Targeting LAMP2 in human cerebrospinal fluid with a combination of immunopurification and high resolution parallel reaction monitoring mass spectrometry

Background: Alzheimer’s disease is the most common form of dementia. An increasing body of evidence suggests that endo-lysosomal dysfunction is a pathogenic mechanism of Alzheimer’s disease. Thus there is a potential for proteins involved in the normal function of endo-lysosomal vesicles to act as biomarkers of disease. Herein we focused on the lysosomal protein LAMP2 that is involved in chaperone mediated autophagy. / Results: Using a combination of immunoprecipitation, digestion and nano-liquid chromatography tandem mass spectrometry we targeted and identified six tryptic LAMP2 peptides in human cerebrospinal fluid. Employing the identified proteotypic tryptic peptides a hybrid immunoprecipitation high resolution parallel reaction monitoring mass spectrometric method was developed for the relative quantitation of LAMP2. The method was evaluated in a number of experiments which defined the overall methodological as well as the analytical micro-liquid chromatography mass spectrometric intra- and inter-day variability. We identified an overall methodological peptide dependent intra-day variability of 8–16 %. The inter-day experiments showed similar results. The analytical contribution to the variation was minor with a coefficient of variation of 0.5–2.1 %, depending on the peptide. Using the developed method, with defined and limited variability, we report increased cerebrospinal fluid levels of three LAMP2 peptides in Alzheimer’s disease subjects (n = 14), as compared to non-Alzheimer’s disease controls (n = 14). / Conclusion: Altered LAMP2 levels in cerebrospinal fluid may indicate endo-lysosomal dysfunction in Alzheimer’s disease. However, further studies in larger cohorts comprised of well-defined patient materials are required. We here present a tool which can be used for exploring the relevance of the level of LAMP2 as a potential measure of lysosomal dysfunction in Alzheimer’s disease or other neurodegenerative diseases

Thermal Impact on Spiking Properties in Hodgkin-Huxley Neuron with Synaptic Stimulus

The effect of environmental temperature on neuronal spiking behaviors is investigated by numerically simulating the temperature dependence of spiking threshold of the Hodgkin-Huxley neuron subject to synaptic stimulus. We find that the spiking threshold exhibits a global minimum in a "comfortable temperature" range where spike initiation needs weakest synaptic strength, indicating the occurrence of optimal use of synaptic transmission in neural system. We further explore the biophysical origin of this phenomenon in ion channel gating kinetics and also discuss its possible biological relevance in information processing in neural systems.Comment: 10 pages, 4 figure

A Parallel Reaction Monitoring Mass Spectrometric Method for Analysis of Potential CSF Biomarkers for Alzheimer's Disease

Scope: The aim of this study was to develop and evaluate a parallel reactionmonitoring mass spectrometry (PRM-MS) assay consisting of a panel ofpotential protein biomarkers in cerebrospinal fluid (CSF).Experimental design: Thirteen proteins were selected based on theirassociation with neurodegenerative diseases and involvement in synapticfunction, secretory vesicle function, or innate immune system. CSF sampleswere digested and two to three peptides per protein were quantified usingstable isotope-labeled peptide standards.Results: Coefficients of variation were generally below 15%. Clinicalevaluation was performed on a cohort of 10 patients with Alzheimer’s disease(AD) and 15 healthy subjects. Investigated proteins of the granin familyexhibited the largest difference between the patient groups. Secretogranin-2(p<0.005) and neurosecretory protein VGF (p<0.001) concentrations werelowered in AD. For chromogranin A, two of three peptides had significantlylowered AD concentrations (p<0.01). The concentrations of the synapticproteins neurexin-1 and neuronal pentraxin-1, as well as neurofascin werealso significantly lowered in AD (p<0.05). The other investigated proteins,β2-microglobulin, cystatin C, amyloid precursor protein, lysozyme C,neurexin-2, neurexin-3, and neurocan core protein, were not significantlyaltered.Conclusion and clinical relevance: PRM-MS of protein panels is a valuabletool to evaluate biomarker candidates for neurodegenerative disorders

Scientific Opinion on Dietary Reference Values for iron

Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies derived Dietary Reference Values (DRVs) for iron. These include Average Requirement (AR) and Population Reference Intake (PRI). For adults, whole-body iron losses were modelled using data from US adults. Predicted absorption values, at a serum ferritin concentration of 30 \ub5g/L, of 16 % for men and 18 % for women were used to convert physiological requirements to dietary iron intakes. In men, median whole-body iron losses are 0.95 mg/day, and the AR is 6 mg/day. The PRI, calculated as the dietary requirement at the 97.5th percentile, is 11 mg/day. For postmenopausal women, the same DRVs as for men are proposed. In premenopausal women, additional iron is lost through menstruation but, because losses are highly skewed, the Panel set a PRI of 16 mg/day to cover requirements of 95 % of the population. In infants and children, requirements were calculated factorially, taking into consideration the needs for growth, replacement of losses and percentage iron absorption from the diet (10 % up to 11 years and 16 % thereafter). PRIs were estimated using a coefficient of variation of 20 %. They are 11 mg/day in infants (7\u201311 months), 7 mg/day in children aged 1\u20136 years and 11 mg/day in children aged 7\u201311 years and boys aged 12\u201317 years. For girls aged 12\u201317 years, the PRI of 13 mg/day is the midpoint of the calculated dietary requirement of 97.5 % of girls and the PRI for premenopausal women; this approach allows for the large uncertainties in the rate and timing of pubertal growth and menarche. For pregnant and lactating women, for whom it was assumed that iron stores and enhanced absorption provide sufficient additional iron, DRVs are the same as for premenopausal women

General scientific guidance for stakeholders on health claim applications

The European Food Safety Authority (EFSA) asked the Panel on Dietetic Products Nutrition and Allergies (NDA) to update the General guidance for stakeholders on the evaluation of Article 13.1, 13.5 and 14 health claims published in March 2011. Since then, the NDA Panel has completed the evaluation of Article 13.1 claims except for claims put on hold by the European Commission, and has evaluated additional health claim applications submitted pursuant to Articles 13.5, 14 and also 19. In addition, comments received from stakeholders indicate that general issues that are common to all health claims need to be further clarified and addressed. This guidance document aims to explain the general scientific principles applied by the NDA Panel for the evaluation of all health claims and outlines a series of steps for the compilation of applications. The general guidance document represents the views of the NDA Panel based on the experience gained to date with the evaluation of health claims, and it may be further updated, as appropriate, when additional issues are addressed

Scientific Opinion on Dietary Reference Values for copper

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) derived Dietary Reference Values (DRVs) for copper. Owing to the absence of appropriate biomarkers of copper status and the limitations of available balance studies, the Panel was unable to derive Average Requirements (ARs) and Population Reference Intakes (PRIs). Hence, Adequate Intakes (AIs) were defined based on mean observed intakes in several European Union (EU) countries, given that there is no evidence of overt copper deficiency in the European population. Data from balance studies were used as supportive evidence. For adults, AIs of 1.6 mg/day for men and 1.3 mg/day for women are proposed. For children, AIs are 0.7 mg/day for children aged 1 to &lt; 3 years, 1 mg/day for children aged 3 to &lt; 10 years, and 1.3 and 1.1 mg/day for boys and girls aged 10 to &lt; 18 years, respectively. For infants aged 7–11 months, based on mean observed intakes in four EU countries, an AI of 0.4 mg/day is proposed, which is supported by upwards extrapolation of estimated copper intake in exclusively breast-fed infants. For pregnant women, an increment of 0.2 mg/day is estimated to cover the amount of copper deposited in the fetus and the placenta over the course of pregnancy and in anticipation of the needs for lactation, and for lactating women the same increment is estimated to cover the amount of copper secreted with breast milk. Thus, for pregnant and lactating women, the Panel derived an AI of 1.5 mg/day

A fixed carbohydrate: protein ratio <= 1.8 on an energy basis consumed in the context of an energy-restricted diet and reduction of body weight: evaluation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006.

Following an application from Marks and Spencer PLC, submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of the United Kingdom, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to a CHO:P ratio &lt;= 1.8 on an energy basis in the context of an energy-restricted diet and body weight. The Panel considers that the food/constituent that is the subject of the health claim is sufficiently characterised. The Panel also considers that reduction of body weight in the context of an energy-restricted diet is a beneficial physiological effect. The target population proposed by the applicant is 'adults between the ages of 18 and 70 years with excess body weight'. No conclusions could be drawn from two unpublished studies investigating the effect of ready-to-eat meals with a CHO: P ratio &lt;= 1.8 on body weight. The remaining 14 human intervention studies investigated the effect of diets targeting a CHO: P ratio &lt;= 1.8 as compared to diets targeting a CHO: P ratio &gt;= 3.0 on overweight and obese adults in the context of energy restriction. Four out of seven studies lasting &lt; 12 weeks reported an effect of a CHO: P ratio &lt;= 1.8 on body weight in overweight/obese subjects, whereas no significant effect was observed in six out of the seven studies lasting 12 weeks or more. The Panel considers that these studies do not provide evidence for a sustained effect of the food/constituent on body weight. The Panel concludes that a cause and effect relationship has not been established between the consumption of a fixed CHO: P ratio &lt;= 1.8 on an energy basis consumed in the context of an energy-restricted diet and reduction of body weight. (C) 2017 European Food Safety Authority

Dietary reference values for vitamin D

Following a request from the European Commission, the EFSA Panel&nbsp;on Dietetic Products, Nutrition and Allergies (NDA) derived dietary reference values (DRVs) for vitamin D. The Panel&nbsp;considers that serum 25(OH)D concentration, which reflects the amount of vitamin D attained from both cutaneous synthesis and dietary sources, can be used as a biomarker of vitamin D status in adult and children populations. The Panel&nbsp;notes that the evidence on the relationship between serum 25(OH)D concentration and musculoskeletal health outcomes in adults, infants and children, and adverse pregnancy-related health outcomes, is widely variable. The Panel&nbsp;considers that Average Requirements and Population Reference Intakes for vitamin D cannot be derived, and therefore defines adequate intakes (AIs), for all population groups. Taking into account the overall evidence and uncertainties, the Panel&nbsp;considers that a serum 25(OH)D concentration of 50&nbsp;nmol/L is a suitable target value for all population groups, in view of setting the AIs. For adults, an AI for vitamin D is set at 15&nbsp;\u3bcg/day, based on a meta-regression analysis and considering that, at this intake, the majority of the population will achieve a serum 25(OH)D concentration near or above the target of 50&nbsp;nmol/L. For children aged 1\u201317&nbsp;years, an AI for vitamin D is set at 15&nbsp;\u3bcg/day, based on the meta-regression analysis. For infants aged 7\u201311&nbsp;months, an AI for vitamin D is set at 10&nbsp;\u3bcg/day, based on trials in infants. For pregnant and lactating women, the Panel&nbsp;sets the same AI as for non-pregnant non-lactating women, i.e. 15&nbsp;\u3bcg/day. The Panel&nbsp;underlines that the meta-regression was done on data collected under conditions of assumed minimal cutaneous vitamin D synthesis. In the presence of cutaneous vitamin D synthesis, the requirement for dietary vitamin D is lower or may even be zero