Exhaled volatilome analysis as a useful tool to discriminate asthma with other coexisting atopic diseases in women of childbearing age

©2021. The authors. This document is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by /4.0/ This document is published version of a Published Work that appeared in final form in Scientifc Reports. To access the final edited and published work see https://doi.org/10.1038/s41598-021-92933-2The prevalence of asthma is considerably high among women of childbearing age. Most asthmatic women also often have other atopic disorders. Therefore, the diferentiation between patients with atopic diseases without asthma and asthmatics with coexisting diseases is essential to avoid underdiagnosis of asthma and to design strategies to reduce symptom severity and improve quality of life of patients. Hence, we aimed for the frst time to conduct an analysis of volatile organic compounds in exhaled breath of women of childbearing age as a new approach to discriminate between asthmatics with other coexisting atopic diseases and non-asthmatics (with or without atopic diseases), which could be a helpful tool for more accurate asthma detection and monitoring using a noninvasive technique in the near future. In this study, exhaled air samples of 336 women (training set (n= 211) and validation set (n= 125)) were collected and analyzed by thermal desorption coupled with gas chromatography-mass spectrometry. ASCA (ANOVA (analysis of variance) simultaneous component analysis) and LASSO+LS (least absolute shrinkage and selection operator+ logistic regression) were employed for data analysis. Fifteen statistically signifcant models (p-value< 0.05 in permutation tests) that discriminated asthma with other coexisting atopic diseases in women of childbearing age were generated. Acetone, 2-ethyl-1-hexanol and a tetrahydroisoquinoline derivative were selected as discriminants of asthma with other coexisting atopic diseases. In addition, carbon disulfde, a tetrahydroisoquinoline derivative, 2-ethyl-1-hexanol and decane discriminated asthma disease among patients with other atopic disorders. Results of this study indicate that refned metabolomic analysis of exhaled breath allows asthma with other coexisting atopic diseases discrimination in women of reproductive ag

Continuous intracellular pH measurement: Escherichia coli culture medium pH dependence

E. coli is the most used host microorganism to high-valuable compounds production in current industry. Cells pH homeostasis is a crucial factor to grow in different environment maintaining an intracellular pH control, however, to our knowledge, there is not a study about E. coli continuous intracellular pH growing in a culture. In this study the intracellular E. coli pH has been determined for cultures growing with glucose or glycerol as carbon sources. To determine intracellular E. coli pH, a novel, simple and accurate method based on pGFPR01 plasmid (kindly provided by Dr. Joan Slonczewski) was employed (1). This vector expressed ratiometric pHluorin. Thus, fluorescence ratio 410/470 showed a perfect lineal dependence with intracellular pH between 5.5 and 9. The results showed a surprising high intracellular pH for E. coli glucose cultures, which raised values of 8.5-9 at exponential growth phase. However, glycerol cultures showed a major regulation of intracellular pH, keeping the values at 6.5-7.5 in exponential and stationary stages. These differences show the dependence between the intracellular pH and the carbon source. In addition, the measurement method developed is a very useful tool for the control of bioprocesses

Engineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains.

©2019. The authors. This document is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by /4.0/ This document is the published version of a Published Work that appeared in final form in Microbial Cell Factories. To access the final edited and published work see https://doi.org/10.1186/s12934-019-1202-1Background: Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overfow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to fnd the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two diferent approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation. Results: We evaluated protein overexpression in E. coli BL21 wt and fve mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efcient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fvefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation. Conclusion: The results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fuxes to diferent pathways and afect the fnal yield of the recombinant protein bioprocess. Thereby, we obtained a fvefold increase in protein production and an efcient use of the resources employing the most suitable strain and culture conditions

Characterization of acetyl-CoA synthetase kinetics and ATP-binding

Background The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. Methods In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. Results The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. Conclusions The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General Significance The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation

Bacterial sirtuins overview: an open niche to explore

Sirtuins are deacetylase enzymes widely distributed in all domains of life. Although for decades they have been related only to histones deacetylation in eukaryotic organisms, today they are considered global regulators in both prokaryotes and eukaryotes. Despite the important role of sirtuins in humans, the knowledge about bacterial sirtuins is still limited. Several proteomics studies have shown that bacterial sirtuins deacetylate a large number of lysines in vivo, although the effect that this deacetylation causes in most of them remains unknown. To date, only the regulation of a few bacterial sirtuin substrates has been characterized, being their metabolic roles widely distributed: carbon and nitrogen metabolism, DNA transcription, protein translation, or virulence. One of the most current topics on acetylation and deacetylation focuses on studying stoichiometry using quantitative LC-MS/MS. The results suggest that prokaryotic sirtuins deacetylate at low stoichiometry sites, although more studies are needed to know if it is a common characteristic of bacterial sirtuins and its biological significance. Unlike eukaryotic organisms, bacteria usually have one or few sirtuins, which have been reported to have closer common ancestors with the human Sirt5 than with any other class. In this work, in addition to carrying out an in-depth review of the role of bacterial sirtuins in their physiology, a phylogenetic study has been performed that reveals the evolutionary differences between sirtuins of different bacterial species and even between homologous sirtuins

Characterization of CobB kinetics and inhibition by nicotinamide

<div><p>Lysine acetylation has emerged as a global protein regulation system in all domains of life. Sirtuins, or Sir2-like enzymes, are a family of histone deacetylases characterized by their employing NAD⁺ as a co-substrate. Sirtuins can deacetylate several acetylated proteins, but a consensus substrate recognition sequence has not yet been established. Product inhibition of many eukaryotic sirtuins by nicotinamide and its analogues has been studied <i>in vitro</i> due to their potential role as anticancer agents. In this work, the kinetics of CobB, the main <i>Escherichia coli</i> deacetylase, have been characterized. To our knowledge, this is the first kinetic characterization of a sirtuin employing a fully acetylated and natively folded protein as a substrate. CobB deacetylated several acetyl-CoA synthetase acetylated lysines with a single kinetic rate. In addition, <i>in vitro</i> nicotinamide inhibition of CobB has been characterized, and the intracellular nicotinamide concentrations have been determined under different growth conditions. The results suggest that nicotinamide can act as a CobB regulator <i>in vivo</i>. A nicotinamidase deletion strain was thus phenotypically characterized, and it behaved similarly to the Δ<i>cobB</i> strain. The results of this work demonstrate the potential regulatory role of the nicotinamide metabolite <i>in vivo</i>.</p></div

Acetate overflow.

<p>Extracellular acetate concentrations (mM) per OD (600 nm) of <i>E</i>. <i>coli</i> wt (black line), Δ<i>cobB</i> (green line) and Δ<i>pncA</i> (blue line) strains grown in minimal medium with 20 mM glucose. <i>E</i>. <i>coli</i> wt growth curve is shown as a discontinuous red line.</p