Identification of metabolic network models from incomplete high-throughput datasets

MOTIVATION: High-throughput measurement techniques for metabolism and gene expression provide a wealth of information for the identification of metabolic network models. Yet, missing observations scattered over the dataset restrict the number of effectively available datapoints and make classical regression techniques inaccurate or inapplicable. Thorough exploitation of the data by identification techniques that explicitly cope with missing observations is therefore of major importance. RESULTS: We develop a maximum-likelihood approach for the estimation of unknown parameters of metabolic network models that relies on the integration of statistical priors to compensate for the missing data. In the context of the linlog metabolic modeling framework, we implement the identification method by an Expectation-Maximization (EM) algorithm and by a simpler direct numerical optimization method. We evaluate performance of our methods by comparison to existing approaches, and show that our EM method provides the best results over a variety of simulated scenarios. We then apply the EM algorithm to a real problem, the identification of a model for the Escherichia coli central carbon metabolism, based on challenging experimental data from the literature. This leads to promising results and allows us to highlight critical identification issues.     

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2442 isolated from inclusion bodies

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni²⁺-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.     

Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30?years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.     

Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer's disease subjects

Phosphorylation on tyrosine, threonine and serine residues represents one of the most important post-translational modifications and is a key regulator of cellular signaling of multiple biological processes that require a strict control by protein kinases and protein phosphatases. Abnormal protein phosphorylation has been associated with several human diseases including Alzheimer's disease (AD). One of the characteristic hallmarks of AD is the presence of neurofibrillary tangles, composed of microtubule-associated, abnormally hyperphosphorylated tau protein. However, several others proteins showed altered phosphorylation levels in AD suggesting that deregulated phosphorylation may contribute to AD pathogenesis. Phosphoproteomics has recently gained attention as a valuable approach to analyze protein phosphorylation, both in a quantitative and a qualitative way. We used the fluorescent phosphospecific Pro-Q Diamond dye to identify proteins that showed alterations in their overall phosphorylation in the hippocampus of AD vs. control (CTR) subjects. Significant changes were found for 17 proteins involved in crucial neuronal process such as energy metabolism or signal transduction. These phosphoproteome data may provide new clues to better understand molecular pathways that are deregulated in the pathogenesis and progression of AD.     

Different factors affecting human ANP amyloid aggregation and their implications in congestive heart failure

Aims

Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis.

Methods and Results

A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric β-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing β-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA.

Conclusions

We established a link between IAA and CHF by ex vivo examination and assessed that β-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that β-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis.     

B-cell epitope prediction for peptide-based vaccine design: towards a paradigm of biological outcomes for global health

Global health must address a rapidly evolving burden of disease, hence the urgent need for versatile generic technologies exemplified by peptide-based vaccines. B-cell epitope prediction is crucial for designing such vaccines; yet this approach has thus far been largely unsuccessful, prompting further inquiry into the underlying reasons for its apparent inadequacy. Two major obstacles to the development of B-cell epitope prediction for peptide-based vaccine design are (1) the prevailing binary classification paradigm, which mandates the problematic dichotomization of continuous outcome variables, and (2) failure to explicitly model biological consequences of immunization that are relevant to practical considerations of safety and efficacy. The first obstacle is eliminated by redefining the predictive task as quantitative estimation of empirically observable biological effects of antibody-antigen binding, such that prediction is benchmarked using measures of correlation between continuous rather than dichotomous variables; but this alternative approach by itself fails to address the second obstacle even if benchmark data are selected to exclusively reflect functionally relevant cross-reactivity of antipeptide antibodies with protein antigens (as evidenced by antibody-modulated protein biological activity), particularly where only antibody-antigen binding is actually predicted as a surrogate for its biological effects. To overcome the second obstacle, the prerequisite is deliberate effort to predict, a priori, biological outcomes that are of immediate practical significance from the perspective of vaccination. This demands a much broader and deeper systems view of immunobiology than has hitherto been invoked for B-cell epitope prediction. Such a view would facilitate comprehension of many crucial yet largely neglected aspects of the vaccine-design problem. Of these, immunodominance among B-cell epitopes is a central unifying theme that subsumes immune phenomena of tolerance, imprinting and refocusing; but it is meaningful for vaccine design only in the light of disease-specific pathophysiology, which for infectious processes is complicated by host-pathogen coevolution. To better support peptide-based vaccine design, B-cell epitope prediction would entail individualized quantitative estimation of biological outcomes relevant to safety and efficacy. Passive-immunization experiments could serve as an important initial proving ground for B-cell epitope prediction en route to vaccine-design applications, by restricting biological complexity to render epitope-prediction problems more computationally tractable.      

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Changes in Cyclins and Cyclin-Dependent Kinases Induced by DNA Damaging Agents in a Human Ovarian Cancer Cell Line Expressing Mutated or Wild-Type P53

The expression of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors was evaluated in clones from a human ovarian cancer cell line transfected with a temperature-sensitive mutant of p53, after treatment with the anticancer agents doxorubicin (DX) and AMSA. The two drugs were selected on the basis of their activity in these clones, since AMSA is equally active in cells expressing mutated or wild-type (wt) p53, while DX was much less cytotoxic in cells expressing wt p53. In untreated cells, the expression of wt p53 induced an accumulation of cells in the G2 and perhaps also the G1 phase of the cell cycle. Concomitantly cyclin B1 and cdc2 increased. Cyclin E and particularly D1 levels were also raised by wt p53 expression. Treatment of mutated p53-expressing cells (SK23a cells kept at 37°C) with DX or, more so, with AMSA, resulted in a strong accumulation of cyclin B1 and cdc2, in accordance with their ability to block cells in G2 phase of the cell cycle. Wt p53-expressing cells (SK23a cells kept at 32°C) treated with the drugs showed an increase in p21 expression and consequently decreased kinase activity after immunoprecipitation with p21 antibodies. Cdc2-associated kinase activity was also reduced in these conditions. We could also observe a decrease in the percentage of cells in G1 and G2 phases and an accumulation of cells in S phase after both DX and AMSA. Cdk2, retinoblastoma, and p27 levels did not change significantly. Treatment with DX or AMSA caused similar effects, suggesting that p53-induced changes in cyclin, cdk, and cdk inhibitors after DNA damage are not responsible for the marked reduction in the cytotoxicity of DX we observed in wt p53-expressing cells.