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Mifepristone (RU486, MIF) is a synthetic steroidal hormone with progesterone and glucocorticoid receptor antagonistic characteristics. MIF is commonly used for pharmalogical abortions, but also for the treatment of endometrial and endocrine disorders. The goal of the study was to establish and validate a targeted HPLC-MS/MS method for the quantification of MIF and one of its active metabolites metapristone (MET) in plasma after subcutaneous implantation of slow-release MIF pellets in female BALB/c mice. Additionally, we aimed to apply the analytical method to tissue of several organs to understand the tissue-specific distribution of both analytes after release into systemic circulation. Sample preparation comprised a simple liquid-liquid extraction with diethylether and required 100 μl of plasma or homogenates of approximately 50 mg of tissue. The presented HPLC-MS/MS method showed high sensitivity with baseline separation of MIF, MET, and the internal standard levonorgestrel within a run time of only 8.0 minutes and comparable limits of quantification for plasma and tissue homogenates ranging from 40 pg ml -1 to 105 pg ml -1 for MIF and MET. The presented study is suitable for murine plasma and tissues and can be easily applied to human samples.

Breastfeeding is the most appropriate source of a newborn's nutrition; among the plethora of its benefits, its modulation of circadian rhythmicity with melatonin as a potential neuroendocrine transducer has gained increasing interest. Transplacental transfer assures melatonin provision for the fetus, who is devoid of melatonin secretion. Even after birth, the neonatal pineal gland is not able to produce melatonin rhythmically for several months (with an even more prolonged deficiency following preterm birth). In this context, human breast milk constitutes the main natural source of melatonin: diurnal dynamic changes, an acrophase early after midnight, and changes in melatonin concentrations according to gestational age and during the different stages of lactation have been reported. Understudied thus far are the factors impacting on (changes in) melatonin content in human breast milk and their clinical significance in chronobiological adherence in the neonate: maternal as well as environmental aspects have to be investigated in more detail to guide nursing mothers in optimal feeding schedules which probably means a synchronized instead of mistimed feeding practice. This review aims to be thought-provoking regarding the critical role of melatonin in chrononutrition during breastfeeding, highlighting its potential in circadian entrainment and therefore optimizing (neuro)developmental outcomes in the neonatal setting.

Competing Interests: Potential conflict of interest None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

Background: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target., Objectives: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC., Methods: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo., Results: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival., Conclusions: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes., (© 2023 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Protein aggregates arise naturally under normal physiological conditions, but their formation is accelerated by age or stress-induced protein misfolding. When the stressful event dissolves, these aggregates are removed by mechanisms, such as aggrephagy, chaperone-mediated autophagy, refolding attempts, or the proteasome. It was recently shown that mitochondria in yeast cells may support these primarily cytosolic processes. Protein aggregates attach to mitochondria, and misfolded proteins are transported into the matrix and degraded by mitochondria-specific proteases. Using a proximity labeling method and colocalization with an established stress granule (SG) marker, we were able to show that these mitochondria-localized aggregates that harbor the "super aggregator" Ola1p are, in fact, SGs. Our in vivo and in vitro studies have revealed that Ola1p can be transferred from mitochondria to lipid droplets (LDs). This "mitochondria to LD" aggregate transfer dampens proteotoxic effects. The LD-based protein aggregate removal system gains importance when other proteolytic systems fail. Furthermore, we were able to show that the distribution of SGs is drastically altered in LD-deficient yeast cells, demonstrating that LDs play a role in the SG life cycle., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite significant improvements in survival following preterm birth in recent years, the neurodevelopmental burden of prematurity, with its long-term cognitive and behavioral consequences, remains a significant challenge in neonatology. Neuroprotective treatment options to improve neurodevelopmental outcomes in preterm infants are therefore urgently needed. Alleviating inflammatory and oxidative stress (OS), melatonin might modify important triggers of preterm brain injury, a complex combination of destructive and developmental abnormalities termed encephalopathy of prematurity (EoP). Preliminary data also suggests that melatonin has a direct neurotrophic impact, emphasizing its therapeutic potential with a favorable safety profile in the preterm setting. The current review outlines the most important pathomechanisms underlying preterm brain injury and correlates them with melatonin's neuroprotective potential, while underlining significant pharmacokinetic/pharmacodynamic uncertainties that need to be addressed in future studies.

It is widely accepted that nine hallmarks-including mitochondrial dysfunction, epigenetic alterations, and loss of proteostasis-exist that describe the cellular aging process. Adding to this, a well-described cell organelle in the metabolic context, namely, lipid droplets, also accumulates with increasing age, which can be regarded as a further aging-associated process. Independently of their essential role as fat stores, lipid droplets are also able to control cell integrity by mitigating lipotoxic and proteotoxic insults. As we will show in this review, numerous longevity interventions (such as mTOR inhibition) also lead to strong accumulation of lipid droplets in Saccharomyces cerevisiae , Caenorhabditis elegans , Drosophila melanogaster , and mammalian cells, just to name a few examples. In mammals, due to the variety of different cell types and tissues, the role of lipid droplets during the aging process is much more complex. Using selected diseases associated with aging, such as Alzheimer's disease, Parkinson's disease, type II diabetes, and cardiovascular disease, we show that lipid droplets are "Janus"-faced. In an early phase of the disease, lipid droplets mitigate the toxicity of lipid peroxidation and protein aggregates, but in a later phase of the disease, a strong accumulation of lipid droplets can cause problems for cells and tissues.

Psoriasis is an inflammatory skin disease characterized by increased neo-vascularization, keratinocyte hyperproliferation, a pro-inflammatory cytokine milieu and immune cell infiltration. Diacerein is an anti-inflammatory drug, modulating immune cell functions, including expression and production of cytokines, in different inflammatory conditions. Therefore, we hypothesized that topical diacerein has beneficial effects on the course of psoriasis. The current study aimed to evaluate the effect of topical diacerein on imiquimod (IMQ)-induced psoriasis in C57BL/6 mice. Topical diacerein was observed to be safe without any adverse side effects in healthy or psoriatic animals. Our results demonstrated that diacerein significantly alleviated the psoriasiform-like skin inflammation over a 7-day period. Furthermore, diacerein significantly diminished the psoriasis-associated splenomegaly, indicating a systemic effect of the drug. Remarkably, we observed significantly reduced infiltration of CD11c + dendritic cells (DCs) into the skin and spleen of psoriatic mice with diacerein treatment. As CD11c + DCs play a pivotal role in psoriasis pathology, we consider diacerein to be a promising novel therapeutic candidate for psoriasis.

