Thirty years development of ¹³C metabolic flux analysis: a review / 生物工程学报

¹³C metabolic flux analysis (¹³C-MFA) enables the precise quantification of intracellular metabolic reaction rates by analyzing the distribution of mass isotopomers of proteinogenic amino acids or intracellular metabolites through ¹³C labeling experiments. ¹³C-MFA has received much attention as it can help systematically understand cellular metabolic characteristics, guide metabolic engineering design and gain mechanistic insights into pathophysiology. This article reviews the advances of ¹³C-MFA in the past 30 years and discusses its potential and future perspective, with a focus on its application in industrial biotechnology and biomedicine.

The acute coronary syndrome revisited: effects and therapeutic modulation of excess metabolic fuel supply

Our proposal is that that metabolic perturbations occurring during and after the onset of the acute coronary syndrome (ACS) require careful management from the moment patients with this diagnosis are admitted to the intensive care unit (ICU). We advocate that insulin treatment should be initiated when blood glucose levels rise to above 11 mM (or 200 mg/dl), thereby providing additional therapeutic benefits. The reasons are as follows

Visual FTBL Generator: Visual Generation Tool for 13C-FLUX File

Abstract 13C metabolic flux analysis (13C-MFA) has achieved increasing significance in quantitative metabolic system analysis in recent years. In 13C metabolic flux analysis, 13C-FLUX software is a major analytical tool. The software's input script is primarily expressed in textual form without visual presentation of the structure of the entire metabolic network, thus error-prone in manual input. To solve this problem, we have developed a visual FTBL generator (VFG, available at http://47.100.98.220/vfg/index.jsp in a Google or Firefox browser)for MFA that eliminates the tedious, error-prone text entry mode and provides a user-friendly graphical interface and simple visual reaction generation functions.

Nitrogen and phosphorus flux from the production of Nile tilapia through the application of environmental indicators / Fluxo de nitrogênio e fósforo na produção de tilápia-do-nilo (Oreochromis niloticus) a partir da aplicação de indicadores ambientais

Abstract We aimed in this study utilize environmental indicators as a quantitative method to evaluate and discuss the nitrogen (TN) and phosphorus (TP) flux by a production stage grow-out (termination) of Nile tilapia (Oreochromis niloticus) in fishpond. The TN and TP load, the mass balance, the input of TN and TP via feed and the converted nutrients in fish biomass are the environmental indicators applied in this study. During the production cycle (128 days), the system exported 15,931 g TN and 4,189 g TP that were related to the amount of feed supplied (r Pearson = 0.8825 and r = 0.8523, respectively), corroborated by the feed conversion ratio (1.61:1). The indicators showed that 26% TN and 45% TP were reversed into fish biomass, 62% TN and 40% TP were retained in the fishpond, and 12% TN and 15% TP were exported via effluent. The largest contribution of nutrients generated by the system and exported via effluent was observed in phase III and IV. This result is supported by the feed conversion ratio 2.14 and 2.21:1 obtained at this phase, a fact explained by the amount of feed offered and the fish metabolism. Application of environmental indicators showed to be an efficient tool to quantify flux of TN and TP produced during the grow-out period of Nile tilapia and therefore, guide management practices more sustainable. Concerning the environmental sustainability of the activity the implementation of best management practices such as the better control of the feed amount offered would lead to a smaller loss of TN and TP to the water. Furthermore, the use of better quality feeds would allow greater nutrient assimilation efficiency.

Resumo Nós objetivamos neste estudo, utilizar indicadores ambientais como método quantitativo para avaliar e discutir sobre o fluxo de nitrogênio (TN) e fósforo (TP) na etapa final de crescimento (terminação) de tilápia-do-nilo (Oreochromis niloticus) em viveiro escavado. A carga de TN e TP, o balanço de massa, a entrada de nutrientes via ração e o TN e TP convertido em biomassa de peixe foram os indicadores ambientais utilizados neste estudo. Durante o ciclo produtivo (128 dias), o sistema exportou 15.931 g NT e 4.189 g PT os quais foram relacionadas às quantidades de alimento fornecido (r Pearson = 0,8825 e r = 0,8523, respectivamente), corroborada pela conversão alimentar (1,61:1). Os indicadores evidenciaram que 26% NT e 45% PT foram revertidos em biomassa de peixe, 62% NT e 40% PT ficaram retidos no viveiro e 12% NT e 15% PT foram exportados via efluente. O maior aporte de nutrientes gerado pelo sistema e exportado via efluente foi verificado nas fases III e IV. Este resultado é corroborado pelas taxas de conversão alimentar de 2,14 e 2,21:1 obtida nestas fases, fato explicado pela quantidade de ração ofertada e pelo metabolismo dos peixes. A aplicação dos indicadores ambientais mostrou ser uma ferramenta eficiente para quantificar o fluxo de TN e TP produzidos durante a etapa final de crescimento de tilápia-do-nilo e com isso orientar práticas de manejo mais sustentáveis. Com vistas à sustentabilidade ambiental da atividade, a implantação de boas práticas de manejo tais como o melhor controle da quantidade de alimento ofertado levaria a menor perda de NT e PT para a água. Além disso, o uso de rações de melhor qualidade permitiria maior eficiência de assimilação desses nutrientes.

