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Long-chain polyunsaturated fatty acids (LCPUFA) are important for brain development and functioning and with that, possibly school performance. Several cross-sectional studies have shown significant positive associations between fish consumption, an important source of LCPUFA and school grades in adolescents. The effect of LCPUFA supplementation on school grades in adolescents has not been investigated yet. The goal of the current study was to investigate (I) the associations between the Omega-3 Index (O3I) at baseline and after 12 months respectively and school grades and (II) the effect of one year krill oil supplementation (source of LCPUFA) on school grades in adolescents with a low O3I at baseline. A double-blind randomised placebo-controlled trial with repeated measurements was executed. Participants received either 400 mg eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per day for the first three months in Cohort 1 and the nine months thereafter 800 mg EPA + DHA per day, Cohort 2 started immediately with 800 mg EPA + DHA per day,or a placebo. The O3I was monitored with a finger prick at baseline, three, six and twelve months. Subject grades for English, Dutch and math were collected, a standardised mathematics test was executed at baseline and at 12 months. Data was analysed with (I) explorative linear regressions to investigate associations at baseline and follow-up and (II) mixed model analyses separately for each of the subject grades and the standardised mathematics test to investigate the effect of supplementation after 12 months. The krill oil group had a small significant increase in the mean O3I at all time points. However, very few participants achieved the intended target O3I range of 8–11%. At baseline a significant association between baseline O3I and English grade was show, additionally a trend for an association with Dutch grade was shown. After 12 months no significant associations were found. Additionally, there was no significant effect of krill oil supplementation on subject grades or standardised mathematics test score. In this study, no significant effect of krill oil supplementation on subject grades or standardised mathematics test performance was found. However, as many participants dropped out and/or were non-adherent, results should be interpreted with caution.

The detection of 2-chloroethanol in foods generally follows an assumption that the pesticide ethylene oxide has been used at some stage in the supply chain. In this situation the Pesticide Residues in Food Regulation (EC) 396/2005 requires 2-chloroethanol to be assessed as if equivalent to ethylene oxide, which has been classified as a genotoxic carcinogen. This review investigated whether this is an appropriate risk assessment approach for 2-chloroethanol. This involved an assessment of existing genotoxicity and carcinogenicity data, application of Structure Activity Based Read Across for carcinogenicity assessment, biological reactivity in the ToxTracker assay and micronuclei formation in HepaRG cells. Although we identified there is an absence of a standard oral bioassay for 2-chloroethanol, carcinogenicity weight-of-evidence assessment along with data on relevant structural analogues do not show evidence for carcinogenicity for 2-chloroethanol. The absence of genotoxicity was demonstrated for 2-chloroethanol and suitable analogues. In contrast, ethylene oxide showed reactivity towards markers indicative of direct DNA damage which is consistent with what is known about its mode-of-action. These data facilitate the understanding of 2-chloroethanol and given that it is not a genotoxic carcinogen suggest it must be assessed relative to non-cancer endpoints and a health protective Reference Dose should be established on that basis.

Long-chain polyunsaturated fatty acids (LCPUFA) are important for brain development and function, maybe especially during adolescence. Observational studies have demonstrated an association between fish consumption (a source of LCPUFA) and cognition in adolescents, but intervention trials are lacking. The goal of the current study was to investigate the effect of one year of krill oil (a source of LCPUFA) supplementation on the cognitive performance of adolescents with a low Omega-3 Index (O3I &le, 5%). A double-blind, randomized, and placebo-controlled supplementation trial with repeated measurements (baseline (T0), three months (T1), six months (T2), and 12 months (T3)) in adolescents (267 randomized) was executed. Participants were randomized to 400 mg eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per day in Cohort I or placebo and 800 mg EPA + DHA per day in Cohort II or placebo. O3I was monitored by a finger prick at all time points. At T0, T2, and T3, participants executed a neurocognitive test battery. Covariate corrected mixed models were run with either condition (krill or placebo) or O3I as predictors. Krill oil supplementation led to a small but significant increase in mean O3I, but few participants increased to the intended O3I range (8&ndash, 11%). There was no significant effect of supplementation on the neurocognitive tests, nor a relationship between O3I and neurocognitive test scores. The increase in O3I was small in most participants, probably due to non-compliance. Possibly the increase in O3I was too small to demonstrate an effect. More research on the influence of LCPUFAs on cognition in adolescents is needed.

Introduction: Observational studies have shown a relationship between omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) and depression in adolescents. However, n-3 LCPUFA supplementation studies investigating the potential improvement in depressive feelings in adolescents from the general population are missing.Methods: A one-year double-blind, randomized, placebo controlled krill oil supplementation trial was conducted in two cohorts. Cohort I started with 400 mg eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) or placebo, after three months this increased to 800 mg EPA and DHA per day, whilst cohort II started with this higher dose. Omega-3 Index (O3I) was monitored via finger-prick blood measurements. At baseline, six and 12 months participants completed the Centre for Epidemiologic Studies Depression Scale (CES-D) and the Rosenberg Self Esteem questionnaire (RSE). Adjusted mixed models were run with treatment allocation/O3I as predictor of CES-D and RSE scores.Results: Both intention-to-treat and assessing the change in O3I analyses did not show significant effects on CES-D or RSE scores.Conclusion: There is no evidence for less depressive feelings, or higher self-esteem after one year of krill oil supplementation. However, due to a lack of adherence and drop-out issues, these results should be interpreted with caution.

PurposeDepression is common in adolescents and long-chain polyunsaturated fatty acids (LCPUFA) are suggested to be associated with depression. However, research in adolescents is limited. Furthermore, self-esteem has never been studied in relation to LCPUFA. The objective here was to determine associations of depression and self-esteem with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), Omega-3 Index (O3I), n-6 docosapentaenoic acid (n-6 DPA, also called Osbond acid, ObA), n-3 docosapentaenoic acid (DPA), and arachidonic acid (AA) concentrations in blood of adolescents attending lower general secondary education (LGSE).MethodsBaseline cross-sectional data from a krill oil supplementation trial in adolescents attending LGSE with an O3I ≤ 5% were analysed using regression models built with the BayesFactor package in R. Fatty acids and O3I were determined in blood. Participants filled out the Centre for Epidemiologic Studies Depression (CES-D) scale and the Rosenberg Self-Esteem scale (RSE).ResultsScores indicative of depression (CES-D ≥ 16) were found in 29.4% of the respondents. Of all fatty acids, we found extreme evidence [Bayes factor (BF) > 100] for a weak negative association between ObA and depression score [− 0.16; 95% credible interval (CI) − 0.28 to − 0.04; BF10 = 245], and substantial evidence for a weak positive association between ObA and self-esteem score (0.09; 95% CI, − 0.03 to 0.20; BF10 = 4). When all fatty acids were put in one model as predictors of CES-D or RSE, all of the 95% CI contained 0, i.e., no significant association.ConclusionNo evidence was found for associations of DHA, EPA and O3I with depression or self-esteem scores in LGSE adolescents with O3I ≤ 5%. The associations of higher ObA status with lower depression and higher self-esteem scores warrant more research.

Szterk et al. (Food Chemistry 243 (2018) 403-409) have recently analyzed the content of menaquinone-7 (MK-7) in eight dietary supplements. The authors concluded that five out of eight were below the declared content. For all samples, the authors used tetrahydrofuran (THF) to extract MK-7 prior to analysis. Two of the tested products that were below the declared content were microencapsulated MK-7 which had a coating with limited solubility in THF. By dissolving the coating with water and ethanol prior to extraction with ethyl acetate, all MK-7 will be made accessible prior to analysis by HPLC. We have repeated the analysis of the two microencapsulated products that Szterk et al. claimed were below the declared content, and have shown they contain 102% and 105% of the label claim. Since Szterk et al. have used a solvent that does not dissolve the coating on microencapsulated MK-7, their conclusion is not justified and is thus misleading.

Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs. These two endosomal proteins are important for specific receptor sorting. Hrs is recruiting ubiquitinated receptors to early endosomes to further facilitate degradation through the ESCRT complex. Upon receptor activation Hrs becomes phosphorylated and is relocated to the cytosol, important for receptor degradation. In this work we have studied the endosomal binding dynamics of Eps15 and Hrs upon EGF-R and PDGF-R stimulation. By analysing the fluorescence intensity on single endosomes after ligand stimulation we measured a time-specific decrease in the endosomal fluorescence level of Eps15-GFP and Hrs-YFP. Through FRAP experiments we could further register a specific change in the endosomal-membrane to cytosol binding properties of Eps15-GFP and Hrs-YFP. This specific change in membrane fractions proved to be a redistribution of the immobile fraction, which was not shown for the phosphorylation deficient mutants. We here describe a mechanism that can explain the previously observed relocation of Hrs from the endosomes to cytosol after EGF stimulation and show that Eps15 follows a similar mechanism. Moreover, this specific redistribution of the endosomal protein binding dynamics proved to be of major importance for receptor degradation.

Objective – To develop and validate a simple patient questionnaire for the detection of overactive bladder (OAB).Design – An open, non-randomized multicentre study.Setting – A pilot study (n=133) was conducted to bring forward five questions from initially 14 questions, for detection of OAB. These five questions were subject to further validation in the main study (n=520).Subjects – 531 adults responding to a newspaper advertisement regarding symptoms of OAB and patients seeing a physician for other reasons were attending 28 general practitioners.Main outcome measures – Agreement rate, sensitivity, and specificity.Results – The agreement rate between the patients’ own diagnosis based on the patient questionnaire, and the physicians’ diagnosis based on medical history, urine analysis, and micturition chart, was 0.78 (κ=0.89). Sensitivity and specificity were 0.98 and 0.90, respectively.Conclusion – The validated questionnaire may become a useful tool to decide whether a patient has overactive bladder. The ...

Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins. It appears that several chemokines must form higher-order oligomers to elicit proper in vivo effects, as mutants restricted to forming dimers have lost the ability to recruit leukocytes to sites of inflammation. Here, we show for the first time that the chemokine RANTES/CCL5 binds to the surface of human endothelial cells in a regular filamentous pattern. Furthermore, the filaments bound to the surface in a heparan sulfate-dependent manner. By electron microscopy we observed labeling for RANTES on membrane projections as well as on the remaining plasma membrane. Mutant constructs of RANTES restricted either in binding to heparin, or in forming dimers or tetramers, appeared either in a granular, non-filamentous pattern or were not detectable on the cell surface. The RANTES filaments were also present after exposure to flow, suggesting that they can be present in vivo. Taken together with the lacking in vivo or in vitro effects of RANTES mutants, we suggest that the filamentous structures of RANTES may be of physiological importance in leukocyte recruitment.

Background: Some of the mechanisms underlying cell division and partitioning of the cellular components into the daughter cells are well known. Within the endomembrane system, there is a general cessation of membrane traffic, including endocytosis and endosome fusion, at the onset of mitosis. However, the fate of endosomes and lysosomes during mitosis has been less well studied. Results: Using video and confocal microscopy of living cells, we show here that endosomes and lysosomes remain intact and separate during mitosis. The segregation into daughter cells takes place by coordinated movements, and during cytokinesis, these organelles accumulate in the vicinity of the microtubule organization center. However, partitioning into daughter cells is not more accurate than a calculated stochastic distribution, despite the apparent order to the process. Conclusion: We conclude that partitioning of endosomes and lysosomes is an ordered, yet imprecise, process, and that the organelle copy number is maintained by the daughter cells.

