Metabolic network visualization eliminating node redundance and preserving metabolic pathways

Background  

The tools that are available to draw and to manipulate the representations of metabolism are usually restricted to metabolic pathways. This limitation becomes problematic when studying processes that span several pathways. The various attempts that have been made to draw genome-scale metabolic networks are confronted with two shortcomings: 1- they do not use contextual information which leads to dense, hard to interpret drawings, 2- they impose to fit to very constrained standards, which implies, in particular, duplicating nodes making topological analysis considerably more difficult.     

Changes in serum markers indicative of health effects in vineyard workers following exposure to the fungicide mancozeb: an Italian study

The aim of this study was to investigate the health effects induced by exposure to the fungicide mancozeb in Italian vineyard workers. Ninety-three Italian subjects entered the study - 48 vine-growers intermittently exposed to mancozeb and 45 healthy controls. The subjects were investigated three times: before the seasonal application of pesticides (T0), 30 days after the beginning of the application period (T30), and 45 days after T0 (T45). At T0 the comparison between agricultural workers and controls showed a higher prevalence of cold or flu symptoms, a statistically significant lower percentage of monocytes, higher absolute count of T lymphocytes, CD4 and natural killer cells, and lower plasma levels of IgA and IgM in workers. Such differences were not confirmed at T30 and T45. In fact at T30 in exposed workers, besides a significant increase of urinary ethylenethiourea, confirming mancozeb exposure, T lymphocytes, CD4 and natural killer cells, IgA and IgM returned to values comparable to those observed in controls. Moreover, no other differences in clinical signs, haematological, and immune parameters, such as the immune functional capability evaluated as a response to hepatitis B vaccination, was observed. Altogether the differences between exposed and controls were not consistently correlated to any clinical impairment and suggest that the seasonal application of mancozeb does not pose a significant health risk to exposed subjects.     

Reproducible and Consistent Quantification of the Saccharomyces cerevisiae Proteome by SWATH-mass spectrometry

Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS technique to quantify changes over time in a large fraction of the proteome expressed in Saccharomyces cerevisiae in response to osmotic stress.We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection data independent acquisition data sets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. Proteins were quantified by the targeted extraction and integration of transition signal groups from the SWATH-MS datasets for peptides that are proteotypic for specific yeast proteins. We consistently identified and quantified more than 15,000 peptides and 2500 proteins across the 18 samples. We demonstrate high reproducibility between technical and biological replicates across all time points and protein abundances. In addition, we show that the abundance of hundreds of proteins was significantly regulated upon osmotic shock, and pathway enrichment analysis revealed that the proteins reacting to osmotic shock are mainly involved in the carbohydrate and amino acid metabolism. Overall, this study demonstrates the ability of SWATH-MS to efficiently generate reproducible, consistent, and quantitatively accurate measurements of a large fraction of a proteome across multiple samples.In systems biology and biomedical studies targeted mass spectrometry via selected reaction monitoring (SRM)¹ (also known as multiple reaction monitoring, MRM) has emerged as a powerful technique for the consistent and reproducible quantification of proteins across numerous complex samples (1–6). Optimal sets of precursor/fragment ion pairs, called transitions, uniquely represent a specific peptide. They constitute a definitive mass spectrometric assay for the detection of targeted peptides, and thus the proteins from which they derive, in the complex matrix of trypsinized biological samples (1, 7). Protein quantification is then performed by relating the intensity of the acquired transition signals to suitable reference signals. Most quantification strategies commonly used in proteomics are compatible with this method (8). Recently, the high-throughput development of S/MRM assays has been achieved via the generation of MS/MS spectral libraries from the measurements of thousands of synthetic peptides representing proteotypic peptides (9). Moreover, many experimental and bioinformatics workflows have been developed for assay generation, assay optimization, data evaluation, and the dissemination of optimized S/MRM assays (10–16). In combination, these developments have supported the creation of mass-spectrometric maps of entire proteomes of selected species including Streptococcus pyogenes, Mycobacterium tuberculosis, and Saccharomyces cerevisae (5, 17–19) and the robust use of these resources to quantify specific protein sets across multiple biological samples.Currently, targeted proteomics by S/MRM can be multiplexed to a maximum set of ∼100 proteins that can be measured in a single LC-S/MRM run at optimal quantitative accuracy, limit of detection and dynamic range. The quantification of higher numbers of proteins per run compromises some of the performance parameters of the method because of well understood tradeoffs (8). Attempts have been made to further increase the degree of multiplexing of S/MRM, either by automated adjustment of the scheduled detection windows (20) or by acquiring, in a data-dependent manner, the complete set of precursor-fragment ion pairs of a given assay (21). Alternatively, parallel reaction monitoring (PRM) approach operated on quadrupole-orbitrap mass spectrometer has shown detection and quantification performances similar or better than those obtained in SRM, because of the increased selectivity of the mass analyzer (22–24). These approaches are promising, but their application relies on prior knowledge of the precursor ions that need to be targeted during the data acquisition, and they still are subject of the above-mentioned tradeoffs.Recently, we developed a novel MS strategy that combines data independent acquisition (DIA) of trypsinized protein samples with S/MRM-like, in silico targeted analysis of the acquired complete fragment ion maps (25). We termed the method SWATH-MS, and applied the sequential isolation window acquisition principle (26) to repeatedly cycle, in a single injection, through 32 consecutive 25-Da precursor isolation windows (swaths). The process acquires fragment ion spectra of all precursors in a space defined by the 400–1200 m/z precursor range and a user-specified retention time window. We used the prior information in MS/MS spectral libraries to extract groups of signals that uniquely identify a specific peptide, and to demonstrate that peptides could be identified and quantified over a dynamic range of four orders of magnitude, even when the precursors were not detectable in a survey MS scan. For the 45 proteins involved in the central carbon metabolism of yeast, we demonstrated that the accuracy of quantification was equivalent to that of S/MRM (25). However, because of the lack of adequate software tools at that time, the extensive high-throughput targeted data analysis of the SWATH-MS maps could not be fully demonstrated in that first study.Here we demonstrate the multiplexing capabilities of SWATH-MS for the detection and quantification of significantly larger fractions of a proteome as compared with S/MRM, without compromising reproducibility, consistency, and quantitative accuracy. We describe the large scale deployment of fragment ion spectral libraries and the use of S/MRM-like analysis tools specifically adapted to SWATH-MS data for the detection and quantification of temporal changes of the S. cerevisae proteome in response to osmotic stress.     

