FiatFlux – a software for metabolic flux analysis from (13)C-glucose experiments

BACKGROUND: Quantitative knowledge of intracellular fluxes is important for a comprehensive characterization of metabolic networks and their functional operation. In contrast to direct assessment of metabolite concentrations, in vivo metabolite fluxes must be inferred indirectly from measurable quantities in (13)C experiments. The required experience, the complicated network models, large and heterogeneous data sets, and the time-consuming set-up of highly controlled experimental conditions largely restricted metabolic flux analysis to few expert groups. A conceptual simplification of flux analysis is the analytical determination of metabolic flux ratios exclusively from MS data, which can then be used in a second step to estimate absolute in vivo fluxes. RESULTS: Here we describe the user-friendly software package FiatFlux that supports flux analysis for non-expert users. In the first module, ratios of converging fluxes are automatically calculated from GC-MS-detected (13)C-pattern in protein-bound amino acids. Predefined fragmentation patterns are automatically identified and appropriate statistical data treatment is based on the comparison of redundant information in the MS spectra. In the second module, absolute intracellular fluxes may be calculated by a (13)C-constrained flux balancing procedure that combines experimentally determined fluxes in and out of the cell and the above flux ratios. The software is preconfigured to derive flux ratios and absolute in vivo fluxes from [1-(13)C] and [U-(13)C]glucose experiments and GC-MS analysis of amino acids for a variety of microorganisms. CONCLUSION: FiatFlux is an intuitive tool for quantitative investigations of intracellular metabolism by users that are not familiar with numerical methods or isotopic tracer experiments. The aim of this open source software is to enable non-specialists to adapt the software to their specific scientific interests, including other (13)C-substrates, labeling mixtures, and organisms

Formação continuada em rede online de professores da educação básica

Ao tratarmos de formação de professores, estamos nos referindo aos processos que contribuem para a constituição de competências do profissional que vai atuar na Educação Básica, compreendendo-a em toda sua complexidade, dados os desafios da contemporaneidade. Se nos voltarmos para a Universidade, enquanto instituição responsável pela formação inicial dos professores, temos que questionar como é que os cursos de licenciatura estão dando conta dessa tarefa. Todas essas questões, entre outras, vão constituindo a identidade profissional dos professores na contemporaneidade e pensar sua formação, portanto, significa pensá-la para além da formação inicial. Este artigo tratará de um projeto realizado com os professores da Rede Jesuíta de Educação no Brasil chamado Programa Loyola que vem proporcionando momentos de formação docente. A formação docente procura, através da modalidade a distância, uma nova forma de se apropriar das bases educativas jesuítas, tendo em vista o apostolado da educação. Com isso, a cibercultura torna-se um meio de capacitação docente nesse contexto, porque proporciona a criação de um espaço comum, onde, com o apoio das Tecnologias Digitais Virtuais - TDVs, é possível a interação entre docentes. O método científico utilizado na pesquisa foi uma abordagem qualitativa, configurando-se de forma exploratória e utilizando os procedimentos técnicos do estudo de caso e classificada como uma pesquisa bibliográfica e documental. Um dos objetivos da pesquisa foi como estimular a aprendizagem online, em rede, de forma que os professores, mesmo estando dispersos geograficamente, sintam-se pertencentes a um mesmo grupo que compartilha pressupostos comuns. Além deste, procurou verificar como propiciar o compartilhamento de experiências e a aprendizagem online, em rede. A aprendizagem em rede se deu de forma desafiadora, mas com um resultado satisfatório. O que moveu o grupo desde o início foi a curiosidade em aprender sobre uma nova tecnologia para trabalhar com os alunos.In addressing teacher training, we are referring to the processes that contribute to the formation of the professional skills that will operate in Basic Education, understanding it in all its complexity, given the challenges of contemporary. If we turn to the University as an institution responsible for the initial training of teachers, we should question how the degree courses are realizing this task. All these questions, among others, will constitute the professional identity of teachers in contemporary thinking and their training, therefore, means to think it beyond the initial training . This article will deal with a project conducted with the teachers of the Jesuit Schools in Brazil called Loyola Program that has provided moments of teacher training. Teacher training demand through the distance mode, a new way of appropriating the bases Jesuit education, considering the apostolate of education. Thus, cyberculture becomes a means of teacher training in this context, because it provides the creation of a common space where, with the support of Digital Virtual Technologies, it is possible the interaction between teachers. The scientific method used in the research approach was qualitative, setting up an exploratory manner and using the technical procedures of the case study and classified as a documentary and bibliographical research. One goal of the research was to stimulate learning online network, so that teachers, even though geographically dispersed, feel belonging to a group that shares common assumptions. Besides this, tried to verify facilitate the sharing of experiences and learning online network. Network learning occurred in a challenging way, but with a satisfactory result. What moved the group from the beginning was the curiosity to learn about a new technology to work with students.Universidade Aberta; Pavilhão do Conhecimento; LEA

