Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli

Background: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of beta-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Results: Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 [DEGREE SIGN]C. The half-life of the enzyme at this temperature was 9.2 h. Conclusions: This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozymeL.T., A.H. and J.B. acknowledge funding from the Spanish Ministry of Science (grants CIT 010000-2009-29, RyC2006-02441) and an institutional grant from Fundación Ramón Areces to CBMS

Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells-a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality

Engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the Km for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site. © 2013, American Society for Microbiology.Spanish Ministry of Science (CIT no. 010000-2009-29, RyC2006-02441, and BIO2010-18875); Fundación Ramón ArecesPeer Reviewe

A novel non-peptidic agonist of the ghrelin receptor with orexigenic activity in vivo

Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand

Evaluation of the conceptual model on shrubbiness-desertification on arid rangelands

Advancement of shrubbiness (increase in plant cover and density of woody species) on different environments world-wide has generated an increasing interest in the scientific community, particularly because of its association with the desertification process which is increasing, also at a world-wide level. There are two opposing positions, both of them validated by scientific evidences. The greatly accepted position recognizes shrubbiness as one of the mechanisms conducive to desertification in natural environments, cataloguing it as a negative process. A different view, based on recent studies, suggests that shrubbiness should be considered as a positive process because it may drive a reversal of desertification, depending on the environment and the species of shrub studied at a site. This review presented within this framework discusses the existing relationship between shrubbiness and desertification and proposes an approach that should be considered in future research.ISSN 0570-179

Influencia del enfriamiento en las propiedades de titanatos de lantano y litio

Se ha estudiado el efecto de los tratamientos a alta temperatura sobre la estructura y la movilidad del litio para la solución sÛlida Li3xLa2/3-xTiO3 (0.03<x<0.167) con difracciÛn de rayos X a alta temperatura (DRXAT), espectroscopia Raman, RMN y de impedancias. Los patrones de difracción de rayos X (DRX) a temperatura ambiente de muestras enfriadas lentamente muestran una estructura tipo perovskita doblada en el eje c con simetrÌas tetragonales u ortorrómbicas, mientras que las muestras con un enfriamiento r·pido muestran una estructura tipo perovskita c ̇bica simple. Sin embargo, el espectro Raman de las muestras analizadas se interpreta, en todos los casos, con una simetrÌa tetragonal en la que el desorden catiónico se incrementa con el contenido de litio y el tratamiento de enfriamiento. La existencia de microdominios de maclado, orientados a lo largo de las tres direcciones de la perovski- ta, favorece la detecciÛn de la fase c ̇bica en los patrones de DRX. A partir de la espectroscopia de RMN del 7Li, se ha detectado un movimiento bidi- mensional del litio en las muestras ordenadas, el cual se convierte progresivamente en un movimiento tridimensional conforme se incrementa el desorden catiÛnico. Asimismo, la presencia de microdominios hace disminuir la conductividad dc de muestras con contenidos bajos de litio

Adiponectin accounts for gender differences in hepatocellular carcinoma incidence

Hepatocellular carcinoma (HCC) is the sixth most common cancer type and the fourth leading cause of cancer-related death. This cancer appears with higher incidence in men and during obesity; however, the specific mechanisms underlying this correlation are unknown. Adipose tissue, a key organ in metabolic syndrome, shows evident gender disparities in the production of adipokines. Levels of the important adipokine adiponectin decrease in men during puberty, as well as in the obese state. Here, we show that this decrease in adiponectin levels is responsible for the increased liver cancer risk in males. We found that testosterone activates the protein JNK in mouse and human adipocytes. JNK-mediated inhibition of adiponectin secretion increases liver cancer cell proliferation, since adiponectin protects against liver cancer development through the activation of AMP-activated protein kinase (AMPK) and p38α. This study provides insight into adipose tissue to liver crosstalk and its gender relation during cancer development, having the potential to guide strategies for new cancer therapeutics.G. Sabio is an investigator on the Ramón y Cajal Program. E. Manieri is a La Caixa Foundation fellow. L. Herrera-Melle is a fellow of the Ministerio de Educación, Cultura y Deporte (FPU15-05802). This study was funded by the following grants: G. Sabio was funded by the European Research Council (ERC 260464), European Foundation for the Study of Diabetes–Lilly, Ministerio de Ciencia, Innovación y Universidades (MICINN/SAF2016-79126-R), Comunidad de Madrid (B2017/BMD-3733), and BBVA Becas Leonardo a Investigadores y Creadores Culturales (Investigadores-BBVA-2017; IN[17]_BBM_BAS_0066); M. Marcos was funded by Instituto de Salud Carlos III and Federación Española de Enfermedades Raras (PI16/01548); and J.L. Torres was funded by Junta de Castilla y León GRS (1587/A/17). F.J. Cubero is a Ramón y Cajal Researcher (RYC-2014-15242) and a Gilead Liver Research Scholar 2018, and his work is supported by the Ministerio de Economia y Competitividad Retos (SAF2016-78711), Comunidad de Madrid (S2017/BMD-3727), The Alan Morement Memorial Fund Cholangiocarcinoma Charity (2018/117), the European Cooperation in Science and Technology Action (CA17112), and the European Foundation for Alcohol Research (EA14/18). L. Moran is a Comunidad de Madrid fellow (S2017/BMD-3727). The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Root traits and arbuscular mycorrhiza on perennial grasses exposed to defoliation after a controlled burning

Plant competition for soil resources is common in semiarid rangelands. Plants have various, alternative mechanisms to deal with soil resource acquisition. They include (1) length, weight and proliferation of roots, (2) root length density, and (3) root colonization by arbuscular mycorrhiza. In rangelands of Argentina, plants are exposed to herbivory after natural fires. As a result, knowledge on how these disturbances impact root traits is important for outlining guidelines focused on rangeland management and improvement. Our aim was to analyze the effects of defoliation after a controlled burning on root traits (1) to (3) on two preferred (Poa ligularis and Nassella tenuis) and one unpreferred (Amelichloa ambigua) perennial grasses. Applied treatments did not affect neither root length nor percentage colonization by arbucular mycorrhiza. The smaller diameter and greater root length found in P. ligularis might contribute to explain its lower root mycorrhizal colonization in comparison to A. ambigua and N. tenuis, respectively. The greatest root length and weight, on P. ligularis will contribute to explain the already known greater competitive ability in this than in the other two study species. Our results suggest that defoliating P. ligularis after fire would not compromise its competitive ability, thus contributing to rangeland management.En pastizales semiáridos, la competencia por los recursos subterráneos es una de las principales formas de competencia entre plantas. Las plantas poseen varios mecanismos alternativos para la adquisición de los recursos del suelo. Éstos incluyen (1) la longitud, peso y proliferación radical, (2) la densidad de longitud de raíces y (3) la colonización del sistema radical por hongos micorrízicos arbusculares. En los pastizales de Argentina las plantas están expuestas a herbivoría luego de fuegos naturales. En consecuencia, resulta crucial conocer cómo impactan dichos disturbios sobre los atributos radicales, con el objeto de emplearlos como herramientas de manejo para hacer un uso sustentable de estos ecosistemas. El objetivo del estudio fue analizar el efecto de la defoliación luego de una quema controlada sobre los parámetros subterráneos mencionados (1 a 3), en dos especies deseables (Poa ligularis y Nassella tenuis) y una indeseable (Amelichloa ambigua). Los tratamientos aplicados no afectaron la longitud radical ni la colonización por hongos micorrízicos arbusculares. El menor diámetro y la mayor longitud radical de P. ligularis, contribuyeron a explicar la menor colonización radical de esta especie respecto de A. ambigua y N. tenuis, respectivamente. Los resultados encontrados sugieren que la defoliación de P. ligularis luego de una quema controlada no comprometería la habilidad competitiva de esta especie, contribuyendo al mejoramiento de los pastizales.Artículo publicado en Phyton, International Journal of Experimental Botany (2018) 87: 51-5

Perceptions and Experiences of Human Papillomavirus (HPV) Infection and Testing Among Low-Income Mexican Women

Background HPV infection causes cervical cancer, a major contributor to morbidity and mortality among low-income Mexican women. Human papillomavirus (HPV) DNA testing is now a primary screening strategy in Mexico’s early cervical cancer detection program (ECDP). Research on Mexican women’s perceptions of HPV and testing is necessary for establishing culturally appropriate protocols and educational materials. Here, we explore perceptions about HPV and HPV-related risk factors among low-income Mexican ECDP participants. Methods We conducted semi-structured interviews with 24 ECDP participants from two primary care health clinics in Michoacán state, Mexico. Interviews addressed women’s understandings of and experiences with HPV and HPV testing. Analysis was inductive and guided by the Health Belief Model with a focus on gender. Results Women’s confusion about HPV and HPV screening caused emotional distress. They understood HPV to be a serious disease that would always cause severe symptoms, often characterizing it as analogous to HIV or inevitably carcinogenic. Women also attributed it to men’s sexual behaviors, specifically infidelity and poor hygiene. Women described both sexes’ desire for sex as natural but understood men’s negative practices of masculinity, like infidelity, as the causes of women’s HPV infection. Some women believed dirty public bathrooms or heredity could also cause HPV transmission. Conclusions These results are consistent with prior findings that geographically and economically diverse populations lack clear understandings of the nature, causes, or symptoms of HPV, even among those receiving HPV testing. Our findings also reveal that local cultural discourse relating to masculinity, along with failure to provide sufficient education to low-income and indigenous-language speaking patients, exacerbate women’s negative emotions surrounding HPV testing. While negative emotions did not deter women from seeking testing, they could be ameliorated with better health education and communication