Prostate cancer is the mostly commonly diagnosed non-cutaneous malignancy and the second leading cause of cancer-related death affecting men in the United States. Moreover, it disproportionately affects the men of African origin, who exhibit significantly greater incidence and mortality as compared to the men of European origin. Since androgens play an important role in the growth of normal prostate and prostate tumors, targeting of androgen signaling has remained a mainstay for the treatment of aggressive prostate cancer. Over the years, multiple approaches have been evaluated to effectively target the androgen signaling pathway that include direct targeting of the androgens, androgen receptor (AR), AR co-regulators or other alternate mechanisms that impact the outcome of androgen signaling. Several of these approaches are currently in clinical practice, while some are still pending further development and clinical evaluation. This remarkable progress has resulted from extensive laboratory, pre-clinical and clinical efforts, and mechanistic learnings from the therapeutic success and failures. In this review, we describe the importance of androgen signaling in prostate cancer biology and advances made over the years to effectively target this signaling pathway. We also discuss emerging data on the resistance pathways associated with the failure of various androgen signaling- targeted therapies and potential of this knowledge for translation into future therapies for prostate cancer. Prostate cancer (PCa) is a deadly malignancy that currently stands as the second leading cause of cancer-related death in American men. American Cancer Society estimates that about 174,650 men will be diagnosed with prostate cancer in 2019 and about 31,620 will die because of it [Androgens play an important role in the growth of prostate and prostate tumors . TherefoThe prostate is a walnut-sized gland in men that produces seminal fluid to nurture and transport the sperms. It is comprised of four zones; anterior fibromuscular stroma, a central zone, a peripheral zone, and the periurethral transition zone ,10. MostMalignant progression of prostate tumors is a multistep process . During Androgen receptor (AR) is a nuclear transcription factor and a steroid hormone receptor. The gene encoding for the AR is located on the chromosome Xq11.2-q12, consists of 8 exons  and spanIt is well established that AR signaling plays a central role in the initiation and progression of prostate cancer. Indeed, the disease progression is closely associated with a relative increase in serum prostate specific antigen (PSA), which is encoded by an androgen-regulated gene, KLK3. Besides, targeting of androgen signaling, although not effective in very long term, provides impressive therapeutic benefit. Even in the resistant prostate tumors, AR signaling is considered to play an active role as evidenced by PSA relapse and other clinical data. Below we describe some common AR aberrations implicating this signaling node in prostate cancer pathogenesis.The point mutation of AR in the ligand-binding domain is reported in prostate cancer and more frequently in patients with castration-resistant disease. This mutation leads to the loss of AR specificity to natural agonists. Mutations at codon 701 (L701H) in exon D and at codon 877 (T877A) in exon H are frequent in metastatic tissues  and assoOverexpression of AR is one of the most common alterations (30–50% patients) reported in castration-resistant prostate cancer (CRPC) . IncreasAR co-regulators interact with AR to modulate gene expression. So far, more than 170 co-regulators have been identified that are associated with CRPC . SRC-2 gAn important mechanism implicating abnormal AR signaling is intra-tumoral synthesis of androgens, which is mostly reported in CRPC ,80. CYP1Since AR signaling is a major driver of prostate cancer growth, its targeting has been exploited for therapeutic benefit since past several decades. In most cases, direct targeting of either the ligand or the receptor has been exploited. In some cases; however, alternative approaches have been evaluated to indirectly suppress AR signaling via targeting of its co-factors. In sections below, we discuss various strategies employed to target this important signaling node along with their success and limitations. see in .Physiologically, hypothalamus holds a major control on the biosynthesis and secretion of androgens by testes. Luteinizing hormone-releasing hormone (LHRH) is released from the hypothalamus, which stimulates the pituitary gland to release follicle stimulating hormone (FSH) and luteinizing hormone (LH). LH then acts on the receptors present on the Leydig cells in the testes to enzymatically regulate the conversion of cholesterol to two key intermediates, dehydroepiandrosterone and androstenedione. With the action of enzyme 17-beta-hydroxysteroid dehydrogenase, androstenedione is then further processed and converted to testosterone. FSH acts on the sertoli cells present within the testes and induces the expression of LH receptors. Testosterone can further be processed enzymatically to a highly potent form i.e., dihydrotestosterone (DHT). Therefore, targeting of LHRH via agonists or antagonists has been exploited to achieve lowering of serum androgen levels .LHRH agonists  were introduced in the United States in 1984 for hormonal therapy of prostate cancer . LHRH agLHRH antagonists control testosterone levels better and faster than agonists . They diAfter it was established that intra-tumoral androgen synthesis and adrenal androgens can be a cause of failure of castration therapy ,146 effoIn recent years, other drugs targeting androgen biosynthesis have also been developed. Orteronel (TAK-700), a new drug, is shown to be five times more selective than abiraterone in inhibiting the 17,20-lyase activity of monkey and human CYP17A1 and it drastically reduced serum androgen levels in monkeys . A phaseHaving observed bypass mechanisms and some adverse effects of ligand-targeted therapy, efforts were initiated to develop novel therapeutic strategies that target the AR directly. Most of the approaches that have been evaluated involve the use of steroidal or non-steroidal anti-androgens. These compounds inhibit the binding of the ligand to the AR and have met with mixed success and failure . The development of steroidal antiandrogens began in 1962. Among the important steroidal antiandrogens are progestogens, cyproterone acetate (CPA), megestrol acetate (MA), dienogest, and chlormadinone acetate (CA). Of these, CPA was the most commonly used drug in the clinics for the treatment of PCa. CPA is derived from hydroxyprogesterone, which has greater binding affinity for the AR as compared to the first generation non-steroidal antiandrogens that are discussed later ,163. CPANon-steroidal agents have narrower target range and thus exhibit fewer side effects compared to the steroidal anti-androgens. Bicalutamide, Flutamide and Nilutamide are the first-generation nonsteroidal anti-androgens. Bicalutamide binds to the allosteric site on the AR inducing a conformational change in the co-activator binding site and thus interferes with its transcriptional activity. A reduction in AR expression is also observed following bicalutamide treatment, but it is transient. It is also shown that bicalutamide, instead of working as antagonist, produces agonistic action as well in some CRPC . FlutamiAZD3514 demonstrated anti-tumor activity in HID28 mouse model of CRPC . MoreoveAR signaling can also be regulated indirectly via the targeting of co-receptors and other chaperon proteins . SteroidHeat shock proteins (HSPs) also play a significant role in AR signaling axis . HSP 90 Bioactive natural products derived from fruits, vegetables, herbs, marine sponges, and edible plants are considered potent chemo-preventive and/or -therapeutic agents and can be used either alone or as adjuvants. Several natural agents have shown good efficacy against PCa in laboratory and preclinical studies and many of them target AR signaling . SeveralTherapeutic landscape of PCa has improved significantly particularly over the last decade or so. This has resulted in longer life for patients benefitting from the widespread use of new generation drugs and combination therapies. We have gathered new molecular insights from failed therapies and laboratory studies that have helped us made newer advances. Significant efforts are now being focused on directly targeting the AR especially after the reports suggesting a critical role of constitutively active AR variants in castration resistance that allows persistent disease progression. There is also an increasing realization that therapy-resistance is often multifactorial and PCa is a highly heterogeneous disease. A number of studies have suggested the involvement of multiple oncogenic signaling pathways other than AR that can either compensate for the loss of AR signaling or contribute to its aberrant activation, especially in advanced stages. Of note, overexpression of HER2/neu and IGF-1 is shown to activate AR signaling . Similar


