Genetically encoded biosensor for engineering branched-chain higher alcohol production pathway in saccharomyces cerevisiae

Branched-chain higher alcohols (BCHAs) including isobutanol, isopentanol, and 2-methyl-1-butanol, are promising alternatives to the first-generation biofuel ethanol. These alcohols have better fuel properties than ethanol, such as higher energy density, ease of refining, and better compatibility with existing gasoline engines and infrastructures 1. We have developed a genetically encoded biosensor to measure the metabolic activity of BCHA biosynthesis in Saccharomyces cerevisiae. This biosensor enables high-throughput screens to identify strains with higher metabolic flux to BCHA synthesis. The versatility of this tool has allowed us to use it in several applications, including in vivo BCHA metabolic pathway engineering/optimization and enzyme engineering. We have been able to screen for isobutanol hyper-producing stains with optimum combinations of genes from the mitochondrial isobutanol pathway (Mito-IbOH-pathway) 2. The ability of this biosensor to monitor the activity of both the mitochondrial 2 and cytosolic isobutanol pathways 3, has allowed us to engineer several enzymes and regulatory proteins involved in the isobutanol pathways in either compartment, boosting enzymatic activity by as much as 400%. Thus, we have demonstrated the use of this new technology to accelerate the development of strains and enzymes to boost BCHA production in mitochondria and the cytosol. Future applications include combining the biosensor with optogenetic regulation of BCHA biosynthesis for closed-loop dynamic control of this pathway, and using the biosensor to empower systems biology studies for gene discovery, enzyme evolving, and enzyme engineering to boost BCHA production. Please click Additional Files below to see the full abstract

Hippocampal Memory Recovery After Acute Stress: A Behavioral, Morphological and Molecular Study

Indexación: Scopus.Laboratory of Neuroplasticity and Neurogenetics, Department of Biochemistry and Molecular Biology, Faculty of Chemistry and Pharmaceutical Sciences, Universidad de Chile, Santiago, Chile, 2Laboratorio Farmacología del Comportamiento, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile, 3Department of Kinesiology, Faculty of Health Sciences, Universidad Católica del Maule, Talca, Chile, 4Facultad de Psicología, Universidad de Talca, Talca, Chile, 5Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Santiago, Chile, 6Escuela de Química y Farmacia, Facultad de Medicina, Universidad Andres Bello, Santiago, Chile.This study was supported by the following grants: Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1120528 (JLF), Fondo Central de Investigación, Universidad de Chile ENL025/16 (JLF).Several studies have shown that a single exposure to stress may improve or impair learning and memory processes, depending on the timing in which the stress event occurs with relation to the acquisition phase. However, to date there is no information about the molecular changes that occur at the synapse during the stress-induced memory modification and after a recovery period. In particular, there are no studies that have evaluated—at the same time—the temporality of stress and stress recovery period in hippocampal short-term memory and the effects on dendritic spine morphology, along with variations in N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits. The aim of our study was to take a multidimensional approach to investigate concomitant behavioral, morphological and molecular changes induced by a single restraint stress exposure (2.5 h) and a recovery period of 6 and 24 h in rats. We found that acute stress elicited a reduced preference to explore an object placed in a novel position (a hippocampal-dependent task). These changes were accompanied by increased activity of LIM kinase I (LIMK; an actin-remodeling protein) and increased levels of NR2A subunits of NMDA receptors. After 6 h of recovery from stress, rats showed similar preference to explore an object placed in a novel or familiar position, but density of immature spines increased in secondary CA1 apical dendrites, along with a transient rise in GluA2 AMPA receptor subunits. After 24 h of recovery from stress, the animals showed a preference to explore an object placed in a novel position, which was accompanied by a normalization of NMDA and AMPA receptor subunits to control values. Our data suggest that acute stress produces reversible molecular and behavioral changes 24 h after stress, allowing a full reestablishment of hippocampal-related memory. Further studies need to be conducted to deepen our understanding of these changes and their reciprocal interactions.Adaptive stress responses are a promising avenue to develop interventions aiming at restoring hippocampal function impaired by repetitive stress exposure. © 2018 Aguayo, Tejos-Bravo, Díaz-Véliz, Pacheco, García-Rojo, Corrales, Olave, Aliaga, Ulloa, Avalos, Román-Albasini, Rojas and Fiedler.https://www.frontiersin.org/articles/10.3389/fnmol.2018.00283/ful

Toxicidad sub crónica y actividad analgésica in vivo del extracto clorofórmico de las hojas de Calea urticifolia (Juanislama

Introduction: The population uses medicinal plants indiscriminately to treat diseases, with the believe that they are safe and lack adverse effects. Objective: To determine the in vivo toxicological and analgesic effect of the chloroform extract of Calea urticifolia leaves. Methodology: The toxicological study was performed using a 90- day sub-chronic toxicity test in NIH mice, at repeated and continuous doses. . Blood biochemistry, hematology and histopathological examination of organs were performed. The analgesic activity was evaluated in vivo using a model of abdominal contortions. Results: The administration of the plant extract caused the appearance of clinical signs of toxicity, alterations in hematic parameters and blood biochemistry, as well as histological alterations in some of organs. The analgesic activity at 100 mg/kg was similar to the Indomethacin drug. Conclusion: Despite the proven analgesic activity, according to the observed toxicological effects in this study, the prolonged use of Calea urticifolia leaves is not recommended for the treatment of diseasesIntroducción: La población utiliza la medicina a base de hierbas de forma indiscriminada basándose en la creencia de que las plantas medicinales carecen de efectos adversos. Objetivo: Determinar in vivo el efecto toxicológico y analgésico del extracto clorofórmico de las hojas de Calea urticifolia. Metodología: El estudio toxicológico fue realizado mediante la prueba de toxicidad subcrónica de 90 días, a dosis repetidas y continuas en ratones NIH. Se realizaron análisis de bioquímica sanguínea, hematología y el examen histopatológico de órganos. La actividad analgésica fue evaluada con el modelo in vivo de contorsiones abdominales. Resultados: La administración del extracto vegetal provocó la aparición de signos clínicos de toxicidad, alteraciones en los parámetros hematólogos y bioquímica sanguínea, además alteraciones histológicas en algunos de los órganos. La actividad analgésica a 100 mg/kg resultó comparable con el fármaco indometacina. Conclusión: Pese a la actividad analgésica demostrada, y de acuerdo a los efectos toxicológicos encontrados, no se recomienda el uso prolongado de las hojas de Calea urticifolia, para el tratamiento de enfermedade

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication

[EN] Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.European funds (European Regional Development Fund, ERDF); Spanish Ministerio de Economı´a y Competitividad; Junta de Andalucí

Childhood acute leukemias are frequent in Mexico City: descriptive epidemiology

<p>Abstract</p> <p>Background</p> <p>Worldwide, acute leukemia is the most common type of childhood cancer. It is particularly common in the Hispanic populations residing in the United States, Costa Rica, and Mexico City. The objective of this study was to determine the incidence of acute leukemia in children who were diagnosed and treated in public hospitals in Mexico City.</p> <p>Methods</p> <p>Included in this study were those children, under 15 years of age and residents of Mexico City, who were diagnosed in 2006 and 2007 with leukemia, as determined by using the International Classification of Childhood Cancer. The average annual incidence rates (AAIR), and the standardized average annual incidence rates (SAAIR) per million children were calculated. We calculated crude, age- and sex-specific incidence rates and adjusted for age by the direct method with the world population as standard. We determined if there were a correlation between the incidence of acute leukemias in the various boroughs of Mexico City and either the number of agricultural hectares, the average number of persons per household, or the municipal human development index for Mexico (used as a reference of socio-economic level).</p> <p>Results</p> <p>Although a total of 610 new cases of leukemia were registered during 2006-2007, only 228 fit the criteria for inclusion in this study. The overall SAAIR was 57.6 per million children (95% CI, 46.9-68.3); acute lymphoblastic leukemia (ALL) was the most frequent type of leukemia, constituting 85.1% of the cases (SAAIR: 49.5 per million), followed by acute myeloblastic leukemia at 12.3% (SAAIR: 6.9 per million), and chronic myeloid leukemia at 1.7% (SAAIR: 0.9 per million). The 1-4 years age group had the highest SAAIR for ALL (77.7 per million). For cases of ALL, 73.2% had precursor B-cell immunophenotype (SAAIR: 35.8 per million) and 12.4% had T-cell immunophenotype (SAAIR 6.3 per million). The peak ages for ALL were 2-6 years and 8-10 years. More than half the children (58.8%) were classified as high risk. There was a positive correlation between the average number of persons per household and the incidence of the pre-B immunophenotype (Pearson's r, 0.789; P = 0.02).</p> <p>Conclusions</p> <p>The frequency of ALL in Mexico City is among the highest in the world, similar to those found for Hispanics in the United States and in Costa Rica.</p

Yeast : the soul of beer’s aroma—a review of flavour-active esters and higher alcohols produced by the brewing yeast

Among the most important factors influencing beer quality is the presence of well-adjusted amounts of higher alcohols and esters. Thus, a heavy body of literature focuses on these substances and on the parameters influencing their production by the brewing yeast. Additionally, the complex metabolic pathways involved in their synthesis require special attention. More than a century of data, mainly in genetic and proteomic fields, has built up enough information to describe in detail each step in the pathway for the synthesis of higher alcohols and their esters, but there is still place for more. Higher alcohols are formed either by anabolism or catabolism (Ehrlich pathway) of amino acids. Esters are formed by enzymatic condensation of organic acids and alcohols. The current paper reviews the up-to-date knowledge in the pathways involving the synthesis of higher alcohols and esters by brewing yeasts. Fermentation parameters affecting yeast response during biosynthesis of these aromatic substances are also fully reviewed.Eduardo Pires gratefully acknowledges the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) for the PhD fellowship support (SFRH/BD/61777/2009). The financial contributions of the EU FP7 project Ecoefficient Biodegradable Composite Advanced Packaging (EcoBioCAP, grant agreement no. 265669) as well as of the Grant Agency of the Czech Republic (project GACR P503/12/1424) are also gratefully acknowledged. The authors thank the Ministry of Education, Youth and Sports of the Czech Republic (MSM 6046137305) for their financial support

Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols.

Publisher's version link: https://doi.org/10.1038/nbt.2509Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains revealed that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves greater local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion and reducing the loss of intermediates to competing pathways