We finally analyzed 294 subjects. VAE occurred in 9.9% and high mortality at 48.3%. Median values of Timing 1 and Timing 2 in the non-VAE and VAE groups were 30.3 ± 24.0 and 30.0 ± 20.7 h, and 125.7 ± 136.6 and 127.9 ± 111.4 h, respectively, and the differences were not significant . We found no significant relationship between the occurrence of VAE leading to high mortality and timing of rehabilitation intervention.The present study aimed to investigate the relationship between the occurrence of ventilator-associated events (VAE) in the intensive care unit and the timing of rehabilitation intervention. We included subjects who underwent emergency tracheal intubation and received rehabilitation. We performed rehabilitation according to our hospital’s protocol. We assessed the mechanical ventilation parameters of inspired oxygen fraction and positive-end expiratory pressure, and a VAE was identified if these parameters stabilized or decreased for ≥2 days and then had to be increased for ≥2 days. We defined time in hours from tracheal intubation to the first rehabilitation intervention as Timing 1 and that to first sitting on the edge of the bed as Timing 2. Data were analyzed by the  After admission to the intensive care unit (ICU), patients are in danger of complications such as sepsis, ICU-acquired weakness, and delirium ,2,3. In However, VAP is problematic because it lacks objective and gold-standard criteria, thus is difficult to diagnose. Therefore, the Center for Disease Control and Prevention (CDC) established a new surveillance definition termed ventilator-associated events (VAE). Although VAE is not used in clinical situations, the definition has been retrospectively used for the objective surveillance of medical records ,7,8.2) and positive end-expiratory pressure (PEEP) need to be stabilized or decreased for a period of ≥2 calendar days . After that, the FiO2 or PEEP is required to have been increased for a period of ≥2 calendar days because of clinical events. Thus, the diagnosis of VAE requires at least four full days to meet the definition.VAE is identified according to the following factors of mechanical ventilation. As prerequisites, the fraction of inspiratory oxygen , physicians, and nurses every morning. Each day, we adjust the intervention time and share information again with the nurse just before the intervention. We intervened with help from the nurse from 9:30–16:30 on Monday to Friday and from 9:30–12:00 on Saturday and Sunday.We defined two types of rehabilitation timing in units of hours: the time from tracheal intubation to the first rehabilitation intervention (regardless of its contents) was defined as Timing 1, and the time from tracheal intubation to first sitting on the edge of the bed (regardless of assistance) was defined as Timing 2.The following data were collected from the subjects’ computerized medical records: age, body mass index (BMI), sex, principal diagnostic reason for tracheal intubation, Acute Physiology and Chronic Health Evaluation II (APACHE II) score , LOS dur2 and PEEP to define VAE. There are two sets of criteria for VAE as defined by the CDC National Healthcare Safety Network [2 should be stable or decreasing for at least two calendar days, followed by an increase of at least 0.20 and maintained at that level for at least two calendar days, and (2) the daily minimum PEEP should be stable or decreasing for at least two calendar days, followed by an increase of at least 3 cm H2O and maintained at that level for at least two calendar days. We diagnosed VAE according to the presence of either condition. We investigated the clinical events that caused the VAE. As we counted all of the events that we considered to be candidates, there was some overlap.We collected the daily values of minimum FiO Network : (1) thet-test, and qualitative variables were compared by the χ2 test between the two groups. A p value of <0.05 was considered to indicate statistical significance. Statistical analyses were performed with IBM SPSS 24.0 J statistical software .We divided the subjects into two groups according to the occurrence of VAE: the non-VAE group and VAE group. All quantitative variables were expressed as the mean ± standard deviation, and all qualitative variables were expressed as the number and percentage of the group. Quantitative variables were compared by the unpaired Student The present study complied with the principles of the Declaration of Helsinki regarding investigations in humans and was approved by the local institutional review board of our hospital .After applying the exclusion criteria to the 851 subjects, we finally analyzed 294 subjects . Diagnosp < 0.001), re-intubation , and weaning mechanical ventilator . All patients who died in the VAE group were difficult to wean from the ventilator.There were 29 (9.9%) subjects with a VAE. t = 0.60, p = 0.95 and t = 0.83, p = 0.93, respectively). The respective mean values of Timing 1 in the non-VAE group and VAE group were 30.3 ± 24.0 and 30.0 ± 20.7 h, and those of Timing 2 were 125.7 ± 136.6 and 127.9 ± 111.4 h, respectively. Diagnostic reasons for tracheal intubation in the VAE group subjects are shown in The top four reasons were pneumonia, followed by stroke, trauma, and infection. The top three clinical events causing VAE were pneumonia in 41.4%, pulmonary edema in 34.5%, and atelectasis in 27.6% of the subjects. Some of these clinical events overlapped.To our knowledge, there have been no reports investigating the relationship between VAE and rehabilitation. Therefore, we investigated the relationship between the occurrence of VAE and the timing of rehabilitation intervention.Among the 9.9% of subjects in whom a VAE occurred, there was a significantly higher rate of re-intubations and lower probability of weaning from the mechanical ventilator. Moreover, mortality in these subjects was increased when compared with the subjects of the non-VAE group. However, there was no significant difference in the timing of rehabilitation intervention between the two groups.We included subjects ventilated for ≥4 calendar days in the present study. Other previous studies used various inclusion criteria such as ≥2 or three calendar days ,19,20. TConsistently high mortality and prolonged mechanical ventilator use due to VAE have been reported in previous studies ,21. We aIn the present study, the first intervention (Timing 1) occurred within approximately 24 h and the first time that the subjects sat on the edge of the bed (Timing 2) occurred within approximately 96 h. In previous definitions, early mobilization was recommended to start within 2–5 days or within 48 h ,15. MendWe found no relationship between VAE and rehabilitation timing. The reasons for this may be as follows. First, we had already intervened in all subjects with sufficient early rehabilitation initiated within two to five days of the onset of illness. Thus, it is highly possible that the initiation of rehabilitation had no effect on VAE.Second, in addition to early rehabilitation, we may require other indices of rehabilitation such as the amount performed, the frequency at which it is performed, and other variables. Yagi et al. reported that early and intensive rehabilitation was important for subjects with stroke . AdditioThird, the disease background in the study subjects was varied. As nursing care and treatment are different according to disease, progress in rehabilitation may also be different. Moreover, various clinical events caused the VAE. The most frequently reported clinical events associated with VAE were pneumonia or aspiration in first place; pulmonary edema, pleural effusion, or heart failure in second place; and atelectasis in third place . The preThe present study was a single-center, retrospective study. The data gathered on timing probably had a 1–2-h error. Moreover, there were data that we could not track such as comorbidities, medical history, and activities of living before hospitalization. We did not consider missing data in the analysis. It may be necessary to consider other characteristics in the analysis. We also did not consider other rehabilitation parameters , and, in particular, changes in posture. It is necessary to investigate how not only physical therapists but also nurses are involved with patients. Although the diagnosis of VAP is made subjectively, we had to collect data on VAE, which is diagnosed objectively; however, VAE is not used clinically, and the events causing it are diverse.We investigated the relationship between the occurrence of VAE and the time from emergency tracheal intubation to rehabilitation intervention in subjects on mechanical ventilation for ≥4 calendar days. VAE occurred in 9.9% of the subjects and caused a poor prognosis and high mortality (48.3%), similar to that reported in previous studies.The first rehabilitation intervention was performed at a median time of approximately 24 h after tracheal intubation, and the subjects first sat on the edge of the bed within approximately 96 h. However, there was no significant relationship between the occurrence of VAE and the timing of rehabilitation intervention. The clinical events causing VAE were varied, with the leading causes being pneumonia, pulmonary edema, and atelectasis. In addition to the early timing of rehabilitation interventions, intensive rehabilitation and ensuring longer times out of bed may also be needed to prevent VAE.


Hip fracture in elderly patients is a rising global public health concern because of population ageing, and increasing frailty. Long-term morbidity related to poor management of hip fracture is associated with decreased quality of life, survival, and increase in healthcare costs. Receiving postoperative rehabilitation is associated with better outcomes and a higher likelihood of returning to pre-existing level of functioning. However little is known about which postoperative rehabilitation pathways are more effective to optimize patient outcomes. Few studies have analyzed postoperative rehabilitation pathways in a universal healthcare system. The aim of this study is to analyze the impact of post-acute rehabilitation pathways on mortality and readmission in elderly patients undergoing surgery for hip fracture in a large metropolitan area in Italy.In this retrospective cohort study, we analyzed 6-month mortality from admission and 6-month readmission after hospital discharge in patients who underwent surgical repair for hip fracture in the hospitals of the Bologna metropolitan area between 1.1.2013 and 30.6.2014. Data were drawn from the regional hospital discharge records database. Kaplan-Meier estimates and multiple Cox regression were used to analyze mortality as a function of rehabilitation pathways. Multiple logistic regression determined predictors of readmission.n = 1677, 76%), with a median age of 83.8 years. Hospital rehabilitation was provided to 519 patients (23.5%), 907 (41.1%) received rehabilitation in private inpatient rehabilitation facilities (IRF) accredited by the National Health System, and 782 (35.4%) received no post-acute rehabilitation. Compared with patient receiving hospital rehabilitation, the other groups showed significantly higher mortality risks  = 2.19, 95%CI = 1.54–3.12, p < 0.001; IRF rehabilitation, HR = 1.66, 95%CI = 1.54–1.79, p < 0.001). The risk of readmission did not differ significantly among rehabilitation pathways.The study population includes 2208 patients, mostly women (Intensive hospital rehabilitation was significantly associated with a lower risk of mortality compared to IRF rehabilitation and no rehabilitation. Our results may help in the development of evidence-based recommendations aimed to improve resource utilization and quality of care in hip fracture patients. Further research is warranted to investigate the impact of the rehabilitation pathway on other outcomes, such as patients’ functional status and quality of life. Hip fracture in elderly patients is a global public health concern because of its frequency and functional consequences on individuals experiencing this event –3. PopulEvidence shows that delivery of surgery in hip fracture patients within 48 h of admission is associated with significant lower mortality rates –8. InterItaly has a universal single-payer health system managed by the state and the regions. The central government oversees the overall system and defines the essential levels of care to be delivered to the citizens, free of charge or after the payment of a fee, with public resources collected through general taxation. The regions are responsible for the actual planning and delivery of services with a certain degree of autonomy , 19. In The aim of this study is to analyze the impact of post-acute rehabilitation pathways on mortality and readmission in elderly patients undergoing surgery for hip fracture in the large metropolitan area of Bologna, located in Emilia-Romagna region, Italy.The data sources for this observational cohort study were the Hospital Discharge Records (HDRs) database of the Emilia-Romagna region , the mortality registry, the regional domiciliary care database (ADI) and outpatient specialty care database (ASA). We extracted from HDRs data for all patients aged > 64 years with a primary diagnosis of hip fracture (ICD-9-CM codes 820.0–820.9) from hospitals in the Bologna Metropolitan Area, Italy , discharged between January 1, 2013 and June 30, 2014. We selected patients from hospitals with at least 100 procedures per year in order to reduce variations related to caseload, which resulted in the exclusion of 16 hospitals and 188 patients. We included patients who underwent a surgical procedure of total or partial hip replacement (ICD-9-CM codes 81.51–81.52) or the reduction of fracture and dislocation (ICD-9-CM codes 79.0–79.5), which resulted in the exclusion of 106 patients. We excluded patients who died within one day from admission  hospital rehabilitation, when rehabilitation was provided in the same hospital where patients received the surgical procedure; 2) IRF rehabilitation, when patients were admitted to private inpatient rehabilitation facilities (IRF), accredited by the National Health System, within 2 days from the index stay discharge and no previous post-acute rehabilitation had been performed. IRFs are private facilities that are accredited to provide inpatient post-acute rehabilitation within the National Health System and are reimbursed by the Region on the basis of a daily admission fee. The accreditation implies that IRFs are allowed to operate within the National Health System because they meet specific parameters of effectiveness and efficiency in terms of staff qualification, procedure volumes and quality of care provided.We set a 2-day threshold for IRF rehabilitation in order to include patients who received inpatient care soon after the index admission. Over 90% of patients who received out-of-hospital rehabilitation were admitted to IRFs within 2 days from the index discharge; 3) no post-acute rehabilitation.A small group of patients (~ 1%) who received both hospital and IRF rehabilitation were included in the hospital rehabilitation pathway.Community rehabilitation was not included in our analysis because it is provided to only 9.6% of cases after discharge. Moreover, many patients seek private outpatient rehabilitation instead of community rehabilitation.The outcomes of interest were mortality at 6 months from index admission, all-cause readmission, and orthopedic readmissions within 6 months from index discharge, in line with other studies –24.Patients’ socio-demographic characteristics (age and gender) and comorbidity were also retrieved from hospital discharge records. Specifically, the presence or absence of myocardial infarction, heart failure, peripheral vascular disease, cerebrovascular disease, dementia, hypertension, neurological disease and renal disease was searched in the 2 years preceding the index admission using Elixhauser et al. algorithm . The tot2 and Kruskal-Wallis tests were used to compare patient characteristics among the three pathways. Following significant tests, post-hoc pairwise comparisons were performed at a Bonferroni-corrected significance level of 0.017 (0.05/3). Six-month mortality was estimated using Kaplan-Meier analysis and compared among rehabilitation pathways using log-rank test for the overall six-month period and then distinguishing the first 30-day period from the 31–180 day period. This was done to take into account the expectation that the majority of mortality events occur in the first month after admission. Multiple Cox regression was used to estimate the mortality risk in the rehabilitation pathways, adjusted for patients’ age, gender, comorbidities and intervention characteristics. The risk of all-cause and orthopedic readmission at 6 months in the three pathways, adjusted for the same set of covariates listed above, was estimated using multiple logistic regression models. In those models, the non-independence of patients within hospitals was taken into account by calculating robust standard errors adjusted for the patient-hospital clusterisation. Both in the Cox regression and in the logistic regression, a backward stepwise procedure was used to remove non-significant predictors until a parsimonious model including only significant covariates was achieved. Stata v.13.1 was used for all analyses.χp = 0.055) and gender (p = 0.542). Patients receiving hospital rehabilitation showed on average more comorbidities  and had a higher percentage of all-cause hospital readmissions at 6 months after discharge . In particular, patients receiving hospital rehabilitation had more frequently heart failure, peripheral vascular diseases, cerebrovascular disease, neurological diseases (other than dementia), and renal diseases (p < 0.001). Compared to the other groups, patients receiving IRF rehabilitation showed a lower incidence of cerebrovascular diseases and dementia (p < 0.001). Length of stay was significantly shorter in the no rehabilitation group (11.5 days) compared with the other two groups . Patients receiving no rehabilitation showed a lower percentage of late (> 48 h) interventions (p = 0.017), more reduction procedures (p = 0.022) and a higher incidence of previous domiciliary care (p = 0.003). The percentage of patients with orthopedic readmissions was not different among subgroups .Table 2 = 20.7, p < 0.001). About one fifth (20.8%) of patients who did not receive rehabilitation died, compared with 15.4% of patients who received IRF rehabilitation and 11.6% of patients receiving inpatient rehabilitation. The Kaplan-Meier estimates of 6-month survival differed significantly among rehabilitation pathways , . Between 30 and 180 days no significant difference in mortality was found among groups .The cumulative 6-month mortality rate was 16.4%, with significant differences among the groups (χ1), Fig. . The surMultiple Cox regression Table  confirmeThe logistic regression of all-cause readmission at 6 months from discharge Table  showed tThis is one of the first studies to assess the effects of different rehabilitation pathways following hip fracture surgery in a universal healthcare system. The results suggest that intensive post-acute rehabilitation within the index hospital was associated with lower risk of mortality compared to other pathways, including IRF rehabilitation, which was the most frequent pathway, although this does not imply a direct causation. Learning from these data, guidelines and recommendations could boost provision of post-acute rehabilitation in the best effective and cost-effective setting, possibly with standardized protocols.Notably, the mortality risk in patients not receiving rehabilitation was more than twofold higher compared with patients who received hospital rehabilitation and it was 66% higher in those who received IRF rehabilitation, after adjustment for patient and intervention characteristics. These results are consistent with recent literature. Ireland et al. reported a 60% mortality risk reduction at 3 months and 40% at 1 year after hospital discharge in patients who received hospital-based rehabilitation compared with no rehabilitation . Pitzul Consistent with other studies, we found that mortality risk at 6 months was higher when surgery was performed more than 2 days after fracture, in males, and in patients with cardiovascular diseases or dementia, regardless of rehabilitation pathway , 30–34. Extending hospital rehabilitation services to all may not be feasible because of costs and lack of resources,  however,The different clinical pathways showed no significant differences in terms of all-cause and orthopedic readmissions. Male gender, length of stay and previous domiciliary care were the strongest predictors of all-cause readmissions. Orthopedic readmissions did not differ among subgroups. These findings may suggest that rehabilitation pathways have no impact on the long-term patients’ functional status.Implementation of effective care pathways for patients with hip fracture is still debated,  and it iOur results should be interpreted in light of several limitations. First, that the study design does not allow to interpret associations between rehabilitation pathways and patient outcomes as causal relationships. Functional outcomes are not available in the administrative databases. A 12-month follow-up would have been more appropriate to study outcomes, such as mortality, still we had a specific focus on the episode of care, in which generally rehabilitation is completed, and there is a general agreement that the time span of the episode is 6 months. Third, administrative data can be affected by variability and lack of completeness in the coding of diagnoses and procedures. However, hospital discharge records from the Emilia-Romagna region showed good accuracy of ICD-9-CM coding, partially mitigating concerns about low coding standards –45. FourIn conclusion, this population-based study of rehabilitation following hip surgery found that immediate rehabilitation within the index hospital was associated with lower risk of mortality. Still, we found that one-third of patients are discharged with no rehabilitation. Future programs focused on improved post-operative care should consider this evidence in planning and policy making. Innovative policies and health plans, aimed at targeting vulnerable populations, such as extremely older patients or patients with impaired cognitive functions, and implementing intermediate care models could be effective tools to expand the access to care and improve the quality of care in a growing high-need and high-cost population.


Augmented reality (AR) comprises special hardware and software, which is used in order to offer computer-processed imaging data to the surgeon in real time, so that real-life objects are combined with computer-generated images. AR technology has recently gained increasing interest in the surgical practice. Preclinical research has provided substantial evidence that AR might be a useful tool for intra-operative guidance and decision-making. AR has been applied to a wide spectrum of orthopedic procedures, such as tumor resection, fracture fixation, arthroscopy, and component's alignment in total joint arthroplasty. The present study aimed to summarize the current state of the application of AR in orthopedics, in preclinical and clinical level, providing future directions and perspectives concerning potential further benefits from this technology. Computer-assisted orthopedic surgery has gained increasing interest, especially in the last two decades , being eAR has been described as “the concept of digitally superimposing virtual objects onto physical objects in real space so individuals can interact with both at the same time” . In contThe basic structure of an AR system used in orthopedic surgery, as it was described by Nikou et al. , comprisThere is a wide variety of AR systems which have been implemented in preclinical and clinical level. An example of a camera-augmented C-arm was described by Navab et al. . A videoBased on preclinical data, Blackwell et al.  remarkedWe aimed to investigate the current state of the implementation of AR in orthopedic surgery. We searched in PubMed and Cochrane Database of Systematic Reviews, using the key words: “AUGMENTED REALITY” AND (“ORTHOPAEDIC” OR “ORTHOPAEDICS” OR “ORTHOPEDIC” OR “ORTHOPEDICS” OR “ARTHROPLASTY” OR “REPLACEMENT” OR “ARTHROSCOPY” OR “FRACTURE” OR “SPINE”). Our inclusion criteria were: studies published after January 2010 and until September 2018 (end of our search), written in English language, with a purpose to investigate the outcomes of the implementation of AR in a specific orthopedic surgical intervention, either in preclinical or in clinical level. We excluded conference papers, expert opinions and review articles. In total, 91 studies were found after the initial search. Finally, 22 preclinical and nine clinical studies met our inclusion and exclusion criteria.Several preclinical studies have been conducted during the last two decades, which have demonstrated that AR could improve the accuracy of interventional procedures. Firstly, there has been substantial evidence published to support the use of AR in fracture surgery. Van Duren et al.  describeLondei et al.  illustraFallavollita et al.  assessedUtilization of AR in spine surgery has been investigated by various researchers. Gibby et al. , who useVarious clinical studies assessing the usefulness of AR in orthopedic surgery have documented satisfactory outcomes . In the Ogawa et al.  evaluateSeveral clinical studies were conducted regarding spine surgery. According to Elmi-Terander et al. , AR surgAs it has been showed, the implementation of AR could possibly lead to improved accuracy in positioning and decreased radiation in a wide spectrum of orthopedic procedures, concerning either trauma or elective orthopedic surgery. However, the cost-effectiveness of AR in orthopedics is a factor which should be critically estimated  before tAlso, concerns have been raised about the extent to which display of information via AR could be misleading or disturbing during surgery . AdequatAlthough it has been shown that AR might have a beneficial impact to orthopedic residents' training, it would be interesting to investigate to what extent AR could play a significant role in providing orthopedic trainees with a valuable learning experience. Moreover, verifying the learning curve would be an interesting aim , which sIn addition, under conditions of real-time surgery, there are factors which might be critical and there are absent in preclinical studies. For example, the effect of patient's respiratory movement has to be taken into account during thoracic spine surgery . MoreoveWe should note that the papers which were found after our search and met our inclusion and exclusion criteria might not cover the whole spectrum of the implementation of AR in orthopedic surgery and, also, could be soon outdated by the ongoing research. In summary, further clinical studies are required to confirm the favorable preclinical outcomes of the use of AR in orthopedics.DC did the literature search and wrote the first draft of the manuscript. M-AM proofread the manuscript and made corrections. VN had the initial idea of writing the manuscript, proofread, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A new molecule induces AR sumoylation and degradation resulting in selective growth inhibition in AR-dependent prostate cancer cells, but its activity is blunted by interference with proteasomes. Aberrant androgen signaling drives prostate cancer and is targeted by drugs that diminish androgen production or impede androgen–androgen receptor (AR) interaction. Clinical resistance arises from AR overexpression or ligand-independent constitutive activation, suggesting that complete AR elimination could be a novel therapeutic strategy in prostate cancers. IRC117539 is a new molecule that targets AR for proteasomal degradation. Exposure to IRC117539 promotes AR sumoylation and ubiquitination, reminiscent of therapy-induced PML/RARA degradation in acute promyelocytic leukemia. Critically, ex vivo, IRC117539-mediated AR degradation induces prostate cancer cell viability loss by inhibiting AR signaling, even in androgen-insensitive cells. This approach may be beneficial for castration-resistant prostate cancer, which remains a clinical issue. In xenograft models, IRC117539 is as potent as enzalutamide in impeding growth, albeit less efficient than expected from ex vivo studies. Unexpectedly, IRC117539 also behaves as a weak proteasome inhibitor, likely explaining its suboptimal efficacy in vivo. Our studies highlight the feasibility of AR targeting for degradation and off-target effects’ importance in modulating drug activity in vivo. Prostate cancer is the most common type of cancer in men in the United States and accounts for 30,000 deaths annually . The abeLike many other transcription factors, AR expression, function, and turnover are tightly regulated at multiple levels, including posttranslational modifications . AR can Current therapies aim at reducing testosterone levels or inhibiting testosterone-AR binding, whereas most therapy escape mechanisms in CRPC rely on altered AR expression or mutations. Directly targeting AR for destruction may, thus, represent a promising approach in fighting therapy-resistant disease . Here, w50 of IRC117539 was determined to be in the low micromolar range. On the other hand, the survival of AR-negative PC3 and Du145 prostate cancer, MiaPaca2 pancreatic carcinoma, HeLa cervical cancer, or WI38 human fibroblast cells was only slightly affected  indicated that IRC117539 promoted physical association between AR and the proteasome . To bettCollectively, our results suggest that IRC117539 antagonizes AR signaling by specifically targeting and binding AR, and tagging it for degradation by the proteasome, most likely through a sequence of posttranslational modifications involving SUMO2/3 and ubiquitin. This catabolic pathway is strikingly similar to the recently uncovered mechanism of therapy-induced PML/RARA degradation in acute promyelocytic leukemia and therapy-induced Tax oncoprotein degradation in adult T-cell lymphoma .50 of IRC117539 on the proteasome was determined to be in the low millimolar range (IC50(tryptic activity) = 878 μM, IC50(chymotryptic activity) > 1 mM). The caspase-like activity of the proteasome was not affected by IRC117539. Critically, protein expression of the catalytic β7, which contains trypsin-like activity, and structural C2 subunits of the 20S particle, sharply declined in LNCaP cells treated with IRC117539  and AR-negative (PC3 and Du145) prostate cancer lines, as well as in most other cell types  and in vivo in mice . On the RC117539 . QuantitRC117539 . ConversRC117539 . Thus, tWe finally analyzed the expression levels of CHOP and BiP, two commonly used markers for ER stress, which mediate cellular unfolded protein response. Analysis of CHOP and BiP expression did not indicate a substantial rise in ER stress in various cells exposed to IRC117539  global accumulation of ubiquitin and SUMO conjugates, which may reflect inhibition of proteolysis, (2) massive stabilization of short-lived proteins , (3) inhibition of proteasome tryptic and chymotryptic activities. Critically, IRC117539’s off-target activity as a proteasome modulator was observed in both AR-positive (LNCaP and VCaP) and AR-negative (PC3 and Du145) prostate cancer cells alike. Other primary and transformed cell lines (WI38 and HeLa) also displayed similar signs of proteasome inhibition upon exposure to IRC117539. However, the compound impaired survival exclusively in AR-positive cell lines , strongly suggesting that loss of viability is uncoupled from proteasome inhibition and most likely a consequence of drug-induced elimination of a driver oncoprotein (AR). Indeed, compared with commonly used proteasome inhibitors such as MG132, IRC117539’s impact on the ubiquitin–proteasome system remains rather weak, not enough to trigger significant ER stress or cause cell death, but possibly sufficient to diminish the rate of AR turnover at the proteasome in vivo (see below). In that respect, we have observed that simultaneous treatment with PD169316, a potent small molecule proteasome activator, drastically enhanced IRC117539-induced AR degradation ex vivo. Importantly, IRC117539 achieves full AR degradation at doses comparable with those required for AR inhibition and survival impairment, whereas a log-fold increase in concentration is needed for full proteasome inhibition .Remarkably, IRC117539 induces loss of viability in AR-dependent, yet androgen-insensitive 22Rv1 cell line. 22Rv1 cells express several AR isoforms: a full-length version with duplicated exon 3 that retains the LBD  and two truncated isoforms lacking the C-terminal LBD that are constitutively active . AlthougHow exactly IRC117539 impairs proteasome function remains to be determined. The compound may directly bind and destabilize defined proteasome subunits, impair proper proteasome assembly or control subunit transcription. Dexamethasone, a glucocorticoid receptor agonist, was recently shown to modify the subunit composition and enzymatic activity of proteasomes , whereasLNCaP, 22Rv1, PC3, and Du145 cells were cultured in 10% FCS RPMI, and MiaPaca2, VCaP, Hela, and WI38 cells were cultured in 10% FCS DMEM media (Gibco) with additional nutrients as stated in ATCC guidelines. For survival assays, cells were treated with 10 μM IRC117539 for up to 6 d. For AR degradation, AR-dependent gene expression, and PLAs, LNCaP cells were treated with indicated doses  of IRC117539 for indicated times (up to 48 h). MG132 (EMD Millipore) was used at a concentration of 2 μM for 24 h before cell lysis and Western blot. Rabbit polyclonal anti-human AR (N-20), p53 (DO-1), and Mdm2 (C-18) antibodies were from Santa Cruz; antibodies for proteasome subunits  were from Abcam; anti-CHOP (L63F7) and anti-BiP (C50B12) antibodies were from Cell Signaling Technologies; and anti-vinculin (7F9) antibody was from Santa Cruz. All other antibodies were described previously . PLAs weFKBP5, PSA, NKX3-1, and AR transcripts were determined by quantitative PCR using TaqMan probe-based gene expression analysis (Applied Biosystems), where a Cyclophilin (Cyc) probe was used as an internal control.Expression levels of human PSMA3, PSMB1, PSMB6, PSMB7, C2, and PSMC3 proteasome subunit transcripts were determined using a TaqMan array for human ubiquitin–proteasome–dependent proteolysis kit from Thermo Fisher Scientific (4414198).Expression levels of human Proteasome’s tryptic, chymotryptic, and caspase-like activities were analyzed in LNCaP cells after a 24-h-long treatment with increasing doses of IRC117539, using the proteasome activity assay kit from Promega (G8660) according to the manufacturer’s instructions. Briefly, LNCaP cells, seeded on 96-well plates, were treated (or untreated) with IRC117539 for 24 h before incubation with relevant fluorescent peptide substrates , the cleavage of which was monitored using a luminometer. The latter is reflective of cellular proteasome enzymatic activities .FRET was determined using LanthaScreen TR-FRET AR Coactivator Assay and LanthaScreen TR-FRET AR T887A Coactivator Assay from Thermo Fisher Scientific according to the manufacturer’s instructions. Assays were run in antagonist mode. Cyproterone acetate or IRC117539 were incubated up to 5 h with 5 nM AR LBD or 5 nM AR T887A-LBD, 500 nM fluorescein D11FxxLF, 5 nM Tb anti-GST antibody, and 5 nM dihydrotestosterone. Curves were generated using XLfit software from IDBS.Mouse handling was performed in accordance with established institutional guidance and approved protocols from the Comité Régional d’Ethique Expérimentation Animale n°4, which enforces the EU 86/609 directive.Probasin mRNA levels were determined by quantitative PCR using TaqMan probe-based gene expression analysis (Applied Biosystems), where Cyclophilin (Cyc) served as internal control.The Hershberger bioassay serves to assess the potency of androgens and anti-androgens in castrated rats upon treatment and is based on determining their effect on ventral prostate weight, an androgen-dependent tissue. Castrated rats were treated with IRC117539, along with dihydrotestosterone, for 10 d before sacrifice. LNCaP xenografts were transplanted subcutaneously into Balb/c nude mice and tumor volume was recorded for up to 50 d upon treatment with IRC117539 or enzalutamide (MDV3100). For orthotopic model, LNCaP tumor cells were injected into ventral prostate of Balb/c nude mice. PSA levels were determined in peripheral blood using ELISA . Once elevation of PSA levels was observed, the mice were castrated. PSA levels decreased and started increasing again approximately 14 d after castration, mimicking CRPC state. Animals were then treated with IRC117539 for 14 d, tumors were harvested, and AR levels were measured in tumors either by IHC  or ELISA .


Non-isocyanate polyurethane (NIPU) was prepared from Jatropha curcas oil (JCO) and its alkyd resin via curing with different diamines. The isocyanate-free approach is a green chemistry route, wherein carbon dioxide conversion plays a major role in NIPU preparation. Catalytic carbon dioxide fixation can be achieved through carbonation of epoxidized derivatives of JCO. In this study, 1,3-diaminopropane (DM) and isophorone diamine (IPDA) were used as curing agents separately. Cyclic carbonate conversion was catalyzed by tetrabutylammonium bromide. After epoxy conversion, carbonated JCO (CJCO) and carbonated alkyd resin (CC-AR) with carbonate contents of 24.9 and 20.2 wt %, respectively, were obtained. The molecular weight of CJCO and CC-AR were determined by gel permeation chromatography. JCO carbonates were cured with different amine contents. CJCO was blended with different weight ratios of CC-AR to improve its characteristics. The cured NIPU film was characterized by spectroscopic techniques, differential scanning calorimetry, and a universal testing machine. Field emission scanning electron microscopy was used to analyze the morphology of the NIPU film before and after solvent treatment. The solvent effects on the NIPU film interfacial surface were investigated with water, 30% ethanol, methyl ethyl ketone, 10% HCl, 10% NaCl, and 5% NaOH. NIPU based on CCJO and CC-AR (ratio of 1:3) with IPDA crosslink exhibits high glass transition temperature (44 °C), better solvent and chemical resistance, and Young’s modulus (680 MPa) compared with the blend crosslinked with DM. Thus, this study showed that the presence of CC-AR in CJCO-based NIPU can improve the thermomechanical and chemical resistance performance of the NIPU film via a green technology approach. Jatropha curcas oil (JCO) is one of the attractive vegetable oils for the oleochemical industry [2 transformation is a sustainable solution for controlling global warming and serves as a reusable resource for CO2 valorization.Raw materials, the petroleum rate, resource depletion, and the awareness of the consumer society have increased worldwide attention on environmental issues and the growing concern for polyurethane (PU) materials. These issues have facilitated research on biodegradable and renewable green resources ,2. Compaindustry ,9,10,11.industry , which mindustry  such as industry ,14, alkyindustry , food paindustry , surfaceindustry , nanocomindustry , etc. Taindustry ,20. Thesindustry . Differeindustry ,22. By c2 into oxiranes, which produce five-member cyclic carbonates, has attracted economic and environmental attention. CO2 fixation into oxiranes is typically performed under various pressures and temperatures in the presence of the tetrabutylammonium bromide (TBAB) catalyst. TBAB is one of the most widely used homogenous catalysts that can decompose toxic and volatile compounds, such as hydrogen bromide, which is highly reactive with epoxy groups, at a high temperature [3PW12O40/ZrO2, and KI with 18-crown-6 ether, have been tested. These catalysts exhibited low efficiency. The performance of TBAB has been enhanced by co-catalysts, such as CaCl2 and SnCl4·5H2O [2 fixation process [The chemical fixation of COperature . HoweverCl4·5H2O . TBAB is process . The dec process .2 to synthesize carbonated soybean oil under atmospheric pressure at 110 °C using 5% of TBAB as a catalyst. They observed viscosity enhancement and a slow reaction rate during this reaction [2 and achieved 94% conversion in 20 h. The aminolysis of a five-member ring cyclic carbonate oligomer was intensified by the catalyst to produce efficient PU or PU-like materials under mild conditions [The nature of the reactor system and its reaction time are interdependent. Tamami et al.  were thereaction ,30. Mazoreaction  intensifnditions . This alnditions .Compared with conventional PU, ocyanate-free PU materials ) have lower permeability, better hydrolytic stability properties, and a safer fabrication process.The formation of intramolecular hydrogen bonds via the hydroxyl group at the β-carbon atom in the PU chain enhances chemical resistance two times higher than the conventional PU . NIPUs aThe major objective of this study is to optimize the reaction parameters, such as temperature and carbonation pressure, of epoxidized JCO, to assess the thermomechanical properties and the chemical resistance properties of cured JCO films and their blends based on NIPU. To the best of our knowledge, industrial JCO has not been previously investigated in NIPU production. Optimizing the cost-effective reaction parameters of CCJO is essential at the scale-up stage to facilitate mass production of JCO-based PU materials at the industrial level. 2O2; 50%), H2SO4, pyridine, CaO, HCl, NaCl, NaOH, TBAB, IPDA, CO2, methyl ethyl ketone (MEK), perchloric acid, denatured ethanol, KOH, phenolphthalein, sodium bicarbonate, anhydrous sodium sulfate, triethylamine, and tetrahydrofuran were purchased from Sigma-Aldrich . All materials were used as received without any further purification.JCO was supplied by the Wahum Edible Oil Sdn. Bhd. . DM, ethyl acetate, acetic anhydrate, benzene, formic acid , hydrogen peroxide .CJCO is obtained through a two-step synthesis, as shown in w/v)) was added to the flask with continuous stirring. The reaction mixture was maintained at 65 °C for 6 h. The resulting mixture was washed with distilled water, followed by sodium bicarbonate solution until it reached a neutral pH. The oily phase was collected and dried with anhydrous sodium sulfate.JCO epoxidation was conducted in a three-necked round-bottom flask that contains formic acid (4.6 g) with JCO (100 g). Hydrogen peroxide  for blank titration; V is a required volume of the NaOH solution (mL) to titrate the hydrochloric acid-acetone solution with dissolved sample; N = normality of NaOH standard aqueous solution and W is the mass (g) of the sample used for the titration.An ultrasonication-assisted titration method was used to determine the epoxy value (Equation (1)). The epoxy conversion was then calculated using Equation (2).2 pressure reactor. EJCO (100 g) was transferred into the reactor. Then, carbon dioxide and TBAB (3.5 g) were added and heated to 120 °C. Complete conversion was monitored by Fourier transformed infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1H-NMR) spectroscopy, and titration measurements. Epoxy conversion was measured at different temperatures and pressures that range from 110 to 140 °C and from 1.0 to 2.0 MPa, respectively.Catalytic carbon dioxide fixation via the carbonation of EJCO was conducted in the presence of TBAB using a COd6) and a specific amount of cyclic carbonate (around 30 mg) were weighted and then put into an NMR tube for carbonate content determination.3 group of the toluene in the 1H-NMR spectra; 1H-NMR tube.The carbonated content of the purified sample was determined from characteristic peaks of cyclic carbonate using the equations given below as described previously by Cornille et al. . A stand1H-NMR spectroscopy. The carbonation yield increased with the increase in temperature with the Arrhenius law. Moreover, the mass transfer kinetics increased with the increase in reaction temperature [The conversion yield of EJCO to CJCO was determined within the temperature range of 100 °C to 140 °C and pressure range of 1.0 to 2.0 MPa with a constant amount of TBAB. The yield of the catalytic carbon dioxide fixation via carbonation of EJCO was monitored by FTIR and 2. However, the carbonation yield of EJCO varied after 30 h. Furthermore, Motokura et al. [2 pressure between 3.0 and 5.0 MPa at 130 °C within a 7 h reaction period. In this study, the high carbonation period of 30 h and the stability of TBAB at low temperature were linked to a high CO2 gradient. 2 pressure, it could be stated that the optimal carbonation conditions would occur at the temperature of 120 °C and under the pressure of 2.0 MPa of CO2 with a fixed amount of 3.5 mol % of TBAB. In the present study, high reaction temperatures of 130 to 140 °C exhibited low conversion yield of five-member cyclic carbonate. However, a high yield was achieved at 120 °C under 2.0 MPa, as shown in a et al.  have obs2 gas inlet, and a thermometer. The flask was immersed in the oil bath placed in a magnetic stirrer. The reaction was maintained at 230 °C for 1.5 h in the presence of 0.03 wt % CaO and was continued until a drop of reaction mixture could be completely dissolved by the methanol. . The reaction mixture was then cooled to 120 °C and 0.12 mol of succinic anhydride was added to the mixture. The temperature of the mixture was increased to 240 °C and was maintained until an acid value in the range of 10–15 was obtained. The synthezised alkyd resin was used for the preparation of EAR and CC-AR in the following steps. A mixture of glycerol and JCO was placed in a three-necked round-bottom flask connected to a reflux tube, a NThe epoxidation and cyclic carbonation of alkyd resin were conducted by following the same procedures as that of the functional group modification of JCO, as shown in JCO-based non-isocyanate polyurethane (NIPU) was prepared using the different compositions of CJCO and CC-AR in the presence of trimethylamine. The calculated amounts of CJCO, CC-AR, and diamine were transferred into a three-necked round-bottom flask that contained MEK. The feed weight of amine was determined based on the mole ratio of amine to carbonate. The reaction mixture was heated to 70 °C for 8 h and to 100 °C for 10 h with continuous stirring at 600 rpm to make NIPU with IPDA, as described previously . Meanwhi−1.The molecular weight distribution of the samples was obtained by gel permeation chromatography (GPC-THF system) at 40 °C in the 510 pump with a flow rate of 1 mL·min−1 and 32 scans per recording. 1H-NMR spectroscopy was performed on a Bruker ARX 300 spectrometer  operating at 600 Hz to study the chemical shifts of the protons and carbonate content using CDCl3 as a solvent, and chemical shifts (δ) were reported in parts per million. The surface morphology of the NIPU film was investigated using a field emission scanning electron microscope  before and after solvent and chemical treatments.FTIR spectroscopy was conducted to analyze the functional groups of the samples and cured NIPU film using the PerkinElmer Spectrum 400 FTIR spectrometer unit using the KBr pellet technique with the resolution of 4 cmTg, wherein 10 mg samples were heated on the TA DSC Q200 calorimeter  from 10 to 250 °C at a heating rate of 10 °C·min−1. The ultimate tensile strength, Young’s modulus (E), and percent elongation at break (εmax) of the blends were obtained by a universal testing machine  at room temperature. Five test specimens were used to obtain the mean value of all tests. The effect of carbonated alkyd resin on the thermomechanical properties of CJCO and its blends based on NIPU was investigated with respect to the mixing ratio of CJCO and CC-AR and the amine content.Differential scanning calorimetry (DSC) was used to determine the glass transition temperature Solvent and chemical resistance tests were conducted in different solvent environments. Water, ethanol (30% aq.), MEK, NaOH, HCl, and NaCl were used for this purpose.−1 (–C–H symmetric stretching vibration of –CH2 groups), 2854 cm−1 (–C–H asymmetric stretching vibration of –CH2 groups), 1745 cm−1 (C=O stretching vibration), 1652 cm−1 (assigned to the –CH=CH–), 1464 cm−1 (–CH2 scissoring), 1243 cm−1 (–CH2 wagging), 1167 cm−1 (–C–O stretching of ester), and the peak at 760 cm−1 is attributed to –CH2 rocking vibration. The chemical shift of the JCO protons was examined by 1H-NMR as shown in a, –CH=CH–, 8H), 5.32–5.25 , 4.34–4.13 , 2.85–2.75 , 2.4–2.3 , 2.1–2.0 , 1.69–1.58 , 1.41–1.24 , and 0.93–0.88  and between 4.36 and 4.12 , 3.16–3.08 , 3.02–2.96 , 2.96–2.89 , 2.4–2.3 , 1.69–1.58 , 1.41–1.24 , and 0.93–0.88 , 5.06–4.52 , 4.36–4.12 , 2.96–2.89 , 2.4–2.3 , 1.69–1.58 , 1.41–1.24 , and 0.93–0.88 . The carbonate content of triglyceride (JCO) and alkyd resin was calculated using the initial and final epoxy contents after the complete removal of TBAB. Ethyl acetate was used to separate TBAB from carbonated JCO. CJCO was obtained as a clear wine red viscous product. The peak at 1805 cm1H-NMR analyses were applied to examine the corresponding peak confirmation and chemical shift of the monoglyceride, AR, EAR, and CC-AR protons. −1 (attributed to the hydroxyl groups of the monoglyceride of JCO) and 2855 cm−1 (–C–H asymmetric stretching vibration of –CH2 groups), 3464 cm−1 , 1740 cm−1 (C=O stretching vibration of monoglyceride), 1738 cm−1 , 1652 cm−1 (assigned to the –CH=CH–), 1464 cm−1 (–CH2 scissoring), and 1243 cm−1 (–CH2 wagging), as illustrated in FTIR and 1H-NMR spectrum of monoglyceride, wherein the corresponding chemical shift of the protons was observed between 5.5–5.32 , 4.20–4.13 , 3.98–3.92 , 3.83–3.80 , 3.64–3.59 , 2.81–2.77 , 2.72–2.67 , 2.4–2.3 , 2.1–2.0 , 1.69–1.58 , 1.41–1.24 , and 0.93–0.88 , 4.34–4.13 , 3.98–3.92 , 3.83–3.80 , 3.64–3.59 , 3.48 , 2.81–2.77 , 2.4–2.3 , 2.1–2.0 , 1.69–1.58 , 1.41–1.24 , and 0.93–0.88 . Figureacid, 9H ).−1 (attributed to epoxy ring formation), 3446 cm−1 (attributed to the hydroxyl groups of EJCO), 2855 cm−1 (–C–H asymmetric stretching vibration of –CH2 groups), 3498 cm−1 (attributed to the hydroxyl groups of CJCO), 1805 cm−1 (C=O attributed to the stretching vibration of carbonyl), 1738 cm−1 , 1652 cm−1 (assigned to –CH=CH–), 1464 cm−1 (attributed to –CH2 scissoring), and 1243 cm−1 (attributed to–CH2 wagging). The 1H-NMR spectrum exhibits the chemical shift of the EAR and CC-AR protons , 3.02–2.96 , and 2.4–2.3 , 1.69–1.58 , 1.41–1.24 , and 0.93–0.88 , respectively. The observed peaks of CC-AR between 5.06–4.52 , 4.36–4.12 , and 2.4–2.3  confirmed the five membered cyclic carbonate fixation of EAR, as illustrated in The functional groups of EAR and the CC-AR were confirmed by FTIR spectroscopy, as illustrated in  protons b. The pe−1 which is greater than the normal vegetable oil . After chemical modification of JCO to CJCO, the molecular weight of JCO increased from 900 g·mol−1 to 1684 g·mol−1. This is because the higher number of unsaturated double bonds in JCO has been transferred to the cyclic carbonate functional groups in the triglyceride molecules of CJCO [The wide use of TBAB is attributed to its faster epoxy conversion than that of heterogeneous catalysts . The pre−1 (attributed to the –N–H– vibration of NIPU), 1704 cm−1 (attributed to the C–O stretching vibration of urethane), 1658 cm−1 (attributed to the C–O–C stretching vibration of NIPU), and 1540 cm−1 (assigned to the N–H– bending and C–N– vibration of NIPU).−1 and the hydroxyl group at 3464 and 3446 cm−1 of CJCO and CC-AR confirmed the formation of the urethane group via the complete conversion of NIPU [Moreover, the disappearance of the corresponding carbonyl peak at 1805 cm of NIPU . In thisTg value (44 °C) with IPDA compared with DM, as shown in Tg compared to those which were crosslinked with DM due to the presence of the cycloaliphetic content in the IPDA material. In this study, the trend of Tg of the CJCO/CC-AR blends crosslinked with DM was not significant. This insignificant trend of Tg might be due to slippage between the chains of the saturated polymer matrix and the excess amount of CC-AR. The blends of CJCO/CC-AR crosslinked with IPDA show a significant drop of Tg with the increase of CC-AR composition due to the amorphous nature of CC-AR [The thermomechanical properties of the prepared CJCO and their blend-based NIPUs are listed in of CC-AR . w/w). It is believed that the saturation and uniform distribution has been achieved in the CJCO/CC-AR blend with the blend ratio of 1:3. Thus, the further increase of CC-AR in the CJCO/CC-AR blend with the ratio of 1:4 (w/w) would result in slipping between the chains of the saturated polymer matrix [This finding can be attributed to the uniform and stoichiometric distribution of the high and low five-member cyclic carbonate functionalities of CJCO and CC-AR, which enhanced the crosslink density of the polymerization network . Howeverr matrix . Thus, tThe solvent and chemical resistant properties of the NIPU films were measured by the swelling test. The effect of different solvents on the interfacial surface of the NIPU film was investigated based on the attractive forces caused by the