metaBIT,an integrative and automated metagenomic pipeline for analysing microbial profiles from high‐throughput sequencing shotgun data

Micro‐organisms account for most of the Earth's biodiversity and yet remain largely unknown. The complexity and diversity of microbial communities present in clinical and environmental samples can now be robustly investigated in record times and prices thanks to recent advances in high‐throughput DNA sequencing (HTS). Here, we develop metaBIT, an open‐source computational pipeline automatizing routine microbial profiling of shotgun HTS data. Customizable by the user at different stringency levels, it performs robust taxonomy‐based assignment and relative abundance calculation of microbial taxa, as well as cross‐sample statistical analyses of microbial diversity distributions. We demonstrate the versatility of metaBIT within a range of published HTS data sets sampled from the environment (soil and seawater) and the human body (skin and gut), but also from archaeological specimens. We present the diversity of outputs provided by the pipeline for the visualization of microbial profiles (barplots, heatmaps) and for their characterization and comparison (diversity indices, hierarchical clustering and principal coordinates analyses). We show that metaBIT allows an automatic, fast and user‐friendly profiling of the microbial DNA present in HTS shotgun data sets. The applications of metaBIT are vast, from monitoring of laboratory errors and contaminations, to the reconstruction of past and present microbiota, and the detection of candidate species, including pathogens.     

Metabolic Tumour Burden Measured by 18F-FDG PET/CT Predicts Malignant Transformation in Patients with Neurofibromatosis Type-1

Background

To investigate the diagnostic and prognostic performances of ¹⁸F-FDG PET/CT measures of metabolic tumour burden in patients with neurofibromatosis type-1 (NF1), suspect of malignant transformation.

Methods

This retrospective study included 49 patients (15–60 years old, 30 women) with a diagnosis of NF1, followed in our Reference Centre for Rare Neuromuscular Diseases, who presented clinical signs of tumour progression (pain, neurological deficit, tumour growth). Quantitative metabolic parameters were measured on 149 tumoral targets, using semi-automatic software and the best cut off values to predict transformation was assessed by Receiver Operating Characteristics (ROC) analysis. Prognostic value of PET/CT metabolic parameters was assessed by Kaplan-Meier estimates of overall survival.

Results

Lesions were histologically documented in 40 patients: a sarcomatous transformation was found in 16, a dysplastic neurofibroma (NF) in 7, and a benign NF in 17; in the remaining 9 patients, a minimal follow-up of 12 mo (median 59 mo) confirmed the absence of transformation. The optimal cut off values for detection of malignant transformation were, in decreasing order of area under the ROC curves, a tumour-to-liver (T/L) ratio >2.5, SUV_max > 4.5, total lesion glycolysis (TLG) > 377, total metabolic tumour volume (TMTV) > 88 cm³, and heterogeneity index (HI_suv) > 1.69. The best prognostic marker was the TLG: the 4-y estimates of survival were 97% [95% CI, 90% - 100%] in patients with TLG ≤ 377 vs. 27% [95% CI, 5% - 49%] in patients with TLG > 377 (P < 0.0001; χ² 27.85; hazard ratio 13.27 [95% CI, 3.72–47.35]). T/L ratio, SUV_max and TMTV demonstrated slightly lower performance to predict survival, with χ² ranging 14.41–19.12. The HI_suv index was not predictive of survival.

Conclusion

Our study demonstrates that TLG and TMTV, as PET/CT measures of metabolic tumour burden, may be used clinically to identify sarcomatous transformation in patients with NF1 and predict overall survival, with a higher specificity for the TLG. Conventional measures such as the SUV_max, and T/L ratio also demonstrate high prognostic value.     

Development of LCA benchmarks for Austrian torrent control structures

The International Journal of Life Cycle Assessment - This study emerged from a research project that aimed to develop a Life Cycle Assessment (LCA) model for torrent control structures. This...     

PAX3 Confers Functional Heterogeneity in Skeletal Muscle Stem Cell Responses to Environmental Stress

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Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.     

Cytosolic hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase from Arabidopsis thaliana: a specific role in early development and stress response

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction. Functionality of cytHPPK/DHPS was addressed by two parallel approaches: first, the cytHPPK/DHPS was able to rescue yeast mutants lacking corresponding activities; second, recombinant cytHPPK/DHPS expressed and purified from Escherichia coli displayed both HPPK and DHPS activities in vitro. In contrast to mitHPPK/DHPS, which was ubiquitously expressed, the cytHPPK/DHPS gene was exclusively expressed in reproductive tissue, more precisely in developing seeds as revealed by histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line. In addition, it was observed that expression of cytHPPK/DHPS mRNA was induced by salt stress, suggesting a potential role of the enzyme in stress response. This was supported by the phenotype of a T-DNA insertion mutant in the cytHPPK/DHPS gene, resulting in lower germination rates as compared with the wild-type upon application of oxidative and osmotic stress.     

Inhibition of nuclear T3 binding by fatty acids liberated from nuclear membranes via phospholipase C.

1. We have investigated whether phospholipase C (PLC) might act on phospholipids in the nuclear membrane of rat liver, resulting in inhibition of nuclear T3 binding (INB activity). 2. Incubation of intact nuclei with PLC caused a time- and dose-dependent increase of INB activity; this was correlated with a rise of the free fatty acid concentration in the nuclear ether extract. 3. Removal of the nuclear membrane resulted in a loss of INB activity of the nuclei. 4. Other compounds liberated by the action of PLC (such as diacylglycerols, IP3 and phosphoalcohols), had no INB activity. 5. We conclude that PLC can liberate fatty acids from the rat liver-nuclear membrane via further degradation of the direct product diacylglycerol. 6. These fatty acids display inhibition of nuclear T3 binding in vitro.     

The Impact of Seeding Method on Diversity and Plant Distribution in Two Restored Grasslands

Previous studies have compared grassland restoration techniques based on resulting species richness and composition. However, none have determined if different techniques generate different plant distributions in space, which may further impact restoration success. This study tests if there are quadrat‐scale (1 m²) differences between paired drilled and broadcast plantings in diversity, composition, and plant distributions. Higher competition intensity in and more contiguous spaces between rows in drill‐seeded restorations were hypothesized to result in larger patches of native grasses and exotic species. Two paired drill‐ and broadcast‐seeded plantings were sampled in June 2007 in Iowa, U.S.A. Within 10 quadrats in each planting, we measured species abundance with point intercept sampling and plant distributions by dividing the quadrat into 64 cells and recording the most abundant species in each cell. Drilled and broadcast plantings at both sites had similar Simpson’s diversity and evenness. However, the effect of planting type on species richness, composition, and plant distribution was site dependent. Native warm‐season grasses in one site, and exotic species in the second, occupied more space and were distributed in larger patches in drilled plantings. Furthermore, drilled canopies consistently captured more light than broadcast canopies. This suggests that initial differences in seed placement can affect resulting plant distributions, resource use, and potentially long‐term species turnover. Mechanisms structuring vegetation in these communities need to be further investigated to determine if this approach can provide more information on long‐term diversity maintenance in restorations than traditional measures.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

A structure-function study of ligand recognition by CD22beta

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.