Objectives: This study quantified the burden of hypoxic respiratory failure (HRF)/persistent pulmonary hypertension of newborn (PPHN) in preterm and term/near-term infants (T/NTs) by examining health care resource utilization (HRU) and charges in the United States.Methods: Preterms and T/NTs  having HRF/PPHN, with/without meconium aspiration in inpatient setting from January 1, 2011-October 31, 2015 were identified from the Vizient database . Comorbidities, treatments, HRU, and charges during index hospitalization were evaluated among preterms and T/NTs with HRF/PPHN. Logistic regression was performed to evaluate mortality-related factors.Results: This retrospective study included 504 preterms and 414 T/NTs with HRF/PPHN. Preterms were more likely to have respiratory distress syndrome, neonatal jaundice, and anemia of prematurity than T/NTs. Preterms had significantly longer inpatient stays (54.1 vs 29.0 days), time in a neonatal intensive care unit (34.1 vs 17.5 days), time on ventilation (4.7 vs 2.2 days), and higher total hospitalization charges  . Similar rates were observed for use of antibiotics (96.2% vs 95.4%), sildenafil (9.5% vs 8.2%), or inhaled nitric oxide (93.8% vs 94.2%). Preterms had a significantly higher likelihood of mortality than T/NTs .Conclusions: The findings of more severe comorbidities, higher HRU, hospitalization charges, and mortality in preterms than in T/NTs underscore the significant clinical and economic burden of HRF/PPHN among infants. The results show significant unmet medical need; further research is warranted to determine new treatments and real-world evidence for improved patient outcomes. Neonatal hypoxic respiratory failure (HRF) is a severe respiratory illness that affects 2% of all live births and is responsible for >33% of all neonatal mortality.The etiology of neonatal HRF includes aspiration of meconium, respiratory distress syndrome, pneumonia, congenital diaphragmatic hernia, and oligohydramnios.The standard treatment for neonatal HRF includes conventional mechanical ventilation, respiratory alkalosis, ionotropic support, systemic infusion of vasodilators, neuromuscular blockade, and sedation.Neonates with HRF may require multiple and concurrent therapies, which maybe suggestive of extensive use of health care resources with potential implications on economic burden in these patients. To date, there is a lack of real-world evidence quantifying the burden of hospitalization in managing these patients. The aim of this retrospective observational study is to understand and quantify the clinical and economic burden of hospitalization in infants with HRF/PPHN. In an attempt to evaluate this burden, we conducted a retrospective study using a large hospital database to describe the clinical characteristics, health care resource utilization (HRU), costs and charges among preterm and term and near-term (T/NT) infants with HRF/PPHN in the United States.This was a retrospective cohort study using the Vizient (formerly MedAssets) Health System Database from January 1, 2011 through December 31, 2015. Vizient is an administrative patient-level database which includes inpatient and hospital-based outpatient information from more than 400 hospitals across 42 US states .The data also include information about various clinical grouping methodologies  as well as Charlson and Elixhauser comorbidity index methodologies.The eligible study population included preterm  or T/NT infants  who had a diagnosis of HRF/PPHN  in the inpatient setting during the identification period .The first hospitalization (from admission date to discharge date) during this period which included an HRF/PPHN diagnosis was defined as the index hospitalization.Preterm and T/NT infants diagnosed with HRF/PPHN during an inpatient visit within the identification period were further stratified as preterm infants with HRF/PPHN and T/NT infants with HRF/PPHN.®, beractant, Survanta®, etc.), as the pharmacy file with National Drug Codes (NDCs) was not available.Patient characteristics including sex, most common comorbid conditions, and provider characteristics  were examined for the index hospitalization period. To depict clinical care for hospitalized infants with HRF/PPHN, clinical procedure and treatments  were also examined. Treatments were identified based on the patient-level charge description using key words , NICU use, time in the NICU, ventilation use  code: 94660; ICD-9/10 procedure codes: 93.90, 5A09357, 5A09457, 5A09557]); invasive mechanical ventilation , time on ventilation, and ECMO . Additionally, LOS was reported according to teaching hospital status and patient gestational age. In-hospital mortality rates during index hospitalization were also evaluated and reported.All study variables including demographics, provider characteristics, and outcomes were analyzed descriptively among the overall infant population and among preterm and T/NT infants in the study sample. Means and standard deviations were provided for continuous variables. Numbers and percentages were provided for categorical variables. Statistical tests of significance  were conducted to assess the differences between the cohorts. Additionally, for the economic outcomes, the p-values were obtained by using the student t-test in the log-transformed costs. A logistic regression model was performed to assess factors associated with mortality. Patient characteristics such as sex, comorbidities, and provider characteristics—including US region and teaching hospital status, and HRU —were included as independent variables. All analyses were conducted using SAS statistical software (Version 9.3).The study included a total of 918 infants with HRF/PPHN during index hospitalization, including 504 preterm (55%) and 414 T/NT (45%). Additionally, the prevalence of HRF/PPHN was 50.1% and 66.9% among preterm and T/NT infants in the NICU, respectively (data not shown). Overall, most infants were male (59.0%) and had respiratory distress syndrome (53.7%), followed by patent ductus arteriosus (50.1%) and neonatal jaundice due to preterm delivery (41.7%) (Table 1).Hospital providers were geographically distributed across the United States, with more than half of the infants treated at hospitals in the Southern US region (66.0%) followed by 13.4% treated at hospitals in the Western region, 10.4% treated at hospitals in the Northeastern region, and 10.2% treated at hospitals in the Midwestern US region (Table 1). Additionally, a majority of the infants were treated at hospitals located in urban locations (95.1%) with only a small proportion treated in rural hospitals (0.3%). Also, about half of the infants were treated at a major teaching hospital (50.2%), 21.8% were treated at minor-teaching hospital, and 23.4% were treated at non-teaching hospital. Most infants were treated at large hospitals, with 52.4% infants treated at hospitals with ≥500 beds and 26.0% infants treated at hospitals with 300-499 beds. Among preterm infants with HRF/PPHN, a majority were born at a gestational age of 27-28 weeks (29.6%) followed by 33-34 weeks (27.6%), 29-30 weeks (21.6%) and 31-32 weeks (21.6%). Most T/NT infants with HRF/PPHN were born at the gestational age of 35-36 weeks (69.3%) followed by ≥37 weeks (30.7%) (data not shown).There were no significant differences associated with patient sex between preterm and T/NT infants with HRF/PPHN  (Table 1). Compared with T/NT infants, preterm infants with HRF/ PPHN were significantly more likely to have respiratory distress syndrome , neonatal jaundice due to preterm delivery , and anemia of prematurity , but less likely to have respiratory failure of newborn . Additional demographic and clinical information for preterm and T/NT infants with HRF/PPHN can be found in Table 1.Most infants with HRF/PPHN were treated with antibiotics (95.9%), inhaled nitric oxide (94%) and surfactants (59.3%) (Table 2). Nearly 95% of infants with HRF/PPHN were treated with invasive or non-invasive mechanical ventilation, with an average time on ventilation of 3.6 days; 5.0% were treated with ECMO. Similar rates were observed between preterm and T/NT infants in the use of antibiotics (96.2% vs 95.4%), sildenafil (9.5% vs 8.2%), or inhaled nitric oxide (93.8% vs 94.2%). A clinically meaningful and significantly higher proportion of preterm infants were treated with surfactants , and fewer preterm infants were treated with ECMO  as compared to T/NT infants. Additionally, preterm infants had significantly longer time on ventilation  and time on inhaled nitric oxide  as compared to T/NT infants with HRF/PPHN. Overall, the inpatient mortality rate among infants with HRF/PPHN was 26.1%, with a significantly higher mortality rate  in preterm infants as compared to T/NT infants with HRF/ PPHN.Among all infants with HRF/PPHN, the average inpatient LOS was 42.8 days (median: 26 days), and 61.4% had NICU use with an average duration of 27 days (median: 15 days) in the NICU . Compared to T/NT infants, preterm infants had significantly longer inpatient LOS  and time in the NICU . The median inpatient LOS was 38 days and 20 days, and the median time in NICU was 21 days and 10 days in the preterm and T/NT infants, respectively (data not shown). Additionally, our results showed that the LOS among infants with HRF/PPHN also varied according to teaching hospital status, with the mean LOS being longest among infants treated at a major teaching hospital (46.0 days), followed by those treated at a minor teaching hospital (37.0 days) and non-teaching hospital (36.1 days) (E-Table 1). Among preterm infants with HRF/PPHN, the mean LOS was longest for those treated at a major teaching hospital (57.7 days) followed by those treated at a non-teaching hospital (47.4 days) and a minor teaching hospital (45.7 days). Also, the mean LOS was longest for T/NT infants treated at a major teaching hospital (32.9 days), followed by those treated at a minor teaching hospital (28.2 days) and non-teaching hospital (16.6 days). Additionally, our results showed that LOS was longest among infants born at a gestational age of 27-28 weeks (76.2 days), followed by those born at 29-30 (55.6 days), 31-32 (48.6 days), 33-34 (34.4 days), 35-36 (27.3 days), and ≥37 weeks of gestation (32.8 days) (data not shown).The total hospitalization costs and charges for infants with HRF/PPHN were $134,412  and $527,306 , respectively. Consistent with hospital LOS, preterm infants incurred higher total hospitalization costs  and charges  than T/NT infants . Similarly, the median hospitalization costs were $91,515 and $54,783, and the median hospitalization charges were $364,723 and $211,023 in the preterm and T/NT infants, respectively. However, it was observed that preterm infants had significantly lower total hospitalization costs per day  and lower hospitalization charges per day  when compared to T/NT infants (data not shown). The average cost-to-charge ratio was 0.26 in the overall population and similar between the preterm and T/NT infants . Subgroup analysis among infants who survived revealed that preterm infants incurred significantly higher average total hospitalization costs  and charges  than T/NT infants.Consistent with hospital LOS, total hospitalization costs and charges also varied by teaching hospital status. The highest costs and charges were incurred by infants treated at a minor teaching hospital  followed by those treated at a major teaching hospital  and a non-teaching hospital  respectively (E-Table 1). Among preterm infants with HRF/PPHN, the total costs and charges were highest among those treated at a minor teaching hospital  followed by those treated at a major teaching hospital  and non-teaching hospital  respectively. Additionally, the total costs and charges were the highest among T/ NT infants treated at a minor teaching hospital  followed by those treated at a major teaching hospital  and non-teaching hospital  respectively.Preterm or T/NT infants with evidence of respiratory failure  or treatment with sildenafil  had a significantly increased likelihood of mortality. Those with neonatal jaundice due to preterm delivery , secundum atrial septal defect , longer hospital LOS , and those born in a non-teaching hospital  had a significantly lower likelihood of mortality . Additionally, preterm infants  had a significantly higher likelihood of mortality compared to T/NT infants with HRF/PPHN after adjusting for the patient characteristics.To our knowledge, this is the first real-world study to evaluate the comorbidities, treatments, HRU, and health care costs among preterm and T/NT infants with HRF/PPHN using a large US hospital database. Given the higher mortality rate and the need for prolonged cardiopulmonary support in the surviving neonates,Our results showed that preterm infants diagnosed with HRF/PPHN were more likely to have evidence of respiratory distress syndrome and associated jaundice, which is in agreement with previous studies that observed respiratory and gastrointestinal infections as common preterm-associated comorbidities.et al showed that inhaled nitric oxide has helped improve oxygenation in both preterm and T/NT infants with HRF/ PPHN, thereby reducing the need for ECMO, which is a complicated procedure used for infants who fail to respond to medical treatment.et al (7.6%),Considering that HRF/PPHN can vary from mild hypoxemia with minimal respiratory distress to severe hypoxemia and cardiopulmonary instability, the overall goal of treatment is to improve oxygen levels in the blood with various vasodilators including inhaled nitric oxide, magnesium sulfate, adenosine, and phosphodiesterase inhibitors.et al in which the excess costs attributable to prematurity in hospitals of Hamilton County, Ohio was estimated as $93 million.et al observed that the incremental cost burden was highest in preterm infants born at 27 weeks. However, Hall et al estimated the costs associated with preterm birth in general and did not focus on any specific comorbidity. Also, the lower hospitalization costs per day in the preterm infants further emphasize that the total costs in preterm infants were driven by the longer LOS in the hospital and NICU. Additionally, the results of our study showed that the health care costs and charges were higher among infants born in a teaching hospital, which is consistent with a study conducted by Hsu et al.et al added that the higher costs related to teaching hospitals may have been attributed to the increased number of procedures, which may have impacted daily HRU, thereby substantially adding to the costs.The results of our study showed that, due to the higher prevalence of comorbidities and the need for highly specialized treatments, the economic burden on preterm and T/NT infants with neonatal HRF/ PPHN, including HRU and cost of care, is substantial. More specifically, the economic burden was ~1.5 times higher among preterm infants as compared to T/NT infants with HRF/PPHN. Our results are in agreement with a retrospective study conducted by Hall The findings from our study should be viewed in the context of some study limitations. Our study relied on the patient-level data. While this data is extremely valuable for the efficient and effective examination of health care outcomes, treatment patterns, and costs, they are collected for payment purposes, and not research. The presence of a diagnosis code on a medical claim is not a positive presence of disease and may have been incorrectly coded or included as rule-out criteria rather than the actual disease. Certain clinical and disease-specific parameters are not readily available in this hospital data, which may affect study outcomes. Additionally, treatments were identified based on the charge description due to the absence of a separate pharmacy file including NDC codes in the Vizient database. Due to the retrospective nature of the study, there may be residual confounding due to unobserved clinical or other differences such as confounding by indication affecting treatment decision and outcomes. Also, the generalizability of the study findings may be limited to infants with HRF/PPHN identified by selected respiratory diseases. Additionally, the Vizient health system database has a disproportionate distribution of one geographical region (predominantly American Southern region), which could significantly bias the conclusions; thus, the findings’ generalizability to the entire country may be limited. Therefore, the results of our study should be interpreted with caution.The findings of more severe comorbidities, higher HRU, hospitalization charges, and mortality among preterm and T/NTs with HRF/PPHN from the latest US data underscore the significant clinical and economic burden of HRF/PPHN among infants. The results show significant unmet medical need; further research is warranted to determine effective treatment options, which could improve patient outcomes and potentially reduce clinical and economic burden in these infants.This work was supported by Mallinckrodt Pharmaceuticals.S. Pandya and L. Wang are employees of STATinMED Research, which is a paid consultant to Mallinckrodt Pharmaceuticals. O. Baser has nothing to disclose. G Wan, B Lovelace, J Potenziano, and X Huang are employees of Mallinckrodt Pharmaceuticals, the study sponsor. A. Pham was an employee of Mallinckrodt Pharmaceuticals at the time of the study.G Wan, B Lovelace, J Potenziano, and X Huang were involved in the conception and design, S. Pandya, L. Wang and O. Baser were involved in the analysis and interpretation of the data. All authors contributed to the drafting of the paper and offering critical revisions for intellectual content. All authors agree to be accountable for all aspects of the work and approve of the final version for publication.The authors thank Sujana Borra of STATinMED Research for medical writing assistance.


The adenoviral early region 1A (E1A) protein has proapoptotic and angiogenic activity, along with its chemosensitizing effect, making it the focus of increased interest in the context of cancer therapy. It was previously shown that E1A-induced chemosensitization to different drugs, including histone deacetylases inhibitors (HDACi), appears to be mediated by Forkhead box O (FoxO) transcription factors. In this study, we explore the relationship between E1A expression and the modulation of FoxO activity with HDACi sodium butyrate (NaBut). We show here that the basal FoxO level is elevated in E1A-expressing cells. Prolonged NaBut treatment leads to the inhibition of the FoxO expression and activity in E1A-expressing cells. However, in E1A-negative cells, NaBut promotes the transactivation ability of FoxO over time. A more detailed investigation revealed that the NaBut-induced decrease of FoxO activity in E1A-expressing cells is due to the NaBut-dependent decrease in E1A expression. Therefore, NaBut-induced inhibition of FoxO in E1A-positive cells can be overcome under unregulated overexpression of E1A. Remarkably, the CBP/p300-binding domain of E1Aad5 is responsible for stabilization of the FoxO protein. Collectively, these data show that the expression of E1A increases the FoxO stability but makes the FoxO level more sensitive to HDACi treatment. The early region 1A (E1A) of adenovirus encodes two major proteins E1A 12S and E1A 13S, translated from two alternatively spliced transcripts, including the 12S and 13S messenger RNAs (mRNAs) encoding 243 (243R) and 289 (289R) amino-acid oncoproteins [E1A is a promising candidate for cancer therapy, due to its proapoptotic , antiangBy changing the expression of different sets of genes, HDACis suppress cell growth and induce cell differentiation, senescence, or apoptosis more effectively in transformed cells than in normal cells ,16,17,18BIM [The molecular mechanisms determining the E1A-mediated cellular sensitivity to apoptosis are not yet fully known. However, E1A-dependent chemosensitization appears to be connected with Forkhead box O (FoxO) transcription factors . A knockBIM . Apart fBIM . This acBIM .FoxOs belong to the Forkhead family of transcription factors sharing the common DNA-binding domain FKH . These tIn this study, we are dealing with FoxO1—the most studied member of the Forkhead family. The functions of FoxO1 and its role in cancerogenesis and tumor progression are quite sophisticated and context-specific. FoxO1 deletions are lethal due to incomplete vascular development in the embryo . ParticiThis study is focused on the stabilizing effects of E1A in relation to FoxO, showing the rise in FoxO level in the presence of E1A and establishing the link between HDACi-induced E1A and FoxO degradation. Taken together, the paper deals with FoxO regulation under the HDACi treatment in relation to E1A expression, and it examines the stabilizing functions of E1A.HindIII-G region of Ad5 viral DNA coding E1A and E1B 19kDa (E1A + E1B). E1A + Ras and E1A + E1B transformed and NIH3T3 normal fibroblasts were cultivated in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics. Cells were treated with 4 mM sodium butyrate (NaBut) for 24–72 h . NIH3T3 mouse fibroblasts, HEK293 human kidney (embryonic) cells, and HCT116 human colorectal carcinoma cells were obtained from the Russian Collection of Cell Cultures . The adenovirus E1A constructs (12S (wild type (wt)), 12S (RG2) and 12S (YH47)) were obtained from Dr. Elizabeth Moran . The hemagglutinin (HA)-tagged full-length p300 was a gift from Elizabeth Wilson . FHRE-Luc was a gift from Michael Greenberg .We used rodent embryonic fibroblasts transformed either with pE1A vector coding the early region of human adenovirus type 5 (E1Aad5) in complementation with pSV-ras-gpt coding the cHa-ras carrying mutations at positions 12 and 61 (E1A + Ras) or with p21/waf1: 5′–GCCGTGATTGCGATGCGCTCA–3′ and 5′–ACAGCGACAAGGCCACGTGGT–3′; mnsod: 5′–GCACTGAAGTTCAATGGTGG–3′ and 5′–ACTGAAGGTAGTAAGCGTGCTC–3′; bim: 5′–GCCAAGCAACCTTCTGATG–3′ and 5′–CACTGAGATAGTGGTTGAAGGC–3′; gapdh (m): 5′–TGTGATGGGTGTGAACCACG–3′ and 5′–CCAGTGAGCTTCCCGTTCAG–3′; gapdh (h): 5′–TCATCAGCAATGCCTCCTGCACC–3′ and 5′–ACAGTTTCCCGGAGGGGCCA–3′). PCRs proceeded for 22–32 cycles: A denaturation step at 950 C for 30 s, an annealing step  for 30 s, and an elongation step at 72 °C for 1 min. PCR products were resolved by electrophoresis in 2% agarose gel. The alpha-tubulin gene was selected as an internal control in the PCR assay.The total cellular RNA was isolated with Trizol . The RT step was performed with 2 µg of RNA and 1 µg of random hexaprimers. The PCR step