Mitochondrial genomes reveal the extinct Hippidion as an outgroup to all living equids

Hippidions were equids with very distinctive anatomical features. They lived in South America 2.5 million years ago (Ma) until their extinction approximately 10 000 years ago. The evolutionary origin of the three known Hippidion morphospecies is still disputed. Based on palaeontological data, Hippidion could have diverged from the lineage leading to modern equids before 10 Ma. In contrast, a much later divergence date, with Hippidion nesting within modern equids, was indicated by partial ancient mitochondrial DNA sequences. Here, we characterized eight Hippidion complete mitochondrial genomes at 3.4–386.3-fold coverage using target-enrichment capture and next-generation sequencing. Our dataset reveals that the two morphospecies sequenced (H. saldiasi and H. principale) formed a monophyletic clade, basal to extant and extinct Equus lineages. This contrasts with previous genetic analyses and supports Hippidion as a distinct genus, in agreement with palaeontological models. We date the Hippidion split from Equus at 5.6–6.5 Ma, suggesting an early divergence in North America prior to the colonization of South America, after the formation of the Panamanian Isthmus 3.5 Ma and the Great American Biotic Interchange.     

Characterization of a hyper-viscoelastic phantom mimicking biological soft tissue using an abdominal pneumatic driver with magnetic resonance elastography (MRE)

The purpose of this study was to create a polymer phantom mimicking the mechanical properties of soft tissues using experimental tests and rheological models. Multifrequency Magnetic Resonance Elastography (MMRE) tests were performed on the present phantom with a pneumatic driver to characterize the viscoelastic (μ, η) properties using Voigt, Maxwell, Zener and Springpot models. To optimize the MMRE protocol, the driver behavior was analyzed with a vibrometer. Moreover, the hyperelastic properties of the phantom were determined using compressive tests and Mooney-Rivlin model. The range of frequency to be used with the round driver was found between 60 Hz and 100 Hz as it exhibits one type of vibration mode for the membrane. MRE analysis revealed an increase in the shear modulus with frequency reflecting the viscoelastic properties of the phantom showing similar characteristic of soft tissues. Rheological results demonstrated that Springpot model better revealed the viscoelastic properties (μ=3.45 kPa, η=6.17 Pas) of the phantom and the Mooney-Rivlin coefficients were C(10)=1.09.10(-2) MPa and C(01)=-8.96.10(-3) MPa corresponding to μ=3.95 kPa. These studies suggest that the phantom, mimicking soft tissue, could be used for preliminary MRE tests to identify the optimal parameters necessary for in vivo investigations. Further developments of the phantom may allow clinicians to more accurately mimic healthy and pathological soft tissues using MRE.     

Structure and pharmacology of pentameric receptor channels: from bacteria to brain

Orthologs of the pentameric receptor channels that mediate fast synaptic transmission in the central and peripheral nervous systems have been found in several bacterial species and in a single archaea genus. Recent X-ray structures of bacterial and invertebrate pentameric receptors point to a striking conservation of the structural features within the whole family, even between distant prokaryotic and eukaryotic members. These structural data reveal general principles of molecular organization that allow allosteric membrane proteins to mediate chemoelectric transduction. Notably, several conformations have been solved, including open and closed channels with distinct global tertiary and quaternary structure. The data reveal features of the ion channel architecture and of diverse categories of binding sites, such as those that bind orthosteric ligands, including neurotransmitters, and those that bind allosteric modulators, such as general anesthetics, ivermectin, or lipids. In this review, we summarize the most recent data, discuss insights into the mechanism of action in these systems, and elaborate on newly opened avenues for drug design.     

Next-generation sequencing offers new insights into DNA degradation

The processes underlying DNA degradation are central to various disciplines, including cancer research, forensics and archaeology. The sequencing of ancient DNA molecules on next-generation sequencing platforms provides direct measurements of cytosine deamination, depurination and fragmentation rates that previously were obtained only from extrapolations of results from in vitro kinetic experiments performed over short timescales. For example, recent next-generation sequencing of ancient DNA reveals purine bases as one of the main targets of postmortem hydrolytic damage, through base elimination and strand breakage. It also shows substantially increased rates of DNA base-loss at guanosine. In this review, we argue that the latter results from an electron resonance structure unique to guanosine rather than adenosine having an extra resonance structure over guanosine as previously suggested.