Many of the cost-effectiveness analyses of apixaban against warfarin focused on Western populations but Asian evidence remains less clear. The present study aims to evaluate the cost-effectiveness of apixaban against warfarin in Chinese patients with non-valvular atrial fibrillation (NVAF) from a public institutional perspective in Hong Kong.We used a Markov model incorporating 12 health state transitions, and simulated the disease progression of NVAF in 1,000 hypothetical patients treated with apixaban/warfarin. Risks of clinical events were based on the ARISTOTLE trial and were adjusted with local International Normalized Ratio control, defined as the time in therapeutic range. Real-life input for the model, including patients’ demographics and clinical profiles, post-event treatment patterns, and healthcare costs, were determined by a retrospective cohort of 40,569 incident patients retrieved from a Hong Kong-wide electronic medical database. Main outcome measurements included numbers of thromboembolic and bleeding events, life years, quality-adjusted life years (QALYs) and direct healthcare cost. When comparing apixaban and warfarin, treatment with incremental cost-effectiveness ratio (ICER) less than one local GDP per capita  was defined to be cost-effective.In the lifetime simulation, fewer numbers of events were estimated for the apixaban group, resulting in reduced event-related direct medical costs. The estimated ICER of apixaban was USD 7,057 per QALY at base-case analysis and ranged from USD 1,061 to 14,867 per QALY under the 116 tested scenarios in deterministic sensitivity analysis. While in probabilistic sensitivity analysis, the probability of apixaban being the cost-effective alternative to warfarin was 96% and 98% at a willingness to pay threshold of USD 33,534 and 100,602 per QALY, respectively.Apixaban is likely to be a cost-effective alternative to warfarin for stroke prophylaxis in Chinese patients with NVAF in Hong Kong. Non-valvular atrial fibrillation (NVAF), the most common sustained cardiac arrhythmia, affects 1–2% of the general population worldwide and its prevalence increases with age . TherefoLong-term anticoagulation management is required for stroke prophylaxis in patients with NVAF. Traditionally, warfarin (a vitamin K antagonist) has been the most commonly used anticoagulant . HoweverIn addition to improved clinical efficacy and safety compared against standard treatment, cost-effectiveness is also an important consideration for new treatments in the setting of increased healthcare costs globally. Apixaban has been found to be cost-effective against warfarin in Europe –10, AustThe research protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster, Hong Kong. No written informed consent was obtained from patients for their clinical records to be used in this study, due to this is a retrospective study based on electronic medical database. All of the patient records/information was anonymized and de-identified prior to analysis. Lifetime cost and health outcomes were estimated from Markov cohort simulations. A retrospective cohort study was conducted to obtain real-life data for the model inputs. Clinical event risks were based on the ARISTOTLE trial [Tables A and B in S1 File. Model inputs were entered, cross-checked and analysed independently by two researchers.This study compares the cost-effectiveness of apixaban against warfarin in Chinese patients with NVAF using a Markov model. Data sources and the retrieval procedure are illustrated in LE trial  and incoFigures A and B in S1 File. A detailed description of the model can be found in previous publications [A previously developed Microsoft Excel Markov model incorporating 12 health states was adapted to simulate lifetime disease progression in 1,000 patients treated by apixaban (5mg BID) or warfarin (any strength). The heaications , 10, 15.The target population of the model are patients with NVAF who are suitable for warfarin therapy. Patient profiles were determined by a retrospective cohort analysis of incident patients with NVAF in the Clinical Data Analysis and Reporting System (CDARS). In brief, CDARS is an electronic medical database in Hong Kong that covers a population of over 7 million utilising public healthcare services and accounts for 80% of inpatient records in the territory . De-idenTable C in S1 File for ICD-9 codes used). Among these patients, those with diagnoses of valvular heart disease, hyperthyroidism or had undergone valvular replacement surgery within 1 year prior to the index date were further excluded according to corresponding ICD-9 codes. The follow-up of each patient was commenced from the index date until the occurrence of any clinical events, death or July 31st 2014, whichever came first. This resulted in the identification of 40,569 eligible patients with 76,196 patient-years of follow-up. The key demographic and clinical profiles of these patients are shown in At the time of the study, CDARS was unable to identify patients with NVAF directly. Target patients were identified by first retrieving patients with documented diagnosis of atrial fibrillation from Jan 1st 2010 to Dec 31st 2013 using ICD-9 diagnosis codes .Healthcare resource consumption regarding anticoagulation management was reflected by the frequency of routine care and monitoring visits, proportion of patients experiencing dyspepsia and proportion of patients requiring annual renal monitoring whilst on treatment from the CDARS retrospective cohort (Table A in S1 File). Following all bleeding events, a certain number of patients were assumed to switch to aspirin as second line treatment. The rate of treatment switch (± 90 days of the event index date) was determined by retrieving prescription records from CDARS and literature. The remaining patients were modelled to have their original treatment interrupted for 6 weeks then restarted [Table A in S1 File, post intracranial haemorrhage, 44% of patients switched to aspirin and the remaining 56% discontinued their original treatment temporarily and restarted the same treatment 6 weeks later). [Treatment patterns post-event were determined from the CDARS retrospective cohort study, published literature or expert opinion wherever appropriate (estarted  (i.e. as later). FollowingTable B in S1 File.Utility for NVAF was adopted from Ho et al , which wThe analysis was conducted from a public institutional perspective in Hong Kong so only direct medical costs were considered. Costs are presented to the value of US dollars in 2014 (1 USD to 7.75 HKD) and discounted at 3.5% per year according to cost-effectiveness analyses (CEA) guideline .Table B in S1 File. Daily drug costs for warfarin and apixaban were based on local retail prices . Annual monitoring and management costs were determined by multiplying the cost for blood and renal tests per visit by number of clinical visits per patient from the CDARS retrospective cohort. Similarly, acute care costs associated with clinical events were calculated by multiplying daily inpatient charges in public hospital [The total cost components are outlined in hospital , 26 by tWe compared the cost-effectiveness of apixaban versus warfarin by assessing the differences in lifetime cost and clinical benefits. Base-case analysis was performed based on model inputs described earlier. Treatment with incremental cost-effective ratio (ICER) less than the willingness-to-pay (WTP) threshold of one local GDP per capita  for eacTables A and B in S1 File) while all the others were constant to project its effect on ICER. All tested parameters were ranked according to ICER variation and the top 15 most influential parameters were presented in Tornado diagrams.The effect of uncertainties in the base-case analysis was evaluated by deterministic and probabilistic sensitivity analyses (SA). In deterministic SA, each of the 116 input parameters varied from a predefined range  and varied simultaneously in 2,000 iterations for the pair-wise comparisons. The cost and health outcomes were further assessed using cost-effectiveness acceptability curves (CEACs). Two willingness-to-pay (WTP) thresholds, one-time and three-times of local GDP per capita  were apTable 2). Compared with warfarin, 36 patients need to be treated with apixaban to avoid one additional stroke, and apixaban is the dominant treatment in reducing bleeding events.From a cohort of 1,000 patients over a lifetime, the use of apixaban was predicted to result in 116 fewer NVAF-related events  and 31 fewer event-related deaths than patients treated with warfarin in the base-case analysis (Table 2). This was predominantly attributed to the higher drug price of apixaban, in contrast to lower costs related to management, monitoring and clinical events. Compared with warfarin, the net cost, net health benefits and incremental cost-effective ratios of apixaban were detailed in The cost of treating one patient with apixaban was USD 1,228 more than warfarin . Uncertainties in drug costs, the risk of cardiovascular hospitalisation for both treatments, risk of ischaemic stroke for apixaban and risk of intracranial haemorrhage for warfarin had greatest impact on the ICER.As illustrated in Fig 3). ICER was below the threshold of USD 33,534 per QALY  in 96% of the trials. When the upper range of the WHO threshold of USD 100,602 per QALY gained  was applied, 98% of the trials supported the cost-effectiveness of apixaban.The probabilistic sensitivity analysis demonstrated that apixiban is more cost-effective against warfarin over a life-time horizon  fewer thromboembolic events were estimated for the apixaban group in the lifetime cohort simulations, resulting in corresponding increased LYs, QALYs and reduced event-related healthcare costs; (ii) compared with warfarin, apixaban presented an ICER of USD 1,061–14,867 per QALY gained which is within the common acceptable WTP of one local GDP per capita as recommended by the WHO; (iii) bThe greater health benefits and cost-effectiveness of apixaban findings in this study are in line with other modeling CE studies conducted in western developed countries –12. Lee 2  score, similar risk factor profiles were shared, except previous stroke is more prevalent in Asians (29% vs. 18%) [In light of the widely reported cost-effectiveness of apixaban in Caucasians, evaluation of the Asian population is still limited. However, from a clinical perspective, Asian and Western patients with AF are not necessarily the same. Firstly, among the five components of the CHADSvs. 18%) . Secondlvs. 18%) , 7. Thirvs. 18%) , 7. Althn Asians % vs. 18%vs. 18%) , which hAnother important differentiator and strength of our study is the use of real-life, local INR control data in our model. It is well documented in the published literature that good INR control, although important for good clinical outcome , is unusOf note, variations in healthcare system and services have been well recognised worldwide  and this2≥2). In addition, the cut-off point of assessing treatment switch was restrict to 90 days, based on Witt et al’s study on the association of warfarin interruption and gastrointestinal tract bleeding [There are limitations in this modeling analysis. Firstly, clinical efficacy and safety parameters were based on the landmark trial with 1.8 years of follow-up. They were assumed to be constant over the lifetime in the model, but might not reflect the long-term performance of treatments in real terms. Secondly, following a major bleeding event, treatment options were limited to switching to aspirin or restarting the original anticoagulant in 6 weeks. In real-life clinical practice, some patients may cease treatment or choose other anticoagulants. However, aspirin is perceived as a safer alternative to oral anticoagulants in the Chinese population  given thbleeding . This mableeding . Thirdlybleeding  which maIn conclusion, the current comprehensive modeling assessment based on real-life, local clinical, economic evidence and ARISTOTLE trial, demonstrate that apixaban is a cost-effective alternative against warfarin for the prevention of stroke and bleeding events in Chinese patients with NVAF. Our results suggest that apixaban is relatively more favourable in terms of health benefits and incremental cost.S1 FileFigure A. Schematic representation of Markov model. NVAF: non-valvular atrial fibrillation. Figure B. Health states transitions from NVAF. CRNM Bleed: Clinically relevant non-major bleed; ICH: Intracerebral haemorrhage; NVAF: Non-valvular atrial fibrillation; NVAF without original anticoagulation: patients with NVAF who discontinue the initially assigned anticoagulation treatment but have not yet experienced any of the events including ischaemic stroke, haemorrhagic stroke, myocardial infarction, systematic embolism or death. Table A. Base-case and sensitivity analyses inputs for clinical events, deaths, post-event treatment and anticoagulation management. Table B. Base-case and sensitivity analyses inputs for utility and cost. Table C. The ICD-9 codes (diagnosis codes unless other specified) used in this study. Supplemental References.(DOCX)Click here for additional data file.


The morphology of his leukemic blasts was consistent with the hypogranular variant of APL. Subsequent FISH and cytogenetic analysis revealed a unique translocation involving five chromosomal regions: 9q34, 17q21, 15q24, 12q13, and 15q26.1. Molecular testing demonstrated PML/RARA fusion transcripts. Treatment with conventional chemotherapy was added and he went into a complete remission. Given his elevated white blood cell count at presentation, intrathecal chemotherapy for central nervous system prophylaxis was also given. The patient remains on maintenance therapy and remains in remission. This is the first such report of a 5-way chromosomal translocation leading to APL. Similar to APL with chromosomal translocations other than classical t which result in the typical PML-RARA fusion, our patient responded promptly to an ATRA-containing regimen and remains in complete remission.Acute promyelocytic leukemia (APL) is classically characterized by chromosomal translocation , resulting in the PML-RARA fusion protein leading to disease. Here, we present a case of a 50-year-old man who presented with signs and symptoms of acute leukemia with concern for APL. Therapy was immediately initiated with all- Chromosomal translocation t15;17)q22;q21) is the hallmark of APL, resulting in the fusion of the promyelocytic gene (PML) on chromosome 15 to the retinoic acid alpha receptor gene (RARA) on chromosome 17. Variant translocations which also result in this PML-RARA fusion have been reported in approximately 9% of cases, with similar sensitivity to all-5;17. He did not have hyperuricemia, renal dysfunction, or electrolyte disturbances suggestive of tumor lysis syndrome. He was admitted for further work-up, which included a bone marrow aspiration and biopsy performed on hospital day number 1. Due to concerns for acute myeloid leukemia, he was transferred to our institution late on a Friday evening on hospital day number 2.The past medical history was significant for coronary artery disease status after drug-eluting stent placed 9 months earlier, hypertension, and some arthritic pains of his shoulder. The patient lived at home with his girlfriend. His 15-year-old daughter stayed with him every other week and his son lived within a 10-mile radius. He reported no occupational or radiation exposures. There was no family history of hematologic malignancies, and he had no siblings.Upon transfer, the peripheral smear revealed nearly 100% blasts with no Auer rods or coarse granularity. Many of the blasts contained dumbbell-shaped nuclei with very fine granules concerning the hypogranular variant of APL . He was The bone marrow aspirate and biopsy showed a hypercellular marrow (90–100% cellular with 64% blasts) and decreased trilineage hematopoiesis . ButyratHe was deemed high-risk due to initial leukocyte count and treated definitively with ATRA and idarubicin. On the second day of ATRA plus anthracycline, he became hypoxic and short of air, with concern for ATRA differentiation syndrome. ATRA was held for two days and symptoms resolved, after which ATRA was restarted. He had no further complications.A month later, repeat bone marrow analysis confirmed complete remission by morphology. Cytogenetics confirmed resolution of the complex translocation and RT-PCR was unable to detect any PML-RARA fusion transcripts. Because of his initial high-risk presentation, he received prophylactic intrathecal chemotherapy with induction and consolidation per Tallman and Altman's recommendation . He contSimilar to our patient, APL without the typical t15;17) but with the common PML-RARA fusion is usually sensitive to ATRA induction therapy. However, ATRA resistance is seen with APL resulting from t, when RARA is fused to the PML-linked zinc finger (PLZF). This entity is characterized by a retinoid-independent binding of corepressors and therefore, a lack of differentiation of promyelocytes when ATRA is attempted [5;17 but Our patient's case is unique given this five-way translocation with 46,XY,t resulting in PML-RARA fusion and APL. Similar to other variant APL chromosomal translocations resulting in PML-RARA fusion, his APL was sensitive to ATRA therapy and he continues to do well clinically.


Molecular imaging also potentially provides useful approaches to explore the pathogenesis of viral infections. Thus, the purpose of this study was to image the pathological features of SFTSV infection in vivo by PET imaging. In a mouse model, we showed that 18F-FDG accumulations clearly identified the intestinal tract site as a pathological site. We also demonstrated that 18F-FDG PET imaging can assess disease progression and response to antiserum therapy within the same individual. This is the first report demonstrating a molecular imaging strategy for SFTSV infection. Our results provide potentially useful information for preclinical studies such as the elucidation of the mechanism of SFTSV infection in vivo and the assessment of drugs for SFTS treatment.Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that causes fever, enteritis, thrombocytopenia, and leucopenia and can be fatal in up to 30% of cases. However, the mechanism of severe disease is not fully understood. Molecular imaging approaches, such as positron-emission tomography (PET), are functional  It has been suggested that SFTSV is transmitted by Ixoded ticks, and humans and animals are infected by tick bites  fluoro-D-glucose (18F-FDG) can be used to assess glucose metabolism and 18F-FDG PET/CT is currently utilized for imaging tumor, infection and inflammation in both basic studies and clinical applications [deoxy-2-1F fluoro-ications .in vivo approach to examine the disease progression of SFTSV infection.Molecular imaging has been developed and applied in research for neurology, oncology, cardiovascular physiology, and immunology. However, molecular imaging for infectious diseases caused by highly pathogenic viruses, including biosafety level (BSL)-3 agents, has not been fully utilized because of the need for a high-level biocontainment facility . It is sin vivo [in vivo imaging of disease progression and antiviral intervention caused by SFTSV infection.Type-I interferon receptor knock-out A129) mice provide a useful model for investigating the pathogenic mechanism of SFTSV infection 9 mice prin vivo . We previn vivo . Howeverin vivo . Thus, win vivo by PET. In a mouse model, we first examined the pathological features of lethal infection with SFTSV. We next assessed whether 18F-FDG PET/CT imaging provides an effective approach for monitoring disease progression of SFTSV infection. We further evaluated whether the therapeutic efficacy of anti-serum treatment could be observed by 18F-FDG PET/CT imaging in the same individual.The purpose of this study was to image the pathological features of SFTSV infection A129 mice were infected with a lethal dose of SFTSV, and we observed the pathological changes at 3 days pi before mice started to die at 4 days pi . The groThe histopathological examination revealed erosion in the gastric mucosa but not in the small intestinal mucosa of SFTSV-infected mice Figure . Viral gThese observations suggest that lethal infection with SFTSV caused severe gastrointestinal disorders. These findings suggest that the gastrointestinal tract is a potential target for PET imaging of SFTSV infection.18F-FDG PET imaging of SFTSV-infected mice at 3 days pi. 18F-FDG accumulation was clearly observed in the intestine of SFTSV-infected mice, but not in the stomach . We previously showed that SFTSV-infected A129 mice exhibited high levels of viral replication in tissue, such as the lung, spleen, liver, kidney, brain, and spinal cord . Howeverin vivo studies of SFTSV infection and preclinical studies such as the assessment of drugs for SFTS treatment.However, SFTSV-infected A129 mice showed apparent gastrointestinal disease. Mice showed acute weight reduction after 2 days pi  and the 18F-FDG accumulated in the intestinal tract but not in the stomach of SFTSV-infected mice. 18F-FDG is a glucose analogue that is absorbed by cells with a high-glucose demand. The FDG is moved by glucose transporters and is phosphorylated to 18F-FDG-6-phosphate once in cells, but it is not further metabolized in cells [18F-FDG PET imaging for the detection of pathological conditions, such as tumors and inflammatory diseases, in the gastrointestinal tract [18F-FDG uptake occurs in indomethacin-induced intestinal ulceration models, and 18F-FDG accumulation was found in inflammatory cells [Interestingly, in cells . There hal tract , 18-20. ry cells .18F-FDG accumulation in SFTSV infected-mice. However, 18F-FDG accumulations were not observed in other sites of gastrointestinal tract and other organs. In particular, gross pathological changes were apparently present in the stomach, but 18F-FDG uptake was not observed. Although it is unclear why the specific 18F-FDG uptake was observed in the intestine but not in the stomach, the elucidation of the mechanism will provide interesting insights into the mechanism of gastrointestinal disease in vivo due to lethal infection with SFTSV.Viral infections and inflammatory reactions in the intestine may be correlated with specific in vivo molecular imaging for highly pathogenic pathogens. Thus, molecular imaging is expected to play an increasing role in the pathological studies and the development of effective treatment strategies for infectious viruses including BSL-3 viruses.Classical approaches to preclinical studies of pathogenic virus infections using animal models involve euthanasia and tissue dissection. As a result, it was impossible to follow the real-time dynamics of disease progression in the same individuals. Molecular imaging techniques are noninvasive and can provide insight into disease progression, prognosis, and responses to therapy in the same individuals. However, there are currently a limited number of reports using The YG-1 strain of SFTSV was kindly provided by Ken Maeda at Yamaguchi University. The