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Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.

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Malolactic fermentation (MLF) positively influences the quality of the wine, and it occurs as a result of a lactic acid bacteria's metabolism, mainly of the Oenococcus oeni species. However, delays and halting of MLF are frequent problems in the wine industry. This is mainly because O. oeni's development is inhibited by different kinds of stress. Even though the sequencing of the genome of the PSU-1 strain of O. oeni, as well as other strains, has made it possible to identify genes involved in the resistance to some types of stress, all of the factors that could be involved are still unknown. With the aim of contributing to this knowledge, the random mutagenesis technique was used in this study as a strategy for genetic improvement of strains of the O. oeni species. The technique proved to be capable of generating a different and improved strain when compared to the PSU-1 strain (the parent from which it descends). Then, we evaluated the metabolic behavior of both strains in three different wines. We used synthetic MaxOeno wine (pH 3.5; 15% v/v ethanol), red wine (Cabernet Sauvignon), and white wine (Chardonnay). Furthermore, we compared the transcriptome of both strains, grown in MaxOeno synthetic wine. The specific growth rate of the E1 strain was on average 39% higher in comparison to the PSU-1 strain. Interestingly, E1 strain showed an overexpression of the OEOE_1794 gene, which encodes a UspA-like protein, which has been described as promoting growth. We observed that the E1 strain was able to convert, on average, 34% more malic acid into lactate than the PSU-1 strain, regardless of the wine being used. On the other hand, the E1 strain showed a flux rate of fructose-6-phosphate production that was 86% higher than the mannitol production rate, and the internal flux rates increase in the direction of pyruvate production. This coincides with the higher number of OEOE_1708 gene transcripts observed in the E1 strain grown in MaxOeno. This gene encodes for an enzyme fructokinase (EC 2.7.1.4) involved in the transformation of fructose to fructose-6-phosphate.

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The Atacama Desert is the most arid desert on Earth, focus of important research activities related to microbial biodiversity studies. In this context, metabolic characterization of arid soil bacteria is crucial to understand their survival strategies under extreme environmental stress. We investigated whether strain-specific features of two Microbacterium species were involved in the metabolic ability to tolerate/adapt to local variations within an extreme desert environment. Using an integrative systems biology approach we have carried out construction and comparison of genome-scale metabolic models (GEMs) of two Microbacterium sp., CGR1 and CGR2, previously isolated from physicochemically contrasting soil sites in the Atacama Desert. Despite CGR1 and CGR2 belong to different phylogenetic clades, metabolic pathways and attributes are highly conserved in both strains. However, comparison of the GEMs showed significant differences in the connectivity of specific metabolites related to pH tolerance and CO2 production. The latter is most likely required to handle acidic stress through decarboxylation reactions. We observed greater GEM connectivity within Microbacterium sp. CGR1 compared to CGR2, which is correlated with the capacity of CGR1 to tolerate a wider pH tolerance range. Both metabolic models predict the synthesis of pigment metabolites (ß-carotene), observation validated by HPLC experiments. Our study provides a valuable resource to further investigate global metabolic adaptations of bacterial species to grow in soils with different abiotic factors within an extreme environment.

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In information security, one way to keep a secret content is through encryption. The objective is to alter the content so that it is not intelligible, and therefore only the intended user can reveal the secret content. With the aim to provide examples of encrypted audio data, we applied a novel method of encryption based on the Collatz conjecture in five hundred speech recordings (50 speakers, 10 different messages), and then five hundred encrypted audio files were obtained. The main characteristics of our encrypted recordings are as follows: the spectrogram is quasi-uniform, histograms have a repetitive pattern, average of samples is around -0.4, standard deviation is around 0.55; Shannon entropy is around 7.5 (for 8-bits per sample). The novelty of the results consists in obtaining a completely different behavior than natural speech recordings, i.e.: spectrogram with higher energy in low frequencies, histogram with Gaussian behavior, average of samples around 0, standard deviation around 0.11, entropy around 5.5. A more comprehensive analysis of our encrypted signals may be obtained from the article "High-uncertainty audio signal encryption based on the Collatz conjecture" in the Journal of Information Security and Applications.

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The effect of ethanol on the metabolism of Oenococcus oeni, the bacterium responsible for the malolactic fermentation (MLF) of wine, is still scarcely understood. Here, we characterized the global metabolic response in O. oeni PSU-1 to increasing ethanol contents, ranging from 0 to 12% (v/v). We first optimized a wine-like, defined culture medium, MaxOeno, to allow sufficient bacterial growth to be able to quantitate different metabolites in batch cultures of O. oeni. Then, taking advantage of the recently reconstructed genome-scale metabolic model iSM454 for O. oeni PSU-1 and the resulting experimental data, we determined the redistribution of intracellular metabolic fluxes, under the different ethanol conditions. Four growth phases were clearly identified during the batch cultivation of O. oeni PSU-1 strain, according to the temporal consumption of malic and citric acids, sugar and amino acids uptake, and biosynthesis rates of metabolic products - biomass, erythritol, mannitol and acetic acid, among others. We showed that, under increasing ethanol conditions, O. oeni favors anabolic reactions related with cell maintenance, as the requirements of NAD(P)+ and ATP increased with ethanol content. Specifically, cultures containing 9 and 12% ethanol required 10 and 17 times more NGAM (non-growth associated maintenance ATP) during phase I, respectively, than cultures without ethanol. MLF and citric acid consumption are vital at high ethanol concentrations, as they are the main source for proton extrusion, allowing higher ATP production by F0F1-ATPase, the main route of ATP synthesis under these conditions. Mannitol and erythritol synthesis are the main sources of NAD(P)+, countervailing for 51-57% of its usage, as predicted by the model. Finally, cysteine shows the fastest specific consumption rate among the amino acids, confirming its key role for bacterial survival under ethanol stress. As a whole, this study provides a global insight into how ethanol content exerts a differential physiological response in O. oeni PSU-1 strain. It will help to design better strategies of nutrient addition to achieve a successful MLF of wine.

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ABSTRACT Introduction: Spontaneous pneumomediastinum (SPM) is defined as the presence of air in the mediastinum. It is a rare entity considered benign and self-limiting, which mostly affects young adults. Its diagnosis is confirmed through clinical and radiological studies. Case description: 21-year-old male patient with cough and greenish expectoration for four days, associated with dyspnea, chest pain, fever and bilateral supraclavicular subcutaneous emphysema. Chest X-ray suggested pneumomediastinum, which was confirmed by tomography. The patient was hospitalized for observation and treatment. After a positive evolution, he was discharged on the sixth day. Discussion: SPM is a differential diagnosis in patients with chest pain and dyspnea. Its prevalence is lower than 0.01% and its mortality rate is low. It should be suspected in patients with chest pain and subcutaneous emphysema on physical examination. Between 70 and 90% of the cases can be identified by chest X-ray, while confirmation can be obtained through chest tomography. In most cases it does not require additional studies. Conclusion: SPM is a little known cause of acute chest pain, and rarely considered as a differential diagnosis; it is self-limited and has a good prognosis.

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BACKGROUND: Nannochloropsis salina (= Eustigmatophyceae) is a marine microalga which has become a biotechnological target because of its high capacity to produce polyunsaturated fatty acids and triacylglycerols. It has been used as a source of biofuel, pigments and food supplements, like Omega 3. Only some Nannochloropsis species have been sequenced, but none of them benefit from a genome-scale metabolic model (GSMM), able to predict its metabolic capabilities. RESULTS: We present iNS934, the first GSMM for N. salina, including 2345 reactions, 934 genes and an exhaustive description of lipid and nitrogen metabolism. iNS934 has a 90% of accuracy when making simple growth/no-growth predictions and has a 15% error rate in predicting growth rates in different experimental conditions. Moreover, iNS934 allowed us to propose 82 different knockout strategies for strain optimization of triacylglycerols. CONCLUSIONS: iNS934 provides a powerful tool for metabolic improvement, allowing predictions and simulations of N. salina metabolism under different media and genetic conditions. It also provides a systemic view of N. salina metabolism, potentially guiding research and providing context to -omics data.

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ABSTRACT Introduction: Pulmonary infarction occurs in 29% to 32% of patients with pulmonary thromboembolism (PTE). The infection of a pulmonary infarction is a complication in approximately 2 to 7% of the cases, which makes it a rare entity. Case Presentation: 49-year-old woman with pleuritic pain in the left hemithorax that irradiated to the dorsal region, associated with dyspnea and painful edema in the left lower limb of two days of evolution. Two weeks prior to admission, the patient suffered from a left knee trauma that required surgical intervention; however, due to unknown reasons, she did not receive antithrombotic prophylaxis. Physical examination showed tachycardia, tachypnea and painful edema with erythema in the left leg. After suspecting a pulmonary thromboembolism, anticoagulation medication was administered and a chest angiotomography was requested to confirm the diagnosis. The patient experienced signs of systemic inflammatory response, and respiratory deterioration. A control tomography was performed, suggesting infected pulmonary infarction. Antibiotic treatment was initiated, obtaining progressive improvement; the patient was subsequently discharged, and continued with anticoagulation medication and follow-up on an outpatient basis. Conclusions: Pulmonary infarction is a frequent complication in patients with PTE. Therefore, infected pulmonary infarction should be suspected in patients with clinical deterioration and systemic inflammatory response. The radiological difference between pulmonary infarction and pneumonia is not easily identified, thus the diagnostic approach is clinical, and anticoagulant and antimicrobial treatment should be initiated in a timely manner.

RESUMEN Introducción El infarto pulmonar ocurre entre un 29 y un 32% de pacientes con tromboembolismo pulmonar (TEP). Por su parte, la infección de un infarto pulmonar complica aproximadamente del 2 al 7% de los casos, lo que hace que el infarto pulmonar infectado sea una entidad poco frecuente. Descripción del caso Mujer de 49 años con dolor pleurítico en hemitórax izquierdo, irradiado a región dorsal, asociado a disnea y edema doloroso de miembro inferior izquierdo de dos días de evolución. Dos semanas antes de su ingreso la paciente sufrió trauma de rodilla izquierda, el cual que requirió intervención quirúrgica; sin embargo, por motivos desconocidos, no recibió profilaxis anti trombótica. En el examen físico se encontró taquicardia, taquipnea y edema doloroso con eritema en pierna izquierda. Al existir alta sospecha de tromboembolia pulmonar se inició anticoagulación y se solicitó angiotomografía de tórax, con la cual fue posible confirmar el diagnóstico. Durante su evolución, la paciente experimentó signos de respuesta inflamatoria sistémica, deterioro respiratorio. Se realizó tomografía de control sugestiva de infarto pulmonar infectado. Se inició antibiótico y la paciente mejoró de forma progresiva; después de esta mejora, fue dada de alta para continuar anticoagulación y seguimiento ambulatorios. Conclusiones El infarto pulmonar es una complicación frecuente en pacientes con TEP. Por lo tanto, debe sospecharse infarto pulmonar infectado en pacientes con deterioro clínico y respuesta inflamatoria sistémica. La diferencia radiológica entre infarto pulmonar y neumonía no es fácil de identificar, su enfoque diagnóstico es clínico y el tratamiento anticoagulante y antimicrobiano debe iniciarse de manera oportuna.

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Oenococcus oeni is the main responsible agent for malolactic fermentation in wine, an unpredictable and erratic process in winemaking. To address this, we have constructed and exhaustively curated the first genome-scale metabolic model of Oenococcus oeni, comprising 660 reactions, 536 metabolites and 454 genes. In silico experiments revealed that nutritional requirements are predicted with an accuracy of 93%, while 14 amino acids were found to be essential for the growth of this bacterial species. When the model was applied to determine the non-growth associated maintenance, results showed that O. oeni grown at 12% ethanol concentration spent 30 times more ATP to stay alive than in the absence of ethanol. Most of this ATP is employed for extruding protons outside of the cell. A positive relationship was also found between specific consumption rates of fructose, amino acids, oxygen, and malic acid and the specific production rates of erythritol, lactate, and acetate, according to the ethanol content of the medium. The metabolic model reconstructed here represents a unique tool to predict the successful completion of wine malolactic fermentation carried out either by different strains of Oenococcus oeni, as well as at any particular physico-chemical composition of wine. It will also allow the development of consortium metabolic models that could be applied to winemaking to simulate and understand the interactions between O. oeni and other microorganisms that share this ecological niche.

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Piscirickettsia salmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with highly adverse impact in the Chilean salmon farming industry. The development of effective treatment and control methods for piscireckttsiosis is still a challenge. To meet it the number of studies on P. salmonis has grown in the last couple of years but many aspects of the pathogen's biology are still poorly understood. Studies on its metabolism are scarce and only recently a metabolic model for reference strain LF-89 was developed. We present a new genome-scale model for P. salmonis LF-89 with more than twice as many genes as in the previous model and incorporating specific elements of the fish pathogen metabolism. Comparative analysis with models of different bacterial pathogens revealed a lower flexibility in P. salmonis metabolic network. Through constraint-based analysis, we determined essential metabolites required for its growth and showed that it can benefit from different carbon sources tested experimentally in new defined media. We also built an additional model for strain A1-15972, and together with an analysis of P. salmonis pangenome, we identified metabolic features that differentiate two main species clades. Both models constitute a knowledge-base for P. salmonis metabolism and can be used to guide the efficient culture of the pathogen and the identification of specific drug targets.

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Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 µM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.

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