Allergic rhinitis (AR) is a major chronic inflammatory disease of the respiratory tract. A large number of epidemiological investigations have shown that the prevalence of AR is increasing, resulting in a large social burden. Importantly, the impact of air pollution on health is a widespread concern. We aim to evaluate association of air pollution and AR risk.2), nitrogen dioxide (NO2), carbon dioxide (CO), and O3, and patient data will be collected. The correlation analysis will be performed in air pollutants and AR risk.This prospective study includes patients undergoing AR. The exclusion criteria will be as follows: Patients with nasal infection, nasal polyps, nasal tumors, mental disorders, and immunodeficiency will be excluded. Air pollution levels of ambient air pollutants including PM2.5, PM10, sulfur dioxide (SO2, SO2, PM10, and PM2.5 for AR in several aspects, including symptom score, drug score, quality of life score, asthma control score, side effects, and laboratory examination such as nasal function test, serum total immunoglobulin E, and nasal secretion smear.This study will provide correlation of NO Researches on the correlation between air pollution and respiratory diseases (such as asthma and COPD) are abundant, while few studies investigated the correlation of allergic rhinitis (AR) with air pollution in China.Rapid economic development and the acceleration of urbanization have aggravated the haze in Sichuan, including Deyang city, an important industrial city in north Sichuan. The impact of air pollution exposure on human health has become a sensitive topic for the public, media, and even the government in China and neighboring countries. Moreover, AR involves a variety of immune-active cells and cytokines after atopic individuals are exposed to allergens, affecting 10% to 40% in the world. In China, the prevalence rate of adults is about 4% to 38%. Due to the easy relapse of AR and the delay in the course of the disease, the life, and work of the patients has been seriously affected. Some AR patients have suffered from asthma, nasal polyps, and rhinosinusitis. Obviously, AR has become a public health problem. Some experts believe that the increase in the prevalence of AR is caused by the increase of air pollutants–19; however, there is still no hard evidence to support it. At present, there are no continuous and dynamic studies to observe the changes of pollutants and the AR risk.The AR is a noninfectious respiratory inflammatory disease of nasal mucosa mediated by immunoglobulin E.This study was designed to assess the relationship between air pollution exposure and the risk of AR.22.1We aim to clarify the relationship between air pollution exposure and the risk of AR. Flow chart of study protocol is shown in Figure 2.1.1Aim 1: By collecting the general clinical data of patients with AR in multicenter outpatient clinics in Deyang region from January 2019 to December 2020, the prevalence of AR in outpatient clinics in Deyang region was investigated.2, and SO2 in Deyang area and discuss the correlation between pollutants and the prevalence of AR.Aim 2: To collect air pollution indicators including PM2.5, PM10, NOAim 3: To understand the quality of life of AR patients in the Asia-Pacific region via analyzing the correlation between AR symptoms and atmospheric pollutants.Aim 4: To provide targeted guidance and Suggestions for government departments to prevent airway inflammation and other diseases caused by air pollution. In this way, medical expenses of the public and health departments can be reduced and the quality of life of patients can be improved.2.2http://www.chictr.org.cn/index.aspx) with a unique ID of ChiCTR2000029027.The protocol scheme matches PRISMA's reporting standards. This study protocol has been registered on Chinese Clinical trial registry (2.3All age groups (newborn to adult) suffering from any form of AR will be included. These may include participants with acute or chronic comorbidities but without immunodeficiency. Participants may also include those on conventional treatment for other health issues. Symptoms of AR include rhinorrhea, nasal obstruction, nasal itching, and sneezing which are reversible spontaneously or with treatment. A diagnosis of AR will include the following symptoms based on the Guideline Summary of Management of Allergic and Non-AR: Nose: nasal discharge (runny nose), sneezing, nasal blockage or congestion, and itchy nose and palate; Eye: bilateral itchy eyes, red eyes , and swollen eyes.There is no specific duration of symptoms required for diagnosis; however, a very short or nonrepetitive history of symptoms generally excludes a diagnosis of AR. The presence of any one of the nasal symptoms will be essential for a diagnosis of AR except when nasal blockage is the only symptom. Nasal blockage on its own rarely indicates allergy. The duration of symptoms will not be present as a criterion since this has not been defined for a diagnosis of AR.2.3.1All participants will be diagnosed as AR in accordance with the diagnosis and treatment guidelines for AR , and will be diagnosed by a senior attending physician who had received unified training. All the patients participating in the questionnaire can understand the questionnaire accurately and cooperate with the successful completion of the questionnaire survey.2.3.2The exclusion criteria are as follows: Patients with nasal infection, nasal polyps, nasal tumors, mental disorders, and immunodeficiency will be excluded.2.3.3Patient data will be collected for all patients with AR, including general data such as sex, age, sex, place of residence, course of disease, and main clinical symptoms.2.4Deyang is the North city of Sichuan Province and closed to Sichuan capital city Chengdu. In recent years, PM pollution has become a serious issue for this city due to its large population and automobile and industrial emissions.2.5Each area in Deyang city  outpatient department of otolaryngology head and neck surgery patients, according to the guide to conform to the diagnosis and treatment of AR  for inclusion criteria, the general clinical data  each year, Deyang area in patients with AR disease registration rates.2), nitrogen dioxide (NO2), carbon dioxide (CO), and O3 will be derived from the China National Environmental Monitoring Center (http://www.cnemc.cn/). Daily data of weather conditions, including relative humidity (%) and mean temperature (°C), will be obtained from the China Meteorological Monitoring Database (http://data.cma.cn/).Air pollution levels of ambient air pollutants including PM2.5, PM10, sulfur dioxide .Continuous variables will be expressed as mean ± standard deviation, while nominal variables are expressed in frequency and percentage. Comparisons of continuous variables will be performed using the 2-tailed The data used for the study will be preserved and analyzed by the primary investigator. The data are accessible only to the primary investigator and study nurse for data safety. The data will be preserved for 2 years after the end of the study.2.7This study has been approved by the ethics committee of Deyang People's Hospital. All participants will sign the informed consent after being informed about the goals and methods of the study. The present study will be conducted in accordance with the tenets of the 1975 Declaration of Helsinki, as revised in 2000. The result of the study will be disseminated by publication as journal articles.3 Individual pollutants are responsible for increased risk of allergic disease in nitrogen oxides (NOx), SO2, O3, particulate aerodynamic diameters of 10 μm or less (PM10), and particulate aerodynamic diameters of 2.5 μm or less (PM2.5). However, for environmental pollutants, only 6 studies have reported the effects of outdoor air pollutants and the prevalence of AR in the Asian atmosphere.Globally, studies have shown an association between automotive and industrial emissions and increased AR risk.2, CO, and NOx levels, but not O3 and PM10 levels. Another Taiwanese study reported that AR in children is associated with nonsummer warmth and traffic-related air pollutant levels, including CO, NOx, and O3. For Asian adults, a cross-sectional population study in Singapore found that outdoor air pollution is an important environmental risk factor for AR. During 2007 to 2008, the concentrations of NO2 and SO2 were associated with an increase in the prevalence of AR among kindergarten children in 7 cities in Liaoning (China), but not with PM10. In addition, a study in Changsha (China) showed a significant positive correlation between the prevalence of AR in children and cumulative individual exposure to age-related PM10, SO2, and NO2. In this protocol, we will pay more attention on the effects of air pollutants  on AR in Deyang, Sichuan.A national cross-sectional study covering Taiwanese school children showed that AR prevalence was associated with SOXXX.Meng Zhang orcid: 0000-0002-3731-7816.


Propagation of cuttings is frequently used in various plant species, including blueberry, which shows special root characteristics that may hinder adventitious root (AR) formation. AR formation is influenced by various factors, and auxin is considered to play a central role; however, little is known of the related regulatory mechanisms. In this study, a comparative transcriptome analysis of green cuttings treated with or without indole-butyric acid (IBA) was performed via RNA_seq to identify candidate genes associated with IBA-induced AR formation.ARFs and SAURs), 13 transcription factors (LOB domain-containing protein (LBDs)), 6 auxin transporters ) and 6 rooting-associated genes (root meristem growth factor 9 (RGF9), lateral root primordium 1 (LRP1s), and dormancy-associated protein homologue 3 (DRMH3)). All these identified DEGs were highly upregulated in certain stages during AR formation, indicating their potential roles in blueberry AR formation.Rooting phenotypes, especially the rooting rate, were significantly promoted by exogenous auxin in the IBA application. Blueberry AR formation was an auxin-induced process, during which adventitious root primordium initiation (rpi) began at 14 days after cutting (DAC), root primordium (rp) was developed at 21 DAC, mature AR was observed at 28 DAC and finally outgrowth from the stem occurred at 35 DAC. Higher IAA levels and lower ABA and zeatin contents might facilitate AR formation and development. A time series transcriptome analysis identified 14,970 differentially expressed genes (DEGs) during AR formation, of which there were 7467 upregulated and 7503 downregulated genes. Of these, approximately 35 candidate DEGs involved in the auxin-induced pathway and AR formation were further identified, including 10 auxin respective genes (The transcriptome profiling results indicated candidate genes or major regulatory factors that influence adventitious root formation in blueberry and provided a comprehensive understanding of the rooting mechanism underlying the auxin-induced AR formation from blueberry green cuttings. Vaccinium corymbosum L.) is a member of the Ericaceae family and a commercially important small fruit crop because of their healthy and flavourful bioactive compounds, and blueberry acreage has been continuously expanded year-by-year worldwide, especially in China [Blueberry  in the IBA treatment Fig. a, adventChanges in plant hormones during blueberry AR formation were analysed. The level of IAA exhibited a significant increase at 21 DAC under the IBA treatment Fig. a-b, whicE-value of 10− 5 against five databases, and the total unigenes annotated by NR database, GO database, KEGG database, eggNOG database and Swiss-Prot database were 107,111, 53,859, 6696, 101,729 and 89,910, respectively  were subjected to total RNA extraction and RNA-seq analysis. Total 40.73–46.23 million (M) pairs of 150 bp raw reads were generated from RNA_sequencing for each library , 21.80% of the annotated sequences showed strong homology , and an additional 82.87% of the annotated sequences showed homology  to available plant sequences  and IBA treatment (T). The DEGs were identified by pairwise comparisons of ten libraries, i.e., CK7 vs. T7, CK14 vs. T14, CK21 vs. T21, CK28 vs. T28, and CK35 vs. T35 , and LAX5) and three efflux carriers (PIN-LIKE 6 (PIL6s)). All these DEGs were upregulated during AR development to some extent  and six auxin transporter genes  were selected to identify the DEG expression profiles by qRT-PCR. RNA_seq showed that ARF7 and ARF9 exhibited a significant transcript peak at 28 DAC in the IBA treatment, while both these ARFs showed a decreasing trend in the control  were suggested to regulate AR formation as downstream target genes of the ARF family. In the present work, thirteen homologue genes of LBDs were identified based on NR annotation  were representatively selected to confirm their expression profiles by qRT-PCR, and the results indicated that LBD16 and LBD37 showed good reproducibility with the RNA-seq data. Moreover, the qRT-PCR indicated that LBD23 was upregulated again at 14 DAC in the IBA treatment and LBD29 showed continuous upregulation from 21 DAC to 35 DAC in the IBA treatment and was also upregulated at 14 DAC and 35 DAC in the control , one putative root meristem growth factor 9 (RGF9), and one dormancy-associated protein homologue 3 (DRMH3)  Fig. . The RNADAC Fig. e, and itDAC Fig. f, DRMH3 ent Fig. g. The qRent Fig. g.Arabidopsis and other plant species, the regulatory pathway that controls blueberry AR formation was derived. It was speculated that IBA would induce the expression of auxin responsive factors ARF7/9 to perceive auxin signalling, whereas ARF7/9 directly or indirectly affected the downstream target LBDs genes to establish AR founder cells with nuclei migration. Then, auxin polar carriers, including influx carriers AUX22 or LAX3/5 and efflux carriers PIL6s, would be upregulated to facilitate the establishment of auxin asymmetric distribution, which includes AR primordium formation. Finally, the AR primordium transforms to the AR apical meristem and outgrowth from the cuttings under the effect of LRP1, RGF9, DRHM3 and other genes  were demonstrated to be involved in regulating AR formation, for which ARFs would activate or repress early responsive genes by banding with auxin response elements in the promoter region of these genes [ARFs have been isolated and identified from Arabidopsis, out of which ARF7 and ARF19 were suggested to participate in AR formation by positively activating the transcription of their downstream genes of LATERAL ORGAN BOUNDSARIS-DOMAIN (LBDs) [ARF7 and ARF19 could enhance AR emergence, while the loss-of-function of arf7, arf19 and arf7/arf19 double mutants led to severe defects in AR formation [LBD16 could finally result in the establishment of adventitious root-primordium identity, the lbd18 mutant exhibited a reduced number of roots and lbd16lbd18 double mutants showed a dramatic reduction in roots in comparison with lbd16 or lbd18; however, over-expression of LBD18 rescued the root formation in lbd18 and lbd16lbd18 mutant and could stimulate root formation in arf7arf19 mutants [ARF7, ARF9 and their downstream LBDs genes, were identified, and both transcriptome and qRT-PCR data showed obviously upregulation of these DEGS, indicating their potential roles in blueberry AR formation. However, the functional roles of these responsive genes in blueberry AR formation have not been clarified and needs to be verified.In the model plant pression , 40. Trase genes . To dateN (LBDs) –44. Overormation . The act mutants –48. In tArabidopsis and other species have indicated that the auxin polar transport (APT) system, which is mediated mainly by influx and efflux carriers, is essential for AR initiation and subsequent development because the APT system is strictly directional to establish auxin asymmetric localization and thus induces ARs initiation and emergence [PIN-FORMED (PIN) protein and its analogous PIN-LIKES (PILS) were believed to be efflux carriers, AUX1 and AUX1-LIKE (LAX) families acted as influx carriers and have been proven to exert great influence on AR formation [PIN1 would enhance AR formation from non-root tissues, with the pin1 mutant failing to establish an auxin gradient and showing developmental disorders in root formation. Similarly, AR development in rice was significantly inhibited in OsPIN1 RNA interference (RNAi) transgenic plants, which exhibited a defect in AR emergence, and exogenously applied NAA could rescue the rooting phenotypes in RNAi-OsPIN1 plants [pils2pils5 double loss of function mutant had higher auxin levels and lateral root numbers than the PILS5 gain-of-function phenotype [AUX22 upregulated at a very early stage i.e., 7 DAC - 21 DAC. Moreover, the auxin influx carriers LAX3/5 and auxin efflux carrier PIN-like6 were also upregulated, suggesting that these auxin-related genes might participate in the blueberry AR induction phase, in which the founder cells began to be dedifferentiated and then the dome-shaped root primordial was formed [Arabidopsis or other plant species, their functional roles in regulating blueberry AR formation remain unclear and need to be further studied.Numerous studies in mergence –51. PIN-ormation –55. Upreormation , 30. It 1 plants . The pilhenotype , 58. An s formed . AlthougLateral root primordial 1 (LRP1), which is one of ten members of the SHI gene family, acts as a transcriptional activator downstream of AUX/IAA, and it is expressed mainly in the early stages of lateral root primordium formation [LRP1-like transcripts were initially detected in the cotyledon and then rapidly became restricted to the upper zone, where cell division and root formation took place [LRP-like genes were identified by transcriptome data and they were certainly upregulated after IBA treatment, especially at 21 DAC and 28 DAC, during which the rp/rpi and AR begin to be formed, indicating the potential role of LRP-like genes in blueberry AR formation. In addition, some other genes, such as root meristem growth factor 9 (RGF9) and dormancy-associated protein homologue 3 (DRMH3), were identified in this present study. RGF, an important peptide hormone, was suggested to regulate root meristem development through its effects on the PLT1/2 stem cell transcription factor or by regulating of the stability of the receptor RGFR1 [RGF9 was upregulated after IBA treatment. DRM, one of auxin-repressed super-family genes, was suggested to be highly expressed in both dormant and non-growing tissues. BrDRM1 was indicated to be negatively associated with root growth, which was reduced by more than 50% in BrDRM1-overexpressing Arabidopsis plant, and the reduction was correlated to an increase in BrDRM1 expression levels [DRMH3 was highly regulated with AR formation, especially at 28 DAC after IBA treatment. This inconstancy might be caused by the varied function of DRM based on the plant species or from the functional differences among each member of the DRM family. Therefore, the molecular function of DRMH3 in blueberry AR formation should be further analysed. Although the molecular functions of the DEGs identified in this study remain unknown, our work offers a foundation for the future characterization of gene functions to ascertain the metabolism of IBA-induced AR formation in blueberry. However, AR formation is a complex developmental process controlled by multiple genes or transcription factors in addition to these DEGs in the auxin signalling pathway; thus, genes in other biological pathways might also play potential roles in regulating AR formation. Therefore, in the future, we will attempt to perform a comprehensive analysis of transcriptome data to broaden our understanding of the regarding regulatory mechanisms involved in AR formation of blueberry, such as the DEGs in the auxin biosynthesis and distribution, secondary metabolism, transport and degradation pathways as well as the DEGs associated with cytokinin, brassinosteroids (BRs), ABA, ethylene, GA and their interactions with the candidate DEGs in the auxin-signalling pathway.ormation –63. LRP1ok place . In thisor RGFR1 , 66. In n levels . HoweverThe adventitious rooting rate was significantly improved by IBA application. AR formation in blueberry was an auxin-induced process, with the adventitious root primordium (rp) initiated at 14 DAC, the rp formed at 21 DAC, the AR formed at 28 DAC and outgrowth from the stem observed at 35 DAC. Higher IAA content and lower ABA or zeatin levels would facilitate blueberry AR formation. To our knowledge, this work was the first to provide a comprehensive transcriptome profiling database for a dynamic view of transcript information in the IBA-induced AR formation in blueberry. A total of 672,606 transcripts and 308,719 unigenes were assembled. Approximately 14,970 DEGs were successfully identified, out of which 7467 were upregulated and 7503 were downregulated. A total of 35 DEGs involved in auxin-signalling and rooting-related pathways were considered as candidate genes based on their expression profiles. Although further research on the functional characteristics of these DEGs is required, our findings offer new insights into the molecular mechanisms underlying blueberry AR formation and provide a relatively complete molecular platform for future studies on the progression of blueberry AR formation. Moreover, the findings in the present work allow for the identification of candidate genes involved in blueberry AR formation and thus represent an important molecular resource for the further fast propagation and breeding of blueberry. Future works should aim to characterize the functional role of these identified individual DEGs and thus their regulatory networks or cross-talks.The southern highbush blueberry cultivar ‘Biloxi’ was used as the material in this study and obtained from the Hongyue Flower Co. LTD, and the plants were then cultivated at the Zhuang-hang Comprehensive Experimental Station of the Shanghai Academy of Agricultural Sciences, Shanghai, China. In spring, approximately 150 green cuttings with a length of 10–15 cm were randomly collected and most of the leaves were removed, retaining the top 1–2 leaves. The cuttings were immersed in deionized water (control) and 1000 ppm indole-butyric acid  for 1 min and then inserted into growth medium that consisted of peat moss, vermiculite and garden soil in a ratio of 3:1:1 (v/v). Water management was previously described in detail in An et al. (2019) [The stem from the base of the cuttings was randomly sampled at a 7-day interval after the trial installation . Approximately 0.5 g of fresh tissues from each sampled time were ground in liquid nitrogen and digested in 5 mL ethyl acetate for 12 h at 4 °C and then centrifuged at 10000 rpm at 4 °C for 10 min. The residue composition was further digested in 3 mL ethyl acetate and centrifuged at 10000 rpm at 4 °C for 10 min. The supernatant was dried by nitrogen flow at 25 °C, dissolved using 300 μL chromatographic methanol and then ultrasonically extracted for 10 min. Finally, the solution was filtered with a 0.22 μm membrane filter, and 5 μL was injected for analysis. The level of endogenous plant hormones  was analysed by liquid chromatography-mass spectrometry (LC-MS) according to Niu et al. (2014)  with minn = 10 for each replicate. The samples were fixed in the FAA solution . Before microstructure observation, the samples were softened for approximately 10 days by 4% ethylenediamine and then dehydrated using graded ethanol . Vitrification was performing using dimethylbenzene, and then the samples were embedded with paraffin. Then, 10-μm-thick transverse sections were cut with a rotatory microtome , and photos were captured and observed under a light microscope (NIKON ECLIPSE E200).After insertion, a 1-cm section from the bottom of the cuttings was sampled at a 7-day interval to observe the formation of adventitious root. All samples were collected from three biological replicates, with The stem of the base of cuttings (1–2 cm) was sampled at a 7-day interval, and then the samples were immediately frozen in liquid nitrogen and stored in − 80 °C before being analysed. Total RNA was extracted by the TRIzol Reagent . RNA integrity was then confirmed using the Agilent 2100 Bioanalyzer . The RNA-seq libraries were generated with the TruSeq RNA Sample Preparation Kit , and were applied to a HiSeq platform (Illumina) for RNA_seq analysis by Shanghai Personal Biotechnology Co., Ltd. Transcriptome de novo assembly was carried out using Trinity.E-value ≤10− 5, and the sequence direction was determined based on the best-aligning results. GO enrichment and the KEGG pathway annotation were obtained using BLAST2GO software and KEGG Automatic Annotation Server, respectively. The significance at P-value ≤0.05 of the DEGs were determined by DESeq software with a threshold of |log2FoldChange| > 1 [All unigenes were annotated by BLASTx searchs against five databases, i.e. NR database, GO database, KEGG database, eggNOG database and Swiss-Prot database with nge| > 1 –72.2O. The PCR conditions were described detail in Jiang et al. (2016) [GAPDH was used as internal control. Each reaction was performed with three biological replicates, and each sample was analysed in triplicate . The special primer sequences for the qRT-PCR analysis are shown detail in Additional file For qRT-PCR, about 1 μg of total RNA were reversed to obtain cDNA using the PrimerScript™ RT reagent Kit with gDNA Eraser . The qRT-PCR were carried out on a LightCycler 480 Real-Time PCR System  in a total volume of 10 μl containing 5.0 μl SYBR Premix ExTaq™ (RR820A), 0.5 μl of each primer (10 uM), 2 μl of cDNA and 2 μl ddH. (2016) . GAPDH wThe data analysis was performed via a one-way analysis of variance (ANOVA) with SPSS 18.0 software . Graphics were created using GraphPad Prism 6.0 .Additional file 1: Figure S1. Characteristics of the homology search of assembled blueberry unigenes against the NR database. Note: a, E-value distribution of the top BLAST hits for each unique sequence with a cut-off E-value of 1.0 E− 5; b, similarity distribution of the top BLAST hits for each unigene; and c, species distribution of all homologous unigenes with an E-value of at least 1.0 E− 5.Additional file 2: Figure S2. Functional annotation of blueberry based on Gene Ontology. A total of 263,143 unigenes were categorized into three main categories: biological process (96615), cellular component (103924) and molecular function (62604).Additional file 3: Figure S3. Statistics of differently expressed genes (DEGs). Note: a, significantly up- or downregulated genes using the threshold of P-value ≤0.001 and log2Ratio ≥ 1 in CK28 vs. T28; b, graphical representation of overall differently expressed genes in the IBA treatment; c, number of upregulated unigenes in the IBA treatment illustrated using a Venn diagram; and d, number of downregulated unigenes in the IBA treatment illustrated using a Venn diagram.Additional file 4: Figure S4 Expression of auxin responsive factor ARF9 during AR formation in the stem of blueberry green cuttings. Note: Control and IBA indicate the treatment used in this study. Axis X indicates the sampled time, e.g., 7d means the samples were collected 7 days after cutting. The bar indicates the standard error (n = 3).Additional file 5: Figure S5. Expression of auxin influx carriers LAX3 and LAX5 during AR formation in the stem of blueberry green cuttings. Note: Control and IBA indicate the treatment used in this study. Axis X indicates the sampled time, e.g., 7d means the samples were collected 7 days after cutting. The bar indicates the standard error (n = 3).Additional file 6: Figure S6. Expression of auxin efflux carriers PIN-like 6b and PIN-like 6c during AR formation in the stem of blueberry green cuttings. Note: Control and IBA indicate the treatment used in this study. Axis X indicates the sampled time, e.g., 7d means the samples were collected 7 days after cutting. The bar indicates the standard error (n = 3).Additional file 7: Figure S7. Expression of four LBDs during AR formation in blueberry green cuttings. Note: Control and IBA indicate the treatment used in this study. Axis X indicates the sampled time, e.g., 7d means the samples were collected 7 days after cutting. The bar indicates the standard error (n = 3).Additional file 8: Figure S8. Expression of four lateral root primordium protein-related LRP genes during AR formation in the stem of blueberry green cuttings. Note: Control and IBA indicate the treatment used in this study. Axis X indicates the sampled time, e.g., 7d means the samples were collected 7 days after cutting. The bar indicates the standard error (n = 3).Additional file 9: Table S1 Regression equation of the calibration curves for IAA, ABA, GA3 and zeatin.Additional file 10: Table S2 Primers for the qRT-PCR analysis used in this study.


Although more than 1,000 androgen receptor (AR) mutations have been identified and these mutants are pathologically important, few theoretical studies have investigated the role of AR protein folding stability in disease and its relationship with the phenotype of the patients. Here, we extracted AR variant data from four databases: ARDB, HGMD, Cosmic, and 1,000 genome. 905 androgen insensitivity syndrome (AIS)-associated loss-of-function mutants and 168 prostate cancer-associated gain-of-function mutants in AR were found. We analyzed the effect of single-residue variation on the folding stability of AR by FoldX and guanidine hydrochloride denaturation experiment, and found that genetic disease-associated mutations tend to have a significantly greater effect on protein stability than gene polymorphisms. Moreover, AR mutants in complete androgen insensitivity syndrome (CAIS) tend to have a greater effect on protein stability than in partial androgen insensitive syndrome (PAIS). This study, by linking disease phenotypes to changes in AR stability, demonstrates the importance of protein stability in the pathogenesis of hereditary disease. The most common cause of monogenic diseases is single-nucleotide variation (SNV) leading to amino acid substitutions. These missense variants can have a strong effect on the stability of a protein, leading to detrimental changes to protein function. Loss of protein stability is a major contributor to this single-gene disease1. More and more attention has been paid in the past few decades to understand the biological principles of protein stability6. Accurately predicting protein stability through theoretical and experimental methods is crucial for academic research and industrial applications.Since most proteins need to be folded to function, protein stability is one of the most basic properties of a protein. The protein stability discussed herein primarily refers to the thermodynamic stability of a protein, which determines whether the protein is in a naturally folded configuration or a denatured (unfolded or extended) state. Protein stability is a fundamental property that affects protein configuration, activity and regulation. It plays an essential role in evolution, a variety of diseases and industrial applications9. During the fetal period, androgens are primarily responsible for sex differentiation by masculinizing the Wolff tube and external genitalia9. During puberty, androgens regulate the growth and function of the male reproductive system9. In adults, androgens play key role in regulating behavior, spermatogenesis and bone metabolism9. Androgens mediate their actions primarily via the androgen receptor (AR), a ligand-dependent nuclear transcription factor expressed in primary/secondary sex organs9. AR is also expressed in non-genital organs such as the skeletal muscle, skin, adrenal gland, kidney and nervous system9.Androgens have a wide range of physiological effects on male reproductive and non-reproductive systems at different stages of development11, spinal and bulbar muscular atrophy (SBMA)13 and benign prostatic hyperplasia15. AR also plays an important role in the development and metastasis of several hormone-related cancers, including prostate cancer12, breast cancer17, liver cancer20.AR is an extensively studied steroid receptors. AR mutations have been identified in various diseases, including hereditary diseases such as androgen insensitivity syndrome (AIS)21. The primary mechanism of action for AR is to directly regulate gene transcription. Androgen binds to the AR, leading to conformational change of AR, dissociation of heat shock proteins, driving the interaction between the N- and C-terminus of AR, and importin-α binds AR to transport AR into the nucleus22. In the nucleus, the AR dimerizes and binds to androgen response elements (ARES) in the promoter region of the target genes23. AR interact with additional proteins in the nucleus, causing the transcription of specific target genes to be up- or down-regulated. Notable target genes for AR are insulin-like growth factor I receptor (IGF-1R)24, prostate-specific antigen (PSA)26, and transmembrane protease serine 2 (TMPRSS2)29.The AR contains three major functional domains: (1) the N-terminal domain (NTD) comprises an activation function 1 (AF-1) region, (2) DNA binding domain (DBD) and (3) the C-terminal ligand binding domain (LBD) comprise an AF-2 region12, few theoretical studies have investigated the impact of mutations on AR protein folding stability in disease and its relationship with the phenotype of the patients. Several algorithms have been developed to predict the effect of mutations on protein stability35. Notable algorithms include FoldX36, Dmutant37, I-Mutant2.038, CUPSAT39, Eris40 and STRUM41. Compared with other methods, FoldX performs well and is the most commonly used protein stability prediction algorithm42. Folding free energy reflects the overall protein stability, and changes in protein stability due to naturally occurring missense mutations often cause disease42. This work calculates the folding free energy of AR variants by FoldX and measures the guanidine hydrochloride (GdmHCl) denaturation curves of different mutants, trying to establish correlation between protein stability and patient phenotype. By correlating the patient's phenotype with changes in AR stability, this study may prove to be diagnostic and/or predictive tools for assessing the effects of mutations on disease outcome.Although more than 1,000 AR mutations have been identified and these mutants are pathologically importanthttps://androgendb.mcgill.ca), HGMD , Cosmic (2018.10.01), 1,000 genome; (2) analyzed mutation frequency, protein stability and relative surface accessibility (RSA) of these variants; (3) summarized the pathogenic mechanism of AR mutants in related diseases.In order to study the pathogenic pattern of AR mutants in related diseases, our computational analysis consist of the following steps as in Fig. 44. The latest human AR reference gene sequence (NM_000044.2) encodes a protein of 920 amino acids in length (instead of the previous 919). Because the reference length of ploy-Q is replaced by 23 instead of the original 21, and the reference length of poly-G is changed from 24 to 23. The length of the ploy-Q repeat is inversely proportional to the AR transcriptional activity, and the longer the polyglutamine repeat, the smaller the AR transcriptional activity45. The AR NTD is an intrinsically disordered proteins (IDP) that lacks a stable structure in aqueous solution  and intramolecularly interact with C terminal LBD domain (N/C interaction of AR)51.The NTD domain accounts for more than half of the entire AR protein as in Fig. 52. AR contains two NLS sequences-one in the DBD domain, the other one in the hinge region between DBD and LBD. NLS consists of two basic amino acid clusters separated by ten residues (617-RKCYEAGMTLGARKLKK-634). The binding of androgens to AR induces the exposure of NLS and result in nuclear import of AR by binding to the importin-α53.AR DBD (amino acids 560–620) consists of two zinc-coordinated modules, and it is highly conserved among steroid hormone receptors . AR LBD consists of 11 α-helices and 4 short β-strands, forming a three-layer anti-parallel α-helical sandwich fold, which is characteristic of AR LBD  was first well characterized in 200012. Severe androgen insensitivity (AI) produces an external female phenotype. Partial AI produce a range of external genital phenotypes, from near-normal females to normal or near-normal males. It has been suggested that the clinical severity of AI be divided into three levels: complete, partial , and mild (for the least severe form). In addition, there are 4 AR loss-of-function mutations associated with premature ovarian failure (POF)12.1,110 mutations were found in the AR gene, of which 905 were possible loss-of-function alterations and caused androgen insensitivity syndrome (AIS) or related with AIS. Different AR mutations impair androgen-dependent male sexual differentiation to varying degrees55. In prostate cancer, AR mutation may reduce the specificity and selectivity of its binding ligands, thereby being activated by a wider range of ligands such as adrenal androgens, estrogens, progesterone and antiandrogens. These gain-of-function AR mutations in prostate cancer tissue may be responsible for the failure of prior anti-androgen therapy. In addition, in spinal cord and bulbar muscular atrophy , poly-Q amplified AR protein is toxic to motor neurons to some extent by gain-of-function56.There are also 168 AR mutations that are possible gain-of-function alterations found in prostate cancer tissues. Aside from skin cancer, prostate cancer is the most common cancer among men and is the second leading cause of cancer-related death in the United StatesThe AR NTD is highly conserved with relatively few deleterious mutations, and only 8% of the residues in the domain are found to have mutations, including 230 variants , in which one amino acid is replaced by another amino acid, account for more than 40% of the unique variants observed in the Exome Aggregation Consortium databaseFrom the HGMD dataset, we downloaded a collection of 337 single residue mutations in the AR that are known to be associated with human inherited disease. All mutations in this set were found in patients with AIS and were associated with loss of AR function. From the Cosmic dataset, we downloaded 323 single residue mutations in the AR that are known to be associated with human cancers, the vast majority of which were associated with prostate cancer. From the 1,000 genome dataset, we downloaded 39 single residue variations in the AR that are not significantly associated with human diseases. The Venn diagram below shows the relations between these three AR mutation sets Fig. .Figure 4FoldX, the most commonly used protein stability prediction algorithm, can estimate the effect of SRV on protein stability based on the three-dimensional structure of the protein. So far, only the DBD and LBD domain in AR has crystal structure available, so we selected SRV of the DBD and LBD domain for analysis. We applied FoldX to analyze single residue variations in the AR DBD and LBD domain to estimate changes in protein folding free energy caused by these variations. We found that the predicted folding free energy change ΔΔG of human inherited disease-associated AR mutations (HGMD) was significantly higher Fig. . The ΔΔG59. Most intracellular protein degradation occurs via the ubiquitin–proteasome system (UPS) or autophagy–lysosomal pathway (ALP). In a folded protein, degradation signals are usually buried in the protein. When a protein is partially or fully unfolded, one or more degrons of the protein may be exposed. E3 ubiquitin ligase scans cells for such degradation signals, binds substrates, and promotes substrate ubiquitination and degradation by the 26S proteasome. A recent study on Lynch syndrome-associated MSH2 mutations found that, as little as 3 kcal/mol was sufficient to trigger protein degradation60. The ALP is usually responsible for the degradation of highly misfolded and insoluble protein aggregates.Structurally unstable or misfolded proteins may form toxic aggregates or inclusions. Organisms control protein quality by refolding or degrading of these unstable or misfolded proteinsWe further analyzed the correlation between the clinical severity of the AIS patient and the folding stability of the related AR mutation. AR mutants that cause severe androgen insensitivity (CAIS) tend to have a significantly greater impact on protein stability compared to partial AI (PAIS). PAIS AR mutants compared to polymorphisms tend to have a slight greater impact on protein stability Fig. .Figure 6Escherichia coli and purified by Ni–NTA affinity column and following Superdex-75 gel filtration column  AR, we randomly select two AR mutants that cause CAIS  and two mutants that cause PAIS . The above proteins with N-terminal his-tag were expressed in In the room termperature, the AR-LBD WT and its mutants have similar CD spectra  of AR-LBD WT is at 3.0 M GdmHCl, which is higher than those C1/2 of disease mutants  at zero GdmHCl concentration gives the extrapolated folding free energy at physiological condition. The slope m represents the sensitivity of protein stability to denaturant. These values are given in Table We further calculated the folding free energies of AR-LBD variants based on the GdmHCl denaturation experiment. The linear relationship between ΔG and GdmHCl concentration is shown in Fig. 0 of AR-LBD WT is − 5.3 kcal/mol, while those of I738T, C807Y, W752R and L813F mutants are higher (less stable) by 2.3–3.8 kcal/mol (ΔΔG). W752R and L813F, mutations of CAIS, compared to PAIS mutations I738T and C807Y, have greater changes in folding free energy. We proposed that AR mutants with less stabilities can lead to more severe androgen insensitivity syndrome. In summary, the experimental results are consistent with the FoldX prediction results.The fitted folding free energy ΔG61. The m values of I738T, C807Y, W752R and L813F mutants were 1.3, 1.1, 0.9 and 0.6, respectively, which were lower than the m value of AR-LBD WT (1.7). The decrease of the m value indicates two possibilities: (1) the mutation increases the solvent accessibility of the hydrophobic residue in the folded state. (2) The deletion of some hydrogen bonds in the mutant will weaken the hydrophobic interaction of certain residues, thereby increasing its accessibility or sensitivity to GdmHCl. The decreasing trend of m value of mutants is also related to the ΔΔG of these mutant proteins. The L813F mutant with the smallest m value possessed the greatest decrease in protein stability . The I738T with the smallest m value reduction has the smallest stability reduction .The m value can be interpreted by the free energy change of the folded and unfolded protein being transferred from water to 1 M GdmHClIn summary, for the first time through computer-based analysis, we found that mutations associated with human inherited diseases tend to have a significantly greater impact on protein stability than polymorphisms. Using computer-based analysis and GdmHCl denaturation experiments, we found that changes in protein folding stability are correlated with patient phenotypes. The change of folding free energy of the AR-LBD mutants predicted by FoldX are consistent with the measured folding free energy changes by GdmHCl denaturation experiments, which further supports the reliability of our conclusion. Therefore, this paper clearly demonstrates the importance of AR protein stability in the pathogenicity of hereditary diseases (AIS), and provides reference for clinic diagnosis.62. For these pathogenic variations, it may be possible to rescue protein function by preventing their recognition or degradation by the protein quality control system62. If the degradation of these protein variants is inhibited, it is possible to avoid pathogenicity. In addition, some pathogenic variations may be so unstable that even inhibition of their degradation is not sufficient to rescue their stability and function. These variants can restore protein function by stabilizing the protein with small molecules that bind directly to the unstable protein variants63. Small molecules compounds have been shown to rescue function of pathogenic variations, such as in mutants CFTR64 and TP5365.In addition to predicting pathogenicity and improving diagnosis, this AR protein stability studies provide new opportunities for the treatment of AIS. Many pathogenic variations might be adequately functional, but are degraded by protein quality control system due to mild instabilityhttps://androgendb.mcgill.ca), HGMD , Cosmic (2018.10.01), 1,000 genome;We extracted AR variations from the following four databases: ARDB  was added with the final concentration of 30 μM. After 15 min, 0.1 mM IPTG was added to induce protein expression at 16 °C overnight. The cells were collected by centrifugation at 5,000g for 10 min at 4 °C. The centrifuged cells were then resuspended and sonicated in 20 mM Tris buffer containing 500 mM NaCl and 50 mM imidazole (pH8.0). Debris was removed by centrifugation at 20,000g for 30 min. The supernatant was loaded into a Ni–NTA column and the desired fraction was eluted with 300 mM imidazole. The eluent was loaded into a Superdex 75 column equilibrated with a buffer . The collected fractions were concentrated to about 0.5–2 mg/ml for CD measurement. The protein concentration was determined by using the calculated extinction coefficient at 280 nm.The sequence of human AR cDNA (NCBI accessiong number: CCDS14387.1) encoding 664-920aa was cloned into pET28a expression vector. Site-directed mutagenesis was prepared using the procedure provided by the QuickChange site-directed mutagenesis kit. All constructs were verified by DNA sequencing. In the expression of WT AR and mutants, when the ODCD measurement is performed on a Chirascan spectrometer. All CD measurements were performed in buffer . For the GdmHCl induced denaturation experiments, 70 μg/ml of AR-LBD WT and mutant proteins with different GdmHCl concentrations were prepared and equilibrated at 25 °C for 1 h. The CD signal was measured at a path of 0.1 cm, and three independent measurement results were averaged.θi] is the ellipticity at the ith gdmHCl concentration, [θF] is the ellipticity of the protein completely folded, [θU] is the ellipticity of the protein in 5 M GdmHCl. It is assumed that the protein in 5 M GdmHCl has been fully unfolded.i is the folding constant of the monomer protein at the ith GdmHCl concentration, which can be calculated by the folding fraction αi. The free energy of protein folding can be estimated by the following equation:KF is the folding constant of monomer protein, which can be calculated by the function KF = [F]/[U], where [F] and [U] represent respectively folded and unfolded fractions.A two-state hypothesis was used to fit the denaturation curve of GdmHCl. The folding free energy of AR-LBD WT and mutant proteins without GdmHCl is estimated by a linear extrapolation:Gi is the free energy of protein at the ith GdmHCl concentration, and ΔG0 is the free energy of protein folding without GdmHCl.The free energy of protein folding is a linear function of GdmHCl concentration, where Δ


The development of prostate cancer (PCa) from androgen-deprivation therapy (ADT) sensitive to castration resistant (CRPC) seriously impacts life quality and survival of PCa patients. Emerging evidence shows that long noncoding RNAs (lncRNAs) play vital roles in cancer initiation and progression. However, the inherited mechanisms of how lncRNAs participate in PCa progression and treatment resistance remain unclear. Here, we found that a long noncoding RNA LINC00675 was upregulated in androgen-insensitive PCa cell lines and CRPC patients, which promoted PCa progression both in vitro and in vivo. Knockdown of LINC00675 markedly suppressed tumor formation and attenuated enzalutamide resistance of PCa cells. Mechanistically, LINC00675 could directly modulate androgen receptor’s (AR) interaction with mouse double minute-2 (MDM2) and block AR’s ubiquitination by binding to it. Meanwhile, LINC00675 could bind to GATA2 mRNA and stabilize its expression level, in which GATA2 could act as a co-activator in the AR signaling pathway. Notably, we treated subcutaneous xenografts models with enzalutamide and antisense oligonucleotides (ASO) targeting LINC00675 in vivo and found that targeting LINC00675 would benefit androgen-deprivation-insensitive models. Our findings disclose that the LINC00675/MDM2/GATA2/AR signaling axis is a potential therapeutic target for CRPC patients. Radical surgery and radiation therapy are the standard treatments for localized PCa. Androgen-deprivation therapy (ADT) is an effective treatment for locally advanced, biochemically recurrent or metastatic PCa2. However, most PCa patients that are initially sensitive to ADT will develop lethal CRPC inevitably. Dysregulation of the androgen receptor (AR) signal pathway is critical for PCa development and progression into CRPC3. Most PCa that relapses during ADT remains dependent on AR activity, in which events such as AR amplification and stabilization of mRNA and protein4, sensitization to low levels of androgen or steroid analog5, intratumoral de novo steroidogenesis6, or AR splice variant production7 may occur. The underlying mechanism and potential target that drive PCa into CRPC need to be illustrated so that novel therapeutics can be developed to be used in combination with ADT, which may be more effective in prolonging survival.Prostate cancer (PCa) is the most commonly diagnosed male malignancy and second frequent cause of mortalityThe ubiquitin–proteasome system plays vital roles in several biological processes, and protein ubiquitylation requires three processes, including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). Mouse double minute-2 (MDM2), as a ring finger-containing E3, has been found to be involved in the regulation of AR ubiquitylation and degradation, by directly binding to the N-terminal and DBD of AR protein.8. LncRNAs used as diagnostic biomarkers have received sustained attention because they are expressed in a more tissue-specific manner than protein-coding genes9. Emerging evidence shows that lncRNAs play vital roles in cancer initiation and progression10. In PCa, lncRNAs such as PCA3 and SChLAP1 were reported as diagnostic and prognostic biomarkers12. PCa-associated lncRNAs were reported to participate in tumor progression13, castration resistance15, and metastasis16 via diverse mechanisms, including recruitment of the chromatin-modifying complex, chromatin remodeling, or transcriptional co-regulation, and by interacting with RNAs or proteins at post-transcriptional levels. Our previous studies revealed that lncRNAs were implicated in PCa progression and AR signaling pathway17. Li et al. found that LINC00675 was highly expressed in pancreatic ductal adenocarcinoma compared with adjacent normal tissue, and was positively correlated to lymph-node metastasis and poor survival18. Finally, LINC00675 was reported to be involved in cell proliferation, metastasis, and invasion in gastric cancer, colorectal cancer, and cervical cancer21. To date, the role of LINC00675 in PCa still remains unsettled.Long noncoding RNAs (lncRNAs) are known as a group of transcripts with lengths exceeding 200 nucleotides and not translated into proteinIn this study, we discovered that LINC00675 is significantly enriched in androgen-insensitive PCa cell lines and tissue specimens of CRPC patients. LINC00675 binds to AR protein and blocks its ubiquitination under androgen-deprivation conditions. Moreover, we demonstrated that LINC00675 could bind to GATA2 mRNA and stabilize its expression level, and then GATA2 can act as a co-activator in AR signaling in the nucleus. Our results suggest that the LINC00675/MDM2/GATA2/AR signaling axis contributes to the castration resistance and progression of PCa and is a promising therapeutic target.LNCaP-SF and its relative control LNCaP-JP human PCa cells were provided and cultured as recommended by Professor Atsushi Mizokami . LNCaP-SF was constructed from the androgen-dependent cell line LNCaP-JP. The culture medium was replaced every 2–3 days with 5% charcoal-stripped fetal bovine serum , then LNCaP-JP cells were cultured in androgen-deficient medium for over 6 months to construct stable LNCaP-SF cell line. LNCaP-SF was cultured in DMEM medium containing 10% charcoal-stripped fetal bovine serum, while its control cell line LNCaP-JP was cultured in DMEM medium containing 10% FBS. LNCaP-Abl cell was kindly provided by professor Jun Qin  and cultured in RPMI-1640 medium containing 10% FBS. Its control cell line LNCaP was cultured as LNCaP-JP. LNCaP-C4–2b was obtained from American Type Culture Collection (ATCC) and cultured in T-medium containing 10% FBS. T-medium was prepared according to standard protocols. Enzalutamide (MDV3100)-resistant C4–2b (LNCaP-C4–2b-ENZ) cell was kindly provided by Dr. Allen Gao  and maintained in DMEM containing 20 μM enzalutamide and 10% FBS. BSA (A8022), triiodo-L-thyronine (T2877), transferrin (T4382), D-biotin (47868), and adenine (A3159) were purchased from Sigma-Aldrich. Insulin (12585–014) was purchased from Gibco.2 atmosphere.The cells were all cultured in a 37 °C water-saturated 5% COThe plasmids used to knock down LINC00675, and its vector GV248 were purchased from Genechem, Inc . Sequences of the shRNAs used to knock down LINC00675 were as follows: (1) forward: 5′- CUCCGAUCUUUCAUGGUAU-3′; reverse: 5′-AUACCAUGAAAGAUCGGAG-3′; (2) forward: 5′-CAUAGGCUAACUGGAUAGA-3′; reverse: 5′-UCUAUCCAGUUAGCCUAUG-3′; (3) forward: 5′-CAGUGGCUCAUAUUGAGGU-3′; reverse: 5′-ACCUCAAUAUGAGCCACUG-3′. The plasmids used to overexpress AR, and its vector LvEKP01 were purchased from Era Biotech, Inc . The Flag-tagged pcDNA3.1-AR, pcDNA3.1-AR-truncated, and LvEKP01-LINC00675 were self-constructed. Full-length LINC00675, AR, and truncated AR  were amplified by PCR and subcloned into the relative sites of the LvEKP01 vector or pcDNA3.1 vector.293T cell was used to generate lentiviruses and seeded in 10-cm culture dishes before transfection. For the knockdown experiment, 293T cells were transfected with shLINC00675 plasmids or its vector with the packaging plasmids psPAX2 and pMD2.0 G using Lipofectamine 3000 . For the overexpression experiment, 293T cells were transfected with pcDNA3.1-AR, pcDNA3.1-AR-truncated, LvEKP01-LINC00675, and its relative vectors with the packaging plasmids pGag-pol, pREV, and pVSVG. Supernatant was collected 48 h after transfection, and filtered through 0.45-μm sterilization filters. Then 1 ml of lentiviruses, 1 ml of culture medium, and 2 μl of polybrene  were applied to the cells. Culture medium was replaced after 8–12 h, and the stable cells were selected with puromycin  or flow cytometry.TM Reagent, and RNA quality was checked using an Agilent 2100 Bioanalyzer . Then the total RNA was purified with the RNA Clean XP Kit  and DNase set . Afterward, 1000 ng of RNA was used as templates to synthesize cDNA with VAHTS® mRNA-seq V2 Library Prep Kit for Illumina . Finally, cDNA was amplified by PCR, quality checked using Bioanalyzer , and sequenced using Illumina HiSeq 4000 platform .The total RNA was extracted using TRIzolTM M-280 Streptavidin  was added to the RNA–protein complex and incubated with the complex at room temperature for 2 h. After washes, the pull-down complexes were denatured and detected by western blot.Full-length LINC00675 was amplified by PCR. The primer sequences are as follows: LINC00675-sense-F: 5′- TAATACGACTCACTATAGGGAGAGTGGCTCCAAGAAGCGCCAG-3′; LINC00675-sense-R: 5′-TTACATAAAAACAAAGGCCAAATCT-3′. Then LINC00675 RNA was biotinylated using Biotin RNA Labeling Mix Kit  and in vitro transcribed using T7 polymerase . After this, 3 μg of biotinylated LINC00675 RNA was incubated with 1 mg of total cell lysates to bind to the protein of interest at 4 °C overnight. Then, 30 μl Dynabeads3 cells per well in 96-well culture plates. Cell viability was determined by CCK-8 at a final concentration of 10% to each well, and the absorbance of the samples was measured at 450 nm using an EpochTM Microplate Spectrophotometer  every 24 h for 5 days. The proliferation curves were plotted using the absorbance.Cell Counting Kit-8 (CCK-8)  was used to assess the long-term cell survival according to the manufacturer’s instructions. Cell suspensions were seeded at 5 × 107 LNCaP-C4–2bshGFP和LNCaP-C4–2bshLINC00675 cells. Tumor growth was monitored every 3 days after a week post inoculation. Tumor volume was calculated according to the formula: volume = length × width2 × 0.5. After 4 weeks, the mice were euthanized, and tumors were harvested, weighed, and subjected to IHC and western blot analyses.Male BALB/c nude mice, 4–5 weeks old, were purchased from the Department of Laboratory Animal Science in Shanghai Jiao Tong University. To evaluate the effect of LINC00675 knockdown on tumor growth in vivo, mice were subcutaneously inoculated with 1 × 107) were mixed with Matrigel  at a ratio of 1:1 and subcutaneously injected into the 6-week-old male BALB/c nude mice. When the tumors were touchable, the mice were treated with enzalutamide and/or antisense oligonucleotides (ASO). In all, 10 mg/kg enzalutamide was intraperitoneal injected every 2 days. The in vivo-jetPEITM DNA & siRNA Delivery Kit  was used to knock down LINC00675 in vivo. In total, 20 μg of ASO and 2.4 μl in vivo-jetPEI reagent were, respectively, dissolved in 12.5 μl 10% glucose solution, then mixed and diluted into 50 μl. After incubation at room temperature for 15 min, the complex was injected into the tumors at a frequency of twice a week. Formalin-fixed paraffin-embedded sections of subcutaneous tumors were subjected to IHC.LNCaP-C4–2b-ENZ cells  kit was used for detection. The quantitative results for IHC images were calculated by ImageJ software.Formalin-fixed paraffin-embedded sections from specimens were baked at 60 °C for 4 h, deparaffinized by three times of 10-min extraction in 100% xylene and rehydrated in a series of graded alcohol . After washing with phosphate buffered saline (PBS), sections were transferred to boiling sodium citrate buffer  to retrieve antigen for 30 min and pre-treated with 3% HThe primary antibodies are as follows: Ki67  and AR  were commercially purchased.Western blots were performed according to standard methods. After relative treatment, cells were washed three times with pre-chilled PBS and lysed for 30 min on ice with RIPA buffer  containing protease and phosphatase-inhibitor mixture . Lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. Protein concentration of the supernatants was determined using a BCA Protein Assay Kit . The protein fractions were resuspended in loading buffer and denatured at 100 °C for 10 min. Equal amount of proteins was resolved by 10% SDS polyacrylamide gels and transferred to PVDF membranes . Membranes were blocked in 5% fat-free milk in TBST buffer (0.1% Tween-20) for 1 h at room temperature and then incubated with specific antibodies for different western blot analyses at 4 °C overnight.The antibodies used in the assays are listed in the Supplementary Materials.t test for variables with normal distribution, or the Mann–Whitney U test for variables without normal distribution; categorical variables were compared by Pearson’s Chi-square test or Fisher’s exact test. Two-sided P-values <0.05 were considered statistically significant.Statistical analyses were conducted using SPSS19.0 software, and graphs were generated using GraphPad Prism 6.0 software. All in vitro experiments were performed in biological triplicate. Data were presented as the mean ± SD. Continuous variables were compared by Student’s 22, we also found that LINC00675 expression was markedly increased in tissues of PCa patients compared with tissues of normal prostate  and respective androgen-dependent parental cell lines (LNCaP-JP & LNCaP) to assay lncRNA expression profiles using RNA sequencing Fig. . Fold chate Fig. . A similate Fig. . Taken tTo explore the potential function of LINC00675 on PCa progression, we constructed three short-hairpin RNAs (shRNAs) to inhibit LINC00675 expression in LNCaP-SF and LNCaP-C4–2b Fig. . KnockdoNext, using Gene Set Enrichment Analysis (GSEA) with the above PCa cell lines RNA-seq data, we found that the AR signaling pathway was upregulated  of AR to promote AR–ubiquitin interaction and AR ubiquitinationMoreover, Flag-AR, HA-MDM2, and different concentrations of LINC00675 were co-transfected into the 293T cells. Exogenous co-IP showed that LINC00675 could repress the interaction between AR and MDM2, and the repression effects were dose-dependent Fig. . In summTo determine specific areas of LINC00675 that could bind to AR, we constructed four truncated LINC00675, including 1–488S, 1–736S, 489–1530S, and 737–1530S as shown in Fig. To evaluate whether LINC00675 silencing would promote ADT, we conducted in vivo subcutaneous xenograft study with antisense oligonucleotides (ASO) targeting LINC00675 with or without enzalutamide , while E3 ubiquitin ligase–MDM2 recruits AR via binding to its NTD and mediates ubiquitination degradation of the protein31. HOTAIR (HOX transcript antisense RNA) was reported as an androgen-repressed lncRNA that could bind AR protein to protect it from ubiquitin-mediated degradation14. The study showed that HOTAIR could bind to AR protein to block its interaction with MDM2. Zhang et al. reported that ARLNC1 could bind to AR mRNA directly and stabilize AR transcript via RNA–RNA interaction32. In this study, we found that LINC00675 could directly bind to NTD of AR in specific regions as a protector to block the binding area between AR and MDM2, and thus decrease ubiquitination levels of AR to increase the stability of AR protein.The AR signaling axis is critical in PCa development and progression, and androgen deprivation represents first-line treatment for late-stage PCa patients34. GATA2 could bind to the upstream promoter region of AR and facilitate AR expression; in addition, GATA2 recruit p300 for H3K27 acetylation to create an active and accessible chromatin environment34. GATA2 expression levels are upregulated in CRPC tumor samples than primary cancer samples33, and GATA2 expression levels increase throughout the transition from androgen-dependent status to castration-resistant PCa xenograft mouse model35. Notably, our findings demonstrate that LINC00675 could bind to GATA2 transcript in addition to AR protein. On one hand, LINC00675 directly binds to the NTD of AR protein to prevent its interaction with MDM2 via the same domain, thus LINC00675 inhibited AR ubiquitination-mediated degradation and increased AR protein stability via a competitive mechanism. On the other hand, LINC00675 promotes AR signaling by binding GATA2 mRNA, increasing GATA2 mRNA stability, causing increased GATA2 co-activation of AR and promoting PCa progression.As a mediator of AR signaling, it has been reported that GATA2 plays a critical role in sustaining both AR-dependent and -independent PCa progression36. Targeting the MALAT1/AR-V7 axis with the drug ASC-J9 might be effective in patients insensitive to enzalutamide treatment37. Also, the drug MS351 as a PRC1 complex inhibitor could disrupt the chromatin-modifying complexes between PRC2 and HOTAIR38. In this study, we treated subcutaneous xenografts models with enzalutamide and ASO-targeting-LINC00675 in vivo and found that targeting LINC00675 would benefit androgen-deprivation insensitive models, which suggest that the LINC00675/AR axis may be a promising drug target in ADT-insensitive cases.LncRNAs could be degraded by siRNAs or ASOs and targeted by small-molecular inhibitors to disrupt downstream pathwaysAlthough we have explored the role of LINC00675 in AR signaling during CRPC, the molecular mechanism leading to LINC00675 upregulation in CRPC remains to be fully illustrated. In addition, whether the physical interaction between LINC00675 and AR protein, and binding of LINC00675 and GATA2 mRNA transcript occurs with additional RNA-binding protein need to be illustrated.In summary, it is our novel discovery that LINC00675 promotes castration resistance by binding AR, blocking its recruitment with MDM2 and protecting it from ubiquitin-mediated degradation. LINC00675 could bind to GATA2 mRNA and enhance its stability, thus GATA2 protein could act as a co-activator of AR (Fig. supplementary materialsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4


More than one billion people worldwide live with a disability, yet rehabilitation professionals are scarce in low- and middle-income countries. Attempts to expand access to rehabilitation services have encountered barriers on multiple levels: limited resources on the systemic level, hierarchies on the professional level, and cultural stigma on the community level.We sought to determine if an academic-community partnership could overcome multiple levels of barriers to expand services for people with disabilities.Toward an All-Inclusive Jordan incorporates community-based rehabilitation with prelicensure health professions education to address the three primary levels of barriers to rehabilitation services in low- and middle-income countries. The yearlong curriculum includes formal training, research, and advocacy with graduate students from the United States and health professions students and community members in Palestinian refugee camps near Amman, Jordan.After two cycles of the program, 14 Jordanian volunteers have partnered with 20 graduate students from the United States. They have delivered over 300 direct rehabilitation sessions, conducted ten workshops with mothers of children with disabilities, and trained 12 community-based rehabilitation workers in the refugee camps.The academic-community partnership model builds on the evidence base for the success of community-based rehabilitation services in low- and middle-income countries. Its components address barriers on multiple levels to create a sustainable expansion of services to people with disabilities. Archives of Physical Medicine and Rehabilitation, Jesus and Hoenig noticed that, in discussing the need for quality improvement, the consensus documents included no mention of rehabilitation services [Several recent international reports on the quality of health care have identified the shortcomings of health care systems globally, particularly in low- and middle-income countries (LMIC). In an editorial in services . Overlooservices . To addrThe 2015 public health emergency in Brazil sparked by the Zika virus illustrated the danger of focusing health care attention primarily on the immediate response without considering access to rehabilitation. When the link between congenital developmental conditions and the Zika virus was established, international research on diagnosis and treatment ramped up to supplement local efforts to contain the mosquito vector . By 2017Of the over one billion people living with a disability, 80% live in a LMIC like Brazil, where access to rehabilitation services is severely limited . DependiSome academics and nongovernmental organizations have recognized the urgent need to expand access to rehabilitation services for the large and growing population of people with disabilities . Their rAny effort to expand access to rehabilitative services in LMIC must address obstacles on three levels: macrosystemic, professional, and community. At the macrosystemic level, the most obvious and pernicious obstacle is the competition for scarce resources. As with other health interventions that require long-term investment, rehabilitation services appear less attractive than measures that produce immediate results . The priAnother barrier to expanding rehabilitative services emerges from embedded hierarchies among the health professions. Physician dominance over health care in many LMIC may lead them to deprioritize rehabilitation, which is traditionally delivered by therapists. In high-income countries, bureaucratization and corporatization of health care has introduced outside administration that erodes physician dominance . By contThe third level of impediment exists among communities. In many LMIC, cultural stigma against people with disabilities discourages them from seeking treatment. People with disabilities face bullying in school, discrimination in employment, and deprecation from family members. Some come to internalize these negative attitudes. In a comparative study of people with disabilities in Cameroon and India, researchers found that interviewees prioritized family members’ medical needs over their own impairments, which they grew to accept or treat with traditional remedies . Even inTo respond to the urgent and unmet need for rehabilitation globally, the WHO announced a call to scale up rehabilitation efforts by 2030. As a response, several frameworks were formed including the Global Rehabilitation Alliance, the rehabilitation competency framework, and an update of the previously formed Community-Based Rehabilitation (CBR) framework. The Global Rehabilitation Alliance was formed in 2018 to supplement the development and delivery of rehabilitation universally. This alliance plays a key role in advocating for rehabilitation on a systemic level but remains advisory and does not address the competition for scarce resources in LMIC that limits rehabilitation services to people with disabilities, nor does it address obstacles at the professional or community levels.Another response to the WHO call for action on the professional level has been the development of the rehabilitation competency framework. In 2018, a task force convened to organize a collection of competency statements for rehabilitation professionals to assist in overcoming workforce barriers. This framework (which is still under development) will assist rehabilitation practitioners worldwide by providing a unified language across disciplines while avoiding variation in terminology, concepts, and structure . While tCommunity-Based Rehabilitation (CBR) was established in 1978 by the WHO as an approach for social inclusion in resource-constrained settings and focused on working with people with disabilities within their communities. Since its establishment, CBR efforts have targeted barriers to access at the community level by equipping families, peers, and professionals as volunteers to deliver services without rehabilitation specialization . Social One promising approach that counters barriers at all three levels is to incorporate CBR with prelicensure health professions education. In 2017, the primary author launched Toward an All-Inclusive Jordan at MGH Institute of Health Professions, an independent, not-for-profit graduate school in Boston, Massachusetts, USA. The yearlong curriculum was designed to engage health professions students from the United States and Jordan in a CBR initiative in AlBaqaa refugee camp near Amman, Jordan, as a pilot. The program has three main components:Education: MGH Institute graduate students in clinical doctorate programs in occupational and physical therapy and master’s students of speech-language pathology learn service delivery models of rehabilitation in LMICs. First, students complete a semester-long course in CBR and then conduct capstone graduation projects. Both elements include working in the field as rehabilitation trainers and providing direct service, including assessment, treatment planning, and rehabilitation intervention for people of disabilities and their families.Research: Doctoral capstone projects of students are tailored to inform areas of rehabilitation and rehabilitation access in Jordan, such as studying the correlates of participation of children with disabilities in Jordanian schools and the prevalence of depression among Jordanians with physical disabilities.Advocacy: Raising awareness about the rights of people with disabilities and educating community members about their legal rights is an integral component of the program. While they are visiting the refugee camp, graduate students help develop workshops to spread awareness among people of disabilities and their families about their rights. Additionally, the students work on developing content for social media platforms to address disability and stigma nationally.The Toward an All-Inclusive Jordan approach counters the three levels of barriers to expanding rehabilitation services in LMIC Figure . First, In two years, 14 Jordanian volunteers have partnered with 20 graduate students from MGH Institute of Health Professions to conduct over 300 direct rehabilitation sessions, ten workshops with over 100 mothers of children with disabilities , and trained over 12 CBR workers in refugee camps. To date, we are still in close contact with our collaborators in different refugee camps in Jordan. We continue to investigate methods of service delivery, and one idea we are piloting is the use of telehealth to support CBR workers.The most noticeable long-term effect of our program was the fruitful collaborative partnerships established between American and Jordanian students and volunteers. Participants’ evaluations reflected that this experience nurtured action toward a meaningful shared cause, such as disability in this example. This common purpose could be the foundation for creating even more change on multiple levels. Some program alumni are already thinking of how they can transform rehabilitation systems through similar CBR programming in their home countries of Sri Lanka and Sudan.The highest cost in running this program is hiring an on-site project manager who can coordinate logistics in the refugee camp. Students from the United States bear the costs of travel, accommodation, and tuition while in Jordan. While the example provided in this commentary is specific to our school, the many lessons learned can be applied to other settings and populations.Despite the potential of this model, it has several limitations. First, faculty members planning global health experiences must rely on collaboration between departments, administrative leadership, and compliance offices. This work is usually not accounted for in the faculty members’ workload and might deter some from creating such opportunities in their institutions. A second limitation of community-based programs is the potential for political conflict or environmental concerns in the host country to interrupt travel from the high-income country, compromising the direct service model. In addition, communities in LMIC may not have the technological infrastructure to support telehealth arrangements. Finally, cultural differences can lead to differences in communication and execution patterns, but this, of course, can be a learning opportunity where the LMIC’s approach to problem-solving can be transferable to high-income countries.Expanding access to rehabilitation services in LMIC will require overcoming barriers on the macrosystemic, professional, and community levels. The academic-community partnership model builds on the proven success of community-based rehabilitation to provide basic rehabilitation services in LMIC while adding elements that address the competition for scarce resources and interprofessional hierarchies. Jordan, with its existing infrastructure of CBR, stakeholder buy-in, and identified need for services in refugee camps, has proven particularly fitting for an academic-community partnership. Each LMIC faces a different context, though the model of arraying challenges on three levels and tackling each of them through enhanced CBR applies broadly. The community-based rehabilitation component brings basic rehabilitation services to meet immediate needs and build advocacy skills. The academic component produces outcomes for research and interprofessional cooperation in a sustainable, cost-effective way. No country is immune from the tendency to favor acute medical interventions over long-term rehabilitative care. In this way, high-income countries can learn from the CBR efforts developed in LMIC to improve treatment for people with disabilities across the globe.


Humans routinely engage in many distinct interactions in parallel. Team members collaborate on several concurrent projects, and even whole nations interact with each other across a variety of issues, including trade, climate change and security. Yet the existing theory of direct reciprocity studies isolated repeated games. Such models cannot account for strategic attempts to use the vested interests in one game as a leverage to enforce cooperation in another. Here we introduce a general framework of multichannel games. Individuals interact with each other over multiple channels; each channel is a repeated game. Strategic choices in one channel can affect decisions in another. With analytical equilibrium calculations for the donation game and evolutionary simulations for several other games we show that such linkage facilitates cooperation. Our results suggest that previous studies tend to underestimate the human potential for reciprocity. When several interactions occur in parallel, people often learn to coordinate their behavior across games to maximize cooperation in each of them. Most evolutionary game theory focuses on isolated games. Here, Donahue et al. present a general framework for ‘multichannel games’ in which individuals engage in a set of parallel games with a partner, and show that such parallel interactions favor the evolution of reciprocity across games. Once there is a “shadow of the future”, people are more hesitant to free ride even if there are strong short run incentives to do so. When individuals interact more than once, they can adopt conditional strategies that take into account the co-player’s past behavior6. With these conditional strategies, cooperation can be enforced more effectively than would be possible in one-shot interactions. To describe direct reciprocity mathematically, researchers use the framework of iterated games8. This framework considers individuals who repeatedly engage in the same strategic interaction. Over the last decades, research on repeated games has identified which strategies can sustain cooperation20, which conditions allow these strategies to spread in a population27, and which of these strategies are used by human subjects31.Many of our social interactions occur in the context of repetition, which enables the evolution of cooperation by direct reciprocity32, firms compete in distinct geographic locations33, and political parties or entire nations need to collaborate on a whole range of different policy areas. If individuals treated all their different games as independent, each game could be analyzed in isolation, and the existing framework of direct reciprocity would continue to make correct predictions. In many scenarios, however, individuals have an incentive not to treat the different games as independent. By conditioning behavior in one game on what happened in another, individuals can increase their bargaining power34. This added leverage can be used to force cooperative behaviors even in those games in which cooperation is particularly difficult to sustain. To capture such strategic spillovers between distinct interactions, we introduce an evolutionary framework for multichannel games  games are coupledOur evolutionary findings suggest that individuals quickly learn to coordinate their own behaviors across different social dilemmas. They tend to use cooperation in more valuable interactions as a means to promote cooperation in those games with a larger temptation to defect. Remarkably, this endogenous coupling of independent games does not need to come at the cost of reduced cooperation in the most valuable game. Instead, individuals often evolve to be more cooperative in all games, including those in which subjects are highly cooperative even without any linkage. To explore this effect in more detail, we explore which strategies can be used to sustain full cooperation in all concurrent games. When each game is a donation game, we provide a complete characterization of these “partner” strategies. Based on these analytical results, we show that the set of partner strategies expands considerably once individuals are allowed to link their different games. Our findings suggest that linkage enhances the influence and flexibility individuals have. This enhanced flexibility is crucial to establish cooperation in some games, and it can further promote the already existing cooperative behaviors in others.7. That is, a player who cooperates in game k transfers a benefit bk to the co-player at own cost ck. Defectors pay no cost and create no benefit. We assume bk > ck > 0, such that each game has the incentive structure of a prisoner’s dilemma. It follows that cooperation does not evolve in either game if players only interact once35. However, here we consider repeated interactions. After each round, there is another one in which players again have to decide whether to cooperate in either game. We refer to such repeated interactions across multiple parallel games as a multichannel game. A player’s payoff in the multichannel game is computed by summing up her average payoffs across all individual games.In the main text, we introduce our framework for the most simple setting, by considering two players who simultaneously interact in two games, as depicted in Fig. k, they only take into account what previously happened in that very game, while ignoring what happened in the other. Such a scenario may reflect, for example, two companies who compete in two different geographic markets, each managed by independent subunits. In contrast, in the linked case , players do so by random strategy exploration. In that case, they choose a new strategy uniformly at random from the set of all available strategies. Otherwise, with probability 1 − μ, players consider imitating the strategy of someone else. To this end, they randomly sample a role model from the population. Then they adopt the role model’s strategy with a probability that increases in the role model’s payoff . Over time, the two elements of imitation and random strategy exploration yield a stochastic process on the space of all possible population compositions. We explore this process through computer simulations in the limit of rare mutations46 . Similarly, we say a strategy is a game-4. In contrast, in the linked case the respective thresholds only need to be met on average, when taking a weighted mean across both games. Players can afford to be more forgiving in one game by being more restrictive in the other. The specific weights depend on how valuable cooperation is in the respective game. The more valuable, the less forgiving a player should be after a co-player’s defection.Supplementary Fig. entclass1pt{minimaConditions  and 5)  5) can aIn a next step, we have investigated to which extent the four strategy classes described above can explain the simulation results in Fig. This analysis also shows how resistant strategies from different strategy classes are to mutant invasions Fig. c, f. In After exploring the effects of linkage among reactive players, we have run simulations for memory-1 strategies Fig. . In addi39. After all, when multiple social dilemmas occur in parallel, this does not only increase the opportunities to retaliate, but also the opportunities to defect in the first place. As a result, merely interacting across several copies of the same social dilemma does not alter the possible equilibrium outcomes39.Herein, we have introduced a general framework to explore the evolution of reciprocity when people interact in multiple games simultaneously. Such multichannel games are different from usual repeated games because they allow players to engage in cross-reciprocity. If a player defects in one game, the co-player may respond by defecting in the same game, a different game, or in all games currently played. Previous work suggests that these added retaliation opportunities do not necessarily enhance cooperationUsing an evolutionary approach, we nevertheless find that multichannel games facilitate cooperation. Even in those cases in which linkage leaves the set of equilibrium outcomes unchanged, it may still affect the number of strategies that give rise to each equilibrium outcome. To illustrate this point, we have considered a multichannel game in which individuals simultaneously interact in multiple social dilemmas. Once players are able to link these games, the set of partner strategies that enforce full cooperation increases substantially . As a consequence, players are more likely to discover and adopt these partner strategies over the course of evolution.Throughout the main text, we have focused on simple instances of multichannel games. We have considered two players who use reactive or memory-1 strategies to interact across two donation games. However, our framework is in no way restricted to these cases. In the “Methods”, we describe how our model can be adapted to cover interactions across arbitrarily many donation games. The respective characterizations of partners, semi-partners and defector strategies immediately carry over, and also the evolutionary dynamics is similar  games, as depicted in Fig. m games. Games are independent in the sense that a player’s one-round payoff in each game only depends on the player’s and the co-player’s action in that game, irrespective of the outcome of the other games. For each game k, we denote the possible one-round payoffs by Rk, Sk, Tk, and Pk. Here, Rk is the reward when both players cooperate, Sk is the sucker’s payoff a cooperator obtains when the co-player defects, Tk is the temptation to defect when the co-player cooperates, and Pk is the punishment payoff for mutual defection. For the game to be a social dilemma53, we assume that Rk > Pk , and that either Tk > Rk or Pk > Sk. The prisoner’s dilemma corresponds to the case where all three inequalities are satisfied. Throughout the main text, we focus on a special case of the prisoner’s dilemma, called donation game7. In the donation game, cooperation means to pay a cost ck > 0 to transfer a benefit bk > ck to the co-player. It follows that Rk = bk − ck, Sk = −ck, Tk = bk, and Pk = 0. However, the general framework is able to capture arbitrary kinds of social dilemmas  or to defect (D) for each of the k may only depend on the previous history of game k. In contrast, in the linked case  ϵ {C, D}m. For m = 2, reactive strategies take the form of eight-dimensional vectors, as represented in Eq. . A player’s payoff in the multichannel game is defined as the sum over all her m repeated-game payoffs.We consider infinitely many rounds in the limit of no discounting. For each game ε, a player who intends to cooperate instead defects, and conversely a player who intends to defect cooperates. In addition to making the model more realistic, implementation errors ensure that payoffs are well-defined, independent of the outcome of the very first round of the game23. Our simulation results are robust with respect to the exact magnitude of this error rate, provided that errors are sufficiently rare for the player’s strategies to have an impact , the chosen player engages in random strategy exploration. In that case, the player randomly picks a new strategy from the set of all available strategies  is C and it is zero otherwise. Using this notation, we obtain . A player with that strategy cooperates if and only if either both players have cooperated in the previous round of the respective game, or if no one did. In the linked case, evolving strategies resemble a different strategy, which we term CIC. Players with this strategy use in each round the same action in all games they participate in. This action is cooperation if and only if in each game, players used the same action in the last round; otherwise they defect.The simulations for memory-1 players in Fig. bk and ck these two strategies are subgame perfect equilibria. A subgame perfect equilibrium is a refinement of the Nash equilibrium: players are required not to have an incentive to deviate after any previous history of play56. We obtain the following conditions  as a means to achieve cooperation in low-benefit games (with bk < 2ck).The two conditions again reflect one reason why full cooperation is easier to sustain in the linked case. Unlinked strategies like Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary