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Gap repair is a fast and efficient method for assembling recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is typically a vector carrying replicative sequences and a selective marker. This technique avoids laborious and costly in vitro purification and ligation of DNA. The DNA repair machinery can also close and ligate the linear vector by mechanisms other than homologous recombination, resulting in an empty vector. The frequency of these unwanted events can be lowered by removing the 5'-phosphate groups using phosphatase, which is the standard method used for in vitro ligation. However, phosphatase treatment is less effective for gap repair cloning than for in vitro ligation, presumably due to the ability of the S. cerevisiae DNA repair machinery to efficiently repair the missing phosphate group to allow religation. We have developed a more efficient method to prevent vector religation, based on treatment of the vector fragment with Taq DNA polymerase and dATP. This procedure prevents vector recircularization almost completely, facilitating the screening for true recombinant clones.

The multidrug resistance (MDR) phenotype, frequently observed during cancer treatment, is often associated with drug efflux pump activity. However, many other factors are also known to be involved. Cancer cells often rely on aerobic glycolysis for energy production; this is known as the "Warburg effect" and is used as a survival mechanism. Associated to this event, a reverse pH gradient across the cell membrane occurs, leading to cytosol alkalinization and extracellular acidification. In the present study, we investigated the role of different mechanisms involved in MDR, such as altered tumor microenvironment and energetic metabolism. The breast cancer cell line MCF-7, used as model, was exposed to two widely used antitumor drugs, paclitaxel (antimitotic agent) and doxorubicin (alkylating agent). Cancer pH regulation was shown to be crucial for malignant characteristics such as cell migration and drug resistance. Our results showed that a lower extracellular pH induced a higher migratory capacity and higher resistance to the studied chemotherapeutical compounds in MCF-7 cells. Besides the influence of the extracellular pH, the role of the tumor metabolism in the MDR phenotype was also investigated. Pre-treatment with different bioenergetic modulators led to cell ATP depletion and altered lactic acid production and glucose consumption, resulting in increased sensitivity to paclitaxel and doxorubicin. Overall, this study supports the potential use of compounds targeting cell metabolism and tumor microenvironment factors such as pH, as co-adjuvants in conventional chemotherapy., The authors thank Dr. Rita Reis for her technical support in microscopy and image analysis. This work was supported by FEDER through POFC-COMPETE and by national funds from FCT through the project PEst-C/BIA/UI4050/2011.

Over recent years, hair has become the ideal matrix for retrospective investigation of chronic abuse, including for tramadol. However, in order to exclude the possibility of external contamination, it is also important to quantify simultaneously its main metabolite, O-desmethyltramadol (M1), which presence in hair reflects systemic exposure. In the present study a methodology aimed at the simultaneous quantification of tramadol and M1 in human hair was developed and validated for the first time. After decontamination of hair samples (60 mg), tramadol and M1 were extracted with methanol in an ultrasonic bath (~5 h). Purification was performed by solid-phase extraction using mixed-mode extraction cartridges. Subsequently to derivatization, analysis was performed by gas chromatography-electron impact/mass spectrometry (GC-EI/MS). The method proved to be selective. The regression analysis for both analytes was shown to be linear in the range of 0.1-20.0 ng/mg with correlation coefficients of 0.9995 and 0.9997 for tramadol and M1, respectively. The coefficients of variation oscillated between 3.85 and 13.24%. The limits of detection were 0.03 and 0.02 ng/mg, and the lower limits of quantification were 0.08 and 0.06 ng/mg for tramadol and M1, respectively. The proof of applicability was performed in hair samples from six patients undergoing tramadol therapy. All samples were positive for tramadol and M1.

The development of analytical techniques that enable the use of hair as an alternative matrix for the analysis of drugs of abuse is useful for confirming the exposure in a larger time window (weeks to months, depending on the length of the hair shaft). In the present study a methodology aimed at the simultaneous quantification of cocaine and morphine in human hair was developed and validated. After decontamination, hair samples (20 mg) were incubated with a mixture of methanol/hydrochloric acid (2:1) at 65 °C overnight (~16 h) in order to extract the drugs of the matrix. Purification was performed by solid-phase extraction using mixed-mode extraction cartridges. After derivatization with N-methyl-N-(trimethylsilyl) trifluoroacetamide, blank, standards and samples were analyzed by gas chromatography/electron impact-mass spectrometry (GC-EI/MS). The method proved to be selective, as there were no interferences of endogenous compounds with the same retention time as cocaine, morphine and ethylmorphine (internal standard). The regression analysis for both analytes showed linearity in the range 0.25-10.00 ng/mg with correlation coefficients ranging from 0.9989 to 0.9991. The coefficients of variation oscillated between 0.83 and 14.60%. The limits of detection were 0.01 and 0.02 ng/mg, and the limits of quantification were 0.03 and 0.06 ng/mg for cocaine and morphine, respectively. The proposed GC-EI/MS method provided an accurate and simple assay with adequate precision and recovery for the quantification of cocaine and morphine in hair samples. The proof of applicability was performed in hair samples obtained from drug addicts enrolled in a Regional Detoxification Treatment Center. The importance of hair samples is highlighted, since positives results were obtained when urine immunoassay analyses were negative.