Dual UHPLC-HRMS Metabolomics and Lipidomics and Automated Data Processing Workflow for Comprehensive High-Throughput Gut Phenotyping.

In recent years, feces has surfaced as the matrix of choice for investigating the gut microbiome-health axis because of its non-invasive sampling and the unique reflection it offers of an individual's lifestyle. In cohort studies where the number of samples required is large, but availability is scarce, a clear need exists for high-throughput analyses. Such analyses should combine a wide physicochemical range of molecules with a minimal amount of sample and resources and downstream data processing workflows that are as automated and time efficient as possible. We present a dual fecal extraction and ultra high performance liquid chromatography-high resolution-quadrupole-orbitrap-mass spectrometry (UHPLC-HR-Q-Orbitrap-MS)-based workflow that enables widely targeted and untargeted metabolome and lipidome analysis. A total of 836 in-house standards were analyzed, of which 360 metabolites and 132 lipids were consequently detected in feces. Their targeted profiling was validated successfully with respect to repeatability (78% CV < 20%), reproducibility (82% CV < 20%), and linearity (81% R2 > 0.9), while also enabling holistic untargeted fingerprinting (15,319 features, CV < 30%). To automate targeted processing, we optimized an R-based targeted peak extraction (TaPEx) algorithm relying on a database comprising retention time and mass-to-charge ratio (360 metabolites and 132 lipids), with batch-specific quality control curation. The latter was benchmarked toward vendor-specific targeted and untargeted software and our isotopologue parameter optimization/XCMS-based untargeted pipeline in LifeLines Deep cohort samples (n = 97). TaPEx clearly outperformed the untargeted approaches (81.3 vs 56.7-66.0% compounds detected). Finally, our novel dual fecal metabolomics-lipidomics-TaPEx method was successfully applied to Flemish Gut Flora Project cohort (n = 292) samples, leading to a sample-to-result time reduction of 60%.

Quantitative food web modeling unravels the importance of the microphytobenthos-meiofauna pathway for a high trophic transfer by meiofauna in soft-bottom intertidal food webs.

Meiofauna are known to have an important role on many ecological processes, although, their role in food web dynamics is often poorly understood, partially as they have been an overlooked and under sampled organism group. Here, we used quantitative food web modeling to evaluate the trophic relationship between meiofauna and their food sources and how meiofauna can mediate the carbon flow to higher trophic levels in five contrasting soft-bottom intertidal habitats (including seagrass beds, mudflats and sandflats). Carbon flow networks were constructed using the linear inverse model-Markov chain Monte Carlo technique, with increased resolution of the meiofauna compartments (i.e. biomass and feeding ecology of the different trophic groups of meiofauna) compared to most previous modeling studies. These models highlighted that the flows between the highly productive microphytobenthos and the meiofauna compartments play an important role in transferring carbon to the higher trophic levels, typically more efficiently so than macrofauna. The pathway from microphytobenthos to meiofauna represented the largest flow in all habitats and resulted in high production of meiofauna independent of habitat. All trophic groups of meiofauna, except for selective deposit feeders, had a very high dependency on microphytobenthos. Selective deposit feeders relied instead on a wider range of food sources, with varying contributions of bacteria, microphytobenthos and sediment organic matter. Ecological network analyses (e.g. cycling, throughput and ascendency) of the modeled systems highlighted the close positive relationship between the food web efficiency and the assimilation of high-quality food sources by primary consumers, e.g. meiofauna and macrofauna. Large proportions of these flows can be attributed to trophic groups of meiofauna. The sensitivity of the network properties to the representation of meiofauna in the models leads to recommending a greater attention in ecological data monitoring and integrating meiofauna into food web models.

Trophic importance of microphytobenthos and bacteria to meiofauna in soft-bottom intertidal habitats: A combined trophic marker approach.

Meiofauna can play an important role in the carbon fluxes of soft-bottom coastal habitats. Investigation of their feeding behavior and trophic position remains challenging due to their small size. In this study, we determine and compare the food sources used by nematodes and benthic copepods by using stable isotope compositions, fatty acid profiles and compound specific isotope analyses of fatty acids in the mudflats, seagrass beds and a sandflat of the Marennes-Oléron Bay, France, and the Sylt-Rømø Bight, Germany. Suspended particulate organic matter was much more 13C-depleted than other food sources and meiofauna, highlighting its poor role in the different studied habitats. The very low proportions of vascular plant fatty acid markers in meiofauna demonstrated that these consumers did not rely on this food source, either fresh or detrital, even in seagrass beds. The combined use of stable isotopes and fatty acids emphasized microphytobenthos and benthic bacteria as the major food sources of nematodes and benthic copepods. Compound specific analyses of a bacteria marker confirmed that bacteria mostly used microphytobenthos as a substrate.

Biogeochemical markers across a pollution gradient in a Patagonian estuary: A multidimensional approach of fatty acids and stable isotopes.

A combined approach merging stable isotopes and fatty acids was applied to study anthropogenic pollution in the Río Negro estuary. Fatty acid markers of vegetal detritus indicated considerable allochthonous inputs at freshwater sites. Correlative evidence of diatom fatty acids, δ13C, chlorophyll and particulate organic matter suggested the importance of diatoms for the autochthonous organic matter production at the river mouth. Low δ15N values (~0‰) and high fatty acid 18:1(n-7) concentrations in the suspended particulate matter, in combination with the peaks of coliforms and ammonium, indicated a strong impact of untreated sewage discharge. The 15N depletion was related to oxygen-limited ammonification processes and incorporation of 15N depleted ammonium to microorganisms. This work demonstrates that the combined use of lipid and isotopic markers can greatly increase our understanding of biogeochemical factors and pollutants influencing estuaries, and our findings highlight the urgent need for water management actions to reduce eutrophication.

