MultiMetEval: comparative and multi-objective analysis of genome-scale metabolic models

Comparative metabolic modelling is emerging as a novel field, supported by the development of reliable and standardized approaches for constructing genome-scale metabolic models in high throughput. New software solutions are needed to allow efficient comparative analysis of multiple models in the context of multiple cellular objectives. Here, we present the user-friendly software framework Multi-Metabolic Evaluator (MultiMetEval), built upon SurreyFBA, which allows the user to compose collections of metabolic models that together can be subjected to flux balance analysis. Additionally, MultiMetEval implements functionalities for multi-objective analysis by calculating the Pareto front between two cellular objectives. Using a previously generated dataset of 38 actinobacterial genome-scale metabolic models, we show how these approaches can lead to exciting novel insights. Firstly, after incorporating several pathways for the biosynthesis of natural products into each of these models, comparative flux balance analysis predicted that species like Streptomyces that harbour the highest diversity of secondary metabolite biosynthetic gene clusters in their genomes do not necessarily have the metabolic network topology most suitable for compound overproduction. Secondly, multi-objective analysis of biomass production and natural product biosynthesis in these actinobacteria shows that the well-studied occurrence of discrete metabolic switches during the change of cellular objectives is inherent to their metabolic network architecture. Comparative and multi-objective modelling can lead to insights that could not be obtained by normal flux balance analyses. MultiMetEval provides a powerful platform that makes these analyses straightforward for biologists. Sources and binaries of MultiMetEval are freely available from https://github.com/PiotrZakrzewski/MetEv​al/downloads

Qualitatively and quantitatively similar effects of active and passive maternal tobacco smoke exposure on in utero mutagenesis at the HPRT locus

BACKGROUND: Induced mutagenesis in utero is likely to have life-long repercussions for the exposed fetus, affecting survival, birth weight and susceptibility to both childhood and adult-onset diseases, such as cancer. In the general population, such exposures are likely to be a consequence of the lifestyle choices of the parents, with exposure to tobacco smoke one of the most pervasive and easily documented. Previous studies attempting to establish a direct link between active smoking and levels of somatic mutation have largely discounted the effects of passive or secondary exposure, and have produced contradictory results. METHODS: Data from three studies of possible smoking effects on in utero mutagenesis at the HPRT locus were compiled and reanalyzed, alone and in combination. Where possible, passive exposure to environmental tobacco smoke was considered as a separate category of exposure, rather than being included in the non-smoking controls. Molecular spectra from these studies were reanalyzed after adjustment for reported mutation frequencies from the individual studies and the entire data set. RESULTS: A series of related studies on mutation at the X-linked HPRT locus in human newborn cord blood samples has led to the novel conclusion that only passive maternal exposure to tobacco mutagens has a significant effect on the developing baby. We performed a pooled analysis of the complete data from these studies, at the levels of both induced mutation frequency and the resulting mutational spectrum. CONCLUSION: Our analysis reveals a more commonsensical, yet no less cautionary result: both active maternal smoking and secondary maternal exposure produce quantitatively and qualitatively indistinguishable increases in fetal HPRT mutation. Further, it appears that this effect is not perceptibly ameliorated if the mother adjusts her behavior (i.e. stops smoking) when pregnancy is confirmed, although this conclusion may also be affected by continued passive exposure

Synthesis and characterisation of alpha-carboxynitrobenzyl photocaged l-aspartates for applications in time-resolved structural biology

We report a new synthetic route to a series of α-carboxynitrobenzyl photocaged ⌊-aspartates for application in time-resolved structural biology. The resulting compounds were characterised in terms of UV/Vis absorption properties, aqueous solubility and stability, and photocleavage rates (τ = μs to ms) and quantum yields (φ = 0.05 to 0.14)

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing polyketides

Non-Invasive Detection of a Small Number of Bioluminescent Cancer Cells In Vivo

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice

Assessment of a continuous blood gas monitoring system in animals during circulatory stress