Caracterização do secretoma de cepas de Xylella fastidiosa / Characterization of the secretome Xylella fastidiosa strains

As doenças causadas pelo fitopatógeno Xylella fastidiosa, uma bactéria Gram-negativa, devem-se aos seus múltiplos fatores de virulência, tais como formação de biofilme, secreção de enzimas de degradação da parede celular do xilema (CWDE), expressão de proteínas de adesão e produção de vesículas de membrana externa (OMVs). Esses fatores de virulência são controlados por uma via de sinalização mediada por DSF (fatores de sinalização difusíveis de natureza lipídica) e relacionada com percepção de quórum. Nesse trabalho, tivemos como objetivo ampliar a caracterização do secretoma de cepas selvagens e mutantes de X. fastidiosa para evidenciar proteínas e metabólitos potencialmente associados à adaptação ao hospedeiro, virulência e patogenicidade. Desenvolvemos, paralelamente, três estudos empregando como abordagens metodológicas a proteômica, a metabolômica e a transcritômica. No primeiro estudo, comparamos o secretoma (exoproteoma) da cepa Temecula1 selvagem (WT) e do mutante no gene da sintase de DSF (ΔrpfF), o qual exibe fenótipo de hipervirulência em videiras. A este estudo associamos a comparação dos transcritomas dessas cepas. Os resultados mostraram que, mesmo no cultivo in vitro, X. fastidiosa expressa e secreta fatores de virulência previamente conhecidos (lipases-esterases e proteases), além de toxinas (microcinas) que, supostamente, teriam papel de controlar bactérias competidoras pelo mesmo nicho. No segundo estudo caracterizamos a composição de OMVs secretadas no cultivo in vitro por X. fastidiosa Fb7 e 9a5c (cepas isoladas de laranjeiras) e Temecula1 (cepa isolada de videira). Demonstramos que Fb7 produz até 57% mais OMVs que 9a5c e Temecula1 e identificamos um total de 202 proteínas distintas nas OMVs produzidas pelas 3 cepas, ampliando consideravelmente o número de proteínas secretadas por meio de OMVs descrito, até então, para X. fastidiosa. Entre as proteínas enriquecidas, citamos adesinas afimbriais, porinas, lipoproteínas, hidrolases (lipases/esterases, proteases e peptidases) e uma pectina-liase putativa. Destacamos a detecção da enzima L-ascorbato oxidase nas OMVs e sugerimos que esta enzima poderia atuar na depleção do ascorbato produzido pelo hospedeiro vegetal. Além disso, demonstramos, pela primeira vez, que OMVs de X. fastidiosa transportam ácidos graxos da família DSF, sugerindo um papel adicional para OMVs nesse fitopatógeno. Finalmente, no terceiro estudo verificamos alterações relevantes no perfil de metabólitos secretados por X. fastidiosa em resposta a sua interação com metabólitos secretados por Burkholderia phytofirmans, proposta como uma cepa para o biocontrole da doença de Pierce de videiras. Confirmamos que o sobrenadante de B. phytofirmans possui um composto de natureza apolar que induz a formação de biofilme em X. fastidiosa, contudo ainda não foi possível decifrar a natureza química deste composto

The diseases caused by the phytopathogen Xylella fastidiosa, a Gram-negative bacterium, are due to multiple virulence factors, such as biofilm formation, secretion of xylem cell wall degradation enzymes (CWDE), expression of adhesion proteins and production of outer membrane vesicles (OMVs). These virulence factors are controlled by a DSF (diffusible signaling factors of a lipidic nature) mediating signaling pathway and related to quorum sensing perception. In this work, we aimed to extend the characterization of the secretoma of wild type and mutants strains of X. fastidiosa to uncover proteins and metabolites potentially associated to host adaptation, virulence and pathogenicity. We developed three studies in parallel using proteomics, metabolomics and transcriptomics as methodological approaches. In the first study, we compared the secretome (exoproteome) of the wild type strain Temecula1 (WT) and of DSF synthase mutant (ΔrpfF) which exhibits hypervirulence phenotype in grapevines. We also compared the transcriptomes of these strains. Our results showed that, even in in vitro culture, X. fastidiosa expresses and secretes previously known virulence factors (lipasesesterases and proteases), as well as toxins (microcins) that might play a role in controlling competing bacteria in the same niche. In the second study, we characterized the composition of OMVs secreted by in vitro cultures of X. fastidiosa Fb7 and 9a5c (strains isolated from orange trees) and Temecula1 (strain isolated from grapevine). We have shown that Fb7 produces up to 57% more OMVs than the 9a5c and Temecula1. Moreover we identified a total of 202 distinct proteins in the OMVs produced by these three strains, increasing considerably the number of OMVs secreted proteins so far described for X. fastidiosa. Among the proteins enriched in OMVs, we point out afimbrial adhesins, porins, lipoproteins, hydrolases (lipases/esterases, proteases and peptidases) and a putative pectin-lyase. We highlight the detection of the enzyme L-ascorbate oxidase in the OMVs and we suggest that this enzyme could act in the depletion of ascorbate produced by the plant host. In addition, we have demonstrated, for the first time, that X. fastidiosa OMVs transport fatty acids from the DSF family, suggesting an additional role for OMVs in this phytopathogen. Finally, in the third study we verified relevant changes in the profile of metabolites secreted by X. fastidiosa in response to the interaction with metabolites secreted by Burkholderia phytofirmans that has been sugested as a biocontrol strain for Pierce's disease in grapevines. We confirm that the B. phytofirmans supernatant has a non-polar compound that induces biofilm formation in X. fastidiosa, but it has not yet been possible to elucidate the chemical nature of this compound

