Inconsistencies remain regarding the effectiveness and safety of leukotriene receptor antagonists (LTRAs) and selective H1-antihistamines (SAHs) for allergic rhinitis (AR). A meta-analysis of randomized controlled trials (RCTs) was conducted to compare the medications.Relevant head-to-head comparative RCTs were retrieved by searching the PubMed, Embase, and Cochrane’s Library databases from inception to April 20, 2020. A random-effects model was applied to pool the results. Subgroup analyses were performed for seasonal and perennial AR.p = 0.003, I2 = 89%) and daytime eye symptoms score , but were superior in terms of the nighttime symptoms score . The effects of the two treatments on the composite symptom score  and rhinoconjunctivitis quality-of-life questionnaire (RQLQ)  were similar. Incidences of adverse events were comparable . These results were mainly obtained from studies on seasonal AR. No significant publication bias was detected.Fourteen RCTs comprising 4458 patients were included. LTRAs were inferior to SAHs in terms of the daytime nasal symptoms score  in AR patients, LTRAs are more effective in improving nighttime symptoms but less effective in improving daytime nasal symptoms compared to SAHs. Allergic rhinitis (AR) is a common allergic disease caused by immunoglobulin E (IgE)-associated inflammation of the nasal membranes as a result of exposure to allergens , 2. AR cThe PRISMA  statement  and the PubMed, Embase, and the Cochrane Library  databases were systematically searched for relevant studies using the following combined strategy: (1) “leukotriene receptor antagonist” OR “LTRA” OR “montelukast” OR “zafirlukast” OR “pranlukast”; (2) “selective H1-antihistamine” OR “SAH” OR “cetirizine” OR “ebastine” OR “loratadine” OR “desloratadine” OR “acrivastine” OR “fexofenadine” OR “levocetirizine” OR “rupatadine”; (3) “allergic rhinitis”; and (4) “random” OR “randomized” OR “randomised” OR “randomly”. Only clinical studies published in English or Chinese were considered. The reference lists for related reviews and original articles were also searched to complement the results. The latest database search was conducted on April 20, 2020.The inclusion criteria were: (1) peer-reviewed articles in English or Chinese; (2) designed as RCTs; (3) included patients with AR who were randomly allocated to receive LTRAs or SAHs with or without concomitant treatments; (4) with a treatment duration of at least 1 week; and (5) at least one of the following outcomes: daytime nasal symptoms score (DNSS), nighttime symptoms score (NSS), daytime eye symptoms score (DESS), composite symptoms score (CSS), RQLQ, and incidence of AEs. No restrictions were applied for the age of the patients or the blindness of the RCTs during the process of study inclusion. The DNSS includes four nasal symptoms  and each symptom domain is scored from 0 to 3, with the highest score indicating the most serious symptoms. The DNSS is calculated as the sum of the scores (0–12) . SimilarThe study search, data extraction, and quality evaluation were performed independently by two of the authors and disagreements were resolved by consensus between them. We extracted data regarding the study information , study design , patient information , treatment regimens , treatment duration, and outcomes reported. Quality evaluation was performed using the Cochrane’s Risk of Bias Tool  accordinp < 0.10 [I2 statistic was also calculated, and an I2 > 50% reflected significant heterogeneity. Pooled analyses were calculated using a random-effects model because this method incorporates the influence of potential heterogeneity and yields a more generalized result [p values < 0.05 were considered statistically significant. RevMan  and Stata software  were applied for statistical analyses.The effects of LTRAs and SAHs on continuous outcomes, including DNSS, NSS, CSS, DESS, and RQLQ were summarized as differences in the changes in each outcome from the baseline between the groups. MD was used as the measure of the effect on the continuous outcome and the CIs were extracted. For categorized outcomes such as the incidence of AEs, OR and corresponding CIs were used. We used the Cochrane’s Q test to assess heterogeneity, and significant heterogeneity was suggested if p < 0.10 . The I2 d result . Sensitid result . p valueIn summary, 322 articles were obtained through the database search after excluding duplicates. Among them, 296 articles were subsequently excluded primarily based on the titles and abstracts because the studies were not relevant. Among the 26 potentially relevant articles, 12 were further excluded after a full-text review due to the reasons shown in Fig. Table Table p = 0.003; Fig. I2 = 89%). Sensitivity analysis by excluding one dataset at a time showed similar results. Subgroup analyses also showed similar results for seasonal AR patients  but not for perennial AR patients . However, the between-subgroup difference was not statistically significant  but not for perennial AR patients . However, the between-subgroup difference was not statistically significant . For the other outcomes, Egger’s regression tests were not performed as < 10 datasets were available.The funnel plots were symmetrical, suggesting a low risk of publication bias for the outcomes of the meta-analyses Fig. A–F. EggeThe main findings of the meta-analysis were: (1) LTRAs are inferior to SAHs for improving the daytime nasal symptoms of AR, including stuffy, runny, and itchy nose and sneezing; (2) LTRAs are superior to SAHs for improving the nighttime symptoms of AR, including nasal congestion on awakening, difficulty going to sleep, and nighttime awakenings; (3) the effects of the two medications on the composite symptoms, daytime eye symptoms, and QoL for AR patients are similar; and (4) the incidence of AEs was comparable for patients in both groups. These results suggested that although the two medications were similar in terms of the overall AR symptoms (CSS), eye symptoms (DESS), quality of life (RQLQ), and incidence of AEs, SAHs are more suited for patients with primarily daytime symptoms, while LTRAs are more suited for patients with nighttime symptoms.A few previous meta-analyses have explored the comparative role of LTRAs and SAHs in the management of AR patients. Xu et al. evaluated nine RCTs published up to 2014 and reported that for seasonal AR patients, LTRAs were inferior to SAHs in terms of the DNSS and CSS, but were superior in terms of the NSS . The autFor patients with AR, nighttime symptoms are bothersome, which usually leads to sleep disturbance and daytime tiredness, thereby significantly decreasing QoL in these patients . In a prWe performed subgroup analyses to explore the potential differences between LTRAs and SAHs in patients with seasonal or perennial AR. The results of our meta-analysis were mainly driven by studies that included patients with seasonal AR. The differences between LTRAs and SAHs became non-significant when only studies with perennial AR were considered (e.g. DNSS). Therefore, the comparative efficacy and safety of LTRAs and SAHs in patients with perennial AR remain to be clarified in large-scale RCTs. Interestingly, the only study that compared the effects of LTRAs and SAHs on RQLQ in patients with perennial AR showed a superiority of LTRAs over SAHs . The reaOur study has several limitations. Firstly, the ages of the included patients varied. Due to the lack of study data stratified by ages, we were unable to compare the safety and efficacy of LTRAs and SAHs in pediatric and adult patients. Secondly, significant heterogeneity remained in some outcomes, which may be explained by the differences in patient characteristics, medication regimens, and follow-up durations. Thirdly, LTRAs are suggested to be effective for asthma. LTRAs are assumed to have better efficacy for patients with AR and asthma. Although some of the patients who were included in the studies had asthma, we were unable to compare the efficacy and safety of LTRAs and SAHs in these patients because stratified results were not reported. Finally, in view of the potential preference of LTRAs and SAHs for AR patients according to their symptoms, combined treatment with the two medications may achieve better symptom improvement, which should be validated in future studies.The results of this meta-analysis of head-to-head RCTs showed that although both medications are safe and effective in improving the QoL of AR patients, LTRAs are more effective in improving nighttime symptoms but less effective in improving daytime nasal symptoms compared to SAHs. These findings were mainly driven by studies that included seasonal AR patients. Further studies are needed to compare the efficacy and safety of LTRAs and SAHs in patients with perennial AR and to determine the efficacy of a combined treatment with the two medications for AR patients.


Disrupted endothelial permeability plays a crucial role in the vasculitis pathogenesis of Kawasaki disease (KD), which leads to pathological vascular leak and facilitates inflammatory cell infiltration in vascular lesions; however, the mechanisms involved in the development of endothelial barrier dysfunction during KD vasculitis are still largely unclear. Here, we found that sera from patients with KD can induce endothelial cell (EC) hyperpermeability compared to sera from healthy controls. We observed that serum vascular endothelial growth factor (VEGF) levels were increased in KD patients and sera from KD patients upregulated the expression of VEGF receptor 2 (VEGFR2) and neuropilin-1 (NRP1) in human coronary artery endothelial cells (HCAECs). Intriguingly, compared with silence of VEGFR2 in HCAECs, NRP1 silence resulted in a marked decrease in EC permeability. Furthermore, soluble NRP1 (sNRP1) remarkably reduced the stimulation of EC permeability by sera from KD patients compared with bevacizumab treatment. Importantly, we showed that besides VEGF, angiopoietin-like-4 (ANGPTL4), a NRP1-binding vasoactive factor, was also increased in KD and contributed to the EC permeability in KD conditions. In addition, levels of both ANGPTL4 and VEGF were inversely correlated with albumin levels in the serum of KD patients. Collectively, the data demonstrated that overexpressed NRP1, along with upregulated VEGFR2, in HCAECs treated with KD sera promotes endothelial permeability via interaction with the increased ANGPTL4 and VEGF in KD. Neutralization of hyperpermeability factors by sNRP1 may be a novel therapeutic strategy for KD vasculitis. Kawasaki disease (KD) is a common febrile pediatric disorder of unknown etiology and unclear pathogenesis with an increasing incidence and a leading cause of acquired heart disease in children under 5 years old . KD is cNRP1, a transmembrane glycoprotein, acts as a coreceptor for VEGF, which enhances VEGF-VEGFR2-mediated bioactivity, such as angiogenesis and endothelial permeability . On the In the present study, we found that sera from patients with KD can induce human coronary artery endothelial cells (HCAECs) to upregulate the expression of VEGFR2 and NRP1. The overexpression of these two receptors, along with the increased ANGPTL4 and VEGF in the serum of KD, is responsible for the EC hyperpermeability. Moreover, compared with bevacizumab , soluble NRP1 (sNRP1), via neutralization of both ANGPTL4 and VEGF in KD sera, remarkably reduced the stimulating effect of KD sera on EC permeability. Our study indicated that ANGPTL4 is implicated in the promotion of vascular permeability in KD, and sNRP1 may be a novel therapeutic candidate for the treatment of KD vasculitis.g and then filtered by sterilizing filters and stored at -80°C until use.This study was approved by the Ethics Committee of the Affiliated Children's Hospital of Xi'an Jiaotong University and was conducted in accordance with the 2013 Declaration of Helsinki. Written informed consent was obtained from the parents of all participants included in this study. All patients diagnosed with KD met the criteria of “Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease” by the American Heart Association in 2017 . In thisThe levels of VEGF, ANGPTL4, and semaphorin3A in KD serum were measured by 2-step sandwich ELISA kits  according to the manufacturer's instructions. Serum albumin concentration was assessed by the automatic biochemical analyzer . Serum samples were analyzed in duplicate.Human coronary artery endothelial cells (HCAECs) were purchased from SCIENCELL  and cultured as previously described . For theTotal RNA was extracted from HCAEC lysates by RNeasy mini kit . cDNA was synthesized with PrimeScript RT Mix . The mRNA expression levels of VEGFR1, VEGFR2, NRP1, and NRP2 were analyzed using SYBR Green Real-Time PCR Mix  in an ABI 7500 analyzer .μm pore, Corning, USA) that were coated with fibronectin for 1 h and allowed to grow as 2-day-old monolayers. After serum-starvation overnight, 500 μl and 200 μl media containing 20% human serum (KD serum or HC serum) were added for 6 h to the lower and upper chambers, respectively. After rinsing, a total of 200 μl 300 μg/ml FITC-dextran  was added to the upper chamber for 30 min. The fluorescence intensity in the lower chamber was measured using a fluorescence microplate reader .HCAECs were seeded on transwells (6.5 mm diameter polycarbonate membranes with 0.4 M ± SD). Unpaired Student's t-test was used to determine statistical differences between two groups. Pearson's method was performed to explore the correlation between levels of VEGF and ANGPTL4 and the concentration of albumin in KD serum. A 2-tailed P value < 0.05 was statistically significant. Statistical analysis was conducted using Prism 7.0 software .Quantitative data were expressed as the means ± standard deviation (Hypoalbuminemia, a condition caused by albumin leakage outside the blood vessels following high vascular permeability, is a characteristic feature of KD . So we fTo explore the mechanism by which KD serum induces EC hyperpermeability, we next measured the levels of VEGF, a well-known vascular permeability factor, in KD patients and the expression of four VEGF receptors, VEGFR1, VEGFR2, NRP1, and NRP2, in HCAECs stimulated by KD sera. We found that serum VEGF levels were higher in KD patients than in HC children . We alsoWe next set out to investigate whether the upregulation of VEGFR2 and NRP1 is responsible for the KD sera-induced EC hyperpermeability. To this end, we blocked the expression of VEGFR2 and NRP1 with specific siRNAs in HCAECs. We found that upon the knockdown of VEGFR2 or NRP1 expression on HCAECs, the enhancement of EC permeability induced by KD sera was significantly reduced . HoweverANGPTL4 and semaphorin3A, two NRP1-binding proteins, have been reported to act as vasoactive mediators for endothelium permeability , 10, 14.To further identify if both ANGPTL4 and VEGF contribute to the hyperpermeability conditions of KD, we analyzed the relationship of the concentrations between the two proteins and albumin in KD sera and found that the levels of both ANGPTL4 and VEGF negatively correlated with albumin levels in the serum of KD patients, which further indicated that the two vasoactive factors are responsible for vascular permeability in KD conditions .Increased vascular permeability is a significant feature of KD pathophysiology, but its underlying mechanism is still largely unclear. Here, we demonstrated that overexpressed NRP1 and VEGFR2 in ECs contribute to the vascular hyperpermeability by binding with ANGPTL4 and VEGF in KD and highlighted the pathological involvement of ANGPTL4/NRP1 interaction in the promotion of EC permeability in KD conditions. Furthermore, our observations showed a potential therapeutic effect of sNRP1 on KD vasculitis.in vitro [Hypoalbuminemia is common and thought to be an indicator of the increased vascular permeability in KD , 15. In in vitro , 16–18. in vitro . Here, fVEGF, also known as VEGF-A, is a well-known vascular permeability factor and acts as a proinflammatory cytokine by increasing the permeability of ECs , 19. AccIntriguingly, the knockdown of NRP1 expression in HCAECs showed a marked decrease in HCAEC monolayer hyperpermeability induced by KD sera compared with VEGFR2 knockdown, suggesting an additional combination between NRP1 and its ligands that contribute to NRP1-mediated EC permeability. Besides binding VEGF, NRP1 has been shown to interact with two other vascular permeability factors, semaphorin3A and ANGPTL4 , 10, 14.Despite a well-described key effect of ANGPTL4 on lipid metabolism, a lot of research evidence has shown that ANGPTL4, as a multifunctional protein, plays an important role in vascular homeostasis and inflammation , 26; howOf note, sNRP1 showed a marked inhibition effect on KD sera-induced EC hyperpermeability. Despite a similar inhibition by bevacizumab, this VEGF-neutralizing antibody was not as powerful as sNRP1, which therefore indicated sNRP1 may be a promising therapeutic candidate for KD treatment and prevent the inflammatory progress of vessels at an earlier stage of acute KD. Additionally, it is noteworthy that IVIG treatment showed no significant inhibition of KD sera-induced EC hyperpermeability, which is similar to Terai et al.'s study  showing α [There are some limitations in this study. First, we did not explore what factors in KD serum contribute to the overexpression of VEGF receptors in HCAEC. In this regard, some proteins, such as CRP and TNF-α , 34, havα –37. WhetIn summary, our study showed that both the upregulation of NRP1 and VEGFR2 in ECs and the increase of ANGPTL4 and VEGF in serum are responsible for the vascular hyperpermeability in KD setting. For the first time, we found that the ANGPTL4/NRP1 axis participates in the promotion of EC permeability in KD conditions. sNRP1 has a potential as a novel therapeutic candidate for KD treatment by neutralizing ANGPTL4 and VEGF and inhibiting vascular hyperpermeability.


Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity. Androgen receptor (AR) signaling is regulated by multiple post-translational modifications. Here, the authors identify the writer and reader enzymes for AR ADP-ribosylation and show how they modulate AR signaling output in prostate cancer cells. Ligand-induced alterations in AR structure facilitate protein–protein interactions fundamental to AR regulation as a transcription factor4. One striking effect of androgen binding to AR is the induction of post-translational modifications. AR contains >20 sites that undergo modification, which include phosphorylation, acetylation, SUMOylation, and ubiquitination6. To date, the most abundant post-translational modification on AR is phosphorylation, which maps primarily to the NTD. Given that the NTD mediates the transactivation function of AR, androgen-regulated LBD–NTD interactions7 could modulate AR output through changes in protein structure and post-translational modifications that specify co-factor recruitment.The androgen receptor (AR) transduces the effects of androgen by acting as a ligand-regulated transcription factor. Androgen binds with nanomolar affinity to the C-terminal ligand-binding domain (LBD) of AR and induces structural changes within the LBD that promote intramolecular interactions with the N-terminal domain (NTD)8. Androgen deprivation and treatment with anti-androgens also seem to mimic the genetic loss of homologous recombination (HR) genes such as BRCA1/2 and ATM which occurs in ~10% of prostate cancer patients9. Reduced HR activity renders solid tumors responsive to poly(ADP-ribose) polymerase (Parp) inhibitors that prevent base excision repair (BER)11. Deficits in HR and BER generate synthetic lethality because tumor cells are forced to repair DNA by error-prone, non-homologous end joining (NHEJ). These relationships have generated strong interest in Parp inhibitors, which improve outcomes in ovarian, breast, and prostate cancer patients who harbor mutations in DNA repair genes12.Because ligand binding plays a crucial role in AR regulation, therapeutic strategies for prostate cancer have largely focused on reducing androgen synthesis and the development of anti-androgen compounds+ as a co-factor for post-translational modification by ADP-ribosylation14. Parp1, Parp2, and the tankyrases, Parp5a and Parp5b, ADP-ribosylate protein substrates, but also extend the initial ADP-ribose conjugate and generate poly(ADP-ribose) chains. While the functions of poly(ADP-ribose) chains are not fully understood, these structures are known to be generated in diverse cellular contexts such as DNA repair and telomere maintenance16. Most Parp family members mediate mono-ADP-ribosylation; this occurs on a variety of amino acids, is reversible by cellular hydrolases, and is predicted to impact protein activity17. But with the exception of bacterial toxins, relatively little is known about how mono-ADP-ribosylation contributes to protein regulation.Clinically used Parp inhibitors such as Olaparib and Veliparib were developed against Parp1, the founding member of an enzyme family that uses NADHere, we describe a pathway that integrates Parp function with androgen signaling and characterize a mechanism that regulates AR output. The pathway is based on androgen induction of the mono-ADP-ribosyltransferase Parp7, which, in turn, ADP-ribosylates AR on multiple cysteine (Cys) residues. ADP-ribosylation by Parp7 results in macrodomain- (MD) mediated assembly of an AR-Dtx3L/Parp9 complex. Parp7 enzyme activity, AR ADP-ribosylation, and assembly of the AR-Dtx3L/Parp9 complex are inhibited by Olaparib. Using depletion approaches and RNA-seq, we show that the Dtx3L/Parp9 complex modulates the expression of AR-regulated genes. Our data identify an androgen-Parp signaling axis that uses an ADP-ribose writer (Parp7) and reader (Parp9) to control the assembly of a transcription factor complex.20. The MS results were validated by probing AR IPs for Dtx3L and Parp9, and by the reciprocal approach, Dtx3L IP, and immunoblotting for AR  AR into PC3 prostate cancer cells, treated the cells with an androgen agonist (R1881), and at multiple timepoints (0–24 h), isolated AR by immunoprecipitation (IP). SDS-PAGE and silver staining revealed the R1881-induced early release of Hsp90 from AR (0 and 2 h comparison), but also a time-dependent association of ~80-kDa proteins with AR at later timepoints  as a biochemical probe since it shows selectivity and relatively high affinity for ADP-ribose (KD = 126 nM)24. We immobilized GST-Af1521 on glutathione beads, performed pull-down assays with extracts from PC3-AR prostate cancer cells, and examined the bound fractions by AR immunoblotting  and androstenedione (ASD) but neither by the adrenal androgen dehydroepiandrosterone (DHEA), estradiol nor by various anti-androgens Fig. . The levent Fig. . These d27. In this case, the mass addition (+212 Da) reflects the phosphoribosyl group. Using these two approaches, we identified a total of 11 AR ADP-ribosylation sites, including seven sites in the NTD , while parallel incubations with chemicals that release ADP-ribose from the side chains of other ADP-ribose acceptors 28 had no effect . We also used the approach of treating IP’d AR with the pyrophosphatase NUDT16 since converting ADP-ribose to phosphoribosyl enables enrichment of the peptide by metal affinity chromatography1 Da. We dition +2 Da refleect Fig. . HgCl2 t330 Fig. . Lastly,nts Fig. . The bioWe performed an androgen treatment time course and Af1521 pulldown, and found that AR ADP-ribosylation is detected after 4 h of R1881 treatment, with higher levels of ADP-ribosylation observed at 9 and 24 hr Fig. . Because32, it is clear that AR undergoes robust androgen-induced binding to the Parp7 gene, which we verified by ChIP-PCR  were apparent for a few Parps, the only Parp whose expression increased >2-fold in both prostate cancer lines was the mono-ADP-ribosyltransferase Tiparp  did not impair ADP-ribosylation or binding to Dtx3L/Parp9, and inhibiting ADP-ribosylation did not prevent phosphorylation assessed with phospho-site-specific antibodies  and cytoplasm (NES-AR), though excluding AR from the nucleus also required mutating its endogenous NLS  prevented ADP-ribosylation and, as expected, eliminated complex formation  in ovarian cancerval Fig. .P value cutoff of <0.001. This identified >7000 genes differentially expressed in response to R1881, and 683 genes with different expression levels between control and Dtx3L knockdown, the latter without R1881 treatment , as well as genes that were repressed by both R1881 treatment and Dtx3L knockdown (208 genes) Fig. , left. RKLK3 and TMPRSS2. We then compared gene expression between the upper and lower deciles based on KLK3 and TMPRSS2, and overlapped the genes with higher or lower expression  with the data from R1881-treated VCaP cells. This revealed an enrichment in the overlap between R1881-induced and higher expression in the KLK3 and TMPRSS2 upper 10%  promoter is used extensively by the prostate cancer field to measure AR-dependent transcription. We employed the PSA-luciferase reporter, which contains proximal and distal androgen response elements (AREs) to test whether AR-Dtx3L/Parp9 complex formation regulates transcription 45. R1881-induced ADP-ribosylation was detected at two AR binding sites in the MYBPC1 gene, but not at an intergenic site . By contrast, treating cells with Veliparib, which inhibits Parp1 but not Parp7, does not significantly change AR ADP-ribosylation nor MYBPC1 gene expression  signaling in immune cells, since Dtx3L/Parp9-STAT1 complexes isolated by IP can be dissociated by the addition of ADP-ribose47.MDs are phylogenetically conserved protein modules that bind mono- and poly-ADP-ribose, and in some cases, show hydrolysis activity20 showed that the Dtx3L/Parp9 heterodimer contains ADP-ribosyltransferase activity, which is in addition to the E3 ubiquitin ligase activity discovered by the Shipp laboratory19. Both activities are utilized in the context of Ub processing and conjugation, where the Dtx3L/Parp9 heterodimer can transfer ADP-ribose to the carboxyl terminus of Ub and block substrate conjugation20. The outcome of the reaction, substrate conjugation or Ub-ADP-ribosylation, is dictated by NAD+ concentration, with the latter favored in the presence of high NAD+. We found that E3 and ADP-ribosyltransferase activities are functional in the AR-Dtx3L/Parp9 complex, suggesting Parp7 modification of AR can target these activities to chromatin sites. This view is consistent with data showing that Dtx3L/Parp9 depletion affects the expression of certain AR-regulated genes, and ADPr ChAP showing ADP-ribosylated AR is recruited to chromatin. We also employed a PSA reporter assay to test whether the Dtx3L/Parp9 binding to AR promotes transcription. Using Parp9 knockout cells, we found that androgen induction of AR-dependent transcription from the PSA promoter was significantly increased by WT Parp9, but not by Parp9 that was deficient for binding ADP-ribosylated AR because of point mutations in its two MDs. These data argue that a physical interaction between the Dtx3L/Parp9 heterodimer and AR is important for transcription. Consistent with this view, Cys-to-Gly mutations in four of the ADP-ribose acceptor sites in the AR NTD reduced ADP-ribosylation, Dtx3L/Parp9 complex formation, and AR-dependent transcription from the PSA promoter. Exactly how Dtx3L/Parp9 promotes transcription remains to be determined. One scenario is that it involves the E3 ligase activity of Dtx3L, which is known to modify histones, but an effect on co-factor interactions with AR is also possible. AR binding to chromatin appears to be independent of ADP-ribosylation, based on our finding that Olaparib inhibition of AR ADP-ribosylation did not detectably reduce AR binding to the MYBPC1 promoter.Previous work from our group48. In another context, Dtx3L/Parp9 mediates STAT-dependent repression of the tumor suppressor IRF1, consistent with the repressive effect of Dtx3L/Parp9 in tethering assays49. By contrast, Dtx3L/Parp9 enhances IFN-dependent gene expression through STAT1 by utilizing its E3 activity towards histone H2BJ50. Our gene expression analysis indicates that Dtx3L/Parp9 depletion results in negative and positive effects on AR-dependent transcription, depending on the gene. Dtx3L/Parp9 levels influence the basal levels of AR-regulated genes, assayed in the absence of androgen, as well as the induced levels of androgen-regulated genes, measured in response to R1881. The crosstalk between IFN and androgen signaling appears to be bidirectional given that IFN can induce Dtx3L/Parp9, and AR can repress IFN signaling through induction of genes such as SOCS3 in prostate cancer cells51. The AR-Dtx3L/Parp9 can therefore be considered a signaling node that is regulated by at least three inputs: (i) androgen, which increases pathway activity by inducing Parp7 expression; (ii) NAD+ levels, which influence the level of substrate conjugation with Ub; (iii) IFN, which controls Dtx3L and Parp9 expression through a STAT-regulated bidirectional promoter52. It seems probable that these diverse inputs help fine-tune transcription of AR-regulated genes. Based on our RNA-seq data, Dtx3L and Parp9 may also exert effects on transcription that are androgen-independent, and AR transcriptional effects that are ADP-ribosylation independent.Dtx3L has been shown to act as an E3 ligase for histone monoubiquitylation and contribute to the regulation of chromatin-based events. Histone modification by Dtx3L, notably histone H4K91-Ub generation, contributes to DNA repair kinetics by enhancing 53BP1 recruitmentPARP7 gene was associated with an overall survival benefit (44.8 versus 15.7 months) in ovarian cancer patients41. Also, a genome-wide association study identified an ovarian cancer susceptibility locus in the PARP7 gene53. These genomic data prompt the hypothesis that Parp7 activity could favor clinical outcome, and that Parp inhibitors lacking off-target effects on Parp7 might be more beneficial than pan-Parp inhibitors.RNA-seq data from prostate adenocarcinoma (PrAd) patients suggest there is an inverse correlation between Parp7 expression and the time period between therapy and biochemical recurrence. We also noted in RNA-seq data that Parp7 levels are lower in prostate cancer metastases than in primary tumors. These observations, which suggest a reduction in Parp7 mono-ADP-ribosylation activity could be associated with disease progression, is interesting given the related findings in ovarian cancer. Amplification of the 54. Parp7 restrains AHR transcription activity through ADP-ribosylation, but the mechanism has not been fully defined. Among the dioxin and AHR-regulated genes restrained by Parp7 are the cytokines II1b, Cxcl2, and Tnf, which point to a role in the inflammatory response54. Parp7 also restrains type I IFN signaling through ADP-ribosylation of the kinase TBK1 and suppression of IFN expression55. With regard to nuclear receptors, liver X receptor (LXR) can be ADP-ribosylated by Parp7, and Parp7 knockout mice show reduced ligand induction of the LXR target gene Srebp156. Ligand regulation of Parp7 expression and transcription factor ADP-ribosylation appear to be a feature of several nuclear receptor pathways. These data raise the question of whether the ADP-ribose reader and E3 functions of the Dtx3L/Parp9 heterodimer contributes to transcriptional regulation in a variety of biological settings.Parp7 makes important contributions to other nuclear pathways. In the liver, Parp7  is induced by the aryl hydrocarbon receptor (AHR) and helps protect against dioxin-induced lethality2 and at 37 °C. For RNA-seq, qPCR, ChIP, and ChAP experiments, cells were switched to phenol red-free medium and FBS depleted of androgens 24 h before R1881 addition.VCaP prostate cells (ATCC CRL-2876) were maintained in DMEM/F12 supplemented with 10% FBS and 1% Pen Strep. HEK293T cells (ATCC CRL-3216) were maintained in medium DMEM supplemented with 5% FBS and 1% Pen Strep. PC3 (ATCC CRL-1435), PC3M (RRID:CVCL_9555), and derivative cell lines were grown in RPMI 1640 supplemented with 5% FBS and 1% Pen Strep. All cells were grown in the presence of 5% COg, and then passed through a sterile 0.45-µm filter, with optional concentrating lentivirus using Takara’s Lenti-X Concentrator. Infection of target cells was carried out in the presence of 8 µg/ml of polybrene. After 2–3 cell doublings, the cells were either selected with 0.2–0.5 mg/ml of hygromycin or 1–2 µg/ml of puromycin based on the antibiotic resistance of the plasmid, or cell sorted for GFP co-expression. PC3-AR cells expressing Flag-tagged AR was generated and selected by growth in hygromycin. PC3M-HA-AR and PC3M-HA-Parp7 were produced from PC3M cells with an HA-AR and HA-Parp7 lentivirus containing a puromycin resistance marker. Additional cell lines generated were VCaP(shGFP), VCaP(shParp7), and VCaP(pLKO-Tet-ON shVector), VCaP(pLKO-Tet-ON/shDtx3L). Cell lines containing two transgenes were produced by sequential transfection and drug selection. The cell lines constructed with two transgenes were PC3AR(pWPI), PC3AR(pWPI/HA-Parp7), PC3AR(pWPI/Parp9 wild type or mutants-Avi/GFP-BirA*), PC3AR(shGFP), PC3AR(shParp7), PC3M-HA-AR(Flag-AR wild-type and Cys-to-Gly mutants), and PC3M-HA-Parp7(Flag-AR wild-type and Cys-to-Gly mutants).HEK293T cells were grown to ~70% confluency and co-transfected with target plasmid and accessory plasmids pMD2g and psPAX2 (ratio 2:1:1) using Fugene-6. After cell incubation at 37 °C for ~16 h, cells were replenished with fresh DMEM supplemented with 35% FBS, and further incubated for another 24 h. The medium containing lentiviruses was transferred to a fresh conical tube, clarified by centrifugation ~700 × To generate PARP9-deficient PC3-AR prostate cancer cells, two single-guide RNAs , M2 , Tubulin , AviTag , Parp1 , HA tag , Alexa Fluor 680 donkey anti-Rabbit IgG(H + L) , IRDye800-conjugated anti-mouse IgG(H&L) , Cy™3 AffiniPure Donkey Anti-Rabbit IgG (H + L)  and Alexa Fluor® 488 AffiniPure Donkey Anti-Mouse IgG (H + L) , and Neutravidin-DyLight-800 . Uncropped scans of blots are available in the Source Data file.The following antibodies were used at the concentrations and dilutions indicated for Western blot (WB) and immunofluorescence microscopy (IF). A rabbit polyclonal was raised against a Parp7 peptide (amino acids 119–132) and affinity-purified before use (WB: 1 μg/ml). The rabbit antibodies to AR (WB: 1 μg/ml), Dtx3L (WB: 0.2 μg/ml), Parp9 (WB: 1 μg/ml), and AR phosphor-Serine-sites  (WB: 1:1000–2000) have been described25 was used to generate a panel of Flag-AR mutants  was obtained from the DNASU Plasmid Repository and cloned into the pGEX-4T-2 bacterial expression vector. To generate tandem Af1521 expression, Af1521 was amplified by PCR and inserted in-frame into the AccI and NotI restriction sites25. Other plasmids used were pWPI (Addgene 12254), pWPI/HA-Parp7, pWPI/Parp9 WT or mutants-Avi/GFP-BirA*, pLKO.1 GFP shRNA , shParp7 (Sigma SHCLNG-NM_015508), pLKO.Tet-On , Tet-shDtx3L with target sequence CGCGTATTAGGAGTCTCAGAT, pKH3 (Addgene 12555), pKH3/HA-Parp9, pKH3/Parp9, pBacPAK8 (Clontech 631402), pBacPAK8/T7-His-Parp7, pET-Duet1/His6-Parp9/Dtx3L, pMBPHis-Parallel 1 plasmid , MBP-Parp9-MD1, MBP-Parp9-MD2, MBP-Parp9-MD1-MD2.A lentiviral plasmid encoding Flag-AR and hygromycin resistanceThe synthetic androgen R1881  was used in most experiments, for treatment times that are specified in the figures. Other agonists and antagonists used were as follows: dihydrotestosterone , 4’-androstane-3,17-dione , Casodex (10 µM), flutamide (10 µM), hydroxyflutamide , dehydroepiandrosterone , estradiol (100 nM), cyproterone acetate , and enzalutamide (20 µM). In cycloheximide (CHX) experiments, cells were initially treated with R1881 for 4 h before adding CHX  and incubating for an additional 6 h. For AviTag labeling, PC3AR(pWPI/Parp9 WT or mutants-Avi/GFP-BirA*) cells were incubated with both R1881 (2 nM) and biotin (50 μM) for 18–24 h. The Parp inhibitors Veliparib and Olaparib were used at the concentrations shown in the figures. For inhibition of Parp1 automodification, Veliparib and Olaparib were added 30 min prior to hydrogen peroxide  treatment. For the CHX and Olaparib chase experiment, PC3-AR were treated with R1881 (2 nM) overnight and followed with a chase by in the same medium containing R1881 (2 nM), CHX (100 µg/ml), and Olaparib (10 µM).g) for 20 min and then subjected to antibody beads (or GST-Af1521 beads) binding for 2.5–4 h. The beads were collected either by centrifugation for Agarose/Sepharose beads, or by magnetic fields for magnetic beads. The beads were washed five times with the wash buffer , and then resuspended in SDS-loading buffer followed with western blot analyses. For silver staining, SDS-PAGE mini gels were incubated in the fixation solution (methanol: acetic acid: Milli Q water = 50:10:40) one time overnight, and the second time for 2 h. The gel was then washed 5 min each for five times with Milli Q water, incubated 90 s in the sodium thiosulfate solution (one pellet per 250 ml Milli Q water), rinsed three times with Milli Q water, incubated 20 min with silver nitrate solution (90 mg of AgNO3 in 50 ml Milli Q water), rinsed again three times with Milli Q water, incubated in the developing solution  until reaching desired bands intensity, and then put into the quenching solution (10% acetic acid).All steps were performed at 4 °C. Pelleted cells were resuspended in the cell extraction buffer , and then end-over-end rotated for 20 min. The extracts were clarified with centrifugation . Chemical release of ADPr from AR was performed using HgCl2 in 100 mM Tris-HCl, pH 7.5 at room temperature for 1–2 h. AR was also incubated with 0.1 M hydroxyamine pH 7.5 or 0.1 M CHES buffer pH 9 at 37 °C for 2 h. Radioactive ADP-ribosylation assays measuring Parp7 activity were conducted in 50 mM HEPES, 5 mM β-Mercaptoethanol (BME), and 50 mM MgCl2. 32P-NAD+ was added (0.05 μM labeled plus 24.95 μM unlabeled NAD+) at 35 °C for 30 min. Autoradiography was used to detect auto-ADP-ribosylation. To measure and quantify ADP-ribosylation, proteins were captured on 0.2 μm nitrocellulose filters , washed, and 32P signal was counted using a scintillation counter (Beckman). For assays that included inhibitors, complete reactions excluding NAD+ were pre-incubated on ice for 30 min. NADase (Sigma) solubilized by lipase was used to make a 1 mM ADP-ribose stock with unlabeled NAD+ and 1.2 μM radiolabeled NAD+. Generation of ADP-ribose by NADase at 37 °C for 30 min was confirmed by thin-layer chromatography. ADP-ribose was purified using a Microcon Centrifugal Filter (Millipore). Binding reactions were incubated at room temperature for 1 h and included 100 μM ADP-ribose and 4 μM recombinant MD protein. Proteins were captured on a 0.2-μm nitrocellulose filter , washed, and 32P signal was counted using a scintillation counter (Beckman). ADP-ribosylated Parp1C was generated in reactions containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 2 mM DTT, 2 μM PARP1C and 4 μM HPF1. The reaction was held on ice for 10 min, supplemented with 0.2 mM β-NAD+, further incubated on ice for 10 min, and finally supplemented with 2  μM Duplex oligo pair and incubated at 37 °C for 30 min. NAD+ was removed by desalting on a spin column (Thermo Scientific 89891), and aliquots were snap-frozen and stored at −80 °C. Dtx3L/Parp9 ubiquitylation/ADP-ribosylation was performed in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2 buffer, containing 0.1 mM ATP, 1 mM DTT, 0.1 mg/ml T7His6-ubiquitin, 0.01 mg/ml each of E1 and E2, recombinant and IP’d Dtx3L/His6-Parp9, β-NAD+ (12.5 μM biotin-NAD+ + 0.1 mM β-NAD+), and optional 1 μM Olaparib. For NUDT16 and ARH3 treatment, Flag peptide-eluted AR, immunoprecipitated AR and AR extract were incubated at 37 °C for 60 min with 3 μM NUDT16 in a buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Triton X-100, 0.2 mg/ml BSA, 2 μM Olaparib and protease inhibitors. ADP-ribosylated PARP1C was incubated at 37 °C for 60 min with 0.1 μM ARH3 in a buffer containing 20–50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 2 mM DTT, 2 μM Olaparib and 0.2 mg/ml BSA. For chemical sensitivity analysis, ADP-ribosylated PARP1C was incubated with 50 mM HgCl2 or 100 mM NaCl in a buffer containing 100 mM Tris-HCl, pH 8 at room temperature for 60 min.Parp7 ADP-ribosylation assays were performed at 30 °C for 30 min in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 2 mM DTT containing ~15 ng/μl Parp7 enzyme along with either 1 mM NADn = 1), the sample was reduced with 10 mM DTT followed by alkylation with 50 mM iodoacetamide—each at room temperature for 1 h. In total, 0.5 µg of Promega alkylated trypsin was added to each tube for overnight digestion. The samples were quenched with 3 μL of acetic acid and reduced in volume to 20 μL. The samples were spun before loading to remove any possible precipitate. The LC-MS system consisted of a Thermo Electron Velos Pro Orbitrap mass spectrometer system with a Protana nanospray ion source interfaced to an in-house, self-packed 8 × 75 µm id (Phenomenex Jupiter 10 µm C18 reversed-phase) capillary column. 10 µL of the extract  was injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µL/min over 1.3 h using an Agilent 1200 quaternary HPLC. The nanospray ion source was operated at 2.5 kV and the heated ion capillary set to 265 °C. The digest was analyzed using the rapid switching capability of the instrument acquiring a full scan mass spectrum to determine peptide molecular weights followed by product ion spectra (20) to determine amino acid sequence in sequential scans. The settings for these scans are as follows—MS collected in the Orbitrap , MS/MS CID collected in ion trap . The instrument control software was set for dynamic exclusion—repeat 1, repeat duration 30, exclusion duration 60, list size 200. The data were analyzed by database searching using the Sequest search algorithm within Thermo Proteome Discoverer 2.1 against human androgen receptor (SwissProt P10275). Search parameters—data extraction setting , parent 10 ppm, fragment 0.7 Da, dynamic modifications , 1 missed cleavage, and tryptic (KR). Peptides were filtered by charge state and xcorr— +1 > 1.8, +2 > 2.0, +3 > 2.2, +4 > 2.5. Peptides that passed these minimal criteria were manually examined by Dr. Sherman to first determine the modified peptide mass was within 5 ppm of predicted and second to determine the site of modification. Initial searches against common modification sites  yielded no obvious results. Four sites—C125, C284, C327, and C406—were identified after consideration of Cys as a potential modification target. Examination of all spectra showed low mass ions 348 (AMP), 428 (ADP), 542(ADP-ribose), and 524 (ADP-ribose—water).For the first MS experiment examining ADP-ribosyl modification (n = 1), NUDT16 was used to treat IP’d AR27. The sample was reduced with 10 mM DTT in 0.1 M ammonium bicarbonate, then alkylated with 50 mM iodoacetamide in 0.1 M ammonium bicarbonate (both room temperature for 0.5 h). The sample was then digested overnight at 37 °C with 0.1 μg  in 50 mM ammonium bicarbonate. Phosphoribosylated peptides were enriched using a TiO column. The LC-MS system consisted of a Thermo Electron Q Exactive HF mass spectrometer system with an Easy Spray ion source connected to a Thermo 75 μm × 15 cm C18 Easy Spray column (E800). In total, 80% of the extract  was injected and the peptides eluted from the column by an acetonitrile/0.1 M formic acid gradient at a flow rate of 0.3 μL/min over 1.0 h using a Thermo nLC1200 HPLC. The nanospray ion source was operated at 1.9 kV, the heated capillary set to 250 °C, and the RF set to 40. The digest was analyzed using the rapid switching capability of the instrument acquiring a full scan mass spectrum to determine peptide molecular weights followed by product ion spectra (10) to determine amino acid sequence in sequential scans. The settings for these scans are as follows—MS collected in the Orbitrap , MS/MS HCD collected in Orbitrap . The instrument control software was set for automatic dynamic exclusion for 20 s and a lock mass of 445.12006. The data were analyzed by database searching using the Sequest search algorithm within Thermo Proteome Discoverer 2.4SP1 against human androgen receptor (SwissProt P10275). Search parameters—data extraction setting , parent 10 ppm, fragment 0.02 Da, dynamic modifications , no enzyme. Peptides were filtered by charge state and xcorr—+1 > 1.8, +2 > 2.0, +3 > 2.2, +4 > 2.5. Peptides that passed these minimal criteria were manually examined by Dr. Sherman to first determine the modified peptide mass was within 3 ppm of predicted and second to determine the site of modification. Initial searches against common modification sites  yielded no obvious results. Eight sites—C131, C290, C406, C519, C596, C602, C620, and C670—were identified after consideration of Cys as a potential modification target.For the second MS experiment examining phosphoribosyl modification (n = 1), which was carried out as described for AR but without NUDT16 treatment, and yielded 82% sequence coverage.MS/MS analysis of recombinant His6-T7-Parp7 (https://www.ebi.ac.uk/pride/archive/) partner repository with the dataset identified PXD018811 (AR ADP-ribosylation sites) and PXD025195 (Parp7 ADP-ribosylation sites).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (BL21(DE3)pLysS with 0.4 mM IPTG at 18 °C overnight. MBP and MBP tagged proteins , GST, GST-Af1521, and GST-Af1521tandem (Cloned in pGex-4T-2) were expressed in the bacterial strain BL21 with 0.4 mM IPTG at 18 °C overnight. Parp7 was cloned into baculoviral transient plasmid pBacPAK8 with N-terminal His6-T7 tag. Plasmids were co-transfected with linearized BacPAK6 DNA (Clontech) to prepare the initial baculovirus, followed with virus amplification and then protein expression in insect cell line Sf21 at 27 °C for 5–6 days. The purification of His6-, GST-, and MBP-fusion proteins were done at 4 °C on TALON (Takara 635502), Glutathione (Sigma G4510) or Amylose beads (New England BioLabs E8021S). Fluorescent labeling of purified GST-Af1521tandem was conducted using the IRDye 800CW Protein Labeling Kit (LICOR #928-38040).The pET-DUET1/His6-Parp9/Dtx3L plasmid was used for co-expression of human His6-Parp9 and untagged human Dtx3L in the bacterial strain ADP-ribosylated AR was harvested from mammalian cells using cell lysis buffer  and clarified by centrifugation prior to incubation with recombinant Af1521 (10 μg GST-Af1521/10 μl Glutathione-agarose beads) or recombinant Parp9 MD fused to MBP. Binding reactions were supplemented with 0.5% Triton X-100, and mixed end-over-end for 4 h at 4 °C. The beads were washed five times with binding buffer, and fractions examined by immunoblotting with antibody.Cells were grown on coverslips, washed once with PBS, fixed with 3.75% formaldehyde for 10 min, washed three times with PBS, and permeabilized with 0.2% Triton X-100 for 5 min. Cells were washed three times with PBS and treated with blocking solution (2% BSA and 2% FBS in PBS) for 30 min. Primary and secondary antibodies were diluted in the blocking solution prior to incubation. The cells were incubated in primary antibodies for 2 h at RT, washed three times in PBS, incubated with secondary antibodies for 1 h at RT, incubated with DAPI solution in PBS (1 μg/ml) for 15 s, and mounted using Vectashield.21 was analyzed using RNA-seq data from prostate cancer lines treated +R1881 for 0, 6, and 12 h (PC3-AR), and 0 and 24 h (VCaP) GSE120660 (Androgen effect on PC3-AR and VCaP cells). The effect of Dtx3L on androgen-regulated gene expression was performed in a similar manner in VCaP control  and Dtx3L-depleted  cell lines by treatment +Dox for 4 days, followed by +R1881 for 24 h GSE133876 (Dtx3L and androgen effect in VCaP cells). RNA was prepared using a kit (Qiagen RNeasy) and library construction and sequencing performed by HudsonAlpha. RNA-seq data were analyzed using the Galaxy server . Transcript quantification was performed using Salmon58 to map to the hg38 human genome build, and DESeq2 within the Galaxy site was used for normalizing count data, estimating dispersion, fitting a negative binomial model for each gene, and comparing expression between groups59. A fold cutoff of + /− 0.5 log2 and an adjusted P value of <0.001 was considered significant. Distributions of genes within overlapping gene sets were compared using a 2 × 2 contingency table and Chi-squared test60, and the degree of overlap compared by Fisher exact or Chi-squared test.Androgen-regulated gene expressionGUS. The results shown are representative of at least three experiments. PSA-luciferase experiments in Parp9 knockout cells were performed using a dual-luciferase reporter assay system (Promega), making use of the complete 6.1-kB promoter for the KLK3 gene (PSA). This involved co-transfecting plasmids for PSA promoter-firefly luciferase, cytomegalovirus promoter-Renilla luciferase, and either WT Parp9 or mutant Parp9 . Assays were performed in triplicate and the data plotted as firefly:Renilla. Immunoblotting for Parp9 was used to establish protein expression levels.For real-time quantitative PCR (RT-qPCR) experiments, RNA was extracted using TRIzol (Thermo Fisher Scientific) or the Qiagen RNeasy kit. The cDNA synthesis was carried out using Bio-Rad iScript reagents. DNA amplification using primer pairs , resuspended in the extraction buffer  with end-over-end rotation at 4 °C for 20 min. Pellets were collected by centrifugation , washed with EDTA-free extraction buffer to remove the EDTA, and then sonicated . Samples were supplemented with CaCl2  and micrococcal nuclease (NEB 500 gel units) to cleave the DNA at 37 °C for 5 min. EGTA  and SDS (0.1%) were added to quench the nuclease reaction. The samples were incubated for 10 min at 4 °C, followed by centrifugation . The supernatant was pre-absorbed with unconjugated Protein G beads in the presence of BSA  and salmon sperm DNA , then subjected to antibody bead binding at 4 °C for overnight, with rabbit IgG beads as the negative control. Beads were collected with centrifugation, washed five times with the extraction buffer supplemented with 0.1% SDS, then resuspended in 100 µl of reverse cross-linking buffer , and incubated at 65 °C for 6 h. DNA was purified using the QIAquick Gel Extraction kit (Qiagen). Standard SensiMix Sybrgreen-based reactions were used to quantify the DNA.Cells were fixed using 1% formaldehyde in growth medium at 37 °C for 10 min and neutralized with 125 mM glycine at room temperature for 5 min. Cells were harvested in cold PBS + 1 mM PMSF at 4 °C by centrifugation . Cells were resuspended in the extraction buffer , and sonicated on ice for three times with 10 pulse each at scale “4” with 50% cycle for each time. Samples were supplemented with CaCl2  and DNA was cleaved using Micrococcal Nuclease (500 gel units) for 5 min at 37 °C. After adding EGTA  and SDS , the mixture was incubated at 4 °C for 10 min, and then centrifugated at 4 °C for 20 min at 16,800 × g. The supernatant was transferred to a fresh siliconized tube with 5 µl of packed Glutathione MagBeads, supplemented with BSA  and salmon sperm DNA (0.2 mg/ml), and incubated at 4 °C for 2 h. The solution was separated from the magnetic beads; 10 µl of the clarified solution was set aside as 5% input, and 200 µl was incubated at 4 °C for 3 h in another siliconized tube with the pre-formed beads . The magnetic beads were separated from the solution, washed 5 times with the wash buffer , and incubated at 65 °C for 6 h in the reverse cross-linking buffer . DNA was purified using the QIAquick Gel Extraction kit (Qiagen). Standard SensiMix Sybrgreen-based reactions were used to quantify the DNA. For re-ChIP assays, the GST-AF1521tandem/glutathione MagBeads with associated DNA were eluted with 100 µl of wash buffer supplemented with 1 mg/ml BSA, 0.2 mg/ml salmon sperm DNA and 0.5 mM ADPr, and the eluted material was further immunoprecipitated with 2 µl of M2-magnetic beads, followed with five times washing with wash buffer. The M2-magnetic beads pre-incubated with 5 μg of Flag peptide were used as a negative control.Cells were washed once with PBS, fixed at room temperature with 3.7% formaldehyde and 1 µM Olaparib in PBS for 5 min, washed twice with cold (4 °C) TBS (20 mM Tris-HCl pH 7.5 and 100 mM NaCl), permeabilized with cold methanol for 10 min, washed twice with cold TBST (TBS and 0.15% Tween-20), and then harvested in cold TBST and 1 µM Olaparib by centrifugation (2200 × 5AIL (Parp9 MD2) with 2BFQ (Af1521 structure) and 3VFQ (Parp14 MD2). In addition, Parp9 MD1 primary sequence was threaded onto 3VFQ (Parp14 MD2 structure) using SwissModel. Further refinement was achieved using PyMol. ADP-ribosylated peptide sequences  from AR and Parp7 were analyzed using Seq2Logo34. PRAD (prostate adenocarcinoma) data for box and whisker plots was downloaded from the publicly available TCGA Data Portal (http://cancergenome.nih.gov/tcga). RSEM (RNA-Seq by Expectation-Maximization) values were used as relative expression. Significance was calculated using a paired t test. Similarly, using another publicly available dataset from Memorial Sloan Kettering Cancer Center (MSKCC)43, Parp7 mRNA was assessed in prostate cancer metastases and primary tumors. For disease recurrence, MSKCC prostate cancer patient data61 was stratified by Parp7 mRNA expression levels and plotted using cBioPortal. Details for the significance tests can be found within the text and was performed using GraphPad Prism6.In silico protein modeling was performed in PyMol by aligning n = 3 biological independent experiments). All other experiments were performed as replicates  with similar results.The MS/MS discovery of ADP-ribosylation sites was performed once, and validated by mutagenesis and chemical sensitivity analysis. RNA-seq and reporter gene assays were both performed as replicates (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Participants with allergic rhinitis had significantly greater sleep disturbance, fatigue, and depressive symptoms than those without allergic rhinitis, and allergic rhinitis was a significant factor influencing sleep disturbance and fatigue among the participants after controlling for confounding variables. Therefore, it is imperative to develop effective interventions to manage allergic rhinitis symptoms and improve sleep and fatigue in affected nurses.Allergic rhinitis (AR) is a common chronic disease that negatively affects physical and mental health. The purpose of this study was to determine the effects of allergic rhinitis on sleep, fatigue, and depressive symptoms among Korean female nurses. This was a cross-sectional study conducted using data from the Korea Nurses’ Health Study (KNHS), and a total of 8645 female nurses was selected for the final analysis. The demographic characteristics, Body Mass Index, alcohol consumption, shift work, comorbidities (atopic dermatitis and asthma), self-rated health, sleep disturbance (Jenkins Sleep Questionnaire), fatigue , and depressive symptoms  were collected. The data were analyzed using chi-square tests,  Allergic rhinitis (AR) is a common chronic disease affecting 10–40% of the population worldwide , while tThe main symptoms of AR are nasal itching, sneezing, rhinorrhea, and nasal congestion , with naAR can affect mental health, as well as physical health. In adults with AR, the more persistent and severe the symptoms, the higher the degree of depression ,18,19. GFew studies have investigated the effects of AR on physical and mental health specifically among nurses, who are frequently exposed to workplace allergens. Sleep, fatigue, and depression, which have been reported to be related to AR, can negatively affect nurses’ job performances. Therefore, this study was conducted to determine the effect of AR on sleep disturbance, fatigue, and depressive symptoms among a nationwide cohort of Korean nurses.This study is a cross-sectional study conducted using data from the Korea Nurses’ Health Study (KNHS), an ongoing web-based cohort study started to identify factors affecting women’s health. KNHS, which started in 2013, uses a similar protocol as the NHS3 in the United States but uses a questionnaire customized for Korea . ParticiSleep disturbance was measured using Jenkins Sleep Questionnaire (JSQ) . The tooFatigue was measured using the Chalder Fatigue Scale (CFS) . This toDepressive symptoms were measured using the Patient Health Questionnaire (PHQ-9) . The 9-iBaseline characteristics, comorbidities, and self-rated health were included as covariates based on a literature review to determine the effect of AR on sleep disturbance, fatigue, and depressive symptoms. The baseline characteristics included age, education level, marital status, body mass index (BMI), alcohol consumption, and shift work. The comorbidities included atopic dermatitis and asthma. Sleep disturbance, fatigue, and depressive symptoms have been reported to affect each other ,27,28, sThis study was conducted after approval by the institutional review board for the ethical protection of participants (IRB No. 201904-0012-03). Anonymity and confidentiality were assured, and informed consent was obtained from the participants as part of the online survey.t-test were used to analyze and confirm the differences in the baseline characteristics and comorbidities between the AR group and the non-AR group. The t-tests were used to check the differences in the degree of sleep disturbance, fatigue, and depressive symptoms between the AR group and the no AR group.A data analysis was performed using SPSS version 26.0 . The chi-square test and p < 0.05.Finally, hierarchical multiple regression analyses were performed to examine the effect of AR on the sleep disturbance, fatigue, and depressive symptoms. Hierarchical multiple regression was utilized using four stage models. The stage 1 model included the AR status. The stage 2 model included age, level of education, marital status, BMI, alcohol consumption, and shift work, while the stage 3 model included atopic dermatitis and asthma as the confounding variables. In stage 4, each model included sleep disturbance, fatigue, or depressive symptoms as the confounders, as well as self-rated health. The threshold of statistical significance for this study was n = 1808). There were significant differences in the age , atopic dermatitis , asthma , and self-rated health  between the AR and the no AR groups . The mean score of total fatigue in the AR group, 17.44 (SD = 5.74), was also higher than that of the no AR group . Participants with AR had significantly higher depressive symptoms (M ± SD = 5.41 ± 4.33) than those without AR  (< 0.001) .Hierarchical multiple regression analyses were performed to identify the effect of allergic rhinitis on the sleep disturbance, fatigue, and depressive symptoms . The VIFp < 0.001) and fatigue  among the participants after controlling for confounders. However, AR was not found to have an influence on depressive symptoms. The explanatory power of model 4 was 35.4%  for sleep disturbance and 46.3%  for fatigue.From the multiple regression analyses, AR was identified as a significant factor influencing sleep disturbance , alcohol consumption , shift work , atopic dermatitis , self-rated health , fatigue , and depressive symptoms . The confounders affecting fatigue were marital status , BMI , shift work , self-rated health , sleep disturbance , and depressive symptoms . Lastly, age , marital status , BMI , self-rated health , sleep disturbance , and fatigue  increased the risk of depressive symptoms  [Few studies have been conducted on the prevalence of AR among nurses. A study involving nurses at university hospitals in Thailand reported the prevalence of AR among nurses to be 7.9% , and a sIn the present study, when the confounding variables were controlled, AR was significantly associated with sleep disturbances. Previous studies on the associations between AR and sleep disturbances have shown relatively consistent results. A study in the United States involving 5563 adults aged 18 years or older demonstrated a significant association between AR and poor sleep parameters, including insomnia . In addiThe results of the present study revealed that, with the confounding variables controlled, AR was significantly associated with fatigue. This result is similar to the results of previous studies regarding the association between AR and fatigue. Specifically, one study on the association between AR and fatigue among patients with obstructive sleep apnea (OSA)  showed tThe present study found no significant association between AR and depressive symptoms. This finding contradicts that of a systematic review on the association between allergic disorders and the risk of depression that showed a higher risk of depression among patients with AR  and thatFinally, among the general characteristics of the participants, shift work was found to be significantly associated with sleep disturbances and fatigue. Previous studies have consistently reported that shift work negatively affects the extent of sleep disturbances and fatigue experienced among nurses ,33,35. TThe present study found that AR negatively affects sleep and fatigue among Korean female nurses. Nurses can be exposed to various allergens, such as drugs, antiseptics, and latex gloves, at work, and such occupational exposures may aggravate their AR symptoms. Therefore, organizational support should be provided so that nurses are not unnecessarily exposed to allergens during work. Efforts made by nurse managers, such as to identify the detailed occupational history of nurses with AR and agents that nurses may be exposed to during work and to develop and implement safety guidelines for reducing harmful exposure ,10, are This study is noteworthy in that it is the first to examine the associations between AR and sleep disturbances, fatigue, and depression among female nurses. In addition, the results will aid in the development and implementation of health promotion activities among Korean nurses, because the study population included nurses working at hospitals nationwide. It may also be possible to extend these health promotion activities to females in the general population, because AR was found to be associated with sleep disturbances and fatigue even after controlling for the nature of nursing work. Despite its strengths, the present study had two limitations that should be addressed. First, the duration of the disease could not be determined due to limitations in the data used. Second, the present study did not identify the severity of the AR symptoms. Considering these limitations, future studies that investigate the effects of AR on sleep disturbances, fatigue, and depression are warranted.The present study analyzed the associations between AR and sleep disturbances, fatigue, and depressive symptoms among Korean nurses. The results indicated that, after adjustment for the general and work-related characteristics of the participants, AR was significantly associated with sleep disturbances and fatigue but not with depressive symptoms. Nurses have high levels of sleep disturbance and fatigue due to the nature of their work compared to those in other occupations, and these issues are likely to be compounded in nurses with AR. Therefore, nurse managers should pay special attention to these situations and develop measures that prevent nurses diagnosed with AR from being unnecessarily exposed to allergens at work.


A significant improvement in Token test (p = 0.021), semantic fluency (p = 0.036), Rey's Figure-Copy (p < 0.001), and Raven's Colored Progressive Matrices (p = 0.004) was observed. The pain intensity perception (p < 0.001) and the risk of developing pressure ulcers (p < 0.001) as assessed, respectively, by the Numeric Rating Scale and by the Norton Scale were improved. With regard to the second aim, in M-Ms group, we found a positive correlation between the number of neuromotor sessions and the change in functional disability and language comprehension; in the Ss group, on the other hand, despite a higher number of hospitalization days, the total number of completed sessions was positively associated with the change in visuoconstructional abilities. Our findings suggest that an intensive, inpatient, and multidisciplinary rehabilitation program may improve functional abilities, some strategic cognitive functions, and geriatric aspects in PwPD with mild–moderate motor impairment.Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor  and nonmotor symptoms . In recent years, several studies demonstrated that neurorehabilitation therapy is an effective treatment in addition to pharmacological personalized interventions in persons with PD (PwPD). The main aim of this study was to explore the short-term changes in functional, cognitive, and geriatric domains after a multidimensional rehabilitation program in PwPD (as primary condition) in mild–moderate (M-Ms) to severe (Ss) stages. Our second aim was to compare the effects of multidimensional rehabilitation in M-Ms versus Ss of PD. Twenty-four PwPD in M-Ms to Ss  were included in a retrospective, observational study. Motor, cognitive, functional, and neuropsychiatric aspects were collected in admission (T0) and in discharge (T1). PwPD were involved in a person-tailored , inpatient, intensive (5–7 days per week), multidisciplinary , comprehensive, and rehabilitative program. According to Movement Disorders Society Unified Parkinson's Disease Rating Scale III cutoff, PwPD were classified in M-Ms or Ss ; 87.50% of our sample reported significant reduction of functional disability at Barthel Index ( Parkinson's disease (PD) is considered a multiple system neurodegenerative disorder with early prominent death of dopaminergic neurons in the substantia nigra pars compacta.de novo drug-naive patients, and they are not represented differently in the three main motor subtypes  , the neurodegenerative process produces a wide range of non-motor symptoms , some of which precede the motor dysfunction by more than a decade . Psychiad mixed) .To date, several drugs and surgical approaches are available for treating motor symptoms such as levodopa, dopamine agonists, monoamine oxidase B (MAO-B)/catechol-O-methyltransferase inhibitors, apomorphine infusion, levodopa–carbidopa intestinal gel infusion, and deep brain stimulation (DBS) .However, the aforementioned strategies fail to prevent or delay the progressive neurodegenerative process, and patients continue to develop a progressive deterioration of their functional status with a loss of independence and a decline in quality of life (QoL). Increased disability is largely related to the increase of “axial” disturbances such as posture, balance, and gait, along with increased risk of falls. Moreover, even with optimal medical or surgical management, non-motor symptoms are suboptimally controlled. Therefore, the appearance over time of motor and non-motor complications induced by dopaminergic drugs  results in detrimental impact on patients' clinical conditions and their QoL, emphasizing the need for non-pharmacological therapies .In particular, cognitive impairment is related to QoL deterioration and functional disability in PwPD and imposes considerable burden on the caregiver .To date, neurosurgical procedures and optimal pharmacological management have been shown ineffective in improving cognitive functions, even though MAO-B inhibitors may improve cognitive symptoms during wearing-off .DBS-related cognitive outcomes are heterogeneous . A recenIn recent years, several interventional studies have reported that the neurorehabilitation therapy is an effective treatment in addition to optimal medical therapy in PwPD , 10–17. Interestingly, action observation therapy has been demonstrated effective in improving cognitive abilities of PwPD if it is used within a dual task framework .To date, there are still few studies in which the rehabilitation program is conducted in an inpatient setting with intensive and multidimensional approach. The global impact of the rehabilitation programs may be greater if implemented in an inpatient setting. In the inpatient environment, PwPD can receive adequate management for several complex motor and non-motor symptoms. An inpatient multidisciplinary rehabilitation program ensures daily therapies in an environment that allows for regular monitoring of functional status and medication response, as well as an integrated and intense tailored physical rehabilitation .Unfortunately, PwPD as the primary diagnosis are not typically referred for inpatient rehabilitation. Indeed, PwPD are often admitted with the aim of receiving inpatient hospital services following a cardiopulmonary, orthopedic, or general medical procedure or more rarely to improve the management of dopaminergic therapy.With this background, the main aim of this study was to explore the short-term changes in functional, cognitive, and geriatric domains of an intensive, inpatient, multidisciplinary, and person-tailored rehabilitative program in PwPD (as primary condition) in mild–moderate (M-Ms) to severe (Ss) stages. Our second aim was to compare the effects of multidimensional rehabilitation in mild–moderate stage vs. severe stage of PD.This is a retrospective, observational study, in which 24 PwPD accessed the NeuroRehabilitation Unit of the S. Maria Nascente Center, IRCCS Fondazione Don Carlo Gnocchi ONLUS in Milan, were included.The study was performed in accordance with the principles of the Helsinki Declaration and by previous approval from the IRCSS Fondazione Don Carlo Gnocchi ONLUS Ethics Committee.All medical records and data extracted and included in the present study concerned patients with the following characteristics: [1] diagnosis of PD according to the United Kingdom Parkinson's Disease Society Brain Bank ; [2] invEvery professional who conducted rehabilitation sessions  recorded the number of sessions given to each PwPD and its contents. A database containing all the clinical information from the patient's medical records was created; all pharmacological treatment was extrapolated, and the levodopa equivalent daily dose (LEDD) was calculated for each patient .The main data and variables extracted from the medical records were [1] the Barthel Index (BI)  as a measure of PwPD's functional disability in daily life ; [2] theThe Movement Disorders Society Unified Parkinson's Disease Rating Scale Part III-Motor Examination (MDS-UPDRS) Part III  was fillAll PwPD were involved in an intensive, tailored to individual's needs, specialized, inpatient, goal-based multidisciplinary rehabilitation program.The primary aim of the rehabilitation program was to increase and promote functional, motor, and cognitive abilities, as well as to optimize the subjects' medication regimens.In order to achieve this aim, all PwPD were involved in a combination of physical, cognitive, occupational, and speech therapy for a minimum of two daily rehabilitative sessions for 5–7 days per week .All admitted patients were enrolled in an intensive rehabilitation program consisting of daily sessions from Monday to Sunday for a total of at least 500 min a week.The program was based on a multidisciplinary evaluation assessing the patients' needs and the possible goals, performed by a neurologist, together with a physical medicine and rehabilitation specialist. Interventions may include physical therapy , occupational therapy , cognitive rehabilitation (paper-and-pencil and computerized activities), and speech and swallowing rehabilitation . The duration of the admission was established following an intermediate reassessment of the program and goals performed by the multidisciplinary rehabilitation team  after 2–3 weeks of admission.https://www.jamovi.org). Parametric and non-parametric statistics were adopted as appropriate. The PwPD sample was characterized in terms of age, sex, and clinical variables at the baseline.Statistical analyses were performed using SPSS 24.0 and jamovi (version 1.2) software , changes or variation scores were calculated from T1 to T0, obtaining Δ values. Paired-samples https://gamlj.github.io/) implemented in the software jamovi (version 1.2.25.0) software (https://www.jamovi.org). Generalized linear mixed models (GLMMs) were performed to evaluate score differences across the two time points (baseline and posttreatment).In order to account for relevant aspects related to delivery of treatment, PwPD's performance on outcome measures was analyzed using the jamovi module  (GAMLj: Different test scores were used as dependent variables (one for each model), and time was considered as a fixed effect. To account for subject-specific variability, each subject was used as a random factor in all the models. The number of rehabilitative sessions, hospitalization days, and MMSE at the baseline were used as covariates as potential confounding variables. GLMMs final models were as follows: outcome measure ~1 + time + hospitalization days + number of rehabilitative sessions + MMSE at the baseline +(1 | subject), and only for MMSE, global cognitive outcome measure ~1 + time + hospitalization days + number of rehabilitative sessions (1 | subject).2 test was calculated to compare proportions between patients that remained stable after the intervention with them who reported an increase in the functional assessment evaluated by the BI.A χThen, Spearman rank correlation coefficients (rS) were computed between changes or variation scores in all clinical outcomes and the seven UPDRS domains scores at the baseline.Moreover, PwPD were classified in mild–moderate stage (M-Ms) or in severe stage (Ss) according to MDS-UPDRS Part III cutoff points  . The numSpearman rank correlation coefficients (rS) between the change scores (Δ) in clinical outcomes and the number of sessions were calculated in both the M-Ms and Ss groups. Spearman rank correlation coefficients (rS) between the change scores (Δ) in LEDD, neuropsychological measures, and BI were also computed. We interpreted the magnitude of correlation (effect size) as follows: 0.1–0.3 as a weak effect, 0.4–0.6 as a moderate effect, and 0.7 and higher as a strong effect .p-values lower than 0.05 were considered statistically significant.All tests were 2-tailed, and Sociodemographic and clinical characteristics as well as the measures of comorbidity and somatic health [Cumulative Illness Rating Scale (Severity Index and Comorbidity Index)]  of our sThroughout their length of stay , all PwPD were involved in a combination of physical therapy (mean number of sessions ± SD) (32.26 ± 11.76), cognitive therapy (16.45 ± 11.14); occupational therapy (13.96 ± 6.79), and speech therapy (10.13 ± 8.83) for a minimum of two sessions per day, 5–7 days per week.Results of GLMMs performed to evaluate score differences across the two time points (T0 and T1) are summarized in F = 48.98; p < 0.001]. Overall, comparison between proportions showed that 87.50% of patients reported higher scores vs. 12.50% of patients who remained stable in terms of the BI total score [p < 0.001]. MMSE at baseline had a significant impact on changes after treatment in BI total score .Significant differences after the multimodal intervention emerged in the BI total score  in visuoconstructional abilities  and in abstract reasoning . MMSE at baseline had a significant impact on changes after treatment in Token test ; phonemic fluency ; semantic fluency ; Copy Rey's Figure ; and Raven's Colored Progressive Matrices .With respect to the neuropsychological evaluation, a significant improvement was observed in language  and for the Norton Scale . There was no statistically significant association between change scores in geriatric aspects and the number of rehabilitative sessions. MMSE at baseline had a significant impact on changes after treatment in the Norton Scale .After the multidisciplinary rehabilitation program, significant differences were also reported for the NRS .Splitting our sample according to MDS-UPDRS Part III cutoff points, 10 patients were classified in the M-Ms group and 14 patients in the Ss group. In relation to the duration and intensity of the multimodal intervention, the total number of rehabilitative sessions  and the total number of hospitalization days was higher in the Ss group than in the M-Ms group  and in language comprehension . In the Ss group, however, the total number of completed sessions was positively associated with the change in visuoconstructional abilities . The change in visuoconstructional abilities was also positively correlated with the number of neuromotor sessions  and with the number of occupational therapy sessions . In this last group, we observed a trend for the correlation between the number of neuromotor sessions and abstract reasoning .In the M-Ms group, the number of neuromotor sessions was positively correlated with the change in functional disability [ΔBI total score (M-Ms group): rS = 0.66 (moderate effect); The main finding of our study is that an intensive, inpatient, multidisciplinary rehabilitation program leads to significant short-term changes in PwPD with mild–moderate motor impairment at baseline, in particular with respect to functional status and in some strategic cognitive domains. In PwPD with severe motor impairment at baseline, the more intensive rehabilitation program was associated only with significant short-term changes in visuoconstructive abilities.Most importantly, the improvement in several activities of daily living such as dressing, bladder control, chair transfer, ambulation, and stair climbing was inversely related with the severity of motor dysfunctions at baseline evaluation, in particular with upper extremity bradykinesia.Several factors determine the functional impairment in PwPD. In particular, rigidity and bradykinesia tend to most greatly affect a patient's ability to work and perform activities of daily living.In particular, as regards the upper limb, the bradykinesia may cause issues in dexterous activities such as using kitchen or work tools. It may also contribute to reducing the coordination in activities such as dressing, toileting, and transferring.Effectively, some activities of daily living evaluated by the BI such as dressing and chair transfer require an appropriate movement speed of the upper limbs and manual dexterity, and this could explain the best rehabilitation treatment response in PwPD with lower bradykinesia severity at baseline.Interestingly, during the rehabilitative hospitalization, the improvement in functional disability has made possible the reduction of the daily dose of dopaminergic medications, albeit not statistically significant. Indeed, it has been demonstrated that intensive rehabilitation, improving motor performances, and autonomy in activities of daily life, reduces the need to increase dopaminergic therapy , 39.In the present study, PwPD showed significant improvements in language comprehension, visuoconstructional abilities, and abstract reasoning following cognitive therapy sessions.Interestingly, we observed a significant effect of baseline global cognitive functions on the reported changes on outcome measures, implying that the treatment, regardless of the intensity, is effective only according to cognitive functioning at baseline.Indeed, patients with lower global cognitive impairment are likely to maintain information about goals to be achieved over time, such as rehabilitation goals .Our findings confirmed the beneficial effects of an intensive cognitive rehabilitation program in combination with multiple integrated interventions . Consistent with previous studies, we suppose that the benefits of cognitive treatments have been enhanced by combining cognitive remediation with motor function training and occupational interventions .To date, few studies have investigated the neurobiological and neurofunctional modifications in PwPD after an integrative cognitive rehabilitation program –44. ThesIn light of our results, it is arguable that the improvement in PwPD's cognitive performances  may result in a significant reduction of disability as indicated by clear BI modification.Additionally, improvement in functional performance driven by high-intensity rehabilitation program was associated with significant improvements in important geriatric aspects such as pain intensity perception, and the risk of developing pressure ulcers as assessed, respectively, by the NRS and by the Norton Scale.Our findings strengthen the evidence about the efficacy of an inpatient multidisciplinary rehabilitation treatment on PwPD , 39, 45.A still debated issue is whether a single or a multidisciplinary and integrated approach  should be proposed for PwPD. Indeed, an excessively multifocused set might be affected by a low intensity of training.In a recent study, a remarkable effect (persisting at 1-year follow-up) on motor symptom severity and QoL was demonstrated combining high intensity in an inpatient setting with a multidisciplinary approach. The multifaceted needs of PwPD vary considerably across disease stages so that different management approaches and interventions tailored to the individual conditions of patients are mandatory .Despite compelling scientific evidence, an inpatient rehabilitation admission is typically not considered for patients with a progressive disease such as PD. An inpatient rehabilitation program can ensure many repetitions of the same task with consequent reinforcement of the beneficial effects. Moreover, it has been amply demonstrated that PwPD may benefit more from blocked practice than from random practice of exercise and physical therapy . Indeed,One of the major future challenges will be the ability to identify PwPD most likely to benefit from inpatient rehabilitation. In particular, it is important to identify at which level of motor impairment the multidisciplinary inpatient rehabilitation program may be of benefit. An important finding of our study is the evidence of beneficial effects of an intensive rehabilitation program in mild–moderate severity of motor symptoms. Indeed, despite the fewer number of rehabilitative sessions and hospitalization days, PwPD in M-Ms group showed the best improvements in degree of disability according to BI. Despite this, the positive association between the number of neuromotor, occupational, and speech therapy sessions and the improvements in the executive-constructional task performances observed in Ss of PwPD suggests that rehabilitation provides benefit in advanced motor impairment as well. However, in our study, the total numbers of rehabilitative sessions and hospitalization days were higher in the Ss group than in the M-Ms group. Indeed, subjects with a more advanced disease needed longer treatments without having the same benefit.In light of our results, we speculate that an intensive inpatient multidisciplinary rehabilitation should be considered in early/moderate PD stages in order to optimize functions and slow progression of disability and to avoid longer hospitalization/rehospitalization and consequently higher financial costs.Given the paucity of studies about the effectiveness of a cognitive, motor, and functional rehabilitation program, we claim that the present article is of great interest. Compared to many of the previous studies, our work examined the effect of an inpatient multidisciplinary rehabilitation treatment on PwPD with different disease severity (from mild to severe). Moreover, we investigated the effectiveness of an inpatient rehabilitation program on PwPD as the primary diagnosis instead of PwPD hospitalized as a result of cardiopulmonary, orthopedic procedure or to manage the dopaminergic therapy.The main strength of the present investigation is the fair number of treated patients, although several methodological issues severely limit generalization of findings beyond the current sample.The lack of a control group limited our ability to attribute the improvements observed specifically to rehabilitation or to the combination of pharmacological and rehabilitation interventions. In addition, improvements attributable to other aspects of an inpatient hospital admission cannot be ruled out. Moreover, this study did not include a short- and long-term follow-up after the discharge. As such, we were unable to verify the maintenance of cognitive and functional improvements over time. Additionally, we did not verify the effectiveness of intensive rehabilitation program on motor functions. Indeed, the MDS-UPDRS Part III was assessed only at baseline.Although we are aware that this is a retrospective study in which we collected data and information from medical records, it is necessary to highlight some further limitations about the assessment phase on admission and discharge: in fact, no important data have been collected in the cognitive field  and in the neuropsychiatric field .The interest to promote inpatient rehabilitation programs in PwPD as a means for eliciting improvement in functional status and cognitive performances has underscored the importance of identifying the tailored multidisciplinary approach in accordance to the disease stage and individual conditions of patients.Our results suggest a potential role of inpatient multidisciplinary rehabilitation in driving activity-dependent functional improvement in mild–moderate PwPD's motor skills.Our data showed that treatment intensity is not a mediating factor in determining posttreatment changes and that only PwPD with mild–moderate motor impairment can improve after a multidimensional treatment. However, it is important to clarify that the potential effect of multidimensionality was not explored.Moreover, the longitudinal maintenance of changes should be examined to consider the periodicity with which PwPD should attend multiple integrated interventions.Future tailored protocols aiming to integrate multimodal rehabilitation programs should also identify optimal parameters of frequency, intensity, duration, and task-specific exercises to improve effectiveness and tolerability.More research is needed to evaluate the long-term effects on the functional outcome with longer periods of time and the possible benefits of booster sessions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRCSS Fondazione Don Carlo Gnocchi ONLUS Ethics Committee. The patients/participants provided their written informed consent to participate in this study.MM, FLS, RN, and FBa: study concept, methodology, and design. FLS, MDC, and FBo: data selection, collection, and data-entry. SDT: methodology and statistical analyses. MM, FLS, and SDT: data interpretation and drafting the first version of the manuscript. RN and FBa: supervision and project administration. MM, FLS, SDT, MDC, FBo, RN, and FBa: critical review and revision of the manuscript. All the authors made changes and approved the final paper.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.