<p>Abstract</p> <p>Background</p> <p>The study was aimed to determine the measurement accuracy of The CDI™ blood parameter monitoring system 500 (Terumo Cardiovascular Systems Corporation, Ann Arbor MI) in the real-time continuous measurement of arterial blood gases under different cardiocirculatory stress conditions</p> <p>Methods</p> <p>Inotropic stimulation (Dobutamine 2.5 and 5 μg/kg/min), vasoconstriction (Arginine-vasopressin 4, 8 and 16 IU/h), hemorrhage (-10%, -20%, -35%, and -50% of the theoretical volemia), and volume resuscitation were induced in ten swine (57.4 ± 10.7 Kg).Intermittent blood gas assessments were carried out using a routine gas analyzer at any experimental phase and compared with values obtained at the same time settings during continuous monitoring with CDI™ 500 system. The Bland-Altman analysis was employed.</p> <p>Results</p> <p>Bias and precision for pO₂were - 0.06 kPa and 0.22 kPa, respectively (r²= 0.96); pCO₂- 0.02 kPa and 0.15 kPa, respectively; pH -0.001 and 0.01 units, respectively ( r²= 0.96). The analysis showed very good agreement for SO₂(bias 0.04,precision 0.33, r²= 0.95), Base excess (bias 0.04,precision 0.28, r²= 0.98), HCO₃(bias 0.05,precision 0.62, r²= 0.92),hemoglobin (bias 0.02,precision 0.23, r²= 0.96) and K⁺(bias 0.02, precision 0.27, r²= 0.93). The sensor was reliable throughout the experiment during hemodynamic variations.</p> <p>Conclusions</p> <p>Continuous blood gas analysis with the CDI™ 500 system was reliable and it might represent a new useful tool to accurately and timely monitor gas exchange in critically ill patients. Nonetheless, our findings need to be confirmed by larger studies to prove its reliability in the clinical setting.</p

Characterization of an IncFII Plasmid Encoding NDM-1 from Escherichia coli ST131

Background: The current spread of the gene encoding the metallo-ß-lactamase NDM-1 in Enterobacteriaceae is linked to a variety of surrounding genetic structures and plasmid scaffolds. Methodology: The whole sequence of plasmid pGUE-NDM carrying the bla NDM-1 gene was determined by high-density pyrosequencing and a genomic comparative analysis with other blaNDM-1-negative IncFII was performed. Principal Findings: Plasmid pGUE-NDM replicating in Escherichia coli confers resistance to many antibiotic molecules including b-lactams, aminoglycosides, trimethoprim, and sulfonamides. It is 87,022 bp in-size and carries the two b-lactamase genes bla NDM-1 and bla OXA-1, together with three aminoglycoside resistance genes aacA4, aadA2, and aacC2. Comparative analysis of the multidrug resistance locus contained a module encompassing the blaNDM-1 gene that is actually conserved among different structures identified in other enterobacterial isolates. This module was constituted by the blaNDM-1 gene, a fragment of insertion sequence ISAba125 and a bleomycin resistance encoding gene. Significance: This is the first characterized bla NDM-1-carrying IncFII-type plasmid. Such association between the bla NDM-1 gene and an IncFII-type plasmid backbone is extremely worrisome considering that this plasmid type is known to sprea

A Bio-Catalytic Approach to Aliphatic Ketones

Depleting oil reserves and growing environmental concerns have necessitated the development of sustainable processes to fuels and chemicals. Here we have developed a general metabolic platform in E. coli to biosynthesize carboxylic acids. By engineering selectivity of 2-ketoacid decarboxylases and screening for promiscuous aldehyde dehydrogenases, synthetic pathways were constructed to produce both C5 and C6 acids. In particular, the production of isovaleric acid reached 32 g/L (0.22 g/g glucose yield), which is 58% of the theoretical yield. Furthermore, we have developed solid base catalysts to efficiently ketonize the bio-derived carboxylic acids such as isovaleric acid and isocaproic acid into high volume industrial ketones: methyl isobutyl ketone (MIBK, yield 84%), diisobutyl ketone (DIBK, yield 66%) and methyl isoamyl ketone (MIAK, yield 81%). This hybrid “Bio-Catalytic conversion” approach provides a general strategy to manufacture aliphatic ketones, and represents an alternate route to expanding the repertoire of renewable chemicals

Mammalian cell transfection: the present and the future

Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration