Background. The polyol pathway, a bypass pathway of glucose metabolism initiated by aldose reductase (AR), has been shown to play an important role in mediating tissue ischemia/reperfusion (I/R) impairment recently. Here, we investigated how and why this pathway might affect the fatty liver following I/R. Methods. Two opposite models were created: mice with high-fat-diet-induced liver steatosis were treated with aldose reductase inhibition (ARI) and subsequent I/R; and AR-overexpressing L02 hepatocytes were sequentially subjected to steatosis and hypoxia/reoxygenation. We next investigated (a) the hepatic injuries, including liver function, histology, and hepatocytes apoptosis/necrosis; (b) the NAD(P)(H) contents, redox status, and mitochondrial function; and (c) the flux through the caspase-dependent apoptosis pathway. Results. AR-inhibition in vivo markedly attenuated the I/R-induced liver injuries, maintained the homeostasis of NAD(P)(H) contents and redox status, and suppressed the caspase-dependent apoptosis pathway. Correspondingly, AR overexpression in vitro presented the opposite effects. Conclusion. The flux through the polyol pathway may render steatotic liver greater vulnerability to I/R. Interventions targeting this pathway might provide a novel adjunctive approach to protect fatty liver from ischemia. Despite the last hope for selective patients with end-stage liver disease, liver transplantation is facing the dilemma of a huge donor deficit. Today, this worry has been further compounded by the increasing proportion of fatty liver grafts . As fattThe polyol pathway, which consisted of the two key enzymes aldose reductase (AR) and sorbitol dehydrogenase (SDH), has been implicated in the pathogenesis of various diabetic complications. In this bypass pathway, glucose is reduced to sorbitol by AR with the concomitant oxidation of coenzyme NADPH to NADP, and then SDH converts sorbitol to fructose with the reduction of NAD to NADH. Recently, accumulating evidences have indicated the detrimental role of polyol pathway in the I/R events, which could be reversed by ARI –5. Till The study was approved by the Nanjing Medical University Experimental Animal Department (number NJMU-AEARIA-4001-20120401) and complied closely with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .β-actin were all purchased from Cell Signaling Technology . The primary antibody from rabbit directed against AR and the secondary antibody from mouse were from Abcam .The AR inhibitor (zopolrestat), oleate, and palmitate were all purchased from Sigma . The triglyceride determination kits were from Sigma Chemical Company. NADP(H), NAD(H), and Annexin V-FITC/PI Quantification Kits were from Biovision. Diagnostic kits for JC-1, malondialdehyde (MDA), glutathione (GSH)/oxidized glutathione (GSSG), reactive oxygen species (ROS), and BCA protein assay kits were all from the Beyotime Institute of Biotechnology . Primary antibodies from rabbit directed against Bcl-2, Bax, cleaved caspase 3, and Wild-type C57BL/6 male mice (aged 2 weeks and weighted 9-10 g) were obtained from the Chinese Academy of Sciences , maintained in 25 ± 2°C under a 12 h dark/light cycle, and fed high-fat diet  to develop fatty livers. Six weeks later, the mice were randomized into three groups containing 11 animals each: AR inhibition (ARI) group, pretreated with intraperitoneal administration of zopol (50 mg/kg/day) for 5 days + 70% liver I/R; control (Ctrl) group, treated with saline control + 70% liver I/R; and Sham group, administrated with intraperitoneal administration of zopol (50 mg/kg/day) for 5 days + sham-operation. Before the intraperitoneal injection, one random animal from each group was sacrificed and hepatic sections were stained with Oil Red O to validate the steatosis as previously described [Frozen liver tissues were homogenized and the crude lipids were extracted with the chloroform : methanol (2 : 1). After the removal and blow-drying of chloroform phase, the samples were resuspended in chloroform-Triton X, redried under nitrogen, and eventually resuspended in distilled water. The concentration of triglyceride (TG) was measured in a 96-well plate using a colorimetric assay kit.Single-hepatocyte suspensions were prepared using a modified enzymatic technique as previously described, and subsequent Trypan Blue exclusion demonstrated that cell viability exceeded 89% for all animals [The cytosolic contents of NAD(P)(H) and ratios of NAD/NADH and NADP/NADPH were determined using quantification kits. Fresh liver samples were homogenized in homologous extraction buffer and centrifuged at 14,000 rpm for 5 min. The supernatants were filtered through 10 kD Spin Columns  to cut off the NAD(P)H-consuming enzyme NADase and then measured according to the manufacturer's instructions.The concentrations of total glutathione (GSH) and its oxidized disulfide (GSSG) were detected using a second-step enzymatic method, as previously described . The conTo measure MDA, frozen liver samples were homogenized in 20 mM trisbuffer (pH 7.4) containing 5 mM butylated hydroxytoluene and centrifuged at 4000 ×g for 15 min as previously described . All furHistological sections stained with hematoxylin and eosin (H&E) were processed routinely. The degrees of necrosis and inflammation in the mouse livers were evaluated by one experienced pathologist using the Suzuki score, which measures the extent of congestion, vacuolization, and necrosis on a four-point scale for a total score of 0–12 . For ultTo construct a hepatocellular line stably overexpressing AR, human L02 hepatocytes  were transfected with pcDNA3.1B-AR-Flag-GFP  using Lipofectamine 2000 (Invitrogen). Before transfection, the following primers were used to amplify the open reading frame of the AR gene: AR-nest-out: 5′-ATTTAAAGGTACGCGCCGCG-3′ (forward) and 5′-CGCTGGCCACTCTACAGGTT-3′ (reverse); AR-nest-in: 5′-ATATCTCGAGATGGCAAGCCGTCTCCTGCT-3′ (forward) and 5′-GCGCGAATTCTAAACTCTTCATGGAAGGGGTAATCC-3′.To induce the steatosis in L02 hepatocyte, a modified method from previous literatures was deployed . In brie2 , as previously described [2 which could absorb oxygen and produce carbon dioxide both to an ultimate concentration of 10% within 1 h (oxygen deprivation). Four hours later, the hepatocytes were reexposed to normoxic conditions (95% O2 and 5% CO2) and total culture medium for a further 6 h.The H/R procedure was performed using an AnaeroPack-COescribed . FirstlyTo evaluate the hepatocytes' apoptosis after H/R exposure, commercial Apo-BrdU-Red In Situ DNA Fragmentation Assay Kit  was deployed. The Br-dUTP in this kit could actively bind DNA strand breaks, then be identified by a red fluorescence labeled anti-BrdU monoclonal antibody, and ultimately be read by FCM (Ex/Em: 488/576 nm). In brief, L02 hepatocytes after H/R treatment were collected, resuspended into PBS containing 1% (w/v) formaldehyde, and stored in 4°C. Further procedures were performed according to the instructions contained in the kit.The apoptosis in L02 hepatocytes was explored using the aforementioned methods when L02 cell underwent H/R treatment.JC-1 probe is a bioimaging dye which dominantly accumulates in the mitochondria as aggregates and emits red fluorescence (580 nm) when mitochondrial membrane potential (ΔΨm) is high. On the contrary, JC-1 flees the mitochondria as a monomer and emits green fluorescence (520 nm) when ΔΨm is low. Here, the mitochondrial depolarization in ischemic liver was observed under a fluorescence microscope using JC-1, as previously described . L02 celβ-actin (1 : 2000), and secondary antibody (1 : 2500). The levels of protein expression were determined with Image J software .Total proteins from liver tissues subjected to reperfusion for 6 h or H/R-treated L02 hepatocytes were routinely analyzed with immunoblotting. Before sample loading, the BCA Protein Assay Kits were used to assure the homogeneity of protein concentrations. The antibodies were used at the following concentrations: AR (1 : 1500), cleaved caspase 3 (1 : 2000), Bcl-2 (1 : 2000), Bax (1 : 2000), t-test using SPSS 18.0 statistical software. A probability level of P < 0.05 was considered statistically significant. All results were obtained from at least 5 independent experiments.The results are expressed as mean ± SEM. Statistical analysis was performed with an unpaired Student's To confirm the homogeneity of steatosis in the mouse livers after HFD feeding, their TG contents were analyzed. As shown in As direct causes for liver dysfunction following I/R, hepatocellular necrosis, apoptosis, and inflammation were analyzed. As shown in Figures The transaminases ALT and AST are generated within hepatocytes and drastic elevations always denote cellular membranous leakage or hepatocyte disruption caused by hepatic inflammation and/or necrosis. In the present study, I/R-insult dramatically increased serum transaminase levels, whereas the ARI administration significantly palliated these changes Figures .It is well known that the proper proportions of Bcl-2 family members, especially the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax, are critical for the maintenance of mitochondrial function and the modulation of the caspase-dependent apoptotic pathway. Meanwhile, caspase 3 is widely accepted as an “executor” for cell apoptosis when it was matured to cleaved form. In this study, ARI markedly enhanced Bcl-2 and the Bcl-2/Bax ratio while it suppressed the activation of caspase 3 at the protein level, although there was no evident change in the Bax protein levels Figures .NADPH is an indispensable coenzyme in the generation of GSH, and the latter acts as the major intracellular ROS-scavenger and may in turn inhibit the formation of MDA, a production of membrane lipid-peroxidation. Therefore, the hepatocellular contents of NAD(P)(H), GSH, GSSG, and MDA as well as the proportion of ROS-positive hepatocytes were measured. After ARI treatment, the I/R-induced decreases in the cytosolic content of NADPH and GSH as well as cytoplasmic NAD were significantly attenuated as compared with the control group, while cytoplasmic NADH and cytosolic NADP and MDA contents presented the opposite trends Figures . On the After steatosis induction, L02 cell from pAR, Ctrl, and GFP groups showed significantly high TG level as compared with Lean group, although there was no marked variance among pAR, Ctrl, and GFP groups . On FCM After H/R treatment and JC-1 incubation, Ctrl and GFP groups presented with similar ΔΨm under the fluorescence microscope, being markedly lower than Sham (Red-fluorescent and cytoplasmic dominance) and higher than pAR (Green-fluorescent and cytomembrane dominance) groups .As shown in Figures  in vivo AR inhibition and in vitro AR overexpression were deployed to investigate the role of polyol pathway in ischemic fatty liver. Our results demonstrated that flux through polyol pathway may render steatosis liver greater vulnerability to I/R events, which mainly showed as decreased liver function, disrupted histological architecture, and declined hepatocytes' fates. To be a potential therapeutic target, the role of AR in ischemic steatotic liver should be sufficiently understood and validated.In this study, two opposite models includingNADPH is an indispensable cofactor in the reducing two important antioxidant agents, GSH and thioredoxin, from their oxidized forms. As a result, NADPH acts as an indirect scavenger of ROS in the body , and alsα-TfR-Tf pathway and the enhanced activation of SIRT1 [During hypoxia, low level of ATP could occur in the liver to sustain the hepatocytes through anaerobic glycolysis mediated by cofactor NAD , 21. It of SIRT1 , 24, 25.of SIRT1 .c, the downstream activation of caspase 3, and so forth [Mitochondrion acts as the fundamental generator of intracellular energy and is also involved in multiple hepatocellular function regulations such as redox homeostasis and cell fate . On the so forth . In the so forth . Therefoso forth . Presumaso forth , 32.Until now, various protocols have been developed to modulate the metabolic processes and/or antioxidant properties of the fatty liver during I/R. As the mitochondrion acts as both major victim and the generator of oxidative stress, pharmacological agents aiming at keeping the hepatic energetic balance and mitochondrial function may represent a novel strategy for protecting the fatty liver from severe I/R-induced impairment. in vivo AR inhibition and in vitro AR overexpression, to investigate the effects of AR on the fatty liver after I/R. Our study demonstrated that the flux through the polyol pathway renders the fatty liver greater vulnerability to I/R in the forms of decreased liver function and deteriorated hepatocytes fates, which could be evidently rehabilitated by ARI pretreatment. This study demonstrated that strategies that inhibit polyol pathway might provide a novel adjunctive approach to protect the steatotic liver from I/R-insult.In conclusion, we used two opposite models,


To evaluate functional outcomes, care needs and cost-efficiency of specialist rehabilitation for a multicentre cohort of inpatients with complex neurological disability, comparing different diagnostic groups across 3 levels of dependency.A multicentre cohort analysis of prospectively collected clinical data from the UK Rehabilitation Outcomes Collaborative (UKROC) national clinical database, 2010–2015.All 62 specialist (levels 1 and 2) rehabilitation services in England.Working-aged adults (16–65 years) with complex neurological disability. Inclusion criteria: all episodes with length of stay (LOS) 8–400 days and complete outcome measures recorded on admission and discharge. Total N=5739: acquired brain injury n=4182 (73%); spinal cord injury n=506 (9%); peripheral neurological conditions n=282 (5%); progressive conditions n=769 (13%).Specialist inpatient multidisciplinary rehabilitation.Dependency and care costs: Northwick Park Dependency Scale/Care Needs Assessment (NPDS/NPCNA). Functional independence: UK Functional Assessment Measure +FAM). Cost-efficiency: (1) time taken to offset rehabilitation costs by savings in NPCNA-estimated costs of ongoing care, (2) FIM efficiency (FIM gain/LOS days), (3) FIM+FAM efficiency (FIM+FAM gain/LOS days). Patients were analysed in 3 groups of dependency.Mean LOS 90.1 (SD 66) days. All groups showed significant reduction in dependency between admission and discharge on all measures (paired t tests: p<0.001). Mean reduction in ‘weekly care costs’ was greatest in the high-dependency group at £760/week (95% CI 726 to 794)), compared with the medium-dependency (£408/week (95% CI 370 to 445)), and low-dependency (£130/week (95% CI 82 to 178)), groups. Despite longer LOS, time taken to offset the cost of rehabilitation was 14.2 (95% CI 9.9 to 18.8) months in the high-dependency group, compared with 22.3 (95% CI 16.9 to 29.2) months (medium dependency), and 27.7 (95% CI 15.9 to 39.7) months (low dependency). FIM efficiency appeared greatest in medium-dependency patients (0.54), compared with the low-dependency (0.37) and high-dependency (0.38) groups. Broadly similar patterns were seen across all 4 diagnostic groups.Specialist rehabilitation can be highly cost-efficient for all neurological conditions, producing substantial savings in ongoing care costs, especially in high-dependency patients. A large 5-year national consecutive cohort analysis representing all specialist (levels 1 and 2) rehabilitation units in England.Prospective routinely collected data are reflective of real clinical practice.Different methods for evaluation of cost-efficiency are compared in the same data set.Owing to evolution of reporting requirements over the data collection period, the outcomes of interest were collected in less than 50% of the full rehabilitation data set, so selection bias cannot be excluded.This highly selected group of patients with complex needs is atypical in comparison to populations described in published analyses from other large data sets, but has potential relevance for other health systems that provide tertiary specialist rehabilitation services.Over 1 million people in the UK (2% of the population) have a disabling neurological condition, of which 350 000 require help for most of their daily activities and it is estimated that 850 000 people care for someone with a neurological condition.5Much of the evidence for effectiveness of rehabilitation comes from the arenas of stroke and care of older people. To date, there has been relatively little focus on younger  adults with complex disability following neurological illness or injury. Specialist rehabilitation is increasingly recognised as an essential component of healthcare for this group of patients.They assume linearity of change and equal weighting of items to the prediction of overall cost of care, which is not necessarily the case.They are frequently confounded by floor and ceiling effects.14The FIM is largely focused on physical disability, which limits its use in the context of complex neurological disability, where cognitive and psychosocial problems are often the principal limiting factors.The Functional Independence Measure (FIM) is the most widely used standardised outcome measure for rehabilitation in the world. Established large rehabilitation data sets in the USA and Australia rely on the FIM, not only as a measure of functional gains during rehabilitation, but as a casemix tool and a measure of cost-efficiency. In the absence of direct costing data, the ‘FIM-efficiency index’ (FIM gain/length of stay (LOS)) is often used as a proxy for cost-efficiency.Rehabilitation services are planned and provided in coordinated regional networks over a relatively small geographical area. Local general (level 3) rehabilitation services provide for the majority of patients, but a smaller number are referred to specialist (level 1 or 2) services, which take a selected population of mainly younger adults with complex needs for rehabilitation that are beyond the scope of their local rehabilitation services.16The statutory commitment to life-long provision of care supports longer periods of rehabilitation in these specialist services, provided that this can be demonstrated to produce meaningful cost-benefits through gains in wider independence and reduction of long-term care needs.The UK National Health Service (NHS) provides one of the most comprehensive health and social service systems in the worldSince 2010, the national UK Rehabilitation Outcomes Collaborative (UKROC) database has collated episode data for all inpatients admitted to specialist rehabilitation services (levels 1 and 2) in England, providing national benchmarking on quality, outcomes and cost-efficiency of rehabilitation. Within the UKROC data set, functional gain is evaluated using the UK Functional Assessment Measure (UK FIM+FAM),20A previously published single-centre analysis using these indices demonstrated the cost-efficiency of rehabilitation for younger adults with complex needs following acquired brain injury (ABI),21The objective of this article is to present the first national cohort analysis of the UKROC database to describe functional outcome, change in care needs and cost-efficiency following specialist rehabilitation for working-aged adults with complex disability arising from neurological conditions. In particular, we wished to determine whether the single-centre findings above were reproducible across multiple centres and across a wider range of neurological conditions.What types of functional gain are made during rehabilitation by patients with different neurological conditions?Can longer lengths of stay for highly dependent patients be justified by savings in ongoing care costs?Are there important differences in outcome and cost-efficiency across different neurological conditions and for different levels of patient dependency that service planners should be aware of?Specific research questions were:A large 5-year multicentre national cohort analysis of prospectively collected clinical data from the UKROC national clinical database 2010–2015. Participants were working-aged adults (aged 16–65) with complex neurological disability undergoing specialist inpatient rehabilitation in England.In England, level 1 rehabilitation units are tertiary services providing for a regionally based catchment population of 3–5million and taking a highly selected caseload of patients with very complex needs. They are subdivided by casemix into hyperacute, 1a , 1c  and 1b (mixed) services. Level 2 services take a mixed caseload providing for a more local population, divided into 2a (supradistrict) and 2b  specialist rehabilitation services. The data reporting requirements have evolved over time and vary somewhat between the different levels of service.The UKROC database was established in 2009 through funding a programme grant from the UK National Institute for Health Research (NIHR),http://www.csi.kcl.ac.uk/ukroc.html.Data collection started formally in April 2010. Reporting was initially voluntary and contributing centres could report any one of three approved outcome measures, the Barthel Index (BI), the FIM or UK FIM+FAM.Since April 2012, levels 1 and 2a services are commissioned centrally by NHS England and are required to report the full UKROC data set for all admitted episodes, including the UK FIM+FAM as the principal outcome measure.Reporting of the Northwick Park Dependency Scale and Care Needs Assessment as a measure of cost-efficiency was optional until April 2013, but is now a requirement for national benchmarking for these levels 1 and 2a services.Locally commissioned level 2b  services may still report only lower level data such as the BI or FIM.The data set comprises sociodemographic and process data  as well as clinical information on rehabilitation needs, inputs and outcomes. Full details may be found on the UKROC website The UK FIM+FAM is a global measure of disability.18The Northwick Park Dependency Score (NPDS) is an ordinal scale of dependency on nursing staff time (number of helpers and time taken to assist with each task) designed to assess needs for care and nursing in clinical rehabilitation settings.Low dependency (NPDS <10): patients are largely independent for basic self-care,Medium (NPDS 10–24): patients generally require help from one person for most self-care tasks,High (NPDS ≥25): patients require help from two or more persons for most care tasks and often also have special nursing needs.The NPDS also translates via a computerised algorithm to the Northwick Park Care Needs Assessment (NPCNA)Although there is no formal accreditation process for use of the UK FIM+FAM and NPDS, the attendance of UK FIM+FAM training by at least a core team of staff is requirement for UKROC registration. All units that are registered with UKROC have access to the national training and update workshops, as well as free telephone support.Within the UKROC data set, the cost-efficiency is calculated as the time taken to offset the cost of rehabilitation by the resulting savings in the cost of ongoing care in the community. This is calculated from the ‘episode cost of rehabilitation’ divided by ‘reduction in weekly cost of care’ from admission to discharge, as estimated by the NPCNA. The episode cost was calculated per patient as ‘bed-day cost×LOS’. The cost per bed-day was calculated on updated data from our previously published cost-analysis.In order to identify plausible admissions for rehabilitation (as opposed to brief inpatient assessment or for long-term care) we selected patients with LOS between 8 and 400 days. Other cohort studies have used similar cut-off points,A neurological condition recorded in the diagnostic category;A LOS 8–400 days;Valid UK FIM+FAM and NPDS ratings completed both within 10 days of admission and within the last week before discharge.De-identified data were extracted for all recorded inpatient episodes for adults aged 16–65 years admitted to level 1 or 2 specialist rehabilitation service and discharged during the 5-year period between 1.4.2010 and 31.3.15, if they had:Data were collated in MS Excel and transferred to SPSS V.22 for analysis.Ninety-five per cent CIs were calculated and multiple comparisons made using bootstrapping with samples of n=1000, to minimise the effect of any skewed data.Paired t tests were used to compare significant differences between admission and discharge.One-way analyses of variances (ANOVAs) with bootstrapped post hoc analysis and Bonferroni correction to correct for multiple tests were used to compare differences for diagnostic groups and for different levels of dependency. Key results from post hoc analyses are summarised in the text, but not given in tables. Further details are available on request from the corresponding author.Because data reporting was initially voluntary, missing data were expected. No data were imputed for missing values. There is continued debate about whether to use parametric or non-parametric statistics for this type of data. In this analysis, given the large size of the data set and long ordinal nature of the measures , we have elected to describe and analyse the data using parametric statistics—although non-parametric analysis gave very similar results and is available from the authors if required.In this non-interventional observational study, size was not predetermined but dictated by the accruals to the national data set over the 5-year period that met the inclusion criteria. Because the data set was dominated by patients with ABI, analysis was also undertaken separately for each diagnostic group.A total of 62 rehabilitation units  provided data, with good representation across all four health regions in England.Demographics are given in The study sample comprised approximately 3:2 males:females, with a mean age at admission of 47.3 (SD=12.6) years. The mean rehabilitation LOS was 90.1 (SD=65.5) days. Nearly three-quarters of the sample (73%) had ABI, the remainder having spinal cord injuries , peripheral neurological conditions, for example, Guillain-Barré syndrome (5%) and progressive conditions (13%). One-way ANOVA tests confirmed significant differences in LOS and episode costs (p<0.001) between the different diagnostic groups. Patients with ABI stayed longest (mean 90 days) with the highest episode costs (mean approximately £43 000), while those with progressive conditions stayed the shortest (mean 56 days) and corresponding lower episode costs (mean approximately £25 000).The UKROC software generates ‘FAM splats’ in the form of radar charts which provide an ‘at a glance’ view of the disability profile and patterns of change during rehabilitation for the 30 FIM+FAM items. The differences in functional outcome across the diagnostic groups are summarised in Between admission and discharge, change in FIM+FAM motor score was significantly different between all groups (p<0.001), except between ABI and SCI (p=1.0). Change in FIM+FAM cognitive score was significantly different between all of the groups (p<0.01) except for SCI and progressive conditions (p=1.0). Mean FIM+FAM efficiency was lowest in progressive conditions (mean 0.44) followed by the SCI group (mean 0.59) while broadly similar in the ABI and peripheral neurology groups at a mean of 0.71 and 0.77, respectively.The differences in dependency are also summarised in Between admission and discharge, reduction in dependency and care costs were significantly different between all groups (p<0.001), except between ABI and SCI (p≥0.1). The mean individually calculated time to offset the cost of rehabilitation was lowest in the progressive conditions, at 8.5 months compared with 19–20 for the other groups, but the data were widely spread with overlapping CIs and post hoc tests did not show any significant between-group differences.The change in dependency, care needs and cost of care in the community are summarised in As anticipated, LOS and the total cost of the rehabilitation episode were greatest in the high-dependency group and smallest in the low-dependency group with some twofold difference between them, and post hoc tests showed significant differences seen between all three groups (p<0.001).The ongoing care hours and costs of care in the community remained high at discharge in the same pattern as on admission, but the reduction in care hours and costs was greater in the higher dependency groups, reflecting the higher starting levels—again with significant differences between all dependency groups (p<0.001).Despite the higher cost of the rehabilitation, the time to offset the costs of treatment through savings in the cost of ongoing community care was shortest in the high-dependency group at 14.2 months, followed by the medium-dependency group at 22.3 months, and longest in the low-dependency group 27.7 months. But, despite the nearly twofold difference between the means for the low-dependency and high-dependency groups, the CIs were wide and the between-group ANOVA only just reached significance at p=0.024.By contrast, FIM efficiency was highest in the medium-dependency group at 0.54 but similar between the low-dependency and high-dependency groups at 0.37 and 0.38, respectively (p=0.15). FIM+FAM efficiency was also highest in the medium-dependency group at 0.83, and again similar in the low-dependency and high-dependency groups at 0.70 and 0.58, respectively (p=0.65).Because the data set was dominated by the ABI group, we also compared the main cost-efficiency parameters between dependency groups separately for each of the diagnostic groups—see Large cohort analyses of routinely collected outcome data make an important contribution to our understanding of the gains that can be made from rehabilitation in the course of real-life clinical practice, and provide the opportunity for comparing different populations and practices. This first multicentre analysis of the UK national clinical data set for specialist rehabilitation demonstrates that patients with complex neurological disability have the potential to gain from specialist rehabilitation across a wide range of conditions. It confirmed that the findings from the previous single-centre study of ABI patientsIt should be noted that ‘specialist rehabilitation’ means something rather different in the UK from other countries. In the USA and Australia, a ‘specialist rehabilitation centre’ would be one in which the central focus of treatment is rehabilitation, often in diagnosis-specific programmes . In the UK, the term ‘specialist rehabilitation’ is reserved for tertiary (levels 1 and 2) centres, serving a large catchment population  and admitting a selected population of patients with highly complex rehabilitation needs, regardless of diagnosis.The time since onset was highly skewed but, on average, very long  compared with other published series.Nevertheless, for a UK population with mean age 47 years in 2015, the average projected life expectancy would be approximately 40 years  and 42 years .Our analysis also demonstrated that cost-efficiency measured in this way was highest in the most dependent group of patients. This not only confirms the results from our previous single-centre study in patients with ABI,These findings are important because, in many countries, these highly dependent patients may be denied rehabilitation if they are not expected to make significant gains on a FIM score. Thus, they emphasise the need for a range of different measures, reflecting different patient groups and their potential for change in during rehabilitation. FIM+FAM efficiency showed a similar pattern to FIM efficiency, so the additional 12 items did not necessarily improve its performance as a proxy for cost-efficiency, but they did provide a more holistic evaluation of the change in cognitive/psychosocial function, in addition to motor function, as illustrated in The data were collected in the course of routine clinical practice. Despite the training provided to all units registered with UKROC, the exact level of expertise of clinicians recording the tools in each of the 62 centres is unknown. Nevertheless, the data set is reflective of real-life clinical practice, where staff experience is expected to vary.Because of the evolution of reporting requirements over the data collection period, the analysed sample represents less than 50% of the full rehabilitation data set. This finding was expected and comparison of demographic and baseline data suggested that the analysed sample was reasonably representative of the total population. Nevertheless, the possibility of selection bias cannot be excluded.The NPCNA estimates of continuing care costs are not true assessments as applied in traditional health economic studies. On the other hand, the instrument has been in use for over 15 years and is now quite widely taken up both in clinical practice and in researchFinally, while rehabilitation is provided through the health sector, the saving in care costs accrues to those responsible for ongoing care . Thus, the actual opportunity for realisation and reinvestment of the savings will depend on the local funding arrangements for health and social care.The authors recognise the following limitations to this study:The above limitations accepted, findings from this study add to the growing body of evidence for the cost-effectiveness of rehabilitation for patients with complex disabilities.


The concept of control is gaining importance in the field of allergic rhinitis (AR), with a visual analogue scale (VAS) score being a validated, easy and attractive tool to evaluate AR symptom control. The doctors’ perception of a VAS score as a good tool for evaluating AR symptom control is unknown, as is the level of AR control perceived by physicians who treat patients.307 voluntarily selected physicians attending the annual (2013) European Academy of Allergy and Clinical Immunology (EAACI) meeting completed a digital survey. Delegates were asked to (1) estimate how many AR patients/week they saw during the season, (2) estimate the proportion of patients they considered to have well-, partly- and un-controlled AR, (3) communicate how they gauged this control and (4) assess how useful they would find a VAS as a method of gauging control. 257 questionnaires were filled out completely and analysed.EAACI delegates reported seeing 46.8 [standard deviation (SD) 68.5] AR patients/week during the season. They estimated that 38.7 % (SD 24.0), 34.2 % (SD 20.2) and 20.0 % (SD 16.34) of their AR patients had well-controlled (no AR symptoms), partly-controlled (some AR symptoms), or un-controlled-(moderate/severe AR symptoms) disease despite taking medication [remainder unknown (7.1 %)]. However, AR control was assessed in many ways, including symptom severity (74 %), frequency of day- and night-time symptoms (67 %), activity impairment (57 %), respiratory function monitoring  and incidence of AR exacerbations (50 %). 91 % of delegates felt a simple VAS would be a useful tool to gauge AR symptom control.A substantial portion of patients with AR are perceived as having uncontrolled or partly controlled disease even when treated. A simple VAS score is considered a useful tool to monitor AR control.The online version of this article (doi:10.1186/s13601-015-0080-9) contains supplementary material, which is available to authorized users. Control of disease is considered one of the key outcomes in several medical domains. Although the concept of control is well-defined in asthma, chronic obstructive pulmonary disease and other conditions such as glycaemic control in diabetes,  it has oA VAS for AR has been shown to assess disease severity according to the Allergic Rhinitis and its Impact on Asthma (ARIA)-guidelines, with a VAS cut off score of 50 mm distinguishing between mild and moderate/severe AR in adults . The VASThe major gaps in the current appreciation of VAS as a tool for the evaluation of symptom control are the following: (1) the level of control reached in patients by actual treatment options as perceived by the medical doctors, (2) how disease control is evaluated, and (3) physician perception on the usefulness of a VAS score for the evaluation of symptom control. Physicians often under-estimate disease severity and impact on patients’ lives, while at the same time over-estimate the effectiveness of treatment , 13. PhyA quantitative, digital survey, designed to collect views of physicians who treat AR routinely in clinical practice, was carried out during the 32nd EAACI Congress  from 22nd to 26th June 2013. The survey content was informed by experts in the field of AR  and conducted at the Meda booth by physicians attending the exhibition assess how useful they would find a VAS as a method of gauging control.Delegates were asked to:A representation of a VAS with marker slider was shown to delegates when considering their response to question 4. Survey questions and representation of the VAS with marker slider are provided in Additional file Descriptive statistics (mean and standard deviation) were used to summarise survey responses.307 EAACI 2013 delegates from 60 different countries and from different specialties  completed the survey. Valid question responses were obtained for 257 of these. Surveys from 50 delegates were not included as they were incomplete.On average, respondents reported seeing 46.8 [standard deviation (SD) 68.5] AR patients/week during the season. They estimated that AR was well-controlled, partly-controlled and un-controlled in 38.7 % (SD 24.0), 34.2 % (SD 20.2) and 20.0 % SD 16.34) of patients, respectively, and unknown for the remainder (7.1 %). Delegates reported assessing disease control in many different ways, including symptom severity (74 %), frequency of day- and night-time symptoms (67 %), activity impairment (57 %), respiratory function monitoring  and incidence of AR exacerbations (50 %) . One of the most striking findings was the extra-nasal symptoms, like frequency of day- and night-time symptoms and impaired activity being reported as key determinants of AR control.91 % of the EAACI delegates who completed this survey agreed upon the validity of a VAS as a useful tool for assessing AR control. In our opinion, it is an intuitive tool for use in clinical studies, and by physicians and patients every day. The VAS is well suited to the task of assessing AR control. It is simple and quick to complete, incorporating assessment of both AR symptoms and quality-of-life . It corrLimitations of this survey relate to the fact that the most respondents were specialists , with relatively few GPs included. Delegates were not provided with an alternative control tool choice and also completed the survey at the Meda booth, which may have introduced bias. However, no financial or any other incentive was given to complete the survey. Also delegate speciality was not consistently recorded which may have yielded interesting insights into how AR control is assessed across specialities. Finally, information on what proportion of AR patients had concomitant asthma was not captured. This would have provided important information on how disease control was assessed and whether the perception of control was better or worse for those patients with co-morbid disease.Allergy Diary’ which is now available for free download in many European countries. Users can assess their disease control daily by simply clicking on the VAS in response to the question ‘overall how much are your allergic symptoms bothering you today?’, from ‘not at all bothersome’ to ‘extremely bothersome’. VAS scores are logged and plotted over time with control assessed as well-, partly- and un-controlled, according to specific VAS score cut-offs. The VAS will also be incorporated into a companion app for healthcare providers as well as into the new AR guideline, and used to guide treatment decisions. Moving to a digital VAS is attractive since in real life, on paper, VAS scores are often wrongly completed by the patient, even after explanation; either by failing to cross the line, putting a cross above or below it or writing a figure. An electronic version would prevent such errors. However, it may not allow for complexity of response such as persistence of a problematical co- morbidity despite good control of AR. The overall aim of ‘Allergy Diary’, the Allergy Diary companion app and the updated AR guideline (and the VAS contained within them) is to facilitate a top down communication, from guidelines to healthcare providers to patients, allowing doctors to more easily comply with the guidelines, to better tailor AR medications to patients’ needs and enable patients to better communicate their needs.The VAS will form the basis of a new contre les MAladies Chronique pour un VIeillissement Actif (MACVIA)-ARIA AR app directed at patients called ‘In short, a common language of AR disease control is needed. A simple VAS to measure and assess disease control could meet this need and is welcomed by physicians. It should enable us to move from the illusion to the confirmation of communication.


P = 0.007). No relationship was established between these predominant symptoms and the aeroallergens used in this study. Intermittent mild and moderate/severe AR were evident in 13.5% and 31.1%, while persistent mild and moderate/severe were seen in 20.3% and 35.1%, respectively. House dust mites allergen yielded the highest number of positive responses (22.6%) followed by tree pollen (16.8%). No relationship was observed between the allergens tested and AR severity. Majority of patients were oligosensitive (33.8%) and polysensitive (35.1%) and were not significantly associated with AR severity (P = 0.07). Most AR patients presenting for treatment in Abuja, Nigeria, had moderate-severe persistent AR and showed similar SPT sensitization pattern with countries having similar climatic conditions. Sensitization patterns were not related to ARIA classification or predominant AR symptoms.Allergic rhinitis (AR) is prevalent in Nigeria, though little information exists on the allergen. We assessed the clinical features of AR patients in our environment based on the allergic rhinitis impact on asthma (ARIA) classification. Only patients with positive skin prick test (SPT) were recruited. Seventy-four patients participated in the study. AR and asthma comorbidity were observed in 13.5%. The proportion of “sneezers-runners” was higher than “blockers” with significantly more “sneezers-runners” having persistent AR ( Allergic rhinitis (AR) is a recurrent or chronic allergen specific, IgE-mediated hypersensitivity disorder affecting the nasal lining and characterized by nasal congestion, rhinorrhea, sneezing, nasal itchiness, and/or postnasal drip . It is cSkin prick test (SPT) is a standardized test widely used in the diagnosis of suspected cases of IgE-mediated allergy. It is considered as the gold standard in the diagnosis of allergy . GeneralTo date, there has been no information regarding the common allergen from the Federal Capital Territory (FCT) of Nigeria, Abuja. The aim of this study is to identify the clinical profile of AR patients according to ARIA guidelines and investigate the common allergens in Abuja, Nigeria.This cross-sectional study was conducted for a period of 18 months (March 2014 to September 2015). Sample population was based on the AR patients referred to the allergy clinic, affiliated to Medicaid Radiodiagnostic Center, Wuse 2, Abuja. Patients were consecutively recruited (convenient sampling method) and fall within the age range of 5 to 65 years. They were patients with a positive history of nasal inflammation  for at least one-year duration. Patients' symptoms were categorized as “sneezers-runners” and “blockers” based on the predominant complaint [SPT was performed on patients who have stopped taking antihistamines at least 5 days before the test, while patients with severe dermatographism were excluded from the study. Informed consent was obtained from all patients. SPT was performed by the same investigator.Dermatophagoides pteronyssinus and Dermatophagoides farinae mix), molds , animal dander (cat hair and dog epithelium), and cockroach extracts. Of the pollen allergen used in this study, about 80% of the plants are present in Abuja environment. SPT was performed according to international guidelines [ørsholm., Denmark) placed at least 2 cm apart to avoid contamination. A positive reaction is a wheal ≥ 3 mm in diameter. Histamine hydrochloride (1%) and normal saline (0.9%) were used as positive and negative controls, respectively. Patients with negative skin prick test were excluded from the study.A total of 22 allergens were used in this study; these allergens make up the subtropical prick test batch of ALK-Abello, Denmark. These include tree pollen , weed pollen , grass pollen ), house dust mites , molds, animal dander, and cockroach extracts. SPSS 16 software (Chicago Illinois) was used in the analysis and A total of 96 new patients with suspected allergic rhinitis presented at the allergic clinic within the study period. Only 74 patients from these had a positive SPT result and were enrolled into the study. There were more females (56.8%) than males. The youngest was 5 years and the oldest was 65 years. Their mean age was 30.8 years (95% CI 26.7 to 34.9 years). Twenty-one respondents (28.4%) were categorized as children (5 to 17 years of age) with a male : female ratio of 1.3 : 1. The majority of the study population resided in Abuja Municipal Area Council (AMAC) territory which is basically the city center.P = 0.007). Based on ARIA guidelines, most patients (67.2%) had moderate-severe AR (intermittent and persistent) and this was significantly related to animal exposure (P = 0.035) and not to age, gender, or family history of atopy. There was no significant relationship between these predominant symptoms and any of the aeroallergens.According to predominant symptoms, the proportion of “sneezers-runners” was higher than “blockers” (56.8% versus 43.2%). “Sneezers-runners” tend to have persistent AR, while “blockers” symptoms were more intermittent (P = 0.005). Moderate-severe persistent AR was more common among “sneezers,” while moderate-severe intermittent AR was common with the “blockers.” There was no significant association between AR severity and the presence of asthma (P = 0.26) or family history of atopy (P = 0.19).There was a significant association between AR severity and the predominant complaints  by the pP = 0.03). Also, house dust mites was significantly related to a positive family history of atopy (P = 0.035). There was no significant difference between the positive skin tests and gender as well as history of asthma. Only 23 of the 74 patients (31.1%) had sensitivity to one allergen. There was no single allergen that has the tendency for monosensitization.House dust mites allergen yielded the highest number of positive responses (22.6%) followed by tree pollen (16.8%). Weed pollen allergen yielded the least (7.4%), while animal dander and fungi allergen both came to 13.1% each . The senFurthermore, no relationship was observed between the allergens tested and duration of AR (intermittent and persistent), as shown in Most of the patients with a positive SPT were in the persistent AR category (66.2%). The number of allergens which produced a positive skin response from each patient was closely distributed. The highest was reaction to 3 or more allergens (35.1%) followed by reaction to 2 allergens (33.8%) and then to one allergen (31.1%), as shown in The prevalence of AR is increasing worldwide, yet it remains underdiagnosed and undertreated especially in developing countries . A self-About a third of the children 33%) in this study reported persistence or recurrence of rhinitis symptoms after adenoidectomy. This was lower than was observed by Colavita et al.  but sign3% in thiP = 0.007). We could not establish any relationship between these predominant symptoms and the aeroallergens used in this study. Studies by Shah and Pawankar [We recorded a higher proportion of “sneezers-runners” to “blockers” similar to the findings by Lee et al.  and ShahPawankar  and Deb Pawankar  observedThis study observed that a majority of AR patients were categorized as moderate-severe persistent AR, according to ARIA classification, while the least were mild intermittent AR. This has been observed by most studies , 5, 12 wNo relationship was established between the type of AR and the allergen to which the patients were sensitized. This was similarly observed in a national, cross-sectional study of AR patients in Mexico . The mosOur study also revealed a tendency for multiple sensitizations for allergens among the patients. This was statistically significant in all except animal dander. Thus a patient with positive sensitization to house dust mites could also be sensitized against pollen, insect, or fungi allergens. This supports the argument that time of exposure  does not properly define AR patients  and alsoIn conclusion, most AR patients presenting for treatment in Abuja, Nigeria, have moderate-severe persistent AR and show similar SPT sensitization pattern with other countries having similar climatic conditions. Sensitization patterns are not related to ARIA classification or any predominant AR symptoms but rather may rely on the environmental condition of study area and genetic makeup of the study population.