was performed in the presence of 100 ng of primers to the complementary DNA (cDNA) of mouse or human genes , and probed with appropriate antibodies. As primary antibodies, we used antibodies to Foxo1 #2880 , E1A (M73) sc-25 (Santa Cruz), pan-Ras #OP40 , and alpha-tubulin T5168 (Sigma). Anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase (Sigma) were used as the secondary antibodies. Visualization of membrane-bound proteins was performed by enhanced chemiluminescence .Every protein of interest was analyzed at least three times on different sets of samples. The band density was evaluated using ImageJ . Then, density values were scaled to load control and converted to relative units. The figure plots represent the mean values of several experiments; error bars indicate the standard error of the mean (SEM).3 cells per well and transfected with the appropriate constructs  using Lipofectamine-2000 (Invitrogen) as recommended by the manufacturer. For stable plasmid integration, E1A + Ras cells were co-transfected with a vector coding the early region of Ad 5 (E1Aad5), and a selective pBABE-puro construct bearing the puromycin resistance gene. For the luciferase assay, cell extracts were prepared, and luciferase activity was determined according to the protocol supplied with the Luciferase Assay kit . Luciferase activity was determined using a TD-20/20 luminometer . Each experimental point was carried out in triplicate, and each experiment was performed no less than three times. Diagrams represent means ± SEM.For transient and stable transfection, cells were plated in 96-well plates (antibiotic-free DMEM with 10% FBS) at a seeding density of 150 × 10Long-term HDACi treatment was shown to induce FoxO1 and FoxO3 degradation in E1A + Ras-transformed mouse cells . In addiImmunoblotting data reveal that NaBut induced FoxO1 accumulation in E1A-negative cells. On the contrary, in E1A-expressing cells, there was a tendency toward declining of the FoxO1 protein level after prolonged NaBut treatment a,b.The expressions of E1A and FoxO1 correlated positively in E1A-expressing cells treated with sodium butyrate. NaBut-induced decrease of FoxO1 protein level was accompanied by a reduced expression of adenoviral protein E1A. The dynamic of FoxO1 level under NaBut treatment is shown more clearly on the plot b, represE1A gene and its endogenous promoter. The control NIH3T3 and E1A-expressing NIH3T3 cells were prolonged treated with NaBut (up to 72 h) and assayed for FoxO1 expression. Immunoblotting data demonstrate that prolonged action of sodium butyrate increased the initially low level of FoxO1 expression in E1A-negative cells NIH3T3. On the contrary, the initially elevated level of FoxO1 decreased in E1A-expressing cells under prolonged sodium butyrate treatment  including the reatment c.To assess the influence of NaBut on the transactivation ability of FoxO in dependence of E1A expression, the ability of FoxO to activate the target gene transcription was analyzed in E1A-expressing and E1A-negative cells under prolonged NaBut treatment using RT-PCR and a luciferase assay. E1A-negative rodent NIH3T3 and E1A-expressing E1A + Ras and E1A + E1B cells were transfected with a vector, coding a luciferase under control of FoxO-responsive promoter (FHRE-Luc). Cells were left untreated or treated with NaBut for 24–72 h and assayed for luciferase activity. Luciferase assay data demonstrate that NaBut enhanced FoxO transactivity in both cell lines, but temporal dynamics in these cells were different a. NaBut The same results were obtained in human cells. The luciferase activity in E1A-negative HCT116 cells kept increasing even after 48 h of NaBut treatment. FoxO activity increased 10-fold and 20-fold after 24 and 48 h, respectively. On the other hand, in E1A-expressing cells HEK293, FoxO-activity, after a temporary increase, decreased under a prolonged action of NaBut b.p21Waf1, encoding the cyclin kinase inhibitor, and MnSod, the product of which is involved in the ROS inactivation, to validate the FoxO transactivation function changes under NaBut treatment. In general, the dynamics of FoxO target gene expression was consistent with the data obtained with a luciferase assay. RT-PCR analysis of the FoxO target gene expression revealed an increased basal level of mRNA in E1A-expressing cells (E1A + Ras), compared with E1A-negative cells (NIH3T3). Expression of the studied genes did not increase in E1A-expressing cells under prolonged exposure to sodium butyrate. On the other hand, in E1A-negative cells, an intense transcription increase was observed  [E1A promoter, we performed a transient and stable transfection of E1A + Ras cells with the plasmid coding the 12S E1A under the control of a strong promoter of cytomegalovirus (CMV). The regulation and activity of CMV and Ad5 viral promoters differ significantly; thus, they can be used in target cells for different purposes. The high-activity CMV promoter is convenient for efficient transgene expression. Moreover, the CMV promoter was reported to be stimulated by HDACi [We previously showed an HDACi-induced decrease in protein and mRNA level of E1A in HEK293 and E1A + Ras-transformed cells . In E1A  type 5) ,48,49. Tby HDACi . FirstlyThus, our results suggest that, for overcoming NaBut-induced inhibition of the FoxO activity in E1A-expressing cells, increased expression of E1A independent of HDACi is required.To elucidate the mechanisms of E1A influence on the stability of FoxO, we tried to determine which regions of adenoviral E1A protein are important for the induction of FoxO activity. For this, NIH3T3 cells were transfected with different adenovirus E1A constructs. At 36 h after transfection, cells were lysed and assayed for FoxO1 expression. As shown in Next, we investigated whether sodium butyrate modulates the interaction of p300 with FoxO in E1A + Ras cells. To assess the dynamic of FoxO/p300 interaction, E1A + Ras cells were transfected with a vector coding HA-tagged full-length p300 acetyltransferase, which was then treated with NaBut for 24 h. The coimmunoprecipitation assay followed with immunoblotting revealed that endogenous FoxO1 and p300 could associate in sodium butyrate-treated cells only b. In untOur data show that the presence of adenoviral E1A significantly alters the cell response to NaBut treatment, specifically by influencing the dynamics of FoxO expression and activity. First of all, the basal level of FoxO1 expression in E1A-positive cells is generally higher. Moreover, we showed that both rodent (NIH3T3 and REF52) and human cancer cells (HCT116) transfected with a vector encoding the E1A are characterized by an increased basal level of FoxO1 protein. Secondly, our experimental data reveal that the transactivation function of FoxO is generally in line with the FoxO protein level. In E1A-negative cells, the low basal level of FoxO1 greatly increases under NaBut treatment, accompanied by enhanced FoxO transactivation function. As for E1A-expressing cells, there is a downward trend in FoxO1 expression and activity under prolonged NaBut treatment. Several mechanisms leading to FoxO stabilization by E1A protein are suggested. E1A can bind to the proteasomes and inactivate their functions ,52, prevWe established the crucial role of the p300-binding region of the E1A protein in the induction of FoxO a. ComparcHa-ras gene. Ras was shown to suppress FoxO1 expression, activating the Ras/PI3K/Akt pathway, leading to FoxO1 degradation [In E1A-expressing cells, different dynamics of FoxO decrease are observed. In E1A + Ras cells, FoxO undergoes complete degradation, while the degradation of FoxO is less evident in E1A + E1B and HEK293 cells. This discrepancy may be attributed to the Ras oncoprotein. There are no Ras mutations in E1A + E1B and HEK293 cells , whereasradation ,31. As sradation , which wradation .Our present study provides findings that HDACi-induced degradation of FoxO protein can be avoided, provided that the level of E1A protein does not decrease significantly following HDACi treatment. However, in cells expressing E1A under the adenoviral promoter, whose activity is inhibited by HDACi, the amount of E1A decreases following the addition of HDACi, which causes the FoxO expression and activity to decrease. In other words, expression of the early E1A region of adenovirus increases FoxO stability, but it makes the FoxO protein level more sensitive to HDACi treatment.In summary, here, we present evidence showing that FoxO expression and stability under NaBut treatment is firmly associated with expression of E1A and Ras. Oncogenic Ras causes the degradation of FoxO through activation of the Ras/Akt signaling pathway, but expression of E1A in cells carrying activated Ras is able to overcome this inhibition and enhance FoxO expression.FasL, and the proapoptotic Bcl-2 family member Bim. Alternatively, FoxO factors can promote cell-cycle arrest by activating the cell-cycle inhibitors p21Waf1 or p27kip1 to induce G1 arrest, or GADD45 to induce G2 arrest.The interpretation of these findings could be challenging since FoxOs are proven to cause both tumor suppression ,63 and pRas gene mutation, long-term HDACi treatment inhibits FoxO expression and activity. We suggest that the E1A/HDACi co-treatment is ineffective in anticancer therapy of tumors with Ras mutations. On the other hand, there is a hypothesis that FoxO acts as a tumor suppressor in normal cells; however, within tumor cells, it can contribute to their survival and tumor growth [Our results suggest that, in E1A-expressing cells with a r growth ,68. If w


Prostate cancer (PCa) is dependent on the androgen receptor (AR). Advanced PCa is treated with an androgen deprivation therapy-based regimen; tumors develop resistance, although they typically remain AR-dependent. Expression of constitutively active AR variants lacking the ligand-binding domain including the variant AR-V7 contributes to this resistance. AR and AR-V7, as transcription factors, regulate many of the same genes, but also have unique activities. In this study, the capacity of the two AR isoforms to regulate splicing was examined. RNA-seq data from models that endogenously express AR and express AR-V7 in response to doxycycline were used. Both AR isoforms induced multiple changes in splicing and many changes were isoform-specific. Analyses of two endogenous genes, PGAP2 and TPD52, were performed to examine differential splicing. A novel exon that appears to be a novel transcription start site was preferentially induced by AR-V7 in PGAP2 although it is induced to a lesser extent by AR. The previously described AR induced promoter 2 usage that results in a novel protein derived from TPD52 (PrLZ) was not induced by AR-V7. AR, but not AR-V7, bound to a site proximal to promoter 2, and induction was found to depend on FOXA1. Metastatic PCa is treated with an androgen deprivation therapy (ADT) based regimen but eventually, these tumors develop resistance through multiple mechanisms that reactivate AR6. These include the expression of constitutively active AR splice variants that lack a ligand-binding domain such as AR-V710. Whether and how AR-V7 differentially regulates transcription relative to AR has been a subject of great interest, but its capacity to regulate splicing has not been studied.The androgen receptor  is a hormone activated transcription factor that is important for the growth, invasion, and progression of prostate cancer (PCa)12. Over 90% of human genes undergo alternative splicing14. Different alternative combinations of exons and splice sites generated during splicing can produce multiple transcripts and different protein sequences from a single locus. This is, in part, responsible for the much greater diversity of the transcriptome and proteome than would be obtained from a single protein isoform derived from each gene16. Dysregulation or defects in the process may lead to disruption of normal cellular function and the eventuality of disease including cancer19.Alternative splicing is broadly recognized for its vital role in the regulation of genes and creating gene diversity in multicellular organisms21. Exon skipping events are reported to be the most prevalent alternative splicing event in the human genome and aberrant splicing associated with it is responsible for the loss of functional domains or a frameshift in the coding sequence23.There are many types of alternative splicing; the most common are exon skipping (ES), mutually exclusive exons (MXE), alternative 5′ splice sites (A5SS), alternative 3′ splice sites (A3SS), alternative first exon (AFE), alternative last exon (ALE), tandem 3′UTRs and intron retention (IR)27. RNA-seq based data of AR activity in response to androgens identified many genes whose transcription is regulated by the AR in prostate cancer26. Transcription also has been coupled with pre-mRNA processing and promoter selection in the presence of androgens in prostate cancer31. Previously, we prepared AR expressing LNCaP and VCaP prostate cancer cell lines that express AR-V7 in response to Doxycycline (Dox)33. Analysis of our RNA-seq data and other studies revealed differential expression of target genes (GEO#GSE14390533). Our current analyses reveal that AR activated by R1881 or AR-V7 induced by Dox drive the production of alternative mRNA isoforms in prostate cancer cell lines. This includes all forms of alternative splicing resulting in changes in both coding and regulatory regions in untranslated regions. Here, we have identified differential alternative splicing events induced by the AR isoforms in LNCaP and VCaP cell line models and evaluated the mechanisms for differential induction.Besides controlling transcriptional levels, steroid hormone receptors play a role in regulating alternative splicing34. AR-V7 lacks the LBD and hinge region, but contains the amino-terminal transactivation domain and DNA binding domain (DBD) encoded by exons 1–3 and 16 unique amino acids derived from a cryptic exon 3b9. We have previously described the LNCaP and VCaP models in which AR-V7 can be induced using Doxycycline33. The levels of expression of full-length AR and induced AR-V7 protein are shown in Fig. 36; whereas Dox induced expression of AR-V7 also represses expression of endogenous AR  and its variant AR-V7 are shown in Fig. Alternative splicing events were identified in the entire read set for the RNA-seq data from our LNCaP-AR-V7 and VCaP-AR-V7 models STable . Splicin22. One of the most differentially regulated events in both cell lines (LNCaP-AR-V7 and VCaP-AR-V7) is an inclusion/exclusion exon event in PGAP2 (Post-GPI Attachment to Proteins 2), a protein involved in GPI remodeling. Figure Skipped Exon events represent the most common alternative splicing event type involved in regulating genesReviewing the Sashimi plots Fig. a revealeOverlap with ChIP-exo data of LNCaP cells expressing either the full-length AR or the AR-V7 isoform showed clear peaks at the location of the novel exon, further suggesting a potential novel transcription initiating site under the control of AR or AR-V7 Fig. a. This w9, so depletion of AR-V7 alone would not be very informative. However, Fig. We next asked whether we can detect the novel exon to exon 4 junction in LN-95 and 22Rv1 cells, which both endogenously express small amounts of AR-V7. In LN-95 cells, we depleted AR-V7 using siRNA and measured expression by qPCR. Figure 39. Androgen driven selection of an alternative promoter in the TPD52 gene switches from promoter 1 (extended) to alternative promoter 2 (short) and this alternative promoter 2 is known to produce a protein isoform known as PrLZ31. Because AR is known to preferentially induce transcription from an alternative first exon31, we sought to determine whether AR-V7 also induced this form. Our RNA-seq data also suggested that TPD52 was induced by R1881 and that there also was preferential usage of the internal (promoter 2) rather than the extended 5′ promoter (promoter 1) resulting in different first exons. Figure 33. An analysis of previously published FOXA1 binding to this gene using IGV showed multiple FOXA1 binding sites40  has a well-characterized role in prostate cancer progression31. The constitutively active AR splice variants that appear in CRPC also regulate transcription and there is evidence that although many of the actions of AR and its variants are similar, each can also regulate unique targets. Thus, we sought to determine whether their capacity to regulate splicing also differs. Our RNA-seq data derived from LNCaP and VCaP cell line models showed that not only do AR and AR-V7 differentially regulate expression of a subset of target genes but that both induce alternative splicing of mRNAs  was chosen because it occurred in both cell line models and the Sashimi plot Fig. a suggest was chos43. Moreover, it is expressed at higher levels in prostate cancer and even in prostatic epithelial neoplasia compared to normal or benign prostate epithelia44. We confirmed the preferential induction from promoter 2 both by examining our RNA Seq tracks and by qPCR of products initiated at promoter 1 or promoter 2. Interestingly, AR-V7 did not induce transcription from promoter 2. The same transcriptional pattern was observed in the VCaP model. Whereas our LNCaP model ChIP-exo data showed that AR exhibited binding to a region within the first intron following promoter 2, AR-V7 did not . Fetal Bovine Serum (FBS) and Charcoal Stripped Serum (CSS) were purchased from Sigma Aldrich, St. Louis, MO. CSS was also prepared in the cell culture core. R1881 and Doxycycline (Dox) were purchased from Sigma Aldrich. Unless specified, all other chemicals are reagent grade.LNCaP, VCaP and 22Rv1 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. LN-95 cells were obtained from Dr. Jun Luo, Johns Hopkins University, and cultured in phenol red-free RPMI 1640 medium with 10% Charcoal Stripped Serum (CSS). The LNCaP-AR-V7-pHage and VCaP-AR-V7-pHage cell lines32, β-tubulin antibody (RRID AB_309885) at a 1:10,000 dilution, or a FOXA1 antibody (RRID: AB_2104842) at a 1:1000 dilution. Proteins were detected using horseradish peroxidase-conjugated secondary anti-mouse (GenDEPOT) antibody at 1:10,000 and ECL2 western blot substrate .LNCaP-AR-V7-pHage, VCaP-AR-V7 pHage and LN-95 cells were lysed by three cycles of freeze/thaw treatment using a lysis buffer containing 0.01 M Tris, 1 mM EDTA, 12 mM monothioglycerol pH 7.6, 0.4 M NaCl and protease inhibitors (Roche) and the supernatant was collected from the lysate after centrifugation at 12,000 rpm for 5 min. The concentration of proteins was determined by the Bradford protein assay reagent (GenDEPOT). A 7.5% SDS gel was used to resolve 20 μg of proteins. Proteins were then transferred to a Protran nitrocellulose membrane  and blots were incubated using the previously described anti-AR antibody AR441 (RRID AB_11000751) at a 1:1000 dilutionKnock-down of AR-V7 and FOXA1 expression was performed using the transient transfection of siRNA against the gene in comparison to non-targeted negative control siRNA. Synthesized oligo duplexes were purchased from Sigma-Genosys, The Woodlands, TX, and the sequences are listed in STable PureXtract RNAzol solution  was used to prepare RNA from the treated LNCaP, LNCaP-AR-V7, VCaP, and VCaP-AR-V7 cells. cDNA Synthesis Master Mix (GenDEPOT) was used to reverse transcribe the total RNA (0.5 μg) to make cDNA to measure target genes. AmfiSure Taq DNA Polymerase (GenDEPOT) was used for PCR amplification. Primers used for amplification are listed in the primer STable PureXtract RNAzol solution  was used to prepare RNA from the treated LNCaP, LNCaP-AR-V7, VCaP, VCaP-AR-V7, 22Rv1 and LN-95 cells. cDNA Synthesis Master Mix (GenDEPOT) was used to reverse transcribe the total RNA (0.5 μg) to make cDNA to measure target genes. Target gene expression was analysed using the primers (STable 33 using AR antibody (RRID: AB_2793341) or rabbit IgG antibody (Invitrogen). Immunoprecipitated DNA fragments were measured by qPCR using the primer sequences listed in the Supplementary file.ChIP assays were performed as previously described46. Gene expression was quantified using feature counts against the GENCODE gene reference. Differentially expressed genes were detected using edgeR, with significance achieved at FDR-adjusted p-value < 0.05 and fold change exceeding 1.25x. The Mixture-of-Isoforms (MISO) model was applied to identify alternatively spliced genes and differential used exons following the developer's guidelines47. We considered alternative splicing effects for a Bayesian Factor (BF) ≥ 3 and a percent spliced in (psi (ψ)) difference of at least 0.2.For each sample, reads were quality trimmed using Trim Galore! and then aligned to the hg19 genome using HISAT2RNAseq and ChIP-exo track expression datasets used are listed in STable t-test was used to calculate statistical significance (P < 0.05) using PASW SPSS 18 software.All experiments have been performed a minimum of three times and a representative experiment was shown. The Student’s Supplementary Information.


The likelihood of active CH was significantly higher in patients with mandibular deviation higher than 6 mm compared with <6 mm (odds ratio (OR): 3.51, confidence interval (CI) 95%: 1.27–9.72). Conclusion: There is a significant correlation between the magnitude of mandibular deviation quantified on CT and metabolic findings obtained by SPECT in patients with UCH. The risk of active UCH is 3.5 times higher in patients with a mandibular deviation ≥6 mm.Objective: To evaluate the correlation between metabolic bone activity measured by single photon emission computed tomography (SPECT) and the anatomic condylar characteristics acquired by computed tomography (CT), in patients with unilateral condylar hyperplasia (UCH). Method and Materials/Patients: Observational, descriptive study in a group of 71 patients with clinical diagnosis of UCH and indication of SPECT/CT. Bone SPECT images obtained in a gamma-camera GE Infina and processed in a station Xeleris 3 with the program Volumetrix MI Evolution for bone. CT images acquired in a PET/CT Biograph mcT20 equipment (Siemens) processed in a station Osirix V 7.5.1 . Results: The sample included 24 men (33.8%) and 47 women (66.2%). Active state UCH was detected in 40 (56.3%) cases (over 55% uptake in the affected condyle) and 38 (53.5%) presented mandibular deviation to the right side. No significant differences related to sex, age, or mandibular deviation side were found. Mandibular deviation was the only morphologic feature related to active/inactive UCH ( Condylar hyperplasia (CH) is a progressive and self-limiting pathology affecting the mandibular condyle growth and compromising the temporomandibular joint (TMJ) anatomy ,2,3.The functional, occlusal, and esthetic effects of CH in patients demand a multidisciplinary intervention to confirm a clinically suspected diagnosis and establish the therapeutic approach . Early dUCH is effectively diagnosed by measurement of bone metabolic hyperactivity in SPECT mandibular TMJ 3D images ,6,7. RecThe correlation of metabolic and anatomic findings by a team of experts in the management and interpretation of SPECT/CT techniques allows the establishment of clear-cut parameters of the pathology to precisely indicate the extension of the altered region. This approach is a recent breakthrough in the procedures of diagnosis and treatment of UCH ,13.The authors’ hypothesis from the existing literature is that the fusion of images and information obtained from SPECT/CT to diagnose UCH improves the precision and specificity of the diagnostic tests and, consequently, allows better therapeutic decisions ,14,15,16This is a retrospective observational study with no intervention or manipulation of variables from the patients. Therefore, it is a no risk investigation and was approved by the Institutional Ethics Committee  and conducted following all the regulations of the Declaration of Helsinki, last version.A population of 153 image sets from patients tested by SPECT/CT , perform99mTc-MDP for patients over 18 year and normalized according to the EANM Pediatric Dosage Card for patients under 18. The images were obtained using a double head gamma-camera GE Infinia , with low energy collimators, and a 128 × 128 high resolution matrix to obtain 45 images, 18 s of exposure, for each 180° of detection.The mandibular bone SPECT procedure was carried out 2 h after the endovenous administration of a dose of 15 mCi 2) [The data were reconstructed in the processing station Xeleris 3  using the program “Volumetrix MI Evolution for Bone”, with the ordered subsets expectations maximization (OSEM) algorithm for iterative reconstruction, applying four interactions and eight subsets with a Butterworth 0.45 filter and Power 12, plus correction of resolution recovery. From the reconstruction, five transaxial images for quantification in the condyles were obtained, extracting the total counts for a fixed-size ROI (1.76 cm2) .The SPECT report provided quantitative information expressed as radionuclide percentage uptake in the condyles. The counts observed within the selected ROI were used to calculate the % uptake using the following equations:Right side counts + left side countsRight side counts + left side countsA difference in percentage uptake between condyles = or >10% was interpreted as a positive result indicating active pathology .CT cranial images were acquired in a PET/CT Biograph mCT20  equipment without contrast enhanced, from vertex to sternal notch, applying the following parameters: section thickness: 1.0 mm, pitch: 1.0, and a 512 × 512 cubic matrix with isotropic voxel (size 0.75 × 0.75 × 0.75 mm) to avoid image distortion in the different planes. The same parameters were applied to adult and pediatric patients. The CT images were reconstructed using a B26F homogeneous low dose filter for anatomic localization. All the patients were positioned with a head fixing device to avoid artifacts owing to movement and facilitate the image fusion.The set of DICOM images was processed in a work station Osirix V 7.5.1  obtaining linear measurements in sagittal and frontal planes. The description of measurements taken in the 3D bone tissue reconstruction is described in The tomographic measurements were taken by a trained and calibrated operator. Each data set was simultaneously revised and classified according to the craniofacial characteristics of the asymmetry , togetheTo assess the reproducibility of the measurements, a duplicate reading was taken by the same observer on a subsample of 20 patients with a four-week interval between the two measurements. The correlation coefficients (Rho) indicate an agreement higher than 0.90 for all of the variables .® . Normality of distribution was tested by the method of Shapiro–Wilk and, according to it and the kind of variable, the results are expressed as average ± standard deviation, median, inter-quartile range, and absolute/relative frequencies. The Chi square test or Fisher test was applied for bivariate analysis of qualitative variables and either Student’s t-test or U-test for quantitative variables, according to the distribution normality. Correlations were evaluated by the Spearman coefficient rho. The level of significance was p < 0.05.The data were processed by one operator expert in the software management. All statistical analyses were carried out in the software Stata13Intraobserver agreement was evaluated by the intraclass correlation coefficient of Lin.Receiver operating characteristics (ROC) curves were determined to establish the best cut-off value of mandibular deviation to classify the hyperplasia as active or inactive. ROC curves were obtained from estimated sensitivity, specificity, and positive and negative predictive values calculating their 95% confidence intervals.p = 0.1), sex (p = 0.22), or side of mandibular deviation (p = 0.99)  women and 24 (33.8%) men, with a median age of 19 years. From the total number of patients, 40 (56.3%) presented active UCH (≥55% uptake in the affected condyle) and 38 (53.5%) presented right side deviation. No significant differences in the frequency of active UCH were detected in relation to age, ( = 0.99) .p = 0.003).The measurements obtained in CT images of the patients were related to the active or inactive state of UCH. The results are presented in The ability of mandibular deviation to classify the state of UCH as active or inactive was studied by ROC analysis. The area under ROC curve (AUC) was 0.695 (CI 95%: 0.57–0.82), indicating acceptable ability to distinguish the states, as the area is >0.5 .Two cut-off values of mandibular deviation were selected. The first was a 6 mm value because it is more specific, that is, it detects inactive UCH with 55% sensitivity and 74.19% specificity, providing a positive predictive value (PPV) of 73.3% and negative predictive value (NPV) of 56.1%. The other cut-off value was 4 mm, which shows the best sensitivity, that is, it detects more active cases of UCH with a sensitivity of 75% and 54.8% specificity, PPV of 68–18%, and NPV of 63% .The likelihood of having active UCH in patients with mandibular deviation equal or higher than 6 mm was 3.5× higher than the likelihood associated with mandibular deviations <6 mm .When the cut-off value was set to 4 mm, the likelihood of inactive UCH was 73% .Image fusion for diagnostic purposes, as in the case of SPECT/CT, is known as a co-register or hybrid technique and it is used to improve the diagnostic precision and, therefore, to aid in the development of a better treatment plan positively determined by the prognosis . In nuclIn connection with this, Jacene et al.  postulatThe radioactive uptake in bone SPECT depends on the blood circulation and the absorption by the structure of hydroxyapatite crystals. The areas of high uptake of the radioactive tracer are correlated with hyperemia and more metabolic bone activity and, additionally, identify activity at the molecular level. Therefore, nuclear medicine images are highly sensitive for early detection of lesions, very much earlier than X-ray or tomographic images. Bone SPECT is very useful and has been validated for early diagnosis of UCH ,6,7,22, 99mTc-MPD. In this context, Suh et al. [The hybrid SPECT/CT technique for the diagnosis of UCH provides detailed morphologic information about the mandibular condyles and other craniofacial structures that may be compromised in the pathology. This information is associated with the data of bone metabolic activity in the condyles , obtaineh et al.  point ouIn the present study, the fusion of data from SPECT/CT to classify UCH conditions as active or inactive detected a significant difference in the magnitude of mandibular deviation (MD) associated with active cases. This finding is concordant with the results of Wang et al. , postulaRegarding the diagnostic added value of SPECT/CT compared with SPECT alone to evaluate UCH, Fokoue et al.  indicateTaking into account these observations, the authors of the present study postulate that the specificity of the SPECT test is improved by the clinical and tomographic pre-diagnostic findings , and by International studies indicate that, in different populations, the prevalence of UCH is higher in women than in men , as was Regarding the age distribution of UCH, the average age in the active UCH group was similar to that of inactive UCH , both including ranges of residual growth . Althougp = 0.003) and clinically relevant.The difference between the average DM (6.3 mm) in active states of UCH and the average for inactive conditions (4.1 mm) is statistically significant  and ROC analysis established that a mandibular deviation >6 mm is a risk factor for active UCH .The correlation between the magnitude of mandibular deviation measured in CT images and the percentage uptake obtained by SPECT is statistically significant (


Background: Allergic rhinitis (AR) is the most common IgE-mediated allergic disease. Multiple clinical trials have demonstrated promising results on the AR treatment with biologics, in particular with the use of omalizumab – an anti-IgE antibody. Omalizumab has also been established in the routine management of allergic asthma and chronic idiopathic urticaria. However, currently there is no approved license for the use of biologics in AR in Germany. Materials and methods: A systematic literature review has been completed including randomized controlled trials, meta-analyses, and reviews on the treatment of AR with omalizumab. Results: The systematic review demonstrates strong evidence supporting the use of omalizumab in the treatment of AR with regard to symptom control, safety profile, and management of comorbidities. Conclusion: Omalizumab is a good and safe option in the treatment of AR in terms of symptom control and the management of pre-existing comorbidities. Further clinical trials with other biologics in the management of AR are needed and are expected to follow soon.  In Germany, AR is the most common allergic disease with a lifetime prevalence of 14.8%, as epidemiological studies of the Robert Koch Institute (RKI) have demonstrated . In addiIn particular, sufficient efficacy of avoidance measures cannot always be realized. Furthermore, the administration of anti-allergic medication cannot provide a thorough symptom relief in all patients. This is especially true for nasal obstruction under antihistamine therapy , 8. TherThe former underlines the important unmet need for further improvement of therapeutic approaches. Among others, biologicals are promising therapeutic candidates directly targeting individual mechanisms of the inflammatory cascade. Between 1997 and 2019, a large number of phase I and II studies were conducted on efficacy and safety of biologics in AR . A firstA second class of biologics targets interleukins (IL) such as IL-4 and IL-13, and has been investigated in AR patients. In a phase II study, a combined therapy with VAK-694 (IL-4 antagonist) and subcutaneous allergen immunotherapy (SCIT) demonstrated an immunomodulatory effect on the TH2 memory cells; however, a clinical response was lacking . In contCurrently, the most advanced biologic in AR in clinical development is the IgE antagonist omalizumab. It has been approved in the EU since 2005 as an add-on therapy for patients with severe persistent allergic asthma and has been established in routine clinical care . HoweverThe literature search was conducted using PubMed, clinicaltrialisregister.eu, and clinicialtrialis.gov. The search terms “biologicals”, “omalizumab”, and “anti-IgE” in combination with “allergic rhinitis” were used to identify relevant study results. Randomized controlled trials (RCT), placebo controlled (DBPC) trials, review articles, and meta-analyses from 1997 to 2020 were considered for the literature search. The most recent literature search was conducted on April 27, 2020 and aimed to specify the effect of omalizumab on symptom control, reduction of concomitant  medications for symptom relief, disease-specific quality of life, safety of SCIT under omalizumab and safety/tolerability in general. As in other atopic diseases, allergen-specific IgE is overexpressed in patients with AR binding to two different receptors (FcεRI and FcεRII) on effector cells. The binding to these receptors results in the initiation of the inflammatory cascade with the typical pattern of AR symptoms. Increased levels of serum allergen specific IgE have been found to correlate with the onset and symptom severity of AR , 42. Omalizumab binds free serum IgE, preventing the interaction of IgE with the FcεRI receptor and further activation of the subsequent inflammatory pathways. Omalizumab has a higher affinity to free IgE in the serum than the FcεRI receptor of effector cells leading to a strong reduction in free circulating IgE . This isHowever, it should be noted that omalizumab, when given at therapeutic doses, does not bind IgE that is already bound to the FcεRI or FcεRII receptor . Since bPrevious study results also indicate that omalizumab not only suppresses the acute inflammatory response in allergic rhinitis, but also may provide a long-term immunomodulatory effect . Early studies on the use of omalizumab in AR aimed to determine the optimal dosage. Casale et al.  comparedOmalizumab has been demonstrated to be beneficial for patients with moderate to severe intermittent and persistent AR and insufficient symptom control despite conventional therapy . SeveralFurther atopic diseases such as allergic asthma and urticaria are often associated with AR , which oAccording to a variety of studies, about half of all patients with AR also suffer from allergic asthma, and concomitant AR has a significant impact on asthma control. Poorly controlled AR is associated with a higher number of asthma exacerbations and hospitalizations . Since bIn addition to trials investigating efficacy of omalizumab monotherapy, some studies combined this treatment with AIT aimed to increase treatment response to AR patients , 38, 39.Various endpoints have been used in clinical studies to assess symptom severity. The most common score used to measure nasal symptoms was the Daily Nasal Symptom Severity Score (DNSSS). A large number of studies also considered the ocular symptoms of allergic rhinoconjunctivitis (ARC) such as the Daily Ocular Symptom Severity Score (OCSSS) , 34, 50.A meta-analysis from 2014 by Tsabouri et al.  evaluateIn general, a significant improvement in nasal symptoms was observed in all randomized controlled trials between 2000 and 2010. A particularly strong treatment effect size of 39% was seen when omalizumab was combined with AIT , 37. A mHowever, the extent of symptom reduction varied between the studies. For example, Adelroth et al.  observedWhile the study designs were consistently uniform in recording of nasal and ocular symptoms, there were notable methodological differences in the use of concomitant anti-allergic medications. For example, in some studies, this medication was paused following the study protocol , whereasTo assess the impact of omalizumab therapy on quality of life, the standardized and validated Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) has frequently been used in AR trials. This questionnaire contains 28 items relating to activity limitations, sleep disturbances, symptoms, practical problems, and emotional functioning . AdelrotOmalizumab therapy has been shown to be safe and well tolerated in routine clinical care. This is also confirmed in a meta-analysis by Yu et al.  which foIn rare cases, antibodies to the drug may be formed during therapy with humanized DNA-derived monoclonal antibodies in general. However, such events have not been observed with omalizumab , 29, 34.Omalizumab is able to form an immune complex with free IgE. Since these complexes are present as trimers or hexamers, they pose a low risk for the development of serum sickness due to their small size . Based oLarge clinical studies showed no increased risk for the occurrence of malignant tumors with omalizumab . In animA clinical study demonstrated a slight increase in parasitic infections under anti-IgE therapy . HoweverIn the treatment of AR, monoclonal antibodies to IgE represent a promising therapeutic option for the future by effectively reducing the free IgE antibodies in the serum. Omalizumab has already been the subject of a large number of clinical studies in AR with supportive evidence from the treatment of other atopic diseases. In AR, this treatment has demonstrated clinical efficacy by reducing the symptomatic burden and need for concomitant allergic medication and by improving the quality of life in patients with AR. In addition, it has an impact on comorbidities and a convincing safety profile. Omalizumab is approved as an add-on therapy for better control of severe persistent allergic asthma, the severe form of chronic rhinosinusitis with polyposis (CRSwNP) and the severe form of chronic spontaneous urticaria. Clinical development programs in AR are also very convincing. However, further clinical studies on omalizumab and other above-mentioned biologics are needed to integrate these molecules in common treatment concepts of AR. None. Oliver Pfaar reports grants and personal fees from ALK-Abelló, grants and personal fees from Allergopharma, grants and personal fees from Stallergenes Greer, grants and personal fees from HAL Allergy Holding B.V./HAL Allergie GmbH, grants and personal fees from Bencard Allergie GmbH/Allergy Therapeutics, grants and personal fees from Lofarma, grants from Biomay, grants from Circassia, grants and personal fees from ASIT Biotech Tools S.A., grants and personal fees from Laboratorios LETI/LETI Pharma, personal fees from MEDA Pharma/MYLAN, grants and personal fees from Anergis S.A., personal fees from Mobile Chamber Experts (a GA2LEN Partner), personal fees from Indoor Biotechnologies, grants and personal fees from Glaxo Smith Kline, personal fees from Astellas Pharma Global, personal fees from EUFOREA, personal fees from ROXALL Medizin, personal fees from Novartis, personal fees from Sanofi-Aventis and Sanofi-Genzyme, personal fees from Med Update Europe GmbH, personal fees from streamedup! GmbH, grants from Pohl-Boskamp, grants from Inmunotek S.L., personal fees from John Wiley and Sons, AS, personal fees from Paul-Martini-Stiftung (PMS), outside the submitted work. Stefan A. Rudhart reports personal fees from UV-SMART outside the submitted work. Boris A. Stuck has received financial support for research, consultant and speaker fees from the following companies: G. Pohl-Boskamp GmbH & Co.KG, GlaxoSmithKlinie GmbH & Co KG and R. Cegla GmbH & Co.KG. He has also received financial support (sponsorship) for events of the Department of Otorhinolaryngology, Head and Neck Surgery, Marburg from: ALK Abello, Merck, Pohl Boskamp, Bristol Myers Squibb, Sanofi Genzyme. The other authors do not report any conflicts of interest.


We propose a “Curry-style” semantics of programs in which a nominal labelled transition system of types, characterizing observable behaviour, is overlaid on a nominal LTS of untyped computation. This leads to a notion of program equivalence as typed bisimulation.Our semantics reflects the role of types as hiding operators, firstly via an axiomatic characterization of “parallel composition with hiding” which yields a general technique for establishing congruence results for typed bisimulation, and secondly via an example which captures the hiding of implementations in abstract data types: a typed bisimulation for the (Curry-style) lazy


Although the United Nations’ Convention on the Rights of Persons with Disabilities enshrines the right to health for all persons with disabilities (PDs), PDs face health disparities in terms of access to rehabilitation resources, which is important for service supply. This study aimed to explore the trends and distribution of rehabilitation resources for PDs in China from 2014 to 2019, explore the main factors that influence equity, and provide suggestions for policymakers. Data were obtained from the annual China Statistical Bulletin on the Development of Disabled Persons and the database of the China Disabled Persons’ Federation. Six types of rehabilitation resources were chosen to measure the trends in allocation and equity. Data on disparities were analyzed based on western, central, and eastern regions. The Health Resource Density Index and Theil Index were calculated to determine the degree and density of unfairness. The findings show a steady increasing trend in the amount of rehabilitation resources in China from 2014 to 2019. The density and equity of allocation of rehabilitation resources have improved greatly in recent years. Regional disparities were principally caused by differences within the regions. Suggestions including expanding investment in rehabilitation resources and developing rehabilitation systems were put forward. Healthy China Strategy 2030, which proposes to “enable all people to have needed, quality, and affordable health services such as prevention, treatment, rehabilitation, and health promotion.” This was particularly apparent during the 2020 COVID-19 pandemic, as the UN proposed A Disability-Inclusive Response to COVID-19 to address the specific issues faced by PDs [It is estimated that approximately 15% of the global population has experienced some form of disability, and that between 110 and 190 million people have a severe physical impairment ,3. Rehabd by PDs . HoweverThe United Nations’ Convention on the Rights of Persons with Disabilities (CRPD) enshrines the right to health for all PDs. All persons have the right to enjoy the highest attainable standard of health without discrimination on the basis of disability, and to have access to high-quality health services, including rehabilitation . Often, As the aging of China’s population continues, the prevalence of chronic diseases and the total number of PDs increase; thus, the demand for rehabilitation is growing rapidly. By the end of 2019, China’s elderly population (aged 60 and above) had reached 254 million, accounting for 18.1% of the total population of China .Growing interest in inequality has brought many discussions on the subject into the public realm. However, few studies concentrate on rehabilitation resource disparities for PDs. Instead, the research has deeply explored the following three topics: (1) specific rehabilitation issues such as cardiac rehabilitation , exercisThis study aimed to explore the trends and distribution of rehabilitation resources for PDs in China, explore the main factors that influence equity, and provide suggestions for policymakers. Then, the results may draw more attention to rehabilitation resource disparity. Regarding the limitation that the national statistics system does not include specific rehabilitation resources and study purposes, the rehabilitation resources data mainly come from the CDPF, which is a non-governmental and a national umbrella organization for persons with diverse disabilities . The rehabilitation resources excluded medical resources such as hospitals, medical facilities, and workforce. In addition, it is estimated that the actual number of PDs is much higher than the RPDs who are registered with CDPF. To ensure a comparative analysis, this research used the official statistics of RPDs. Based on to data availability, the national analysis period was 2014–2019 and the regional analysis period was 2014–2018.In this study, we collected the relevant data from the annual China Statistical Bulletin on the Development of Disabled Persons (2014–2019) and the CDPF database (2014–2018). All data are openly accessible on the CDPF’s public website. To analyze regional disparities, the 31 provinces, autonomies, and municipalities of mainland China were divided into three regions. According to geographical position and gross domestic product per capita, the three regions are described as western, central, and eastern, which represent undeveloped, developing, and developed regions in mainland China, respectively. Beijing, Tianjin, Hebei, Liaoning, Shanghai, Jiangsu, Zhejiang, Fujian, Shandong, Guangdong, and Hainan belong to the eastern region. Shanxi, Jilin, Heilongjiang, Anhui, Jiangxi, Henan, Hubei, and Hunan belong to the central region. Inner Mongolia, Guangxi, Chongqing, Sichuan, Guizhou, Yunnan, Tibet, Shaanxi, Gansu, Qinghai, Ningxia, and Xinjiang belong to the western region.Generally, health resource-related indicators include three basic aspects: workforce, financing, and property. After scanning the database, we chose six indicators: Staff and Professionals in RIs to represent the rehabilitation workforce; Total Completed Projects and Total Investment of Projects Completed on RSF to represent financing; and Rehabilitation Institution and Total Construction Area of Projects Completed on RSF to represent property. Then, we described and analyzed the six indicators. The indicator descriptions are as follows: (1) Staff in RIs refers to the persons who work at rehabilitation institutions, including professionals , managerial personnel (responsible for management and coordination), and other support personnel; (2) Total Completed Projects on RSFs refers to tailored construction and services projects, such as the establishment of specific rehabilitation training centers, and Total Investment of Projects Completed on RSFs refers to the annual investment on projects; and (3) Rehabilitation Institutions refers to institutions for different types of disabilities, such as visual, hearing and speech, physical, intellectual, mental, and autism in children. In this study, we included assistive technology institutions in RIs in statistics. Total Construction Area of Projects Completed on RSFs refers to the scale of the construction for projects completed on RSFs yearly.HRDI was used to assess equity both in demographic and geographic dimensions. The HRDI was combined with the data to demonstrate differences in rehabilitation resources for PDs in 31 provinces and municipalities. In addition, the HRDI could avoid bias and influences caused by a single aspect of population or geographic area . The valir represents the rehabilitation resource, ip represents RPDs, and ia represents the square of the unit I.In the above formula, ip represents the proportion of the unit RPDs accounting for the aggregation in one region and iy represents the unit’s rehabilitation resource accounting for the aggregation in one region.The Theil Index, named for the researcher who first proposed it in 1967 , is an iThe Theil Index can be decomposed into two components: to describe inequality “within” and “in between” subgroups. The formulas are as follows: w represents the degree of rehabilitation resource allocation fairness in a targeted region; Tb represents the degree of rehabilitation resource allocation equity between the different regions; and gp and gy have the same meaning as ip and iy above.TChanges in the amount of rehabilitation resources for PDs are shown in 2 in 2018 . The results are shown in To further understand the rehabilitation resource distribution in China, we calculated the rehabilitation resources per 10,000 RPDs and per 10,000 kmFrom the perspective of per 10,000 RPDs, we could acquire a basic understanding of provincial resource distribution and density. In terms of the rehabilitation institutions for PDs, the majority of the values were concentrated below 5, except for Shanghai, which reached almost 19. Guangdong had the highest value of staff and professionals in distribution of RIs with more than 100 persons per 10,000 RPDs. Additionally, we found extreme disparities between the highest and lowest values: the number of RIs per 10,000 RPDs in Shanghai (18.96) was 30 times higher than that in Tibet (0.58), while the eastern regions were 1.8 times higher than the western regions.2, the resources were concentrated in five eastern areas , all of which ranked among the top 10 in almost all six indicators. The geographic distribution gap was larger than that of the demographic. For the other indicators, and apart from the projects of RSFs for PDs, Shanghai was the highest ranked and Tibet was the lowest.Regarding the aspect of regional distribution per 10,000 kmTo avoid the bias and influences caused by a single aspect of population or geographical area, HRDI was applied to assess equity both in demographic and geographic dimensions. We calculated formula (1), which indicated that the 31 provinces and municipalities had large regional disparities. The highest HRDI in all indicators was at least 30 times higher than the lowest. The largest disparity regarding HRDI was evident within the RI indicator. Geographically, the eastern regions’ HRDI was 4.2 and 1.8 times higher than those of the western and central areas, respectively. Shanghai ranked first in five indicators, while Fujian ranked first in one indicator. Tianjin, Guangdong, Beijing, Jiangsu, Chongqing, Liaoning, Shandong, Shanxi, etc. ranked within the top 15 HRDI.The results shown in The Theil Index of the eastern regions showed a higher value than the central or western regions. In terms of the rehabilitation workforce, the Theil Index of staff and professionals in RIs presented a first decreasing and then increasing trend (both 0.09 in 2018), but their values were obviously lower than those of the other four indicators, which were more than 0.15. Apart from the five other indicators, the western region’s Theil Index for professionals in RIs for PDs was higher than that of the central and eastern regions: the western region was the least equitable with regard to workforce distribution. Compared to personnel, fairness in the allocation of other rehabilitation resources clearly improved as the values decreased.As shown in 2. Although the United Nations’ CRPD enshrines the right to health for all PDs, PDs face health disparities in terms of access to rehabilitation services. However, our findings indicate that the opportunities for PDs to obtain rehabilitation services are expanding. With the development of the social economy, the aging of the population, and an increase in both the number of groups with disabilities and the various public health initiatives, there has also been an increase in the awareness of not only the general public but also the government of opportunities within the rehabilitation sector. This trend is clearly related to the rapid development of both China’s social economy and its medical reform policies.Rehabilitation is a fundamental health service for people with a variety of health conditions, and primarily focuses on improving their ability to function well in society and on reducing the impact of disability. This was a nationwide study that comprehensively evaluated the trend of rehabilitation resources allocation tailored for PDs in China. We found a steady rising trend in the total amount of rehabilitation resources in China, annually per 10,000 RPDs and per 10,000 kmThe growth rate of the total rehabilitation workforce was lower than that of other selected rehabilitation resources. Furthermore, the personnel density of rehabilitation institutions for PDs has decreased by almost 10% compared to five years ago. Together, these data indicate that the focus on rehabilitation for PDs may be more concentrated on property, infrastructure, and projects, particularly as investment and rehabilitation resources have increased significantly. In addition, because of the new round of health system reforms enacted in 2009, the growth in health spending has also increased rapidly to support the development of the rehabilitation sector.2 show that there was a large gap between different provinces and regions. Furthermore, there was a larger disparity in the geographic distribution of rehabilitation resources than in the distribution of RPDs. With respect to existing research on health resources [This study explored trends, density, distribution, and equity analysis using data from 2014 to 2019 with data from the CDPF. Based on the results of this study, we found that the overall fairness of rehabilitation resource allocation has improved in China. However, further results for the distribution of rehabilitation resources per 10,000 RPDs and per 10,000 kmesources , this st2), and the increasing rates were lower than RPDs. There are three reasons for this finding. First, compared to international training of rehabilitation professionals, China only started systematic rehabilitation education after 2001, and the educational level was mainly focused on vocational or college education. The university major of rehabilitation therapy was not divided into specific professional directions, such as physical therapy, occupational therapy, and speech therapy. Thus, the rehabilitation workforce entering the job market is limited, including rehabilitation institutions for PDs. Second, there is no professional certification system; professionals, such as rehabilitation therapists, cannot practice as medical practitioners. Third, the general public has a low perception of the occupation and regards it as similar to “massage”, leading to low career attraction and high turnover. However, the government has recognized these challenges by expanding higher education on rehabilitation [The value for staff or professionals of rehabilitation institutions showed a downward trend regarding density . The results are not surprising given that the projects, construction area, and investment goals are relatively easy to achieve compared to workforces whose employees require a long training period. However, as the rehabilitation sector for PDs is to some extent a part of public welfare, rehabilitation resources should be allocated scientifically and without bias. Another finding indicates that the causes of inequality in the eastern, central, and western regions principally came from the within-group rather than between-group rate. Compared to health resources, the Theil Index values of rehabilitation resources were higher; however, health resources, such as doctors, experienced the same declining trend during the same time period . The levAs a result, policy makers should focus on the allocation of rehabilitation resources and guide policy in weak provinces in the central and western regions to achieve overall coordinated development for improving access to rehabilitation services for PDs. Our suggestions are as follows: First, the rehabilitation sector should receive more investment. Second, all levels of rehabilitation education should be expanded. Third, the rehabilitation-related statistics system should be further improved, including by inserting it into the national statistics system. Fourth, the government should motivate the market to boost careers in rehabilitation for PDs. Fifth, accreditation of education and regulation of the rehabilitation workforce should be standardized. Sixth, national and sub-national plans regarding the allocation of rehabilitation resources should be created or inserted into a multi-level plan, such as the 14th Five-Year Plan. Finally, the rehabilitation system should be developed and improved within the existing health system. Only in this way can we achieve the goals of Healthy China 2030, Universal Health Coverage, and of SDG3.Although this study specifically described the data of rehabilitation resources from 2014 to 2019, there are some limitations of this data source. Due in part to the data content and indicators having changed in recent years, some data in this study were predicted and estimated, and, furthermore, provincial analysis was only available for a specific period of time. Thus, there is some undue influence on the results. In addition, to discuss the rehabilitation resources for PDs, we used RPDs as the population, weighted on density and the Theil Index analysis. The actual number of potential PDs may be far higher than the RPDs. Moreover, unlike hardware, such as investment and projects, which could be improved in a short period of time, human resources  are key for rehabilitation services and require a longer period of time to be cultivated and trained. Therefore, our next study will continue to follow the updated data from the CDPF. The important results have inspired further analysis on the allocation of human resources for the rehabilitation of PDs, and the rehabilitation resources require evaluation: these areas could represent a future avenue for research.The density and equity of rehabilitation resource allocation has improved markedly in recent years. Compared to the four other indicators in this study, staffs and professionals in RIs for PDs demonstrated lower density and equity in all regions. The distribution of accumulated projects completed of RSFs was found to be the most inequitable when compared to the other five types of rehabilitation resources. Regional disparities were typically caused by differences within the regions. As the population ages and the number of disabilities expands, the need for rehabilitation will only continue to increase. The inequality phenomenon might be caused by various factors; thus, we could not make perfectly accurate measurements and assessments of fairness via one kind of tool or unilateral indices. However, we hope that this study can draw attention to the rehabilitation aspect of PDs to improve their access to basic and necessary services.