polarity of the film surface and the solvent systems . On one Water adsorption on the NIPU film surface was initiated, followed by chemisorption and physisorption via cohesive interaction (intermolecular forces) of the water molecules ,51. ThesThis phenomenon might occur due to the high steric hindrance of the cycloaliphatic content of the IPDA. The swelling behavior of the NIPU films was significantly reduced in the ethanol–water system b compareHowever, the adhesive attractions of the polar ethanol molecules might be enhanced by the β-hydroxyurethane groups of the NIPU film surface via weak van der Waals forces  toward tMoreover, the polarized MEK altered the morphology of the film, as shown in The chemical resistance property of the NIPU films was investigated in different chemical environments, such as diluted HCl (10%), aqueous NaCl (10%), and NaOH (5%). The carbonate content, crosslinking density, β-hydroxyl moiety, and intermolecular rigidity of the blends show a significant effect on the chemical resistance behavior of the cured films. Moreover, the soft segments contribute to alteration of the film surface by chemisorption, whereas the hard segments enhance physisorption via β-hydroxyurethane hydrogen bonds. By contrast, all NIPU films exhibited a significant weight augmentation in their swelling–time diagram under the NaCl solvent system, as shown in + ions toward SCI [2O)17 cluster presents four water molecules coordinated to the −OH oxygen and one water molecule coordinated to hydrogen, thus making the total solvation number of −OH in this cluster equal to 5 [The weight augmentation might also be due to the electrophilic attraction of the solvated Naward SCI . The optual to 5 ,58, as sual to 5 .2 pressure. The carbonate conversion of CJCO and CC-AR was observed at 99% and 97%, respectively, within a 30 h reaction period. The carbonate content of CJCO was higher than that of CC-AR. Compared with the CJCO-based NIPU, the blend of CJCO and CC-AR-based NIPU exhibits excellent thermal, mechanical, solvent, and chemical resistance properties when cured with DM and IPDA. Furthermore, the amine and cycloaliphatic contents of IPDA play a major role in surface interfacial phenomena via sorption processes. In the present study, the strong solvent and chemical resistance properties exhibited by cured blends in different solvent environments can be ascribed to the inclusion of CC-AR in the blend. The amine content, intermolecular attractions of the polymer chains, β-hydroxyurethane hydrogen bonds, and cycloaliphatic content of IPDA can contribute to the enhancement of the interfacial surface of the CJCO/CC-AR blend. The NIPU film with high amine content (30.8% of IPDA) exhibited better solvent and chemical resistance compared with the CJCO/CC-AR blend (ratio of 1:3) and the CJCO film cured with DM.In this study, the carbonation of EJCO and monoglyceride-based carbonated alkyd resin was conducted successfully. The conditions for epoxidation and carbonation reactions will significantly influence the carbonate content of CJCO and CC-AR. The optimal reaction condition was set at 120 °C and 2.0 MPa of CO


Study goal was to elucidate the effect of NOD on rhinomanometric parameters in this group of patients. Atopic bronchial asthma (BA) in children is associated with upper airways pathology (UAP). Among them, a combination of allergic rhinitis (AR) and nasal obstructive disorders (NOD), including hypertrophy of the pharyngeal tonsil (HPT) and anomalies of the intranasal structures (AINS), is abundant. In such patients, anterior active rhinomanometry (AARM) is an important method of examining nasal patency. However, NOD can influence the AARM parameters in children with BA and nasal symptoms, and this effect must be taken into account in clinical practice.Total of 66 children with BA and AR were examined with AARM, rhinovideoendoscopy, spirometry, and standard clinical tests allowing revealing the structure of comorbid pathologies. In order to avoid the influence of anthropometric parameters of children and their age on AARM parameters, a special index of reduced total nasal airflow was used.It has been established that NOD, especially HPT, have a significant negative impact on the indices of anterior active rhinomanometry during the periods of both AR remission and AR exacerbation. The effect of AINS is much weaker and was remarkable only in combination with HPT. Bronchial asthma (BA) is a common chronic airway disease in children which, in most patients, is associated with allergic rhinitis (AR) –4. AR asThe standard method for assessing bronchial patency is the method of spirometry, which is included in current recommendations for the management of patients with asthma , 11. WheThere are only few studies of nasal patency evaluation with the objective methods in asthmatic patients , 16. TheA discussion remains about the association of nasal symptoms and rhinomanometric indicators in patients with AR. In the studies of Ciprandi et al.  and MozzThus, the available results leave unclear the question on how accurately the results of AARM method determine the objective status of asthmatic patients with AR under conditions of comorbid pathologies including NOD. Timely objectification and precise diagnosis of the effect of these pathological conditions on the respiratory function of the nose would enable us to assess the real clinical picture and prospects of both conservative therapy and the need for surgical correction in children with torpid nasal symptoms due to the combination of AR and NOD , 7, 26.An additional complication in the interpretation of rhinomanometric parameters in children is the potential dependence of these parameters on the age and anthropometric parameters of a child. At present, there are studies that characterize rhinomanometric indicators in children in selected populations –29, but Thus, the current lack of data on the NOD influence on the parameters of AARM in children with asthma and nasal symptoms is an objective factor that reduces the reliability and diagnostic significance of AARM in conditions of real clinical practice in monitoring nasal respiratory function. In this study, we investigate this issue by comparing the AARM parameters in children with asthma and nasal symptoms, taking into account the presence or absence of a combination of AR and NOD, as well as the period (remission or exacerbation) of AR in these patients. In addition, we pay special attention to the influence of the age of patients on the characteristics under study and introduce additional characteristics that allow account for the influence of the size of the nasal cavity on monitoring parameters.The study was conducted according to the Helsinki Declaration adopted in June 1964  and revised in October 2000 . The study was approved by the Ethics Committee of Privolzhsky Research Medical University, protocol No 13 of 10.10.16. Informed consent was obtained from the patients between 15 and 17 years old and from the parents of patients under the age of 15, according to the Federal Law “Fundamentals of the Legislation of the Russian Federation on the Protection of Health of Citizens” of July 22, 1993, No. 5487-1.A total of 66 children and adolescents aged from 4 to 17 years were examined, the average age was 8.2±4.3, and boys amounted for 60.6% (40/66), who were on treatment for asthma at the Children's City Clinical Hospital No. 1 of Nizhny Novgorod, Russia, and who had nasal or sinonasal complaints. All the children had a symptomatic complex that was characteristic of BA and AR, family anamnesis associated with atopy was evaluated , positive skin test results were obtained, or high titers of specific class E immunoglobulins were detected at least for one of the most common aeroallergens of the Volga-Vyatka region of the Russian Federation.Criteria for inclusion were the diagnosis of asthma, made in accordance with the existing international and national conciliation documents, the presence of nasal or sinonasal complaints and symptoms in patients . ExclusiDepending on the comorbid UAP pathologies, four groups of patients were considered: Group 1 (BA patients with isolated AR (without NOD)), Group 2 (BA patients with AR+HPT), Group 3 (BA patients with AR+AINS), and Group 4 (BA patients with a combination AR+HPT+AINS). In order to assess the effect of AR severity, two groups of patients were additionally considered in a whole cohort: Group A (patients with AR in the remission period) and Group B (patients with AR in the exacerbation period).Due to the presence of nasal symptoms, all the patients were examined by an otorhinolaryngologist, who conducted a routine examination and a rhino video-endoscopic examination of upper airways. Rigid rhinoscopes from the company Karl Storz (Germany) with a viewing angle of 0 and 30°, diameter of 2.7 and 4.0 mm, and a flexible nasopharyngoscope 3.2 mm were used. Video recording was done by means of Atmos camera (Germany). Rhinovideoscopic examination was performed after instillation of a 2% solution of lidocaine on the nasal mucosa and application of anesthesia using 0.1% solution of epinephrine hydrochloride and 10% lidocaine. AR and allergic rhinosinusitis were diagnosed in accordance with the available international recommendations. The involvement of sinuses in the pathological process was noted using the criteria of the European Position Paper on Rhinosinusitis and Nasal Polyps (EPOS), 2012 . In asse3/s. Below, only the data for the pressure of 150 Pa/cm3/s corresponding to the reference anthropometric data of , where Me is the median and  is 95% confidence interval. The differences between continuous variables of two groups were compared using the ANOVA criteria and Mann-Whitney U test. The differences between three groups were analyzed using multiple ANOVA criteria and the Kruskal-Wallis test. Correlations between the two sets were estimated using the Spearman determination coefficient. The values of p<0.05 were considered statistically significant.In all examined patients with asthma, AR was verified as persistent 86.4% (57/66) or intermittent 13.6% (9/66). This strong association of BA and AR is consistent with our previously published data, as well as with the data provided by Blaiss et al. , 39. Acc3/s. Differences between TNAF in boys (n = 40) of 565.8  cm3/s and in girls (n = 26) of 593.4  cm3/s were not detected, p = 0.64. In contrast, the groups of patients in different AR periods had significantly different TNAF values, p < 0.0001. Group A of patients in the remission of AR had the median TNAF values of 734.5.1  cm3/s which corresponded to a mild degree of nasal congestion. For Group B (exacerbation period of AR), these indicators were 409.0  cm3/s, which corresponds to the congestion of an average degree of severity.In the AARM evaluation of the respiratory function, the median TNAF value was 576.7  cmn=66, R=0.299, p=0.0148) and in the patients with different AR period. In the period of exacerbation, the correlation between TNAF and body height is most pronounced  whereas in the period of remission the correlation is significantly lower . The linear regressions for patients of Groups A and B are shown in The measured TNAF values depending on their body height are shown in 3/s) and body heights h (in cm) for mixed (boys+girls) population. Although this dependence was published in a logarithmic form, it has essentially linear character and we approximate it by the linear expressionOne of the most important issues when discussing the AARM results in children is the question of the reference rhinomanometric values and their dependence on anthropometric parameters of patients , 27, 28.The confidence interval of the reference TNAF values for healthy patients  is shownIt is also evident from 3/s, h is the body height in cm, and the measurement units of RTNAF are cm2/s. This quantity includes the body height, not the age of the children, since the nasal flow obviously depends on the size of the patient's nasal cavity, which is primarily related to the length of the child's body  cmi et al. , where t3/s. It should be noted that the causes of nasal obstruction were not elucidated in their study and the children's age was from 6 to 15 years. At the same time, Chen I. et al.  cm3/s during the AR remission and 409.0  cm3/s during the AR exacerbation. Thus, our results demonstrate clear and statistically significant correlation with the severity period of comorbid pathology. Design of the study [It was found that the nasal respiratory flow values, evaluated both by the direct measurement (TNAF) and using the relative RTNAF index, are significantly higher in children during AR remission than in children with acute AR. Although this fact is expected, there are some contradictions in the available publications about the significance of TNAF in children with BA. So, Yukselen et al.  observed. et al.  observedhe study  excludedThe differences in the nasal flow rate in children with BA, given in different sources, can be associated with several factors. First, the activity of allergic inflammation  was not considered. Second, the anthropometric and age parameters of the child were not taken into account. Third, the structure of comorbid UAP, including NOD, that potentially can occur in these children was not accounted. This indicates the relevance of standardizing the evaluation of indicators of AARM in children, including patients with asthma, which has objective difficulties in comparison with the category of adults.We have established that the studied indicators  both in the period of remission of AR and in the period of acute AR have the highest values in children with isolated AR compared with children who have a combination of AR and NOD. In the period of AR remission, the most significant limitations of the nasal respiratory function were observed in children with the combination AR+HPT+AINS, as evidenced by both TNAF measurements and RTNAF index. In the AR exacerbation period, any NOD types as a whole have a negative impact on the nasal respiratory function in comparison with isolated AR, but the most pronounced decrease in nasal respiratory function is observed in children with HPT, as in the case of a combination of AR+HPT and with the combination of AR+HPT+AINS. This is evidenced by the analysis of both TNAF and RTNAF values. Thus, in children with asthma and nasal symptoms in the period of AR remission, significant deterioration in nasal respiratory function is observed mainly in children who have a combination of AR with HPT and AINS. In the period of exacerbation of AR, the most significant contribution to the disruption of the nasal respiratory function is made by HPT, both in the form of a combination of AR+HPT and in the form of AR+HPT+AINS.3/s in children with HPT, which was significantly lower than in children who did not have HPT  [3/s. Studies that reflect the effect of HPT on rhinomanometric parameters in children with asthma have not been found.We found single publications, mainly Zicari A.M. and coauthors, indicating a decrease in TNAF to 629.1 ± 146 cmlergies) . These dDuring the acute phase of AR, children with HPT had the lowest values of both TNAF and RTNAF, which indicates a significant negative effect of HPT on the respiratory function of the nose in children during acute AR. Similar patterns were obtained in patients who have a combination of AR+HPT+AINS (somewhat higher than in children with AR+HPT). In the period of exacerbation of AR, patients with HPT show the most significant decrease in nasal respiratory flow.We have not established a significant influence of AINS  on the parameters of AARM in the period of remission of AR in children with asthma, both in the analysis of absolute TNAF values and in the analysis of the RTNAF index in comparison with children who had isolated AR. This, perhaps, may be due to the compensatory capabilities of one of the half of the nose, having a larger volume with AINS. When analyzing the values of the RTNAF index, it was established that, in the period of AR remission, the value of the nasal flow when leveling the effect of anthropometric parameters in children with HPT is statistically not distinguished from the nasal flow, of patients having isolated AR.Analysis of TNAF in a group of children who had a combination of AR with AINS in the acute phase of AR showed a tendency to decrease nasal patency in patients with AINS, in comparison with children with isolated AR. In the period of exacerbation of AR, the absolute values of TNAF, but not RTNAF rates, are decreased as compared to the isolated AR. Thus, the effect of AINS on the nasal flow during the AR exacerbation period is not obvious.The influence of AINS on the rhinomanometric parameters of the respiratory function of the nose in the children's population, and in particular in patients with asthma, is not adequately covered in the literature. We found mainly studies performed in adults as a criterion for assessing the respiratory function in surgical intervention for the correction of AINS . In chilRhinomanometric studies in children with asthma and nasal symptoms show a clear dependence of the severity of nasal obstruction from the AR phase (remission or exacerbation). In addition, a negative effect on the state of nasal respiratory function of HPT, more pronounced with exacerbation of AR, was found, which should be taken into account when managing these patients. The effect of HPT on the decrease in nasal flow in the period of remission of AR is less significant than in the period of exacerbation. The effect of AINS on the nasal respiratory function of the nose is not found. Our results demonstrate that the interpretation of the AARM indices in children with asthma and nasal symptoms should take into account the entire spectrum of the existing pathology of UAP in this category of patients. At the same time, when analyzing the AARM parameters, it is important to take into account the influence of the patient's anthropometric indices on the parameters of the nasal respiratory flow.


Resistance to androgen receptor (AR)-targeted therapies in prostate cancer (PC) is a major clinical problem. A key mechanism of treatment resistance in advanced PC is the generation of alternatively spliced forms of the AR termed AR variants (AR-Vs) that are refractory to targeted agents and drive tumour progression. Our understanding of how AR-Vs function is limited due to difficulties in distinguishing their discriminate activities from full-length AR (FL-AR). Here we report the development of a novel CRISPR-derived cell line which is a derivative of CWR22Rv1 cells, called CWR22Rv1-AR-EK, that has lost expression of FL-AR, but retains all endogenous AR-Vs. From this, we show that AR-Vs act unhindered by loss of FL-AR to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrate that AR-Vs drive expression of a cohort of DNA damage response genes and depletion of AR-Vs sensitises cells to ionising radiation. Moreover, we demonstrate that AR-Vs interact with PARP1 and PARP2 and are dependent upon their catalytic function for transcriptional activation. Importantly, PARP blockade compromises expression of AR-V-target genes and reduces growth of CRPC cell lines suggesting a synthetic lethality relationship between AR-Vs and PARP, advocating the use of PARP inhibitors in AR-V positive PC. AR gene (Prostate cancer (PC) is the second most common malignancy in men with approximately 1.3 million new cases reported worldwide in 2018 (World Cancer Research Fund). At presentation, PC growth is androgen-dependent hence current treatments act to attenuate the androgen receptor (AR) signalling axis via the use of hormonal therapy, including anti-androgens . AlthougAR gene , as wellAR gene ,13, whicCritically, AR-Vs represent a major clinical challenge. Unlike wild-type and mutant FL-AR isoforms that are generally repressed by next-generation anti-androgens enzalutamide and apalutamide ,15, AR-VA major limitation in the study of AR-V biology, however, is the paucity of models that permit discriminate AR-V-specific functional and phenotypic read-outs that are not influenced by FL-AR. Utilising either FL-AR siRNA-mediated knockdown or enzalutamide treatment in FL-AR- and AR-V-expressing CWR22Rv1 and VCaP cell lines, several groups have attempted to establish models for interrogating splice variant transcriptomics and co-regulator requirements . AlthougExon Knockout) is dependent upon AR-Vs for growth, is refractory to all FL-AR-targeting