Delivery of BMP-2 by two clinically available apatite materials: In vitro and in vivo comparison

Bone morphogenetic proteins (BMPs) are deposited in bone and responsible for osteoinduction. The interplay between delivery system and BMP, resulting in a characteristic release profile, is crucial for clinical success. We here report on two apatite based commercially available granules which could potentially be used in a combination product with recombinant human BMP-2 (rhBMP-2). Regardless of their similar chemistry, their interaction with rhBMP-2 differs. Deproteinized bovine bone matrix (DBBM), a clinically well-established bone substitute, has a high affinity to rhBMP-2 and releases only 50% of the growth factor during the first 2 weeks in vitro. Activity of the physio-adsorbed rhBMP-2 is indicated by an enhanced bone augmentation in vivo. In contrast, all rhBMP-2 delivered in combination with synthetic hydroxyapatite/β-tricalcium phosphate (HA/TCP) granules is released during the first 24 h. For both HA/TCP and DBBM, the released rhBMP-2 is active in vitro. Our results suggest that the different release behavior from these two apatite granules is due to the 1000-fold higher specific surface area of DBBM compared to HA/TCP

Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model

Purpose: The L1 cell adhesion molecule (L1CAM) is considered a valuable target for therapeutic intervention in different types of cancer. Recent studies have shown that anti-L1CAM radioimmunotherapy (RIT) with 67Cu- and 177Lu-labelled internalising monoclonal antibody (mAb) chCE7 was effective in the treatment of human ovarian cancer xenografts. In this study, we directly compared the therapeutic efficacy of anti-L1CAM RIT against human ovarian cancer under equitoxic conditions with the radiolanthanide 177Lu and the potential alternative 161Tb in an ovarian cancer therapy model. Methods: Tb was produced by neutron bombardment of enriched 160Gd targets. 161Tb and 177Lu were used for radiolabelling of DOTA-conjugated antibodies. The in vivo behaviour of the radioimmunoconjugates (RICs) was assessed in IGROV1 tumour-bearing nude mice using biodistribution experiments and SPECT/CT imaging. After ascertaining the maximal tolerated doses (MTD) the therapeutic impact of 50% MTD of 177Lu- and 161Tb-DOTA-chCE7 was evaluated in groups of ten mice by monitoring the tumour size of subcutaneous IGROV1 tumours. Results: The average number of DOTA ligands per antibody was 2.5 and maximum specific activities of 600MBq/mg were achieved under identical radiolabelling conditions. RICs were stable in human plasma for at least 48h. 177Lu- and 161Tb-DOTA-chCE7 showed high tumour uptake (37.8-39.0 %IA/g, 144h p.i.) with low levels in off-target organs. SPECT/CT images confirmed the biodistribution data. 161Tb-labelled chCE7 revealed a higher radiotoxicity in nude mice (MTD: 10MBq) than the 177Lu-labelled counterpart (MTD: 12MBq). In a comparative therapy study with equitoxic doses, tumour growth inhibition was better by 82.6% for the 161Tb-DOTA-chCE7 than the 177Lu-DOTA-chCE7 RIT. Conclusions: Our study is the first to show that anti-L1CAM 161Tb RIT is more effective compared to 177Lu RIT in ovarian cancer xenografts. These results suggest that 161Tb is a promising candidate for future clinical applications in combination with internalising antibodies

Comparison of Recombinant Human Haptocorrin Expressed in Human Embryonic Kidney Cells and Native Haptocorrin

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies

Bordetella pertussis Infection Exacerbates Influenza Virus Infection through Pertussis Toxin-Mediated Suppression of Innate Immunity

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers