A collaborative evaluation of LC-MS/MS based methods for BMAA analysis: soluble bound BMAA found to be an important fraction.

Exposure to β-Ν-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery ( < 10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis

Effect of the toxin (microcystin) content of Microcystis on copepod grazing

Although phytoplankton chemical defense may regulate plankton dynamics, demonstrating an ecologically relevant anti-grazer cue is challenging. Presented here is a novel approach to evaluate the quantitative effect of microcystin (MC), the most studied group of cyanobacterial metabolites, on grazing by the common copepod Eudiaptomus gracilis. A temperature-induced gradient in the intracellular MC concentration of three different Microcystis strains enabled the comparison of grazing pressure on cells of the same cyanobacterial strain producing different amounts of MC, in a diet with alternative food (Chlamydomonas). In all treatments, grazing pressure on Microcystis was inversely related to its MC-LR content, while selection for alternative prey was positively related to the MC-LR content of Microcystis. Moreover, grazing on Chlamydomonas also declined with increasing Microcystis MC-LR content, suggesting toxicity related inhibition of E. gracilis. The negative relation between cellular MC-LR concentration and feeding responses supported the anti-grazer hypothesis. Not all MC variants responded to temperature, and some were therefore not associated to grazing responses. Using an induced gradient in the concentration of a suspected phytoplankton defense metabolite to evaluate its quantitative relationship with grazing pressure offers an improved inference on the ecological roles of toxins. Results suggest that either MC-LR or a correlating trait may be inversely linked to the grazer pressure on Microcystis

Toxicity Overrides Morphology on Cylindrospermopsis raciborskii Grazing Resistance to the Calanoid Copepod Eudiaptomus gracilis

Toxicity and morphology may function as defense mechanisms of bloom-forming cyanobacteria against zooplankton grazing. Yet, the relative importance of each of these factors and their plasticity remains poorly known. We tested the effects of chemical and morphological traits of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii on the feeding response of the selective feeder Eudiaptomus gracilis (Calanoida, Copepoda), using a saxitoxin-producing strain (STX+) and a non-saxitoxin (STX−)-producing strain as food. From these two chemotypes, we established cultures of three different morphotypes that differed in filament length (short, medium, and long) by incubating the strains at 17, 25, and 32 °C. We hypothesized that the inhibitory effects of saxitoxins determine the avoidance of C. raciborskii, and that morphology would only become relevant in the absence of saxitoxins. Temperature affected two traits: higher temperature resulted in significantly shorter filaments in both strains and led to much higher toxin contents in the STX+ strain (1.7 μg eq STX L−1 at 17 °C, 7.9 μg eq STX L−1 at 25 °C, and 25.1 μg eq STX L−1 at 32 °C). Copepods strongly reduced the ingestion of the STX+ strain in comparison with STX− cultures, regardless of filament length. Conversely, consumption of shorter filaments was significantly higher in the STX− strain. The great plasticity of morphological and chemical traits of C. raciborskii and their resultant contrasting effects on the feeding behavior of zooplankton might explain the success of this cyanobacterium in a variety of aquatic environments

Intra- and inter-individual variability of longitudinal daytime melatonin secretion patterns in depressed and non-depressed individuals

Disrupted melatonin secretion is regarded as a link between circadian rhythm and major depression, but results have been contradictory. We hypothesize that this might be due to averaging across individuals and too short measurements periods. In this study, pair-matched depressed and non-depressed individuals sampled their saliva three times a day, 30 days, in their natural environment. The depressed group showed significantly more variance and higher melatonin levels (p <0.05). Substantial interindividual heterogeneity and day-to-day variability was found. The individual time-series approach allowed us to reveal this variability. Important information remains unnoticed when analyzing melatonin only at the group level

Imbalance in the blood antioxidant system in growth hormone-deficient children before and after 1 year of recombinant growth hormone therapy

The aim of our study was to examine the effects of 12-month therapy with recombinant growth hormone (rGH) on the blood antioxidant system in children with growth hormone deficiency (GHD). Total antioxidant capacity (TAC) of plasma was measured by FRAP (ferric reducing antioxidant power or ferric reducing ability of plasma); activities of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes were assessed; non-protein thiols (NT) and ceruloplasmin (CP) levels were also measured. These parameters were determined before and after 12 month of rGH treatment. Eleven treatment-naive prepubertal children with growth hormone deficiency were included in the study. Another 11 prepubertal children comprised a control group. Before rGH treatment, TAC of plasma and NT level in the control group were significantly lower (726 ± 196 vs. 525 ± 166 µmol/L, P = 0.0182 and 0.92 ± 0.18 vs. 0.70 ± 0.22 µmol/ml, P = 0.0319, before and after the therapy, respectively). The only parameter that significantly (19.6 ± 4.7 vs. 14.5 ± 3.4 Units/g Hb, P = 0.0396) exceeded the same in the control group after rGH therapy was SOD activity. However, none of the measured parameters of antioxidant system in GHD children, except for TAC (525 ± 166 vs. 658 ± 115 µmol/L, P = 0.0205), exhibited significant improvement toward the end of the 12-month treatment period, although non-significant changes in CAT activity and CP level were also observed. This work has demonstrated that some parameters of the blood antioxidant system are out of balance and even impaired in GHD children. A 12-month treatment with rGH resulted in a partial improvement of the antioxidant system

Imbalance in the blood antioxidant system in growth hormone-deficient children before and after 1 year of recombinant growth hormone therapy

The aim of our study was to examine the effects of 12-month therapy with recombinant growth hormone (rGH) on the blood antioxidant system in children with growth hormone deficiency (GHD). Total antioxidant capacity (TAC) of plasma was measured by FRAP (ferric reducing antioxidant power or ferric reducing ability of plasma); activities of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes were assessed; non-protein thiols (NT) and ceruloplasmin (CP) levels were also measured. These parameters were determined before and after 12 month of rGH treatment. Eleven treatment-naive prepubertal children with growth hormone deficiency were included in the study. Another 11 prepubertal children comprised a control group. Before rGH treatment, TAC of plasma and NT level in the control group were significantly lower (726 ± 196 vs. 525 ± 166 µmol/L, P = 0.0182 and 0.92 ± 0.18 vs. 0.70 ± 0.22 µmol/ml, P = 0.0319, before and after the therapy, respectively). The only parameter that significantly (19.6 ± 4.7 vs. 14.5 ± 3.4 Units/g Hb, P = 0.0396) exceeded the same in the control group after rGH therapy was SOD activity. However, none of the measured parameters of antioxidant system in GHD children, except for TAC (525 ± 166 vs. 658 ± 115 µmol/L, P = 0.0205), exhibited significant improvement toward the end of the 12-month treatment period, although non-significant changes in CAT activity and CP level were also observed. This work has demonstrated that some parameters of the blood antioxidant system are out of balance and even impaired in GHD children. A 12-month treatment with rGH resulted in a partial improvement of the antioxidant system