agents and displays a gene expression programme similar to parental CWR22Rv1 cells consistent with FL-AR and AR-V transcriptional mimicry. Furthermore, we demonstrate for the first time that AR-Vs regulate a DNA damage response (DDR) gene network encompassing a FL-AR-like ‘BRCAness’ signature  and the Nucleofector® II device (programme T-009) according to manufacturer's instructions. Editing efficiency of the two distinct CRISPR complexes was assessed in CWR22Rv1 cells using the SURVEYOR assay (Integrated DNA Technologies) according to manufacturer's instructions. This methodology enables the efficiency of CRISPR-mediated DNA insertions and deletions (indels) to be estimated by firstly mixing (at 1:1 ratio) wild-type and genome-edited amplicons, derived from PCR amplification of the target locus, and then digesting mis-annealed duplexes using the Surveyor nuclease. Resultant DNA is then subject to electrophoresis and CRISPR efficiency is calculated by comparing relative intensities of the wild-type amplicon to CRISPR-derived lower molecular weight species using ImageJ. Additionally, PCR products were sequenced and chromatograms were analysed by the TIDE (Tracking of Indels by DEcomposition) algorithm  to accurately calculate the editing efficiency of Cas9.Two custom gRNAs were designed to target distinct loci within exon 5 of the Mse I restriction enzyme site which was used in restriction fragment length polymorphism (RFLP)-based analysis to enable detection of successful knock-in clones. CWR22Rv1 cells were co-nucleofected with the donor template (1 μg) and Cas9/gRNA_2 construct (6 μg) for 48 h prior to puromycin selection (2 μg/ml) for 5 days and subsequent single cell sorting using a FACS Aria II flow cytometer (BD Biosciences) to clone out single, DAPI negative cells.To knock-in a stop codon into exon 5 of the AR locus, a 180 bp ssODN template was custom designed (Sigma) containing a central TAA sequence and flanked by 75 bp 5′ and 3′ termini 100% complementary to the AR gene sequence. Additionally, the stop codon generated an 1. All cells were maintained in RPMI 1640 media (Sigma) supplemented with 10% foetal calf serum (FCS) and 5% l-glutamine at 37°C and subject to regular mycoplasma testing. Enzalutamide (Selleckchem) and dihydrotestosterone (DHT) (Sigma) were utilised at 10 μM and 10 nM, respectively. PARP inhibitors rucaparib, olaparib and talazoparib were all purchased from Selleckchem and used at 0.5 and 1 μM for assay-specific durations.LNCaP, CWR22Rv1 and VCaP cells were all purchased from ATCC and authenticated prior to conducting the study (see Quantitative PCR (qPCR) was used to assess expression of candidate AR-V target genes and those identified from RNA sequencing experiments see . RNA purWestern blotting was performed as described in  using thTransient transfection of siRNA to deplete CWR22Rv1-AR-EK and CWR22Rv1 cells of all AR isoforms or discriminately of FL-AR and AR-Vs was performed in dextran-coated charcoal-stripped foetal bovine serum (HyClone) containing media  in six-well plates (Corning) using 25nM of siRNA per well and Lipofectamine RNAiMax reagent (Life Sciences) according to manufacturer's instructions. All siRNAs were purchased from Sigma and sequences are listed in cis-regulatory elements of AR target genes  assays (as described in ) or tryp5 cells grown on glass coverslips in six-well plates were transiently transfected with 3 μg of the appropriate pLV-U6g-EPCG vector for 24 h prior to fixing in 4% paraformaldehyde and mounting cells on glass slides using DAPI-containing mounting media (Vectashield) prior to fluorescence microscopy (Nikon TE2000). AR-V localisation was assessed in CWR22Rv1-AR-EK cells seeded in steroid-depleted media in chamber slides (4000 cells/chamber) (Thermo Fischer Scientific). Staining was carried out using the AR (D6F11) primary antibody  at 1:1000 for 1 h at room temperature. Following three PBS washes, cells were incubated with an AlexaFluor 488 secondary antibody for 1 h at room temperature prior to mounting with DAPI and imaging. Finally, to assess γH2AX foci formation after 2 Gy ionising radiation treatment, cells grown on glass coverslips were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.1% Trinton X-100/PBS for 10 min and then blocked in 4% BSA (Sigma) for 1 h prior to incubation with γH2AX antibody  at 1:1000 dilution at 4°C overnight. Cells were washed in PBS and then incubated with an AlexaFluor 546 secondary antibody (Life Technologies) for 1 hour prior to mounting. Images were captured as z-stacks using a Leica upright DM6 fluorescence microscope. Automated foci analysis was performed using a macro in ImageJ as described in  threshold was set at 0.01; hence up- and down-regulated genes were identified as those with FDR ≤ 0.01 and respective FC of ≥1.5 and ≤–1.5.CWR22Rv1-AR-EK cells were transiently transfected in triplicate for 48 hours with control or AR-targeting siRNAs before RNA extraction using RNeasy Mini Kit according to manufacturer's instructions (Qiagen) and QC. 500 ng RNA of each triplicate sample was subject to library preparation using TruSeq Standed mRNA library prep kit (Illumina). Resultant libraries were subject to paired-end (100–125 bp) sequencing on an Illumina HiSeq 2500 sequencer, generating an average of 30 million reads per sample. Each data-set was mapped against the human reference genome (Hg19) utilising STAR and then analysed with HTSeq to extract counts and DESeq2 to perform the differential gene expression comparisons between control and AR-depleted samples utilizing the n = 12), localized PC (n = 49) and metastatic CRPC (n = 27) using the Gene Expression Microarray Analysis dataset from Grasso et al. . For analyzing the effect of siRNA-mediated knockdown or PARP inhibitor treatment on AR-mediated gene expression, chromatin enrichment and cell viability by qRT-PCR, ChIP and clonogenics experiments, respectively, one-way ANOVA and two-tailed student T-tests were conducted depending on the number of variables and *AR gene to ablate cellular FL-AR levels while maintaining expression of all AR-Vs endogenous to the parental cell line  analysis (AR gene locus (data not shown). This CWR22Rv1 cell derivative was named CWR22Rv1-AR-EK (Exon Knockout) and, as predicted, FL-AR was not detectable by western blot analysis utilizing multiple N-terminal-binding antibodies, but AR-V levels were maintained and could be depleted using exon 1-targeting siRNAs  were sequenced and showed no mutations compared to wild-type suggesting that any alteration to this cell line is a consequence of FL-AR loss and not aberrant CRISPR-mediated mutations  demonst  reveale  indicat  and the  before quantifying γH2AX foci 24 h post-treatment to measure DNA damage repair proficiency. Consistent with compromised expression of the DDR gene signature, cells deficient of AR-Vs (siARexon1) demonstrated elevated γH2AX foci compared to control (siScr) cells indicating AR-V-driven DDR-associated gene expression is important for maintaining DNA integrity in CRPC cells post IR treatment Figure  and is cBRCA1, BRCA2 and RAD54B. Moreover, several ‘BRCAness’ genes were downregulated in CWR22Rv1-AR-EK cells upon AR-V depletion using an N-terminal-targeting siRNA , we show that PARP1/2 inactivation reduces AR-V signalling both in 22Rv1-AR-EK cells and, with the exception of UBE2C, in the parental cell line grown in steroid-depleted media  inhibitors  suggestia Figure . Expandin levels  indicaticis-regulatory elements of AR target genes. As shown in Figure PSA and CCNA2 in both cell lines grown in steroid-depleted media suggesting that the enzymes co-associate with the constitutively chromatin-bound AR-Vs at target loci, as previously demonstrated  suggesting discriminate binding capacities of the enzymes to AR target genes. Consistent with down-regulation of AR-V transcriptional activity, we demonstrated that short-term treatment of the CWR22Rv1-AR-EK and parental derivatives with 1 μM talazoparib significantly reduced AR-V enrichment at a number of androgenic loci, including PSA, CCNA2 and KLK2  has an edited AR gene containing a knock-in stop codon to prevent synthesis of FL-AR protein, and wild-type exons encoding the N-terminal transactivation and DNA-binding domains, to enable expression of all AR-Vs nascent to the parental cell line. Key validation experiments have indicated that FL-AR is not detectable in this new derivative using a host of anti-AR antibodies in IP and immunoblotting experiments, while also demonstrating no off-target CRISPR activity at several of the highest-ranked off-target loci. One potential issue with the use of CWR22Rv1 cells is the presence of an intragenic duplication within the AR gene, encompassing exon 3, that could impact on synthesis or activity of AR isoforms. Indeed, a TALEN-engineered CWR22Rv1 cell derivative lacking this duplication (termed 22Rv1-undup1–3) demonstrated elevated FL-AR levels and reduced expression of AR-Vs suggesting that generation of AR isoforms may be distinctly regulated in the CWR22Rv1 model containing the aberrant AR gene , which   in the clinic. Our data and that of others , howevercis-regulatory elements of AR-V-target genes in parental and CRISPR-modified CWR22Rv1 cells and PARP inactivation down-regulates both AR-V and PARP1/2 chromatin-binding. Further interrogation of our transcriptomics data derived from CWR22Rv1 and CWR22Rv1-AR-EK cells identified that both PARP2 and PARBP were down-regulated in response to AR-V knockdown suggesting the existence of a feed-forward regulatory loop between PARP1/2 and AR-Vs that can help elevate and sustain the expression of AR-V-mediated androgenic and DDR gene signatures. Consistent with these findings, cellular PARP activity was shown to be diminished in cells depleted of AR-Vs, suggesting that down-regulated expression of both PARP2 and PARPBP contributes to compromised cellular enzymatic activity. Intriguingly, this finding is at odds with recent data demonstrating that anti-androgen treatment up-regulates PARP activity in FL-AR-expressing C4–2 cells which was suggested to be a redundancy mechanism driven by loss of AR-mediated HR-associated gene expression  (accession number: GSE126306).gkz286_Supplemental_FileClick here for additional data file.