Context.—: Underuse of laboratory testing has been previously investigated in preselected populations, such as documented malpractice claims. However, these numbers might not reflect real-life situations., Objective.—: To evaluate the underuse and misuse of laboratory follow-up testing in a real-life hospital patient population with microcytic anemia, using laboratory results ordered during routine patient care., Design.—: From all patients in whom a microcytic anemia was detected during routine diagnostics in 2018, all available laboratory data were collected and screened for appropriateness of diagnostic workup of iron deficiency and thalassemia. Subgroup analysis was performed for patient groups with mean corpuscular volume values 75 to 79 μm3 (group 1), 65 to 74 μm3 (group 2), and <65 μm3 (group 3)., Results.—: A total of 2244 patients with microcytic anemia were identified. Follow-up testing for iron deficiency was not performed in 761 cases (34%). For inconclusive ferritin levels due to elevated C-reactive protein results (n = 336), reticulocyte hemoglobin content or soluble transferrin receptor levels were missing in 86 cases (26%). In patients with suspected thalassemia (n = 127), follow-up testing for hemoglobin variants was not performed in 70 cases (55%). Subgroup analysis showed that the frequency of underuse of iron status as well as thalassemia/hemoglobinopathy testing decreased from group 1 to group 3. When considering relevant preexisting anemia diagnoses, laboratory tests were underused in 904 cases (40.3%)., Conclusions.—: Because 40% (n = 904) of the patients with microcytic anemia were potentially not followed up correctly, laboratory specialists are advised to act by implementing demand management strategies in collaboration with clinicians to overcome underuse of laboratory tests and to improve patient safety., (© 2023 College of American Pathologists.)

Background/aims: Cells adapt to chronic extracellular hypotonicity by altering metabolism. Corresponding effects of sustained hypotonic exposure at the whole-person level remain to be confirmed and characterized in clinical and population-based studies. This analysis aimed to 1) describe changes in urine and serum metabolomic profiles associated with four weeks of sustained > +1 L/d drinking water in healthy, normal weight, young men, 2) identify metabolic pathways potentially impacted by chronic hypotonicity, and 3) explore if effects of chronic hypotonicity differ by type of specimen and/or acute hydration condition., Materials: Untargeted metabolomic assays were completed for specimen stored from Week 1 and Week 6 of the Adapt Study for four men (20-25 years) who changed hydration classification during that period. Each week, first-morning urine was collected after overnight food and water restriction, and urine (t+60 min) and serum (t+90 min) were collected after a 750 mL bolus of drinking water. Metaboanalyst 5.0 was used to compare metabolomic profiles., Results: In association with four weeks of > + 1 L/d drinking water, urine osmolality decreased below 800 mOsm/kg H 2 O and saliva osmolality decreased below 100 mOsm/kg H 2 O. Between Week 1 and Week 6, 325 of 562 metabolic features in serum changed by 2-fold or more relative to creatinine. Based on hypergeometric test p-value <0.05 or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway impact factor >0.2, the sustained > + 1 L/d of drinking water was associated with concurrent changes in carbohydrate, protein, lipid, and micronutrient metabolism, a metabolomic pattern of carbohydrate oxidation via the tricarboxylic acid (TCA) cycle, instead of glycolysis to lactate, and a reduction of chronic disease risk factors in Week 6. Similar metabolic pathways appeared potentially impacted in urine, but the directions of impact differed by specimen type., Conclusion: In healthy, normal weight, young men with initial total water intake below 2 L/d, sustained > + 1 L/d drinking water was associated with profound changes in serum and urine metabolomic profile, which suggested normalization of an aestivation-like metabolic pattern and a switch away from a Warburg-like pattern. Further research is warranted to pursue whole-body effects of chronic hypotonicity that reflect cell-level effects and potential beneficial effects of drinking water on chronic disease risk.

