Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as Escherichia coli and Bacillus subtilis, or the yeast Saccharomyces cerevisiae. Difficulties arise when transferring these tools to nonmodel organisms. Synthetic biology strategies relying on genome transplantation (GT) aim at using yeast cells for engineering bacterial genomes cloned as artificial chromosomes. However, these strategies remain unsuccessful for many bacteria, including Mycoplasma pneumoniae (MPN), a human pathogen infecting the respiratory tract that has been extensively studied as a model for systems biology of simple unicellular organisms. Here, we have designed a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology for editing the MPN genome. Using this strategy, we have introduced a 15 kbp fragment at a specific locus of the MPN genome and replaced 38 kbp from its genome by engineered versions modified either in yeast or in E. coli. A strain harboring a synthetic version of this fragment cleared of 13 nonessential genes could also be built and propagated in vitro. These strains were depleted of known virulence factors aiming at creating an avirulent chassis for SynBio applications. Such a chassis and technology are a step forward to build vaccines or deliver therapeutic compounds in the lungs to prevent or cure respiratory diseases in humans.

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

Bacteria of the genus Mycoplasma have recently attracted considerable interest as model organisms in synthetic and systems biology. In particular, Mycoplasma pneumoniae is one of the most intensively studied organisms in the field of systems biology. However, the genetic manipulation of these bacteria is often difficult due to the lack of efficient genetic systems and some intrinsic peculiarities such as an aberrant genetic code. One major disadvantage in working with M. pneumoniae is the lack of replicating plasmids that can be used for the complementation of mutants and the expression of proteins. In this study, we have analysed the genomic region around the gene encoding the replication initiation protein, DnaA, and detected putative binding sites for DnaA (DnaA boxes) that are, however, less conserved than in other bacteria. The construction of several plasmids encompassing this region allowed the selection of plasmid pGP2756 that is stably inherited and that can be used for genetic experiments, as shown by the complementation assays with the glpQ gene encoding the glycerophosphoryl diester phosphodiesterase. Plasmid-borne complementation of the glpQ mutant restored the formation of hydrogen peroxide when bacteria were cultivated in the presence of glycerol phosphocholine. Interestingly, the replicating plasmid can also be used in the close relative, Mycoplasma genitalium but not in more distantly related members of the genus Mycoplasma. Thus, plasmid pGP2756 is a valuable tool for the genetic analysis of M. pneumoniae and M. genitalium.

e21063 Background: Breast cancer is the principal malignancy in Mexican women. Breast neoplasias develop chaotic and fragile neovasculature originating intermittent hypoxia which in turn, leads to an adaptive response coordinated by the transcriptional factor HIF-1a (hypoxia-inducible factor-1a). In the present work the expression of HIF-1a was analyzed in different female breast cancer biopsies in order to determine the relationship between HIF-1a expression and cancer prognosis. To determine the functional role of HIF-1a, proteomic and kinetic analysis of several well-established HIF-1 targets such as the glycolytic proteins glucose transporters (GLUT1), hexokinase (HKII), and lactate dehydrogenase (LDH-A) were also determined. In addition, to evaluate the role of HIF-1a expression on mitochondrial function, content of mitochondrial enzymes were also assayed. METHODS Twenty tru-cut biopsies were obtained from female patients (30-45 years old) without previous clinical treatment. After patho-histological assessment of breast cancer incidence, samples were processed by Western-blot analysis (for proteomic analysis) and for enzyme activity determination. RESULTS Significant increased content of HIF-1a was determined in all human solid tumor biopsies analyzed. In consequence, enhanced GLUT1, HKII and LDH-A contents and activities (12 and 314 mU/mg protein for HK and LDH, respectively) were also detected. On the contrary, mitochondrial enzymes (cytochrome c oxidase, ATP/ADP translocator, glutaminase-K and 2-oxoglutarate dehydrogenase) were not detected, whereas ATP synthase content significantly increased in all analyzed samples. CONCLUSIONS These results clearly indicated that HIF-1a, together with their target glycolytic genes and possibly the mitochondrial enzyme ATP synthase, may be useful to depict a breast cancer metabolic marker pattern.

