Simulating Intestinal Transporter and Enzyme Activity in a Physiologically Based Pharmacokinetic Model for Tenofovir Disoproxil Fumarate

Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37°C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration (Cmax; 335 ng/ml), time to Cmax (Tmax; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC0–24; 3,045 ng · h/ml), and concentration at 24 h (C24; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated Cmax (238 ng/ml), Tmax (3 h), AUC0–24 (3,036 ng · h/ml), and C24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p<0.05). These data support that TDF absorption is limited by the action of intestinal lipases. Our results suggest that bioavailability may be improved by protection of drug from intestinal transporters and enzymes, for example, by coadministration of enzyme-inhibiting agents or nanoformulation strategies

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.European Journal of Human Genetics advance online publication, 4 February 2015; doi:10.1038/ejhg.2014.283

Explaining telecoms and electricity internationalization in the European Union: a political economy perspective

One consequence of the liberalization of certain services in the European Union was that a number of formerly inward-looking incumbents in telecommunications and electricity rapidly transformed themselves into some of the world’s leading Multinationals. However, the precise relationship between liberalization and incumbent internationalization is contested. This article tests three persuasive arguments derived from the political economy literature on this relationship. The first claims that those incumbents most exposed to domestic liberalization would internationalise most. The second asserts the opposite: incumbents operating where liberalization was restricted could exploit monopolistic rents to finance their aggressive internationalisation. The third argument claims that a diversity of paths will be adopted by countries and incumbents vis-à-vis liberalization and internationalization. Using correlation and cluster analysis of the sample of all major EU telecoms and electricity incumbent Multinationals evidence is found in favour of the third hypothesis. Internationalization as a response to liberalization took diverse forms in terms of timing and extent and this is best explained using a country, sector and firm logic

Direct observation of topoisomerase IA gate dynamics

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA

Methanosarcina acetivorans C2A Topoisomerase IIIα, an Archaeal Enzyme with Promiscuity in Divalent Cation Dependence

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided

Proof of concept novel low toxicity obscurant (SERDP WP2405)

A significant health and possibly environmental problem is associated with military smokes and obscurants. Besides the fact that these obscuration munitions pose a significant toxicity hazard to the user, the environmental impact of these munitions can also be significant (e.g. due to the use of hexachlorethane). The objective of the SERDP project WP-2405, Proof of Concept Novel Low-toxicity Obscurant, is to investigate the technical feasibility to develop a new effective and environmentally benign obscurant. The project is a collaboration between TNO and Edgewood Chemical and Biological Center. As starting point the result of SERDP project WP-2148 [1] will be used, a sea salt based composition. The focus of the new project will be on the additional steps needed to gain a performance of approximately 70% (or better) of that of traditional HC smoke. The newly developed compositions vary in different additives, as to improve the overall performance of the obscurant while maintaining the low toxicity. Mass extinction coefficients were determined for different compositions by igniting 5 grams of pressed compositions in sub scaled grenades in a smokebox (V~2m3), at different relative humidity. The best five performing compositions were then studied to determine the acute toxicity for human lung cells in culture with an air-liquid interface system, VITROCELL®. This was done by igniting 10 grams in sub scaled grenades in a bio aerosol chamber (V~12m3)

Modified supracricoid laryngectomy: oncological and functional outcomes in the elderly

Eugenia Allegra, Teresa Franco, Serena Trapasso, Rossana Domanico, Alessandro La Boria, Aldo GarozzoDepartment of Otolaryngology- Head and Neck Surgery, University of Catanzaro, Catanzaro, ItalyBackground: Supracricoid laryngectomy is an organ preservation surgical technique for early-stage glottic tumors. Modified supracricoid laryngectomy using sternohyoid muscles for neoglottis reconstruction is a new surgical technique. This report evaluates oncological and functional outcomes of this new technique and its feasibility in elderly patients.Methods: Clinical records from 21 consecutive patients affected by glottic cancer and treated by modified SCL between 2004 and 2009 were retrospectively reviewed. Postoperative parameters and quality of voice after modified SCL were retrospectively reviewed. Actuarial overall survival, disease-specific survival rates, and recurrence-free survival rates were assessed. The functional and oncological outcomes of patients over 65 years were compared with those of patients younger than 65 years of age.Results: There were no postoperative complications and all of the patients had complete swallowing rehabilitation. Twenty of the 21 patients had decannulation. Two patients received total laryngectomy for locoregional relapse. Overall survival and disease-specific survival rates were 100%. Recurrence-free survival rates were 90.1% and 90% in patients younger and older than 65 years of age, respectively. The larynx preservation index was lower in patients who were older than 65 years of age. The postoperative courses with regard to functional outcome and voice quality in elderly patients were similar to those of patients younger than 65 years of age.Conclusion: Modified SCL is a new open organ preservation surgical technique that is oncologically safe. The positive functional and oncological outcomes of this surgical procedure allow it to be performed in elderly patients.Keywords: supracricoid laryngectomy, modified, early glottic cancer, cricohyoidoepiglottopexy, cricohyoidopexy, elderly, larynx, cance