Prostate cancer (PCa) is the most common malignant tumor with a high global incidence in males. The mechanism underlying PCa progression is still not clear. This study observed that NRP1 was highly expressed in PCa and associated with poor prognosis in PCa patients. Functionally, NRP1 depletion attenuated the proliferation and migration ability of PCa cells in vitro and in vivo, while NRP1 overexpression promoted PCa cell proliferation and migration. Moreover, it was observed that NRP1 depletion induced G1 phase arrest in PCa cells. Mechanistically, HIF1α is bound to the specific promoter region of NRP1, thereby regulating its transcriptional activation. Subsequently, NRP1 interacted with EGFR, leading to EGFR phosphorylation. This study also provided evidence that the b1/b2 domain of NRP1 was responsible for the interaction with the extracellular domain of EGFR. Moreover, EGFR mediated NRP1-induced activation of the AKT signaling pathway, which promoted the malignant progression of PCa. In addition, the administration of NRP1 inhibitor EG01377 significantly inactivated the EGFR/AKT signaling axis, thereby suppressing PCa progression. In conclusion, the findings from this study highlighted the molecular mechanism underlying NRP1 expression in PCa and provide a potential predictor and therapeutic target for clinical prognosis and treatment of PCa. Prostate cancer (PCa) is the most common malignant tumor with a high incidence in males. According to the 2020 global cancer statistics, PCa is the second most frequent cancer diagnosis made and the fifth leading cause of cancer death in men, with an estimated 1.4 million new cases and 375,000 deaths occurring annually [+ T cells. Therefore, NRP1 can be a checkpoint target with significant implications for cancer immunology and immunotherapy [Neuropilin 1 (NRP1) is a single-pass transmembrane glycoprotein, consisting of extracellular, transmembrane, and intracellular domains . The extotherapy , 18.Few researchers have previously revealed the oncogenic role of NRP1 in PCa. A retrospective case-control study reported that NRP1 endothelial expressions predicted distant relapse after radical prostatectomy in clinically localized PCa . AnotherIn this study, NRP1 was identified as a novel marker for PCa progression through transcriptome profiling, and the overexpression of NRP1 was correlated with unfavorable prognosis in PCa patients. The b1/b2 domain of NRP1 interacts with the extracellular domain of EGFR in a mechanistic manner to induce the activation of EGFR, which in turn, activates the AKT pathway, thereby promoting the proliferation and migration of PCa cells. Moreover, HIF1α is involved in the transcriptional regulation of NRP1. The findings from this study highlighted that the inhibition of the NRP1/EGFR axis can be a potential therapeutic approach for PCa.https://www.ncbi.nlm.nih.gov/geo/) on the Illumina platform, and the “limma” R package was used to process the gene microarray data. Gene set enrichment analysis (GSEA), Gene set variation analysis (GSVA) and KEGG pathway analysis were carried out to evaluate the pathways enriched in the NRP1 high- and low-expression subgroups. The msigdbr, enrichplot, clusterProfiler, and ggplot2 packages in R were used for analysis and visualization.The RNA-Seq data of the TCGA-PRAD and HRA000099 cohorts were downloaded from the TCGA database and The Genome Sequence Archive for Human, respectively. The RNA sequence analysis (siControl and siNRP1) was performed at Beijing Genomic Institute (BGI). The “EdgSeq” R package was used for the normalization of mRNA-Seq data. The microarray datasets of GSE10645 and GSE70769 were downloaded from the GEO database (http://www.biotechwell.com/index.php?m=home&c=View&a=index&aid=4610). During the experiments, five tissue sections were missing from the tissue microarray. Therefore, 75 tissue specimens were included for the statistical analysis. Ten PCa tissue specimens were generated from patients who underwent surgery at Zhongnan Hospital of Wuhan University. These tissues were formalin-fixed and paraffin-embedded for subsequent experiments. The pathological diagnosis of PCa tissue specimens was independently validated by two pathologists. This study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University , and the specimens met the defined standards of patient information and specimens. All the patients had signed informed consent before the study.Tissue microarray (TMA) containing samples from paired 80 tumor and paracancerous tissue specimens were purchased from Shanghai Weiao Biotechnology Co., Ltd  and colored according to the technical manual containing the TMA pathologic information, which is available from the website , Cycloheximide , Dactinomycin , and EG01377  were purchased from commercial sources indicated.All the cell lines were purchased from the Stem Cell Bank, Chinese Academy of Sciences in Shanghai, China, and verified by the China Centre for Type Culture Collection in Wuhan, China. PCa cell lines  were cultured in Gibco RPMI-1640 medium . HEK293T cell line was cultured in Gibco DMEM medium . All the cell cultures were supplemented with 10% fetal bovine serum (FBS)  and cultured at 37 °C in a humidified incubator containing 5% COTo construct expression plasmids encoding the human full-length NRP1 (NM_003873.7), HIF1α (NM_001243084.2), and EGFR (NM_001346897.2), the full lengths of NRP1, HIF1α, and EGFR protein-coding sequences were commercially synthesized and subcloned into pcDNA3.1 vector (Obio Technology). Then, full-length NRP1 and the corresponding truncated plasmids were subcloned into a pcDNA3.0-HA vector, whereas full-length EGFR and the corresponding truncated plasmids were subcloned into a pcDNA3.0-Flag vector. The small interfering RNAs targeting NRP1  were obtained from Genomeditech . The lentiviral vector containing short hairpin RNA for NRP1 was also constructed and identified by Genomeditech . Lipofectamine 3000  was used for cell transfection in accordance with the manufacturer’s instructions.Total RNA extraction was performed using RNeasy plus mini kits  following the manufacturer’s instructions. Subsequently, the quality of the extracted RNA was determined using the NanoDrop instrument . Then, complementary DNA was synthesized using the ReverTra Ace qPCR RT Kit . Finally, the qRT-PCR was performed on the QuantStudio 6 Flex System (Life Technologies). The specific primer sequences are shown in Supplementary Table 3 cells were seeded into 96-well plates (6 replicate wells per plate) for growth. Then, the 96-well plates were taken out of the incubator every 24 h for five consecutive days, and 20 μL MTT reagent  was added to each well. The plates were then placed again inside the incubator for 4 h, then 150 μL DMSO was added to each well. Finally, the absorbance of each well at 570 nm was tested using a microplate reader  to assess cell viability.The clone formation assay and methyl thiazolyl tetrazolium (MTT) assay were used to detect cell proliferation and viability. For the colony-formation assay, the cells were seeded into six-well plates at 800 cells/well in the desired medium. After 14 days, cells were washed with PBS, fixed with 4% paraformaldehyde (PFA), and stained with 0.5% crystal violet. For the MTT assay, 3 × 10The transfected PCa cells were sequentially digested and centrifuged. For cell cycle analysis, 1 × DNA staining solution  supplemented with propidium iodide was used to treat the cells for 30 min at room temperature. FITC Annexin V Apoptosis Detection Kit I  was used to stain cells for analysis of cell apoptosis. Then, the stained cells were incubated for 30 min at room temperature. The flow cytometry  was applied in the analysis of cell cycle and cell apoptosis.4 cells in 200 μL serum-free medium were seeded into transwell inserts  and placed in 24-well plates with 800 μL medium containing 10% FBS. After 24 h incubation, the cells were fixed with 4% PFA and stained with crystal violet. Finally, the samples were observed for recording images of cell migration. For the wound-healing assay, the transfected PCa cells were cultured in six-well plates to 80% density. Then, the cells were vertically scratched using a 200 mL pipette, and the scratched cells were then washed with PBS. 2 mL of culture medium was then added for further cultivating the cells for 12 h. Finally, the cells were observed to calculate the average wound gap between wound edges.Wound-healing assay and transwell assay were applied for detecting cell migration ability. For the transwell assay, 1 × 10IHC assay was performed according to the following protocol. Briefly, tissue specimens were fixed with 4% formalin and embedded in paraffin. Further, thin sections of tissue embedded in paraffin were cut, dewaxed, and rehydrated. The sections were then blocked in 10% goat serum at room temperature for 10 min and incubated overnight at 4 °C in primary antibodies. Then the sections were incubated in the appropriate secondary antibody and processed with streptavidin-peroxidase before counterstaining with hematoxylin. Specific primary antibodies against NRP1 , EGFR , p-EGFR , p-AKT , p-mTOR , and KI67  were used for IHC. The images were scanned and analyzed using a molecular microscope (Olympus BX53). TMA and IHC analyses were generally performed as described previously [PC-3 and DU-145 cells were washed with PBS followed by fixation with 4% PFA for 20 min at room temperature. The cells were again washed thrice with PBS and then incubated overnight at 4 °C in desired antibodies. After washing thrice in PBS again, the cells were incubated in fluorescein-labeled secondary antibodies for 40 min at 37 °C. After washing thrice again with PBS, cells were incubated in DAPI  at room temperature for 30 min and mounted in the mounting medium. The slides were then analyzed using a laser scanning confocal microscope .The total cellular protein was isolated using RIPA lysis buffer  supplemented with protease inhibitors . Equal amounts of total proteins were loaded on SDS-PAGE gels for separation of proteins according to their molecular weights and then transferred onto PVDF membranes . The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 and then incubated with primary antibodies overnight at 4 °C, followed by incubation with corresponding secondary antibodies at room temperature for 2 h. The protein bands were visualized using enhanced chemiluminescence. Immune response bands were exposed on a Bio-Rad ChemiDoe XRS + Imaging System . The primary antibodies used were NRP1 , EGFR , p-EGFR , CDK2 , CDK4 , CDK6 , CyclinD1 , E-cadherin , N-cadherin , AKT , p-AKT , GSK3β , p-GSK3β , mTOR , p-mTOR , FLAG , HA , and GAPDH .Co-immunoprecipitation (Co-IP) was performed as described in a previous study . The celThe possible binding sites of HIF1α on the promoter region of the human NRP1 gene were predicted using JASPAR matrix models. Based on the predicted sequences of the binding sites, full-length fragments containing the putative human NRP1 promoter region or a negative control region (Obio Technology) were cloned into the pGL4.1-basic plasmid. The pcDNA3.1-HIF1α overexpressing plasmid was also constructed (OBIO Technology). The constructed vectors were verified by sequencing. The PC-3 and DU-145 cells were co-transfected with specific pGL4.1-basic NRP1 promoter plasmids and Renilla luciferase reporter plasmid (pRL-TK) or pcDNA3.1-HIF1α expression plasmid. The luciferase activities were measured 48 h after transfection using the Dual-Luciferase Reporter Assay System . The ratio of firefly luciferase activity/Renilla luciferase activity was also calculated.6 in 100 µL serum-free medium) infected with LV-control (NC group) or LV-shRNA (sh-NRP1 group) lentivirus vectors were injected subcutaneously in the dorsal flank of each mouse. The tumor length and width were measured by a caliper every 5 days using the equation, i.e., tumor size (mm3) = (length × width2)/2. The xenograft growth was monitored for 6 weeks after injection. All the mice were euthanized to obtain fresh tumors and the tumor weights were recorded. Further, the xenografts were analyzed for the desired antibodies by hematoxylin and IHC staining. For the pulmonary metastasis model, the above cells (1 × 106 cells in 100 µL serum-free medium) were injected into the tail vein of each mouse. After growth for another 8 weeks, the fluorescence intensity of tumors was measured using the Living Image software . H&E staining of the lung tissues obtained from mice was used to evaluate lung morphology. The investigator was blinded to the treatment group to which the animals were allocated during the experiment.Animal experiments were performed following the National Institutes of Health Animal Use Guidelines. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Wuhan University. The 3-week-old male BALB/c-nu mice  were raised under a specific pathogen-free (SPF) environment. After 1-week adaption, the mice were randomly divided into 2 groups. For the xenograft mice model, PC-3 cells (5 × 10https://www1.rcsb.org/). The central site of the docking box was determined using AutoDock Tools (version 1.5.6), and AutoDock Vina was used for the virtual molecular docking. Then, the docking image of the best binding pose was generated by PyMOL (version 1.7.2.1).The three-dimensional structures of NRP1 and EGFR were drawn in Chem3D Ultra 14.0 software . The crystal structures of NRP1 (PDB ID: 2QQI) and EGFR (PDB ID:2GS2) proteins were obtained from the RCSB PDB database  and GraphPad Prism version 6.0  were used for statistical processing and plotting. Continuous data were presented as the mean ± SD. The differences in mean values between the two groups were compared using a two-tailed P < 0.05). Moreover, NRP1 expression gradually increased with the increase in Gleason score, and NRP1 expression was significantly higher in PCa patients with lymph node metastasis when compared with those without lymph node metastasis . Further, the correlation between NRP1 and clinicopathological characteristics in the TCGA-PRAD cohort was evaluated. As shown in Table P = 0.0011; Fig. P < 0.0001; P < 0.0001; Fig. P = 0.037; Table n = 44) and the high NRP1 group (n = 31). The chi-square analysis indicated that high NRP1 expression was positively associated with higher Gleason score, advanced stage, and lymphatic metastasis  was used to predict the binding site of HIF1α on the NRP1 promoter region. Based on the canonical binding motifs of HIF1α  were chosen to investigate the biological function of NRP1 Fig. . As NRP1Further, the role of NRP1 in tumor growth and migration in the xenograft mouse model was investigated. Firstly, PC-3 cell lines with stable depletion of NRP1 by lentivirus-based shRNA were obtained Fig. , and theGSEA analysis was performed based on the TCGA-PRAD dataset to investigate the potential mechanism of NRP1 in malignant PCa progression. This indicated enrichment of EGFR-related signaling in the samples with highly expressed NRP1 Fig. . FurtherThe N-terminal region of NRP1 contains three domains, each of which binds to different proteins and mediates different signaling pathways. EGFR contains an extracellular ligand-binding domain at the N-terminus, a transmembrane lipophilic region, and an intracellular region containing the tyrosine kinase domain at the C-terminus. To identify the key region of NRP1 interacting with EGFR, three domain truncation mutants of NRP1 were cloned Fig. , and EGFRNA-Seq analysis was performed using NRP1-depleted DU-145 cells and control cells to investigate the downstream signaling pathways and functional processes that were affected by NRP1-mediated transcriptional regulation. The GSVA result indicated significant downregulation of the AKT pathway in NRP1-depleted DU-145 cells compared to controls Fig. . The KEGTo investigate the clinical significance of the NRP1 inhibitor, the effects of a novel inhibitor of NRP1 (EG01377) were further evaluated in PCa progression. Different concentrations of EG01377 were used for the treatment of PC-3 and DU-145 cells. MTT and clone formation assay demonstrated that EG01377 significantly attenuated cell proliferation and clone formation ability in a concentration-dependent manner Figs. . The traPCa, one of the most common urinary tumors, is the fifth leading cause of cancer-related deaths in men and has become a global threat to men’s health. However, the molecular mechanisms underlying PCa progression are still unknown, leading to poor prevention and treatment strategies for advanced PCa, especially CRPC and mCRPC. Therefore, a comprehensive study of molecular biological mechanisms and cellular pathways underlying PCa progression can help to explore novel intervention strategies and drug targets for PCa.In this study, the focus was on NRP1 and its oncogenic role in PCa. It was observed that NRP1 was overexpressed in PCa, positively associated with adverse clinicopathologic factors, and an independent predictor for poor survival. In PCa cells, NRP1 promoted malignant phenotypes, including cell viability and migration, tumor growth, and metastasis. Mechanistically, NRP1 binding triggered EGFR phosphorylation, thereby activating the AKT/mTOR pathway to promote malignant PCa cell behavior. Moreover, HIF1α is involved in the transcriptional regulation of NRP1, as shown in Fig. NRP1 is a single-pass transmembrane glycoprotein belonging to the neuropilin family. A previous study reported that NRP1 is highly expressed in several tumors, and an increase in its level correlated with tumor progression –30, thusHypoxia is a common feature of solid malignancies including PCa . Under hAlthough high expression of NRP1 is caused by HIF1α, the exact mechanism involved in the promotion of cell growth in PCa by NRP1 needs to be investigated. In this study, GSEA revealed that NRP1 regulated EGF/EGFR signaling axis in PCa. EGFR is a member of the ErbB transmembrane growth factor receptor family . It is pAberrant EGFR can modulate several downstream pro-oncogenic signaling pathways, including PI3K/AKT pathway, Ras/Raf/MAPK pathway, and JAK/STAT pathway –46. ThesIn conclusion, this study demonstrated a novel signaling regulatory axis in PCa progression. As shown in Fig. Original Data FileSupplementary FiguresTable S1reproducibility checklist


Nanostructure of TiC and TiO2 in the anatase and rutile phases was obtained through heat treatment of TiC at different times and temperatures (TiC@AR) which led to a reduction in energy bandgap from UV to visible light, in addition to the enhancement of the surface activity. After TiC@AR polymerization by xylan and polyvinyl alcohol and obtaining TiC@AR/PX, the energy bandgap reduced to IR range (52% of the sunlight) while showing an enhancement in the surface activity. The photocatalytic activity of the compounds was tested by studying the decomposition of BPB solution under visible light. The result illustrated the ability of TiC and TiC@AR to decrease the concentration of BPB after 150 min by 35% and 37%, respectively, while this reduction was 72% for TiC@AR/PX. Considering the effective parameters, the energy bandgap and the surface layer played key roles in photocatalytic degradation.In this study, a composite bearing titanium carbide (TiC), titanium dioxide (TiO Various approaches have been developed to combat these health problems, among which coagulation  and biol2 and H2O . Th. Tha). Nb,e,h). Based on the result, the surface activity of TiC is 5.67 m2 g−1 which exhibited a fourfold enhancement (20.195 m2 g−1) after converting to TiC@AR (2 g−1). Based on a/mol g−1, while this parameter was 0.001 and 0.0035 na/mol g−1 for TiC@AR and TiC@AR/PX, respectively, implying a step-wise increase in the surface of the samples.BET analysis was used to determine the surface activity of the samples b,e,h. Bao TiC@AR . Upon coc,f,i). While total pores of TiC and TiC@AR are, respectively, 0.011 and 0.031 with three times growth, this parameter became 0.11 cm3 g−1 for TiC@AR/PX, showing a 10-fold enhancement compared to TiC. This difference is also significant when analysing the mean pore diameter  to 30.5 m2 g−1 (TiC@AR/PX).The data extracted from BJH determine the significant influence of the conversion (TiC to TiC@AR/PX) process . The surTwo parameters, including adsorbate and adsorbent, affect the adsorption. Based on the Langmuir method, single layer adsorption keeps a few numbers of adsorption sites with the same method and no transmigration of adsorbate in the plane surface. After filling the site, adsorption stopped, implying surface saturation . The res2, polyvinyl alcohol and xylene positively affected the surface activity and porosity, which not only improved light absorption but also provided a better platform for the reaction between electrons of the conduction band and the oxygen of the solvent to form free radicals. In addition, the high porosity of TiC@AR/PX means that electrons react longer whereas one of the main drawbacks of a conventional nano-photocatalyst is its fast recombination rates (a fraction of a second) and termination of the reaction.According to the results, the modification by different phases of TiOR is the reflectance. UV–visible spectroscopy was carried out at 200–800 nm to calculate the energy bandgap of the samples . On the other hand, TiC@AR showed a peak at 416 nm, suggesting activity in the first region of visible light (violet range) (b). The peaks corresponding to TiC determine the effect of TiC on TiC@AR.According to the results, TiC had some sharp peaks between 240 and 290 nm, with the highest peak at 290 nm, implying that the sample cannot absorb visible light a. On thet range) b. The pec). To find the best activating region, the Tauc plot was used to find the energy bandgap:α is the absorption coefficient, hv is the photon energy, α0 and h are constants, gE represents the optical bandgap of the material and n is in the range 0.5–3 (depending on the electronic transition).On the other hand, TiC@AR/PX exhibited many peaks in both UV and visible regions, due to the presence of different functional groups of polyvinyl alcohol and xylan c. To finhv. The hv value at the intersection point of the tangent and the x-axis shows the bandgap. According to figure 10d, the presence of several sharp peaks makes it difficult to determine an accurate place of the dominant peak. Based on the results of UV–visible spectroscopy, the highest peak is located at 220 nm; therefore, it can be claimed that the energy bandgap is around 5.74 eV.The energy bandgap was determined from the curves of e). The obtained graph shows the energy bandgap of 2.96 eV for TiC@AR.In contrast to TiC, the energy bandgap of TiC@AR can be calculated with more accuracy e. The obf), showing a drastic decline. It is worth noting that TiC@AR/PX even showed peaks at longer wavelengths with higher absorption due to possessing different functional groups. Therefore, it can absorb in the IR region of sunlight (accounting for 52% of the sun spectrum intensity). Both TiC and TiC@AR exhibited a decrease in absorption after their highest peak in UV and visible regions. This was the opposite in the case of TiC@AR/PX, and it showed further growth at the longer wavelengths. Another point is the absorption of the samples. Both TiC and TiC@AR have the same absorption between 0 and 45, while this parameter ranged between 80 and 110 for TiC@AR/PX, exhibiting almost a twofold increment.The energy bandgap of TiC@AR/PX was also calculated as 1.57 eV f, showinEHOMO = −(Ered + 4.8); the factor of 4.8 in the formula is a correction, which is related to Ag/AgCl couple and NHE. Both TiC and TiC@AR showed almost the same behaviour and the oxidation peak appeared around 0.7 V so the EHOMO is obtained as −5.5 V versus vacuum and consequently the potential of the samples is 1.1 V versus NHE through the formula of φLUMO = −[EHOMO − (−4.4)].Cyclic voltammetry has been done for TiC and TiC@AR to find the position of the valence and conduction bands . The pot. 4−) located in the valence band can be excited by a light source and transferred to the conduction band, leaving holes (h+) in the valence band. Electron–hole pairs promote photocatalytic degradation.The photocatalytic activity of the samples was tested using BPB dye . The ele2 phase on TiC (TiC@AR), the photocatalytic activity of TiC@AR increased insignificantly (40%). Upon modifying with polyvinyl alcohol and xylan (TiC@AR/PX), a remarkable enhancement occurred in photodegradation of BPB (2 times more than TiC@AR) (75%) (a).According to the results, TiC showed better degradation (15%) . After hR) (75%) a.FigureKobs values are presented in figure 13b. The real degradation kinetics cannot be regarded as a first-order rate (ln(C0/C) = Kobst; in which Kobs is the rate constant (min−1), t is the reaction time (min), C0 is the aqueous concentration of BPB before the degradation and C is the concentration of BPB at different times after degradation). The best explanation for the degradation rate of each sample can be found in K increased from TiC to TiC@AR/PX (around five times).The 2 g−1) is above six times more than TiC (11.9 m2 g−1) and two times more than TiC@AR (23.13 m2 g−1).Energy bandgap and surface activity play important roles in the degradation of BPB. After heat treatment of TiC and obtaining TiC@AR the energy bandgap reached 2.96 eV which is in the violet region. After modification by polyvinyl alcohol and xylan, it remarkably reduced to 1.57 eV (IR region). On the other hand, the surface activity showed a fourfold enhancement after TiC conversion into TiC@AR; upon modifying by polymers, it did not change dramatically. The significant difference in the BPB degradation ability of TiC@AR/PX and two other compounds can be attributed to its energy bandgap. However, the result of the Langmuir model illustrates that the surface activity of the first layer is also involved. Based on the Langmuir result, the surface activity of TiC@AR/PX . Finally, both radical agents react with BPB to reduce its concentration and produce H2O and CO2  and times  to find the optimal condition to achieve the best TiOThe energy bandgap was reduced to visible (TiC@AR) and IR (TiC@AR/PX) ranges, while the surface activity exhibited a sixfold increase. These parameters made TiC@AR/PX a powerful platform to reduce the concentration of BPB dye (by approx. 75%) under sunlight, which is a promising method in the photocatalytic field.


Allergic rhinitis (AR) is very commonly caused by pollens. The symptoms of AR consist of sneezing, nasal congestion, rhinorrhea, nasal itching and airflow obstruction. The diagnosis has long been based on clinical history, skin prick tests and in vitro measurement of specific IgE, but the innovative approach of precision medicine has made diagnostic tools of much greater accuracy available.This review covers the advances in the treatment of seasonal AR concerning the drugs to be used according to the grade of disease and the characteristics of the patients, and the role of allergen immunotherapy (AIT), which is the only treatment capable of acting, in addition to the symptoms, on the cause of AR and therefore to modify its natural history.Drug treatment of AR include a large number of agents, the choice of which depends on the severity of the disease. AIT has high evidence of efficacy demonstrated by meta-analyses, and further improvement is currently apparent, as for diagnosis, applying the means of precision medicine. However, when AIT is performed in current practice, without the strict rules of controlled trials, long-term low adherence is a major problem to be solved. Allergic rhinitis (AR) is very commonly caused by pollens. The major culprit, due to its presence in all temperate zones, is grass pollen. A recent study on a large birth cohort found that the prevalence of sensitization to Phleum pratense was 9.7% at 4 years, 28.4% at 8 years and 37.1% at 16 years .Several clinical variants of AR have been classified in the past, but the first edition of the document “Allergic Rhinitis and its Impact on Ashtma” (ARIA) reduced them to only the intermittent and persistent forms . As far Precision medicine, which has tools such as component resolved diagnosis (CRD), able to detect specific single-allergen molecules, allow to discern the causative allergens from the simply cross-reactive ones, while defining the patient's treatable traits addressing genetic and phenotypic features, and also omics, to predict the patient's response to the therapy  Fig. 1)1).Fig. 1The treatment approach for AR may be based on drugs aimed at repealing or reducing allergic symptoms or on allergen immunotherapy (AIT), which is instead designed to modify the immunological response to the causative allergen inducing its tolerance. However, these two treatments have synergistic effect on patients and are not alternatives. Symptomatic treatments for AR are commonly dispensed as over the counter drug by pharmacists. In 2015, a survey performed in Italy showed that 87% of pharmacists, compared to 49% of general practitioners, were unaware of the ARIA guidelines [Since the estimated prevalence of AR ranges from 20 to 30% of the population in both Europe and United States , it goesThe International Consensus Statement on allergic rhinitis dedicated a section to the evidence of efficacy for the different drugs used to treat AR, including intranasal and oral antihistamines, intranasal, oral, and injectable corticosteroids, oral and intranasal decongestant, leukotriene receptor antagonist, oral cromolyn, intranasal anticholinergics and biologics , as measured through the Aggregate Grade of Evidence (AGE) . Table 1Another interesting approach is based on optimizing the treatment and disease management by detecting patients with severe AR. A large multicenter French study on seasonal AR assessed the patients with a 17 items questionnaire (Allergic Rhinitis Physician Score (ARPhyS) and the commonly used total symptom score (TSS-17). Patients were stratified according to AR severity and categorized into “mild”, “moderate”, and “severe”, through five different methods. The ARPhyS scale proved to be the best in discriminating the patients' AR severity, with reported cut-offs at the score of 8 to 9 for mild to moderate AR, and of 11 to 12 for moderate to severe AR. TSS-17 also had excellent score reliability (0.864), while Cronbach’s α coefficient was considered only acceptable (0.626). The authors then concluded that the ARPhyS scale could be a useful tool in the general practitioner daily practice, to identify those patients with severe AR in need of the specialist’s interventions .The concept of personalized medicine, which is based on treating the patient and not just the disease, as previously done for centuries, is currently deeply changing the practice of medicine and particularly the therapy .The first study using the personalized medicine approach analyzed the outcome of different drugs to treat AR in elderly patients. The results highlighted important matters: first and second generation antihistamines demonstrated a high incidence of adverse effects and drug to drug interactions; oral decongestants were risky when a variety of comorbidities common in older people were present. Leukotriene receptor antagonists were as effective as antihistamines, but less effective than intranasal corticosteroids, which showed the greatest safety and efficacy profile. The authors concluded that the approach for the diagnosis and treatment of AR in elderly patients should be tailored to their specific age-related factors . Further+ T cell, from patients at different time points and from a healthy control group. The first results showed complex changes subsequent to AIT, consistent with the authors’ hypothesis of an interferon signaling pathway involving a different number of genes. However, the authors claimed the need for the result's validation in a larger group of patients [4 hydrolase was consistent with the proteomics data, showing a significant increase in responders after 1 year of AIT, while non-responders showed no significant changes. Based on these results, the authors suggested that serum LTA4H could be used as a valid biomarker for early prediction of effective AIT [4, and IgG allergen recognition was analyzed in samples of serum collected from AR subjects undergoing AIT for pollen allergy. The study showed wide-ranging induction of Phl p 1 and Bet v 1-specific humoral immunity in subjects after 3 years of AIT for grass and birch allergy. Different profiles based on the different epitopes detected were found mainly after 1 year of AIT, indicating that the leading allergen-specific clones persists as a fundamental supplier to humoral immunity even after their preliminary creation during the first phases of the therapy. Pattern of different allergen isoforms specific for different subjects were detected as group cross-reactivities, which might imply different grades of protection against various allergen sources. The authors suggested that the deconvolution of the epitopes could be an important topic for future attempts to evaluate the results of AIT in a personalized practice [The literature on personalized medicine applications for allergen immunotherapy (AIT) is significantly more abundant. Actually, AIT is perfectly suited to the three needs to be met in personalized medicine: identification of the disease's molecular mechanism, availability of a diagnostic tool able to recognize such mechanism, and a treatment capable of blocking the mechanism itself . Indeed,patients . More retive AIT . A Europtive AIT . Mikus epractice . The mospractice .Adherence to medical therapy, as the degree to which a subject correctly follows medical advice , is of c, even if both drug treatment and AIT have been used successfully for a very long time, has still room for improvement. The progress ensured by the innovative precision medicine has already brought enhancements in diagnosis, pharmacological therapy as well as AT, but it is apparent that the road ahead is still longer than the one traveled so far. In particular, the serious problem of poor adherence must be tackled with the utmost commitment, to avoid that an AIT treatment duration shorter than the recommended one of three consecutive years nullifies its effectiveness.The treatment of seasonal ARSeasonal AR has been known for centuries, and in 1911, when effective drugs were not available for its treatment, an innovative therapy called desensitization was proposed in the UK, which consisted of initially administering low and then progressively increasing doses of an extract of the grass Phleum pratense, to induce tolerance to the allergen . Today tAs for any drug, side effects are possible. The greatest risk is borne by systemic corticosteroids, which must be reserved for the most serious cases. A recent consensus document addressed the efficacy of systemic steroids in the treatment of upper airway diseases as well as highlighting the possible harms of this therapy, providing recommendations for their use. Although with less frequency and severity than systemic corticosteroids, other drugs are also affected by side effects . A recen


The androgen receptor (AR) plays an essential role in prostate cancer progression and is a key target for prostate cancer treatment. However, patients with prostate cancer undergoing androgen deprivation therapy eventually experience biochemical relapse, with hormone-sensitive prostate cancer progressing into castration-resistant prostate cancer (CRPC). The widespread application of secondary antiandrogens, such as enzalutamide, indicates that targeting AR remains the most efficient method for CRPC treatment. Unfortunately, neither can block AR signaling thoroughly, leading to AR reactivation within several months. Here, we report an approach for suppressing reactivated AR signaling in the CRPC stage. A combination of the protein phosphatase 1 subunit α (PP1α)-specific inhibitor tautomycin and enzalutamide synergistically inhibited cell proliferation and AR signaling in LNCaP and C4-2 cells, as well as in AR variant-positive 22RV1 cells. Our results revealed that enzalutamide competed with residual androgens in CRPC, enhancing tautomycin-mediated AR degradation. In addition, the remaining competitive inhibitory role of enzalutamide on AR facilitated tautomycin-induced AR degradation in 22RV1 cells, further decreasing ARv7 levels via a full-length AR/ARv7 interaction. Taken together, our findings suggest that the combination of tautomycin and enzalutamide could achieve a more comprehensive inhibition of AR signaling in CRPC. AR degraders combined with AR antagonists may represent a new therapeutic strategy for CRPC. Prostate cancer is the most common cancer and the second leading cause of cancer-related deaths in Western men . For higAR is a member of the steroid receptor family of transcription factors that share structurally conserved domains, consisting of an N-terminal domain (NTD), a DNA-binding domain, a ligand-binding domain (LBD), and a hinge region containing a nuclear localization sequence . The impRecently, some attempts have been made to further block the reactivated AR in CRPC. EPI-001 and its analogs are small molecular inhibitors of the NTD, which could overcome the shortcomings of current therapies targeting the LBD . SelectiHere, we explored a newly identified drug combination of enzalutamide and a protein phosphatase 1 subunit α (PP1α) inhibitor tautomycin, which contributed to the increased stability and nuclear accumulation of AR in CRPC. These two drugs synergically inhibited cell proliferation, especially for AR variant-positive cells. The enzalutamide could significantly enhance the tautomycin-mediated degradation of AR and AR-v7 via the ubiquitin–proteasome pathway. These findings suggest the potential of a combination of enzalutamide and tautomycin to develop novel therapeutics for CRPC.To explore the effect of the tautomycin and enzalutamide combination, an MTT assay was performed to detect cell viability in LNCaP and C4-2 cells. As shown in Fig. The mechanism underlying the inhibition of proliferation by tautomycin and enzalutamide mainly relies on targeting and decreasing the AR signaling activity. Therefore, to investigate the synergistic effect of tautomycin and enzalutamide, we evaluated AR signaling using Western blotting and real-time quantitative reverse transcription PCR (RT-qPCR). As shown in Fig. PP1α reportedly regulates AR protein stability via MDM2, an E3 ubiquitin ligase responsible for the intracellular degradation of AR . As showThe increased AR degradation after tautomycin and enzalutamide combination treatment overcame AR reactivation such as AR overexpression or stability evaluation. We further aimed to identify whether this combination could prevent AR reactivation caused by structural alterations. 22RV1 cells are known to express both full-length AR (FL-AR) and AR variants (ARVs) that are enzalutamide-resistant. The MTT, EdU, and colony formation assays were repeated. As shown in Fig. To assess the AR signaling alteration after tautomycin and enzalutamide combination treatment, we next detected the protein and mRNA levels of AR, ARv7, and their downstream targets. Tautomycin decreased the protein levels of both FL-AR and ARVs, while enzalutamide acted as an enhancer of tautomycin effects Fig. . With thIn Fig. Prostate cancer is an endocrine-related cancer, and the AR signaling pathway, which participates in the entire disease process, is indispensable for cancer growth and distant metastasis . Thus, uTautomycin is a PP1α inhibitor. As a protein phosphatase, PP1α negatively regulates E3 ligase MDM2 and SKP2 activity by decreasing their phosphorylation . AccumulEnzalutamide is a secondary antiandrogen developed for the treatment of CRPC. Compared with first-generation antiandrogens, enzalutamide shows approximately 8-fold greater affinity than bicalutamide. Previous studies have reported that enzalutamide could block ligand–receptor binding, nuclear translocation, DNA-binding, and coactivator peptide recruitment, thereby affecting the inhibition efficiency of AR downstream target transcription . HoweverIn 22RV1 cells, in addition to the finding that enzalutamide could facilitate tautomycin to further decrease AR signaling, we noticed that enzalutamide also helped block ARVs signaling. Most ARVs are truncations of AR and do not have the LBD; therefore, they are constitutively activated in the absence of androgen or the presence of an AR competitive inhibitor, including enzalutamide. Recently, numerous studies have reported the interaction between AR and ARVs. Xu et al. observed that ARv7 and ARv567es heterodimerize with FL-AR in an androgen-independent manner . MoreoveIn summary, we provide a new potential strategy for CRPC treatment. Our results revealed that the combination of tautomycin and enzalutamide inhibits prostate cancer cell proliferation by synergistically promoting AR and ARv7 protein degradation via the ubiquitin–proteasome pathway. These findings also raise the possibility of combining AR degraders with AR antagonists to treat CRPC and AR antagonists resistant CRPC.2 incubator at 37 °C. FL-AR siRNA (CGUGCAGCCUAUUGCGAGAUU) was synthesized by GenePharma . Hemagglutinin (HA)-ubiquitin was a gift from Edward Yeh . The Flag-AR plasmid was synthesized by GeneChem . Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) was used to transfect both siRNA and plasmids according to the manufacturer’s instructions. All cell lines were authenticated by STR profiling and tested negative for mycoplasma contamination.The prostate cancer cell lines LNCaP, C4-2, and 22RV1 were purchased from Procell Life Science & Technology Co. Ltd. . All the cell lines were cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS  or charcoal-stripped FBS  and 1% penicillin/streptomycin (Thermo Fisher Scientific) in a CO4 cells/well) were seeded in 96-well plates. After 24 h, cells were treated with enzalutamide  and tautomycin (Wako) at various concentrations. Cells were then grown for 48 h, and cell viability was evaluated using the MTT (Sigma) assay, as previously described [Cells (1 × 10escribed .4 cells/well) were seeded in 96-well plates. After 24 h, cells were treated with enzalutamide (MedChemExpress) and tautomycin  at various concentrations, followed by incubation with 5-ethynyl-20-deoxyuridine  for 2 h, and processed according to the manufacturer’s protocols. The 1× Apollo reaction cocktail and Hoechst 33342 (5 μg/mL) were used to stain the cells for 30 min and then visualized using a fluorescence microscope. EdU-positive cell density was calculated using ImageJ software (RRID:SCR_003070) according to the presence or absence of nuclear-specific staining (Hoechst 33342) compared with negative controls.LNCaP, C4-2, and 22RV1 cells , and after 2 days, they were treated with enzalutamide and tautomycin at various concentrations and then cultured in a cell incubator for another two weeks. The cells were fixed with 4% paraformaldehyde for 10 min. The cells were then stained with 0.5% crystal violet for 20 min. The number of cells was calculated using ImageJ software (RRID:SCR_003070).® III All-in-one RT SuperMix (Vazyme Biotech Co. Ltd.). Taq Pro Universal SYBR qPCR Master Mix  was used for real-time quantitative PCR on a QuantStudio™ 6 Flex system (Thermo Fisher Scientific). The data were analyzed using the 2–ΔΔCt method.Prostate cancer cells were seeded in 12-well plates and treated with enzalutamide and tautomycin at the indicated concentrations. After 48 h, TRIzol reagent (Invitrogen) was used for RNA extraction, according to the manufacturer’s instructions. Reverse transcription was performed with 1 μg of RNA using HiScriptProstate cancer cells were lysed in RIPA buffer containing a proteasome inhibitor cocktail , following the manufacturer’s instructions. The protein concentration was determined using the Pierce™ BCA Protein Assay Kit . Proteins were mixed with protein loading buffer and boiled at 100 °C for 10 min. From each group, 30 μg of protein were separated using 10% SDS-PAGE and subjected to Western blot analysis. The primary antibodies used in this study were as follows: AR , PSA , DYKDDDDK Tag horseradish peroxidase conjugate , AR-v7 , HA Tag , and GAPDH . Full-length western blots are provided in supplementary materials.Cells were seeded in 6-well plates and, after 24 h, were treated with cycloheximide , enzalutamide, and tautomycin at the indicated concentrations. RIPA buffer containing a proteasome inhibitor cocktail was used to lyse cells after 0, 4, 8, and 12 h. Then, the protein was subjected to Western blot analysis to evaluate AR or AR-v7 protein levels.Prostate cancer cells transfected with HA-ubiquitin and Flag-AR were treated with MG132 (10 µM), enzalutamide, or tautomycin at the indicated concentrations. After 24 h, the cells were lysed, incubated with anti-Flag magnetic beads , and incubated overnight at 4 °C. The anti-Flag magnetic beads were pelleted, washed three times with immunoprecipitation wash buffer, and then eluted with 1× protein sample loading buffer at 100 °C for 5 min.g for 20 min, and 5× protein sample loading buffer was added to the supernatant after boiling at 100 °C for 10 min and detected using Western blotting.The cells were seeded into a 100 mm dish, and after they reached 80% confluence, they were digested and divided into separate aliquots and exposed to enzalutamide (50 µM) or dimethyl sulfoxide at a gradient concentration of dihydrotestosterone  for 1 h in a cell incubator. 46 °C for 3 min was used to perform heat shock and the lysed by freeze-thaw cycle three times using liquid nitrogen. The cell lysate was centrifuged at 20,000 × g for 20 min. Supernatants were transferred to new tubes, boiled at 100 °C for 10 min, and detected by Western blot.CETSA experiments were performed according to the general CETSA protocol . Brieflyt-test was used for data comparison between the two groups. One-way analysis of variance (ANOVA) was used to compare two or more groups. Two-way ANOVA tests, the bliss-independent model [Statistical analyses were performed using GraphPad Prism . Data are presented as the mean ± SEM from at least three biological replicates. The two-tailed Student’s nt model , and Chont model  were useSupplementary materialssupplementary full length western blotTable S1. Determination of synergy


Although several therapeutic options have been shown to improve survival of most patients with prostate cancer, progression to castration-refractory state continues to present challenges in clinics and scientific research. As a highly heterogeneous disease entity, the mechanisms of castration-resistant prostate cancer (CRPC) are complicated and arise from multiple factors. Among them, noncoding RNAs (ncRNAs), the untranslated part of the human transcriptome, are closely related to almost all biological regulation, including tumor metabolisms, epigenetic modifications and immune escape, which has encouraged scientists to investigate their role in CRPC. In clinical practice, ncRNAs, especially miRNAs and lncRNAs, may function as potential biomarkers for diagnosis and prognosis of CRPC. Therefore, understanding the molecular biology of CRPC will help boost a shift in the treatment of CRPC patients. In this review, we summarize the recent findings of miRNAs and lncRNAs, discuss their potential functional mechanisms and highlight their clinical application prospects in CRPC. Prostate cancer (PCa) is the most prevalent male-related malignancy in the western world and is driven largely by androgen receptor (AR) signaling ,2. GivenFor decades, studies on the molecular mechanism of prostate cancer have focused on protein-coding genes. Quite recently, the doctrine of molecular biology was changed with a deeper understanding of noncoding RNAs (ncRNAs). In recent decades, a series of studies have revealed the scientific secrets of ncRNAs, which have pivotal regulatory roles in almost all cellular and biological functions, such as apoptosis, migration, drug resistance , cellulaHistorically, ncRNAs can be classified as short ncRNAs  and long noncoding RNAs  based on their sizes. The most investigated ncRNA types include microRNAs (miRNAs), lncRNAs and circular RNAs (circRNAs) . miRNAs In contrast to miRNAs, lncRNAs are a group of transcripts with sizes greater than 200 nt that play regulatory roles at both the transcriptional and posttranscriptional levels ,33. StruIn the present review, we attempt to clarify the major contribution of ncRNA-regulated mechanisms, including cancer metabolism, epigenetic modifications and abnormal AR signaling, to the occurrence of castration-resistant prostate cancer .The diagnostic criteria of CRPC status are serum testosterone <50 ng/dL or 1.7 nmol/L plus biochemical progression or radiologic progression . BiochemAndrogens are not only involved in the normal development of the prostate gland, but also in the occurrence of PCa. Given this, androgen deprivation therapy (ADT), including chemical and surgical castration, is the first line of defense or an important adjunctive therapy to radiotherapy for men with PCa . DespiteMetabolic remodeling is considered a common hallmark of the biological capabilities of cancer obtained during the multistep initiation of human tumors, including PCa . CurrentGiven the key regulatory function of ncRNAs in cancers, ncRNAs have been reported to be involved in multiple cancers, including metabolic abnormalities . A previSimilarly, in prostate cancer, an androgen-induced prostate-specific LncRNA named prostate cancer gene expression marker 1 (PCGEM1) was discovered to increase glucose uptake and glycolysis for adequate energy supply and the proliferation and survival of LNCaP cells . PCGEM1-Previous studies have confirmed that AR signaling may limit glycolysis and enhance lipogenesis in PCa, hence driving the proliferation and migration of PCa cells ,91,92. TIn recent decades, biologists have become increasingly concerned about the epigenome. Epigenetic alteration has become a driving factor of malignant cell proliferation and the development of cancers. Conventional epigenetic aberrations at the transcriptional level include methylation and acetylation modifications of DNA and histones, as well as chromatin remodeling . MoreoveThere is an increasing amount of evidence that regulatory ncRNAs play a significant role in epigenetic control, and these ncRNAs highlight the prominent role in gene expression. Recently, a novel LncRNA, LncRNA NXTAR, has been discovered to be repressed and been identified to act as a tumor suppressor in PCa . LncRNA Cell signaling plays an important role in a variety of biological processes, including carcinogenesis. Numerous signaling pathways have been identified in CRPC, such as the WNT pathway ,127, thePI3K/AKT signaling is among the pivotal pathways responsible for driving the process of malignancy in many solid tumors and is emerging as a potential target for cancer . Shang eAR is an androgen-activated steroid hormone receptor produced by the AR gene, which is located on the long arm of the X chromosome ,141. TheThe AR signaling pathway is initiated by androgen  after binding to AR. Then, AR separates from Hsp90 and forms an AR dimer, which elicits its translocation into the nucleus . AR signAlternative splicing is a ubiquitous regulatory mechanism that brings about the production of proteome complexity from a limited number of genes . Not surAberrant AR signaling is fundamental to prostate cancer growth, metastasis, metabolic reprogramming and ultimately to its lethality ,162,163.In the test, the last step of androgen biosynthesis involves two critical sequential reactions that are catalyzed by a single enzyme, the cytochrome P450 monooxygenase 17α-hydroxylase/17,20-lyase (collectively referred to as CYP17) . ObviousEnzalutamide is a second-generation competitive inhibitor of AR. It weakens the AR pathway on three levels—through inhibition of androgen binding, nuclear translocation and DNA binding—and thus induces apoptosis, which makes it an attractive option in CRPC . FinasteCurrently, the usual first-line therapies for CRPC are abiraterone and enzalutamide. These AR-targeting agents can extend the survival of patients with CRPC for an extra 5 months but are not curative due to a series of drug resistance mechanisms, including restored androgen receptor signaling, androgen receptor splice variants, androgen receptor bypass signaling and complete androgen receptor independence . AccordiNXTAR upstream region [Over the past decades, noncoding transcripts have been increasingly recognized as targets or mediators of the AR signaling axis and may play a central role in the enhancement and maintenance of AR signaling activity  37,52,1,152,182.m region . This nem region .Additionally, as AR mRNA contains a long 3′-UTR, it is highly probable that the AR signaling pathway or AR expression is affected by a range of miRNAs . Indeed,Although current targeted drugs for PCa have greatly improved the prognosis of men with PCa, patients also inevitably develop resistance to these therapies. While the exact mechanisms of drug resistance have not been fully elucidated, ncRNAs have been found to play a critical role in reversing ADT resistance and improving the sensitivity of targeted drugs with the deepening understanding of ncRNAs recent years . TherefoAlthough most men with prostate cancer benefit from ADT, progression to a castration-resistant state remains inevitable within 2–3 years of initiation of ADT and this poses a major challenge in clinical management. Therefore, a thorough elucidation of the intrinsic mechanisms of CRPC may help to solve this dilemma. An increasing number of ncRNAs have been identified to be involved in epigenetic modifications, tumor metabolism and AR regulation, which are all closely related to the occurrence and progression of CRPC. Clinically, several studies have recognized their roles as promising diagnostic, prognostic and even therapeutic biomarkers, especially miRNAs on account of their relative stability, tiny size and significant control of gene expression. It is conceivable that ncRNAs signatures in circulating cancer cells or macrovesicles secreted by cancer cells could provide personalized treatment for patients with PCa.However, despite these favorable prospects, circulating ncRNA detection faces technical challenges due to the low abundance of ncRNA in circulation. Therefore, there is an urgent need for other more accurate and convenient non-invasive tests to diagnose PCa. Due to the ease of collection of PCa cells and their direct release into the urethra via the prostate duct, urine is expected to become the non-invasive biomarker test of choice for diagnosis and prognosis. Unfortunately, until now, only Lnc PCA3 has been utilized as a urinary biomarker in the clinical diagnosis of PCa . Additio


The sense of presence in augmented reality (AR) has been studied by multiple researchers through diverse applications and strategies. In addition to the valuable information provided to the scientific community, new questions keep being raised. These approaches vary from following the standards from virtual reality to ascertaining the presence of users’ experiences and new proposals for evaluating presence that specifically target AR environments. It is undeniable that the idea of evaluating presence across AR may be overwhelming due to the different scenarios that may be possible, whether this regards technological devices—from immersive AR headsets to the small screens of smartphones—or the amount of virtual information that is being added to the real scenario. Taking into account the recent literature that has addressed the sense of presence in AR as a true challenge given the diversity of ways that AR can be experienced, this study proposes a specific scope to address presence and other related forms of dimensions such as immersion, engagement, embodiment, or telepresence, when AR is used in games. This systematic review was conducted following the PRISMA methodology, carefully analysing all studies that reported visual games that include AR activities and somehow included presence data—or related dimensions that may be referred to as immersion-related feelings, analysis or results. This study clarifies what dimensions of presence are being considered and evaluated in AR games, how presence-related variables have been evaluated, and what the major research findings are. For a better understanding of these approaches, this study takes note of what devices are being used for the AR experience when immersion-related feelings are one of the behaviours that are considered in their evaluations, and discusses to what extent these feelings in AR games affect the player’s other behaviours. Among the latest computing technologies disseminated across society, we find virtual reality (VR), augmented reality (AR), and the concept of mixed reality—the avant-garde of interactive experiences. Thus, for developers and researchers in the area of all these extended realities, these are frequently drawn on to improve various applications in several fields.According to our research in recent years, with AR implementations and experiences, in addition to their irrefutable advantages, a vast number of questions have arisen when trying to provide objective data related to these types of applications.A previous study conducted a literature review focused on VR and AR systems and their effects on learning . This stThe current systematic review provides a detailed analysis of immersion-related feelings in AR games and aims to answer the questions below. Note that, in order to identify feelings that may be related to immersion, the term “immersion-related feelings” is considered in the current study to designate the most common states that may be related to immersion, such as presence, engagement, embodiment, or telepresence.Q1: What are the immersion-related feelings that are being reported in AR games?Q2: How are immersion-related feelings being evaluated in AR games?Q3: What are the devices that are being used for AR games when immersion-related feelings are being considered in the experience?Q4: To what extent do immersion-related feelings in AR games affect other feelings and behaviours in the player?This systematic review aimed to answer the following questions:The following subsections provide the context and clarify why this review is needed for all those who intend to use and explore augmented reality solutions.An overview of augmented technology is presented as well as an outline of AR game considerations.AR has undergone rapid growth in terms of technological advances and gaining popularity across the world, becoming a frequent approach in different areas for different purposes. From medical aid to learning experiences at schools and at museums to pure entertainment, AR is an interesting and attractive way to innovate in the digital world. In this case, since AR can be considered a variant of VR and as Ronald T. Azuma sustained back in the 1990s , AR can The technological development of the last decade brought new possibilities for AR to succeed as in the case of smartphones or head-mounted displays such as HoloLens. Studies aiming to enhance our understanding of the impact of AR largely support its usage as can be observed in systematic reviews focused on the advantages of AR in education , in mediDepending on the intended use of the AR implementation, it is important to understand how users react and what the impact of their experience with AR is. Some authors have highlighted the blurred boundaries in AR experiences between the activity itself and the surroundings as non-players and elements of the real space can affect the experience . This chA systematic review targeting serious games has been partially undertaken, yielding useful insights for all those who intend to develop a game for learning purposes in science education . The redThis subsection aims to provide context on immersion feelings, including feelings that may be related to immersion, such as the sense of presence, engagement, embodiment, or engagement with the experience. Initially, an overview of the different definitions of immersion is presented, with the aim to provide some context of the different feelings that may be related to immersion. Then, some of the determinants that may affect immersion are clarified and finally, evaluation insights are exposed.According to the literature, immersion is a powerful experience of gaming and it is frequently regarded as a significant matter of interaction . JennettAnyhow, the scope of this research is not to navigate across the different definitions of immersion, given that this has already been established in previous studies ,27. RathWhen observing presence studies, frequently considered for immersion-related studies, it is important to mention that the definition of presence is still under substantial discussion and, as explained above, it is not the purpose of this study to discuss the different definitions provided in the literature. This section aims to provide some context to better understand the analysed studies in the future. The sense of presence can be related to the sense of being in a virtual element . HoweverIn line with this insight, and as highlighted in the literature, to explore how different realities may be blended and how people feel present is foundational . FurtherThis brief overview of immersion-related concepts demonstrates several directions that immersion-related feelings may take. Participants often confuse the concepts of presence with immersion in their responses when participating in this type of study . Thus, tImmersion-related feelings may vary according to a wide variety of factors presented in the literature such as realism , interacAccording to Rebollo et al., immersion in AR games triggers the feeling of being physically located in the place where players are playing the game . ImmersiThis study is important to clarify the relationship between AR and immersion. Across the literature, it is common to conduct evaluations related to immersion using similar procedures and tools that are being used for VR environments. However, as indicated in the literature, studies of AR immersion-related feelings have revealed contradictory results when evaluated and compared to VR approaches. For example, while participants pointing to something can be interpreted as an indicator of presence, according to Slater et al.  within tThe lack of validated, theory-based instruments for measuring immersion is well known in the literature, and as Georgiou et al. highlighted, it is even more evident for AR implementations . These aThe current literature review followed the PRISMA method  and thenA randomised search of AR games that considered immersion-related feelings in their studies was performed, with the aim to provide relevant data taking into account its implementations, usage, and evaluation. The inclusion criteria are specified in The strategy followed to obtain a full analysis regarding the specified topics included two search stages: a search of online searches, and a search analysing the records obtained from the previous search stage.For the first search stage, the literature was identified through online searches by conducting an extensive search with the EndNote 20 software, through the Science Citation Index on Web of Science (Clarivate) conducted on 3 February 2022. The search was performed to be equivalent to the following logical expression: Title/Keywords/Abstract containing  AND (“Game” OR “Games”) AND (“Immersion” OR “Engagement” OR “Presence” OR “Embodiment” OR “Telepresence”).For the second search stage, carried out when reviewing full-text articles , not being a game (7 records), not presenting an AR experience (1 record) or for not being possible to obtain the full-text version of the record (1 record). After this process, a total of 20 studies remained for the qualitative and quantitative analyses.After removing all duplicated records, the search performed in the identified database returned 79 articles. The titles and abstracts of the unique 79 records were analysed, taking into account the eligibility criteria, among which 37 records were excluded for not meeting such criteria. As a result, 42 records were obtained as eligible for a full-text analysis. Among those 42 records, 22 more records were excluded based on the previously defined exclusion criteria, resulting in a total of 20 studies carefully analysed for this survey. Please refer to This first subsection of results depicts the most relevant qualitative data gathered. A list of the analysed studies is presented, a word cloud based on the titles was generated, and an overview of the major findings is emphasised in this subsection.The analysed articles included in this systematic review are listed in Several other studies seemed to fit the inclusion criteria as they presented interesting approaches in AR games such as a game targeting aesthetic engagement with a city , an AR aOn the other hand, some interesting studies investigated immersion-related feelings in their work, such as the supernumerary hand illusion , the infwww.jasondavies.com (accessed on 28 Febrary 2022).Considering the 20 records which were carefully analysed, a word cloud was generated based on the titles of those studies, which is presented in Regarding the technical concepts and approaches used, we observed a preference for “augmented reality” while “Games” and “game” were predictably frequent words as well. There was a tendency for the frequent use of “presence” and “immersion”. An interesting concept that appeared quite frequently was “learning”.The major findings are highlighted hereinafter.McCall et al.  reportedFollowing the study by McCall et al. , Putten Lee et al. conducted their study in an identical collaborative scenario to understand the effects of latency . Their rDatcu et al. also used a collaborative game to compare presence in physical and AR environments . Their rIn a serious game approach, Furió et al.  studied Furthermore, in their study focused on devices, Sekhavat et al. compared the results obtained from different types of camera, namely VR, AR, and stereo-AR . They obKoh et al.  collecteSocial presence as being an item of continuance intention—hedonic gratification—was studied by Bueno et al. also using the AR game Pokémon Go as a case study . In theiSocial presence was also the focus in the research study performed by Oriti et al. which compared interfaces. They observed that the AR interface obtained lower scores when compared to the VR interface, and noticed that behavioural involvement was higher for VR. Despite these insights, they realised that both interfaces obtained relatively low results for social presence in general .Aiming to understand the role of AR to stimulate students, Estudante et al. observed that students are immersed in the story by being able to use AR games as a teaching method in a manner complementary to traditional methods . GeorgioSimilarly, with the narrative as a determinant of the experience in mind, Jin et al. conducted a comparison between a natural user interface with a graphical user interface using AR-based head-mounted displays . For thiLin et al. linked the immersion feelings to effective learning and stated that, according to their results, learners were more deeply immersed during the AR gameplay, achieving more effective learning . EngagemEngagement—more specifically, game engagement—was an element studied for psychosocial well-being by Seaborn et al. . Their rIt was previously stated that a study linked usability with immersion without providing results . Shin etA quantitative analysis is now presented, highlighting devices used in the AR games studies analysed herein, as well as the variables that were under study and under what condition they were evaluated.As illustrated in In Following the data presented in The discussion of this systematic review focuses on the questions previously presented in the introduction section.This research concludes that 90% of the analysed studies evaluated immersive-related feelings with questionnaires, since it was not clear which evaluation was used for the remaining 10%. Interviews and observations were used 20% and 10% of the time, respectively. The sample sizes of the conducted evaluation varied between 18  and 1183Most studies selected their evaluation tools based on validated questionnaires. This provides useful information for further evaluations.Immersion was evaluated by using: (1) the Immersive Experience Questionnaire (IEQ)  twice—seSense of presence was evaluated by using: (1) a modified MEC spatial presence questionnaire  by addinEngagement had several approaches: self-engagement was evaluated by: (1) using a custom questionnaire ; (2) theSocial presence was evaluated by using a custom questionnaire  while coIt was observed that the sense of presence is positively affected by audio elements, the nature of tasks and the actions of the player in the game, while realism may negatively affect presence . While cEngagement appears to not be influenced by the device used when comparing a tablet to a smartphone . This isImmersion has been reported as being higher in AR scenarios than in non-AR virtual scenarios . In lineIn general, social presence has a positive impact on the continuation of one’s intention to play Pokémon Go ; nonetheAs discussed in According to analysed records, there are no guidelines available for tracing a methodology to evaluate immersion/related feelings in AR games. Different tools were used and a wide variety of sample sizes were identified. Questionnaires, however, seem to be undeniable tools for evaluation. Therefore, to answer the question “How are immersion-related feelings being evaluated in AR games?”, the answer is not standardised. Immersion-related feelings are evaluated in AR games with questionnaires—mainly validated questionnaires—some specifically targeting AR experiences, whilst others are being evaluated with tools targeted for VR or games.When trying to understand what devices are being used for AR games when immersion-related feelings are being considered in the experience, i.e., question number three, it was observed that most studies opted for implementing them in mobile platforms. Smartphones and tablets together correspond to more than half of the evaluated games, followed by head-mounted displays with 32% of occurrences. Mobile computers were used in two situations and a video projector was used in one.As discussed in As for the limitations of this current systematic review, we addressed the main limitation which was the search conducted on a single database. According to the literature, in addition to the Web of Science and the underlying databases and the indices it contains, there were at least 13 other academic search systems that were identified as well suited for systematic reviews, such as the ACM Digital Library or Scopus . NotwithThe current systematic literature provided a global overview of AR games that considered immersion-related feelings in their studies. The analysed data allow us to understand what immersion-related feelings are being reported in AR games and it is now clearer how immersion-related feelings are being evaluated in AR games. It was observed that a wide variety of users’ feelings related to immersion were also identified as links between these feelings with others, such as engagement or learning skills.This systematic review also exposed the different approaches that were conducted to evaluate immersion-related feelings. The way it has been evaluated is similar across studies, frequently making use of questionnaires. Validated questionnaires or customised versions of validated questionnaires are commonly used, but several studies have opted to create their own versions of questionnaires which are not validated. Some of the validated questionnaires are being used to ascertain AR feelings even if previously validated for VR scenarios. The differences between immersion feelings and presence feelings are interesting and considerably different depending on the desired outcome. For example, when users felt more present while playing an AR game, they walked longer during the day , while iAn identification of the devices used for AR games, when immersion-related feelings are being considered in the experience, was pointed out to support the use of different technologies, depending on the target of each game. For example, the size of the screen is often pointed out in the literature as a hampering factor to engage in AR experiences .An overview of immersion-related feelings in AR games and how they affect other feelings and behaviours in the player was conducted, and it was observed that these feelings in AR games can affect a wide range of other feelings and behaviours in the player. Overall, these feelings have been linked to engagement, learning abilities, the intention to continue playing, and the interest in it.Major findings were also outlined as the different outcomes observed in the analysed studies are interesting and useful for further implementations and research. The research herein also demonstrated that there are topics raising some new questions, especially related to immersion and presence variables, which reveal that there are several research opportunities for AR games in the scientific community.


Allergic rhinitis (AR) is a major non‐communicable disease that affects the health‐related quality of life (HRQoL) of patients. However, data on HRQoL and symptom control in AR patients with comorbid asthma (AR + asthma) are lacking.In this multicentre, cross‐sectional study, patients with AR were screened and administered questionnaires of demographic characteristics and health conditions . HRQoL was assessed using a modified version of the RHINASTHMA questionnaire  and symptom control was evaluated by a modified version of the Control of Allergic Rhinitis/Asthma Test (CARAT) .Out of 643 patients with AR, 500 (78%) had asthma as a comorbidity, and 54% had moderate‐severe intermittent AR, followed by moderate‐severe persistent AR (34%). Compared to the patients with AR alone, patients with AR + asthma had significantly higher RHINASTHMA  and a significantly lower CARAT score . Upon stratifying asthma based on severity, AR patients with severe persistent asthma had worse HRQoL and control than those with mild persistent asthma. The association was significantly higher among non‐obese participants compared to obese ones, with RHINASTHMA‐upper symptoms score but not with CARAT.Our observation of poorer HRQoL and symptoms control in AR patients with comorbid asthma supports the importance of a comprehensive approach for the management of AR in case of a comorbid allergic condition. In this multicentre study, using validated questionnaires, we aimed to assess the differences in symptom control and HRQoL between AR patients with or without comorbid asthma.22.1In the international multi‐centre  cross‐sectional AIS Life + study, conducted between 2013 and 2014, we enrolled participants suffering from nasal allergy. A convenient sample of individuals with an active condition of pollen‐induced AR was selected from pre‐existing epidemiological study databases or through web advertisement , clinics of general practitioners  or public health databases and pulmonary clinics  and invited to participate in this epidemiological survey. All potential participants were administered a screening questionnaire through a telephone interview to check whether they were eligible for the study. We included participants who: (1) were adults (≥18 years of either sex); (2) reported a physician‐diagnosed pollen‐induced allergic rhinitis/hay fever or nasal symptoms or positive clinical tests to pollen (skin prick tests or specific immunoglobulin E [IgE]) in the last 12 months; (3) spent most of the week (at least 5 days/week) living, studying, or working in the areas where this study was conducted; and (4) were not treated with allergen immunotherapy over the previous 6 years.Comite´ Consultatif sur le Traitement de l’Information en Matière de Recherche dans le domaine de la sante´) and by the National Commission on Informatics and Health . In France, the CNIL approved in 2016 that all data acquired before 2016, without the previous need for authorization from an Ethics Committee, could still be utilized. In any case, patients were seen in the frame of routine care (“soins courants”). The AIS study was conducted according to the Declaration of Helsinki and reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.The study was approved by the ethics committees of the participating centres in Italy  and in Austria (Berlin Charite University Ethics: EA1/119/12), and signed informed consents were obtained from all the participants before recruitment. In France, the approval by an external ethic committee was declared as not applicable at that time: instead, the study was approved by the hospital ethics committee, by the National Committee for Information Management on Medical Research , and potential risk factors , on symptoms/diagnosis of AR and asthma, disease severity level, as well as on previous clinical tests .The HRQoL of the participants was assessed using a modified version of the validated RHINASTHMA questionnaire , that is, the only available instrument that allows evaluating the concomitant impact of AR and asthma on HRQoL.The control of AR and asthma was evaluated by a modified version of the Control of Allergic Rhinitis/Asthma Test (CARAT) .2.2.1Data were described as frequency (%), mean (standard deviation [SD]), or median (interquartile range [IQR]) for categorical, continuous, and ordinal variables, respectively. To test the association between QoL and control  scores, and AR + asthma (independent variable), we first used a bivariate analysis using Wilcoxon rank‐sum test. Then, we constructed univariable (unadjusted) and multivariable (adjusted) regression models among the independent variable and HRQoL and control scores using a mixed effect Poisson regression model. As potential confounders, we tested fixed factors  and a random factor (the country). To include confounders in the regression models, we used a priori evidence criteria, that is, covariates were considered as confounders if were found consistent in previous literature. However, confounders were retained in the model if they modified the estimates of the remaining variables by more than 10%. We checked the collinearity of the confounders using the variance inflation factor (VIF). The parsimony of the models was confirmed by Akaike information criterion.p‐value <0.05 was considered statistically significant.We also performed two secondary analyses. Firstly, we tested if there was any effect modification by obesity on the association between AR + asthma, and the HRQoL and control scores. Secondly, we performed meta‐analyses to determine if there were any heterogeneity in the HRQoL and control  scores between the participating countries. All analyses were conducted using a complete case approach in Stata V.16 , and a 3The demographic and clinical characteristics of all the participants, stratified by country, are presented in Table In the multivariable analysis, we observed that, compared to AR alone, AR with comorbid asthma was significantly associated with poorer QoL . The association was persistent in RHINASTHMA subdomain scores; however, the magnitude of the estimates was different, being the highest for lower airways  and the lowest for upper airways . Upon stratifying asthma, based on the 2017 GINA grades, the magnitude of the association was the highest in AR patients with severe persistent asthma , and the lowest in AR patients with mild persistent asthma  ; the lower airway symptoms were more poorly controlled  than the upper airway symptoms . Upon stratifying asthma according to GINA grade, AR patients with severe persistent asthma had the poorest control  than those with mild persistent asthma  . The difference was more pronounced for upper airways, the association being significantly higher among non‐obese participants  than obese ones  . However, we did not observe significant differences in other subdomains. We did not observe any effect modification by obesity on CARAT scores  across the participating countries   Figure . However4In our study, we found a significantly worse QoL  and symptoms control  in patients with AR + asthma than in patients with AR alone. We also found that the association was significantly higher among non‐obese participants compared to obese ones, when assessed through RHIN‐Upper symptoms score but not with CARAT. We also observed country‐specific variations in the RHINASTHMA and CARAT Total scores. Although one previous study compared the individual/social burden of disease between asthmatics and asthmatics with concomitant AR, unlike ours, that study did not compare the difference in disease control and HRQoL between the two groups of patients.CLC, EMR4P, IL5RA, FRRS1, HRH4, SLC29A1, SIGLEC8, IL1RL1) that are presumed to describe the link for multimorbidity.It is well‐known that several triggers such as seasonal meteorological changes, pollen season, air pollution, or even occupational exposures may lead to poor QoL of asthmatic patients with or without AR.We found that the non‐obese patients with AR + asthma had poorer QoL (RHIN‐Upper score) than their obese counterparts. Reports citing the role of higher BMI on the QoL in patients with an upper airway disease have been mixed. While some studies reported poorer asthma QoL in asthmatics with higher BMI,We found significant country‐wise heterogeneity in RHINASTHMA and CARAT‐Total scores between AR alone and AR + asthma. This could be explained by a higher number of AR + asthma patients in France than in the two other countries. Another reason for such heterogeneity could be due to significant variation in allergen sensitivity between the countries, which has also been previously reported describing a significant difference in aeroallergens and allergies between European countries.e.g., allergies, rhinitis, tobacco smoke) and tools to control the disease have been developed. However, we are still uncertain how to prevent AR patients from developing asthma, allergen immunotherapy being the current only attempt. Preventive measures should be able to change the natural history of the disorder, avoiding asthma development in patients with AR and/or evolution by providing its control.Our findings add important clinical knowledge to the existing strategies for the management of AR with concomitant asthma. Although AR and asthma are two different diseases with distinct clinical features, when AR persists with asthma, either condition is often overlookedOur study has some limitations. Firstly, considering that subjective symptom‐rating scales may not be entirely accurate, the risk for potential bias could not be completely avoided. However, we used standardized instruments, and therefore the possibility of such bias was marginal. Secondly, the considered period might be insufficient to evaluate the QoL and the control appropriately. Thirdly, other comorbidities might have modified the patients' responses. Despite these limitations, our findings are derived from incident patients drawn from the general population of three European countries in which AR and asthma diagnoses were made by a doctor. However, due to the small sample size, it is not possible to indicate whether these results may be generalized. Further studies, after controlling for potential confounders and biases in larger populations, are therefore warranted.5In summary, using combined assessment tools for AR and asthma, we found that AR patients with comorbid asthma have a poorer quality of life and symptom control than those with AR alone. This finding highlights the importance of a comprehensive approach for the management of AR in case of a comorbid allergic condition for optimum care, and such strategies would be the gateway to reducing the global burden of these diseases.Subhabrata Moitra: Formal analysis (Lead); Software (Lead); Writing – original draft (Lead), Writing – review & editing . Marzia Simoni: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Sandra Baldacci: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Sara Maio: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Anna Angino: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Patrizia Silvi: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Giovanni Viegi: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Stefania La Grutta: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Franco Ruggiero: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Gianni Bedini: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Francesca Natali: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Lorenzo Cecchi: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Uwe Berger: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Maria Prentovic: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Amir Gamil: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Nour Baiz: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Michel Thibaudon: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Samuel Monnier: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing–review & editing . Davide Caimmi: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Luciana K. Tanno: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Pascal Demoly: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Simone Orlandini: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing–review & editing . Isabella Annesi‐Maesano: Conceptualization (Lead); Data curation (Lead); Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources (Lead); Supervision (Lead); Validation ; Visualization ; Writing – review & editing .Subhabrata Moitra reports personal fees from Synergy Respiratory & Cardiac Care (Canada), Permanyer Inc. (Spain), Elsevier Inc., and Apollo Gleneagles Hospital (India) outside this submitted work. Uwe Berger reports grants from CAMS‐32 outside the submitted work. Pascal Demoly received grants from ALK, Stallergenes Greer, AstraZeneca, ThermoFisherScientific, Ménarini, GlaxoSmithKline, Zambon, Viatris, and personal fees from Chiesi, and Puuressentiel, outside the submitted work. Pascal Demoly is also the Vice President of the French Allergy Society and Immediate Past President of the French Allergy Council. Isabella Annesi‐Maesano reports grants from the European Council and other public bodies outside the submitted work. Isabella Annesi‐Maesano is also the President of the Institut Recherche et Dévelopment Ethics Committee and a Member of the European Respiratory Society Ethics and Integrity Committee. Other authors do not have any conflict of interest to declare.Supplementary Material 1Click here for additional data file.


Tea plant breeding or cultivation mainly involves propagation via cuttings, which not only ensures the inheritance of the excellent characteristics of the mother plant but also facilitates mechanized management. The formation of adventitious root (AR) determines the success of cutting-based propagation, and auxin is an essential factor involved in this process. To understand the molecular mechanism underlying AR formation in nodal tea cuttings, transcriptome and endogenous hormone analysis was performed on the stem bases of red (mature)- and green (immature)-stem cuttings of ‘Echa 1 hao’ tea plant as affected by a pulse treatment with naphthalene acetic acid (NAA).IAA and GH3 genes may explain the decrease of endogenous IAA. NAA reduced endogenous cytokinin levels and further downregulated the expression of cytokinin signalling-related genes. By the use of weighted gene co-expression network analysis (WGCNA), several hub genes, including three  that are highly related to root development in other crops, were identified that might play important roles in AR formation in tea cuttings.In this study, NAA significantly promoted AR formation in both red- and green-stem cuttings but slightly reduced callus formation. External application of NAA reduced the levels of endogenous indole-3-acetic acid (IAA) and cytokinin . The number of DEGs (NAA vs. CK) identified in the green-stem cuttings was significantly higher than that in the red-stem cuttings, which corresponded to a higher rooting rate of green-stem cuttings under the NAA treatment. A total of 82 common DEGs were identified as being hormone-related and involved in the auxin, cytokinin, abscisic acid, ethylene, salicylic acid, brassinosteroid, and jasmonic acid pathways. The negative regulation of NAA-induced CSLD2, SVL1 and SAUR21, were identified that might play important roles in AR formation in tea cuttings.NAA promotes the formation of AR of tea cuttings in coordination with endogenous hormones. The most important endogenous AR inductor, IAA, was reduced in response to NAA. DEGs potentially involved in NAA-mediated AR formation of tea plant stem cuttings were identified via comparative transcriptome analysis. Several hub genes, such as The online version contains supplementary material available at 10.1186/s12870-022-03701-x. Camellia sinensis (L.) O. Kuntze], an important industrial crop species of the Camellia genus, originates from subtropical areas but is currently cultivated and consumed worldwide +or[M-H]-)