Unlike cytarabine, cellular entry of Elacytarabine, the elaidic acid ester derivative of cytarabine, is independent of the human equilibrative nucleoside transporter 1 (hENT1). This phase II study tested whether the hENT1 blast expression level can be used as a predictive marker for cytarabine response and if the efficacy of elacytarabine is independent of hENT1 expression. A total of 51 patients with acute myeloid leukemia (AML) induction failure were given elacytarabine-idarubicin as a second induction course. The hENT1 expression level was analyzed prior to first induction and/or prior to treatment with elacytarabine. The overall response rate (ORR) was 41% and the safety profile was manageable. There is a trend suggesting that hENT1 expression influences response to cytarabine, but not sufficient to support it as a biomarker for guiding treatment. Further, we conclude that the activity of elacytarabine is not significantly predicted by the hENT1 expression level.

Organelles in the endocytic pathway interact and communicate through the crucial mechanisms of fusion and fission. However, any specific link between fusion and fission has not yet been determined. To study the endosomal interactions with high spatial and temporal resolution, we enlarged the endosomes by two mechanistically different methods: by expression of the MHC-class-II-associated chaperone invariant chain (Ii; or CD74) or Rab5, both of which increased the fusion rate of early endosomes and resulted in enlarged endosomes. Fast homotypic fusions were studied, and immediately after the fusion a highly active and specific tubule formation and fission was observed. These explosive tubule formations following fusion seemed to be a direct effect of fusion. The tubule formations were dependent on microtubule interactions, and specifically controlled by Kif16b and dynein. Our results show that fusion of endosomes is a rapid process that destabilizes the membrane and instantly induces molecular-motor-driven tubule formation and fission.

Elacytarabine is a novel cytotoxic nucleoside analogue, independent of nucleoside transporters (e.g. human Equilibrative Nucleoside Transporter 1 [hENT1]) for cell uptake, and mechanisms of action similar to those of cytarabine. This Phase II study assessed the efficacy and safety of elacytarabine in patients with advanced stage acute myeloid leukaemia (AML). Patients received 2000 mg/m(2) per d continuously i.v. during days 1-5 every 3 weeks. Patients were matched by six risk factors with historical controls; remission rate (assessed after 1 or 2 cycles) and 6-month survival were compared. Sixty-one patients, median age 58 years, were enrolled; 52% had five or six risk factors. The remission rate was 18% (95% confidence interval: 9-30%) vs. 4% in controls (P < 0·0001), 6-month survival rate was 43%, median overall survival was 5·3 months (vs. 1·5 months); 10 patients (16%) were referred for stem cell transplantation after treatment. Side effects were predictable and manageable. The most common grade 3/4 non-haematological adverse events were febrile neutropenia, hypokalemia, fatigue, hyponatraemia, dyspnoea and pyrexia. Thirty-day all-cause mortality, after start of treatment, was 13% vs. 25% in controls. Elacytarabine has monotherapy activity in patients with advanced AML. This study provides proof-of-concept that lipid esterification of nucleoside analogues is clinically relevant.

Early endosomal antigen 1 (EEA1) is a cytosolic protein that specifically binds to early endosomal membranes where it has a crucial role in the tethering process leading to homotypic endosome fusion. Green fluorescent protein-tagged EEA1 (EEA1-GFP) was bound to the endosomal membrane throughout the cell cycle, and measurements using fluorescent recovery after photobleaching showed two fractions: one rapidly exchanging with the cytosolic pool, and the other with a long half-life. The exchange consists of a release and binding process, and we have separated these two by using GFP and photoactivable GFP. The release rate was identical to the exchange rate, showing that the dissociation characteristics determine the cycling of this molecule. During mitosis, we found that the dissociation rate was markedly accelerated and, in addition, the long-lived fraction was markedly reduced. This indicates that a fusion arrest in mitosis is not the result of EEA1 not binding to early endosomes, but rather due to the marked shift in membrane-binding characteristics. This might be a general mechanism to fine-tune and control tethering and fusion of early endosomes.

