telemedicine. You could walk someone through like ventilator settings or frankly, do actual true physical interventions. Like put a central line in, intubate someone, take the gallbladder out.Participant 6In addition, some respondents described use cases for AR in labor and delivery.Respondents saw the capability of AR to help educators teach remote learners, both nonmedical and medical, about the important cues to recognize, especially with the ability of AR to create persisting labels and instructions in the environment. Rather than relying on verbal instruction, AR allows the learner to visually experience and later recognize a previously unfamiliar signal as a finding associated with a specific outcome or danger. For those without medical training, such as patients and caregivers, AR-assisted visualization of specific signs or symptoms may better inform them about the outcomes to expect and the required action:The general categories are education, prevention, ongoing instructions and management. A lot of times when we’re discharging people from the emergency department, we’ll say to people, “Return if something worsens.” So I think even to illustrate signs and symptoms that should raise a flag or alarm them, that they need to get evaluated. [Participant 7] Nonphysicians could also use AR remotely to teach cue recognition, with a respondent describing how patients could learn to recognize fall hazards at home:Maybe the patient puts on some special glasses, and then the occupational therapist can see everything that the patient sees, and then the patient is kind of just walking around their house...and the occupational therapist can be looking at all these different features of the environment that might be a fall risk, and then advising them on how to reorganize.Participant 21In the case of health care trainees, remote teaching goes beyond foundational knowledge such as anatomy, as some respondents commented about the use of AR to provide experience-focused learning through empathy. Through AR, trainees could identify unspoken signs or even experience the circumstances that affect a patient’s health. A provider explained the following:For a third party to receive instruction, I think it would be nice to be able to see things from another person’s perspective...There’s this concept called embodiment that I think we really need in the health care setting in order to better understand your patients and the people around you.Participant 20Interviewees saw the potential for AR in long-distance education programs and global health, with the most cited example being teleultrasound. Respondents also mentioned the increased capability and capacity of remote hospitals and long-term care facilities as a result of AR enhancing what advanced centers can share with less specialized facilities. A participant described how rural hospitals could expand their services to avoid transferring patients:Rural areas do not have enough specialists. You can get full-on neurology—we talk about stroke all the time doing the full neuro exam, but they [rural areas] are doing it. You could probably even keep more patients than they already do. They keep a lot of patients within their hospitals already with telestroke programs and with this, can take it even further.Participant 8The adoption of any technology requires overcoming challenges related to the users, infrastructure, and technology itself. The interviewed providers described barriers that apply to both AR and innovations in general. First, the addition of AR to telemedicine may potentially exacerbate existing disparities. Second, providers require both evidence of clinical value and support to adopt AR. Finally, providers view the acceptability of AR to also be influenced by consumer preferences.Respondents described how the addition of AR to telemedicine would underscore the social determinants of health that already stratify patients in using general telemedicine, such as technology literacy, financial costs, equipment availability, and access to high-speed internet. Differences in technology literacy among providers may also lead to unequal care, as providers with more familiarity and access to AR devices may use them more effectively than others. An individual asked the following:Do you want your 60-year-old attending that’s been practicing for 20 years doing this because they’re the most experienced provider? Or do you want your intern, who’s spent their life playing video games and using Oculus, doing this? Even though they don’t have the same clinical knowledge?Participant 4Concerns more specific to AR devices include those related to users with disabilities or susceptibility to motion sickness and the responsibility of cost, storage, and upkeep of AR devices. Given that AR may require unique hardware to be used, participants described how some populations that already struggle to access health care may also lack the means to fix and receive education about how to properly care for the device. Some commented about whether it would be necessary to develop dedicated facilities to overcome barriers regarding storage space, maintenance costs, and financial limitations of patients that prevent travel. However, the space needed to use AR may also be challenging to establish in rural areas that lack access to specialized health care or in densely populated urban areas where dedicated spaces would be costly to create.Interviewees described that studies in the areas of efficacy, safety, patient satisfaction, and profit would influence providers’ acceptability of new technologies such as AR. Respondents described that efficiency also played a role, especially when considering the burden of wearing or being seen with an AR headset; difficulty in building rapport with patients; time consumed to set up tools; and availability of preexisting, less complex methodologies. An interviewee described the following:Actually having a clinical benefit is not enough. Clinical benefit, and it either needs to make me more money or make my day easier and more efficient. You need one plus one, and ideally you need all three. Otherwise, adoption will be heavily limited. [Participant 5] Respondents also commented about the need for use infrastructure and institutional support, particularly with the use of AR tools occurring at an early stage of a provider’s training:I think if you start this in med school, obviously you’re going to get a higher rate of acceptance that we can grow into a residency, and then attendingship, just like ultrasound. I grew up when ultrasound was still beginning, and now you can’t imagine being without it. I’m still lousy at it because I didn’t grow up with it in residency...Participant 17Interviewees commented that consumer adoption and awareness about AR would play a role in the acceptability of AR in health contexts, with some interviewees drawing comparisons with technologies adopted by consumers such as smartphones and VR devices:I think we’ve shown that well-designed technology is engaging and can easily be adopted by large numbers of people. We all have mobile devices now—we didn’t have them 15 years ago and I think that the barriers are going to be how well the technology is developed to be intuitive and engaging. It needs to be at the point where it’s the way an Apple product is, right?Participant 3Interviewees anticipate mixed responses from providers and patients to AR in telemedicine, with one-third  of the respondents expressing a sense of inevitability for AR to appear in the future of remote health care, if given time:For all technology adoption, there’s the early adopters and then on the other side, the laggards...They’ll be exceptions and it’ll get to the point where it’s so effective and makes you so efficient, and your job so much easier that you can’t not use it, and at that point the laggards will join and I just, personally, believe that point is somewhere within 10 to 20 years from now...When the pandemic hit, everybody was like, “you can use telehealth because it works and it’s great” and “we really need to use this,” and so they just snap their fingers and let everybody use it. So something like that will happen again when the people basically just demand it.Participant 19We conducted semistructured interviews with emergency medicine providers with various levels of experience in telemedicine and AR technology to understand the opportunities and concerns for the intersection of the 2 aspects. We identified several areas of interest in which AR can assist in conducting remote medical communications, including the process of information collection and dissemination in various clinical settings. Furthermore, we identified multiple considerations for the implementation of AR, including barriers and facilitating factors both common to health care innovations and unique to AR.see-as-I-see perspective to remote users prompted the interest in using such technology to enhance the sharing of observed information. According to our interviews, this enhanced interaction facilitates evaluation and supervision, especially for step-by-step processes requiring active feedback, including procedures inside and beyond the operating room. Surgical specialties have explored this potential extensively, with telesurgery tools such as Proximie and Virtual Interactive Presence and AR allowing remote experts to see the operating field of surgeons and interact using real-time annotation and labeling [Although modern platforms used in telemedicine allow both audio and video communication, the video aspect does not always replicate the experience of human vision in usual in-person interaction. The ability of AR-enabled headsets and devices to provide a labeling -39. The labeling ,41 and wlabeling ,43. For labeling ,44,45. Rlabeling , with moAccess to useful views of the local user is not always sufficient, as swift clinical decision-making also requires the timely review of relevant past information and standard references. From our study, one of AR’s potential advantages is its real-time access to essential data that are conventionally obtained from a workstation or device elsewhere. Few studies have explored AR’s role in displaying visual aids to facilitate remote services related to emergency telemedicine ,44, but According to our study, AR is also perceived to enhance telecommunication with a remote expert capable of providing further insight or instruction crucial for health care delivery and education. Given the diversity of pathology encountered by emergency medicine providers, access to specialty opinion is paramount to providing accurate, quality care while creating the opportunity to expand the consulting provider’s capabilities. Some studies so far that have implemented AR technology for communicating with remote specialists involve telestroke , teleultAnother potential use perceived by interviewees involved the educational opportunities between the more and less clinically experienced users, as they use AR to interact remotely—the literature includes numerous papers on AR in surgical telementorship and training ,54 and fRegardless of the variety of uses that AR technology may offer for telemedicine, further investigation is needed to overcome the barriers that prevent its integration into general health care and education. Our study underscores the concerns regarding obstacles related to known social determinants of health  and limitations of the current infrastructure and design of AR devices. For those introducing AR into health care, the research necessary to support its adoption extends beyond potential clinical and educational benefits. Evidence of patient satisfaction and financial gains is also perceived to be essential, with an example of the latter being the use of AR-enabled devices for telecommunication to conserve personal protective equipment ,65,66. Fadjunct such as AR would likely encounter resistance from a user population whose attention is already divided. This study emphasizes AR’s potential to improve efficiency in acquiring and exchanging knowledge, suggesting that an AR-enabled, portable communication device that integrates clinical decision-making tools into a single space would offer advantages over the current, stationary workstations and pose a unique direction for innovation. Past devices such as Google Glass had the potential to contribute to many in-person and remote medical services but had limitations such as limited video quality and battery life that prevented further adoption [Another area of potential investigation involves the question of whether AR serves as an adjunct tool versus an independent, novel means for communication. Telemedicine providers often juggle multiple applications while engaging with the other user, such as the audio-video platform itself, EMR, and other internet-related databases the provider may use. Unifying these sources of data into a single interface has yet to be achieved, and the addition of a new adoption . A devicadoption .This study has several noteworthy limitations in its design. First, the interviews were conducted over the internet with brief video demonstrations of tools that interviewees would likely find unfamiliar. This form of elicitation, rather than an in-person session using the equipment, may have influenced their responses. Of note, despite only seeing 2 examples of AR to remotely interact with an at-home patient and caregiver, interviewees responded with a wide spectrum of settings and users when prompted. A second limitation relates to the population of study; participants were mostly practicing emergency medicine physicians recruited from urban, academic medical centers in the northeast region of the United States. Therefore, the perspectives of those interviewed in this study may not be generalizable to other regions; specialties; and different types of health care workers, such as nurses, technologists, and hospital administrators, to name a few. Several studies focusing on the caregiver, patient, and trainee perspectives of AR devices for specific telemedicine-related uses have shown positive acceptability -72, but We conducted semistructured interviews with emergency medicine providers to identify areas of interest and possible challenges for AR in telemedicine. Overall, 2 common themes that emerged included uses for AR to improve remote information collection and distance education. AR enhances the collection of observational and medical data through its ability to share another individual’s perspective and access references and consultants, all of which facilitate active feedback and clinical decision-making. AR supplements distance learning by enhancing step-by-step procedural guidance and teaching of important cues, with educational opportunities available for interactions among providers and between providers and nonproviders. Participants recognized multiple barriers to implementation, some familiar to telemedicine and others specific to AR devices, and the need for institutional support and consumer familiarity to facilitate adoption. On the basis of these findings, we discuss future directions for the research and design of AR devices in the remote, real-time health care setting.


In addition, Cu12a-π can be transformed into cluster-based nonporous adaptive crystals (Cu12b-NACs) via ligand-exchange following a dissociation-reassembly mechanism. Moreover, Cu12a-π can efficiently remove 97.2% of iodine from saturated iodine aqueous solutions with a high uptake capacity of 2.96 g·g−1. These prospective results positioned at cluster-based porous supramolecular framework and enlighten follow-up researchers to design and synthesize such materials with better performance.The assembly of cluster-based π-stacked porous supramolecular frameworks presents daunting challenges, including the design of suitable cluster building units, control of the sufficient C-H···π interactions, trade-off between structural dynamics and stability as well as understanding the resulting collective properties. Herein, we report a cluster-based C-H···π interaction-stacked porous supramolecular framework, namely, Cu12a-π, consisting of Cu The assembly of cluster-based π-stacked porous supramolecular frameworks presents daunting challenges. Here, the authors report a dynamic and transformable supramolecular framework based on Cu12 cluster and C-H···π-stacking with permanent porosity Recently, a series of crystalline porous materials (CPMs) were assembled through intermolecular weak non-covalent interactions, which involve hydrogen bond10, π···π or C-H···π stacking16, electrostatic interactions, van der Waals forces18, etc. Wherein, π-stacked organic frameworks (πOFs), one of the newly emerging CPMs, have greatly broadened the boundaries of porous science, but few examples associated to such CPMs have been reported. Moreover, most of the reported πOFs are stabilized not merely by pure π···π stacking interactions, whereas other forces are also involved to a greater or lesser extent, e.g. C-H···π stacking interactions12. As a result, it is hard to figure out how much the C-H···π stacking interactions contribute the stability of CPMs. Therefore, the construction of supramolecular networks with permanent pores stabilized by C-H···π stacking interactions is meaningful to investigate the performance of such a non-covalent interaction in CPMs.Inspired by fascinating molecular pores that nature has evolved, scientists have made great progress in constructing porous materials21. The rational construction of such CPMs may require the following two main factors: 1) the robust building unit; 2) the medium strength of the bridge linker, where the frame structure can balance the stability and flexibility. However, the most reported πOFs or HOFs (hydrogen-bonded organic frameworks) are constructed by organic building units, which may limit the stability on the process of dynamically structural transformation under drastic physical stimulation23. In addition, the further post-transformation of such OFs must be done through the redesign of the building unit. Therefore, the development of suitable building unit to rationally construct the C-H···π interaction-stacked porous supramolecular framework (PSF) with both dynamic structure and post-transformation is an important but currently challenging subject.In general, the CPMs stabilized by C-H···π interactions may show the similar dynamic behavior like third-generation MOFs due to the characteristic of non-covalent interactions, and can reversibly transform their structures in response to chemical guests via physical stimulationCu12a-π, based on non-covalent C-H···π interactions between cluster-based building units that are protected by 9-(prop-2-yn-1-yl)−9H-carbazole (Cbz-PrAH) and p-tert-butylthiacalix[4]arene (H4TC4A). Of these, the Cbz-PrAH contains extended aromatic ring, which is conducive to the formation of intra- and intermolecular non-covalent C-H···π interactions24; H4TC4A is a kind of macrocyclic ligand with intrinsic cavity structure29. As displayed in Fig. Cu12a), which can transform to guest-free PSF (Cu12a-π) through a dynamical single-crystal-to-single-crystal (SC-SC) fashion by removing guest molecules under heating and vacuum conditions. The monomer Cu12 nanocluster as a zero-dimensional (0D) cluster-based building unit, can be further supramolecularly assembled into C-H···π interactions-stacked PSF. Even more impressively, the post-transformation of Cu12a-π followed a dissociation-reassembly mechanism, giving the cluster-based nonporous adaptive crystals (NACs) Cu12b-NACs with updates of both structure and properties.To address these challenges, we designed a three-dimensional (3D) PSF, namely Cu12a, we selected copper(I) precursor, Cbz-PrACu  to react with H4TC4A via solvothermal method, giving the red rhombic crystals of Cu12a in a quite high yield of 91%  nanoclusters protected by Cbz-PrA‒ and TC4A4‒ ligands. The infrared (IR) spectroscopy confirmed the existence of Cbz-PrA‒ in Cu12a. Besides, elements of Cu12a were confirmed by energy-dispersive x-ray (EDX) spectroscopy. More synthetic details and basic characterizations are listed in the Supplementary Figures and Tables 4(TC4A)2]·(CH3COOCH2CH3). The structural anatomy of the monomer was shown in Fig. 4 units rotated by 45° in sequence, the equatorial copper atoms are bulged from the centres of four vertical faces of the cube about 0.446 Å. The Cu12 kernel exhibits a C4 symmetry with a C4 axis passing through the centres of two horizontal planes, and is consolidated by short Cu···Cu contacts (average distance of 2.76 Å), which are shorter than the sum of Bondi’s van der Waals radii of two Cu atoms (2.80 Å)30, implying an appreciable cuprophilic interactions. Furthermore, the types of capping ligands in the monomer of Cu12a are somewhat reminiscent of the Cu13a and Cu13b, where Cbz-PrA− and TC4A4− capped on the periphery of the copper kernel in our previous work31.Single-crystal X-ray diffraction (SCXRD) analyses revealed that the monomer of − ligands like nails hold only one coordination mode .The difference is that four Cbz-PrA36. Inspired by this, the guest EA molecules in Cu12a may endow it with similar behavior. Thus, the crystalline sample of Cu12a has been heated at 338 K upon vacuum for two days. Surprisingly, the activated sample of Cu12a, termed Cu12a-π, can still be analyzed via SCXRD, in which the guest EA molecules had been successfully removed to form permanent voids  layers of Cu12a and Cu12a-π both adopt AA stacking mode, but the latter occurred with a distinct displacement between each layers along the ab plane, resulting in a slight reduction in the porosity (from 11.5% to 9.1%). Impressively, upon further soaking Cu12a-π in EA solvent for three days, the lattice again recovered to P21/c accompanied with the re-adsorption of the EA molecules. This indicates that the Cu12a and Cu12a-π as host matrixes can achieve reversible SC-SC transformation by removal and re-adsorption of the guest molecule, thus demonstrating the crystalline structural flexibility of Cu12a. Thermogravimetric analysis (TGA)  for at least 72 h at room temperature, their PXRD patterns were highly consistent well with the theoretical patterns of Cu12a, which demonstrated the framework highly dynamic stability to chemical solvents  Fig.  indicate8 K Fig. . Besidesure Fig. , which ints Fig. . This ex12 cluster shows significant cavity structures above the poles of copper kernel, which is conductive to the close arrangement of cavity structures in one plane. And the higher rim of the cavity in Cu12 cluster shows a maximum pore diameter of ~8.9 Å. Therefore, Cu12 cluster can be regarded as a porous 0D cluster-based building unit, associated with two kinds of C-H···π interactions (C(sp3)-H···π(Cbz)=3.1 Å and C(sp2)-H···π(Cbz)=3.1 Å) between a carbazole ring and other carbazole ring, as well as between an allylene and a carbazole ring  stacking via almost negligible interlayer H···H interactions (2.26 and 2.93 Å) of tertiary butyl to form a final 3D supramolecular framework -H···π(Cbz)=3.2 or 3.4 Å and C(sp2)-H···π(Cbz)=2.8 Å) due to the incorporation of guest EA solvents.In Fig. ing Fig. , togetheork Fig. . Of thesCu12a-π, it is clearly seen that the small cavities provided by the thiacalix[4]arene and permanent voids between each layers effectively endow the Cu12a-π with certain porosity  surface area of 31.93 (45.29) m2·g−1, and a wide pore size distribution in the range of 0.8–1.6 nm calculated by Horvath-Kawazoe (HK) model  at normal pressure, further confirming the real existence of permanent porosity in Cu12a-π.On the basis of packing characteristic of 41. Inspired by this, the Cu12 nanocluster acting as a building unit may feature ligand-exchange to remodel the PSFs. Therefore, we tried to transform Cu12a-π by dissolving crystalline samples in an isopropanol/tetrahydrofuran (v:v = 3:3) mixture, to which an excess of H4PTC4A  was added, and then the above solution continued to react under solvothermal condition to generate red block crystals of Cu12b. Except for the p-phenyl-thiacalix[4]arene CT in Cu12b leading to the variation in the absorption intensity of the two nanoclusters at ~300 nm (Please see details in the Supplementary Methods); (ii) the position of the maximum absorption peaks of the two ligands (H4TC4A and H4PTC4A) . As shown in Fig. Cu12a and Cu12b in mixed CH3OH and CHCl3 exhibit a series of +1 species  at the m/z range of 3010-3090 and 3170-3345, respectively, which correspond to their intact cluster bound with different cations or solvents. The most dominant peak 1b at m/z 3036.9040 and peak 2b at m/z 3196.6478 can be attributed to [Cu12(TC4A)2(Cbz-PrA)4Na]+  and [Cu12(PTC4A)2(Cbz-PrA)4Na]+ , respectively. However, as the reaction of H4PTC4A and Cu12a proceeded in CHCl3, the parent peaks of Cu12a (1a−1e) progressively faded away within 20 min  appeared at 30 min, and can be assigned to [Cu12(PTC4A)(TC4A)(Cbz-PrA)4H]+. This species 3a is the mono-substituted product of Cu12a, in which one of the TC4A4- ligands is replaced by PTC4A4-. After 80 min, we observed a species 2j, [Cu12(PTC4A)2(Cbz-PrA)4(CH3OH)(H2O)2K]+, which can be identified as the bis-substituted product of Cu12a. This result indicates that Cu12b is more thermodynamically stable than Cu12a, which also was confirmed by DFT calculations . More formula assignments for the above species in ESI-MS were performed as listed in Supplementary Table To verify the assumption on the transformation mechanism, we monitored the ligand-exchange process in the structural transformation from min Fig. , which i12 clusters was initially presumed to be a simple ligand-exchange process based on the single crystal structure and optical monitoring. However, further investigation using ESI-MS tracking revealed that this transformation was not a straightforward ligand-substitution, but rather a kinetically driven dissociation of the initial cluster, followed by a reassembly to form a thermodynamically stable new cluster. Thus, we have established the dissociation-reassembly (DR) mechanism to describe above transformation process, highlighting the dynamic nature of the dissociation and subsequent reassembly steps. The DR mechanism provides a conceptual framework to understand the intricate dynamics involved in the transformation of Cu12a to the more stable Cu12b50.The transformation between the two Cu4‒, we further checked the packing formed by Cu12 cluster in Cu12b. There is one THF molecule residing in the cavity of each PTC4A4‒ in Cu12b, which reduces the void produced by PTC4A4‒ ligand itself, so the Cu12b is a nonporous structure. The activated experiment of Cu12b was further carried out, and the activated sample namely Cu12b-NACs had been successfully removed the guest THF solvents , which are relatively nonporous but with medium surface areas56. In light of this, the analysis of N2 adsorption isotherm of Cu12b-NACs gives an experimental Brunauer-Emmett-Teller (Langmuir) surface area of 14.88 (12.63) m2·g-1, which is less than half of that of Cu12a-π, and the pore-size distribution exhibits broad and ambiguous peak  image of I2@Cu12a-π showed the crystal-like morphology  and Cu12a-π, which has been reported previously for iodine adsorption materials66. Moreover, the I3d X-ray photoelectron spectrum (XPS) of I2@Cu12a-π was measured, and two broad peaks at ~620 and ~632 eV indicate the presence of the I3‒ and I5‒ anions, respectively  with cluster-based building units held together through intralayer non-covalent C-H··π interactions. The Cu12a-π is successfully constructed through the SC-SC transformation by the removal of guest molecules from pristine Cu12a, showing unexpected structural dynamics and superior thermal stability than most reported π-stacked frameworks. Moreover, the changing in upper rim of thiacalix[4]arene, Cu12a-π can be transformed into the nonporous adaptive crystal Cu12b-NACs following a DR mechanism, realizing the structural and performance updates. Based on the permanent porosity, Cu12a-π can absorb iodine in aqueous solution reaching a high capacity of 2.96 g·g‒1. Overall, all unique structural features as well as the iodine adsorption performance of such cluster-based PSF are indicative of a bright future of this type of porous materials.In summary, we have successfully developed a dynamic and transformable 3D PSF  were mixed in 6.00 mL methanol/ethyl acetate (v:v = 3:3). The resulting suspension was sealed and heated at 80 °C for 2000 min. After cooling to room temperature, red crystals of Cu12a were formed . Selected IR peaks (cm−1): 2950 (s), 2906 (w), 2860 (w), 1732 (s), 1584 (m), 1450 (s), 1364 (m), 1315 (m), 1257 (s), 1208 (m), 1037 (m), 926 (w), 885 (w), 840 (s), 751 (s), 711 (s), 625 (w), 531 (m).Cbz-PrACu  and HCu12a  were dissolved in mixed solvents of 6.00 mL isopropanol/tetrahydrofuran (v:v = 3:3), then H4PTC4A  was added successively. The resulting suspension was sealed and heated at 80 °C for 2000 min. After cooling to room temperature, red crystals of Cu12b were formed . Selected IR peaks (cm−1): 2955 (s), 2901 (w), 2865 (w), 1777 (w), 1589 (m), 1454 (s), 1364 (m), 1311 (s), 1255 (s), 1202 (m), 916 (w), 876 (w), 831 (s), 741 (s), 714 (s), 616 (w), 526 (m).The crystals of Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1-3Source Data


During the last few decades, the field of rehabilitation has experienced substantial development, growth and acceptance. Rehabilitation addresses the impact of a health condition on a person’s everyday life by optimising their functioning and reducing their experience of disability. Rehabilitation expands the focus of health beyond preventative and curative care to ensure people with a health condition can remain as independent as possible and participate in education, work and meaningful life roles .We also now have a better understanding of the growing global need, demand and recognition of rehabilitation around the world. For example, it has been estimated that 2.41 billion people in the world could benefit from rehabilitation services. This means that at least one in every three persons in the world needs rehabilitation at some point during the course of their disease or injury  seeks to understand and improve how societies organise themselves in achieving collective health goals and how different actors interact in the policy and implementation processes to contribute to policy outcomes   invite researchers in the field of HPSR for rehabilitation to submit their manuscripts to our journals for peer review and possible publication, (2) create a special journal section, series or designation dedicated to HPSR for rehabilitation, (3) appoint editorial board members with expertise in HPSR for rehabilitation and (4) disseminate research articles among funding agencies and policymakers. These actions by our academic journals will help the WRA achieve its goal of strengthening rehabilitation services for all.