Temperature-dependent lipid levels and components in polar and temperate eelpout (Zoarcidae).

Total lipid content, lipid classes and fatty acid composition were analysed in tissues from two eelpout species fed on the same diet, the Antarctic Pachycara brachycephalum and the temperate Zoarces viviparus, with the aim of determining the role of lipids in fishes from different thermal habitats. The lipid content increased with decreasing temperature in the liver of both species, suggesting enhanced lipid storage under cold conditions. In P. brachycephalum, lipid composition in the liver and muscle was strongly dominated by triacylglycerols between 0 and 6 degrees C. In contrast, in the temperate species, lipid class composition changed with changes in the temperature. When acclimatized to 4 and 6 degrees C Z. viviparus not only displayed a shift to lipid anabolism and pronounced lipid storage, as indicated by high triacylglycerol levels, but also a shift to patterns of cold adaptation, as reflected by an increased content of polyunsaturated fatty acids in the lipid extract. Unsaturated fatty acids were also abundant in the Antarctic eelpout, but when compared to Z. viviparus at the same temperatures, the latter had significantly higher ratios of polyunsaturated to saturated fatty acid levels, whereas the Antarctic eelpout showed significantly higher ratios of monounsaturated to saturated fatty acid levels. High delta-15N values of the Antarctic eelpout reflect the high trophic level of this scavenger in the Weddell Sea food web. Stable carbon values suggest that lipid-enriched prey forms a major part of its diet. The strategy to accumulate storage lipids in the cold is interpreted to be adaptive behaviour at colder temperatures and during periods of irregular, pulsed food supply.

Automated leucocyte differentials in 292 patients with leucopenia: an evaluation of the Abbott CELL-DYN 3500 (CD3500) haematology analyser.

The widespread use of intensive therapies and the need to haematologically monitor patients on a frequent basis means that the proportion of blood samples with moderate to severe leucopenia is significant and increasing. From a laboratory perspective, particularly because of the need to spend significant amounts of time in obtaining manual differentials from stained smears with low leucocyte numbers, these clinical trends have created additional pressures on what is often a limited manpower resource. Moreover in such situations, differentials obtained from examination of only 20 or 50 cells are not uncommon and the statistical consequences of this will be clearly apparent. Currently, there is general user confidence for automated leucocyte differentials for blood samples with normal WBC parameters, but there has been some reluctance to extend this to samples with leucopenia. In order to explore this further, we examined the efficiency of a modern automated five-part differential analyser (Abbott CELL-DYN 3500) in an unselected series of 292 samples with leucopenia (WBC count range range; 0.28-2.48 x 10(9)/l). Of these, 49 were from leucopenic sero-positive HIV patients with the remaining 243 samples originating from haematological oncology clinics, patients receiving radiotherapy for non-haemopoietic malignancies, and from patients with various chronic diseases. Morphologically, 204 of these samples did not show any blast cells or NRBC, 48 had blast cells but no NRBC, 29 had NRBC but no blasts, and the remaining 11 showed both blasts and NRBC. For 277 cases with less than 5% blasts, there was an excellent correlation between the manual and CD3500 automated differential, with no obvious bias between manual and automated subpopulation estimates at any percentage level. Linear regression analyses comparing absolute neutrophil, eosinophil, lymphocyte and monocyte counts for these same samples further revealed impressive correlations (r > 0.92) for all leucocyte populations and the absolute neutrophil count in particular (r = 0.986). Manual and CD3500 leucocyte differential comparisons for 11 cases with > 5% blasts showed good correlations for absolute neutrophil and eosinophil counts although, when the blast cell percentage was high, correlations for lymphocyte and monocyte counts were less consistent (an operator alert in the form of a 'Blast Flag' was, however, given in 10/11 of these particular cases). Four additional cases where manual differentiation between lymphoid cells and monocytes was recorded as difficult also showed consistently good correlations for manual vs automated neutrophil and eosinophil estimates. Not surprisingly, and essentially as a result of the low confidence noted for the manual differential itself, correlations for lymphoid and monocytic cells were relatively poor. In conclusion, this study has demonstrated that the CD3500 provides reliable and accurate absolute neutrophil and eosinophil counts in leucopenic samples irrespective of the presence of blasts or NRBC. These observations are particularly important in terms of monitoring patients who are liable to develop neutropenia as a result of chemotherapy and radiotherapy, and provide evidence that the routine use of automated leucocyte differentials may be confidently extended to the analysis of leucopenic samples.

Quantified EEG in different G situations.

The electrical activity of the brain (EEG) has been recorded during parabolic flights in trained astronauts and non trained volunteers as well. The Fast Fourier analysis of the EEG activity evidenced more asymmetry between the two brain hemispheres in the subjects who suffered from motion sickness than in the others. However, such a FFT classification does not lead to a discrimination between deterministic and stochastic events. Therefore, a first attempt was made to calculate the dimensionality of "chaotic attractors" in the EEG patterns as a function of the different g-epochs of one parabola. Very preliminary results are given here.