Meneco, a Topology-Based Gap-Filling Tool Applicable to Degraded Genome-Wide Metabolic Networks

International audienceIncreasing amounts of sequence data are becoming available for a wide range of non-model organisms. Investigating and modelling the metabolic behaviour of those organisms is highly relevant to understand their biology and ecology. As sequences are often incomplete and poorly annotated, draft networks of their metabolism largely suffer from incompleteness. Appropriate gap-filling methods to identify and add missing reactions are therefore required to address this issue. However, current tools rely on phenotypic or taxonomic information, or are very sensitive to the stoichiometric balance of metabolic reactions, especially concerning the co-factors. This type of information is often not available or at least prone to errors for newly-explored organisms. Here we introduce Meneco, a tool dedicated to the topological gap-filling of genome-scale draft metabolic networks. Meneco reformulates gap-filling as a qualitative combinatorial optimization problem, omitting constraints raised by the stoichiometry of a metabolic network considered in other methods, and solves this problem using Answer Set Programming. Run on several artificial test sets gathering 10,800 degraded Escherichia coli networks Meneco was able to efficiently identify essential reactions missing in networks at high degradation rates, outperforming the stoichiometry-based tools in scalability. To demonstrate the utility of Meneco we applied it to two case studies. Its application to recent metabolic networks reconstructed for the brown algal model Ectocarpus siliculosus and an associated bacterium Candidatus Phaeomarinobacter ectocarpi revealed several candidate metabolic pathways for algal-bacterial interactions. Then Meneco was used to reconstruct, from transcriptomic and metabolomic data, the first metabolic network for the microalga Euglena mutabilis. These two case studies show that Meneco is a versatile tool to complete draft genome-scale metabolic networks produced from heterogeneous data, and to suggest relevant reactions that explain the metabolic capacity of a biological system

Dynamics of ampicillin-resistant Enterococcus faecium clones colonizing hospitalized patients: data from a prospective observational study

<p>Abstract</p> <p>Background</p> <p>Little is known about the dynamics of colonizing <it>Enterococcus faecium </it>clones during hospitalization, invasive infection and after discharge.</p> <p>Methods</p> <p>In a prospective observational study we compared intestinal <it>E. faecium </it>colonization in three patient cohorts: 1) Patients from the Hematology Unit at the University Hospital Basel (UHBS), Switzerland, were investigated by weekly rectal swabs (RS) during hospitalization (group 1a, n = 33) and monthly after discharge (group 1b, n = 21). 2) Patients from the Intensive Care Unit (ICU) at the University Medical Center Utrecht, the Netherlands (group 2, n = 25) were swabbed weekly. 3) Patients with invasive <it>E. faecium </it>infection at UHBS were swabbed at the time of infection (group 3, n = 22). From each RS five colonies with typical <it>E</it>. <it>faecium </it>morphology were picked. Species identification was confirmed by PCR and ampicillin-resistant <it>E. faecium </it>(ARE) isolates were typed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). The Simpson's Index of Diversity (SID) was calculated.</p> <p>Results</p> <p>Out of 558 ARE isolates from 354 RS, MT159 was the most prevalent clone (54%, 100%, 52% and 83% of ARE in groups 1a, 1b, 2 and 3, respectively). Among hematological inpatients 13 (40%) had ARE. During hospitalization, the SID of MLVA-typed ARE decreased from 0.745 [95%CI 0.657-0.833] in week 1 to 0.513 [95%CI 0.388-0.637] in week 3. After discharge the only detected ARE was MT159 in 3 patients. In the ICU (group 2) almost all patients (84%) were colonized with ARE. The SID increased significantly from 0.373 [95%CI 0.175-0.572] at week 1 to a maximum of 0.808 [95%CI 0.768-0.849] at week 3 due to acquisition of multiple ARE clones. All 16 patients with invasive ARE were colonized with the same MLVA clone (<it>p </it>< 0.001).</p> <p>Conclusions</p> <p>In hospitalized high-risk patients MT159 is the most frequent colonizer and cause of invasive <it>E. faecium </it>infections. During hospitalization, ASE are quickly replaced by ARE. Diversity of ARE increases on units with possible cross-transmission such as ICUs. After hospitalization ARE are lost with the exception of MT159. In invasive infections, the invasive clone is the predominant gut colonizer.</p

Amphibole and apatite insights into the evolution and mass balance of Cl and S in magmas associated with porphyry copper deposits

Chlorine and sulfur are of paramount importance for supporting the transport and deposition of ore metals at magmatic–hydrothermal systems such as the Coroccohuayco Fe–Cu–Au porphyry–skarn deposit, Peru. Here, we used recent partitioning models to determine the Cl and S concentration of the melts from the Coroccohuayco magmatic suite using apatite and amphibole chemical analyses. The pre-mineralization gabbrodiorite complex hosts S-poor apatite, while the syn- and post-ore dacitic porphyries host S-rich apatite. Our apatite data on the Coroccohuayco magmatic suite are consistent with an increasing oxygen fugacity (from the gabbrodiorite complex to the porphyries) causing the dominant sulfur species to shift from S2− to S6+ at upper crustal pressure where the magmas were emplaced. We suggest that this change in sulfur speciation could have favored S degassing, rather than its sequestration in magmatic sulfides. Using available partitioning models for apatite from the porphyries, pre-degassing S melt concentration was 20–200 ppm. Estimates of absolute magmatic Cl concentrations using amphibole and apatite gave highly contrasting results. Cl melt concentrations obtained from apatite (0.60 wt% for the gabbrodiorite complex; 0.2–0.3 wt% for the porphyries) seems much more reasonable than those obtained from amphibole which are very low (0.37 wt% for the gabbrodiorite complex; 0.10 wt% for the porphyries). In turn, relative variations of the Cl melt concentrations obtained from amphibole during magma cooling are compatible with previous petrological constraints on the Coroccohuayco magmatic suite. This confirms that the gabbrodioritic magma was initially fluid undersaturated upon emplacement, and that magmatic fluid exsolution of the gabbrodiorite and the pluton rooting the porphyry stocks and dikes were emplaced and degassed at 100–200 MPa. Finally, mass balance constraints on S, Cu and Cl were used to estimate the minimum volume of magma required to form the Coroccohuayco deposit. These three estimates are remarkably consistent among each other (ca. 100 km3) and suggest that the Cl melt concentration is at least as critical as that of Cu and S to form an economic mineralization

PET/CT Imaging of c-Myc Transgenic Mice Identifies the Genotoxic N-Nitroso-Diethylamine as Carcinogen in a Short-Term Cancer Bioassay

Background: More than 100,000 chemicals are in use but have not been tested for their safety. To overcome limitations in the cancer bioassay several alternative testing strategies are explored. The inability to monitor non-invasively onset and progression of disease limits, however, the value of current testing strategies. Here, we report the application of in vivo imaging to a c-Myc transgenic mouse model of liver cancer for the development of a short-term cancer bioassay. Methodology/Principal Findings: mCT and 18 F-FDG mPET were used to detect and quantify tumor lesions after treatment with the genotoxic carcinogen NDEA, the tumor promoting agent BHT or the hepatotoxin paracetamol. Tumor growth was investigated between the ages of 4 to 8.5 months and contrast-enhanced mCT imaging detected liver lesions as well as metastatic spread with high sensitivity and accuracy as confirmed by histopathology. Significant differences in the onset of tumor growth, tumor load and glucose metabolism were observed when the NDEA treatment group was compared with any of the other treatment groups. NDEA treatment of c-Myc transgenic mice significantly accelerated tumor growth and caused metastatic spread of HCC in to lung but this treatment also induced primary lung cancer growth. In contrast, BHT and paracetamol did not promote hepatocarcinogenesis. Conclusions/Significance: The present study evidences the accuracy of in vivo imaging in defining tumor growth, tumor load, lesion number and metastatic spread. Consequently, the application of in vivo imaging techniques to transgeni

Human Glycolipid Transfer Protein (GLTP) Expression Modulates Cell Shape

Glycolipid transfer protein (GLTP) accelerates glycosphingolipid (GSL) intermembrane transfer via a unique lipid transfer/binding fold (GLTP-fold) that defines the GLTP superfamily and is the prototype for GLTP-like domains in larger proteins, i.e. phosphoinositol 4-phosphate adaptor protein-2 (FAPP2). Although GLTP-folds are known to play roles in the nonvesicular intracellular trafficking of glycolipids, their ability to alter cell phenotype remains unexplored. In the present study, overexpression of human glycolipid transfer protein (GLTP) was found to dramatically alter cell phenotype, with cells becoming round between 24 and 48 h after transfection. By 48 h post transfection, ∼70% conversion to the markedly round shape was evident in HeLa and HEK-293 cells, but not in A549 cells. In contrast, overexpression of W96A-GLTP, a liganding-site point mutant with abrogated ability to transfer glycolipid, did not alter cell shape. The round adherent cells exhibited diminished motility in wound healing assays and an inability to endocytose cholera toxin but remained viable and showed little increase in apoptosis as assessed by poly(ADP-ribose) polymerase cleavage. A round cell phenotype also was induced by overexpression of FAPP2, which binds/transfers glycolipid via its C-terminal GLTP-like fold, but not by a plant GLTP ortholog (ACD11), which is incapable of glycolipid binding/transfer. Screening for human protein partners of GLTP by yeast two hybrid screening and by immuno-pulldown analyses revealed regulation of the GLTP-induced cell rounding response by interaction with δ-catenin. Remarkably, while δ-catenin overexpression alone induced dendritic outgrowths, coexpression of GLTP along with δ-catenin accelerated transition to the rounded phenotype. The findings represent the first known phenotypic changes triggered by GLTP overexpression and regulated by direct interaction with a p120-catenin protein family member

Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices

Overexposure to apoptosis via disrupted glial specification perturbs Drosophila macrophage function and reveals roles of the CNS during injury

Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. Here, we seek to understand how apoptotic cells affect macrophage function in the context of a genetically tractable Drosophila model in which macrophages encounter excessive amounts of apoptotic cells. Loss of the glial-specific transcription factor Repo prevents glia from contributing to apoptotic cell clearance in the developing embryo. We show that this leads to the challenge of macrophages with large numbers of apoptotic cells in vivo. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells, and their developmental dispersal and migration is perturbed. We also show that the requirement to deal with excess apoptosis caused by a loss of repo function leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within a repo mutant background. In investigating the underlying cause of these impaired inflammatory responses, we demonstrate that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together, these results demonstrate important insights into macrophage biology and how repo mutants can be used to study macrophage–apoptotic cell interactions in the fly embryo. Furthermore, this work shows how these multipurpose cells can be ‘overtasked’ to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo

Hair Cell Bundles: Flexoelectric Motors of the Inner Ear

Microvilli (stereocilia) projecting from the apex of hair cells in the inner ear are actively motile structures that feed energy into the vibration of the inner ear and enhance sensitivity to sound. The biophysical mechanism underlying the hair bundle motor is unknown. In this study, we examined a membrane flexoelectric origin for active movements in stereocilia and conclude that it is likely to be an important contributor to mechanical power output by hair bundles. We formulated a realistic biophysical model of stereocilia incorporating stereocilia dimensions, the known flexoelectric coefficient of lipid membranes, mechanical compliance, and fluid drag. Electrical power enters the stereocilia through displacement sensitive ion channels and, due to the small diameter of stereocilia, is converted to useful mechanical power output by flexoelectricity. This motor augments molecular motors associated with the mechanosensitive apparatus itself that have been described previously. The model reveals stereocilia to be highly efficient and fast flexoelectric motors that capture the energy in the extracellular electro-chemical potential of the inner ear to generate mechanical power output. The power analysis provides an explanation for the correlation between stereocilia height and the tonotopic organization of hearing organs. Further, results suggest that flexoelectricity may be essential to the exquisite sensitivity and frequency selectivity of non-mammalian hearing organs at high auditory frequencies, and may contribute to the “cochlear amplifier” in mammals

A method for the isolation of human gastric mucous epithelial cells for primary cell culture: A comparison of biopsy vs surgical tissue

We have developed a method for the isolation and growth of normal human gastric mucous epithelial cells using biopsies or surgically resected tissues as the source of the cells. The attachment and growth of cells were dependent upon: (1) cell planting density, ∼50,000 cells/cm 2 ; (2) extracellular matrix (fibronectin); and (3) and the use of a porous filter. In all experiments we found better cells attachment and growth of human gastric mucous cells isolated from surgical specimens compared with those gastric mucous cells isolated from gastric biopsies. The initial cell viability (as measured by Trypan-blue) was the same in both populations of gastric mucous epithelial cells isolated from either gastric biopsies or surgical specimens. After 4–5 days in culture one could detect various amounts of mucin in all the cells using either periodic acid Schiff (PAS) staining or a specific anti-mucin antibody. A similar pattern of much straining was also found in primary cultures of guinea pig gastric mucous epithelial cells. Immunohistochemical staining for chief cells (anti-pepsinogen) or parietal cells (anti-H + /K + ATPasc) in the gastric mucous cuboidal-like epithelial cells with tight junctions, desmosomes,short microvilli, a filamentous terminal web, mucous granules, and basal lamina-like structure. We could not detect the presence of fibroblasts during the 7–9 days that the primary cells were in culture. This cell culture method will prove useful in the isolation of normal human gastric mucous epithelial cells for in vitro studies of gastric mucosal injury and repair.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43235/1/11022_2004_Article_BF00127904.pd