Metabolic profiling of serum using Ultra Performance Liquid Chromatography and the LTQ-Orbitrap mass spectrometry system

Advances in analytical instrumentation can provide significant advantages to the volume and quality of biological knowledge acquired in metabolomic investigations. The interfacing of sub-2mum liquid chromatography (UPLC ACQUITY((R))) and LTQ-Orbitrap mass spectrometry systems provides many theoretical advantages. The applicability of the interfaced systems was investigated using a simple 11-component metabolite mix and a complex mammalian biofluid, serum. Metabolites were detected in the metabolite mix with signals that were linear with their concentration over 2.5-3.5 orders of magnitude, with correlation coefficients greater than 0.993 and limits of detection less than 1mumolL(-1). Reproducibility of retention time (RSD<3%) and chromatographic peak area (RSD<15%) and a high mass accuracy (<2ppm) were observed for 14 QC serum samples interdispersed with other serum samples, analysed over a period of 40h. The evaluation of a single deconvolution software package (XCMS) was performed and showed that two parameters (snthresh and bw) provided significant changes to the number of peaks detected and the peak area reproducibility for the dataset used. The data were used to indicate possible biomarkers of pre-eclampsia and showed both the instruments and XCMS to be applicable to the reproducible and valid detection of disease biomarkers present in serum.     

Metabolite profiles of interacting mycelial fronts differ for pairings of the wood decay basidiomycete fungus, Stereum hirsutum with its competitors Coprinus micaceus and Coprinus disseminatus

The paper presents the first proof-of principle study of metabolite profiles of the interacting mycelial fronts of a wood decomposer basidiomycete, Stereum hirsutum, paired with two competitor basidiomycetes, Coprinus disseminatus and C. micaceus, using TLC and GC-TOF-MS profiling. GC-TOF-MS profiles were information rich, with a total of 190 metabolite peaks detected and more than 120 metabolite peaks detected per sample. The metabolite profiles were able to discriminate between the interactions of S. hirsutum with the two species of Coprinus. In confrontation with C. micaceus, where S. hirsutum mycelial fronts always overgrew those of C. micaceus, there were down-regulations of metabolites in the interaction zone, compared to monocultures of both S. hirsutum and C. micaceus. In contrast, in pairings with C. disseminatus, whose mycelia overgrew those of S. hirsutum, there were some up-regulations compared with monoculture controls, the majority of the metabolites being characteristic of the S. hirsutum monoculture profile. These differences indicate that up-regulation of metabolites in the mycelia of S. hirsutum may be connected to a defensive role or to stress. The results also show proof of principle for the employment of metabolic profiling for biological discovery studies of metabolites produced by fungi that could be applied to natural product screening programmes.     

Foraging mechanics and their outcomes for cattle grazing reproductive tropical swards

For grazing animals an important determinant of animal performance is the rate of nutrient intake (RNI) which depends on diet quality and instantaneous intake rate (IIR). In turn, diet quality and IIR are the outcome of the interaction between the morphology and behaviour of the animal and the structure of the sward. Using artificial microswards of Panicum maximum we evaluated the effect of four levels of the tensile resistance of stems in tropical swards on the grazing behaviour of cattle of two age classes (1- and 3-year-old steers) faced with a stem barrier either in a vertical (experiment 1, E1) or horizontal plane (experiment 2, E2). The animals did not select against low tensile resistance stems (LTRS) but did avoid high tensile resistance stems (HTRS) which resulted in a significant difference (P < 0.001) between diet DM (dry matter) digestibility and forage DM digestibility in swards with HTRS. IIR decreased (P < 0.001) on average 62% in E1 and 67% in E2 as stem tensile resistance increased, 144% and 177% in E1 and E2, respectively. This led to a reduction (P < 0.001) in digestible DM IIR in both young and mature cattle respectively of 56% and 68% in E1, and 45% and 79% in E2, as stem tensile resistance increased. The decline in IIR was due to an increase in time per bite and a reduction in bite dimensions that were the result of different mechanisms in the two experiments: when leaves were taller than the stems (E1) the decrease in bite area was associated with an asymptotic increase (P < 0.001) in bite force in mature animals, and a maintenance (P = 0.456) of bite force in young animals, suggesting that maximum bite force regulated bite area in E1. This was not the case in E2 because both bite area and bite force decreased (P < 0.05) with the increase in stem tensile resistance suggesting that the animals were not able to gather enough plant material with the tongue in order for bite force to regulate bite area. We conclude that bite force and tongue force regulate selectivity and bite dimensions, which are the ultimate determinants of the RNI; IIR is the primary determinant of the RNI with diet quality being of lesser importance; and HTRS act as deterrents to achieving maximum IIR in tropical swards, particularly in mature cattle.     

Influence of BCG vaccine strain on the immune response and protection against tuberculosis

The Bacille Calmette–Guérin (BCG) vaccine has been used for more than 80 years to protect against tuberculosis. Worldwide, over 90% of children are immunized with BCG, making it the most commonly administered vaccine, with more than 120 million doses used each year. Although new tuberculosis vaccines are under investigation, BCG will remain the cornerstone of the strategy to fight the worsening tuberculosis pandemic for the foreseeable future. The recent delineation of genetic differences between BCG vaccine strains has renewed interest in the influence of the vaccine strain on the protective efficacy against tuberculosis. This review critically examines the data from animal and human studies comparing BCG vaccine strains. Although there is good evidence to support the notion that the induced immune response and protection afforded against tuberculosis differs between BCG vaccine strains, currently, there are insufficient data to favour or recommend one particular strain. Identifying BCG strains with superior protection would have a dramatic effect on tuberculosis control at a population level: a small increment in protection provided by BCG immunization will prevent large numbers of cases of severe tuberculosis and deaths, particularly in children.     

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean

(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.     

Inhibition of Wnt and PI3K Signaling Modulates GSK-3β Activity and Induces Morphological Changes in Cortical Neurons: Role of Tau Phosphorylation

Glycogen synthase kinase GSK-3β has been identified as one of the major candidates mediating tau hyperphosphorylation at the same sites as those present in tau protein in brain from Alzheimer′s disease (AD) patients. However, the signal transduction pathways involved in the abnormal activation of GSK-3β, have not been completely elucidated. GSK-3β activity is repressed by the canonical Wnt signaling pathway, but it is also modulated through the PI3K/Akt route. Recent studies have suggested that Wnt signaling might be involved in the pathophysiology of AD. On the other hand, modulators of the PI3K pathway might be reduced during aging leading to a sustained activation of GSK-3β, which in turn would increase the risk of tau hyperphosphorylation. The role of Wnt and PI3K signaling inhibition on the extent of tau phosphorylation and neuronal morphology has not been completely elucidated. Thus, in the present investigation we analyzed the effects of different negative modulators of the Wnt and the PI3K pathways on GSK-3β activation and phosphorylation of tau at the PHF-1 epitope in cortical cultured neurons and hippocampal slices from adult rat brain. Changes in the microtubule network were also studied. We found that a variety of Wnt and PI3K inhibitors, significantly increased tau phosphorylation at the PHF-1 site, induced the disarrangement of the microtubule network and the accumulation of tau within cell bodies. These changes correlated with alterations in neuronal morphology. Special issue article in honor of Dr. Ricardo Tapia.     

Biogeography of copepods in lakes and ponds of subarctic Québec,Canada

We aimed to determine whether the copepod assemblages in lakes and ponds of northern Québec, Canada, were closer in composition to those found in southern Québec, or to those reported from the subarctic and arctic. Six calanoid and five cyclopoid species were identified from 37 ponds and lakes located in the region between 55° N and 59° N. Species diversity was generally low, ranging from 0 to 4 species per lake. Dominant species were Leptodiaptomus minutus and Acanthocyclops vernalis. The species assemblages showed high affinity with those found in forested regions of southern Québec. Exceptions were Leptodiaptomus tyrrelli, previously only recorded west of Hudson Bay, and Hesperodiaptomus arcticus, hitherto recorded north of 58° N. Relationships between the lakes, species, and environmental variables were explored using multivariate analysis. Lakes situated along the coast and on two offshore islands clustered together and were characterised by higher conductivity and pH than those lakes located further inland. Leptodiaptomus tyrrelli was common in these coastal lakes. Canonical correspondence analysis revealed statistically significant relationships between copepod distributions and conductivity, dissolved organic carbon and pH. These three variables accounted for 70% of the variation in the species' distribution.     

An Investigation on Platelet Transport during Thrombus Formation at Micro-Scale Stenosis

This paper reports on an investigation of mass transport of blood cells at micro-scale stenosis where local strain-rate micro-gradients trigger platelet aggregation. Using a microfluidic flow focusing platform we investigate the blood flow streams that principally contribute to platelet aggregation under shear micro-gradient conditions. We demonstrate that relatively thin surface streams located at the channel wall are the primary contributor of platelets to the developing aggregate under shear gradient conditions. Furthermore we delineate a role for red blood cell hydrodynamic lift forces in driving enhanced advection of platelets to the stenosis wall and surface of developing aggregates. We show that this novel microfluidic platform can be effectively used to study the role of mass transport phenomena driving platelet recruitment and aggregate formation and believe that this approach will lead to a greater understanding of the mechanisms underlying shear-gradient dependent discoid platelet aggregation in the context of cardiovascular diseases such as acute coronary syndromes and ischemic stroke.