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Background: Long-term opioid use after surgery is a crucial healthcare problem in North America. Data from European hospitals are scarce and differentiation of chronic pain has rarely been considered., Methods: In a mixed surgical cohort of the PAIN OUT registry, opioid use and chronic pain were evaluated before surgery, and 6 and 12 months after surgery (M6/M12). Subgroups with or without opioid medication and pre-existing chronic pain were analysed. M12-chronic pain was categorised as chronic postsurgical pain (CPSP) meeting the ICD-11 definition, chronic pain related to surgery not meeting the ICD-11 definition, and chronic pain unrelated to surgery. Primary endpoint was the rate of M12 opioid users. Variables associated with M12 opioid use and patient-reported outcomes were evaluated., Results: Of 2326 patients, 5.5% were preoperative opioid users; 4.4% and 3.5% took opioids at M6 and M12 (P<0.001). Chronic pain before operation and at M6/M12 was reported by 41.2%, 41.8%, and 34.7% of patients, respectively (P<0.001). The rate of M12 opioid users was highest in group unrelated (22.3%; related 8.3%, CPSP 1.5%; P<0.001). New opioid users were 1.1% (unrelated 7.1%, related 2.3%, CPSP 0.7%; P<0.001). M12 opioid users reported more pain, pain-related physical and affective interference, and needed more opioids than non-users. The predominant variable associated with M12 opioids was preoperative opioid use (estimated odds ratio [95% confidence interval]: 28.3 [14.1-56.7], P<0.001)., Conclusions: Opioid use was low in patients with CPSP, and more problematic in patients with chronic pain unrelated to surgery. A detailed assessment of chronic pain unrelated or related to surgery or CPSP is necessary., Clinical Trial Registration: NCT02083835., (Copyright © 2023 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Acute respiratory distress syndrome (ARDS) has high mortality (~40 %) and requires the lifesaving intervention of mechanical ventilation. A variety of systemic inflammatory insults can progress to ARDS, and the inflamed and injured lung is susceptible to ventilator-induced lung injury (VILI). Strategies to mitigate the inflammatory response while restoring pulmonary function are limited, thus we sought to determine if treatment with CNP-miR146a, a conjugate of novel free radical scavenging cerium oxide nanoparticles (CNP) to the anti-inflammatory microRNA (miR)-146a, would protect murine lungs from acute lung injury (ALI) induced with intratracheal endotoxin and subsequent VILI. Lung injury severity and treatment efficacy were evaluated via lung mechanical function, relative gene expression of inflammatory biomarkers, and lung morphometry (stereology). CNP-miR146a reduced the severity of ALI and slowed the progression of VILI, evidenced by improvements in inflammatory biomarkers, atelectasis, gas volumes in the parenchymal airspaces, and the stiffness of the pulmonary system., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Fetal growth restriction is strongly associated with impaired placentation and abnormal uteroplacental blood flow. Nitric oxide donors such as pentaerythritol tetranitrate are strong vasodilators and protect the endothelium. Recently, we demonstrated in a randomized controlled pilot study a 38% relative risk reduction for the development of fetal growth restriction or perinatal death following administration of pentaerythritol tetranitrate to pregnant women at risk, identified by impaired uterine perfusion at midgestation. Results of this monocenter study prompted the hypothesis that pentaerythritol tetranitrate might have an effect in pregnancies with compromised placental function as a secondary prophylaxis., Objective: This study aimed to test the hypothesis that the nitric oxide donor pentaerythritol tetranitrate reduces fetal growth restriction and perinatal death in pregnant women with impaired placental perfusion at midgestation in a multicenter trial., Study Design: In this multicenter, randomized, double-blind, placebo-controlled trial, 2 parallel groups of pregnant women presenting with a mean uterine artery pulsatility index >95th percentile at 19+0 to 22+6 weeks of gestation were randomized to 50-mg Pentalong or placebo twice daily. Participants were assigned to high- or low-risk groups according to their medical history before randomization was performed block-wise with a fixed block length stratified by center and risk group. The primary efficacy endpoint was the composite outcome of perinatal death or development of fetal growth restriction. Secondary endpoints were neonatal and maternal outcome parameters., Results: Between August 2017 and March 2020, 317 participants were included in the study and 307 were analyzed. The cumulative incidence of the primary outcome was 41.1% in the pentaerythritol tetranitrate group and 45.5% in the placebo group (unadjusted relative risk, 0.90; 95% confidence interval, 0.69-1.17; adjusted relative risk, 0.90; 95% confidence interval, 0.69-1.17; P=.43). Secondary outcomes such as preterm birth (unadjusted relative risk, 0.73; 95% confidence interval, 0.56-0.94; adjusted relative risk, 0.73; 95% confidence interval, 0.56-0.94; P=.01) and pregnancy-induced hypertension (unadjusted relative risk, 0.65; 95% confidence interval, 0.46-0.93; adjusted relative risk, 0.65; 95% confidence interval, 0.46-0.92; P=0.01) were reduced., Conclusion: Our study failed to show an impact of pentaerythritol tetranitrate on the development of fetal growth restriction and perinatal death in pregnant women with impaired uterine perfusion at midgestation. Pentaerythritol tetranitrate significantly reduced secondary outcome parameters such as the incidence of preterm birth and pregnancy-induced hypertension in these pregnancies., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Pain after paediatric appendectomy and tonsillectomy is often undertreated. Benchmarking of hospitals could reveal which measures are associated with improved patient- or parent-reported pain-related outcomes., Methods: A total of 898 anonymised cases from 11 European hospitals participating in PAIN OUT infant were analysed. The children completed a questionnaire on patient-reported outcomes (PROs) 24 h after surgery. According to a composite PRO measure, including pain intensity and pain-related interference, hospitals were allocated to Group I (favourable results), II (average results), and III (unfavourable results). Benchmarking of hospital groups was performed investigating process variables (dosing of non-opioid analgesics, opioids, and dexamethasone) associated with PROs, side-effects, and children's perception of care. Variables associated with PROs were analysed using multinomial regression analysis with the PRO score-related hospital group as a dependent variable (estimated odds ratios [OR], 95% confidence interval [CI])., Results: During the first 24 h after surgery, 1.2 (1.1-1.3) full daily doses of non-opioid analgesics (non-steroidal anti-inflammatory drug [NSAID], paracetamol, metamizole) were administered in group I and 0.7 (0.6-0.8) in group III (P<0.001). Intraoperative dexamethasone was administered to 70.1 and 52.6% of the children in Group I and Group III, respectively (P<0.001). A lower number of full daily doses of non-opioid analgesics: 0.22 [0.15-0.31]), less dexamethasone (0.49 [0.33-0.71]), fewer non-opioid analgesics before the end of surgery (0.37 [0.22-0.62]) and higher opioid doses were associated with hospital allocation to group III vs group I (Nagelkerke's R 2 =0.433)., Conclusions: The results indicated substantial deficits in the concept, application, and dosing of analgesics in paediatric patients after surgery. Timely administration of adequate analgesic doses can easily be introduced into daily clinical practice., Clinical Trial Registration: clinicaltrials.gov NCT02083835., (Copyright © 2022 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Objective: Diabetes affects the lives of patients and their close relatives. Considering the proven benefit of patient education programs, DiaLife was elaborated as the first German education program addressing the needs of relatives. The objective of this study was to investigate its efficacy., Methods: The evaluation was implemented in the form of a cRCT with longitudinal design and waiting list condition.In total, 179 relatives were recruited. Participants' diabetes-related knowledge was defined as the primary outcome. Diabetes-related strains, family interaction and other psychosocial factors were determined as secondary outcomes., Results: A generalized estimating equation model showed a persistent increase of diabetes-related knowledge in the intervention group compared to the control group regardless of the type of diabetes. Concerning secondary outcomes, mixed linear models showed an improvement for relatives of people with type 2 diabetes who participated in the DiaLife program., Conclusion: This study provides evidence of DiaLife's efficacy regarding a persistent increase of diabetes-related knowledge and a positive effect on psychosocial outcomes in relatives of people with type 2 but not in type 1 diabetes. Adding (an)other psychosocial module(s) might improve their well-being and psychosocial outcomes., Practice Implications: Diabetes centers should consider implementing an education program for relatives, such as DiaLife, in their curriculum., Trial Registration: The study was registered at the German Clinical Trials Register (DRKS00015157; date of registration: 24.08.2018)., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Insufficiently treated pain after paediatric appendectomy and tonsillectomy is frequent. We aimed to identify variables associated with poor patient-reported outcomes., Methods: This analysis derives from the European PAIN OUT infant registry providing information on perioperative pharmacological data and patient-reported outcomes 24 h after surgery. Variables associated with the endpoint 'desire for more pain treatment' were evaluated by elastic net regularisation (odds ratio [95% confidence interval])., Results: Data from children undergoing appendectomy (n=472) and tonsillectomy (n=466) between 2015 and 2019 were analysed. Some 24.8% (appendectomy) and 20.2% (tonsillectomy) wished they had received more pain treatment in the 24 h after surgery. They reported higher composite pain scores (5.2 [4.8-5.5] vs 3.6 [3.5-3.8]), more pain-related interference, and more adverse events than children not desiring more pain treatment, and they received more opioids after surgery (morphine equivalents (81 [60-102] vs 50 [43-56] μg kg -1 ). Regression analysis revealed that pain-related sleep disturbance (appendectomy odds ratio: 2.8 [1.7-4.6], tonsillectomy 3.7 [2.1-6.5]; P<0.001) and higher pain intensities (1.5-fold increase) increased the probability of desiring more pain treatment. There was an inverse association between the number of different classes of non-opioids administered preventively, and the desire for more analgesics postoperatively. Children not receiving any non-opioid analgesics before the end of a tonsillectomy had a 3.5-fold (2.1-6.5-fold) increase in the probability of desiring more pain treatment, compared with children receiving at least two classes of different non-opioid analgesics., Conclusions: Preventive administration of at least two classes of non-opioid analgesics is a simple strategy and may improve patient-reported outcomes., Competing Interests: Declarations of interest UMS received honoraria and reimbursement for travel costs from Syntetica and Grünenthal. WM received honoraria from Bionorica, BioQPharm, Böhringer, Grünenthal, Kyowa, Mundipharma, Northern-Swan, Sanofi, TAD, and Tilray. The other authors declare that they have no conflicts of interest., (Copyright © 2021 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Background: Fevipiprant, an oral antagonist of the prostaglandin D 2 receptor 2, reduced sputum eosinophils and improved lung function in phase 2 trials of patients with asthma. We aimed to investigate whether fevipiprant reduces asthma exacerbations in patients with severe asthma., Methods: LUSTER-1 and LUSTER-2 were two phase 3 randomised, double-blind, placebo-controlled, parallel-group, replicate 52-week studies; LUSTER-1 took place at 174 clinical sites in 25 countries and LUSTER 2 took place at 169 clinical sites in 19 countries. Fevipiprant or placebo was added to Global Initiative for Asthma Steps 4 and 5 therapy in adolescents and adults with severe asthma. Patients aged 12 years or older with uncontrolled asthma on dual or triple asthma therapy were randomly assigned by use of interactive response technology to one of three treatment groups (once-daily fevipiprant 150 mg, fevipiprant 450 mg, or placebo) in a 1:1:1 ratio within each of the randomisation strata: peripheral blood eosinophil counts (<250 cells per μL or ≥250 cells per μL), patient age (<18 years or ≥18 years), and use or non-use of oral corticosteroids as part of their standard of care asthma therapy. The primary efficacy endpoint was the annualised rate of moderate to severe asthma exacerbations with 150 mg or 450 mg doses of fevipiprant once daily compared with placebo over 52 weeks, in patients with high blood eosinophil counts (≥250 cells per μL) and in the overall study population. All patients who underwent randomisation and received at least one dose of study medication were included in efficacy and safety analyses. These trials are registered with ClinicalTrials.gov, NCT02555683 (LUSTER-1) and NCT02563067 (LUSTER-2), and are complete and no longer recruiting., Findings: Between Dec 11, 2015, and Oct 25, 2018, 894 patients were randomly assigned to fevipiprant 150 mg (n=301), fevipiprant 450 mg (n=295), or placebo (n=298) in LUSTER-1. Between Dec 3, 2015, and July 10, 2018, 877 patients were randomly assigned to fevipiprant 150 mg (n=296), fevipiprant 450 mg (n=294), or placebo (n=287) in LUSTER-2. In the high eosinophil population, in LUSTER-1 the annualised rate ratio of moderate to severe exacerbations compared with placebo was 1·04 (95% CI 0·77-1·41) for fevipiprant 150 mg and 0·83 (0·61-1·14) for fevipiprant 450 mg, and in LUSTER-2 it was 0·69 (0·50-0·96) for fevipiprant 150 mg and 0·72 (0·52-1·01) for fevipiprant 450 mg. In the overall population, in LUSTER-1 the annualised rate ratio of moderate to severe exacerbations compared with placebo was 0·96 (95% CI 0·75-1·22) for fevipiprant 150 mg and 0·78 (0·61-1·01) for fevipiprant 450 mg and in LUSTER-2 it was 0·82 (0·62-1·07) for fevipiprant 150 mg and 0·76 (0·58-1·00) for fevipiprant 450 mg. In the overall pooled population of both studies, serious adverse events occurred in 53 (9%) patients in the fevipiprant 150 mg group, 50 (9%) in the fevipiprant 450 mg group, and 50 (9%) in the placebo group. Adverse events leading to death occurred in two (<1%) patients in the fevipiprant 450 mg group and three (<1%) in the placebo group., Interpretation: Although neither trial showed a statistically significant reduction in asthma exacerbations after adjusting for multiple testing, consistent and modest reductions in exacerbations rates were observed in both studies with the 450 mg dose of fevipiprant., Funding: Novartis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria (P-score), in line with the results of the EuroFlow quality assessment rounds performed by the EuroFlow expert laboratories(Kalina et al., 2015). Central analysis of data allowed identification of deviations from the standardized procedures and technical issues (e.g. failure to perform correct instrument setup and improper compensation). In summary, here we show that inter-laboratory cross-platform standardization of 8-color flow cytometric measurements in clinical laboratories is feasible and allows for fully comparable MedFI results across BD FACSCanto II and BC Navios instruments. However, adherence to standardized protocols is crucial. Thus, training of the laboratory personnel in the EuroFlow standardized procedures is highly recommended to prevent errors in instrument setup and sample preparation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial and human proteins which might be used later for an easier diagnosis. Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used. Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase. SIGNIFICANCE: Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Myelodysplastic syndromes (MDS) are emerging disorders of the elderly with an increasing burden on healthcare systems. He we report on the first population-based, epidemiological analysis of patients diagnosed with MDS in Switzerland between 2001 and 2012. The aim of this study was to characterize the extent and limitations of currently available population-based, epidemiological data and formulate recommendations for future health services research. The investigated outcomes comprised trends of annual case frequency, classification of morphological subtypes, incidence, mortality and survival. Annual case frequency increased by 20% (from 263 to 315 cases per year), whereas age-standardized incidence-/mortality-rates remained stable (2.5/1.1 per 100'000 person-years). This observation reflects population growth as well as higher diagnostic awareness and not an increase of age-specific risk. However, it will inevitably influence the future prevalence of MDS and the impact on healthcare systems. Reporting of classification in MDS subtypes was poor with modest improvement from 20% to 39% and increased awareness for mainly higher-risk diseases. Relative survival for all patients at 5-years (RS) ranged between 37 and 40%. Significant better RS was found for younger compared to older higher-risk MDS patients (48% vs. 17%), reflecting the effect of allogeneic hematopoietic stem-cell transplantation. However, no survival advantage was found in elderly patients after introduction of hypomethylating agents as standard for care in this patient group. Our data is in line with results from other MDS and cancer registries. It allows formulating recommendations for future collaborative health services research on MDS patients with national and international partners., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Free-range chickens are a good indicator of soil contamination with oocysts because they feed from the ground and they are also an important source of infection for cats that in turn shed oocysts after eating tissues of intermediate hosts. Little is known of the epidemiology of toxoplasmosis in chickens. In the present study 90 Toxoplasma gondii seronegative, sentinel chickens were placed on three (30 each) swine farms in New England in November, 2003. Chickens were bled monthly and their sera were tested for T. gondii antibodies by the modified agglutination test (MAT, cut-off 1:25). Chickens that seroconverted were euthanized and their tissues were bioassayed in mice, cats, or both. Over the course of the experiment (7 months), 31 of 71 chickens seroconverted (MAT 1:100 or higher). Tissues of 26 seropositive chickens were bioassayed in both cats and mice; viable T. gondii was isolated, by bioassay in mice, from hearts (whole) of all 26 chickens, brains (whole) of 3 chickens and leg muscles (25 g) of 11 chickens; 21 of 26 cats fed 250 g of muscle from seropositive chickens excreted T. gondii oocysts. Results indicated that the density of T. gondii in poultry muscle is low but heart is the tissue of choice for isolation of viable parasites., (Published by Elsevier B.V.)

Objective: This study characterized thalamo-cortical communication by assessing the effect of context-dependent modulation on the very early somatosensory evoked high-frequency oscillations (HF oscillations)., Methods: We applied electrical stimuli to the median nerve together with an auditory oddball paradigm, presenting standard and deviant target tones representing differential cognitive contexts to the constantly repeated electrical stimulation. Median nerve stimulation without auditory stimulation served as unimodal control., Results: A model consisting of one subcortical (near thalamus) and two cortical (Brodmann areas 1 and 3b) dipolar sources explained the measured HF oscillations. Both at subcortical and the cortical levels HF oscillations were significantly smaller during bimodal (somatosensory plus auditory) than unimodal (somatosensory only) stimulation. A delay differential equation model was developed to investigate interactions within the 3-node thalamo-cortical network. Importantly, a significant change in the eigenfrequency of Brodmann area 3b was related to the context-dependent modulation, while there was no change in the network coupling., Conclusion: This model strongly suggests cortico-thalamic feedback from both cortical Brodmann areas 1 and 3b to the thalamus. With the 3-node network model, thalamo-cortical feedback could be described., Significance: Frequency encoding plays an important role in contextual modulation in the somatosensory thalamo-cortical network., (Copyright © 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Balanced levels of proteases and anti-proteases are essential in host defense systems. In CF patients' lungs, elevated protease/anti-protease-ratios contribute to damage of airway tissue and premature death with the inherited disease. Little is known about upper airway protease equilibrium in CF., Methods: Neutrophil elastase (NE), Secretory leukocyte protease inhibitor (SLPI), matrix metalloproteinase (MMP)9, tissue inhibitors of metalloproteinase (TIMP)1, cathepsin S (CTSS) and the corresponding cellular distribution were assessed in the nasal lavage (NL) and sputum of 40 CF patients., Results: Concentrations of all proteases and anti-proteases were markedly higher in sputum than in NL (NE: 10-fold, SLPI: 5000-fold). Interestingly, the NE/SLPI ratio was 726-fold higher in NL compared to sputum, while the MMP9/TIMP1 ratio was 4.5-fold higher in sputum compared to NL., Discussion: This first study to compare protease/anti-protease networks of CF upper and lower airways by NL and sputum reveals substantial differences between both compartments' immunological responses. This finding may have implications for sinonasal and pulmonary treatment, possibly leading to new therapeutic approaches., (Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Myeloproliferative neoplasms (MPNs) are a group of clonal disorders characterized by aberrant hematopoietic proliferation and an increased tendency toward leukemic transformation. We used targeted next-generation sequencing (NGS) of 104 genes to detect somatic mutations in a cohort of 197 MPN patients and followed clonal evolution and the impact on clinical outcome. Mutations in calreticulin (CALR) were detected using a sensitive allele-specific polymerase chain reaction. We observed somatic mutations in 90% of patients, and 37% carried somatic mutations other than JAK2 V617F and CALR. The presence of 2 or more somatic mutations significantly reduced overall survival and increased the risk of transformation into acute myeloid leukemia. In particular, somatic mutations with loss of heterozygosity in TP53 were strongly associated with leukemic transformation. We used NGS to follow and quantitate somatic mutations in serial samples from MPN patients. Surprisingly, the number of mutations between early and late patient samples did not significantly change, and during a total follow-up of 133 patient years, only 2 new mutations appeared, suggesting that the mutation rate in MPN is rather low. Our data show that comprehensive mutational screening at diagnosis and during follow-up has considerable potential to identify patients at high risk of disease progression.

Adult hematopoiesis requires tightly regulated cell-cell interactions between hematopoietic cells and the bone marrow stromal microenvironment. We addressed the question if the ectodomain sheddase ADAM10 is essential to regulate adult hematopoiesis. Induced ADAM10 deletion in hematopoietic cells resulted in morphological and histological abnormalities that resemble an unclassified myeloproliferative disorder (MPD). The MPD was characterized by an expansion of granulocytic subpopulations and their infiltration of peripheral hematopoietic tissues, the development of hepatosplenomegaly with extramedullary erythropoiesis, lymphnodepathy and death of the mice around 20weeks after induction. ADAM10 expression analysis during the different stages of the MPD revealed that non-targeted hematopoietic cells repopulated the immune system of the ADAM10-deficient mice. Examination of mice with a myeloid- or epidermis-specific deletion of ADAM10 and bone marrow transplantation (BMT) experiments indicated that the development of the MPD can be triggered by non-cell autonomous effects. We found that plasma levels of clinical markers for MPD such as G-CSF, TIMP-1 and IL-16 were significantly elevated in ADAM10-deficient mice. Our findings indicate that a tightly controlled ADAM10 expression is needed to balance hematopoietic cell-fate decisions in adult mice., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Proteomic profiling and biomarker search are analytical tools as many other. Nevertheless, in the proteomic discovery phase considerable sample fractionation is inevitable before readout. Since these procedures are of notable complexity, proteomic tools need in particular analytical quality validation standards as prevail for other analytical methods. With acceptance of the rule of error propagation the values of imprecision and yield of each preparation step determine overall reproducibility and therewith information harvest of a propagated method series. Thereto, we examined recent proteomic reports with reproducibility data and with parallelization, and automation approaches. Based on the data available from literature it is highly probable, that at least a part of current proteomic platforms actually suffer from high technical variance. The general need for quantification and the impact of precision and recovery levels with each step of typical multi-step workflows are illustrated. All sample preparation approaches that maximize yields (percent recoveries) and minimize overall imprecision should be suitable to improve reliability of biomarker search. This has to be realized by technical innovations that (a) minimize the imprecision of each serial sample preparation step, that (b) minimize the number of serial sample preparation steps, and that (c) parallelize all procedures with a maximum of automation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The 5'-noncoding region (5'NCR) of the HCV-genome comprises an internal ribosome entry site essential for HCV-translation/replication. Phosphorothioate oligodeoxynucleotides (tS-ODN) complementary to this region can inhibit HCV-translation in vitro. In this study, bile acid conjugated tS-ODN were generated to increase cell-selective inhibition of 5'NCR-dependent HCV-translation., Methods: Different bile acid conjugated tS-ODN complementary to the HCV5'NCR were selected for their inhibitory potential in an in vitro transcription/translation assay. To analyze OATP (organic anion transporting polypeptides)-selective uptake of bile acid conjugated ODN, different hepatoma cells were stably transfected with the OATP1B1-transporter and primary human hepatocytes were used. An adenovirus encoding the HCV5'NCR fused to the luciferase gene (Ad-GFP-NCRluc) was generated to quantify 5'NCR-dependent HCV gene expression in OATP-overexpressing hepatoma cells and in vivo., Results: A 17mer phosphorothioate modified ODN (tS-ODN4&#95;13) complementary to HCV5'NCR was able to inhibit 5'NCR-dependent HCV-translation in an in vitro transcription/translation test system by more than 90% and it was also effective in Huh7-cells containing the HCV subgenomic replicon. Conjugation to taurocholate (tS-ODN4&#95;13T) significantly increased selective ODN uptake by primary human hepatocytes and by OATP1B1-expressing HepG2-cells compared to parental HepG2-cells. Correspondingly, tS-ODN4&#95;13T significantly inhibited HCV gene expression in liver-derived OATP1B1-expressing HepG2- or CCL13-cells up to 70% compared to unconjugated tS-ODN and compared to mismatch taurocholate coupled tS-ODN. In vivo, tS-ODN4&#95;13T showed also a trend to block 5'NCR-dependent HCV gene expression., Conclusions: The tested taurocholate conjugated 17mer antisense ODN complementary to HCV5'NCV showed an increased and selective uptake by hepatocytes and liver-derived cells through OATP-mediated transport resulting in enhanced specific inhibition of HCV gene expression in vitro and in vivo. Thus, this novel approach may represent a promising strategy to improve antisense approaches with ODN in the control of hepatitis C infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The African malaria mosquito, Anopheles gambiae, is widespread south of the Sahara including in dry savannahs and semi-arid environments where no surface water exists for several months a year. Adults of the M form of An. gambiae persist through the long dry season, when no surface waters are available, by increasing their maximal survival from 4 weeks to 7 months. Dry season diapause (aestivation) presumably underlies this extended survival. Diapause in adult insects is intrinsically linked to depressed reproduction. To determine if reproduction of the Sahelian M form is depressed during the dry season, we assessed seasonal changes in oviposition, egg batch size, and egg development, as well as insemination rate and blood feeding in wild caught mosquitoes. Results from xeric Sahelian and riparian populations were compared. Oviposition response in the Sahelian M form dropped from 70% during the wet season to 20% during the dry season while the mean egg batch size among those that laid eggs fell from 173 to 101. Correspondingly, the fraction of females that exhibited gonotrophic dissociation increased over the dry season from 5% to 45%, while a similar fraction of the population retained developed eggs despite having access to water. This depression in reproduction the Sahelian M form was not caused by a reduced insemination rate. Seasonal variation in these reproductive parameters of the riparian M form population was less extreme and the duration of reproductive depression was shorter. Blood feeding responses did not change with the season in either population. Depressed reproduction during the dry season in the Sahelian M form of An. gambiae provides additional evidence for aestivation and illuminates the physiological processes involved. The differences between the Sahelian and riparian population suggest an adaptive cline in aestivation phenotypes between populations only 130 km apart., (Published by Elsevier Ltd.)

Introduction: NO-donors reduce the impedance in uteroplacental vessels. Consequently Lees et al. pilot study demonstrated that transdermal nitroglycerin positively influences pregnancy outcome within a high-risk collective. Furthermore the NO-donor pentaerythriltetranitrate additionally expresses cell stabilizing effects in endothelial cells., Objectives: Hypothesizing an effect on endothelial health and uterine perfusion in pregnancies presented with pathological uterine perfusion at 20weeks of gestation we performed a randomized, prospective, and placebo-controlled, double-blind study implemented with the aim to investigate whether the oral NO-donor Pentalong(®) (PETN) is suitable as a prophylactic drug in abnormal placentation., Methods: We included 111 pregnancies presenting with abnormal placental perfusion (bilateral notch or mean RI>0.7) between the 19th and 24thweek of gestation (w.o.g.). Further risk factors (high-risk group: history of HELLP/preeclampsia/IUGR/IUFD/placental abruption, type I diabetes mellitus, hypertension, thrombosis/thrombophilia) were identified in 78 study participants. Fifty-four women received PETN 57 received placebo. Doppler velocimetry measurements of uteroplacental and fetal vessels and fetal growth scans were monitored biweekly with primary endpoints being the occurrence of preeclampsia, IUGR and/or premature birth., Results: Within the first week of intake, PETN improved uteroplacental perfusion significantly in comparison to placebo (mean PI 1.26±0.36 vs. 1.49±0.44; p< 0.01). Overall frequency of premature birth <32nd w.o.g.(4 vs. 12), IUGR <10th percentile (15 vs. 29) and preeclampsia (11 vs. 14) were reduced. Although reduction in preeclampsia was comparably low in those 25 patients developing preeclampsia the outcome was markedly improved in the PETN study group by reducing the frequency of IUGR (4 vs. 10), diagnosis of preeclampsia before 32 w.o.g. (3 vs. 7) and premature birth before 32 w.o.g. (1 vs. 6). Furthermore 4 fetal losses occurred in the study group all in the placebo group., Conclusion: NO-donors constitute an interesting option in the prophylaxis of adverse pregnancy outcome related to abnormal placentation., (Copyright © 2012. Published by Elsevier B.V.)

The solution structure of Fe(II)-peplomycin was determined from NMR data collected for this molecule. As found previously for Fe(II)- and Co(II)-bound bleomycin; the coordination sphere of the metal is composed of the primary and secondary amines in β-aminoalanine, the pyrimidine and imidazole rings in the pyrimidinylpropionamide, and β-hydroxyhistidine moieties, respectively, the amine nitrogen in β-hydroxyhistidine, and either the carbamoyl group in mannose or a solvent molecule. The two most discussed coordination geometries for the aforementioned ligands in metallo-bleomycins have been tested against the NMR data generated for Fe(II)-peplomycin. The interpretation of the experimental evidence obtained through molecular dynamics indicates that both geometries are equally likely in solution for this compound in the absence of DNA, but arguments are offered to explain why one of these geometries is preferred in the presence of DNA., (Published by Elsevier Inc.)

Adrenocortical tumors (ACTs) are common, and most are benign adrenocortical adenomas (ACAs). Malignant adrenocortical carcinoma (ACC) is a rare tumor type and is observed at the rate of one or two cases per million annually. ACTs are classified as either ACAs or ACCs by histopathologic methods that are based on nine Weiss scoring criteria, including the nuclear grade, mitotic rate, presence of necrosis, and others. In this review, we describe the findings of studies that have examined the molecular basis of ACTs, and we compare transcriptome analysis with other diagnostic approaches. ACTs are occasionally difficult to classify. Therefore, molecular techniques, such as microarray analysis, have recently been applied to overcome some of these diagnostic problems. We also discuss the likelihood of the diagnosis and discernment between ACAs and ACCs based on the molecular tests. To show the recent progress in understanding the etiology of ACTs, we highlight the relationship between genetic analysis and transcriptome analysis. We attempt to understand the role of abnormal cell growth and steroid hormone secretion. Genetic and transcriptome analyses have improved our understanding of ACTs considerably, yet many unanswered questions remain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although approximately 95% of patients with polycythemia vera (PV) harbor the V617F mutation in JAK2 exon 14, several mutations in exon 12 have been described in the remaining patients. We conducted a European collaborative study to define the molecular and clinical features of patients harboring these mutations. Overall, 106 PVs were recruited and 17 different mutations identified. Irrespective of the mutation, two-thirds of patients had isolated erythrocytosis, whereas the remaining subjects had erythrocytosis plus leukocytosis and/or thrombocytosis. Compared with JAK2 (V617F)-positive PV patients, those with exon 12 mutations had significantly higher hemoglobin level and lower platelet and leukocyte counts at diagnosis but similar incidences of thrombosis, myelofibrosis, leukemia, and death. In a multivariable analysis, age more than 60 years and prior thrombosis predicted thrombosis. These findings suggest that, despite the phenotypical difference, the outcome of JAK2 exon 12 mutations-positive PV is similar to that of JAK2 (V617F)-positive PV.

During the course of evolution plants have evolved a complex phytohormone-based network to regulate their growth and development. Herein auxins have a pivotal function, as they are involved in controlling virtually every aspect related to plant growth. Indole-3-acetic acid (IAA) is the major endogenous auxin of higher plants that is already known for more than 80 years. In spite of the long-standing interest in this topic, IAA biosynthesis is still only partially uncovered. Several pathways for the formation of IAA have been proposed over the past years, but none of these pathways are yet completely defined. The aim of this review is to summarize the current knowledge on the indole-3-acetamide (IAM)-dependent pathway of IAA production in plants and to discuss the properties of the involved proteins and genes, respectively. Their evolutionary relationship to known bacterial IAM hydrolases and other amidases from bacteria, algae, moss, and higher plants is discussed on the basis of phylogenetic analyses. Moreover, we report on the transcriptional regulation of the Arabidopsis AMI1 gene., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Somatic mutations in TET2 occur in patients with myeloproliferative neoplasms and other hematologic malignancies. It has been suggested that TET2 is a tumor suppressor gene and mutations in TET2 precede the acquisition of JAK2-V617F. To examine the order of events, we performed colony assays and genotyped TET2 and JAK2 in individual colonies. In 4 of 8 myeloproliferative neoplasm patients, we found that some colonies with mutated TET2 carried wild-type JAK2, whereas others were JAK2-V617F positive, indicating that TET2 occurred before JAK2-V617F. One of these patients carried a germline TET2 mutation. However, in 2 other patients, we obtained data compatible with the opposite order of events, with JAK2 exon 12 mutation preceding TET2 mutation in one case. Finally, in 2 of 8 patients, the TET2 and JAK2-V617F mutations defined 2 separate clones. The lack of a strict temporal order of occurrence makes it unlikely that mutations in TET2 represent a predisposing event for acquiring mutations in JAK2.

In follicular lymphoma (FL) and mantle cell lymphoma (MCL) the monoclonal antibody rituximab (R) improves the prognosis when combined with chemotherapy. The present study investigated R-maintenance after R-chemotherapy. Patients with recurring or refractory FL and MCL were randomized to 4 courses of fludarabine, cyclophosphamide, and mitoxantrone (FCM) alone or combined with R (R-FCM). Responding patients underwent a second randomization for R-maintenance comprising 2 further courses of 4-times-weekly doses of R after 3 and 9 months. The first randomization was stopped after 147 patients, when R-FCM revealed a significantly better outcome. All subsequent patients received R-FCM. Of the 176 patients who are currently evaluable (as of October 2005), 138 received R-FCM for remission induction. Response duration was significantly prolonged by R-maintenance after R-FCM, with the median not being reached in this evaluation versus an estimated median of 16 months (P = .001). This beneficial effect was also observed when analyzing FL (P = .035) and MCL (P = .049) separately. Hence, R-maintenance is effective after salvage with R-chemotherapy and significantly prolongs response duration in patients with recurring or refractory FL or MCL.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 77 free-range chickens (Gallus domesticus) from Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 32 (44.4%) of 72 chickens with titers of 1:5 in 4, 1:10 in 3, 1:20 in 1, 1:40 in 1, 1:80 in 8, 1:160 in 8, 1:320 in 3, and 1:640 or higher in 4. Hearts and brains of 31 seropositive chickens were pooled and bioassayed in mice. Tissues from 32 (16+16) seronegative chickens were pooled and fed to two, T. gondii-free cats, and tissues from nine chickens without matching sera were fed to one T. gondii-free cat. Feces of cats were examined for oocysts. T. gondii oocysts were excreted by a cat that was fed tissues of 16 seronegative chickens. T. gondii was isolated by bioassay in mice from 23 chickens with MAT titers of 1:20 or higher. All infected mice from 16 of the 23 isolates died of toxoplasmosis. Overall, 82 (81.1%) of 101 mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 24 isolates using polymorphisms at the SAG2 locus indicated that seven T. gondii isolates were Type I, 17 were Type III, and none was Type II. Phenotypically, T. gondii isolates from chickens from Colombia were similar to isolates from Brazil but different from the isolates from North America; most isolates from chickens from Brazil and Colombia were lethal for mice whereas isolates from North America did not kill inoculated mice. Genetically, none of the T. gondii isolates from Colombia and Brazil was SAG2 Type II, whereas most isolates from chickens from North America were Type II. This is the first report of genetic characterization of T. gondii isolates from Colombia, South America.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in free-range chickens (Gallus domesticus) from 11 Bio-farms in Austria was determined. Antibodies to T. gondii assayed by the modified agglutination test (MAT) were found in 302 of 830 (36.3%) chickens with titers of 1:10 in 50, 1:20 in 69, 1:40 in 53, 1:80 in 40, 1:160 or higher in 90. Hearts of 218 chickens with MAT titers of 10 or higher were bioassayed individually in mice. Tissues from 1183 chickens were pooled and fed to 15, T. gondii-free cats. Feces of the cats were examined for oocysts; 11 cats shed T. gondii oocysts. T. gondii was isolated from 56 chickens by bioassay in mice. Thus, there were 67 isolates of T. gondii from these chickens. Genotyping of these 67 isolates using the SAG2 locus indicated that all 33 were Type II. Phenotypically and genetically these isolates were different from T. gondii isolates from Brazil. None of the isolates was virulent for mice. This is the first report of isolation of T. gondii from chickens from Austria.

The expression of MDR1b coding mRNA is increased in alveolar type II cells from juvenile rat lung in culture. Hydrogen peroxide and paraquat-induced further upregulation supporting that oxidative stress mediated mechanisms are involved in the regulation of MDR1b in rat lung. The expression rates of mRNA for catalase, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Mn-superoxide dismutase (Mn-SOD) remains constant during culture and were not modulated by hydrogen peroxide or paraquat. Thus, antioxidative enzymes in primary A II cells from rat lung are not regulated by reactive oxygen species dependent mechanisms. Primary A II cells were substantially more sensitive towards paraquat-induced cytotoxicity and lipid peroxidation than the permanent human lung tumor cell lines H322 and H358. A 100 microM hydrogen peroxide for 2h induces substantial DNA damage which is not paralleled by an increased rate of lipid peroxidation. The expression rate of mRNA coding for catalase and Mn-SOD was not changed and almost the same is true for the activity of catalase and Cu/Zn-SOD. Only 50 microM paraquat induced a significant decrease in catalase activity and an increase in Cu/Zn-SOD activity.

The present study was undertaken to isolate and genotype Toxoplasma gondii from free-range chickens (Gallus domesticus) from villages in Maharashtra and Tamil Nadu states of central and south India, respectively. Blood, heart, and brain from a total of 741 chickens were examined for T. gondii infection. Antibodies to T. gondii, as assayed with the modified agglutination test (MAT >or = 1:5) were found in 133 (17.9%) chickens. Hearts and brains of 186 chickens were bioassayed in mice. Additionally, hearts and/or brains of most of the seronegative (MAT < 1:5) chickens were fed to 20 T. gondii-free cats, while 32 seropositive chickens (MAT 1:5) were fed to 3 cats. T. gondii was not isolated from any of the chickens by mouse bioassay. Five of the cats that were fed seronegative chickens shed oocysts, while isolates were not obtained from any of the other cats fed seropositive chickens. These five isolates, along with the two that were previously isolated in India through cat bioassay, were genetically analyzed. Genotyping using the SAG 2 locus indicated that two isolates were type II and five were type III. Microsatellite analysis revealed allelic differences between and within the lineages. This is the first report of genetic characterization of any T. gondii isolate from India.

The prevalence of Toxoplasma gondii in free range chickens is a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 40 free range chickens (Gallus domesticus) from a rural area surrounding Paraná, Brazil was assessed. Blood, heart, and brain from each chicken were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT> or =1:5) were found in 16 chickens. Hearts and brains of seropositive (MAT> or =1:5) chickens were bioassayed in mice. Additionally, hearts and brains of seronegative (MAT<1:5) chickens were bioassayed in two T. gondii-free cats (12 chickens per cat). T. gondii was isolated from 13 of 16 (81%) seropositive chickens. Of the two cats fed tissues pooled form seronegative chickens, one shed T. gondii oocysts. Nine of the 13 T. gondii isolates killed 100% of infected mice. The T. gondii isolate from the cat was also virulent for mice. Genotyping of 13 chicken isolates of T. gondii using the SAG2 locus indicated that seven isolates were type I and six were type III; three of these type III isolates killed all infected mice suggesting that all strains virulent for mice are not type I. The isolate from the feces of the cat fed chicken tissues was type I.

The prevalence of Toxoplasma gondii in free range chickens is a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 121 free range chickens (Gallus domesticus) and 19 ducks (Anas sp.) from a rural area surrounding Giza, Egypt was assessed. Blood, heart, and brain from each animal were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 49 (40.4%) chickens in titers of 1:5 in 11, 1:10 in four, 1:20 in four, 1:40 in eight, 1:80 in 10, and 1:160 or more in 12 chickens. Antibodies were found in three ducks each with a titer of 1:80. Hearts and brains of seropositive (MAT > or = 1:5) chickens and ducks were bioassayed in mice. Additionally, hearts and brains of seronegative (MAT<1:5) animals were bioassayed in T. gondii-free cats. T. gondii was isolated from 19 of 49 seropositive chickens (one with a titer of 1:5, two with a titer of 1:20, one with a titer of 1:40, five with a titer of 1:80, three with a titer of 1:160, and seven with a titer of > or = 1:360). One cat fed tissues pooled from 15 seronegative chickens shed T. gondii oocysts, while two cats fed tissues of 34 seronegative chickens did not shed oocysts. T. gondii was isolated from one of the seropositive ducks by bioassay in mice. The two cats fed tissues from 16 seronegative ducks did not shed oocysts. Genotyping of 20 chicken isolates of T. gondii using the SAG 2 locus indicated that 17 isolates were type III and three were type II. The duck isolate of T. gondii was type III. The mice inoculated with tissue stages of all 21 isolates of T. gondii from chickens and ducks remained asymptomatic, indicating that phenotypically they were not type I because type I strains are lethal for mice. Infections with mixed genotypes were not found.

Multidrug resistance type 1 P-glycoproteins (P-gp) and multidrug resistance associated proteins (MRP) were studied in differentiated primary human lung cells in culture, in comparison with permanent human lung cell lines and primary alveolar type II cells from rat lung. AII cells exhibited low basal levels of mdr1b mRNA, that increased over time and after oxygen radical production induced by paraquat. mRNAs coding for antioxidative enzymes catalase (CAT), maganese superoxide dismutase (Mn-SOD) and copper/zinc superoxide dismutase (Cu/Zn-SOD) were not changed. H358, A549, H322 cells expressed low levels of MDR1 mRNA, but the mdr1 substrate rhodamine 123 (Rh 123) was transported out of H358 and H322 cells in a non-invasive, single cell fluorescence assay. The dye efflux could be inhibited by the chemosensitizer, verapamil. Normal human bronchial epithelial cells (NHBEC) expressed immuno-reactive MDR1 P-gp and the MPR protein that was active in the fluorescence assay using the MRP substrate carboxy-dichlorofluorescein (CDF) and MK-571 as an inhibitor. We did observe inter-individual variation of MRP in both the mRNA and the immunoreactive protein in NHBEC culture. Over time (12 weeks) the protein was relatively stable in NHBEC and epithelial cells from peripheral lung (PLC), but the mRNA level was drastically increased when explant cultures were continued (18 weeks).

In this study, we have retrospectively (2 years) assessed the efficacy of the nutritional education (NE) program provided to 83 diabetic patients during an in-hospital training week. Two years after NE, type 1 diabetic patients have decreased significantly their total caloric intake, protein (P<0.05) and fat consumption. Type 2a diabetic patients disclosed a significant increase in CHO consumption while type 2b diabetic patients decreased their fat consumption, SFA consumption and total caloric intake. The percentage of patients following the European Association Study for Diabetes (EASD) nutritional recommendations regarding fat intake has increased significantly in all three diabetic groups and in type 2a diabetic patients regarding CHO consumption. In conclusion, a psycho-educational nutritional education program of 1 week allows a significant improvement of the alimentary behavior 2 years thereafter with 45% of the diabetic patients following the current EASD dietary recommendations.

4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) is a chromophoric and fluorogenic substrate of aldehyde dehydrogenase. Fluorescence of DACA is enhanced by binding to aldehyde dehydrogenase in the absence of catalysis both in the presence and absence of the coenzyme analogue 5'AMP. DACA binds to aldehyde dehydrogenase with a dissociation constant of 1-3 microM and stoichiometry of 2 mol mol(-1) enzyme. Incorporation of DACA during catalysis was also investigated and found to be 2 mol DACA mol(-1) enzyme. Effect of pH on the stoichiometry of DACA incorporation during catalysis has shown that DACA incorporation remained constant at 2 mol DACA mol(-1) enzyme, despite a 74-fold velocity enhancement between pH 5.0 and 9.0. Increase of pH increased decomposition of enzyme-acyl intermediate without affecting the rate-limiting step of the reaction. At pH 7.0 the pH stimulated velocity enhancement was 10-fold over that at pH 5.0; further velocity enhancement (11.5-fold that of pH 7.0) was achieved by 150 microM Mg(2+) ions. The velocity at pH 7.0 with Mg(2+) exceeded that of pH 9.0, and that at maximal pH stimulation at pH 9.5. It was observed that level of intermediate decreased to about 1 mol mol(-1) enzyme, indicating that Mg(2+) ions increased the rate of decomposition of the enzyme-acyl intermediate and shifted the rate-limiting step of the reaction to another step in the reaction sequence.

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly., (Copyright 2000 Academic Press.)

A system for automatic quantification of morphological changes of cell lines, proposed for cytotoxicity tests of biomaterials, is presented. Light-micrographs of cultured cells are segmented by adaptive thresholding within a local adaptive window. Connected cells in binarized micrographs are separated by a novel morphological multiscale method, treating cells in their size-specific scale and hence resulting in scale-independent separations. Significant shape descriptors correlating well with cell toxicity are extracted from single cells. Size and compactness distributions turned out to be reliable and useful parameters, providing an alternative to the common subjective grading of shape deformations by visual inspection. The system is evaluated for several standardized toxical reference substances and is now in use for clinical biocompatibility testing.

A rapid and selective reversed-phase high-performance liquid chromatographic assay with gradient elution and diode-array detection for diacetylmorphine, morphine, codeine, and their free and glucuronidated metabolites in plasma, was developed. After addition of ethylmorphine as internal standard the plasma samples were extracted using C18 ODS-2 solid-phase columns with a recovery better than 80%. The limit of quantitation using an injection volume of 2 microl was 25 ng/ml for each compound. The intra- and inter-day precision was better than 5%. The described method cannot only be used for pharmacokinetic studies but also for intoxication cases to monitor a wide range of opiates.

Subtraction is useful in detecting small changes in sequentially acquired radiographs. Even if the imaging geometry is constant, radiographs must be registered after their digitization. To compare different algorithms for image registration and to register digital X-rays themselves, various similarity measures have been proposed. This study compares eight mathematical similarity standards using 172 radiographs acquired in different, but exactly known projection. Whenever the computation time is a critical factor, e.g. registering images using methods similar to correlation techniques, the entropy of the subtraction image's histogram function (EHDI) is found to be the best similarity standard. If not, e.g. comparative assessing different image registration techniques, the cross covariance coefficient (CCC) is appropriate.