Background: Recommendations on the optimal preservation of 24 h urine for the metabolic work-up in urolithiasis patients are very heterogeneous. In case two such tests with different storage condition recommendations are being analysed, multiple collections would be needed, challenging especially elderly and very young patients. We therefore aimed to evaluate the stability of urine constituents under different storage conditions., Material and Methods: We collected urine samples from ten healthy volunteers and prepared aliquots to be stored either at room temperature or 4 °C. Some aliquots were preserved using hydrochloric acid prior to storage, some thereafter, some using the BD Urine preservation tube and some were not preserved at all. Storage duration was 0, 24, 48 or 72 h. In all samples calcium, magnesium, phosphorus, creatinine, oxalate, citrate and uric acid were measured and compared to the according reference sample., Results: We could not find any significant deviation for any of the analytes and preanalytical treatment conditions compared to the associated reference sample., Conclusion: Preservation of 24 h urine for the metabolic evaluation in stone formers might not be necessary for sample storage up to 72 h., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objectives: Although laboratory result presentation may lead to information overload and subsequent missed or delayed diagnosis, little has been done in the past to improve this post-analytical issue. We aimed to investigate the efficiency, efficacy and user satisfaction of alternative report formats., Methods: We redesigned cumulative (sparkline format) and single reports (improved tabular and z-log format) and tested these on 46 physicians, nurses and medical students in comparison to the classical tabular formats, by asking standardized questions on general items on the reports as well as on suspected diagnosis and follow-up treatment or diagnostics., Results: Efficacy remained at a very high level both in the new formats as well as in the classical formats. We found no significant difference in any of the groups. Efficiency improved in all groups when using the sparkline cumulative format and marginally when showing the improved tabular format. When asking medical questions, efficiency and efficacy remained similar between report formats and groups. All alternative reports were subjectively more attractive to the majority of participants., Conclusions: Showing cumulative reports as a graphical display led to faster detection of general information on the report with the same level of correctness. Considering the familiarity bias of the classical single report formats, the borderline-significant improvement of the alternative tabular format and the non-inferiority of the z-log format, suggests that single reports might benefit from some improvements derived from basic information design., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Introduction: Intoxications are common in intensive care units (ICUs). The number of causative substances is large, mortality usually low. This retrospective cohort study aims to characterize differences of intoxicated compared to general ICU patients, point out variations according to causative agents, as well as to highlight differences between survivors and non-survivors among intoxicated individuals in a large-scale multi-center analysis., Methods: A total of 105,998 general ICU patients and 4,267 individuals with the admission diagnoses "overdose" and "drug toxicity" from the years 2014 and 2015 where included from the eICU Collaborative Research Database. In addition to comparing these groups with respect to baseline characteristics, intensive care measures and outcome parameters, differences between survivors and non-survivors from the intoxication group, as well as the individual groups of causative substances were investigated., Results: Intoxicated patients were younger (median 41 vs. 66 years; p<0.001), more often female (55 vs. 45%; p<0.001), and normal weighted (36% vs. 30%; p<0.001), whereas more obese individuals where observed in the other group (37 vs. 31%; p<0.001). Intoxicated individuals had a significantly lower mortality compared to general ICU patients (1% vs. 10%; aOR 0.07 95%CI 0.05-0.11; p<0.001), a finding which persisted after multivariable adjustment (aOR 0.17 95%CI 0.12-0.24; p<0.001) and persisted in all subgroups. Markers of disease severity (SOFA-score: 3 (1-5) vs. 4 (2-6) pts.; p<0.001) and frequency of vasopressor use (5 vs. 15%; p<0.001) where lower, whereas rates of mechanical ventilation where higher (24 vs. 26%; p<0.001) in intoxicated individuals. There were no differences with regard to renal replacement therapy in the first three days (3 vs. 4%; p=0.26). In sensitivity analysis (interactions for age, sex, ethnicity, hospital category, maximum initial lactate, mechanical ventilation, and vasopressor use), a trend towards lower mortality in intoxicated patients persisted in all subgroups., Conclusion: This large-scale retrospective analysis indicates a significantly lower mortality of intoxicated individuals compared to general ICU patients., (© 2022. The Author(s).)

Colorectal cancer (CRC) is a major public health burden and one of the leading causes of cancer-related deaths worldwide. Screening programs facilitate early diagnosis and can help to reduce poor outcomes. Serum metabolomics can extract vital molecular information that may increase the sensitivity and specificity of colonoscopy in combination with histopathological examination. The present study identifies serum metabolite patterns of treatment-naïve patients, diagnosed with either advanced adenoma (AA) or CRC in colonoscopy screenings, in the framework of the SAKKOPI (Salzburg Colon Cancer Prevention Initiative) program. We used a targeted flow injection analysis and liquid chromatography-tandem mass spectrometry metabolomics approach (FIA- and LC-MS/MS) to characterise the serum metabolomes of an initial screening cohort and two validation cohorts (in total 66 CRC, 76 AA and 93 controls). The lipidome was significantly perturbed, with a proportion of lipid species being downregulated in CRC patients, as compared to AA and controls. The predominant alterations observed were in the levels of lyso-lipids, glycerophosphocholines and acylcarnitines, but additionally, variations in the quantity of hydroxylated sphingolipids could be detected. Changed amino acid metabolism was restricted mainly to metabolites of the arginine/dimethylarginine/NO synthase pathway. The identified metabolic divergences observed in CRC set the foundation for mechanistic studies to characterise biochemical pathways that become deregulated during progression through the adenoma to carcinoma sequence and highlight the key importance of lipid metabolites. Biomarkers related to these pathways could improve the sensitivity and specificity of diagnosis, as well as the monitoring of therapies.

Prognostication after cardiopulmonary resuscitation (CPR) is complex. Novel biomarkers like soluble suppression of tumorigenicity 2 (sST2) may provide an objective approach. A total of 106 post-CPR patients were included in this single-center observational prospective study. Serum sST2 levels were obtained 24 h after admission. Individuals were assigned to two groups: patients below and above the overall cohort's median sST2 concentration. Primary outcome was a combined endpoint at 6 months (death or Cerebral Performance Category > 2); secondary endpoint 30-day mortality. A uni- and multivariate logistic regression analysis were conducted. Elevated sST2-levels were associated with an increased risk for the primary outcome (OR 1.011, 95% CI 1.004-1.019, p = 0.004), yet no patients with poor neurological outcome were observed at 6 months. The optimal empirical cut-off for sST2 was 46.15 ng/ml (sensitivity 81%, specificity 53%, AUC 0.69). Levels above the median (> 53.42 ng/ml) were associated with higher odds for both endpoints (death or CPC > 2 after 6 months: 21% vs. 49%, OR 3.59, 95% CI 1.53-8.45, p = 0.003; death after 30 days: 17% vs. 43.3%, OR 3.75, 95% CI 1.52-9.21, p = 0.003). A positive correlation of serum sST2 after CPR with mortality at 30 days and 6 months after cardiac arrest could be demonstrated., (© 2021. The Author(s).)

Inappropriate laboratory test selection in the form of overutilization as well as underutilization frequently occurs despite available guidelines. There is broad approval among laboratory specialists as well as clinicians that demand management strategies are useful tools to avoid this issue. Most of these tools are based on automated algorithms or other types of machine learning. This review summarizes the available demand management strategies that may be adopted to local settings. We believe that artificial intelligence may help to further improve these available tools.

Introduction: Serum levels of FGF23 have been associated with adverse outcomes in cardiovascular diseases in patients with and without impaired renal function. Hence, this study aimed to explore the prognostic relevance of intact FGF23 (iFGF23) and its derivate C-terminal FGF23 (cFGF23) in patients undergoing transcatheter aortic valve replacement (TAVR) with regard to renal function., Methods: A total of 274 patients undergoing transfemoral TAVR were enrolled in this study. Blood samples were obtained preinterventionally and analyzed for iFGF23 and cFGF23 by means of enzyme linked immunosorbent assay (ELISA). Follow-up was obtained for 12 months., Results: Serum levels of cFGF23 and iFGF23 both correlated positively with serum creatinine and inversely with estimated glomerular filtration rate (eGFR). Cox regression analysis revealed a significant association of cFGF23 with 1-year-mortality in patients with eGFR ≥45ml/min/1.73m², but not in patients with an eGFR <45ml/min/1.73m². A cut-off was calculated for cFGF23 (6.82 pmol/l) and patients with eGFR ≥45ml/min/1.73m² were retrospectively divided into two groups (above/below cut-off). Patients above the cut-off had a significantly worse 1-year-mortality than patients below the cut-off (33.3% vs. 19.6%; OR 2.05 (95%CI 1.03-4.07), p= 0.038). The association of cFGF23 with 1-year-mortality in patients with eGFR ≥45ml/min/1.73m² remained statistically significant even after correction for possible confounders in a multivariate Cox regression analysis., Conclusion: cFGF23 could be an individual risk factor for mortality in patients undergoing TAVR with an eGFR ≥45ml/min/1.73m²., (Copyright © 2020 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

Lipid droplets (LDs) were considered as a mere lipid storage organelle for a long time. Recent evidence suggests that LDs are in fact distinct and dynamic organelles with a specialized proteome and functions in many cellular roles. As such, LDs contribute to cellular signaling, protein and lipid homeostasis, metabolic diseases and inflammation. In line with the multitude of functions, LDs interact with many cellular organelles including mitochondria, peroxisomes, lysosomes, the endoplasmic reticulum and the nucleus. LDs are highly mobile and dynamic organelles and impaired motility disrupts the interaction with other organelles. The reduction of interorganelle contacts results in a multitude of pathophysiologies and frequently in neurodegenerative diseases. Contacts not only supply lipids for β-oxidation in mitochondria and peroxisomes, but also may include the transfer of toxic lipids as well as misfolded and harmful proteins to LDs. Furthermore, LDs assist in the removal of protein aggregates when severe proteotoxic stress overwhelms the proteasomal system. During imbalance of cellular lipid homeostasis, LDs also support cellular detoxification. Fine-tuning of LD function is of crucial importance and many diseases are associated with dysfunctional LDs. We summarize the current understanding of LDs and their interactions with organelles, providing a storage site for harmful proteins and lipids during cellular stress, aging inflammation and various disease states.

Laboratory analyses are crucial for diagnosis, follow-up and treatment decisions. Since mistakes in every step of the total testing process may potentially affect patient safety, a broad knowledge and systematic assessment of laboratory errors is essential for future improvement. In this review, we aim to discuss the types and frequencies of potential errors in the total testing process, quality management options, as well as tentative solutions for improvement. Unlike most currently available reviews on this topic, we also include errors in test-selection, reporting and interpretation/action of test results. We believe that laboratory specialists will need to refocus on many process steps belonging to the extra-analytical phases, intensifying collaborations with clinicians and supporting test selection and interpretation. This would hopefully lead to substantial improvements in these activities, but may also bring more value to the role of laboratory specialists within the health care setting., Competing Interests: Potential conflict of interest: None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

Introduction: The appropriate use of laboratory diagnostics is increasingly at stake. The aim of this study was to depict some paradigmatic examples of under- and overutilization, as well as possible solutions across Europe., Methods: We collected six examples from five European countries where a rise or decline of orders for specific laboratory parameters was observed after organizational changes but without evidence of changes in patient collective characteristics as source of this variation., Results: The collected examples were the following: 1-Germany) Switch from a Brain-Natriuretic-Peptide assay to NT-pro Brain-Natriuretic-Peptide assay, resulting in a 374% increase in these analytics; 2-Spain) Implementation of a gatekeeping strategy in tumor marker diagnostics, resulting in a 15-61% reduction of these diagnostics; 3-Croatia) Stepwise elimination of creatine-kinase-MB assay from the laboratory portfolio; 4-UK) Removal of γ-glutamyl transferase from a "liver function" profile, resulting in 82% reduction of orders; 5-Austria) Implementation of a new device for rapid Influenza-RNA detection, resulting in a 450% increase of Influenza testing; 6-Spain) Insourcing of 1,25-(OH) 2 -Vitamin D measurements, leading to a 378% increase of these analyses., Conclusion: The six paradigmatic examples described in this manuscript show that availability of laboratory resources may considerably catalyze the demand, thus underscoring that inappropriate use of laboratory resources may be commonplace in routine laboratories all across Europe and most probably beyond. They also demonstrate that the application of simple strategies may assist in overcoming this issue. We believe that laboratory specialists need to refocus on the extra-analytical parts of the testing process and engage more in interdisciplinary patient-care., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Too frequent HbA1c measurements may lead to unnecessary treatment modifications of diabetic patients. The aim of this study was to estimate the percentage of falsely elevated HbA1c results in two hospitals, Landeskrankenhaus/Uniklinikum Salzburg (LKH) and Landesklinik St. Veit (STV), as well as to retrospectively investigate the effect of an automated and an educative 60-day re-testing interval (RTI)., Methods: The amount of estimated falsely elevated results (eFER), based on odds calculated using the baseline and the follow-up values and the time between these measurements, the number of HbA1c re-testings within 60 days as well as the overall number of ordered and performed HbA1c analyses were calculated. In LKH, an automated algorithm cancelling inappropriate HbA1c testing was applied, and in STV, educational actions were taken., Results: Before RTI-implementation, eFER were 0.9% and 2.1% and within-60-days-re-testing were 15.0% and 7.4% of cases in LKH and STV, respectively. After RTI-implementation, these numbers decreased to 0.2% (p < .001) and 1.8% (p = .869) and within-60-days-re-testing decreased to 1.1% (p < .001) and 3.6% (p = .003) in LKH and STV, respectively. Median monthly HbA1c measurements decreased by 15.8% (p < .001) and 21.1% (p = .002) in LKH and STV, respectively., Conclusion: Both the educational and the automated 60-day-RTI were proven to be efficient in reducing overall HbA1c measurements, re-testing within 60 days and eFER., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Laboratory overutilization is associated with diagnostic error and potential patient risk. We applied a demand management strategy in collaboration with the local Department of Cardiology to reduce the cardiac markers high-sensitive troponin T (hsTropT) and N-terminal pro brain natriuretic peptide (NTproBNP) in laboratory ordering profiles (LOPs). The present study aimed to retrospectively evaluate the implemented strategies., Methods: Strategies included educational measures and evidence-guided, active test de-selection from all cardiology ward LOPs, and/or permanent removal from LOPs. Tests remained available at all times. We evaluated overutilization by reductions in monthly orders, and assessed differences in 30-day all-cause readmission rate and length of patients' hospital stay., Results: Overall, we observed a mean reduction of 66.1% ± 7.6% (n = 277 ± 31) in hsTropT tests. Educational measures effectively reduced NTproBNP orders by 52.8% ± 17.7% (n = 60 ± 20). Permanent removal of tests from LOPs additionally decreased orders to a final extent of 75.8% ± 8.0% (n = 322 ± 31) in NTproBNP tests. The 30-day readmission rate and overall length of hospital stay did not increase., Conclusions: Our results indicate that cardiac markers in routine care are subject to extensive overutilization when used within LOPs. Educational measures are an effective strategy to overcome the overutilization of cardiac markers but may be more effective when combined with the removal of cardiac markers from LOPs.

Background and Aims: Anti-mitochondrial antibodies (AMA) are closely linked to primary biliary cholangitis (PBC). The prevalence of AMA in the general population is low, and AMA positivity may precede PBC. We aimed to determine the natural history of subjects with positive AMA., Methods: In total, 302 patients were tested AMA-positive over a ten-year period. Of these, immunoblotting confirmed specific AMA in 184 (29 male, 155 female, age 59.6 ± 14.1 years). These subjects were invited to our liver outpatient clinic for clinical and biochemical re-evaluation. Detailed clinical history data were additionally collected from the hospital computer system and by telephone. The subsequent course with regard to mortality, liver-related morbidity, extrahepatic co-morbidities and effectiveness of PBC treatment was determined in 150 subjects (81.5%)., Results: After 5.8 ± 5.6 years of follow-up (FU), of 184 AMA-positive subjects, 28 subjects (15.2%; liver-related mortality n = 5) were deceased, and 122 subjects (66.3%) completed FU while 34 subjects (18.5%) were not available for FU. The 122 patients who completed FU were 63 patients with established PBC, six de novo cases of PBC (10.2% of 59 initially at risk), 42 (34.4%) subjects were still AMA-positive without PBC, and 11 (9.0%) subjects were AMA-negative at FU., Conclusions: Anti-mitochondrial antibodies-positive patients without PBC at baseline infrequently developed PBC over six years of FU. AMA positivity represented a transient serological autoimmune phenomenon in a significant proportion of subjects., (© 2019 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call 'lipid-droplet-accumulating microglia' (LDAM), are defective in phagocytosis, produce high levels of reactive oxygen species and secrete proinflammatory cytokines. RNA-sequencing analysis of LDAM revealed a transcriptional profile driven by innate inflammation that is distinct from previously reported microglial states. An unbiased CRISPR-Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin (GRN), are causes of autosomal-dominant forms of human neurodegenerative diseases. We therefore propose that LDAM contribute to age-related and genetic forms of neurodegeneration.

Background: Heart failure (HF) remains one of the leading causes of death to date despite extensive research funding. Various studies are conducted every year in an attempt to improve diagnostic accuracy and therapy monitoring. The small cytoplasmic heart-type fatty acid-binding protein (H-FABP) has been studied in a variety of disease entities. Here, we provide a review of the available literature on H-FABP and its possible applications in HF. Methods: Literature research using PubMed Central was conducted. To select possible studies for inclusion, the authors screened all available studies by title and, if suitable, by abstract. Relevant manuscripts were read in full text., Results: In total, 23 studies regarding H-FABP in HF were included in this review., Conclusion: While, algorithms already exist in the area of risk stratification for acute pulmonary embolism, there is still no consensus for the routine use of H-FABP in daily clinical practice in HF. At present, the strongest evidence exists for risk evaluation of adverse cardiac events. Other future applications of H-FABP may include early detection of ischemia, worsening of renal failure, and long-term treatment planning., Competing Interests: The authors declare no conflict of interest.

Background Published evidence on the risk of additive carryover during phlebotomy remains elusive. We aimed to assess potential carryover of citrated and heparinized blood and the relative volume needed to bias clinical chemistry and coagulation tests. Methods We simulated standardized phlebotomies to quantify the risk of carryover of citrate and heparin additives in distilled water, using sodium and lithium as surrogates. We also investigated the effects of contamination of heparinized blood samples with increasing volumes of citrated blood and pure citrate on measurements of sodium, potassium, chloride, magnesium, total and ionized calcium and phosphate. Likewise, we studied the effects of contamination of citrated blood samples with increasing volumes of heparinized blood on heparin (anti-Xa) activity, lithium, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). We interpreted these results based on measurement deviations beyond analytical, biological and clinical significance. Results Standardized phlebotomy simulations revealed no significant differences in concentration of surrogate markers. Clinically significant alterations were observed after contamination of heparinized blood samples with volumes of citrated blood beyond 5-50 μL for ionized calcium and beyond 100-1000 μL for sodium, chloride and total calcium. Investigations of pure citrate carryover revealed similar results at somewhat lower volumes. Heparinized blood carryover showed clinically significant interference of coagulation testing at volumes beyond 5-100 μL. Conclusions Our results suggest that during a standardized phlebotomy, heparin or citrate contamination is highly unlikely. However, smaller volumes are sufficient to severely alter test results when deviating from phlebotomy guidelines.

The chroman-like chalcone Xanthohumol C, originally found in hops, was demonstrated to be a potent neuroregenerative and neuroprotective natural product and therefore constitutes a strong candidate for further pharmaceutical research. The bottleneck for in vivo experiments is the low water solubility of this chalcone. Consequently, we developed and validated a suitable formulation enabling in vivo administration. Cyclodextrins were used as water-soluble and nontoxic complexing agents, and the complex of Xanthohumol C and 2-hydroxypropyl- β -cyclodextrin was characterized using HPLC, HPLC-MS, NMR, and differential scanning calorimetry. The water solubility of Xanthohumol C increases with increasing concentrations of cyclodextrin. Using 50 mM 2-hydroxypropyl- β -cyclodextrin, solubility was increased 650-fold. Furthermore, in vitro bioactivity of Xanthohumol C in free and complexed form did not significantly differ, suggesting the release of Xanthohumol C from 2-hydroxypropyl- β -cyclodextrin. Finally, a small-scaled in vivo experiment in a rat model showed that after i. p. administration of the complex, Xanthohumol C can be detected in serum, the brain, and the cerebrospinal fluid at 1 and 6 h post-administration. Mean (± SD) Xanthohumol C serum concentrations after 1, 6, and 12 h were determined as 463.5 (± 120.9), 61.9 (± 13.4), and 9.3 (± 0.8) ng/mL upon i. v., and 294.3 (± 22.4), 45.5 (± 0.7), and 13 (± 1.0) ng/mL after i. p. application, respectively. Accordingly, the formulation of Xanthohumol C/2-hydroxypropyl- β -cyclodextrin is suitable for further in vivo experiments and further pharmaceutical research aiming for the determination of its neuroregenerative potential in animal disease models., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Introduction: As a small proportion of obese individuals do not develop metabolic complications and non-alcoholic fatty liver disease (NAFLD), this study aimed to provide a comprehensive clinical, metabolic and genetic description of obese subjects with healthy livers., Methods: A total of 183 subjects were stratified, according to BMI, presence of metabolic syndrome, biochemical liver tests and hepatic steatosis on ultrasound, into: (i) lean controls (n = 69); (ii) obese healthy (n = 50); and (iii)obese NAFLD (n = 62) groups. Detailed clinical, genetic and metabolic evaluations were then performed., Results: Obese healthy subjects did not differ in glucose parameters from lean controls, and had a lower rate of minor TM6SF2 gene variants compared with obese NAFLD (2/49 vs. 11/60, respectively; P = 0.035) and lean controls (13/64; P = 0.035), but significantly higher leptin concentrations than lean controls (P < 0.001); they also higher adiponectin concentrations (P < 0.001), and lower TNF-α and IL-6 concentrations (P = 0.01 and P < 0.001, respectively), than obese NAFLD subjects. Also, metabolomic studies identified ether- and ester-containing phospholipids [PC ae C44:6, PC ae C42:5, PC aa C40:4; P < 0.001, corrected by the false discovery rate (FDR) method] and found that the amino-acids lysine, glycine and isoleucine (FDR < 0.001) differed between the two obese groups, but not between lean controls and obese healthy subjects., Conclusion: Obese people with healthy livers are characterized by intact glucose homoeostasis, lower pro-inflammatory cytokine levels, and higher adiponectin and leptin concentrations compared with obese people with NAFLD. In addition, the major allele of TM6SF2, a set of phosphatidylcholines and several amino acids are associated with healthy livers in obesity., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Marinobufagenin (MBG) is an endogenous cardiotonic steroid (CTS) that inhibits the Na+/K+-ATPase. Human MBG is significantly increased in end-stage renal disease and immunization against MBG attenuates cardiovascular fibrosis in a rat model of uremic cardiomyopathy. Mineralocorticoid antagonists (MRA) block MBG binding sites and decrease proteinuria in chronic kidney disease (CKD) patients. We therefore aimed to investigate the association of MBG and albuminuria, as a marker of renal damage, as well as MBG and decline of glomerular filtration rate (GFR)., Methods: The Graz endocrine causes of hypertension (GECOH) study is a single center study of adults routinely referred for screening of endocrine hypertension. Plasma MBG was measured by an enzyme-linked immunoassay, and in a post-hoc analysis, follow-up creatinine levels were obtained. Patients with proteinuria >3.5g/day at baseline were excluded from further evaluation., Results: We measured MBG concentrations in 40 hypertensive subjects and excluded one patient due to pre-existing proteinuria. Plasma MBG was significantly correlated with albuminuria (Spearman ρ = .357; p = .028) and proteinuria (ρ = .336; p = .039). In linear regression analysis, the association remained significant after adjustment for age, sex, and BMI (β = .306; p = .036), and for mean systolic blood pressure (β = .352; p = .034). In follow-up analyses (N = 30), MBG was significantly associated with decline in GFR after adjustment for time-to-follow-up (β = -.374; p = .042)., Conclusion: The findings suggest that MBG plasma concentrations were associated with albuminuria as well as decline in kidney function. Whether MBG predicts hard renal endpoints warrants further investigations., Competing Interests: The authors have declared that no competing interests exist.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17 kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17 kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (P = .0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Epidermolysis bullosa simplex (EBS) is a rare genetic, blistering skin disease for which there is no cure. Treatments that address the pathophysiology of EBS are needed., Objective: Compare the impact of 1% diacerein cream with placebo in reducing the number of blisters in EBS., Methods: In a randomized, placebo-controlled, phase 2/3 trial we used a 1% diacerein topical formulation to treat defined skin areas in 17 patients. In a 2-period crossover trial, patients were randomized to either placebo or diacerein for a 4-week treatment and a 3-month follow-up in period 1. After a washout, patients were crossed over during period 2. The prespecified primary end point was the proportion of patients with a reduction of number of blisters by more than 40% from baseline in selected areas over the treatment episode., Results: Of the patients receiving diacerein, 86% in episode 1 and 37.5% in episode 2 met the primary end point (vs 14% and 17% with placebo, respectively). This effect was still significant after the follow-up. Changes in absolute blister numbers were significant for the diacerein group only. No adverse effects were observed., Limitations: Low patient numbers and no invasive data acquisition because of clinical burden in children., Conclusion: This trial provides evidence of the impact of 1% diacerein cream in the treatment of EBS., (Copyright © 2018 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Inappropriate utilization of laboratory resources is an increasing concern especially in high-throughput facilities. Until now, no reliable information has been published addressing to which extent laboratory results are actually used for clinical decision-making. Therefore, we aimed to close this gap using a novel retrospective approach including a survey of clinicians and nurses., Methods: We retrospectively evaluated the number of re-orders for potassium (K), lactate dehydrogenase (LD), aspartate-aminotransferase (AST), activated partial thromboplastin-time (APTT) and prothrombin-time/INR (PT/INR), after the initial order had to be cancelled due to preanalytical non-conformities. We analyzed subgroups regarding time to re-order, ward and sample priority (urgent vs. routine). Subsequently, we surveyed clinicians and nurses, asking for their estimate of the amount of failed re-orders as well as for possible reasons., Results: From initially cancelled tests, only ~20% of K, LD, AST and ~30% of APTT and PT/INR tests were re-ordered within 24 h. 70% of the investigated clinical chemistry and 60% of coagulation tests were re-ordered one week after cancellation or not at all. Survey participants quite accurately estimated these numbers. Routine laboratory panels, short stay of out-patients, obsolete test results and avoiding additional phlebotomies were the main reasons for not re-ordering cancelled tests., Conclusions: Overall, 60-70% of test results in the investigated assays ordered in a high throughput laboratory are potentially inappropriate or of doubtful clinically importance. Although clinicians and nurses are aware of this situation, it is the duty of laboratory specialists to overcome overutilization in close collaboration with all involved healthcare workers., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Introduction: Although centrifugation is performed in almost every blood sample, recommendations on duration and g-force are heterogeneous and mostly based on expert opinions. In order to unify this step in a fully automated laboratory, we aimed to evaluate different centrifugation settings and their influence on the results of routine clinical chemistry analytes., Materials and Methods: We collected blood from 41 healthy volunteers into BD Vacutainer PST II-heparin-gel- (LiHepGel), BD Vacutainer SST II-serum-, and BD Vacutainer Barricor heparin-tubes with a mechanical separator (LiHepBar). Tubes were centrifuged at 2000xg for 10 minutes and 3000xg for 7 and 5 minutes, respectively. Subsequently 60 and 21 clinical chemistry analytes were measured in plasma and serum samples, respectively, using a Roche COBAS instrument., Results: High sensitive Troponin T, pregnancy-associated plasma protein A, ß human chorionic gonadotropin and rheumatoid factor had to be excluded from statistical evaluation as many of the respective results were below the measuring range. Except of free haemoglobin (fHb) measurements, no analyte result was altered by the use of shorter centrifugation times at higher g-forces. Comparing LiHepBar to LiHepGel tubes at different centrifugation setting, we found higher lactate-dehydrogenase (LD) (P = 0.003 to < 0.001) and lower bicarbonate values (P = 0.049 to 0.008) in the latter., Conclusions: Serum and heparin samples may be centrifuged at higher speed (3000xg) for a shorter amount of time (5 minutes) without alteration of the analytes tested in this study. When using LiHepBar tubes for blood collection, a separate LD reference value might be needed., Competing Interests: Potential conflict of interest: Janne Cadamuro received a personal fee as guest member of a Becton Dickinson Advisory board meeting.

Background: Blood collection through intravenous (IV) catheters is a common practice at emergency departments (EDs). This technique is associated with higher in vitro hemolysis rates and may even be amplified by the use of vacuum collection tubes. Our aim was to investigate the association of five different vacuum tubes with hemolysis rates in comparison to an aspiration system under real-life conditions and to propose an equation to estimate the amount of hemolysis, depending on the vacuum collection tube type., Methods: We retrospectively evaluated hemolysis data of plasma samples from our ED, where blood is drawn through IV catheters. Over the past 5 years, we compared 19,001 hemolysis index values amongst each other and against the respective vacuum pressure (Pv) of the collection tubes, which were used within the six observational periods., Results: The highest hemolysis rates were associated with full-draw evacuated tubes. Significantly reduced hemolysis was observed for two kinds of partial-draw tubes. The hemolysis rate of one partial-draw blood collection tube was comparable to those of the aspiration system. Regression analysis of Pv and mean free hemoglobin (fHb) values yielded the formula fHb (g/L)=0.0082*Pv2-0.1143*Pv+ 0.5314 with an R2 of 0.99., Conclusions: If IV catheters are used for blood collection, hemolysis rates directly correlate with the vacuum within the tubes and can be estimated by the proposed formula. By the use of partial-draw vacuum blood collection tubes, hemolysis rates in IV catheter collections can be reduced to levels comparable with collections performed by aspiration systems.

The ketogenic diet (KD), a high-fat low-carbohydrate diet, has shown some efficacy in the treatment of certain types of tumors such as brain tumors and neuroblastoma. These tumors are characterized by the Warburg effect. Because renal cell carcinoma (RCC) presents similar energetic features as neuroblastoma, KD might also be effective in the treatment of RCC. To test this, we established xenografts with RCC 786-O cells in CD-1 nu/nu mice and then randomized them to a control diet or to KDs with different triglyceride contents. Although the KDs tended to reduce tumor growth, mouse survival was dramatically reduced due to massive weight loss. A possible explanation comes from observations of human RCC patients, who often experience secondary non-metastatic hepatic dysfunction due to secretion of high levels of inflammatory cytokines by the RCCs. Measurement of the mRNA levels of tumor necrosis factor alpha (TNFα) and interleukin-6 revealed high expression in the RCC xenografts compared to the original 786-O cells. The expression of TNFα, interleukin-6 and C-reactive protein were all increased in the livers of tumor-bearing mice, and KD significantly boosted their expression. KDs did not cause weight loss or liver inflammation in healthy mice, suggesting that KDs are per se safe, but might be contraindicated in the treatment of RCC patients presenting with Stauffer's syndrome, because they potentially worsen the associated hepatic dysfunction., Competing Interests: CONFLICTS OF INTEREST The authors state no conflicts of interest.

Objectives: Regular aerobic exercise provides beneficial effects on human health and reduces all-cause mortality. Aerobic exercise has profound metabolic effects, and specific metabolites may reflect physiological changes. We aimed to identify endogenous metabolites that distinguish the trained from the untrained state to increase the spectrum of analytes amenable for hypothesis testing and to expand understanding of putative beneficial pathways., Design: Cross sectional laboratory repeated measures study., Methods: Exercise testing was performed in 37 healthy male participants and serum samples were obtained before and after completion of a ten-week standardized exercise program. Samples were analyzed for routine clinical parameters and for 188 endogenous metabolites by LC-MS/MS., Results: Indicating the effectiveness of the intervention program, parameters of sport physiology were different after training. After correcting for multiple testing, serum concentrations of several metabolites differed between the trained and untrained state. Serine and glutamate decreased in response to exercise, whereas sarcosine and kynurenine increased. Phosphatidylcholines showed a mixed response in that four species increased and three decreased. However, all seven lysophosphatidylcholines and all four plasmalogens that differed between the trained and untrained state, increased. One short-chain acylcarnitine also decreased. In receiver operator characteristics analyses, sarcosine displayed the highest AUC value (0.839; 95% CI: 0.734-0.926) in discriminating the pre- from the post-trained state., Conclusions: Our study detected metabolites that clearly differentiate the trained from the untrained state. These metabolites may be targeted in mechanistic studies to understand underlying biochemical pathways and could serve to improve the design, monitoring and individualization of training programs., (Copyright © 2017 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

We report on a preanalytical issue we encountered during routine clinical chemistry analyses, potentially leading to deviated analysis results and believe that it might help other laboratories to overcome similar problems. In a heparin-gel tube we measured an implausible glucose value of 0.06 mmol/L. Re-measurement of the same sample resulted in a glucose value of 5.4 mmol/L. After excluding an analytical error, we inspected the sample closer and found a white material as well as fatty droplets floating on the surface of the plasma tube. Evaluation of these structures revealed that the white particulate matter (WPM) consisted of fibrinogen, platelets and leukocytes and the fatty droplets most probably originated from the separator gel. We concluded that these structures formed a temporary clot in the instruments pipetting needle thereby altering the sampling volume and subsequently the measured glucose value. The formation of WPM might be attributable to high speed centrifugation, high cholesterol levels, the gel formulation or a combination of several issues such as temperature, heparin concentration, pH and patient-specific factors. The gel droplets were most probably caused by an aberrant gel formulation in combination with an improper storage of the empty tubes on the wards prior to phlebotomy. After adding an additional instrument cleansing cycle and changing to another batch of heparin tubes the problems could be significantly reduced., Competing Interests: Potential conflict of interest: None declared.

NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis.

Objectives: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity; however, 5-8% of lean subjects also have evidence of NAFLD. We aimed to investigate clinical, genetic, metabolic and lifestyle characteristics in lean Caucasian subjects with NAFLD., Methods: Data from 187 subjects allocated to one of the three groups according to body mass index (BMI) and hepatic steatosis on ultrasound were obtained: lean healthy (BMI≤25 kg/m 2 , no steatosis, N=71), lean NAFLD (BMI≤25 kg/m 2 , steatosis, N=55), obese NAFLD (BMI≥30 kg/m 2 , steatosis; N=61). All subjects received a detailed clinical and laboratory examination including oral glucose tolerance test. The serum metabolome was assessed using the Metabolomics AbsoluteIDQ p180 kit (BIOCRATES Life Sciences). Genotyping for single-nucleotide polymorphisms (SNPs) associated with NAFLD was performed., Results: Lean NAFLD subjects had fasting insulin concentrations similar to lean healthy subjects but had markedly impaired glucose tolerance. Lean NAFLD subjects had a higher rate of the mutant PNPLA3 CG/GG variant compared to lean controls (P=0.007). Serum adiponectin concentrations were decreased in both NAFLD groups compared to controls (P<0.001 for both groups) The metabolomics study revealed a potential role for various lysophosphatidylcholines (lyso-PC C18:0, lyso-PC C17:0) and phosphatidylcholines (PCaa C36:3; false discovery rate (FDR)-corrected P-value<0.001) as well as lysine, tyrosine, and valine (FDR<0.001)., Conclusions: Lean subjects with evidence of NAFLD have clinically relevant impaired glucose tolerance, low adiponectin concentrations and a distinct metabolite profile with an increased rate of PNPLA3 risk allele carriage.

Objective: Elevated serum ferritin has been linked to type 2 diabetes (T2D) and adverse health outcomes in subjects with the Metabolic Syndrome (MetS). As the mechanisms underlying the negative impact of excess iron have so far remained elusive, we aimed to identify potential links between iron homeostasis and metabolic pathways., Methods: In a cross-sectional study, data were obtained from 163 patients, allocated to one of three groups: (1) lean, healthy controls (n = 53), (2) MetS without hyperferritinemia (n = 54) and (3) MetS with hyperferritinemia (n = 56). An additional phlebotomy study included 29 patients with biopsy-proven iron overload before and after iron removal. A detailed clinical and biochemical characterization was obtained and metabolomic profiling was performed via a targeted metabolomics approach., Results: Subjects with MetS and elevated ferritin had higher fasting glucose (p < 0.001), HbA1c (p = 0.035) and 1 h glucose in oral glucose tolerance test (p = 0.002) compared to MetS subjects without iron overload, whereas other clinical and biochemical features of the MetS were not different. The metabolomic study revealed significant differences between MetS with high and low ferritin in the serum concentrations of sarcosine, citrulline and particularly long-chain phosphatidylcholines. Methionine, glutamate, and long-chain phosphatidylcholines were significantly different before and after phlebotomy (p < 0.05 for all metabolites)., Conclusions: Our data suggest that high serum ferritin concentrations are linked to impaired glucose homeostasis in subjects with the MetS. Iron excess is associated to distinct changes in the serum concentrations of phosphatidylcholine subsets. A pathway involving sarcosine and citrulline also may be involved in iron-induced impairment of glucose metabolism.