SFTSV stock virus was prepared from cell culture medium of Vero E6 cells. Vero E6 cells were maintained in Eagle's Minimal Essential Medium  containing 10% fetal bovine serum (FBS). The viral titers were determined using the focus forming assay described previously . All exp6 focus-forming units (ffu) of SFTSV diluted in EMEM containing 2% FBS. Animal experiments were performed in accordance with the recommendations in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology. The Animal Care and Use Committee of the Nagasaki University approved all of the experimental protocols .A129 mice were purchased from B & K Universal Limited and were mated in the facility at Nagasaki University. Adult mice were subcutaneously inoculated with 10SFTSV-infected A129 mice were anesthetized and then perfused with 10% phosphate-buffered formalin at 3 days pi. The fixed tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Viral RNA was detected using ISH in formalin-fixed, paraffin-embedded (FFPE) sections using the AT-tailing method for high-sensitivity detection as described previously . The AT-18F-FDG  intravenously via the tail vein. The mice were anesthetized with 1.5% isoflurane, and CT acquisitions were performed for anatomical reference. The PET acquisitions were performed for 30 min starting 30 min after 18F-FDG injection. The PET data were reconstructed using a 3D-Maximum-Likelihood Expectation Maximization (MLEM) algorithm (60 iterations). The acquired PET and CT data were processed using VIVID™  and OsiriX MD .PET/CT images were acquired using Triumph combined PET/SPECT/CT systems . SFTSV-infected A129 mice were administered approximately 10 MBq of 6 ffu of SFTSV. Saline was used for mock treatment. Our previous data indicated that antiserum treatment by 3 days pi completely protected mice against lethal infection. Thus, we observed PET imaging after the antiserum treatment to compare survivals between treated and non-treated mice. In addition, to examine the course of disease progression, we observed the sequential imaging in individual mice. Furthermore, to minimize the influence of the intravenous damages by the injection, we examined the mice every second day. As a result, we performed PET imaging at 2 and 4 days pi for antiserum treatment.Antiserum was collected from a patient who contracted SFTS in 2005. The serum collection was performed eight years after the patient recovered. The experiment using human serum was performed with the approval of the Ethics Committee of the Institute of Tropical Medicine, Nagasaki University . The serum was diluted tenfold in saline, and 500 μl of diluent was intraperitoneally injected in mice 1, 24, 48 and 72 hours after inoculation with 10


Aboriginal Australians have a substantially higher frequency of ischaemic heart disease (IHD) events than their non-Aboriginal counterparts, together with a higher prevalence of comorbidities. The pattern of health service provision for IHD suggests inequitable delivery of important diagnostic procedures. Published data on disparities in IHD management among Aboriginal Australians are conflicting, and the role of comorbidities has not been adequately delineated. We compared the profiles of Aboriginal and non-Aboriginal patients in the metropolitan area undergoing emergency IHD admissions at Western Australian metropolitan hospitals, and investigated the determinants of receiving coronary angiography.Person-linked administrative hospital and mortality records were used to identify 28-day survivors of IHD emergency admission events  commencing at metropolitan hospitals in 2005–09. The outcome measure was receipt of angiography. The Aboriginal to non-Aboriginal risk ratio (RR) was estimated from a multivariable Poisson log-linear regression model with allowance for multiple IHD events in individuals. The subgroup of myocardial infarction (MI) events was modelled separately.IHD 0.77, 95% CI 0.72-0.83; RRMI 0.81, 95% CI 0.75-0.87) but in the full multivariable model this disparity was accounted for by comorbidities as well as IHD category and MI subtype, and private health insurance . When stratified by age groups, this disparity was not significant in the 25–54 year age group  but was significant in the 55–84 year age group .Compared with their non-Aboriginal counterparts, Aboriginal IHD patients were younger and more likely to have comorbidities. In the age- and sex-adjusted model, Aboriginal patients were less likely than others to receive angiography (RRThe disproportionate under-management of older Aboriginal IHD patients is of particular concern. Regardless of age, the disparity between Aboriginal and non-Aboriginal Australians in receiving angiography for acute IHD in a metropolitan setting is mediated substantially by comorbidities. This constellation of health problems is a ‘double-whammy’ for Aboriginal people, predisposing them to IHD and also adversely impacting on their receipt of angiography. Further research should investigate how older age and comorbidities influence clinical decision making in this context. Ischaemic heart disease (IHD) is the highest ranking contributor to the substantial life expectancy gap between Aboriginal and non-Aboriginal populations in Australia . Coupledper se, as provision of CARP is dependent on patients’ angiographic findings, information which is not available from administrative records. We have previously reported on the rural population [This aim of this administrative linked data study was to investigate disparities in provision of coronary angiography to Aboriginal and non-Aboriginal IHD patients undergoing emergency admissions to metropolitan hospitals in Western Australia (WA). WA’s only metropolitan area  had a population of around 1.6 million in 2011, of whom approximately 1.6% identify as Aboriginal . The outpulation  and in tpulation -11. Therpulation . Comparepulation . We sepaA person-linked file of all metropolitan residents admitted to metropolitan hospitals from 2005 to 2009 with a principal discharge diagnostic code of IHD, incorporating their previous (15-year history) and subsequent hospital admissions, was extracted from the Hospital Morbidity Data Collection (HMDC) and Deaths datasets of the Western Australian Data Linkage System (WADLS). The WADLS is a comprehensive system linking population-based administrative health data from several statutory datasets through probabilistic matching, with the proportions of invalid  or missed links  both estimated at 0.1% of matches .We identified IHD events in metropolitan residents aged 25–84 years who were admitted to metropolitan public and private hospitals from 2005 to 2009 Figure . The staFor each event, patient demographic variables and 15-year admission histories of specified comorbidities  were recorded. Comorbidities were identified using International Classification of Diseases Australian Modification 10th revision (ICD-10-AM) codes defined by Quan et al. ,17. The The outcome examined was coronary angiography. For each event, we determined if the patient received coronary angiography  within 28 days of the event admission date. Angiography was also assumed to have been performed if the patient had a CARP (Blocks 669–679) recorded within 28 days of the event admission date where angiography was not separately recorded in the procedure fields. Sensitivity analyses were performed comparing outcomes (receipt of coronary angiography among those surviving) for events defined by a 90-day instead of a 28-day interval, in order to investigate the effects of unexpected delays in cardiac catheterisation.Analyses were performed using SAS 9.4 (SAS Institute). Baseline demographic and comorbidity characteristics of events involving Aboriginal and non-Aboriginal subjects were summarised separately. Chi-square tests were used to test for significance of categorical variables. To model event-based receipt of coronary angiography allowing for multiple events in each patient, a Poisson distribution for the dependent variable, together with a log link function, exchangeable variance structure across multiple events for a patient, maximum-likelihood method and robust standard errors was used. When the outcome is binary, the exponentiated coefficients from the Poisson regression represent risk ratios (RR) rather than incidence-rate ratios and the robust standard errors take into account both the use of the Poisson model as a working model for binary data and the clustering of events within individuals . In addiEthics approval was obtained from Human Research Ethics Committees of WA Aboriginal Health, The University of Western Australia and WA Department of Health.Of the 21,433 IHD events identified in metropolitan hospitals from 2005 to 2009  , private health insurance  and IHD category and coronary angiography in the last year . Adjustment for individual comorbidities (Models 5a-e), especially HF  and kidney disease  substantially reduced the gap to 9% less likely. The disparity was not significant in the full model  which included all five comorbidities together. A similar pattern of reduction in the investigation gap was seen in the MI patient subgroup . There was no statistically significant difference in receipt of coronary angiography between Aboriginal and non-Aboriginal patients in the full model .After adjusting for age and sex, Aboriginal people with IHD were 23% less likely than non-Aboriginal people to receive coronary angiography  and MI  but not for either in the younger age group (25–54 years). As previous events and prior angiography may influence receipt of this procedure in future events, analyses were repeated with restriction to (i) first event only and (ii) events without prior angiography in the previous year, producing similar results for both all IHD and MI . Even when stratified by two age groups (25–54 years and 55–84 years), comorbidities accounted for much of the disparity in receiving coronary angiography (Model 4a versus 6a) Table . DispariMI Table .MI 1.00, 95% CI 0.97-1.04) and having pre-existing IHD  were not associated with a lower likelihood of receiving angiography, whereas having diabetes was independently associated with this outcome . In the older age group (55–84 years), apart from the lower likelihood of Aboriginal patients receiving angiography, estimates for all variables were similar to those for the whole cohort. SES was not independently associated with the outcome in either the whole cohort or the two age-groups.In the full multivariable model for MI events, factors independently associated with a lower likelihood of receiving coronary angiography in the whole cohort were older age 65–84 years), being female, not having private health insurance, having coronary angiography in the previous year, and having chronic pulmonary disease, HF or kidney disease, while having transmural MI was associated with higher likelihood of receiving this procedure  conducted throughout Australia and New Zealand  and thosustralia . This liustralia ,9,29,30.In relation to Aboriginal status, the results of our full model are consistent with Randall’s study , in whicAs there was an interaction between Aboriginal status and age, we modelled age by dichotomous stratification concomitantly with multivariate adjustment by age and age-squared as continuous variables. In doing so, we added to the findings of Randall , documenRegardless of age, the greatest contributors to the reduced likelihood of having an angiogram were comorbidities, especially HF and kidney disease, and to a lesser extent chronic pulmonary disease. HF and pre-existing kidney disease, which are substantially over-represented in Aboriginal patients, are important risk factors for contrast-induced acute kidney injury in the setting of coronary angiography  and hencThe strength of this study was its use of person-linked hospital and mortality data with state-wide coverage, allowing us to follow patients throughout their 28-day events. The use of 28-day events was based on previous studies and ICD-10 coding standards -42; repeEvidence-based risk versus benefit considerations justify a somewhat more conservative approach to acute IHD in older patients, ramifying in a lower likelihood of receiving coronary angiography. However, departure from clinical guidelines in actual practice resulting in under-management of older patients is a well described problem ,35. The


Patients with stable disease (SD) at core study end (lanreotide/placebo) or PD (placebo only) continued or switched to lanreotide in the OLE. In total, 88 patients  participated: 38% had pancreatic, 39% midgut and 23% other/unknown primary tumours. Patients continuing lanreotide reported fewer adverse events (AEs)  during OLE than core study. Placebo-to-lanreotide switch patients reported similar AE rates in OLE and core studies, except more diarrhoea was considered treatment-related in OLE . Median lanreotide PFS  was 32.8 months . A sensitivity analysis, addressing potential selection bias by assuming that patients with SD on lanreotide in the core study and not entering the OLE (n=13) had PD 24 weeks after last core assessment, found median PFS remaining consistent: 30.8 months . Median time to further PD after placebo-to-lanreotide switch (n=32) was 14.0 months . This OLE study suggests long-term treatment with lanreotide Autogel 120 mg maintained favourable safety/tolerability. CLARINET OLE data also provide new evidence of lanreotide anti-tumour benefits in indolent and progressive pancreatic/intestinal NETs.In the CLARINET study, lanreotide Autogel (depot in USA) significantly prolonged progression-free survival (PFS) in patients with metastatic pancreatic/intestinal neuroendocrine tumours (NETs). We report long-term safety and additional efficacy data from the open-label extension (OLE). Patients with metastatic grade 1/2 (Ki-67 ≤10%) non-functioning NET and documented baseline tumour-progression status received lanreotide Autogel 120 mg ( CLARINET was a landmark 96-week study that confirmed anti-tumour effects for the long-acting somatostatin analogue lanreotide Autogel (depot in the USA) 120 mg. Lanreotide prolonged progression-free survival (PFS) over placebo in patients with metastatic grade 1 or 2  somatostatin-receptor positive pancreatic, intestinal or of unknown primary origin neuroendocrine tumours (NETs) with prior stable disease (SD), irrespective of hepatic tumour volume , 2015. FHere, we present data from the pre-planned interim analysis of the ongoing single-arm (lanreotide Autogel 120 mg) open-label extension (OLE) to the core study. The primary objective of the OLE study was to investigate the long-term safety of lanreotide in patients with pancreatic and intestinal NETs. Safety/tolerability of lanreotide was investigated not only in patients receiving lanreotide in both the core and OLE studies but also in patients switching from placebo (core study) to lanreotide (OLE). Another objective was to further examine anti-tumour efficacy. In the core study, median PFS for placebo was 18.0 months, but had not been reached with lanreotide after 96 weeks. This analysis provided an opportunity to estimate the median PFS for lanreotide (in continued lanreotide patients) and to determine the median time to subsequent progression in placebo-to-lanreotide patients who had progressive disease (PD) in the core study.Inclusion and exclusion criteria for the core study have been published .The core study included adult patients with sporadic, well- or moderately differentiated NETs that had proliferation index (Ki-67) of up to 10% and were non-functioning, except for gastrinomas adequately controlled with proton pump inhibitors for ≥4 months. Primary tumours were located in the pancreas, midgut or hindgut, or were of other/unknown origin. Patients were also required to have an unresectable locally advanced tumour or metastatic disease (or had declined surgery), target lesion(s) classified as at least grade 2  within the previous 6 months and a World Health Organization (WHO) performance score of ≤2. Core study exclusion criteria comprised treatment with interferon, chemoembolisation or chemotherapy within 6 months before study entry, radionuclide therapy at any time or a somatostatin analogue at any time (unless received over 6 months previously for <15 days); major surgery related to the NET within the previous 3 months; multiple endocrine neoplasia; or previous cancer  1.0) at the end of the 96-week core study (while receiving lanreotide or placebo) or had centrally assessed PD during the core study while receiving placebo . Patients were enrolled within 4 weeks of their last core study visit and received lanreotide Autogel 120 mg by deep s.c. injection every 28 days. The study started in February 2009 and will continue in each country until registration for tumour control. This pre-planned interim analysis was undertaken at the time of last patient's last visit in the core study (data cut-off: March 2013).Supplementary Materials and methods, see section on ClinicalTrials.gov (NCT00842348) and EudraCT (2008-004019-36).Patients provided written informed consent. Study protocol, amendments (amendments occurring after the start of the study are summarised in the The OLE study assessments (multiphase CT or dynamic contrast-enhanced MRI scans) were scheduled for week 1 (OLE study baseline) and every 24 weeks, as well as at the time of early withdrawal (if applicable) or at any time if PD was suspected. Adverse events (AEs) were recorded throughout the study on 4-weekly treatment visits. Other safety assessments included physical examination, assessment of vital signs and clinical laboratory tests , and electrocardiography and ultrasonography of the gallbladder conducted at baseline, weeks 48 and 96, or early withdrawal visit. Scans were assessed locally for the signs of PD according to RECIST 1.0, compared to core study baseline scan or a subsequent scan indicating a nadir in core or OLE studies (patients with SD in core study), or OLE study baseline or a subsequent scan indicating a nadir in OLE study (patients with PD in core study).The primary objective of the OLE study was to investigate the long-term safety of lanreotide Autogel 120 mg in patients with pancreatic and intestinal NETs. The secondary objective was to further investigate its efficacy. This interim analysis provides an opportunity to estimate median PFS for lanreotide in patients originally randomised to and continuing to receive lanreotide, both overall (main efficacy endpoint) and for pre-specified as well as clinically relevant subgroups . The interim analysis also facilitates estimation of the time to subsequent PD in patients switching to open-label lanreotide after PD while receiving placebo in the core study . Insufficient numbers precluded evaluation of subsequent PD in subgroups.Descriptive statistics were calculated for safety data from the safety population . Descriptive rather than inferential statistics were performed because this was a non-comparative OLE study, and there were some inherent differences between patients who switched to active drug from placebo or continued on active drug, notably in SD/PD status at the start of this study. Safety analyses were described for groups according to the sequence of treatment received during the core and OLE studies .PFS (main efficacy end point) and the time to subsequent PD with lanreotide  were described using Kaplan–Meier curves with events defined as deaths and PD ; all other outcomes were censored as per Food and Drugs Administration guidance . Two senStatistical analyses were performed using SAS software version 9.3 .n=138) eligible for the OLE study, 79 patients had SD at the core study end (53 on lanreotide and 26 on placebo) and 59 had PD while receiving placebo during the core study. In total, 52 (37.7%) patients who were eligible did not enrol, mostly  because their study centres did not participate in the OLE study. Reasons for non-enrolment were not documented for the remaining 15 patients.Of patients from the core study , 41 continued on lanreotide and 47 patients switched from placebo to lanreotide. At the time of the pre-planned interim analysis, none of the 88 participants had died.Of 88 patients enrolled in the OLE study  at baseline in the OLE study and a greater proportion with a NET originating in the pancreas .The medians (ranges) for combined lanreotide exposure in the core and OLE studies were 40.0 (26.0–74.3) months in the continued lanreotide group, 18.1 (1.0–49.9) months in the placebo-to-lanreotide group with SD at the end of the core study and 13.0 months (2.0–52.0) in the placebo-to-lanreotide group with PD during the core study.Supplementary Tables S1 and S2, see section on In the continued lanreotide group, incidences of overall and individual AEs  were generally lower for the OLE study compared with the core study . This diSupplementary Table S1), the incidence considered to be treatment-related was higher in the OLE study (most common treatment-related AEs occurring in ≥5% of patients shown in Supplementary Table S2). Incidences of severe and serious AEs were similar between groups and across studies. Only one patient was withdrawn due to a treatment-related AE .In the placebo-to-lanreotide group, the overall incidence of AEs, whether treatment-related or not, was similar across the core and OLE studies . This paThe gallbladder echography was conducted on at least two occasions during the core and OLE studies combined in 29/41 (71%) patients in the continued lanreotide group, and 34/47 (72%) patients in the placebo-to-lanreotide group. Among these patients, new lithiasis and/or new sludge were detected in four patients each in the placebo-to-lanreotide group and six patients each in the continued lanreotide group.No clinically significant trends were observed in other safety assessments.Median PFS was not reached for lanreotide in the 96-week core study . In totaa priori sensitivity analysis, data from 15 patients who were withdrawn from the core study due to investigator assessment of PD  were added as events. Compared with the main analysis, median (95% CI) PFS for lanreotide in the sensitivity analysis was similar ). In the post hoc sensitivity analysis, data from 25 patients (13 receiving lanreotide) with SD at the end of the core study but who did not enter the OLE study were added to the analysis, and all were assumed to have had PD at the first OLE study assessment. Median PFS for lanreotide in this sensitivity analysis was consistent with the main analysis: 30.8 months .Supportive analysis (based on the per-protocol population) provided the same estimate of median PFS for lanreotide as the main (intention-to-treat (ITT)) analysis: 32.9 months . In the n=32) while receiving placebo in the core study and continued into the OLE study (receiving open-label lanreotide), 17 had subsequent PD during the OLE; a median time from first PD in the core study with placebo to subsequent PD in the OLE study with lanreotide was 14.0 months  . For patients without PD on placebo after completing the full 96 weeks of the core study (n=15), three patients experienced PD with lanreotide Autogel 120 mg in the OLE study . Nine patients were continuing lanreotide treatment without PD at the time of interim analysis.For patients who had PD (reached) . Nine paSupplementary Table S3, see section on For continued lanreotide patients, PFS results in the core plus OLE studies across subgroups based on tumour origin, grade, hepatic tumour load, PD and previous therapy status at core study baseline and region were generally consistent with the main analysis . Among patients who received lanreotide in the core and OLE studies, the pattern of most AEs suggests amelioration with increasing duration of treatment exposure, particularly for diarrhoea (whether treatment-related or not). In the group switching from placebo in the core study to lanreotide in the OLE study, the pattern of AEs between studies shows generally comparable rates, including treatment-related AEs except for diarrhoea, which was higher in the OLE study. The latter finding was not unexpected, as diarrhoea is a common transient side effect after the initiation of somatostatin analogue therapy.The safety and tolerability profile of lanreotide is well-evidenced from its use as a treatment for symptoms of carcinoid syndrome in clinical studies  pancreatic and intestinal NETs and indolent disease (96% SD according to RECIST), irrespective of hepatic tumour load; median PFS for lanreotide was not reached  receiving lanreotide for ≤92 weeks  31% of .post hoc sensitivity analysis that included all patients with SD in the core study who did not continue into the OLE study and designated them as having PD at the first OLE assessment. Reassuringly, median PFS from this sensitivity analysis accorded well with the main analysis. Secondly, the OLE study was not specifically designed to measure efficacy, and in contrast to the core study, it was open-label, lacked a control group and based PFS estimates on locally rather than centrally assessed PD. Nevertheless, the estimate of median PFS for lanreotide was based on the ITT population from the randomised controlled core study and, in large part, on events confirmed centrally during the core study.Undoubtedly, extensions of large randomised controlled trials such as CLARINET are important; however, the OLE study is not without limitations. Firstly, not all eligible core study patients continued to be followed into the OLE study, principally owing to non-participation of some core study centres; nonetheless, a degree of selection bias may have occurred. However, as noted, this issue was addressed in a In summary, the CLARINET OLE study demonstrated that the long-term treatment with lanreotide Autogel 120 mg was well-tolerated, with no indication of new safety concerns. Notwithstanding study limitations, the CLARINET OLE data suggest that lanreotide Autogel 120 mg has anti-tumour effects in patients with PD. Additionally, the CLARINET core and OLE studies together provide evidence for long-term PFS benefits of lanreotide Autogel 120 mg in patients with indolent pancreatic and intestinal NETs.http://dx.doi.org/10.1530/ERC-15-0490.This is linked to the online version of the paper at


Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding.The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant  The plant cell wall and its underlying plasma membrane are required to perceive environmental changes like biotic and mechanic stresses and also represent the first layers of defense to invading pathogens. In this regard, cells of the epidermal tissue have a predominant function as they form the outer barrier and are first in interacting with the surrounding environment. Epidermal plant cells usually differ in shape, function and physiological reactions to other plant tissues. Therefore, a specific analysis of separated epidermal tissue can provide new insights especially into the early regulation and organization of stress-related changes and adaption. This is of special interest in the field of plant–microbe interactions where cell wall thickenings, so-called papillae, are one of the most prominent and long-studied responses to invading pathogens in epidermal cells . They coTo overcome these general limitations in sample preparation from tissues, laser microdissection (LM) techniques have been developed to separate specific cells or cellular structures from the surrounding tissue by using a focused laser . After iArabidopsis thaliana. Therefore, we aimed to establish a LM method that would be relatively easy to operate and would not require tissue fixation. In addition, we wanted to avoid tissue cross section as early stage epidermal cell wall alterations, like papillae, would be difficult to identify, extending sample acquisition, and increasing possible protein degradation. A main prerequisite of our LM method development was to ensure an application on papillae generated in host epidermal leaf cells of the model pathosystem A. thaliana – powdery mildew, which would allow to study different mutants showing papillae formation and penetration resistance  as well as the powdery mildew Golovinomyces cichoracearum (strain UCSC1) used for leaf inoculation were cultivated and used as described by Brachypodium distachyon (inbred line Bd21) followed the description from A. thaliana and B. distachyon leaf sections were placed with their adaxial side on the coated microscope slide. For the preparation of A. thaliana leaf sections, the petiole and about 1–2 mm of the adjacent part of the lamina were cut off; B. distachyon leaves were cut into 1 cm sections. Leaf sections on coated microscope slides were then laminated with a self-adhesive tape . During this procedure, the formation of air pockets should be avoided to obtain optimal results of epidermis preparation, which was achieved by a fast tear-off of the tape . In case of an incomplete epidermis preparation, additional tear-offs of the adhesive tape containing the remaining leaf tissue on previously unused positions of the coated microscope slide allowed a preparation of the remaining epidermis . ACT-prepared epidermal tissue layers were dried in a desiccator for 24 h to prepare samples for a long-term storage.For the preparation of single layered epidermal leaf tissue, we developed a combined adhesive tape – liquid cover glass technique (ACT). First, microscope slides were coated with liquid cover glass . To optimize adhesive surface capacity, slides were spray-coated with liquid cover glass, excess liquid removed, and after 2 min of drying again spray-coated. In case of a non-uniform drying of the liquid cover glass coat, which might impair epidermis preparation quality, a short heating of the coated slide at 30°C promoted the formation of an optimal adhesive surface. After a final drying period of 2–3 min, 2HPO4, pH 9]. Small droplets of the aniline blue solution were directly placed on the tissue. During pipetting of the staining solution, the tissue should not float off because this would prevent a subsequent laser capture microdissection. Evaporation of water and drying of the tissue was done in a dark compartment for 16 h at room temperature (22–24°C) without humidity control (room humidity ranging from 40 to 60%). Aniline blue stained-cell wall structures of ACT-prepared epidermis were visualized via epifluorescence microscopy -β-glucan callose, ACT-prepared epidermal tissue was stained with aniline blue , placed in the lid of collection tubes (Carl Zeiss). Collected samples were stored at -20°C until subsequent processing.g for 60 min. The supernatant was used for protein analysis, the pellet for cell wall analysis. Before protein analysis by liquid chromatography coupled with tandem spectrometry (LC-MS/MS) using a nanoLC-electrospray iontrap MS/MS system  μg protein per 10,000 disks and were analyzed by liquid chromatography coupled with tandem spectrometry (LC-MS/MS). Results from ACT- and LMPC-prepared epidermal leaf samples were compared with control protein samples from whole A. thaliana leaf tissue.After successfully establishing ACT preparation of epidermal tissue with subsequent LMPC, we tested whether the collected samples would generally allow a proteome analysis. Moreover, we also wanted to evaluate the appropriate amount of dissected disks, which would be required to perform significant comparable proteome analysis of different samples. Therefore, we randomly dissected 11,000, 35,000, and 50,000 disks from ACT-prepared Figure 4A). Even though these numbers of proteins were only about a sixth of the amount detected in the control protein samples , they allowed a comparative analysis to evaluate the efficiency of the ACT epidermis preparation in accumulating proteins from the plasma membrane. A qualitative analysis of the subcellular localization of the detected proteins revealed that this was especially effective in the 50,000-disks sample. We observed a relative reduction of proteins originating from the cytosol, mitochondria, and plastids, whereas the relative fraction of proteins deriving from membranes, and especially the plasma membrane, was strongly enriched . These results supported our microscopic observation that ACT of epidermal tissue results in a relatively efficient removal of the cytosol and cellular organelles. However, this method was not as effective in removing the nucleus from ACT-prepared epidermal samples as the relative fraction of nucleus-localized proteins was expanded in ACT-prepared epidermis samples . Because the requested effects in accumulating plasma membrane-localized proteins and reducing proteins from the cytosol and organelles was not as distinct in the 35,000-disks sample as in the 50,000-disks sample, we propose sample sizes of about 50,000 disks (∼25 mm2) to be appropriate for downstream proteome analysis by LC-MS/MS. The observed effect of ACT epidermis preparation in accumulating membrane proteins was not only restricted to a qualitative analysis but also reflected in a quantitative analysis. A Top 15 list of protein abundance (Table 1) revealed a quantitative predominance of chloroplast localized proteins like ribulose bisphosphate carboxylase in the control sample from whole leaf protein extracts whereas the LMPC-prepared 50,000-disks epidermis sample was enriched in membrane and nuclear proteins. Remarkably, a -β-glucosidase, which is known to be anchored to the plasma membrane (Table 1).Whereas LC-MS/MS only identified 14 individual proteins in the sample deriving from 11,000 disks , which we considered not sufficient for significant comparative analysis, 47 proteins derived from the 35,000-disks and 54 proteins from the 50,000-disks protein samples . To test HPAEC-PAD on ACT-prepared epidermal leaf cell layers, we dissected 6,000 disks and extracted the mono-carbohydrates from the hydrolyzed hemicellulosic cell wall fraction. As a control, we applied the same method on LMPC samples from liquid cover glass-coated microscope slides without epidermis preparation.A. thaliana cell walls . We also detected residual amounts of glucose as well as galacturonic and glucoronic acid in the control liquid cover glass sample . Because it is most likely that these mono-carbohydrate contaminations derive from liquid cover glass, it would not be possible to avoid this contaminations based on the developed ACT for epidermis preparation. Hence, it would be required to analyze the same sample size of liquid cover glass-coating in parallel to plant samples to facilitate precise mono-carbohydrate quantification. In this regard, we consider a sample size of 6,000 disks (∼8 mm2) as a lower limit for significant and precise mono-carbohydrate quantification.In the 6,000-disks sample, we detected the mono-carbohydrates galactose, glucose, mannose, galacturonic, and glucoronic acid that are typical for ll walls  in such In addition to proteome analysis, the successful detection of hemicellulose-derived mono-carbohydrates clearly revealed that ACT- and LMPC-prepared epidermal leaf samples can be used for the quantitative analysis of the cell wall composition of defined cell wall structures using HPAEC-PAD.A. thaliana have been facilitated. ACT epidermis preparation neither requires tissue embedding nor cross sections. Therefore, complete, defined cell wall structures or areas of modification can be readily identified and isolated using LMPC. This is of special importance in plant–microbe interactions where pathogen-induced cell wall modifications are decisive for plant defense. We successfully demonstrated further processing of ACT- and LMPC-prepared epidermis samples in downstream applications for proteome analysis and carbohydrate composition, opening new perspectives in the quantitative analysis of stress-induced cell wall modification.The developed ACT allows a simple preparation of complete adaxial epidermal tissue layers from large-scale leaf sections. Because the developed method is also applicable on soft tissue, preparations from the most important model plant CF, DE, BvH, RH, CV: conception and design of the research; CF, DE, BvH, RH: acquisition and analysis of the data; CF, DE, CV: interpretation of data; CF, DE, BvH, RH, CV: drafting 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.


Over-expression of the PPP1R Protein Targeting to Glycogen (PTG), under the control of the aP2 promoter in mice, inactivated glycogen phosphorylase (GP) and enhanced basal- and starvation-state glycogen storage. Total interscapular BAT glycogen synthase and the constitutive activity of GS were conditionally affected. During starvation, glucose-6-phosphate (G-6-P) levels and the relative phosphorylation of Akt (p-Ser-473-Akt) were both increased in PTG-overexpressing (Tg) mice, suggesting that elevated glycogen storage during starvation modifies broader cellular metabolic pathways. During refeeding, Tg and WT mice reaccumulated glycogen similarly despite altered GS and GP activities. All observations during refeeding suggest that the phosphorylation states of GS and GP are not physiologically rate-controlling, despite there being a clear balance of endogenous kinase- and phosphatase activities. The studies presented here reveal IBAT glycogen storage to be a tightly-regulated process at all levels, with potential effects on nutrient sensing in vivo.Glycogen storage in brown adipose tissue (BAT) is generally thought to take place through passive, substrate-driven activation of glycogenesis rather than programmatic shifts favoring or opposing the storage and/or retention of glycogen. This perception exists despite a growing body of evidence suggesting that BAT glycogen storage is actively regulated by covalent modification of key glycogen-metabolic enzymes, protein turnover, and endocrine hormone signaling. Members of one such class of covalent-modification regulators, glycogen-binding Phosphoprotein Phosphatase-1 (PP1)-regulatory subunits (PPP1Rs), targeting PP1 to glycogen-metabolic enzymes, were dynamically regulated in response to 24 hr of starvation and/or 24 hr of starvation followed by  BAT is a multi-depot thermogenic organ, responsive to changes in ambient temperature or nutritional status. Though BAT has historically been recognized for its role in thermal homeostasis during hibernation , the medThe observation that interscapular BAT (IBAT) in mice possesses high concentrations of glycogen, second only to those of the liver under basal conditions , 6, has Glycogen particle expansion and degradation are enzymatically regulated by glycogen synthase (GS) and glycogen phosphorylase (GP), respectively. GS catalyzes the addition of uridine diphosphate-glucose (UDP-glucose) onto the terminal glucosyl moiety of glycogen chains, a reaction that is subject to regulation at the levels of substrate availability (modulated via glucose transport), total GS protein levels, spatial localization of GS to glycogen particles, and constitutive GS activity –13. GS cDephosphorylation events targeting regulatory serines on GS and GP are carried out by protein phosphatase-1 (PP1). In glycogen metabolism, scaffolding proteins known as glycogen-targeting PP1 regulatory subunits (PPP1Rs) co-localize the catalytic subunit of PP1 (PP1c) with specific glycogen-metabolic enzymes –24, fineThe PTG gene , coding for the peptide PTG, is expressed in all tissues that have been examined except the testis . PTG posad libitum fed, 24 hr of starvation, and 1–2 hr of refeeding following starvation. Evidence is presented that glycogen storage in the ad libitum fed or starved state is modulated by the phosphorylation state of GP, regulated by glycogen-targeted PP1. Conversely, during refeeding, glycogen replenishment proceeds primarily through the allosteric activation of GS that overrides the regulatory phosphorylation status of the enzyme. Data also suggests that glycogen itself may serve as an intracellular metabolic rheostat, feeding back into broader metabolic control pathways via alterations in glucose-6-phosphate levels and the phosphorylation of Akt.Despite the wide range of glycogen storage observed in IBAT, little is known about the mechanisms by which GS and GP are controlled in this tissue. In the current work, the relative contributions of covalent vs. allosteric regulation of these enzymes under three physiological conditions were investigated: CD1 mice possessing an additional copy of the endogenous gene for Protein Targeting to Glycogen (PTG), under the control of the aP2 promoter (detailed in ), were bMice were maintained on a 12:12-hr light:dark cycle (lights on at 6:00 AM) under specific pathogen-free conditions in the Gwen and Jules Knapp Center for Biomedical Discovery animal facility at the University of Chicago at 22°C. Experimental interactions with the mice were performed under dim red lighting during the dark phase to minimize circadian disruption. Only male mice were used for all experiments to eliminate potential sex-specific variation. All animal procedures for this study were approved by the University of Chicago Institutional Animal Care and Use Committee, and all efforts were made to minimize animal suffering. For sacrifice, mice were anaesthetized with Isoflurane vapor  and death was ensured by cervical dislocation. IBAT was immediately harvested and placed in liquid nitrogen.ad libitum for 1, 2, or 4 hr  prior to sacrifice. The cages of S and SR mice were changed at the time of food withdrawal. Except during starvation, all mice were provided ad libitum access to irradiated Harlan Teklad Global 18% Protein Rodent Diet , referred to as chow and kept on corn-cob bedding.Male, group-housed, wild-type (WT) or Tg littermates were either sacrificed at 19:30 (Fed), or starved for 24 (S) beginning at 19:30 and then either sacrificed or allowed to refeed Glycogen was measured in IBAT exactly as described previously .Whole individual IBAT pads were homogenized by sonication (Sonics VibraCell Ultrasonic Processor) in 15 uL homogenization buffer  containing 1% v/v protease inhibitor cocktail (Sigma P8340) per mg tissue. Samples were centrifuged at 4°C for 15 minutes at 13,400xg to separate lipids and insoluble components. Infranatants were transferred to fresh tubes and centrifuged at 4°C for 15 minutes at 13,400xg. Protein concentration was determined by BCA assay (Pierce BCA Protein Assay Kit Prod #23227). Homogenate drawn from the post-spin infranatant was mixed with Laemmli buffer at a volumetric ratio of 3:1 homogenate:Laemmli and heated to 100°C for 10 minutes. Each well of an SDS-polyacrylamide gel was loaded with protein homogenate containing equal amounts of total protein in Laemmli buffer or 2–5 uL of protein ladder . Separation took place at room temperature by electrophoresis at 120V for 1–2 hr. Proteins were then transferred onto polyvinylidene fluoride membranes  in a wet transfer apparatus at 20V for 14–18 hours at room temperature and then immunoblotted with either rabbit anti-pan-GP (generated and purified as described in ) or rabbref = 2-(delta Ct). This manipulation normalized the expression values, allowing us to scale the data with accurate error bars and perform statistical comparisons between conditions. To calculate fold-change between conditions and genotypes, each Fold Changeref value was divided by the mean Fold Changeref of the control condition to obtain a measure of relative expression.For each biological replicate, one whole snap-frozen IBAT pad was pulverized while frozen and mRNA was isolated using the DNeasy blood and tissue spin column kit (Qiagen). cDNA was prepared from 500 ng of mRNA using the qScript cDNA synthesis DNA kit. Perfecta SYBR Green Fastmix (Quanta Biosciences) was used to perform quantitative real-time PCR . See One whole IBAT pad from each mouse was homogenized by sonication in 15 volumes of ice-cold PBS and placed on ice. Homogenates were centrifuged at 16,100xg for 3 minutes at 4°C and the infranatant was transferred to 10 kDa spin columns . Samples were centrifuged at 16,100xg for approximately 20 minutes at 4°C. The concentration of G-6-P in the filtrate was determined using a commercially-available high-sensitivity G-6-P assay kit .p<0.05.Each comparison made between two treatments or conditions was performed with a Mann-Whitney rank sum test. Significance was set at IBAT has the second highest basal concentration of glycogen in the mouse, second only to that of the liver , making ad libitum (Fed) and in mice starved for 24 hr and then refed for 0, 1, 2, or 4 hr followed by sacrifice and tissue harvest . Over-expression of PTG in IBAT significantly increased basal glycogen storage  were robustly expressed and dynamically-regulated in both WT and Tg IBAT, decreasing in response to starvation and rebounding during refeeding . UCP1 expression decreased in response to starvation and remained suppressed after 2 hr of refeeding with no genotype-specific differences observed  or actively regulated  of key glycogen-metabolic enzymes such as GS and GP. To determine whether GS was actively regulated by nutritional status, GS activities  were meaTotal GS activity was 66% higher in starved Tg mice over starved WT littermates but not significantly different under other conditions examined . ConstitThe GP-catalyzed breakdown of glycogen into G-1-P is a rate-limiting step in glycogenolysis. Despite this fact, GP is commonly overlooked as a major player in the regulation of glycogen storage in insulin-sensitive, glycogenic tissues such as muscle , 13. TheL, PPP1R3D, and PTG were all robustly expressed and dynamically regulated in IBAT, while the skeletal muscle glycogen-targeting PP1-regulatory subunit, GM, was poorly expressed or undetectable was surprising. IBAT shares a precursor cell population with skeletal myocytes, which utilize GM for catecholamine-sensitive promotion of glycogen synthesis. Despite this fact, GM, at least at the expression level, was not consistently expressed or dynamically regulated in IBAT. The combination of PPP1R expressions observed in IBAT more closely mirrored that of the liver, where GL, PPP1R3D, and PTG are the primary (though not the only) actors in the regulation of glycogen metabolism [L are allosterically inhibited by GPa (which is not the case for GM), the phosphorylation state of GS in IBAT may be more dependent upon the regulation of GP than in skeletal muscle. In IBAT, where catecholamines induce thermogenesis  this allows for both GS and GP to be coordinated at the level of targeted PP1-mediated dephosphorylation, enabling both enzymes to be up- or downregulated simultaneously.There were several results in this study that challenge the commonly-held view of BAT glycogen storage from that of a passive, overflow pathway, supporting instead a model of multi-layered, active regulation, in which both substrate-, covalent-, and expression-level mechanisms govern glycogen storage. Firstly, our finding that Gtabolism , 38. Thiin vivo, changes in glycogen storage were at least partly driven by glucose uptake and intracellular substrate availability [Though PTG over-expression reduced the phosphorylation of GP in all conditions examined, its effects on GS were conditional. Total GS in the starved state was elevated, which was likely an indirect effect of PTG over-expression. PTG has been shown in cell culture to increase GS localization to glycogen particles, which stabilizes GS, reducing its turnover . Becauselability . It is na, combined with the reduction in total GP, may have alleviated GPa-mediated repression of GL. This would have allowed GL to exert greater PP1-targeting effects towards GS, producing the observed phenotype. Further study is required to delineate these potential mechanisms.That constitutive GS activity was elevated during refeeding in Tg mice is in agreement with prior reports in which the ability of PTG overexpression to activate GS was demonstrated , 28, 32.in vivo, but that kinase and phosphatase activities balance each other out, creating a stable tension between activating and deactivating forces. It appears that the primary mechanisms by which glycogen is variably regulated in vivo during refeeding are independent of changes to the phosphorylation states of GS and GP, leaving substrate availability, localization effects, and decreased glycogenolysis as the prime suspects. It is therefore plausible to hypothesize that IBAT is constantly primed to synthesize glycogen at all times, and relying on substrate availability and the rate of glycogenolysis to govern the net concentration of glycogen.Our collective observations support the hypothesis that the phosphorylation states of GS and GP are regulated properties In Tg mice, the relative phosphorylation of Akt was unaffected by elevated glycogen levels under basal conditions (Fed), but the starved state yielded multiple indicators of altered signal transduction and glucose metabolism. The enhanced phosphorylation of Akt in the starved state, combined with the elevation of G-6-P, suggests alteration to a major nutrient response signaling pathway. If this is the case, then it challenges the prevailing notion that glycogen acts simply as an overflow pathway for glucose storage during times of excess glucose uptake; that instead, it plays a regulatory role in Akt phosphorylation and potentially broader metabolic pathways such as lipogenesis, lipolysis, glucose uptake, or thermogenesis.Glycogen levels during starvation may feed back on endocrine and nutrient-sensing pathways via Akt. Ser-473 on Akt is a target of nutrient-sensing mTORC2 kinase activity . When phThe described studies, taken in the context of past reports, suggest that BAT possesses a covalently-regulated glycogen storage program controlled by changes in GP activity, acting as a feed-forward metabolic regulator by altering Akt phosphorylation during starvation and rapidly over-accumulating during recovery from starvation independently of the phosphorylation states of GS and GP.


Furthermore, the EGFR pathway plays a role in the transmission of the miR-424 signal that regulates cell growth and the epithelial-to-mesenchymal transition. These results highlight a potential role for miR-424 as a molecular predictor and therapeutic target in bladder cancer.The aim of this study was to examine the role of miRNAs regulation by DNMT1 and its underlying mechanisms in bladder cancer. The choice of target miRNAs was based on the analysis of a TaqMan MicroRNA Panel assay. The role of target miRNA in tumor behavior and the related signaling pathways were assessed using the human bladder cancer cell lines. We also evaluated the predictive power of the target miRNA and its link to DNMT1 from 124 clinical bladder cancer specimens. Our results revealed that the miR-424 level is significantly increased when blocking DNMT1 in bladder cancer cells. From the clinical specimen analysis, the staining of miR-424 was inversely correlated with DNMT1 immunoreactivity. The lack of miR-424 expression was significantly linked to aggressive tumor growth, advanced clinical stage and poor prognosis in bladder cancer. Increased miR-424 suppressed the tumor growth rate and invasion ability determined  Bladder cancer is a significant public health issue worldwide and manifests itself in two distinct forms with different clinical and biological behaviors. Approximately 70% of patients presented with non-muscle-invasive tumors with good prognosis, and the remaining 30% with muscle-invasive tumors have a poor five-year survival rate , 2. AlthMicroRNAs (miRNAs) are single-strand RNA molecules with a length of 20-23 nucleotides that post-transcriptionally control gene expression . A growiin vitro. Next, we performed real-time RT-PCR to quantify the expression levels of miR-424, 98, 145, 223 and 202 in bladder cancers with or without DNMT1 inhibition. The level of miR-424 was more significantly increased in bladder cancer cells with DNMT1-silencing vectors, compared to the others . In contrast, 29% (14/49) of the non-muscle-invasive bladder cancer and 57% (43/75) of the muscle-invasive bladder cancer specimens overexpressed DNMT1. Accordingly, our findings suggest that the downregulation of miR-424 that is associated with increased DNMT1 expression significantly correlated with the clinical stage. Regarding the clinical outcome, the staining of miR-424 was significantly linked to a lower recurrence rate and a longer survival in patients after treatment  have lower expression of miR-424 in tumor tissues compared with adjacent non-malignant epithelial tissues Figure . Of the ast, 29% 4/49 of tnt Table . As detent Table . The finin vitro data in Figure in vitro findings. Moreover, a significant negative correlation was observed in the clinical specimens between EGFR and miR-424  had positive miR-424 immunoreactivity in bladder cancer tissues and there was a negative correlation between the expression levels of miR-424 and DNMT1. Accordingly, we suggested that DNMT1 activity is critical for the downregulation of miR-424 expression in bladder cancer.Although alterations in miRNA expression are thought to play an important role in carcinogenesis , 22, 23,in vitro and a decreased proliferation index in vivo were noted in bladder cancer cells with miR-424 expression vectors. Additionally, we found that the miR-424 expression vector significantly attenuated the invasive ability of cells, as determined through cellular invasion assays and mouse orthotopic models. The molecular and phenotypic changes involved in the EMT appear to be functionally relevant to the invasive characteristics of epithelial tumors, including bladder cancer [It has been shown that miR-424 controls many crucial biological activities, including cellular differentiation and proliferation, cell-cycle progression and angiogenesis, all of which are often perturbed in malignancies -32. Aberr cancer , 38. At r cancer . We demoin vitro and in vivo findings together suggest that miR-424 functions as a tumor suppressor and that the inhibition of miR-424 expression contributes to the progression of bladder cancer by inducing or promoting malignant cellular behaviors and may even predict poor patient prognosis.The identification and selection of molecular targets are important in cancer therapy. The cell line data presented herein highlight the potential pivotal roles of miR-424 in both the development and progression of bladder cancer. We further evaluated the predictive role of miRNA 424 in patients with bladder cancer. Our ISH data obtained from clinical samples reveal that miR-424 is expressed at lower levels in bladder cancer specimens compared with adjacent non-malignant specimens. The predictive powers of miRNA 424 and DNMT1 were further examined in terms of the clinical outcomes of bladder cancer. The data demonstrated a negative correlation between DNMT1-positive samples and those expressing miR-424. The underexpression of miR-424 associated with a positive staining for DNMT1 was preferentially associated with muscle-invasive bladder TCC relative to non-muscle-invasive disease. Moreover, we uncovered a parallel association of low miR-424 expression with poor prognostic factors, such as advanced clinical stage, node metastasis, and high pathological grade, suggesting that miR-424 may play a suppressive role in the progression of bladder cancer. Our data also showed that an enhanced expression of miR-424 is a significant predictor of progression-free survival in bladder cancer. Consistent with the clinical data, the restoration of miR-424 expression in bladder cancer cells markedly affects the cellular biological behaviors, including suppressed proliferation, enhanced apoptosis and inhibited migration and invasion. Thus, our in vitro and in vivo indicated that the miR-424 expression vector attenuated EGFR and p-EGFR expression associated with decreases in phosphorylated AKT. Moreover, the decrease in EGFR and p-AKT induced by blocking DNMT1 was similar to that induced by miR-424. Through protein analysis, we also found that the expression of MDM2, an oncogene downstream of AKT phosphorylation, was decreased in association with increased p53 in cancer cells with miR-424 expression vectors. The IHC data obtained from clinical samples revealed that miR-424 expression is inversely correlated with EGFR staining, confirming the in vitro and in vivo findings. Accordingly, it is likely that the inhibition of the EGFR-mediated PI3K-AKT pathway plays an important role in the transmission of the miR-424 signal to downstream targets that regulate cell growth, EMT and invasiveness. The data obtained from the present study reveal that increased miR-424 production plays an important tumor-suppressive role in bladder cancer. We outlined the main signaling pathways that are thought to link DNMT1 and miR-424 to bladder cancer , including EGFR, Twist and DNA damage-repair genes. Many urothelial cancer cell lines, including HT1376 and HT1197, overexpress EGFR . EGFR isTaken together, our findings show that miR-424 is downregulated in bladder cancer tissues and correlated with progression of the disease and that miR-424 regulates multiple cellular biological behaviors, such as retarding growth, inducing apoptosis, and reducing invasion, by directly targeting EGFR in bladder cancer cells. MiRNAs that play an important role in tumor formation and/or metastasis can serve as targets for therapeutic intervention, such as miRNA replacement therapy. Therefore, these results highlight a potential role for miR-424 as a molecular predictor and therapeutic target in bladder urothelial carcinoma.The Institutional Review Board of our hospital approved the present study. Informed written consent for the acquisition and storage of medical information and tissue specimens was obtained from all patients. A total of 124 patients with bladder TCC, including 75 with muscle-invasive tumors (the cancer has grown through the connective tissue into the muscle layer) and 49 with non-muscle-invasive tumors , were enrolled in the study. The characteristics of the enrolled patients are listed in Table in situ hybridization detection kit  according to the manufacturer's instruction.Formalin-fixed, paraffin-embedded tissues were cut into 4-μm sections, mounted on slides, deparaffinized with xylene and dehydrated using a graded ethanol series. Antigen retrieval using citric acid (pH 6.0) at 97°C for 30 min was followed by treatment with 3% hydrogen peroxide. The slides were incubated overnight at 4°C with antibodies against specific proteins. After three washes in phosphate-buffered saline (PBS), the sections were incubated with biotinylated secondary antibody for 10 min, stained with peroxidase-avidin and washed again in PBS, and then 3-amino-9-ethylcarbazole solution was added. DNMT1 immunoreactivity was considered positive if the cells showed positive nuclear staining, regardless of cytoplasmic staining. The sections were counterstained with hematoxylin. The IRS was calculated by multiplying the staining intensity by the percentage of positively stained cells. The criterion for positive staining was an IRS score of at least 2. The expression of miR-424 in bladder cancer specimens was determined using a biotin-labeled probe  and an The total RNA from the cells stably transfected with DNMT1-silencing vectors or control vectors was extracted with Trizol reagent solution (Ambion) to quantify microRNAs. Expression of miRNAs was analyzed using a TaqMan MicroRNA array (Life Technologies). Briefly, 1 mg of total RNA was reverse-transcribed using a Megaplex Pools kit , after which miRNAs were amplified and detected using polymerase chain reaction (PCR) with specific primers and TaqMan probes. U6 was used as an endogenous control. The gene expression is expressed as the ratio of expression relative to that in the control.The total RNA from the cells transfected with DNMT1-silencing vectors or control vectors with or without 10 μM DNMT inhibitor for 24 h was extracted with Trizol reagent solution (Ambion) to quantify microRNAs. Expression of selected miRNAs was analyzed using a TaqMan Human MicroRNA Assay kit . Briefly, 5 ng of total RNA were reverse-transcribed using specific stem-loop real-time primers, after which they were amplified and detected using PCR with specific primers and TaqMan probes. U6 snRNA  served as an endogenous control.Four human bladder cancer cell lines, namely HT1376 and HT1197, were obtained from the American Type Culture Collection (ATCC). We maintained the bladder cancer cell lines in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The DNMT inhibitor 5-aza-2′-deoxycytidine (5-AZDC) and miR424 inhibitor were purchased from Sigma  and Life Technologies, respectively. The DNMT1-GFP silencing vector and GFP-control vector were purchased from InvivoGen, as previously described . The miR7 tumor cells were subcutaneously implanted by injection into the dorsal gluteal region . The tumor size was measured every three days after implantation (day 0). The tumor volume was calculated assuming an ellipsoid shape. In the orthotopic tumor implantation model, we performed intravesical instillation of cancer cells as previously described  [This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals as promulgated by the Institutes of Laboratory Animal Resources, National Research Council, USA. The protocol was approved by the Committee on the Ethics of Animal Experiments at our Hospital. Eight-week-old female athymic nude mice were used as the xenograft tumor implantation model. In the ectopic tumor implantation model, 1 × 10s/group) . The extThe invasiveness of cells was determined using a Cell Invasion Assay . After incubation for 24 h, the number of cells in the bottom chamber was determined by measuring the fluorescent anion calcein, which was released from intracellular calcein acetoxymethylester.4 cells/ml in six-well plates for IF staining with or without treatment. After treatment for the specified times, the cells were fixed with 2% paraformaldehyde for 5 min and washed in phosphate buffered saline (PBS) with Tween-20 (PBST). The slides were incubated for 1 h at room temperature with antibodies against E-cadherin and cleaved caspase 3 and then with a FITC-conjugated secondary antibody for 1 h and counterstained with DAPI to visualize the nuclei. The expression of miR-424 in bladder cancer cells in vitro was determined using a fluorescein-labeled probe  according to the manufacturer's instructions.The cells were seeded onto glass coverslips at a density of 5 × 10p < 0.05 was adopted throughout the study to determine significance unless otherwise stated.The survival probabilities were analyzed using the Kaplan-Meier method. Survival was calculated from the date at which treatment was started until the date of death or the most recent follow-up. The significance of the differences between groups was assessed using the log-rank test. The data are presented as the means ± standard error of the mean (SEM). A probability level of