Castration-resistant progression of prostate cancer after androgen deprivation therapies remains the most critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor (AR) activity is an established driver of castration-resistant progression, and upregulation of the full-length AR (AR-FL) and constitutively-active AR splice variants (AR-Vs) has been implicated to contribute to the resurgent AR activity. We reported previously that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) can reduce the abundance of both AR-FL and AR-Vs. In the present study, we further showed that the effect of PPD on AR expression and target genes was independent of androgen. PPD treatment resulted in a suppression of ligand-independent AR transactivation. Moreover, PPD delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation and inhibited the growth of castration-resistant 22Rv1 xenograft tumors with endogenous expression of AR-FL and AR-Vs. This was accompanied by a decline in serum prostate-specific antigen levels as well as a decrease in AR levels and mitoses in the tumors. Notably, the 22Rv1 xenograft tumors were resistant to growth inhibition by the next-generation anti-androgen enzalutamide. The present study represents the first to show the preclinical efficacy of PPD in inhibiting castration-resistant progression and growth of prostate cancer. The findings provide a rationale for further developing PPD or its analogues for prostate cancer therapy. However, progression to the presently incurable stage, termed castration-resistant prostate cancer (CRPC), invariably occurs i.e., the androgen biosynthesis inhibitor abiraterone and the potent AR antagonist enzalutamide Several new drugs targeting AR reactivation in CRPC have been developed, and two of these have been approved by the FDA for treatment of metastatic CRPC, The ginseng root is one of the most commonly-used medicinal herbs in the Western world, particularly for cancer intervention LNCaP and 22Rv1 cells were obtained from American Type Culture Collection at Passage 4. CWR-R1 cells were provided by Dr. Elizabeth M. Wilson and cultured as described The procedure was described previously qRT-PCR was performed as described trans-activating activity. It was transiently co-transfected into cells with the pRL-TK Renilla luciferase construct (Promega) at 20∶1 ratio as described Renilla luciferase.The androgen-responsive element (ARE)-luciferase reporter plasmid, containing three repeats of the ARE region ligated in tandem to the firefly luciferase reporter 6 LNCaP cells suspended in 50% Matrigel on the right flank after one week of adaptation. When the tumor size reached ∼100 mm3, castration was performed via a scrotal approach. The day following castration, the mice were randomly assigned to two groups and received 40 mg/kg PPD in olive oil or olive oil as control through oral gavage 6 days weekly. For the castration-resistant 22Rv1 tumor model, mice were surgically castrated after one week of adaptation, and allowed to recover for 3 days before they were inoculated subcutaneously with 4×106 22Rv1 cells on the right flank. The day following inoculation, the mice were randomized and placed on the same treatment regimens as described for the LNCaP model. The tumor dimensions and body weights were measured biweekly and weekly, respectively. Tumor volume was calculated as 2 x length0.524 x width2. Tumors were removed, weighed, and fixed in 10% formalin for paraffin embedding and histological analyses. All animal procedures were approved by the Tulane University Institutional Animal Care and Use Committee.Male nude mice were obtained from the NCI Animal Production Center at 5–6 weeks of age. For the castration-resistant progression model of LNCaP tumors, mice were inoculated subcutaneously with 4×10Immunohistochemistry was conducted as described previously The Student's two-tailed t test was used to determine the mean differences between treatment and control. Data are presented as mean ± SEM.We first assessed the effect of PPD on AR-FL and AR-V protein levels in castration-resistant 22Rv1 and CWR-R1 cells cultured in androgen-deprived condition. These two cell models express AR-FL along with three ∼80-KDa major AR-Vs, namely AR-V7, AR-V1 (aka AR4), and AR-V4 (aka AR5) AR-Vs lack the ligand-binding domain and possess androgen-independent constitutive activity 3. Castrated mice were then divided into two groups receiving either olive oil as control or 40 mg/kg PPD through oral gavage 6 days per week. Treatment was initiated the day following castration.We then evaluated the effect of PPD on prostate cancer recurrence after androgen deprivation therapy in the LNCaP xenograft model. LNCaP is an androgen-dependent human prostate cancer cell line and expresses predominantly AR-FL 3 in the control group, but 330 mm3 in the PPD group, indicating ∼67% inhibition of tumor regrowth by PPD. In line with this result, the average tumor weights were 0.76 g and 0.32 g in control and PPD groups, respectively (Compared to LNCaP tumors grown in gonad-intact mice ectively . During ectively .in vitro results, showing the effectiveness of PPD in downregulating AR level and activity in CRPC cells in vivo.Prostate tumor relapse after castration is associated with elevated serum PSA levels We further evaluated the effect of PPD on mitosis in the tumors via immunohistochemistry using an antibody against phospho-histone H3, a marker of mitosis 3. As shown in We next assessed the ability of PPD, in comparison with the next-generation antiandrogen enzalutamide, to inhibit the growth of prostate tumors that are already castration resistant by using the 22Rv1 xenograft model. 22Rv1 cells were inoculated into the right dorsal flank of castrated male nude mice. Administration of 40 mg/kg PPD or 10 or 30 mg/kg enzalutamide through oral gavage 6 days per week was initiated when the tumors reach ∼100 mmInterestingly, enzalutamide, at neither dose, was able to inhibit the growth of 22Rv1 tumors , suppresWe demonstrated previously that PPD suppresses androgen signaling through decreasing the expression of both AR-FL and AR-Vs Rise in serum PSA level is used as a marker for biochemical recurrence after therapy. We showed that PPD supplementation leads to a reduction in serum PSA levels. Moreover, the reduction is not simply a consequence of decrease of tumor sizes by PPD, since the effect remains significant after normalization by tumor weight. The data, instead, indicate the ability of PPD to suppress AR reactivation in tumors relapsed after androgen deprivation and in tumors that are castration resistant, therefore providing a mechanistic basis for the ability of PPD to inhibit castration resistance.The dose of PPD that was used in our xenograft studies was 40 mg per kg of mouse body weight. Based on the body surface area conversion Despite the long appreciation of the importance of targeting AR signaling for prostate cancer treatment, no therapy has been developed to date to target AR directly to reduce its availability. In addition to PPD, several other compounds have been shown pre-clinically to reduce the levels of AR-FL and AR-Vs and to inhibit the growth of CRPC cells


Twenty-five compounds were defined as hits with activity in all leukemia subgroups  at 10 μM drug concentration. Only one of these compounds, quinacrine, showed low activity in normal PBMCs and was therefore selected for further preclinical evaluation. Mining the NCI-60 and the NextBio databases demonstrated leukemia sensitivity and the ability of quinacrine to reverse myeloid leukemia gene expression. Mechanistic exploration was performed using the NextBio bioinformatic software using gene expression analysis of drug exposed acute myeloid leukemia cultures (HL-60) in the database. Analysis of gene enrichment and drug correlations revealed strong connections to ribosomal biogenesis nucleoli and translation initiation. The highest drug–drug correlation was to ellipticine, a known RNA polymerase I inhibitor. These results were validated by additional gene expression analysis performed in-house. Quinacrine induced early inhibition of protein synthesis supporting these predictions. The results suggest that quinacrine have repositioning potential for treatment of acute myeloid leukemia by targeting of ribosomal biogenesis.To find drugs suitable for repositioning for use against leukemia, samples from patients with chronic lymphocytic, acute myeloid and lymphocytic leukemias as well as peripheral blood mononuclear cells (PBMC) were tested in response to 1266 compounds from the LOPAC During the past decades, the traditional drug discovery process has been increasingly lengthy, expensive and the rate of new drug approvals has been slow.1Thus, new strategies for drug discovery and development are needed. One such strategy is drug repositioning (or repurposing) in which a new indication for an existing drug is identified. In this approach, known on-patent, off-patent, discontinued and withdrawn drugs with unrecognized cancer activity can be rapidly advanced into clinical trials for this new indication, as much or all of the required documentation to support clinical trials can rely on previously published and readily available data.3 However, the relatively poor outcome with respect to identification of clinically novel and significantly improved cancer drugs have led to a renewed and growing interest for cancer drug screening based on compound-induced changes in cellular phenotypes.4 Monolayer cultures of human tumor cell lines have been the general model in these efforts. Although these are important models for prediction of mechanisms of action, they are less predictive of clinical activity.6Many screening approaches for identification of new cancer drug candidates have utilized cell-free assays for detection of specific interactions with known or emerging molecular targets.8 However, non-clonogenic in vitro assays performed on primary cultures of patient tumor cell from different diagnoses can detect tumor-type-specific activity of standard and investigational cancer drugs10 and has successfully been utilized in compound library screening.11Primary cultures of patient tumor cells represent an alternative tumor model system that has received very little attention in the context of cancer drug screening and development. Research efforts using primary cultures of patient tumor cell models have mainly focused on prediction of clinical activity of cancer drugs for individual patients.In the present study 12 samples of leukemia and 4 samples of normal mononuclear cells were tested in response to 1266 mechanistically annotated compounds including Food and Drug Administration-approved drugs. Quinacrine was the only compound found with activity in the three leukemia subtypes tested with concurrent low toxicity in normal mononuclear cells, and was, therefore, selected for further preclinical evaluation.Twelve samples of leukemia , four chronic lymphocytic leukemia), as well as peripheral blood mononuclear cells (PBMC) from four healthy donors were used for the compound screen. An additional 9 AML and 10 PBMC samples were used for validation experiments. Clinical characteristics and genetic information of these nine AML patients are summarized in 12 Cell viability was determined by trypan blue exclusion test and the proportion of tumor cells in the preparation was judged by inspection of May-Grünwald-Giemsa stained cytospin slides. All samples used in this study contained >70% tumor cells. The sampling was approved by the Ethics Committee of Uppsala University (Ns 21/93 and 2007/237).Leukemic samples were obtained by bone marrow/peripheral blood sampling. The leukemic cells and PBMCs were isolated by 1.077 g/ml Ficoll-Paque centrifugation and cryopreserved as described previously.in vitro),13 Kasumi-1  mutation),14 KG1a (high content of immature CD34+ cells)15 and MV4-11 16 were all obtained from American Type Culture Collection, ATCC, Rockville, MD, USA. The cell lines were grown in Dulbecco's Modified Eagle's Medium or Roswell Park Memorial Institute-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/l l-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin  and were cultured at 37 °C in humidified air containing 5% CO2.The AML cell lines HL-60 (with ability to differentiate 1280 substance library (Sigma-Aldrich) were used. The compounds were dissolved in dimethyl sulfoxide and further diluted with phosphate-buffered saline. For further testing, quinacrine was purchased from Sigma-Aldrich. For the screening, 384-well plates were prepared with final test concentrations of 10 μM using a Biomek 2000 as described previously.17 For dose–response studies at seven concentrations in twofold dilutions, final test plates were prepared from source plates containing drug solutions at 10 mM using an acoustic dispensing Echo 350 instrument equipped with Labcytes Access robotics .For screening, 1266 mechanistically annotated compounds, many of them previously used in the clinical setting, from the LOPAC18 was used for measurement of the cytotoxic effect of library compounds. The Fluorometric Microculture Cytotoxicity Assay is based on measurement of fluorescence generated from hydrolysis of fluorescein diacetate to fluorescein by cells with intact plasma membranes. Cells were seeded in 384-well drug containing plates (screening) or blank plates (dose–response) using the pipetting robot Precision 2000  and drugs were then added 12 h later using the acoustic Echo liquid dispenser.The Fluorometric Microculture Cytotoxicity Assay, described in detail previously,4 cells/well. For cell lines the number of cells per well was 2500–5000, adjusted individually for each cell line. In each plate, two columns without drugs served as controls and one column with medium only served as blank. Quality criteria for a successful assay included a mean coefficient of variation of <30% in the control and a fluorescence signal in control wells of >5 × the blank. Results are expressed as Survival Index, defined as fluorescence of experimental wells in percent of unexposed control wells with blank values subtracted.Primary cultures of leukemic and PBMC samples were seeded at a density of 5–10 × 10et al.19 In brief, cells were seeded in a six-well plate at a density of 0.4 × 106 cells per well. Cells were left for 24 h, followed by exposure to either quinacrine at a final concentration of 1 or 10 μM, or to vehicle control (dimethyl sulfoxide). After 6 h the cells were washed with phosphate-buffered saline and total RNA was prepared using RNeasy miniprep kit . Starting from two micrograms of total RNA, gene expression analysis was performed using Genome U133 Plus 2.0 Arrays according to the GeneChip Expression Analysis Technical Manual .We followed the original protocol using HL-60 cells as described by Lamb Raw data were normalized with MAS5 (Affymetrix) and gene expression ratios for drug treated vs control cells were calculated to generate lists of regulated genes. Filter criteria were present call for all genes in the treated cell line and an expression cutoff of at least 100 arbitrary expression units. The results were uploaded to the NextBio software.20 The NextBio data mining strategy was divided into two parts. In the first part, public data were collected from diverse sources, such as NCBI GEO, Array Express, SMD, Broad Cancer Genomics, Cancer Biomedical Informatics Grid and other repositories. A data analysis step produced sets of differentially expressed gene signatures associated with each experimental or clinical comparison, such as disease versus normal. In the final step of part one, all signatures were tagged with relevant ontology terms that reflected associated tissue types, disease/phenotype, compound treatment, or genetic perturbation .The NextBio research platform was used for bioinformatic analysis.In the second part, rank-based enrichment statistics were applied to compute pairwise correlation scores between all signatures followed by a meta-analysis to compute individual signature-ontology concept correlation scores. After application of additional statistical criteria, such as correction for multiple hypotheses testing, the ontology concepts were ranked by statistical significance. A numerical score of 100 was assigned to the most significant result, and normalize the other results' scores to the top-ranked result, for details see ref. 21 The Metadrug database contains both public and proprietary functional ontologies that collect genes/proteins into biologically meaningful categories. In the present study the Metadrug Process ontology was used.Enrichment analysis based on gene expression results was also performed with Metadrug software (Thomson Reuters).In Situ mRNA Cytostar-TH assay' kit, Amersham International, Buckinghamshire, UK) using 14C-labeled thymidine and leucine. A Cytostar-TH plate has scintillants molded into the transparent polystyrene bottom. When labeled substrate is absorbed into the intracellular compartment of the cells at the bottom of the wells, the radioisotope spatially approaches the scintillant, thereby generating a detectable signal, whereas free radiolabelled substrate in the supernatant is unable to stimulate the scintillant.22Effects on DNA and protein synthesis were monitored in Cytostar-TH plates  or 14C-leucine , yielding final radioactivity in the wells of 20 and 40 nCi, respectively. Quinacrine (1–10 μM), Tween (2%) or phosphate-buffered saline was added in duplicate. Radioactivity was measured with a computer-controlled Wallac 1450 MicroBetaH trilux liquid scintillation counter  immediately after addition of the cell suspension and at different time points during a 72 h cell culture period.HL-60 cells were suspended in fresh medium containing 23 was used for screening data management. Raw fluorescence data files were loaded into the Small Laboratory Information and Management System software, which calculates percent inhibition according to the formula: Percent inhibition=100 × (x-negative control/positive control-negative control)−1, where x denotes fluorescence from experimental wells. Small Laboratory Information and Management System also identifies and corrects systematic spatial errors. Screening data were subsequently exported to Vortex  software for further analysis. More than or equal to 50% mean inhibition in the leukemic samples was set as the criterion for qualifying as hit compound.Small Laboratory Information and Management System50-values .Dose-response data were analyzed using calculated Survival Index values and the software program GraphPadPrism4 . Data were processed using non-linear regression to a standard sigmoidal dose–response model to obtain IChttp://discover.nci.nih.gov/cellminer/).Analysis of data from the National Cancer Institute growth inhibitory screen in 60 cell lines (NCI-60 GI 50 data) was performed using the NCI Cellminer database .Subsequently, we validated the screening results by dose–response testing in four AML cell line models as well as in nine additional patient samples of acute myeloid leukemia . In the ectively . In the  2.30 μM , statist24 Quinacrine also induced early inhibition of both DNA and protein synthesis  present in the database. Gene enrichment analysis revealed strong connections to ribosomal biogenesis nucleoli and translation initiation . This waynthesis  supportiynthesis . To subsynthesis .8 Indeed good correlations with clinical outcome have been observed using these model systems.25 Quinacrine appeared most active in AML, which consequently emerge as a candidate diagnosis for quinacrine treatment.In the present study, unbiased screening in primary cultures of human leukemias and PBMCs in response to a mechanistically annotated compound library identified quinacrine as an active agent. The use of primary cultures of human tumor cells from patients rather than commonly used cell lines may have distinct advantages by mimicking the clinical situation more closely.26 Quinacrine is no longer commercially available in the United States or European Union but is still marketed in parts of Asia.Quinacrine is an acridine derivative, which was developed in the early 1920s and used extensively as an antimalarial during the Second World War. It was used by over three million soldiers for up to 4 years in a controlled setting, making it one of the most extensively studied drugs ever. Quinacrine has also been used for the treatment of giardiasis and shows therapeutic activity against several autoimmune disorders such as systemic lupus erythematosis and rheumatoid arthritis.28Although quinacrine has been shown to exert antitumor activity in several cell line models of solid tumors, this is the first paper to show significant activity also in leukemia. This was not only demonstrated in AML cell lines but primarily in more clinically relevant cell cultures obtained directly from patients. Quinacrine stands out as an attractive candidate for repositioning as an anti-leukemic agent because of the wealth of existing data about its safety, tolerance and pharmacokinetics available.28 However, in contrast to many other intercalating drugs, quinacrine does not induce DNA damage and is not considered to be mutagenic in human cells.28 Several other mechanisms of action potentially related to the interaction with DNA have been postulated including inhibition of topoisomerase II29 and NF-kappaB30 as well as activation of the p53 signal pathway.32 Moreover, quinacrine have a profound effect on the cellular transcription machinery, particularly targeting cell stress-related transcription. Thus, quinacrine inhibits heat shock factor-1,33 a major transcriptional regulator of the unfolded protein response and hypoxia inducible factor 1-α, a transcription regulator that promotes tumor cell survival under the conditions of limited oxygen supply.28 Furthermore, quinacrine has been shown to interact with phospholipid bilayers leading to inhibition of phospholipase A2 and C, which in turn may affect many membrane-spanning channels and transporters that require the presence and correct architecture of phospholipids.28 One example is the blockage of P-glycoprotein.34Quinacrine intercalate into double-stranded DNA, which is considered to be an important primary target for the observed antitumor activity.35 In particular, the ellipticine drug family, which has demonstrated antitumor activity in clinical trials, was previously proposed to be the result of DNA breakage following the formation of an ellipticine–topoisomerase II–DNA complex.36 However, recently ellipticine was shown to specifically inhibit pol-I transcription.24 In this context, it is notable that the present data show that quinacrine induces gene expression with highest correlations to that of ellipticine. Also, following inhibition of ribosome biogenesis, several ribosomal proteins are released from the nucleolus, which bind MDM2 and inhibit its ubiquitin ligase activity toward p53, resulting in p53 accumulation.35In the present paper, the bioinformatic analysis pointed toward pol-I as a potential molecular target mediating quinacrine activity. This was further strengthened by measurements of gene expression changes induced after quinacrine treatment. Pol-I genes were downregulated, whereas pol-II genes were considerably less affected. Indeed, inhibition of pol-I could possibly explain and contribute to some previously described actions of quinacrine such as topoisomerase II inhibition and p53 activation. Blockers of topoisomerase I and II activity have been reported to disrupt pol-I transcription.35 Therefore, quinacrine can potentially target ribosome biogenesis through both direct inhibition of ribosomal DNA transcription by DNA intercalation and downregulation of expression of transcriptional activation of Myc, resulting in enhanced antiproliferative activity especially in malignant cells expressing a high level of the Myc protein. Indeed, overexpression of Myc has been reported to be associated with adverse clinical outcome and drug resistance in AML.38 However, definitive conclusions about quinacrine being a selective pol-I inhibitor will require further more detailed molecular studies.Interestingly, quinacrine was also found to downregulate expression of the Myc oncogene , which is a known activator of pol-I transcription.26 (http://www.micromedexsolutions.com). Reversible yellow discoloration of the skin, conjunctiva and urine may occur during long-term use or after large doses. High doses used in the treatment of giardiasis may occasionally cause transient acute toxic psychosis reversible upon cessation of quinacrine26 (http://www.micromedexsolutions.com). Other rare but serious side effects of the compound are different forms of dermatitis and aplastic anemia. These side effects develop gradually, and are reversible if quinacrine is discontinued. Hepatitis and hepatic necrosis occur rarely26 (http://www.micromedexsolutions.com).Although the wide use of quinacrine demonstrates that it is a generally safe compound, some side effects have been identified. The most common adverse effects associated with quinacrine are dizziness, headache and gastrointestinal disturbances such as nausea and vomitingM for quinacrine have been documented on a standard malaria regimen.26 The highest concentrations are found in the liver, spleen, lungs and adrenal glands. Interestingly from a repositioning perspective, quinacrine also accumulates in white blood cells reaching intracellular concentrations exceeding 23 μM.27 The major route of quinacrine elimination is via the renal system.26The typical route of quinacrine administration is oral but it can also be administrated by other routes. It is rapidly absorbed from the gastrointestinal tract with plasma levels increasing 2–4 h after administration and reaching a peak in 8–12 h. Plasma concentration increases during the first week and reaches steady-state by the fourth week. The plasma levels of quinacrine are low in comparison with tissue concentrations. Peak plasma concentrations of up to 0.32 μIn conclusion, the overall results indicate that quinacrine, being a multi-targeted agent with a favorable safety profile have repositioning potential for treatment of AML and should be further evaluated in a clinical trial context in this disease.


According to Wikipedia, an editorial is an ‘opinion piece written by the senior editorial staff’ and supposed ‘to reflect the opinion of the periodical’. This editorial is then somewhat different, as it opens the floor to a new journal, which has not yet any periodicity. However, this new journal deserves an introduction, and an editorial ever more.EJNMMI Physics was launched in late 2013 at the Annual Meeting of the European Association of Nuclear Medicine in Lyon [EJNMMI Research journal, edited by Angelika Bischof-Delaloye. Both, the Research and the Physics journal, are open-access associates to EJNMMI that is edited by Ignasi Carrio and recently reported an impact factor of 5.113 [EJNMMI Physics is far away from any impact factor, yet. We appreciate the appeal and the need of impact factors, and we are certain that together we can build this journal into a scientific platform for applied physics, which in due course can be awarded an impact factor. Our certainty stems from the fact that physics, while being an integral part of medicine, and nuclear medicine in particular, is underrepresented in the relevant medical journals. Therefore, EJNMMI Physics intends to support the presentation of physics and physics-related matters as an integral part of nuclear medicine in particular. It aims at providing a forum to scientifically minded people engaged and interested in nuclear medicine and associate imaging and therapeutic applications. As such, EJNMMI Physics complements its partner journal EJNMMI Research and supports EJNMMI, which focuses mainly on clinical perspectives of nuclear medicine.EJNMMI Physics welcomes original materials and studies with a focus on applied physics, mathematics, as well as imaging system engineering and prototyping in nuclear medicine. This includes physics-driven approaches or algorithms supported by physics that foster early clinical adoption of nuclear medicine imaging and therapy regimens. More specifically, we introduce a number of manuscript categories to better reflect the current scope of physics-driven research in this particular field of medicine:‘Original articles’ are manuscripts that describe unique scientific contributions, starting with a hypothesis, clearly defined materials and methods, presenting results and an unbiased discussion and conclusion. A special category of this type of manuscripts is ‘Short communications’ which describe unique scientific contributions based on an abbreviated study presenting first results of promising nature that appeals to the field of physics in nuclear medicine. Further to this, it is planned to introduce a category of ‘Young Investigator reports’ , which provides a platform for young investigators at an early stage in their career who seek to present findings from their scientific work as part of their diploma thesis and alike. It is planned that the EANM Physics and Dosimetry Committees select highlight contributions from this category to be awarded at the Annual Meeting of the EANMMI.‘Artifact reports’ represent brief communications on frequent and infrequent artifacts of significant magnitude observed in routine applications of nuclear medicine imaging together with a narrative on the origin and solutions of these artifacts. This document could serve as guidance to picking up any such described image distortions and to take preventive measures or to apply illustrated correction measures.Other contributions include standard formats, such as ‘opinion papers’ , ‘invited perspectives’ , ‘editorials’ , ‘letters to the editor’ , and ‘teaching files’ , of which the latter may be regarded as an expanded case review, a category that starts to disappear from other, more traditional journals, but which, we feel, provides relevant information in times when imaging is gaining ground and information is presented mainly visually.Today, we are very happy to announce the first round of accepted manuscripts with this issue. This journal starts out with a series of invited perspectives on the role of Physics in Nuclear Medicine –6. We haEJNMMI Physics, making this a respectable arena for physicists and friends of physics: Soren Baarsgaard Hansen , Dale Bailey , Wolfgang Birkfellner , Ronald Boellaard , Ciprian Catana , Alberto del Guerra , Gaspar Delso , Glenn Flux , Dietmar Georg , Gerhard Glatting , Hans Herzog , Brian Hutton , Marc Kachelriess , Claudia Kuntner , George Loudos , Stan Majewski , Osama Mawlawi , Ewald Moser , Otto Muzik , Uwe Pietrzyk , Harald Quick , Bernhard Sattler , Piotr Slomka , Joerg van den Hoff , Dimitris Visvikis , and Charles Watson . Without a doubt, their scope of expertise covers a wide range of Physics in the realms of applications in medicine, and nuclear medicine in particular.Finally, we like to thank those people who volunteer to serve on the Editorial Board of And now, the floor is open to all who like to see Physics grow in within Nuclear Medicine and to get applied in real-world scenarios.


The results revealed that a decrease in particle size led to an increased rate and extent of lipolysis. Particle size had a crucial impact on lipid bioaccessibility, since it is an indicator of the proportion of ruptured cells in the almond tissue. Separated almond cells with intact cell walls showed the lowest levels of digestibility. This study underlines the importance of the cell wall for modulating lipid uptake and hence the positive health benefits underlying almond consumption.Although almonds have a high lipid content, their consumption is associated with reduced risk of cardiovascular disease. One explanation for this paradox could be limited bioaccessibility of almond lipids due to the cell wall matrix acting as a physical barrier to digestion in the upper gastrointestinal tract. We aimed to measure the rate and extent of lipolysis in an  In order to be digested in the human stomach and small intestine, intracellular lipids have to be released from the tissue and emulsified to increase susceptibility of the lipid to lipase action. Previously, it was assumed, by many researchers, that most of the lipid and other macronutrients are released during mastication and available for digestion and absorption in the upper gastrointestinal (GI) tract . HoweverThe composition of almond seeds varies according to a number of factors, including the variety and the harvest; but, typically, the seeds contain approximately 50% of lipids and 12% of dietary fibre, which is mainly derived from cell wall polysaccharides . The lipin vitro gastric digestion  in raw and roasted almond materials. The samples were manipulated such that they exhibited marked differences in lipid bioaccessibility, and included ground and chewed particles of almond and also intact almond cells. To address this main objective, two methods were employed to measure the free fatty acids release: the pH-stat titrimetric method and gas chromatography (GC) analysis. As far as we are aware, however, this is the first time that the pH-stat method has been used to study the effects of lipid bioaccessibility on lipolysis. This study focussed on simulating the digestion in the duodenal compartment only, because the majority of lipid hydrolysis is considered to take place in the duodenum  were produced by Hughson Nut and supplied by the Almond Board of California. The roasted almonds were produced by using a standardised method of hot air (dry) roasting (150 °C for 15 min). Powdered beta-lactoglobulin (β-Lg) was donated by Davisco Foods International . Almond oil, glyceryl tributyrate (99%), glyceryl trioleate (65%), sodium dihydrogen phosphate (99%), disodium hydrogen phosphate (99%), trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid , sodium metabisulphite (⩾99%), sodium chloride (99.5%), calcium chloride (93%), sodium glycodeoxycholate  and pancreatin from porcine pancreas  were purchased from Sigma–Aldrich Chemical Co. . The oil of roasted almond was obtained from Huilerie Croix Verte  and sodium taurocholate hydrate  was obtained from Alpha Aesar . Internal standards for gas chromatography analysis, C15:0 , were purchased from Nu-Chek-Prep, Inc .Natural raw and roasted almonds (2.2Almond oil emulsion was included as a reference sample with a high lipid bioaccessibility (100%). β-Lg solution was prepared by dissolving 1 wt% of powdered β-Lg in 10 mM phosphate buffer (pH 7.0 at 37 °C) and stirring for at least 2 h. Emulsions were made from either synthetic lipids commonly used to determine lipase activity, namely, tributyrin and triolein, or almond oil. Raw and roasted almond oils contained approximately 64.1% and 63.1% of oleic acid (O), 26.1% and 25.9% of linoleic acid (L), and 6.8% and 7.2% of palmitic acid (P), respectively . The emulsions were obtained by pre-emulsifying 1.6 wt% of oil in β-Lg solution using a homogeniser  for 1 min at 1100 rpm. The pre-emulsion was then sonicated with an ultrasonic processor  at 70% amplitude for 2 min. The lipolysis of unemulsified oils, for raw and roasted almond samples, as shown in Section in is the number of droplets of diameter id. Values are presented as the means ± SEM of at least three replicates.The droplet size distributions of the emulsions were measured with a laser light scattering instrument . Water was used as a dispersant (refractive index of 1.330), and the absorbance value of the oil droplets was 0.001. Almond oil has a refractive index of 1.471, tributyrin 1.435 and triolein 1.470, as measured using a refractometer . The particle size measurements were reported as the average volume diameter: 2.32S2O5) at pH 7.0  and a preservative  were obtained by grinding raw and roasted almonds in a blender  before sieving and collecting the particles from sieves of 1000, 500 and 250 μm aperture, as well as a sieve base (size < 250 μm).Masticated samples (triplicate boluses) were obtained, following a protocol described in detail elsewhere . This st2.4in vitro duodenal digestion model used was adapted from previous studies (2 (0.375 M in deionised water). The system was adjusted to pH 7.0 with 0.15 M NaOH and then 1.5 ml of freshly prepared lipase solution were added (40 mg/ml in 10 mM phosphate buffer). The volume of the reaction system in the vessel was 37.5 ml and its final composition was 0.8 wt% lipid, 12.5 mM bile salts, 2.4 mg/ml of lipase, 150 mM NaCl and 10 mM CaCl2.The pH-stat method is a rapid and convenient tool to study lipolysis occurring in the duodenal compartment and on synthetic lipids (e.g. tributyrin and triolein) and olive oil . This me studies  with a rAs a control, the pH fluctuation of the assay mixture alone was determined by running the titration without any lipase; the volume obtained was then deducted from the volume data produced from the subsequent test lipid samples. This step of the titration was repeated for each sample and it ensured the accurate quantification of newly formed FFA derived from the test samples. Since the modelled duodenal digestion conditions were the same for all materials, primarily the amount of lipid, a difference in lipolysis rates would indicate that there were differences in lipid bioaccessibilities among those materials. Each digestion reaction was repeated at least three times.VNaOH corresponds to the volume of NaOH required to neutralise the FFA produced, mNaOH is the concentration of the NaOH solution used (in M), wlipid is the total mass of TAG initially present in the reaction vessel (in g), and Mlipid is the molecular weight of oil (in g/mol) (The volume of NaOH solution (in ml), added as a function of time to keep the pH constant, is equivalent to the amount of FFA released. Also, since the hydrolysis of TAG leads to one molecule of monoacylglycerol (MAG) and two of FFA, the percentage of FFA released can be calculated as follows:n g/mol) . The moln g/mol) . The ini2.5Digestion of almond materials was performed in the pH-stat, as described in Section Each fraction was evaporated to dryness in the heated centrifugal evaporator. Fatty acid methyl esters (FAME) were obtained by dissolving each sample in 1.8 ml of a solution containing methanolic HCl (3 M) and toluene , based on a method published by 2.6The structural changes of the separated lipid-rich cells, pre- and post-digestion, were studied, using either optical  or confocal laser scanning  microscopes, with 40× (N.A. 1.25) and 63× (N.A. 1.32) oil immersion objective lenses. For optical microscopy, samples of almond cells (unstained) were either suspended in buffer (undigested samples) or collected from the reaction vessel (digested samples), and then mounted on a glass slide, covered and viewed immediately. Cells observed with CLSM were stained with Nile red (1 mg/ml in dimethyl sulphoxide). The samples were excited with an argon laser at 488 nm, and the fluorescence emitted by the samples was detected at 630–680 nm.2.7P < 0.05 (2 tailed). Percentages of FFA release, fatty acid concentrations and reaction rates were assessed by repeated-measures analysis of variance (ANOVA) with time and materials  as ‘within-sample’ factors. Differences between raw and roasted samples were analysed by Student’s paired t-test. The relationship between predicted lipid release, using the theoretical model and percentages of FFA release, was analysed by regression analysis.The data were analysed, using SPSS version 17.0. For all tests, the significance level was set at 33.1Pancreatic lipase catalyses the hydrolysis of TAG and generates free fatty acids (FFA). The reaction can be monitored by maintaining a constant pH by automated addition of NaOH, which neutralises the newly formed FFA. The volume of NaOH solution thus added corresponds to the amount of FFA released. Values for the extent of digestion were calculated as the amount of FFA produced relative to the total amount of TAG in the sample over 60 min. Digestion results, expressed as a percentage FFA at 60 min, and for the initial reaction rates (μmol/min) are presented in In our experiments, between 67% and 70% of the almond oil emulsion initially present in the reaction vessel was digested in one hour; which accords with the extent of lipid digestion of oils occurring in the human GI tract .The initial rates of reaction, as well as the quantity of FFA produced in 60 min, were greater for emulsions than for non-emulsified oils and this was found regardless of the oil type . As previn vitro digestion model is a closed system without an absorption phase to remove excess lipolytic product. It contains a finite concentration of bile salts, capable of “solubilising” a limited quantity of FFA, thus limiting the extent of FFA released. As the ability of the bile salt micelles to “solubilise” FFA diminishes, the interface becomes saturated with FFA, which then inhibits further lipolysis .As lipase is only active at the interface, it was expected that a lag phase would be observed, during which the enzyme adsorbs onto the emulsion surface. The “interfacial quality”, governed notably by the interfacial molecular organisation, including the presence of lipolytic products, and the interfacial conformations of lipids, are highly relevant to the kinetics of the lipase . In the 3.2R2 ≈ 0.65 for both raw and roasted almonds). The higher concentration of FFA generated from smaller particles, compared with the largest particles, is attributed to the greater number of ruptured cells, and therefore increased lipid bioaccessibility, as previously shown  or reaction rate (P = 0.064) were observed between raw and roasted particles, indicating that thermal processing had negligible effects on digestion kinetics. The micrographs in No statistically significant differences in the amount of FFA produced over 60 min  generated from the duodenal digestion of raw and roasted almond emulsions, chewed almonds and almond cells, as measured by pH-stat and GC, are presented in 4in vitro study showed that, depending on the physical state of the almond materials, modified by mastication and mechanical processing, the availability of lipids for lipolysis, and the amount of fatty acids produced from the hydrolysis of TAG varied significantly. The amount of FFA release was greater for smaller particles than for larger particles, due to the greater proportion of ruptured cells, and therefore the amount of released (free) lipid available for digestion. The marked differences in lipid digestion between almond emulsion and lipid-rich almond particles, including separated cells, confirm the crucial role played by plant cell walls as a physical barrier against nutrient release and digestion. Despite their small size (∼30–35 μm), almond cells gave less FFA release than did almond particles, supporting the idea that lipid bioaccessibility relies not only on the available surface area but more importantly on the extent of damaged cells. No significant differences in lipid digestibility were observed between raw and roasted almond samples.The current The structural characteristics of ingested almonds and the mechanism(s) by which lipid is released from almond cells are of crucial importance, since these properties have a direct impact on physiological functions  and subsequently on the risks of developing certain conditions, such as cardiometabolic diseases. This has important implications for many other plant foods where structurally intact dietary fibre slows down and/or restricts energy release, digestion and absorption. The concept of an encapsulation mechanism could be employed in the design of novel food ingredients and foods targetted at consumers needing to reduce energy intake and improve body weight management.


Morning hyperglycemia in patients with type 1 diabetes (T1D) may be caused by the dawn phenomenon, the Somogyi effect or poor glycemic control, diagnoses that lead to different clinical management in each case. Previous reports considered the Somogyi effect as a rare entity in clinical practice.This study aimed to evaluate the frequency and characteristics of fasting hyperglycemia by continuous glucose monitoring system (CGMS) in patients with T1D.We analyzed the glycemic profile measured by 72h-CGMS in 85 patients with T1D , resulting in 255 overnight periods. We assessed the nocturnal glycemic profile and the accuracy of the CGMS sensor per night. Fasting hyperglycemia was categorized into dawn phenomenon when there was no nocturnal hypoglycemia followed by a minimum 10mg/dL rise plasma between 4: 00h-7: 00h; Somogyi effect when nocturnal hypoglycemia (< 70mg/dL) between 0: 00h-3: 00h was followed by morning hyperglycemia and poor glycemic control when there was sustained overnight hyperglycemia. Clinical data  were obtained by chart review and compared to the different causes of fasting hyperglycemia.Mean age (SD) and disease duration of participants were 30.7 (15.6) yrs. and 15 (11.9) yrs., respectively. Mean HbA1c was 8.1% (1.97%). Mean sensor glucose value was 162.6mg/dL (46.5) and mean absolute difference (MAD%) was 10.8 , with a correlation coefficient (CC) of 0.92 (0.1). MAD% was similar between all nights but there was a significant difference in CC of night 1 vs. nights 2 (P=0.001) and 3 (P<0.001). Fasting hyperglycemia was observed in 82.4% of patients. Silent nocturnal hypoglycemia was observed in 61.2% of patients. The main cause of elevated morning glucose levels was Somogyi effect (60%), followed by poor glycemic control (27.1%) and dawn phenomenon (12.9%). Comparing clinical characteristics of patients with dawn phenomenon and Somogyi effect yielded no significant differences.CGMS is a valid diagnostic tool to identify the causes of fasting hyperglycemic status. Somogyi effect was the most common cause of fasting hyperglycemia in T1D patients with poor glycemic control. Therefore, assessing the occurrence of nocturnal hypoglycemia might be important before increasing overnight basal insulin dose.