Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems.

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast.

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates.

Thermal analysis of laser interstitial thermotherapy in ex vivo fibro-fatty tissue using exponential functions.

A therapeutic procedure to treat small, surface breast tumours up to 10 mm in radius plus a 5 mm margin of healthy, surrounding tissue using laser interstitial thermotherapy (LITT) is currently being investigated. The purpose of this study is to analyse and model the thermal and coagulative response of ex vivo fibro-fatty tissue, a model for breast tissue, during experimental laser interstitial thermotherapy at 980 nm. Laser radiation at 980 nm was delivered interstitially through a diffusing tip optical fibre inserted into a fibro-fatty tissue model to produce controlled heating at powers ranging from 3.2 to 8.0 W. Tissue temperature was measured with thermocouples placed at 15 positions around the fibre. The induced coagulation zone was measured on gross anatomical sections. Thermal analysis indicates that a finite sum of exponential functions is an approximate solution to the heat conduction equation that more accurately predicts the time-temperature dependence in tissue prior to carbonization (T < 100 degrees C) during LITT than the traditional model using a single exponential function. Analysis of the ellipsoid coagulation volume induced in tissue indicates that the 980 nm wavelength does not penetrate deep enough in fibro-fatty tissue to produce a desired 30 mm diameter (14.1 x 10(3) mm3) coagulation volume without unwanted tissue liquefaction and carbonization.

Laser interstitial thermotherapy (LITT) monitoring using high-resolution digital mammography: theory and experimental studies.

Laser interstitial thermotherapy (LITT) is a minimally-invasive laser hyperthermia procedure for the treatment of localized tumours. Real-time monitoring of LITT is essential to control the extent of tumour destruction and ensure safe and effective treatments. The feasibility of using high-resolution digital x-ray mammography to monitor LITT of breast cancer was evaluated. Tissue phantoms including polyacrylamide hydrogel and cadaver porcine tissue were heated using a 980 nm diode laser delivered through optical fibres with diffusing tips. Digital images of the tissue phantoms were recorded with a high-resolution digital stereotactic breast biopsy system during heating. The recorded images were processed and analysed to detect heat-induced changes. No changes were detected during heating of the hydrogel. Pixel-by-pixel subtraction of the initial image from images taken during laser heating shows observable thermally-induced changes around the fibre during laser irradiation that correlate with the thermal denaturation zone observed by gross anatomy. These experiments demonstrate that high-resolution digital x-ray mammography can be used to detect heat-induced tissue changes during experimental LITT in fibro-fatty tissue.

Cancer primario de mama localizado: opciones terapeuticas y pautas para su seleccion / Localized primary breast cancer: therapeutic options and standards for its selection

A medida que nuestros conocimientos sobre la historia natural del cancer mamario han ido en aumento, la mastectomia radical ha venido cediendo lugar a procedimientos quirurgicos que conservan el seno y a una mayor confianza en la radioterapia y la quimioterapia. Probablemente, el factor mas importante en la seleccion del tratamiento es la consulta con un equipo multidisciplinario, con experiencia en los diferentes procedimientos y tecnicas que pueden ser eficaces. La clave para el manejo exitoso es la seleccion de un curso de tratamiento apropiado al cual se pueda adaptar la paciente

Pruebas de tamizaje en el diagnostico precoz del cancer mamario / Screening tests in the early diagnosis of breast cancer

Las mujeres en quienes se diagnostica el cancer mamario de manera precoz ya sea por autoexamen, examen medico o por mamografia, tienen una enorme ventaja sobre aquellas en quienes el cancer se descubre en una etapa mas avanzada. Muchas pacientes con tumores pequenos son candidatas para reseccion parcial del seno y su pronostico es tan bueno como el de aquellas tratadas con mastectomia. No cabe duda alguna de que una paciente, que ha recibido adecuada educacion sobre el autoexamen de los senos y acude periodicamente a examen medico y mamografico, tiene mejores posibilidades, en el caso de que desarrolle un cancer mamario no solo desde el punto de vista estetico, sono por la supervivencia mas prolongaday mejor calidad de vida