Determinar en el ámbito de Atención Primaria la frecuencia de las potenciales interacciones farmacológicas de los medicamentos antihipertensivos. Estudio observacional transversal. Centro de salud de características urbanas. Trescientos veintitrés pacientes seleccionados mediante muestreo consecutivo a partir de tarjetas de largo tratamiento. Nivel de confianza del 95 %; precisión ± 5 %; proporción esperada de interacciones del 30%. Sujetos: pacientes hipertensos que consumen medicación antihipertensiva. Variables: medicación antihipertensiva, otros fármacos de uso crónico y datos sociodemográficos. Para valorar las combinaciones inadecuadas se utilizó la Guía de Interacciones de Fármacos 2002 de la Sociedad Española de Farmacia Hospitalaria. Análisis de datos: descripción de variables, pruebas de comparación de medias y proporciones en grupos independientes. Consumo de antihipertensivos, medicación concomitante, presencia de interacciones según las fichas técnicas de la Sociedad Española de Farmacia Hospitalaria y variables sociodemográficas. Edad media de 64,33 años ± 12,24 DE (rango: 32-97), porcentaje de mujeres del 57 %. Número medio de otros medicamentos 3,56 ± 1,45 DE. La distribución con porcentajes de medicamentos antihipertensivos fue: calcioantagonistas: 29 (13,2 %); inhibidores de la enzima conversora de la angiotensina: 225 (70 %); antagonistas del receptor de la angiotensina II: 47 (19,6%); betabloqueantes: 24 (7,2%); alfabloqueantes: 47 (14,6%); alfa-betabloqueantes: 4 (1,2%), y diuréticos: 75 (38,7%). Presentaron alguna interacción moderada o grave el 19,5 % de los pacientes (IC 95 %: 15,2-23,8). Las principales interacciones fueron: enalapril y diuréticos ahorradores de potasio: 8 (2,4 %); captopril y ácido acetilsalicílico: 7 (2,2%); captopril y alopurinol: 3 (0,9 %); nifedipino y omeprazol: 4 (1,2%); nifedipino y antidiabéticos orales: 2 (0,6 %); verapamil y calcio: 2 (0,6 %); doxazosina y diuréticos: 11 (3,4 %); doxazosina y digoxina: 1 (0,3 %); inhibidores de la enzima conversora de la angiotensina o antagonistas del receptor de la angiotensina II y diuréticos como enalapril y tiacidas: 20 (6,2%). La proporción de interacciones no fue significativamente diferente en ambos sexos y tampoco la edad media fue diferente desde el punto de vista estadístico en los pacientes con o sin alguna interacción. Es elevada la proporción de pacientes consumidores de medicación antihipertensiva que presentan interacciones moderadas o graves, especialmente en el caso del enalapril, captopril, nifedipino, verapamil y doxazosina. Determinadas interacciones pueden ser la causa de un mal control de las cifras tensionales o descompensación de otras patologías. Frente al riesgo potencial de las interacciones de los medicamentos antihipertensivos el médico de Atención Primaria debe considerar, cada vez más por su mayor utilización, la compatibilidad de los mismos con el resto de los fármacos. To determine the frequency of potential drug interactions of antihypertensive drugs in the Primary Health Care Setting Cross-sectional observational study. Urban health care center. 323 patients selected by consecutive sampling from long treatment cards. 95 % confidence interval. Accuracy±5%. Expected proportion of interactions 30 %. Subjects: hypertensive patients who take antihypertensive drugs. Endpoints: antihypertensive drugs, other chronic use drugs, and sociodemographic data. To assess inadequate combinations, the 2002 Drug Interactions Guide of the Spanish Society of Hospital Drugs was used. Data analysis: description of endpoints, mean comparison tests and proportions in independent groups. Use of antihypertensive drugs, concomitant medications, presence of interactions according to Spanish Society of Hospital Drug data sheets and sociodemographic endpoints. Mean age of 64.33 years ± 12.24 SD (range: 32-97), percentage of women 57 %. Mean number of other drugs 3.56 ± 1.45 SD. Distribution with percentages of antihypertensive drugs was: calcium antagonists: 29 (13.2 %); ACEIs: 225 (70 %); ARA II: 47 (19.6 %); beta blockers: 24 (7.2 %); alpha blockers: 47 (14.6%); alpha-beta blockers: 4 (1.2 %) and diuretics: 75 (38.7%). A total of 19.5 % of the patients had some moderate or serious interaction (95 % CI: 15.2-23.8). The main interactions were: enalapril and potassium saving diuretics: 8 (2.4 %); captopril and ASA: 7 (2.2%); captopril and allopurinol: 3 (0.9 %); nifedipine and omeprazole: 4 (1.2 %); nifedipine and oral antidiabetics: 2 (0.6 %); verapamil and calcium 2 (0.6 %); doxazosine and diuretics: 11 (3.4%); doxazosine and digoxine 1 (0.3 %), ACEI or ARA II and diuretics as enalapril and thiazides: 20 (6.2 %). The proportion of interactions was not significantly different in both genders and the mean age was also not different from the statistical point of view in patients with or without some interaction. The percentage of patients using antihypertensive drugs who have moderate or serious interactions is high, especially in the case of enalapril, captopril, nifedipine, verapamil and doxazosine. Certain interactions may be the cause of poor control of tension values or decompensation of other diseases. Faced with the potential risk of the interactions of antihypertensive drugs, the Primary Health care physician should increasingly consider the compatibility of these with the remaining drugs due to its greater use.

OBJECTIVE:To determine in primary care the frequency of pharmacological interactions of statins.DESIGN:Transversal observational study.Setting. Urban health centre.Participants. 384 patients taking statins who were chosen by systematic sampling based on long-treatment cards (95% CI, accuracy 5% and expected proportion of possible interactions unknown).MAIN MEASUREMENTS:Consumption of statins, the accompanying medication taken, presence of interactions according to the technical details of statins (Spanish Medication Agency, Ministry of Health and Consumption) and social and demographic variables.RESULTS:In 55 patients (14.3%) (95% CI, 10.8%-17.8%) one of the statin interactions with the other drugs was checked, especially with acenocoumarol, digoxin and anti-acid drugs. In patients with some interaction, the mean number of other drugs was significantly higher (4.51.5 vs 3.31.9 SD; P<.001). 19.1% of men and 10.8% of women showed interactions, the difference being statistically significant (P=.02). By means of logistic regression, both masculine gender (OR=1.8) and taking of other medication in quantities of 5 or more (OR=2.7) appeared as variables associated with the presence of interactions.CONCLUSIONS:The potential pharmacological interactions of statins reach 14.3% of patients with hypercholesterolaemia who take medication long-term. The possibility of reaching high plasma concentrations of statins and/or of modifying the therapeutic effect of various drugs enables a more appropriate use of statins to be recommended, with prescription of those statins that metabolise less through the P450 cytochrome.

Determinar en el ámbito de atención primaria la frecuencia de potenciales interacciones farmacológicas de las estatinas. Estudio observacional transversal. Centro de salud de características urbanas. Se seleccionó a 384 pacientes consumidores de estatinas mediante muestreo sistemático a partir de las cartillas de largo tratamiento (nivel de confianza del 95%, precisión del ± 5% y proporción esperada de posibles interacciones desconocida). Consumo de estatinas, medicación concomitante, presencia de interacciones según las fichas técnicas de las estatinas (Agencia Española del Medicamento. Ministerio de Sanidad y Consumo) y variables sociodemográficas. Resultados. En 55 pacientes (14,3%; IC del 95%, 10,8–17,8%) se comprobó alguna de las interacciones de las estatinas con el resto de los fármacos, sobre todo con acenocumarol, digoxina y antiácidos. Entre los pacientes con alguna interacción, el número medio de otros medicamentos fue significativamente superior (DE, 4,5 ± 1,5 frente a 3,3 ± 1,9; p < 0,001). Presentaron interacciones el 19,1% de los varones y el 10,8% de las mujeres, resultando la diferencia estadísticamente significativa (p = 0,02). Mediante regresión logística, tanto el sexo masculino (OR= 1,8) como el consumo de otros medicamentos en número de 5 o más (OR = 2,7) aparecieron como variables asociadas a la presencia de interacciones. Conclusiones. Las potenciales interacciones farmacológicas de las estatinas alcanzan al 14,3% de los pacientes con hipercolesterolemia que consumen medicación de forma crónica. La posibilidad de alcanzar concentraciones plasmáticas elevadas de estatinas o de modificar el efecto terapéutico de diversos fármacos permite aconsejar un uso más apropiado de aquéllas, prescribiendo las que utilizan en menor medida el citocromo P-450 para su metabolismo. To determine in primary care the frequency of pharmacological interactions of statins. Transversal observational study. Urban health centre. 384 patients taking statins who were chosen by systematic sampling based on long-treatment cards (95% CI, accuracy ±5% and expected proportion of possible interactions unknown). Consumption of statins, the accompanying medication taken, presence of interactions according to the technical details of statins (Spanish Medication Agency, Ministry of Health and Consumption) and social and demographic variables. In 55 patients (14.3%) (95% CI, 10.8%-17.8%) one of the statin interactions with the other drugs was checked, especially with acenocoumarol, digoxin and anti-acid drugs. In patients with some interaction, the mean number of other drugs was significantly higher (4.5±1.5 vs 3.3±1.9 SD; P<.001). 19.1% of men and 10.8% of women showed interactions, the difference being statistically significant (P=.02). By means of logistic regression, both masculine gender (OR=1.8) and taking of other medication in quantities of 5 or more (OR=2.7) appeared as variables associated with the presence of interactions. Conclusions. The potential pharmacological interactions of statins reach 14.3% of patients with hypercholesterolaemia who take medication long-term. The possibility of reaching high plasma concentrations of statins and/or of modifying the therapeutic effect of various drugs enables a more appropriate use of statins to be recommended, with prescription of those statins that metabolise less through the P450 cytochrome.