Determination of Serotonin and Dopamine Metabolites in Human Brain Microdialysis and Cerebrospinal Fluid Samples by UPLC-MS/MS: Discovery of Intact Glucuronide and Sulfate Conjugates

An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain.     

The impact of thermal seasonality on terrestrial endotherm food web dynamics: a revision of the Exploitation Ecosystem Hypothesis

Many terrestrial endotherm food webs constitute three trophic level cascades. Others have two trophic level dynamics (food limited herbivores; plants adapted to tackle intense herbivory) or one trophic level dynamic (herbivorous endotherms absent, thus plants compete for the few places where they can survive and grow). According to the Exploitation Ecosystems Hypothesis (EEH), these contrasting dynamics are consequences of differences in primary productivity. The productivity thresholds for changing food web dynamics were assumed to be global constants. We challenged this assumption and found that several model parameters are sensitive to the contrast between persistently warm and seasonally cold climates. In persistently warm environments, three trophic level dynamics can be expected to prevail almost everywhere, save the most extreme deserts. We revised EEH accordingly and tested it by compiling direct evidence of three and two trophic level dynamics and by studying the global distribution of felids. In seasonally cold environments, we found evidence for three trophic level dynamics only in productive ecosystems, while evidence for two trophic level dynamics appeared in ecosystems with low primary productivity. In persistently warm environments, we found evidence for three trophic level dynamics in all types of ecosystems. The distribution of felids corroborated these results. The empirical evidence thus indicates that two trophic level dynamics, as defined by EEH, are restricted to seasonally cold biomes with low primary productivity, such as the artic–alpine tundra and the temperate steppe.     

Quantitative characterization of clumping in Scots pine crowns

Background and Aims

Proper characterization of the clumped structure of forests is needed for calculation of the absorbed radiation and photosynthetic production by a canopy. This study examined the dependency of crown-level clumping on tree size and growth conditions in Scots pine (Pinus sylvestris), and determined the ability of statistical canopy radiation models to quantify the degree of self-shading within crowns as a result of the clumping effect.

Methods

Twelve 3-D Scots pine trees were generated using an application of the LIGNUM model, and the crown-level clumping as quantified by the crown silhouette to total needle area ratio (STAR_crown) was calculated. The results were compared with those produced by the stochastic approach of modelling tree crowns as geometric shapes filled with a random medium.

Key Results

Crown clumping was independent of tree height, needle area and growth conditions. The results supported the capability of the stochastic approach in characterizing clumping in crowns given that the outer shell of the tree crown is well represented.

Conclusions

Variation in the whole-stand clumping index is induced by differences in the spatial pattern of trees as a function of, for example, stand age rather than by changes in the degree of self-shading within individual crowns as they grow bigger.     

Long-Term Experiments Reveal Strong Interactions Between Lemmings and Plants in the Fennoscandian Highland Tundra

Both the theory and the observations suggest that, there are strong links between herbivores and plants in terrestrial ecosystems; although, the effect of herbivores on plant community biomass is often attributed to variations in plant palatability. The existence of a strong link is commonly tested by constructing exclosures that exclude herbivores during a period of time. We here present data from two long-term (9 and 20 years, respectively) herbivore exclosure studies in lemming habitats on arctic tundra in northernmost Norway. The exclusion of all mammalian herbivores triggered strong increases in community level plant biomass and substantial changes in plant community composition. Palatable plants like graminoids and large bryophytes, as well as unpalatable plants like evergreen ericoids, deciduous shrubs, and lichens were all favored by excluding lemmings. These results reveal that a substantial increase in community biomass which occurs only when plant species capable of accumulating biomass are present, and palatability is a poor predictor of long-term responses of plants to excluding herbivores.     

Biocompatibility of mannan nanogel—safe interaction with plasma proteins

Background

Self-assembled mannan nanogels are designed to provide a therapeutic or vaccine delivery platform based on the bioactive properties of mannan to target mannose receptor expressed on the surface of antigen-presenting cells, combined with the performance of nanogels as carriers of biologically active agents.

Methods

Proteins in the corona around mannan nanogel formed in human plasma were identified by mass spectrometry after size exclusion chromatography or centrifugation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Structural changes and time dependent binding of human apolipoprotein A-I (apoA-I) and human serum albumin (HSA) to mannan nanogel were studied using intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The mannan nanogel effect on blood coagulation and fibrillation of Alzheimer's disease-associated amyloid β peptide and hemodialysis-associated amyloidosis β2 microglobulin was evaluated using thrombin generation assay or thioflavin T fluorescence assay, respectively.

Results

The protein corona around mannan nanogel is formed through a slow process, is quite specific comprising apolipoproteins B-100, A-I and E and HSA, evolves over time, and the equilibrium is reached after hours to days. Structural changes and time dependent binding of apoA-I and HSA to mannan nanogel are minor. The mannan nanogel does not affect blood coagulation and retards the fibril formation.

Conclusions

Mannan nanogel has a high biosafety and biocompatibility, which is mandatory for nanomaterials to be used in biomedical applications.

General Significance

Our research provides a molecular approach to evaluate the safety aspects of nanomaterials, which is of general concern in society and science.     

Contributions of leaf photosynthetic capacity, leaf angle and self-shading to the maximization of net photosynthesis in Acer saccharum: a modelling assessment

Background and Aims

Plants are expected to maximize their net photosynthetic gains and efficiently use available resources, but the fundamental principles governing trade-offs in suites of traits related to resource-use optimization remain uncertain. This study investigated whether Acer saccharum (sugar maple) saplings could maximize their net photosynthetic gains through a combination of crown structure and foliar characteristics that let all leaves maximize their photosynthetic light-use efficiency (ɛ).

Methods

A functional–structural model, LIGNUM, was used to simulate individuals of different leaf area index (LAI_ind) together with a genetic algorithm to find distributions of leaf angle (L_A) and leaf photosynthetic capacity (A_max) that maximized net carbon gain at the whole-plant level. Saplings grown in either the open or in a forest gap were simulated with A_max either unconstrained or constrained to an upper value consistent with reported values for A_max in A. saccharum.

Key Results

It was found that total net photosynthetic gain was highest when whole-plant PPFD absorption and leaf ɛ were simultaneously maximized. Maximization of ɛ required simultaneous adjustments in L_A and A_max along gradients of PPFD in the plants. When A_max was constrained to a maximum, plants growing in the open maximized their PPFD absorption but not ɛ because PPFD incident on leaves was higher than the PPFD at which ɛ_max was attainable. Average leaf ɛ in constrained plants nonetheless improved with increasing LAI_ind because of an increase in self-shading.

Conclusions

It is concluded that there are selective pressures for plants to simultaneously maximize both PPFD absorption at the scale of the whole individual and ɛ at the scale of leaves, which requires a highly integrated response between L_A, A_max and LAI_ind. The results also suggest that to maximize ɛ plants have evolved mechanisms that co-ordinate the L_A and A_max of individual leaves with PPFD availability.     

Differential metabolic consequences of fumarate hydratase and respiratory chain defects

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.     

Surveying biodiversity in protected and managed areas: Algae, macrophytes and macroinvertebrates in boreal forest streams

Extensive surveys of biodiversity in protected and managed areas have not been conducted for a majority of taxonomic groups and ecosystem types, which makes it difficult to assess how large a portion of biodiversity is at least potentially under protection. The situation is the same in boreal regions, and only preliminary analyses of the biodiversity patterns of less well-known organism groups, including many freshwater taxa, within the protected area network have been conducted. We studied patterns of species richness and community composition of algae, macrophytes (bryophytes and vascular plants), and macroinvertebrates of headwater streams draining protected areas and managed forests in a boreal drainage basin in Finland. We found no significant differences in the species richness and community composition of these organism groups between the protected and managed streams. Gamma- and beta-diversity varied strongly among the protected and managed stream groups, yet this variation was contingent on the organism group and the beta-diversity measure used. In general, there was much species turnover within both protected and managed stream groups, masking any between-group differences. However, we found a number of redlisted and rare species in our surveys. Of these species, several macrophyte species occurred more frequently in the protected streams. By contrast, rare species of algae and macrophytes did not generally show such inclinations to the protected streams. We found no strong congruence in species richness or community dissimilarity between algae, macrophytes, and macroinvertebrates, suggesting that the main anthropogenic gradient in terms of forestry is not strong enough to modify stream environmental conditions and thereby shape biodiversity in the focal drainage basin. This finding also suggests that surveys of aquatic biodiversity across protected and managed landscapes should not rely too heavily on the surrogate taxon approach, but instead should consider patterns shown by multiple taxonomic groups that represent biologically and ecologically disparate organisms. Our results indeed suggest that the levels of alpha-, beta-, and gamma-diversity show differing among-taxon responses to forest management and naturalness of headwater streams.     

N-glycoproteomics - an automated workflow approach

Glycan decorations dictate protein functions and thus have crucialimportance in life sciences. Previously glycoprotein analysiswas mainly focused on the analysis of the liberated glycansallowing detailed structural, but lacking positional information.Analysis of intact glycopeptides required purified glycoproteinsand manual interpretation of spectra. We developed an approachwhere mixtures of native glycopeptides were analyzed with tandemmass spectrometry and the spectra were analyzed with automatedin silico workflows. The latter included combination of theoriginal spectra, generation of a human N-glycopeptide library,matching the glycopeptide spectra to the theoretical peptidefragments, scoring the observations, predicting the glycan composition,which were then matched against the observed spectra, statisticalvalidation of the results with target–decoy filtering,and finally the calculation of glycan structures. We verifiedthis approach with the 150 serotransferrin glycopeptide spectra,where we automatically generated 10⁵ putative interpretationsfrom >10⁹ theoretical glycopeptides. After scoring 62 glycopeptidespectra obtained validated interpretation with concomitant aminoacid sequences, glycan compositions, and structures. When applyingthis method to an unknown mixture of human plasma glycoproteinswe identified 80 glycopeptides with their glycan compositionsor structures. Instead of weeks and months of interpretationwork of mass spectrometry files our automated workflow can beexecuted in few hours and provide information concomitantlyfrom both the amino acid and glycan moieties of intact glycopeptidesin mixtures. No advanced computational skills were needed touse these preformed and tested workflows. In case users wantto add complexity to the analysis they are allowed to alterall parameters and rebuild the workflows.