Identification of CYP3A4 and CYP2C8 as the major cytochrome P450 s responsible for morphine N -demethylation in human liver microsomes

1. The aim was to identify the cytochrome P450 (CYP) enzymes responsible for the N -demethylation of morphine in vitro. 2. In human liver microsomes, normorphine formation followed Michaelis-Menten kinetics with mean K m and V max of 12.4 ± 2.2 mM and 1546 ± 121 pmol?min ? 1?mg ? 1, respectively. In microsomes from a panel of 14 human livers phenotyped for 10 CYP enzymes, morphine N -demethylation correlated with testosterone 6 β -hydroxylation (r = 0.91, p <0.001) and paclitaxel 6- α hydroxylation (r = 0.72, p <0.001), two specific markers of CYP3A4 and CYP2C8, respectively. Normorphine formation decreased when incubated in the presence of troleandomycin or quercetin (by 46 and 33-36%, respectively), which further corroborates the contribution of CYP3A4 and CYP2C8. 3. Among eight recombinant human CYP enzymes tested, CYP3A4 and CYP2C8 exhibited the highest intrinsic clearance. More than 90% of morphine N -demethylation could be accounted for via the action of both CYP3A4 and CYP2C8. 4. The in vitro findings suggest that hepatic CYP3A4, and to a lesser extent CYP2C8, play an important role in the metabolism of morphine into normorphine.     

Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy

Background

Dmd^mdx, harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies.

Methods

We generated a Dmd^EGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision.

Results

Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation.

Conclusion

Intravital imaging of Dmd^EGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.

     

Why HIV Infections Have Increased Among Men Who Have Sex with Men and What to Do About It: Findings from California Focus Groups

A resurgence of sexual risk taking, STDs, and HIV incidence has been reported among men who have sex with men (MSM) in several countries. We asked 113 MSM in 12 focus groups conducted in five California cities to identify factors leading to increased risk taking and assess prevention messages to reduce risk in this population. Participants perceived that HIV risk taking has increased because (1) HIV is not the threat it once was due to more effective therapies, (2) MSM communicate less about HIV, and social support for being safe has decreased, and (3) community norms have shifted such that unsafe sex is more acceptable. The prevention messages ranked most likely to motivate risk reduction encouraged individuals to seek social support from friends. Themes ranked least likely to succeed were those that described the negative consequences of HIV or reinforced existing safer sex messages.     

Physics and dosimetry in computed tomography

CT data acquisition and image reconstruction techniques are closely related to image quality and patient radiation dose. There is little question that technological developments currently underway will change the nature of both, and result in improved quality and diagnostic value of cardiovascular CT images while hopefully minimizing radiation dose to the patient.     

Folic acid deficiency and neutrophil dysfunction

Polymorphonuclear leukocyte functions were studied in 92 patients with protein-calorie malnutrition. Serum folic acid levels were higher than 3 ng/ml in 38 patients and 3 ng/ml or less in 54 patients. Significant differences were found between these two groups of patients with regard to phagocytosis (81.5 +/- 1.9 versus 69.2 +/- 2.0 percent, p less than 0.001) and bactericidal ability (90.6 +/- 1.1 versus 84.5 +/- 2.3 percent, p less than 0.05). Correction of folic acid deficiency in 22 patients was associated with recovery of normal phagocytosis (p less than 0.001) but not bactericidal function. Adding folic acid to the serum of eight patients also restored normal phagocytic function (p less than 0.001). A correlation was found in vivo and in vitro between changes over time in folic acid levels and in phagocytosis.