Abstract 46 Background Elacytarabine, a fatty acid derivative (elaidic acid ester) of cytarabine, has pharmacokinetic and pharmacodynamic properties that may lead to improved clinical outcomes (Adema et al., 2011) compared to cytarabine. A phase I study had established 1000 mg/m2/d continuous infusion on d 1 – 5 as a safe and effective dose of elacytarabine given in combination with idarubicin at 12 mg/m2/d IV d 1 – 3 (Giles et al., 2012). Mechanism of Action The mechanism of action is similar to cytarabine, but unlike cytarabine, plasma elimination half-life is long, intracellular distribution is prolonged and activity is independent of membrane nucleoside transporters. Resistance to cytarabine has been associated with decreased expression of the human equilibrative nucleoside transporter 1 (hENT1) (Hubeek et al., 2005). Aims The aim of the study is to determine the efficacy and safety of elacytarabine given in combination with idarubicin; to explore the relationship between the hENT1 status in AML cells and response to cytarabine and to elacytarabine; to evaluate the safety and toxicity of the combination therapy. Methods Adult patients with AML who have not attained blast clearance after the first induction course with a standard dose cytarabine based regimen, assessed from day 12 on, were enrolled in the study. Patients received a combination of elacytarabine and idarubicin scheduled as described above, as second induction course. Patients treated with further courses received either the combination therapy or elacytarabine monotherapy at 2000 mg/m2/d on d 1 – 5, at the investigator‘s discretion. hENT1 expression level was analysed retrospectively by immunocytochemistry at time of initial AML diagnosis (pre-therapeutic) and/or before elacytarabine/idarubicin treatment (baseline). Results In the on-going study which will recruit up to 50 evaluable patients, 47 patients [27 male, 20 female, median age 60 years (range 18–78), ECOG 0–2] have currently been treated. 40 patients have been evaluated for response, of whom 35% suffered from secondary leukemia. Median time from start of first induction course to start of elacytarabine treatment was 36 days (range 13 – 189). After treatment with elacytarabine and idarubicin, 18/40 evaluable patients attained a CR/CRi. Median time to remission was 36 days (range 25 – 60). Incidence of low hENT1 expression was approximately 50%. Preliminary data shows a higher response rate of elacytarabine compared to cytarabine in this low hENT1 population (47% versus 37%). The most frequently reported non haematological adverse events grades 3 and 4 were fatigue, infections/sepsis, hypoalbuminaemia, hypokalaemia and hypoxia. Three deaths occurred within 30 days after start of treatment, all due to sepsis. Additional two patients died from progressive AML within 60 days after start of treatment. All these five patients suffered from secondary leukaemia. Summary/Conclusion At the time of this abstract (August 2012) elacytarabine in combination with idarubicin shows a promising clinical activity with a CR/CRi rate of approximately 45% in patients failing a first induction course. Low expression of the hENT1 transporter is associated with a lower probability of achieving remissions following cytarabine treatment. Preliminary data indicates that assessment of hENT1 expression in blast cells could be used to identify patients less likely to benefit from cytarabine and for whom elacytarabine could be an effective therapy. The emerging safety profile is as expected for these groups of cytotoxic therapies. Results of the completed study will be presented at the meeting. Disclosures: Kindler: Novartis: Honoraria. Johansen:Clavis Pharma: Employment. Bergeland:Clavis Pharma: Employment. Gianella-Borradori:Clavis Pharma: Employment. Flem Jacobsen:Clavis Pharma: Employment. Rao:Clavis Pharma: Honoraria.

Measuring rapid kinetics of proteins in living cells requires the capability for fast, accurate measurements. Researchers hoping to obtain precise kinetic data from fluorescence recovery after photobleaching or photoactivation experiments need an easily controllable system for stimulation of a specific region and subsequent imaging, and the Olympus FluoView FV1000 confocal laser scanning microscope (cLSM) with SIM scanner makes this possible. We describe here how to precisely measure off rates using a cytosolic photoactivatable probe that binds endosomal membranes.