Effects of high flux versus low flux on serum c-reactive protein a as an inflammatory biomarker in hemodialysis patients

Background: Traditional low-flux dialysis cannot improve micro-inflammatory status, while new high-flux dialysis can improve the micro-inflammation and lipid metabolism, it helps to improve the quality of life and survival rate of patients, so how to improve the micro-inflammatory status are a focus for researchers

Objective: was to observe the effect of high flux hemodialysis [HFHD] with Gambro polyflux 170H dialyser and low flux hemodialysis [LFPD] with polyflux 17L dialyser on high-sensitivity C-reactive protein in patients with maintenance hemodialysis

Methods: 60 patients with maintenance hemodialysis were randomly divided into HFHD group and LFHD group. Another 20 cases for physical examinations served as normal control group. The maintenance hemodialysis patients were treated with HFHD using 170H dialyser dliahyser and LFHD using 17L dialyser, three times per week, 4 hours once. After 6 months of the treatment, high-sensitive C-reactive protein was determined in patients as well as normal controls before and after treatment

Results and Conclusion: in two groups, the levels of high-sensitive C-reactive protein before the treatment were higher than normal control [P< 0.001]

In HFHD group, serum high-sensitive C-reactive protein markedly decreased [P <0.01]. In LFHD group, these indices remained unchanged after the dialysis for 6 months. HFHD with 170H polysulfone dialyser is effective in improving micro-inflammation in maintained hemodialysis patients

Topological analysis of carbon flux during multi-stress adaptation in Halomonas sp. AAD12

Background Osmolytes with their effective stabilizing properties are accumulated as protectants not only against salinity but also against denaturing harsh environmental stresses such as freezing, drying, high temperatures, oxygen radicals and radiation. The present work seeks to understand how Halomonas sp. AAD12 cells redirect carbon flux specifically to replenish reactions for biomass and osmolyte synthesis under changing salinity and temperature. To accomplish this goal, a combined FBA-PCA approach has been utilized. Results Experimental data were collected to supply model constraints for FBA and for the verification of the model predictions, which were satisfactory. With restrictions on the various combinations of selected anaplerotic paths (reactions catalyzed by phosphoenolpyruvate carboxylase, pyruvate carboxylase or glyoxylate shunt), two major phenotypes were found. Moreover, under high salt concentrations, when the glucose uptake rate was over 1.1 mmoL DCW- 1 h- 1, an overflow metabolism that led to the synthesis of ethanol caused a slight change in both phenotypes. Conclusions The operation of the glyoxylate shunt as the major anaplerotic pathway and the degradation of 6-phosphogluconate through the Entner-Doudoroff Pathway were the major factors in causing a distinction between the observed phenotypes.

Optimization and application of chemically defined medium for 13C metabolic flux analysis of Streptomyces rimosus M4018 / 生物工程学报

The aim of this study is to develop a synthetic medium suitable for 13C metabolic flux analysis (13C-MFA) of Streptomyces rimosus. The cell growth rate and oxytetracycline production by S. rimosus M4018 were compared when M4018 cells were growth on the optimized chemically defined media with organic nitrogen sources or inorganic nitrogen sources. First, a synthetic medium contained KNO3 as the main nitrogen source was screened, then optimized by a response surface method. Using this new medium, the oxytetracycline yield was increased from 75.2 to 145.6 mg/L. Furthermore, based on the 13C-MFA, we identified that Entner-Doudoroff pathway does not exist in S. rimosus cells cultured in a chemically defined medium with feed of 100% 1-13C labeled glucose. This study is helpful for subsequent 13C-MFA application of S. rimosus.