Background: The role of the androgen receptor (AR) in renal cell carcinoma (RCC) is unclear. We aimed to analyze the expression of AR and its splice variants (SVs) and their correlation with relaxin 2 (RLN2) and cytokines in RCC. Methods: We investigated the expression of RLN2 and AR variants in 25 clear cell RCC (ccRCC) and 9 papillary (pRCC) tumor tissues and the corresponding controls using quantitative PCR and serum RLN2, testosterone and cytokine levels in matched samples using ELISA and chemiluminescent immunometric assay, respectively. Results: ccRCC tissues but not pRCC tissues more frequently expressed AR and the SVs than did normal tissues. All pRCC samples expressed more AR than did ccRCC samples. The highest expression of all AR variants except AR-V12 was found in low-stage tumors, with dominant expression of AR-V7. In males in the ccRCC cohort, the expression of AR-FL, AR-V1 and AR-V3 was significantly correlated with that of RLN2. The secretion pattern of proinflammatory IL-6 was higher in ccRCC than in pRCC. Conclusions: The results highlight additional molecular differences between ccRCC and pRCC, suggesting the influence of external factors on the whole kidney or genetic predispositions to developing certain types of renal cancer, and may support further pathological analysis and studies of targeted hormone therapy. Renal cancer is one of the most common cancers worldwide, reaching over 403,000 new cases in 2018 and representing 2.2% of newly diagnosed cancers [The two most common RCC subtypes are clear cell RCC (ccRCC) and papillary RCC (pRCC). The first type, ccRCC, accounts for 75% of all RCCs, while pRCC (subtype 1 and 2) accounts for approximately 10%. Both tumors differ in vascularization. Clear cell RCC is highly vascularized, while pRCCs are hypovascularized when compared to the surrounding parenchyma ,4. The cn = 571) was associated with longer survival probability (at least 16 years), while lower expression (n = 306) was associated with shorter survival probability (13 years). Patients in this collective were not analyzed with regard to sex or age [According to clinical observations, the incidence of RCC is twice as high in men as in women, which suggests the involvement of steroid hormone receptors in tumor development. Expression analyses often reveal relatively high levels of androgen receptors (AR) in normal kidneys and lower levels in tumorous kidneys. Several reports have described that AR expression tends to decrease with increasing pT stage and Fuhrman’s grade, while others have presented contradictory results. Moreover, primary tumors display higher expression of AR than metastases ,6,7. Sucx or age . Analysix or age . A graphx or age , althougAfter hormone binding and dimerization of monomers, the AR translocates to the nucleus and initiates activation of target genes. This mechanism is well studied in prostate cancer. This acquired knowledge was fundamental in developing AR target therapy and medicines for prostate cancer patients .In addition to its analysis in prostate cancer, AR expression has been analyzed in other tumors, such as bladder , breast Target therapies are also administered to advanced or metastatic RCC patients. These therapies target, for example, vascular endothelial growth factor (VEGF) and tyrosine kinase inhibitors (TKIs) ; howeverDetecting the splice variants (SVs) of AR and their constitutive expression and functionality has increased the significance of AR in terms of pathogenesis. Over 30 variants have been identified to date . Most ofThe androgen receptor can be activated in an androgen-independent manner. One of the proteins that influence the AR signaling pathway is relaxin 2 (RLN2) . RLN2 isThe physiological expression of relaxin (and its receptor RXFP1) in the kidneys is not high. Even if its functional significance is still not clear, its protective activity has been suggested . In partSeveral reports indicate that the expression and activity of AR and RLN2 correlate with the synthesis of proinflammatory interleukins IL-6 and IL-8 under physiological and pathological conditions ,39,40,41In our studies, we investigated the expression of AR and AR-SVs and their correlation with RLN2 expression in both ccRCC and pRCC. Additionally, matched serum samples were investigated for the levels of the AR ligand- testosterone, RLN2 and two cytokines, IL-6 and IL-8.n = 25; pRCC n = 9; papillary adenoma n = 1) and stages (pT1–pT4)  obtained from patients diagnosed with RCC that underwent nephrectomy in our clinic. The tumor tissues collected from treatment-naïve patients were of different types (ccRCC pT1–pT4) , and theThe renal carcinoma cell line Caki-1  was grown in RPMI medium  and the PCa cell line LNCaP (ATCC)-was grown in DMEM enriched with 10% FCS . The medium was changed every 2–3 days and both cell lines were passaged every 4–5 days.−ΔΔCT method.Total RNA was isolated from homogenized frozen tissues using an RNeasy Plus Mini Kit  according to the manufacturer’s instructions. Subsequently, cDNA was synthetized from 500 µg RNA with SuperScript IV VILO Master Mix . Quantitative PCR (qPCR) was performed using 5× Hot FirePol Eva Green qPCR Mix Plus  with a QuantStudio5 Thermocycler (Thermo Fisher Scientific). Expression of the target genes was analyzed using specific primers, and β-actin served as endogen control . The resWe analyzed the presence of two cytokines (IL-6 and IL-8) and testosterone in the serum of 25 ccRCC and 9 pRCC patients using Solid-Phase Chemiluminescent Immunometric Assay kits with an Immulite 1000 Immunoassay System and ADVIA Centaur CP  according to manufacturer’s instructions.Serum levels of RLN2 were analyzed with a Human Relaxin-2 Quantikine ELISA Kit  according to manufacturer’s instructions. Assay Diluent was mixed with the standard, control or samples in a provided 96-well plate and incubated for 2 hrs at RT. After washing, the Human Relaxin-2 Conjugate was added to each well and incubated for 2 hrs at RT. Following repeated washing and 30 min incubation with Substrate Solution, the reaction was stopped with Stop Solution and the optical density was determined using an Infinite M Plex microplate reader .p-values are two-sided, and p ≤ 0.05 was considered to indicate significance.Prism 9  was used to perform statistical analyses and produce figures. All data were tested for normal distribution (Shapiro–Wilk test). The data are presented as the median ± range, and possible correlations between different markers for the different tumor types were identified with Spearman’s rank test. Associations between the expression of AR  and other markers were tested using the Mann–Whitney test. Reported n = 25, pRCC n = 9), one papillary adenoma and corresponding nontumor tissues were subjected to qPCR analyses. The median age of the ccRCC patients was 67.5 years old  and that of the pRCC patients was 70 years old . Our transcriptome studies revealed expression of the full-length AR (AR-FL) as well as four of five tested SVs in normal and tumor tissue. The transcript variant AR-V12 was not detected in any sample , AR-V1  and AR-V3  than normal and tumor tissues of ccRCC patients, as well as significantly higher expression of AR-V4  than normal tissues of ccRCC patients. No significant difference between tumor and normal tissues of any type of RCC was detected .p ≤ 0.02), followed by AR-V7  and AR-V1 . The lowest difference in the same group was noted for AR-FL   displayed similar expression patterns, showing rarer expression in ccRCC tumors than in control tissue  . The ave ≤ 0.04) . Among a ≤ 0.04) . In rega ≤ 0.04) . Analysi ≤ 0.04) .Comparison of the male and female groups among the ccRCCs cohort did not show any significant differences. Analysis of the male patient group revealed slightly higher expression of AR-FL and AR-V1 in pT2 (median 0.94 (range 0.19–1.25) and 0.98 (range 0.24–1.7), respectively) and pT3 tumors (median 1.24 (range 0.32–2.67) and 0.83 (range 0.14–1.63) respectively), while the expression of AR-V3, AR-V4 and AR-V7 dominated in pT1 tumors , 1.08 (range 0.23–2.07), 2.24 (range 0.47–3.86)) . It mustWe analyzed the expression of RLN2 mRNA in ccRCC and pRCC . In our p ≤ 0.1, pT2 p ≤ 0.48)Furthermore, we normalized the RLN2 expression values of both RCC subtypes, by defining the normal tissue values as “1” analyzed expression in different pT stages and compared the male and female populations (ccRCC). We did not find any significant differences between these groups. Results of the whole group of pRCC samples display alternations in mean (pT2 1.97 vs. pT3 0.64) and median (pT2 2.25 (range 0.2–3.2) vs. pT3 0.37 (0.01–1.82)) values; however, the low number of samples does not allow us to state the significant differences. The papillary adenoma and pT3 tumor stage samples of pRCC were statistically not analyzed because of the limited number of samples . No signp ≤ 0.002), AR-V1 and RLN2 expression  and AR-V4 and RLN2 expression   .n = 1). Generally, the expression in papillary tumors was significantly weaker than in clear cell tumors (* p ≤ 0.02)  . No corrSerum samples were investigated for the level of secreted proteins, which may influence the renal activity of AR and RLN2. The secreted levels of the main activator of AR testosterone were not different between ccRCC and pRCC or based on tumor size, and were in the normal range . The serum levels of RLN2 in both tumor types were under the detection limit (≤7.8 pg/mL). Among the interleukins, 70% of pRCC samples but only 45% of ccRCC samples lacked IL-6 secretion . The meaThis study clearly demonstrates differences in AR expression between two main types of RCC—ccRCC and pRCC—and between pT stages of ccRCC tumors. Additional correlations between the expression of three types of AR  and its potential modulator RLN2 suggest that AR may serve as a promising target for therapy in patients with RCC.Signaling related to AR is multistage and complex and may affect other processes, such as inflammation, epithelial–mesenchymal transition, cell migration or proliferation, which are crucial for the development and metastasis of tumors.Extensive analysis has classified RCC as a hormone-related disease ; therefoIn many cancers, the expression of AR is related to tumor stage. For example, bladder cancer is characterized by high expression of AR in tumors of low stage and grade and these features positively affect the survival time of patients . Our resIn PCa, the crosstalk between AR signaling and other signaling pathways is well described. Androgen-independent growth of tumor cells can be mediated by the hormone RLN2, which can activate the AR signaling pathway by inducing the formation of the β-catenin–AR complex and its translocation to the nucleus ,55. AnalAs previously mentioned, targeted therapies administered to RCC patients may achieve disappointing results due to adaptation of the tumor microenvironment and, consequently, resistance . Recentlp ≤ 0.002) in the male cohort. These data suggest the possibility of indirect dependency or direct crosstalk between relaxin and AR in renal carcinoma, especially in men with ccRCC.Renoprotective effects of relaxin, including attenuation of fibrosis, have been noted in patients with several diseases, such as dilated cardiomyopathy or age-related renal fibrosis ,33,34. IOur investigations were not limited to AR-FL only. We strengthened the study by analyzing the expression of five SVs  in patient samples. The exact function of the SVs is not clear. Similar to previously described reports, we found that the expression of AR-FL and all the SVs, except AR-V12, differed between ccRCC tumors of different pT stages with pT1 tumors showing the highest expression and pT4 tumors showing the lowest expression. Additionally, in both pT1 and pT4 but not in pT2 and pT3 tumors, the levels of AR-V7 were increased when compared to those of AR-FL. AR-V12 was not detected in any tissue. Strikingly, in prostate cancer, expression of AR-V7 is considered as a marker limiting treatment or predicting poor prognosis ,22 or asConstitutively expressed variants of AR were analyzed mainly in PCa. Cytoplasm-localized AR-V4 and AR-V1 dimerize with AR-FL and AR-V7; however, only binding to androgen-bound AR or AR-V7 induces transfer to the nucleus. Additionally, AR-V1 can enhance AR-FL but weaken AR-V7 transactivation in androgen-independent actions . The preCytokines, particularly IL-6, may play an important role in RCC. According to Favaro et al. , IL-6 isOur results provide deeper insight into the possible roles and crosstalk of AR and RLN 2 in both ccRCC and pRCC. We demonstrate that AR-FL and the constitutive splice variants AR-V1, AR-V3, AR-V4 and AR-V7 are more highly expressed in pRCC than in ccRCC in both tumor and corresponding control tissues. Moreover, the expression is lower in advanced ccRCC tumors than in early-stage ccRCC tumors. The male cohort showed a positive correlation between AR and RLN2 expression. Analysis of secreted cytokines revealed a differential pattern in the two types of RCC.Our study was performed on human tissues originating from patients with different stages and subtypes of renal carcinoma. We must address several limitations. In the investigations were used a low number of samples. Studies with larger collectives are planned to be performed. The lack of functional correlation analysis should be addressed in the future. For better validation of our results, studies with more samples are planned.


Sex hormones, including androgens and estrogens, play an important role in autonomic, reproductive and sexual behavior. The areas that are important in these behaviors lie within the spinal cord and brainstem. Relevant dysfunctional behavior in patients with altered androgen availability or androgen receptor sensitivity might be explained by the distribution of androgens and their receptors in the central nervous system. We hypothesize that autonomic dysfunction is correlated with the androgen sensitivity of spinal cord and brainstem areas responsible for autonomic functions. In this study, androgen receptor immunoreactive (AR‐IR) nuclei in the spinal cord and brainstem were studied using the androgen receptor antibody PG21 in four uncastrated young adult male cats. A dense distribution of AR‐IR nuclei was detected in the superior layers of the dorsal horn, including lamina I. Intensely stained nuclei, but less densely distributed, were found in lamina X and preganglionic sympathetic and parasympathetic cells of the intermediolateral cell column. Areas in the caudal brainstem showing a high density of AR‐IR nuclei included the area postrema, the dorsal motor vagus nucleus and the retrotrapezoid nucleus. More cranially, the central linear nucleus in the pons contained a dense distribution of AR‐IR nuclei. The mesencephalic periaqueductal gray (PAG) showed a dense distribution of AR‐IR nuclei apart from the most central part of the PAG directly adjacent to the ependymal lining. Other areas in the mesencephalon with a dense distribution of AR‐IR nuclei were the dorsal raphe nucleus, the retrorubral nucleus, the substantia nigra and the ventral tegmental area of Tsai. It is concluded that AR‐IR nuclei are located in specific areas of the central nervous system that are involved in the control of sensory function and autonomic behavior. Furthermore, damage of these AR‐IR areas might explain related dysfunction in humans. In this study, androgen receptor immunoreactive (AR‐IR) nuclei in the spinal cord and brainstem were studied using the androgen receptor antibody PG21 in four uncastrated young adult male cats. It is concluded that AR‐IR nuclei are located in specific areas of the central nervous system that are involved in the control of sensory function and autonomic behavior. Furthermore, damage of these AR‐IR areas might explain related dysfunction in humans. The spinal cords and brainstems were removed and post fixed for 2 h at 4°C.2.2202 for 30 min, blocked with 5% normal donkey serum for 30 min at room temperature, and incubated with the primary antibody against androgen receptors, PG21  diluted in 1% normal donkey serum at 4°C, for three days . One of four sections was pretreated with 0.3% H2.3AR‐IR nuclei were plotted with a computerized X‐Y stage on a Zeiss Axioplan microscope connected to a PC with the Neurolucida system (MicroBrightField). The drawings of the sections of the spinal cord and brainstem containing plotted nuclei were converted to Encapsulated PostScript (EPS) files and further processed in Adobe Illustrator CC 2018. Example drawings of representative sections at specific levels are presented in the results section Figures . The res3AR‐IR was predominantly found in the nuclei of neurons, but occasionally also in nuclei of glial and ependymal cells. AR‐IR cells were present throughout the spinal cord and brainstem. The immunostaining varied from light gray to intense black. AR‐IR nuclei in the white matter represented, in all likelihood, glial cells. No staining was found in control sections. Two outcome measures were determined for each spinal cord and brainstem area: the density of the distribution of AR‐IR nuclei and the nuclear intensity of the staining. The density of the distribution of AR‐IR nuclei and the intensity of nuclear staining in areas of the spinal cord and brainstem are presented in Table 3.1The distribution of AR‐IR nuclei was moderate to low in density at every level of the spinal cord Figures  and 2. MIntensely stained nuclei were observed in the central (lamina X) and intermediate zone at all spinal levels, and in the preganglionic sympathetic and parasympathetic cells of the intermediolateral cell column at thoracic, lumbar, and sacral levels.3.2The distribution of AR‐IR nuclei was dense in the area postrema, the dorsal motor vagus nucleus, and the retrotrapezoid nucleus , and the ventral tegmental area of Tsai Figure . The PAGIntensely stained nuclei were observed in the central tegmental field, the dorsal raphe nucleus, the Edinger Westphal nucleus, the mesencephalic tegmental field, the retrorubral nucleus, and the substantia nigra. The intensity of nuclear AR‐IR staining was less in the brachial nucleus of the colliculus inferior, the medial pretectal area, the oculomotor nucleus, the PAG, the superior colliculus, and the ventral tegmental area of Tsai.4This study provides the distribution of AR‐IR nuclei in the spinal cord and brainstem of the cat. The immunohistochemical results overlap with those of earlier studies in other species such as the rat. Here, we will focus on novel findings and on the overall significance of the distribution of AR‐IR nuclei. We firstly describe the similarities and differences with the distribution of androgen receptors as described in other species. Secondly, we discuss the involvement of the areas that contain AR‐IR nuclei in autonomic function and dysfunction. And thirdly, the distribution of androgen receptors in the cat is compared to that of estrogen receptors in the spinal cord and the brainstem.4.1The density of AR‐IR nuclei in the spinal cord of the cat was moderate. A relatively dense distribution of AR‐IR nuclei was observed in the superior layers of the dorsal horn such as in layer I, in which primary afferent fibers terminate. Nuclei in the intermediate zone, lamina X, and the intermediolateral cell column were also AR‐IR. The present study shows that the intermediomedial cell column contains AR‐IR nuclei indicating that pre‐ganglionic sympathetic and parasympathetic motoneurons might express androgen receptors. These pre‐ganglionic motoneurons innervate the pelvic smooth musculature such as those in the bladder and bowel. Some of these AR‐IR areas at the lumbosacral level integrate visceral and somatic afferent and efferent information of the pelvic and pudendal nerves (Roppolo et al., In the caudal brainstem, a dense distribution of AR‐IR nuclei was found in the area postrema, which is consistent with previous results in the rat (Hamson et al., In the mesencephalon, we observed a dense distribution of AR‐IR nuclei in the dorsal raphe nucleus in cats. The dorsal raphe nucleus is involved in the sleep‐wake state of the organism and it encodes reward signals (Li et al., 4.2Several areas with a high density of AR‐IR nuclei are involved in respiration. The areas that were identified in the present study are the area postrema, the dorsal motor vagus nucleus, the Kölliker‐Fuse nucleus, the nucleus of the solitary tract, the PAG, the parabrachial nucleus, and the retrotrapezoid nucleus. The area postrema has been implicated to alter cardiopulmonary responses in rats (Yang et al., The areas involved in cardiovascular function that contained AR‐IR nuclei in the present study are: the area postrema, the dorsal motor vagus nucleus, the nucleus of the solitary tract, and the PAG. The area postrema and the nucleus ambiguus have been implicated to control heart rate (Gourine et al., Furthermore, androgens play an important role in sexual function. Here, were described areas in the central nervous system that might be androgen sensitive indicated by a high density of AR‐IR nuclei. We showed a high density of AR‐IR nuclei in areas involved in sexual reflexes such as erection and orgasm. These areas include the PAG and the dorsal horn of the sacral spinal cord. The nucleus retroambiguus also contained AR‐IR nuclei. It has connections to the PAG and the lumbosacral spinal cord and is thought to be involved in mating behavior. The involvement of androgens in sexual function is supported by evidence that men with a decreased level of testosterone can experience a decrease in sexual desire and arousal, erectile dysfunction, reduced ejaculate, incontinence on orgasm, and reduced orgasm (Basaria et al., 4.3The distribution of estrogen receptors in the central nervous system is comparable between male and female rodents (Vanderhorst et al., 5In this study, we showed specific areas in the spinal cord and brainstem of the cat in which AR‐IR nuclei are present. In the spinal cord of the cat the most dense distribution of AR‐IR nuclei was observed in the dorsal horn, which is involved in sensory processing. However, androgen receptor expression was more pronounced in supraspinal areas. AR‐IR nuclei in the brainstem of the cat were most prominent in the area postrema, the central linear nucleus, the dorsal motor vagus nucleus, the dorsal raphe nucleus, the PAG, the retrorubral nucleus, the retrotrapezoid nucleus, the substantia nigra, and the ventral tegmental area of Tsai. These areas are important for various autonomic functions such as cardiovascular function, micturition, respiration, and sexual function. These results underline the importance of androgen receptors in the central nervous system and their possible roles in sensory and autonomic functions. For clinical practice this means that patients with an altered availability of androgens or their receptors as is the case in hypogonadism, might experience autonomic dysfunction due to a decreased activation of androgen receptors in the spinal cord and brainstem.The authors declare no conflicts of interest.RC contributed to data analysis/interpretation, drafting of the manuscript, and critical revision of the manuscript. JCC contributed to acquisition of data, data analysis/interpretation, and critical revision of the manuscript. PS and EA contributed to data analysis/interpretation and critical revision of the manuscript. BFMB contributed to the concept and design of the study, acquisition of data, data analysis/interpretation, and critical revision of the manuscript.


A few small chemical compounds are undergoing clinical trials combined with existing AR antagonists. AR protein elimination by enhanced protein or mRNA degradation is a realistic solution for avoiding AR reactivation during androgen deprivation therapy in prostate cancers.Prostate cancer is a major health issue in western countries and is the second leading cause of cancer death in American men. Prostate cancer depends on the androgen receptor (AR), a transcriptional factor critical for prostate cancer growth and progression. Castration by surgery or medical treatment reduces androgen levels, resulting in prostatic atrophy and prostate cancer regression. Thus, metastatic prostate cancers are initially managed with androgen deprivation therapy. Unfortunately, prostate cancers rapidly relapse after castration therapy and progress to a disease stage called castration-resistant prostate cancer (CRPC). Currently, clinical treatment for CRPCs is focused on suppressing AR activity with antagonists like Enzalutamide or by reducing androgen production with Abiraterone. In clinical practice, these treatments fail to yield a curative benefit in CRPC patients in part due to AR gene mutations or splicing variations, resulting in AR reactivation. It is conceivable that eliminating the AR protein in prostate cancer cells is a promising solution to provide a potential curative outcome. Multiple strategies have emerged, and several potent agents that reduce AR protein levels were reported to eliminate xenograft tumor growth in preclinical models  Prostate cancer is the second most common type of cancer diagnosed in men worldwide and the second leading cause of male cancer-related deaths in the U.S. . The AmeCurrently, localized prostate cancer is primarily treated with surgical removal of the gland or radiation therapy if a patient’s condition is not permissive for surgery. Distal metastasis occurs in high-risk patients, including locally advanced  or high-grade (Gleason sum score ≥ 8) tumors, which is the sole cause of death from prostate cancer . This shMetastatic prostate cancers are initially treated with androgen deprivation therapy (ADT) because prostate tissue  expresses androgen receptor (AR) protein that is critical for prostate cancer development and progression , 4. Castde novo androgen synthesis by the malignant prostate cells  are synthetic complementary single-stranded deoxyribonucleotides used to target messenger RNA (mRNA) of targeted genes, resulting in RNase H endonuclease-dependent mRNA cleavage or blockage of protein translation . Dr. Kloude mice . These iude mice , 10. Posude mice –14. A re tissues .in vivo  technology in 2001 , 98, knoin vivo , 17. Thein vivo –23. ThesPROTAC stands for proteolysis targeting chimera. It uses a small bifunctional molecule with two binding moieties connected by a linker to bring together a targeted protein and cellular proteolytic machinery, ubiquitin E3 ligase-mediated proteasome degradation system , 100. ThThe first AR-targeted PROTAC approach was reported in 2004, which used a synthetic peptide targeting the E3 ligase fused to either an artificial FKBP12 ligand or dihydrotestosterone (DHT) . After s50 < 0.1 nM) oral agent and has at least 10-fold more potent than ARV-110 (The AR degrader (ARD) series of PROTAC molecules  were reported from Dr. Wang’s group at the University of Michigan –31. Thei ARV-110 . These m50 = 12.5 nM) (50 < 0.25 μM) (50 = 0.37-2 μM)   and A0310.25 μM)  that pro37-2 μM) . In addi37-2 μM) , 36 and in vitro (1-10 μM) and in vivo via ubiquitin-proteaseom pathway  and in vivo xenograft models (2 mg/kg/day) of prostate cancer , DNA-binding (DBD), hinge region, and C-terminal ligand-binding (LBD). In prostate cancers, the transcriptional splicing variants of the AR gene have been linked to castration-resistance of prostate cancer after ADT and anti-AR therapy with Enzalutamide and Abiraterone –112. Becvia the ubiquitin-proteasome pathway in prostate cancer cells at 0.5-1.0 μM without affecting the full-length AR protein , epidermal growth factor receptor (EGFR) and HER2/Neu. It was recently found to inhibit the transcriptional activities of the full-length AR and AR-v7 protein (0.3 μM for 24 h) V1 cells . It alsoV1 cells . Howevervia the ubiquitin-proteasome pathway in prostate cancer cells  with multiple protein targets –125, incer cells  and supper cells , 45. It er cells  and senser cells . HoweverThailanstatins are bacteria-derived natural products with potent inhibitory activity toward pre-mRNA splicing events . Since AEvodia rutaecarpa  and suppressed VCaP cell-derived xenograft tumor growth in nude mice at a dose of 25 mk/kg/day  within the LBD region  was another small molecule identified from the 2GFP-AR fusing protein screening assay with a robust inhibitory effect at 2.0 μM concentration on AR nuclear translocation and its downstream target PSA gene expression, plus downregulation of AR gene expression at a higher concentration of 10 μM . FurtherJJ-450 is an IMPPE scaffold analog with higher potency and better physicochemical properties . JJ-450 in-silico drug design approach  based on the findings reported about 80-years ago. However, this androgen removal approach is not curative for prostate cancers, and the diseases often relapse and progress to the CRPC stage. Since most of these CRPCs are still AR addictive, current clinical therapies mainly focus on blocking androgen to bind with the AR LBD (AR antagonists) or reducing androgen production (CYP17a1 inhibitors) in non-testis tissues, including prostate cancer tissues. However, treatment resistance eventually develops in part due to AR gene mutation and mRNA splicing events  in virtually all CRPC patients. Furthermore, after long-term treatment with AR antagonists, up to 20% of CRPC patients will develop an even more aggressive subtype, neuroendocrinal prostate cancer (NEPC). Therefore, the androgen removal and blockage approach are non-curative and leads to a more aggressive disease.The AR protein is critical for prostate cancer progression by transcriptionally modulating gene expression after activation by androgens To overcome this obstacle of treatment resistance, research has shifted from androgens to the AR protein in the last 20 years Figure 1Targeting AR protein stability has emerged in recent years as the hotspot in developing new therapeutics for advanced prostate cancers, and several small molecules were reported to reduce AR protein stability. The curcumin analog ASC-J9, Ailanthone, HG122, and CUDC-101 induced AR protein degradation in prostate cancer cells. However, the AR or prostate cancer tissue specificity is not established with these small molecules. The PROTAC technique for AR-specific degradation showed a promising result. The AR PROTAC ARV-110 is tested as a combinational treatment with Abiraterone in a clinical trial. However, these AR CTD-targeting PROTACs utilized AR LBD ligands, and therefore, they are inactive on AR CTD splicing variants, a critical mechanism for treatment resistance in CRPC patients. Interestingly, some other agents specifically targeted the AR-V7 variant for degradation, including Niclosamide, CUDC-101, Thailanstatins, Rutaecarpine, Indisulam, and Nobiletin. Combining AR antagonists, PROTAC molecules, and AR-V7 inhibitors might provide synergistic effects in the clinic.Targeting AR NTD is another approach to bypass AR CTD splicing defects. The first generation of AR NTD inhibitor EPI compounds was failed in clinical trials due to excessive bill burden. The second generation of EPI compound with 20-fold higher potency is being tested as a combinational treatment with Enzalutamide in a clinical trial. UT-34 targets the AR NTD and is also waiting for a clinical test.AR nuclear translocation is an important event for its activity as a transcription factor. Two novel compounds, IMPPE and JJ-450, were recently developed to block AR nuclear translocation. These two compounds showed a very permissive result in animal models. In addition, an AR DBD blocking agent VPC-14449 was reported to suppress AR interaction with its target gene promoter in the nuclear compartment and was found to suppress tumor growth in mice. These compounds are all needed for clinical testing.AR activity is only temporally suppressed during prostate cancer treatment by androgen deprivation and AR antagonists. Due to these treatment stresses, prostate cancer cells used other cellular signal pathways and/or splicing variants for AR reactivation, resulting in treatment resistance. Therefore, complete removal of the AR protein from prostate cancer cells will eliminate all events of AR reactivation after ADT and anti-AR therapy. Especially in the early phase of treatment, most prostate cancer cells are still AR-dependent. Simultaneously removal of the AR protein and androgens will result in robust cell death, leading to a possible curative result or long-term disease-free survival. In addition, early reduction of the AR protein in the androgen-responsive phase of prostate cancer will reduce the likelihood of transcriptional reprogramming , 93, 147All authors participated in drafting the manuscript. All authors contributed to the article and approved the submitted version.This work was partially supported by a grant from KUMC Lied pilot program and DoD PCRP PC190026 to Benyi Li, MD/PhD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Citrus fruits, exerts a potent anticancer activity via inducing G0/G1 phase arrest and enhancing the sensitivity of cells to enzalutamide in AR-V7 positive PC cells. Mechanically, we unravel that nobiletin selectively induces proteasomal degradation of AR-V7 (but not AR). This effect relies on its selective inhibition of the interactions between AR-V7 and two deubiquitinases USP14 and USP22. These findings not only enrich our understanding on the mechanism of AR-V7 degradation, but also provide an efficient and druggable target for overcoming CRPC through interfering the stability of AR-V7 mediated by the interaction between AR-V7 and deubiquitinase.Androgen receptor splice variant 7 (AR-V7), a form of ligand-independent and constitutively activating variant of androgen receptor (AR), is considered as the key driver to initiate castration-resistant prostate cancer (CRPC). Because AR-V7 lacks ligand-binding domain, the AR-targeted therapies that aim to inactivate AR signaling through disrupting the interaction between AR and androgen are limited in CRPC. Thus, the emergence of AR-V7 has become the greatest challenge for treating CRPC. Targeting protein degradation is a recently proposed novel avenue for cancer treatment. Our previous studies have been shown that the oncoprotein AR-V7 is a substrate of the proteasome. Identifying novel drugs that can trigger the degradation of AR-V7 is therefore critical to cure CRPC. Here we show that nobiletin, a polymethoxylated flavonoid derived from the peel of  Prostate cancer (PC) is the second most common male cancer with high lethality worldwide. In 48 countries, mainly in sub-Saharan regions, the Caribbean, Central and South America, and Northern Europe, it is the leading cause of cancer deaths among the male , 2. AndrReactivation of AR signaling is frequently observed in CRPC. Despite a variety of schemes, including repression of androgen synthesis, blockade of AR nuclear translocation, and inhibition of AR function, etc., are used to inhibit the AR signaling pathway, the reactivation of this signaling served for the growth of CRPC cells can still be detected –6. In teCitrus fruits [Nobiletin  is a polymethoxylated flavonoid extensively derived from the peel of s fruits . Multipls fruits , antioxis fruits , anti-ins fruits , 18, ants fruits , 20, cars fruits , and neus fruits , etc. Ho2, which were purchased from the American Type Culture Collection , and authenticated by short tandem repeat (STR), morphology, cell viability, and routine mycoplasma assays. Antibodies and chemicals were shown in Tables Based on our previous reports , 24, WPMCell proliferation assays were performed as previously reported , 24, incThe EdU assay was used to detect DNA replication in cells. The cells were planted on a detachable chamber slide for 24 h, then treated with different concentrations of nobiletin indicated as the figure, and then stained according to the instructions of the EdU kit. After mounting the slides, the images of three independent experiments were randomly captured by Olympus microscope for positive rate statistics.For colony formation assay, the cells were seeded in 35 mm dishes, and treated with the specified dose of nobiletin for 24 h, then washed with PBS and digested with trypsin. Then, the cells were resuspended, seeded on six-well plate and cultured 2 weeks. The cells were fixed with 4% paraformaldehyde for 10 min, then washed with PBS, stained with 1% crystal violet solution for 5 min. Finally, the number of colonies was counted.Flow cytometry analysis was used to detect the cell cycle distribution and apoptosis. This assay was performed as previously reported .As previously described , we extrPrimers were listed as follows: ACTB forward: 5′-TTCTACAATGAGCTGCGTGTG-3′, ACTB reverse: 5′-GGGGTGTTGAAGGTCTCAAA-3′ ; AR-V7 fThe RNA interfering assay by siRNA was used to downregulate the expression levels of the targeted gene. Briefly, mix 500 μl RPMI opti-MEM , 6 μl lipofectamine RNAiMax (Invitrogen), and 10 μl USP22 siRNAs or control siRNAs were added into the cells that have been preinoculated and cultured in dishes for 24 h with a final concentration of the siRNAs at 30–50 nM. Cells were treated for 48 h for other analysis. The USP14 and USP22 siRNAs were purchased from RiboBio .The siRNA sequences are listed below: USP14 siRNA-1: 5′-CTGGCATATCGCTTACGTT-3′;USP14 siRNA-2: 5′-TTGCCGAGAAAGGTGAACA-3′;USP22 siRNA-1:5′-GCAGCGAAAAGCTTGGAAA-3′;USP22 siRNA-2:5′-GTACGGAGGCATCTACTGT-3′.The plasmids and lentivirus transfection assays were used to upregulate the expression levels of the contained gene. For plasmid transfection, the full length of human USP14 CDS plasmids (CMV-MCS-Flag-SV40-neomycin), the full length of human USP22 CDS plasmids (CMV-MCS-6His-SV40-neomycin), the full length of human AR-V7 CDS plasmids (CMV-MCS-HA-SV40-neomycin), and their control plasmids were constructed and purchased from GeneChem . Mix Lipofectamine 3000 (Invitrogen) (2 μl P3000 and 2 μl lipofectamine 3000) and 1 μg plasmids in 500 μl RPMI opti-MEM (Gibco) were added into the cells that have been preinoculated and cultured in dishes for 24 h with a final concentration of the plasmids at 0.75 μg/ml.Cells were treated for 48 h for other analysis.For lentivirus transfection, lentivirus (Ubi-MCS-HA-SV40-puromycin) containing the full length of human AR-V7 CDS and its control lentivirus were also constructed and purchased from GeneChem . After the cells were seeded in dishes or plates and cultured for 24 h, the fresh medium containing 5 μg/ml polybrene and lentivirus at a multiplicity of infection of 10 was used to culture for 48 h. Puromycin at a concentration of 2 μg/ml was used to select and retain the successfully infected cells.Since the existing antibodies (anti-AR-V7 and anti-USP22) do not support the immunofluorescence assay, we used the tag antibody to bind the exogenous labeled target molecule in the cell, and then used the fluorescent secondary antibody to identify them. This assay was mentioned in a previous study . The imaWestern blotting, a routine assay, was performed as we described before . For Co-6 cells/100 μl PBS/mice were inoculated subcutaneously on the backflanks of nude mice aged 5–6 weeks. After 1 week, the nude mice with successful xenogeneic tumor transplantation were randomly divided into four groups (eight mice per group), then treated with 40 mg/kg/2d (i.p.) of nobiletin, 25 mg/kg/2d (p.o.) of enzalutamide, combination of nobiletin and enzalutamide, and drug solvent for 25 days. The body weight of nude mice, tumor volume, and weight were measured. Tumor tissues and liver and kidney tissues were taken for further experiments.All animal experiments were approved by institutional animal care and used committees of Guangzhou Medical University and performed as previously described . Male BATumor tissues and liver and kidney tissues were fixed with Formalin, embedded in paraffin and sectioned according to standard techniques. For IHC assay, tumor section samples of 22Rv1 were immunohistochemically stained with MaxVision kit (Mixin Biotech) and AR-V7, Ki67 antibodies, according to the kit and antibody instructions. Then, the slides were counterstained with hematoxylin, and the primary antibodies were detected by the DAB method. For H&E staining assay, the paraffin sections of liver and kidney tissue of nude mice were deparaffinized in xylene and washed with ethanol solution of decreasing concentration  and distilled water, and then stained by Hematoxylin and Eosin Staining Kit  according to the kit and antibody instructions.t-test or one-way ANOVA is used where appropriate. GraphPad Prism 9 software and SPSS 16.0 were used to statistical analysis. A P value <0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Experimental data are presented as mean ± S.D. from three independent experiments where applicable. In order to determine statistical probabilities, unpaired Student’s To explore the antitumor effect of nobiletin on PC, first of all, cell viability assay was performed in four PC cell lines  and a prostatic stromal myofibroblast cell line WPMY-1. We found that nobiletin significantly reduced the viability of these PC cells, but not the viability of normal prostate cells. Meanwhile, we unexpectedly found that the inhibitory effect of nobiletin in 22Rv1 and C4-2 cells was more obvious than that in PC3 and DU145 cells Fig. . It is wIn addition, this study also ruled out the possibility of nobiletin on inducing apoptosisin the current concentration range and exposure time through Annexin V-FITC/PI staining analyzed by flow cytometry Fig. , indicatNext, we further studied the mechanism by which nobiletin inhibits the growth progression of PC cells. Cell cycle checkpoint is a key regulatory factor in the proliferation process of cancer cells.Therefore, we tested whether nobiletin caused changes in cell cycle progression Fig. . The floAs mentioned above, nobiletin showed a certain degree of AR/AR-V7 dependence on suppressing the proliferation of PC cells in vitro.In order to explore the mechanisms, we first selected the 22Rv1 (a classic cell line with high AR and AR-V7 levels) as the experimental object, and then used western blot assay to detect whether nobiletin affected the protein expression of AR/AR-V7. We were pleasantly surprised to find that although nobiletin had no significant effect on the protein level of the full-length AR (AR-FL), it downregulated AR-V7 in a dose- and time-dependent manner Fig. . Then imNext, we wondered how nobiletin downregulates AR-V7, such as by inhibiting AR-V7 protein synthesis or promoting its degradation. On one hand, real-time quantitative PCR assay was performed to detect the changes of AR-V7 mRNA in 22Rv1 cells treated with various concentrations of nobiletin, but the results showed that nobiletin did not downregulate AR-V7 mRNA levels Fig. , althougFurthermore, Co-IP assay was used to confirm that nobiletin instigated the K48-linked ubiquitinated form of AR-V7 Fig. . FormatiIn our previous research, the GRP78-AR-V7-SIAH2 axis has been reported in detail as the degradation pathway of AR-V7 . Here weDeubiquitylating enzymes (DUBs) mediate the reverse process of protein ubiquitination and is of great significance in the regulation of oncoprotein stability in various cancer cells –35. To iNext, to further explore the subcellular locations of the interactions between these proteins, we showed that the subcellular locations of USP14 and AR-V7 interaction were mainly in the cytoplasm, and small amount in the nucleus through immunofluorescence colocalization assay Fig. , and theTo address whether USP14 and USP22 is critical in nobiletin-induced selective degradation of AR-V7, 22Rv1 cells were treated with nobiletin in the concentration gradient and time gradient as shown in the figure. Next, Co-IP and western blot experiments were performed to detect the interactions of AR-V7 with USP14 and USP22, respectively. We were pleasantly surprised to find that nobiletin notably prevented the interaction not only between USP14 and AR-V7, but also between USP22 and AR-V7, without affecting the levels of USP14 and USP22 Fig. . In addiOur previous study has been demonstrated that rutaecarpinecan selectively trigger the GRP78-dependent AR-V7 degradation . Hence, In summary, in addition to the reported USP14, USP22 was identified as another DUB that can interact with AR-V7. More importantly, their interactions were notably suppressed by nobiletin, which also propelled us to further explore the role of USP22 in the degradation pathway of AR-V7.First, different siRNAs of USP14/USP22 and Flag/His-Tagged plasmid of USP14/USP22 were applied to evaluate the effect of USP14/USP22 on AR-V7 protein levels. To our expectation, the results showed that the knockdown of USP22 decreased AR-V7 expression. And correspondingly, overexpression of USP22 stabilized AR-V7 expression in a manner similar to USP14 Fig. , demonstIn fact, the regulation of AR expression and function by USP22 in prostate cancer has been reported , but wheTo further verify our hypothesis, a series of reversal experiments were performed. Western blot and CCK8 assays showed that the overexpression of USP14/USP22 not only reversed the downregulation of AR-V7 level by nobiletin to a certain extent, but also partly weakened nobiletin-induced inhibition of the CRPC cell viability Fig. . These fAR-V7 is a driver to boost the occurrence and development of CRPC , 38, 39.Immunohistochemistry assay further verified the effect of nobiletin in the downregulation of AR-V7 and Ki67 in vivo Fig. . MoreoveTo further explore the relationship between the antitumor effect of nobiletin and its promotion of AR-V7 protein degradation, an AR-V7-overexpressing 22Rv1 cell line that stably expressing AR-V7 was constructed by using lentiviruses containing AR-V7 plasmids. CCK8, colony formation, and immunoblot assays were performed in these cells exposed to nobiletin. The results showed that nobiletin-induced inhibition of AR-V7, cell viability, and colony formation ability can be significantly reversed by overexpressing AR-V7 Fig. , suggestIn addition, we also would like to verify whether the inhibitory effect of nobiletin on CRPC proliferation is different with or without androgen stimulation, although we deem that nobiletin does not pose a sufficient threat to full-length AR that is activated by androgens to promote CRPC progression. We pretreated 22Rv1 cells with or without Dihydrotestosterone (DHT) and the synthetic androgen Metribolone (R1881), then performed CCK8 analysis for quantitating the inhibitory effect of nobiletin on 22Rv1 cell viability. The results showed that androgen stimulation weakened the suppression of CRPC by nobiletin to a certain extent Fig. , which mNatural products have become one of the historically rich sources of drug research and development. A large number of natural compounds were proposed to display unexpected anticancer effects and low side effects , 40–42, It has been reported that nobiletin inhibits the growth of prostate cancer cells by suppressing TLR4/TRIF/IRF3, TLR9/IRF7  and AKT In order to explore why AR/AR-V7-positive prostate cancer cells are more sensitive to nobiletin, western blot and immunofluorescence assays were performed. We showed that nobiletin at relatively low concentrations (10 and 20 μM) can significantly decrease the expression levels of AR-V7 in CRPC cells, but has no obvious impact on AR-FL. In addition, the real-time quantitative PCR assay help us rule out the possibility of nobiletin on downregulating the transcription level of AR-V7. Furthermore, the CHX-tracking and BTZ-protein degradation reversal experiments consistently confirmed that nobiletin induced AR-V7 protein degradation through the ubiquitin-proteasome pathway. More directly, we found that nobiletin elevated the level of K48-linked polyubiquitination of AR-V7 via Co-IP assay.DUBs, a series of catalytic enzymes, mediate the ubiquitination progression through removing the ubiquitin or polyubiquitin chain from their specific substrate. So it is essential for regulating the stability and function of the substrate protein . In our We also confirmed that nobiletin can significantly decrease the interactions between AR-V7 and USP14/USP22 in a time and concentration-dependent manner, but did not affect the interactions between AR-FL and USP14/USP22, which may explain why nobiletin exhibits a certain selectivity in inducing the degradation of AR-V7. Furthermore, the overexpression of USP14/USP22 reversed the degradation of AR-V7 induced by nobiletin to a certain extent, and partly recovered the viability of CRPC cells. Our previous research also revealed that the GRP78-AR-V7-SIAH2 protein complex mediates AR-V7 degradation . We therIn conclusion, this research not only demonstrates the reason why nobiletin suppressed the growth process of CRPC through the selective degradation of AR-V7, but also enriches our understanding of the degradation mechanism of AR-V7 and provides an efficient treatment target to overcome CRPC via targeting the interaction between AR-V7 and USP14/USP22 Fig. .Supplementary Data


Pennisetum purpureum into bioethanolor/and biomethane, the effects of ensiling pretreatment, NaOH pretreatment,and their combination on digestion performance and mass flow werecomparatively investigated. The coproduction of bioethanol and biomethaneshowed that 65.2 g of ethanol and 102.6 g of methane could be obtainedfrom 1 kg of untreated Pennisetum purpureum, andpretreatment had significant impacts on the production; however, thereis no significant difference between the results of NaOH pretreatmentand ensiling-NaOH pretreatment in terms of production improvement.Among them, 1 kg of ensiling-NaOH treated Pennisetum purpureum could yield 269.4 g of ethanol and 144.5 g of methane, with a respectiveincrease of 313.2% and 40.8% compared to that from the untreated sample;this corresponded to the final energy production of 14.5 MJ, withthe energy conversion efficiency of 46.8%. In addition, for the ensiling-NaOHtreated Pennisetum purpureum, the energy recoveryfrom coproduction (process III) was 98.9% higher than that from enzymatichydrolysis and fermentation only (process I) and 53.6% higher thanthat from anaerobic digestion only (process II). This indicated thatcoproduction of bioethanol and biomethane from Pennisetumpurpureum after ensiling and NaOH pretreatment is an effectivemethod to improve its conversion efficiency and energy output.Toovercome the structural complexity and improve the bioconversionefficiency of  Pennisetumpurpureum.We investigated the effects of pretreatmenton the coproduction(ethanol and methane) performance and material flow from  Amongbiological pretreatment methods, ensiling pretreatment has receivedwide research interests due to its advantages in low external energydemand, preserving the nutrients of raw material, and providing along-term stable supply of material.13 Themain effect of ensiling pretreatment is that hemicellulose is hydrolyzedto monosaccharides such as glucose and xylose, which are then convertedto short-chain organic acids such as lactate and acetate through anaerobicmicrobial fermentation; as such the recalcitrance of lignin-carbohydratecomplexity is reduced, increasing the accessibility of cellulose tomicroorganisms and enzymes.15 Moreover, intermediatesproduced from ensiling pretreatment such as lactic acid can be easilyconverted into gaseous biofuel (such as biogas). Zhao et al. reportedthat, compared with raw switchgrass, biomethane production from theanaerobic digestion (AD) of ensiling-treated switchgrass was improvedby 33.6%.16 It was also reported that ensilingpretreatment increased the ethanol yield from sugar beet pulp by nearly50%.17 Except for ensiling pretreatment,other biological pretreatments have also been proven to improve thefermentation performance of biomass. For example, the biogas productionfrom microalgae increased by more than 21% after enzymatic pretreatment;18 Wyman et al. reported that the biogas productionfrom corn stover pretreated with white-rot fungi increased by 19%.19 Chemical pretreatment methods refer to the useof chemicals  during theprocess, which have been widely accepted to increase the methane yieldin the AD process;20 among these methods,alkaline pretreatment, with NaOH as the most employed and effectivechemical reagent,21 has drawn extensiveattention with following advantages: (1) it can be carried out undermild conditions (atmospheric pressure under 100 °C);7 (2) the hydroxide ion (OH–) can remove most ligninby breaking the ester bonds between xylan and lignin, and partialhemicellulose by weakening the hydrogen bond between cellulose andhemicellulose,22 which facilitates theaccessibility of cellulose to microorganisms and enzymes. Kang etal. investigated the effect of NaOH pretreatment on the digestionperformance of Pennisetum Hybrid, reporting thatthe methane yield increased from 249.3 mL/g volatile solid (VS) to301.7 mL/g VS under the condition of 35 °C for 24 h.23 Kataria et al. reported that the ethanol yieldfrom NaOH treated Kans grass under the condition of 120 °C for120 min increased to 0.38 g/g substrate.24 To the best of our knowledge, although both ensiling and NaOH pretreatmentare popular methods employed for improving bioenergy and biofuel productionfrom raw lignocellulosic material, the effect of integrated ensiling-NaOHpretreatment on biofuel production has not been studied yet. Integratedensiling pretreatment and NaOH pretreatment can not only ensure thelong-term supply of raw materials but also remove lignin and improvebiofuel production efficiency.Pretreatment methods are usually classifiedinto physical, chemical,and biological categories and their combination.Pennisetum purpureum, a kind of herbaceous plants,has characteristics of high biomass production, strong adaptabilityto the environment, and high cellulose content, making it a potentialcandidate for biofuel production.25 Previousstudies have proved that Pennisetum purpureum isa promising feedstock for ethanol production, with a yield of 4.3mg/mL.26 Stillage, the residues after ethanolfermentation, contains high nondegraded carbohydrates content andother organic matters, which has a high chemical oxygen demand (COD)load and is considered to have a considerable pollution potential,especially for industrial-scale bioethanol plant;27 it is reported that the amount of stillage produced isabout 10 times that of bioethanol production.28 Thus, treating stillage with the well-established AD technique cannot only reduce pollution but also produce renewable biogas, so asto make full advantage of pentose and other organic matters that cannotbe utilized in ethanol fermentation.29 Kaparajuet al. found that the methane yield of 324 mL/g VS was obtained whenthe wheat straw stillage was used as raw material with a concentrationof 12.8 g VS/L.30 Alkan-Ozkaynak et al.used corn stillage as raw material for AD and found that the biogasyield was 763 mL/g VS. These suggest that stillage is a potentialsubstrate for AD.31 Moreover, Liu et al.found that the energy conversion efficiency of sugarcane bagasse aftersequential bioethanol and biogas production increased up to 59.5%;this was only 35.7% for ethanol production and 23.8% for biogas production,respectively.32 Several studies comparingthe energy output of pretreated feedstocks in ethanol production alone,methane production alone, and ethanol–methane coproductionare presented in Pennisetum purpureum would greatly improveits conversion efficiency; as such it would make this feedstock morefeasible for biofuel production on an industrial scale, which necessitatesfurther study to verify this hypothesis. In addition, the introductionof material flow analysis (MFA) into a biofuel production processcould provide some useful and insightful information regarding thematerial utilization efficiency and digestion efficiency, making itpossible to concisely control the process.33 Previous studies focused on the mass and energy flow of ethanol–methanecoproduction, however, with the carbon and nitrogen flow yet not investigated.Pennisetum purpureum; (2) analyze and compare the material and energy flow of Pennisetum purpureum under different processes, and (3)calculate and compare the energy conversion efficiency of Pennisetum purpureum to optimize the feasible pretreatmentand biological conversion process.Therefore, the objectives of thisstudy were to (1) investigatethe effects of ensiling pretreatment, NaOH pretreatment, and combinedpretreatment on the physicochemical structure and ethanol-methanecoproduction conversion efficiency of Pennisetum purpureum,were harvested from Zengcheng district, Guangzhou City (China), inSeptember 2019, with a plant height of 2–3 m. The harvestedsamples were chopped to 1–2 cm by hay cutter and smashed witha pulverizer, and then a part of the samples was frozen at −20°C before use. The other part was vacuumed and sealed in plasticsilo bags and ensiled at ambient temperature for 90 days. The compositionof all samples is listed in Penicillium sp..Saccharomyces cerevisiae Y2034 used for ethanol productionwas obtained from the National Center for Agricultural UtilizationResearch (U.S.A.),39 which can only utilizehexose with an ethanol fermentation efficiency of 85–90%.NaOH was purchased from MacklinBio-Chem Technology Co. Ltd. Thecommercial cellulase with an activity of 144 FPU/g powder was purchasedfrom Imperial Jade Biotechnology Co. Ltd. , whichwas extracted from the fermentation broth of Pennisetum purpureum, three processes were designed in this study, which was illustratedin Pennisetum purpureum, respectively.Process III integrated AD of stillage (including both solid and liquidfractions) from Process I after ethanol fermentation to achieve coproductionof ethanol and biogas.To compare the effectsof ensiling pretreatment, NaOH pretreatment, and their combinationon the bioconversion efficiency of Ensilingpretreatment was conducted ina vacuumed and sealed plastic silo bag, and then stored at ambienttemperature for 90 days.40 Sequential ensiling and NaOH pretreatment were set up underthe same condition, with the ensiled samples underwent the NaOH pretreatment.After pretreatment, the samples were centrifuged for solid–liquidseparation. The solid fraction was washed with distilled water toa neutral pH and then stored at −20 °C for the enzymatichydrolysis and fermentation. The liquid fraction was stored to determinethe concentration of total carbon (TC) and total nitrogen (TN).According to a previous study wherethe ethanol production from NaOH pretreated sugarcane bagasse increasedto nearly 20 g/L, the alkaline pretreatment condition was chosen tobe 2% NaOH (w/v), a solid-to-liquid ratio of 1:20 (w/v) based on totalsolids (TS), at 80 °C for 2 h with 150 rpm.2PO4, 0.2 g/L (NH4)2SO4, and0.4 g/L MgSO4·7H2O. The enzymatic hydrolysisexperiment was carried out at the cellulase loading of 20 FPU/g celluloseof substrate and shaken at the condition of 50 °C and 150 rpmfor 72 h. The hydrolysates (including both solid and liquid fractions)after enzymatic hydrolysis were inoculated with the activated yeaststrain which was cultivated at 30 °C and 150 rpm for 16 h ina medium containing 20 g/L peptone, 10 g/L yeast extract, and 20 g/Lglucose; the reaction system was then placed in a shaker incubatorat 30 °C and 150 rpm for 72 h. For high-performance liquid chromatography(HPLC) analysis, samples were taken at intervals of 12 h during enzymatichydrolysis and 24 h during ethanol fermentation.Beforefermentation, enzymatic hydrolysis of the pretreated samples was performedto achieve the conversion of polysaccharides into monosaccharides.The sterilized pretreated samples were added into the aseptic 0.05M acetate buffer (pH 4.8) at a solid concentration of 10%; the buffercontains 5 g/L yeast extract, 5 g/L peptone, 5 g/L KHAll fermentationslurries were distilled with a rotary vacuum evaporator at 70 °Cand −0.1 MPa for 7 min to separate the ethanol from the fermentedliquid.41 The untreated or pretreated samples that performed anaerobic digestiondirectly were denoted AD. For example, AD of samples after ensilingpretreatment, NaOH pretreatment, and ensiling-NaOH pretreatment arecoded AD-ensiling, AD-NaOH, and AD-ensiling-NaOH, respectively. Whilethe stillage (including both solid and liquid fractions) from theprocess I performed AD was denoted ensiling, NaOH, and ensiling-NaOHafter ensiling, NaOH, and ensiling-NaOH pretreatment. The inoculawere added according to the VS content of the solid part after distillationthe untreated/pretreated sample, and the ratio of samples to inoculumwas 1:2 based on VS. The inocula-only (400 mL) reactors were usedas controls. The actual methane yield was calculated by Yactual isthe specific methane yield (mL/g VS) from samples, Ysample is the sample measured methane yield (mL/g VS), Ycontrol is the methane production of the inocula-only(mL/g VS), Vsample is the volume of inoculaadded in the sample reactor (mL), and 400 is the volume of inoculaadded in the control reactor. The inocula for biogas production weregranule sludge which were cultivated with cellulose and peptone inthe laboratory before use; the pH value, TS content and VS contentof the inocula were 7.5, 1.7 ± 0.1%, and 1.5 ± 0.0%, respectively.The anaerobic digestion experimentsof untreated or pretreated samples (process II) and stillage (processIII) were performed at an automatic methane potential test system.42 Thesamples were dried at 105 °C to a constant weight and then placedin a muffle furnace and incinerated at 550 °C for 2 h to calculateTS and VS. C and N content in the solid fraction were analyzed byVario EL cube . A calorimeter was used to measure the calorific value (CV) of the samples.43 The TC and TN concentrations of the liquid fractionafter NaOH pretreatment, ethanol fermentation, and AD were analyzedby Vario TOC  at 850 °C with an oxygen flowrate of 230 mL/min. The carbohydrates (glucan and xylan) and lignincontents of all samples were analyzed in accordance with the descriptionof National Renewable Energy Laboratory (NREL).44 The monomeric sugars and ethanol concentration were testedby the HPLC system  equipped with SH1011 (Shodex)at 50 °C with 5 mM H2SO4 as the mobilephase at a flow rate of 0.5 mL/min. The surface morphologies of untreatedand pretreated samples were imaged by scanning electron microscopy at an accelerating voltage of 2.0 kV.45The content of TS, VS, C, and Nin the solid fraction from different processes was determined accordingto the previously described method.46 A software for substance flow analysis (STAN 2.6.801) wasused to establish the MFA system model, and the data were processedand optimized by IAL-IMPL2013 algorithm to achieve material balance.The results of MFA were presented by graphics according to the studyby Niu et al.43The flow of mass,element (C and N), and energy was systematically evaluated by MFAaccording to the study by Brunner and Rechberger.m1 and m2 are the mass of the sample before and afterpretreatment (g), G1 and G2 are the percentages of glucan in the sample before andafter pretreatment (%), and L1 and L2 are the percentages of lignin in the samplebefore and after pretreatment (%).Glucan recoveryand lignin removal were calculated as per Cglucose is the concentration ofglucose during enzymatic hydrolysis (mg/mL), Cethanol is the concentration of ethanol duringfermentation (mg/mL), glucan content is the mass of glucan in samples(mg), V is the volume of fermentation liquid (mL),0.9 is the dehydration coefficient of glucose converted to glucan,0.51 is the theoretical coefficient for converting glucose into ethanol,and 1.11 is the theoretical coefficient of conversion of glucan toglucose.47Enzymatic hydrolysis yieldand ethanol yield were respectively calculated as follows:A software of SPSS 19.0was applied to analyze the statisticaldifference of the data, and a one-way analysis of variance was used.The variance level was 0.05%.p > 0.05). For theNaOHpretreated sample, the glucose concentration was 52.8 g/L (p < 0.05). For the ensiling-NaOH pretreated sample, the glucoseconcentration was 53.7 g/L after 72 h enzymatic hydrolysis (p < 0.05), while the xylose and arabinose concentrationswere almost constant throughout the enzymatic hydrolysis.The concentration of monomericsugars and ethanol during the respective enzymatic hydrolysis andfermentation process (process I) are shown in 52.8 g/L 2C, whichdrolysis 2D, resul48 who reported that the yeast strain can timelyferment glucose into ethanol but cannot assimilate xylose. In addition,the ethanol concentration from the ensiling-NaOH treated sample wasincreased by 318.8% compared to that from the untreated sample, 310.9%compared to that from the ensiling treated sample, and 8.6% comparedto that from the NaOH treated sample (p < 0.05).It was worth mentioning that the ethanol yield from the NaOH treatedsample and the ensiling-NaOH treated sample was three times that fromthe ensiling treated sample, indicating that NaOH pretreatment andsequential ensiling and NaOH pretreatment of Pennisetum purpureum both are effective pretreatment methods to increase the ethanolyield. Meanwhile, it also suggested that ensiling-NaOH pretreatmentis a feasible method that can not only realize the continuous supplyof raw material but also improve the fermentation performance. Thiscan be attributed to the removal of lignin after NaOH pretreatment,as the lignin removal was 57.2% for ensiling treated samples, andthe relative glucan content was up to 62.1% (25 and 15.0 g/L.49After the enzymatic hydrolysis experiments (72h), the hydrolysates(including solid and liquid fraction) were added into a fermentationreactor with activated yeast strain for ethanol fermentation. As alsoshown in to 62.1% 3. In addPennisetum purpureum (processII) are shown in p < 0.05). After ensiling-NaOH pretreatment (AD-ensiling-NaOH),the highest daily methane yield was 89.0 mL/g VS/d on day 2, whichwas increased by 90.3%, when compared to that from the untreated sample(p < 0.05). As shown in p > 0.05). After NaOH pretreatment,the specific methane yield was 274.5 mL/g VS, corresponding to anincrease of 34.1% compared to that of the untreated sample (p < 0.05). The specific methane yield of ensiling-NaOHtreated samples was 266.1 mL/g VS, which increased by 30.1% when comparedwith that of the untreated sample (p < 0.05).These results agreed with Costa et al.,50 who investigated the effect of NaOH pretreatment on the digestionperformance of sugar cane bagasse and reported that the methane productionfrom NaOH treated samples increased to 313.4 mL/g dry substrates.The daily and cumulative methane yieldof untreated and pretreated p < 0.01). This might be explainedby the different lignin content in untreated and pretreated samples. After NaOH pretreatment,the specific methane yield was 398.6 mL/g VS, corresponding to anincrease of 61.4% compared to that of the untreated sample (p < 0.05). The specific methane yield of the ensiling-NaOHtreated sample was 454.8 mL/g VS, which increased by 84.2% when comparedwith that of the untreated sample (p < 0.05).In addition, compared with the corresponding sample directly performAD under process II, the methane yield of stillage samples under processIII was respectively increased by 20.7% for the untreated sample,20.5% for the ensiling treated sample, 45.2% for the NaOH treatedsample, and 70.9% for the ensiling-NaOH treated sample. These resultswere similar to the literature in which the methane yield of stillagewas 13.4–34.0% higher than that of pretreated barley straw.54 The enhanced maximum daily methane yield andcumulative methane yield could be explained by that the stillage containsmore soluble pentose such as xylose and other degradable compounds,which can be easily converted to methane during the AD process.55 A previous study reported that utilization ofxylose for biogas production could outpace its use for ethanol interms of energy recovery; as such the AD of stillage achieved thehighest energy recovery from acetic acid-pretreated corn stover afterethanol fermentation.56 Therefore, to betterunderstand the mass balance and energy conversion efficiency of thesubstrate under different processes, it necessitates the comparisonsin terms of mass flow including C and N content and energy recoveryin this study.The dailyand cumulative methane yield from the AD of stillage after ethanolfermentation (process III) are also shown in Pennisetum purpureum duringthe coproduction process isshown in Pennisetum purpureum, the carbon contentswere 44.7% for the untreated sample and 43.9% for ensiling treatedsample, and after NaOH pretreatment, the values respectively decreasedto 42.7% and 42.9% (p < 0.05). For the untreatedsample, the carbon content in the solid part decreased to 41.0% afterenzymatic hydrolysis and ethanol fermentation and further decreasedto 38.2% after AD. For the ensiling treated sample, the carbon contentin the solid part decreased to 40.5% after enzymatic hydrolysis andethanol fermentation and further decreased to 38.6% after AD (p < 0.05). The carbon content in the solid part decreasedto 39.5% for NaOH treated sample and 37.9% for ensiling-NaOH treatedsample after enzymatic hydrolysis and ethanol fermentation, and furtherdecreased to 37.6% and 35.4% after AD (p < 0.05),respectively. The nitrogen content in the solid part of Pennisetumpurpureum was 0.7% for the untreated sample and 0.8% forensiling treated sample and completely removed after NaOH pretreatment(p < 0.05), indicating that the NaOH pretreatmentused in this study had an obvious effect on nitrogen removal. A significantincrease of nitrogen content in the solid part was observed afterenzymatic hydrolysis and ethanol production (p <0.05); the nitrogen content increased to 2.1% for the untreated sampleand 2.0% for the ensiling treated sample. Similarly, the values alsoincreased to 2.3% for NaOH treated sample and 2.2% for ensiling-NaOHtreated sample, respectively; this could be attributed to (1) theaddition of cellulase, inoculum, and seed medium and (2) the proteinwas dissolved under alkaline condition and then precipitated in enzymatichydrolysis and ethanol fermentation process (acidic condition). AfterAD, the nitrogen content in the solid part decreased to 2.0% for theuntreated sample, 2.0% for the ensiling treated sample, 2.2% for theNaOH treated sample, and 2.1% for the ensiling-NaOH treated sample,respectively. The decreased nitrogen content could be attributed tothe growth of methanogens during AD process which needs nitrogen,and consequently, the protein was degraded into free ammonia remainingin the liquid part.58The variation in the contents of TS, VS,C, N, TC, TN, and CV of p <0.05) and then increased to 17 145.3 and 11 056.6 mg/Lafter ethanol fermentation, respectively. After AD, the TC concentrationsignificantly decreased to 1177.8 mg/L for the untreated sample and851.7 mg/L for ensiling treated sample (p < 0.05).The TC concentration in the liquid fraction was 7240.0 mg/L for theNaOH treated sample and 5428.1 mg/L for the ensiling-NaOH treatedsample and then increased to 30 989.9 and 33 125.6 mg/Lafter enzymatic hydrolysis and ethanol fermentation (p < 0.05). After AD, the TC concentration decreased to 1536.8 mg/Lfor NaOH treated sample and 1381.4 mg/L for ensiling-NaOH treatedsample (p < 0.05). The TN concentration in theliquid part of Pennisetum purpureum was 1778.3 mg/Lfor the untreated sample and 1524.8 mg/L for ensiling treated sampleafter enzymatic hydrolysis and ethanol fermentation and then decreasedto 915.0 and 829.7 mg/L after AD (p < 0.05), respectively.The TN concentration in the liquid fraction was 404.9 mg/L for NaOHtreated sample and 471.8 mg/L for the ensiling-NaOH treated sample(p < 0.05) and then respectively increased to1527.9 and 1678.1 mg/L after enzymatic hydrolysis. During the fermentationprocess, the TN concentration decreased from 1696.1 mg/L after ethanolfermentation to 1123.6 mg/L after AD for NaOH treated sample and from2040.0 to 1043.3 mg/L for ensiling-NaOH treated sample (p < 0.05).The TC concentration in the liquid fraction was 16 957.2mg/L for the untreated sample and 10 645.6 mg/L for the ensilingtreated sample after enzymatic hydrolysis  could be explained by the hydrolysisof glucan into glucose (p > 0.05); this is because that ligninis poorly degradable in those processes.60As shown in to 18.1% 4B; for tto 18.1% 4C; for tto 18.1% 4D. The s glucose 2. It shoCombiningthe content of TS, VS, C, N, TC and TN during the threeprocesses, the mass flow including C and N flow could be achieved5–7.For process I, the flow of mass,carbon and nitrogen element areshown in The mass, carbon and nitrogen flow of process II areshown in Pennisetum purpureum samples under process III are shownin 35 who reported that 121.6 g of ethanoland 110.6 g of methane could be obtained from 1 kg of dried Pennisetum purpereum. For the carbon flow .Theenergy flow under different production processes is shown in Pennisetumpurpureum after ensiling, NaOH and ensiling-NaOH pretreatment.Results showed that there were no significant differences betweenNaOH pretreatment and ensiling-NaOH pretreatment in terms of the enhancementin ethanol production and methane production. However, the highestenergy output was obtained via the coproduction of ethanol and methaneprocess after ensiling-NaOH pretreatment; the production of ethanoland methane from 1 kg of ensiling-NaOH treated Pennisetumpurpureum was 269.4 g of ethanol and 144.5 g of methane,respectively, which resulted in an energy output of 14.5 MJ with theenergy conversion efficiency of 46.8%. These results demonstratedthat the coproduction of ethanol and methane from Pennisetumpurpureum outpaced the single ethanol and methane production,which may provide useful information for optimally exploiting itsuse for renewable biofuels production.Ethanolproduction, methane production, and coproduction of ethanoland methane were comparatively investigated from


Allergic rhinitis (AR) is a complex disease involving both mucosal and systemic immune compartments. Greater understanding of the immune networks underpinning AR pathophysiology may assist with further refining disease‐specific biomarkers.To compare immune gene expression profiles in nasal mucosa and peripheral blood samples between adults with AR and controls without AR.SD) and 24 adults without AR (36.6 ± 10.2). Gene expression analysis was performed using the NanoString nCounter PanCancer Immune profiling panel (n = 730 immune genes) in combination with the panel plus probe set  with purified RNA from peripheral blood and cell lysates prepared from combined nasal lavage and nasal brushing.This cross‐sectional study included 45 adults with moderate‐severe and persistent AR . In contrast, 14 genes were differentially expressed in nasal lysate samples between groups (p < .05). Upregulation of allergy‐related genes in nasal mucosa samples in the AR group was observed. Namely, chemokines CCL17 and CCL26 are involved in the chemotaxis of key effector cells and TPSAB1 encodes tryptase, an inflammatory mediator released from activated mast cells and basophils. Six differentially expressed genes (DEGs) were in common between the nasal mucosa and blood samples. In addition, counts of specific DEGs in nasal mucosa samples were positively correlated with eosinophil and dust mite‐specific immunoglobulin E (IgE) counts in blood.One hundred and thirteen genes were significantly differentially expressed in peripheral blood samples between groups (Distinct gene expression profiles in blood and nasal mucosa samples were observed between AR sufferers and controls. The results of this study also provide evidence for a close interaction between the local site and systemic immunity. The genes identified in this study contribute to the current knowledge of AR pathophysiology and may serve as biomarkers to evaluate the effectiveness of treatment regimens, or as targets for drug discovery. A series of genes were differentially expressed in both peripheral blood and nasal mucosal samples from adults with moderate‐severe persistent allergic rhinitis compared with adults without a history of allergic rhinitis. Additionally, gene expression experiments are well suited to identifying genes involved in the pathophysiology of specific diseases and in identifying potential drug targets. Traditionally, microarray experiments using nasal mucosal samples have typically relied on nasal biopsy specimensWe have applied this gene expression technology to identify AR‐specific genes and characterize immune‐gene expression profiles of nasal mucosal and peripheral blood samples of participants with AR compared with a cohort of healthy non‐AR controls. The combined approach of analyzing both the local  and systemic immune system  provides a powerful tool to further understand immunological networks underpinning AR pathophysiology.22.1n = 45) and adults with no history of AR (n = 24 controls). Participants attended a screening appointment at the Queensland Allergy Services Clinic in Southport for evaluation of allergen sensitization and clinical characteristics. Following screening, eligible participants were instructed to cease use of all intranasal and immune modulating medications for 14 days before their follow‐up appointment. Participants then attended an appointment at the Clinical Trial Unit at Griffith University for provision of blood and nasal mucosal samples and completion of symptom surveys. This study was approved by the Griffith University Human Research Ethics Committee  and completed in accordance with the Declaration of Helsinki. All participants provided written and informed consent before participation.This study was designed as a cross‐sectional study to characterize differences in the immune gene expression profiles in peripheral blood and nasal mucosa samples between adults with existing AR  for characterization of the cohort.Participants were both male and female aged between 18 and 65 years of age with a more than 2‐year history of AR symptoms. Participants had persistent AR and moderate‐to‐severe symptoms as defined by the Allergic Rhinitis and Its Impact on Asthma guidelines (ARIA) with symptoms occurring for more than 4 days per week and more than 4 weeks in a row, and one or more of the following conditions present (1) sleep disturbance, (2) impairment of daily activities, (3) impairment of school or work, or (4) troublesome symptoms. Participants had a positive allergic response to dust mites determined via a skin prick test and/or serum‐specific immunoglobulin E (IgE) radioallergosorbent test (RAST) (QML Pathology) to AR individuals were excluded from participating if they suffered from non‐AR (vasomotor rhinitis), consumed probiotics in the previous 8–12 weeks, were treated with oral corticosteroids within the previous 6 months or antibiotics within the previous 30 days, used anti‐inflammatory or immune‐modulating medications, had existing respiratory disease including asthma, nasal polyposis, or chronic obstructive pulmonary disorder, had existing immune dysfunction , had recent nasal surgery or nasal trauma that could affect nasal mucosal sampling, were ill or had infectious disease at the time of enrollment, reported hepatic impairment, or excessive alcohol consumption as per the NHMRC alcohol guidelines AustraliaIndividuals were recruited to the study as control group (CG) if they reported no history of AR, tested negative to dust mites, grass and tree pollen, and were free from any chronic disease. Participants were excluded from the CG if they consumed probiotics in the previous 8–12 weeks, had taken antibiotics within the previous 30 days, used anti‐inflammatory or immune‐modulating medications, had existing respiratory disease, immune dysfunction or gastrointestinal disease or disorder, were ill or had an infectious disease at the time of enrollment or were pregnant at the time of enrollment.2.3The severity of AR symptoms was evaluated using a collection of self‐reported symptom surveys completed before sample collection. The AR‐specific symptom severity surveys included the mini Rhinoconjunctivitis Quality of Life Questionnaire (mRQLQ), Total nasal symptom score (TNSS) survey, Total Ocular Symptom Score (TOSS) survey, The Other Allergic Rhinitis Symptom Score (OARSS), and overall symptom severity measured using a 100 mm Visual Analogue Scale. The mRQLQ consists of 14 questions separated into five domains: activities, practical problems, nose symptoms, eye symptoms, and other symptoms. All items on the questionnaire were rated on a 7‐point likert scale (0‐6) with each item averaged to give a maximum overall score of six. The TNSS consisted of nasal congestion, runny nose, itchy nose, and sneezing. Symptoms were scored on a four‐point scale: 0, no symptoms; 1, mild symptoms; 2, moderate symptoms; and 3, severe symptoms; such that the maximum daily TNSS score was 12. The TOSS consisted of itchy eyes, watery eyes, eye redness, scored on the same four‐point scale as the TNSS such that the maximum daily TOSS was 9. The other symptom score consisted of postnasal drip, unrefreshed sleep, itchy throat/palate or ears and sinus pain, scored on the same four‐point scale, such that the daily maximum score was 12.2.4Venous blood samples were collected for full blood count including white cell differential and specific IgE for dust mites and grass pollen mix. In addition, erythrocyte sedimentation rate (ESR) over 1 h was measured using fresh blood samples collected in sodium citrate tubes and using commercially available Vacuette ESR pipettes (Greiner Bio‐One) as per the Westergren method.Nasal washing, brushing, and whole blood samples were collected as described previously.2.5Immune gene expression analysis of nasal cell lysate and extracted RNA from blood was performed using a commercially available NanoString nCounter PanCancer Immune Profiling panel (NanoString Technologies) as described previously.2.6p < .05 padjust) with significant pathway enrichment accepted at p < .05 Bonferroni padjust.Functional gene annotation and pathway analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID) using differentially expressed genes (DEGs) (2.7http://string-db.org/). STRING is an online database for predicting functional interactions between proteins. The minimum required interaction score was defined at 0.40.The DEGs were analyzed with Search Tool for the Retrieval of Interacting Genes (STRING)  with the Bonferroni Hochberg p value correction (padjust) with significance accepted at p < .05. Correlations between clinical features and DEG counts were performed using the Pearson correlation coefficient with significance accepted at p < .05.Differences in demographic and clinical measures between groups was assessed with an independent 33.12 receptor antagonist/5‐HT3 receptor antagonist/5‐HT4 receptor agonist]). Nasal lysate samples from 37 AR participants and 21 CG participants met the quality control guidelines and were included in gene expression analysis. The demographic and clinical characteristics of the participants whose nasal lysate samples were included in the gene expression analysis were similar to that of the entire study cohort and are included in Tables The demographic and clinical characteristics of the cohort are included in Table 3.23.2.1p < .05 and 113 genes were differentially expressed after controlling for false discovery rate (FDR) (padjust)  Table . The top3.2.2p < .05 and 14 genes were differentially expressed after controlling for FDR  pathways. The top four KEGG pathways were toll‐like receptor signaling pathway, cytokine–cytokine receptor interaction, osteoclast differentiation, and chagas disease (American trypanosomiasis) Table . The DEG3.3.2The DEGs were significantly enriched into three KEGG pathways. These KEGG pathways include cytokine–cytokine receptor interaction, hematopoietic cell lineage, and chemokine signaling pathway Table . The DEG3.43.4.1The DEG list was submitted to the STRING database to provide a better understanding of the biological relationship between the DEGs. A total of 112 genes were included in the PPI network with an average node degree of 4.98. As shown in Figure 3.4.2A total of 14 genes were included in the PPI network Figure  with an 3.53.5.1R = 0.30). Whole blood cell counts of total white cells, eosinophils, neutrophils, and lymphocytes as well as specific IgE and IgG4 were on average weakly (mean R = 0.1–3) associated with the DEG counts. In contrast, self‐reported symptom severity was very weakly (mean R = 0–1) associated with the DEGs. The top 10 individual correlations are shown in Figure The relationship between clinical measures and the DEGs were explored. As shown in Figure 3.5.2R = 0.45) and weakly correlated with total white cells (R = 0.22), lymphocytes (R = 0.22), and basophils (R = 0.22). In addition, the DEGs in nasal lysate samples were on average weakly correlated with specific IgE for dust mites (R = 0.28 and 0.29) and IgG4 for dust mites (R = 0.25 and 0.26). None or very weak (R = 0–0.1) correlations between BMI, neutrophils, monocytes, ESR, and self‐reported symptom severity and DEG counts was observed. The top 10 individual correlations are shown in Figure Correlation analysis between the clinical factors and the nasal lysate DEGs was also performed. As shown in Figure 4The results presented here provide further insights into the pathophysiology of AR at both the local site of the allergic response  and the systemic immune system. In this study, genes and pathways that separate AR individuals from those without AR were identified and can be explored as potential biomarkers of disease. This study provides new information about the pathophysiology of AR through the identification of genes that have not been previously associated with AR or atopy. In addition, this study supports the findings of previous studies and confirms the role of specific genes encoding prostaglandin receptors, chemokines, and eotaxin, and tryptase in the pathophysiology of AR. Evidence for interactions between the mucosal and systemic immune system during active disease is also provided in this study.A key finding of this study was the large proportion of DEGs in blood samples from AR sufferers compared with non‐AR controls who were recruited specifically based on extensive clinical characterization of disease history, pathology confirmation of responsiveness to a single aeroallergen class (dust mites), and without the confounding effects of different intranasal medication use. A total of 113 DEGs were identified in blood samples which represents over 24% of genes that were expressed above background, thereby indicating that the gene‐expression profiles of blood samples from AR sufferers is vastly different from that of the control samples. In contrast, only 14 DEGs (~3% of genes expressed above background) were observed between the groups in the nasal mucosal samples. The relatively low number of DEGs in nasal lysate samples compared with blood samples is likely due to the large variability in mRNA expression across nasal lysate samples. The total mRNA quantity, quality, and numbers of infiltrating immune cells varied across samples and this occurrence has been previously reported using this methodology.The top three DEGs that separate AR participants from non‐AR participants in the blood samples of patients with AR include Mitogen‐Activated Protein Kinase 1 (MAPK1), TANK binding kinase 1 (TBK1), and the Prostaglandin D2 receptor (PTGDR2), suggesting their likely involvement in the pathophysiology of AR. Both MAPK1 and TBK1 genes were downregulated in blood samples from AR participants compared with the controls. Notable functions include TBK1 which is associated with the activation and nuclear translocation of the NFκB transcription factor complexIn this study, we identified DEGs that were unique to either nasal lysate samples or blood samples and genes that were differentially expressed in both sample types Figure . A largeThe DEGs in blood and nasal lysate samples were further analyzed for enrichment into KEGG and GAD pathways. The DEGs in blood were enriched into 66 KEGG pathways. Of these enriched KEGG pathways, the top four pathways included cytokine–cytokine receptor interaction, toll‐like receptor signaling, Chagas disease, and osteoclast differentiation. These results were not entirely unexpected given that atopic diseases have been previously associated with cytokine–cytokine receptor interaction, toll‐like receptor signaling, osteoclast differentiation and pathways related to parasitic infections.Protein–Protein Interaction networks predict direct and indirect functional interactions between expressed proteins and provide a system‐wide understanding of cellular function under selected conditions. The DEGs in nasal lysate and blood samples were analyzed with the STRING database to identify the key genes as drivers of the allergic response. The top three genes in the protein–protein interaction network for blood samples based on degree were UBC, MAPK1 and APP, and have been previously associated with atopic disease. The metalloproteinase 33 enzyme encoded by the ADAM33 gene is a recognized allergy candidate gene and is known to cleave peptides of the amyloid precursor protein (APP).The relationships between clinical measures, including white cell differential, specific IgE and IgG4, ESR, demographic features and symptom severity, and the DEG counts in blood and nasal lysate samples were explored to provide a greater understanding of the role of these genes in the pathogenesis of AR. None of the patient‐reported symptom severity measures  correlated with counts of the DEGs in both blood and nasal lysate samples. Response bias in self‐reported data is well‐recognized event known to confound data interpretationOverall, investigation of the peripheral blood and nasal mucosa with the NanoString nCounter system revealed distinct gene expression profiles in our AR cohort compared with controls. Notably, the peripheral blood DEGs represented a large proportion of the total immune genes, indicating the unique nature of the systemic immune system in AR compared with controls. The shared DEGs in the nasal lysate and blood samples and the associations between clinical markers in blood and gene expression in the nasal mucosa, points to a strong interaction between these immune compartments in the pathogenesis of AR. The AR‐specific genes and pathways identified in this study have the potential to be targeted in new drug therapies for the reduction of AR symptoms and as markers to evaluate the effectiveness of systemic and topical AR drugs.Design: Annabelle M. Watts, Nicholas P. West, Amanda J. Cox. Data collection; Annabelle M. Watts, Amanda J. Cox. Data analysis: Annabelle M. Watts, Nicholas P. West. Review of outcomes: Allan W. Cripps, Peter K. Smith. Drafting of manuscript: Annabelle M. Watts Amanda J. Cox. Manuscript revision and approval: all authors.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.


Methotrexate (MTX) is the most commonly used DMARD in the treatment of juvenile idiopathic arthritis (JIA) . RecommeNine hundred seventy-four laboratory sample results from 445 patients with pediatric rheumatologic diseases  admitted to our centre between 2018 and 2021 and treated with MTX orally or subcutaneously for at least 3 months were extracted from the database of the German Center of Pediatric Rheumatology. Weekday of blood sampling and last methotrexate dose was determined to calculate the time difference in days. Laboratory values for GOT, GPT, lymphocyte and neutrophil count were determined and normalized. Statistical analysis using analysis of variance of the time difference between MTX and laboratory sampling of these four parameters was performed, as well as Chi-square analysis for values above the normal limit for GOT and GPT, and below the normal limit for lymphocytes and neutrophils.p < 0.0001), GPT  = 3.657, p = 0.001) and neutrophil count  = 4.841, p < 0.0001) in days of difference, with the highest/lowest values on day 2 after adminstration of MTX . However, abnormal values were not found significantly more frequently on any day for GOT (p = 0.708), GPT (p = 0.243), lymphocytes (p = 0.566) and neutrophils (p = 0.368).A one-way ANOVA revealed that there was a statistically significant difference for GOT  = 8.535, MTX in the treatment of pediatric rheumatic diseases requires regular monitoring of blood parameters . Changes