Evidence that drug flux across synthetic membranes is described by normally distributed permeability coefficients.

Over recent decades, the use of in vitro diffusion cell studies to assess skin permeability has evolved into a major research tool, providing key insights into the relationships between skin, drug and formulation. Sometimes, such studies involve synthetic membranes as this approach can yield useful inferences with respect to drug-skin partitioning and diffusion phenomena. Yet despite the popularity of such studies, it is still not at all known whether typical solute transport across synthetic barriers results in a normal distribution of permeability coefficients or alternatively some type of skewed distribution. The present study aims to shed light on this issue. To this end, five compounds (testosterone, oestradiol, corticosterone, aldosterone and adenosine) exhibiting a broad range of octanol-water partition coefficient values were selected as test penetrants. The protocol involved taking multiple replicate measurements of each drug's passive steady state flux through poly(dimethylsiloxane) membrane. Each penetrant's resultant permeability coefficient database was subjected to a Kolmogorov-Smirnov (KS) test for normality. It was found that the permeability coefficients of all five drugs were distributed in a Gaussian-normal fashion. The theoretical significance and practical impact of these findings are discussed.     

Working conditions and health behavior as causes of educational inequalities in self-rated health: an inverse odds weighting approach

Objective:Using a novel mediation method that presents unbiased results even in the presence of exposure–mediator interactions, this study estimated the extent to which working conditions and health behaviors contribute to educational inequalities in self-rated health in the workforce.Methods:Respondents of the longitudinal Survey of Health, Ageing, and Retirement in Europe (SHARE) in 16 countries were selected, aged 50–64 years, in paid employment at baseline and with information on education and self-rated health (N=15 028). Education, health behaviors [including body mass index (BMI)] and working conditions were measured at baseline and self-rated health at baseline and two-year follow-up. Causal mediation analysis with inverse odds weighting was used to estimate the total effect of education on self-rated health, decomposed into a natural direct effect (NDE) and natural indirect effect (NIE).Results:Lower educated workers were more likely to perceive their health as poor than higher educated workers [relative risk (RR) 1.48, 95% confidence interval (CI) 1.37–1.60]. They were also more likely to have unfavorable working conditions and unhealthy behaviors, except for alcohol consumption. When all working conditions were included, the remaining NDE was RR 1.30 (95% CI 1.15–1.44). When BMI and health behaviors were included, the remaining NDE was RR 1.40 (95% CI 1.27–1.54). Working conditions explained 38% and health behaviors and BMI explained 16% of educational inequalities in health. Including all mediators explained 64% of educational inequalities in self-rated health.Conclusions:Working conditions and health behaviors explain over half of the educational inequalities in self-rated health. To reduce health inequalities, improving working conditions seems to be more important than introducing health promotion programs in the workforce.     

Characterization of xenobiotic-metabolizing enzymes and nitrosamine metabolism in the human esophagus

Esophageal cancer has been associated with tobacco smoking, and nitrosamines are possible causative agents for this cancer. The present study investigated the metabolism of the tobacco carcinogens N'- nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), and N-nitrosodimethylamine (NDMA), as well as the presence of xenobiotic-metabolizing enzymes in human esophageal tissues from individuals in the United States and Huixian, Henan Province, China (a high-risk area for esophageal cancer). All esophageal microsomal samples activated NNN and the metabolic rate was 2-fold higher in the esophageal samples from China than the USA. All microsomal samples activated NDMA. However, most of the microsomal samples did not activate NNK. Troleandomycin (an inhibitor of cytochrome P450 3A) decreased the formation of NNN-derived keto acid by 20-26% in the esophageal microsomes. The activities for NADPH: cytochrome c reductase, ethoxycoumarin O-deethylase, NAD(P)H: quinone oxidoreductase and glutathione S-transferase were present in the esophageal samples. Coumarin 7-hydroxylase (a representative activity for P450 2A6) activity was not detected in the esophageal microsomal samples. The activities for nitrosamine metabolism and xenobiotic- metabolizing enzymes were decreased (by 30-50%) in the squamous cell carcinomas compared with their corresponding non-cancerous mucosa. The presence of activation and detoxification enzymes in the esophagus may play an important role in determining the susceptibility of the esophagus to the carcinogenic effect of nitrosamines. Our results suggest that P450s 3A4 and 2E1 are involved in the activation of NNN and NDMA, respectively, in the human esophagus.      

The efficacy and safety of early supplementation of iron polymaltose complex in preterm infants

The purpose of this study was to examine the efficacy and safety of early nonionic iron supplementation in preterm infants. Infants with gestational age < or = 32 weeks who were fed enriched human milk were assigned concurrently to receive 5 mg/kg/d enteral iron polymaltose complex (IPC) at 2 or 4 weeks of age. The levels of hemoglobin, reticulocytes, serum iron, ferritin, and soluble transferrin receptor were recorded at 2, 4, and 8 weeks of age. The incidence of morbidities associated with prematurity and the need for red blood cell transfusions (RBCTs) were recorded. The 2-week group (n = 32) had a better iron status than the 4-week group (n = 36) at 4 weeks and at 8 weeks of age. The incidence of morbidities associated with prematurity was not different among the groups ( P = 0.26). RBCT was required in one infants of the 2-week group and in 10 infants in the 4-week group ( P = 0.045). The number needed to treat to prevent one RBCT was five. Supplementation of 5 mg/kg/d enteral IPC to preterm infants fed enriched human milk as early as 2 weeks of age was more beneficial to iron status than at 4 weeks of age, and was associated with decreased need for RBCTs and no increase in the incidence of morbidities associated with prematurity.     

Immunologic status of hemophilia patients treated with cryoprecipitate or lyophilized concentrate

We evaluated 37 patients with moderate or severe hemophilia A and six patients with severe factor IX deficiency for clinical or laboratory evidence of immune abnormalities. Patients were assigned to one of four groups according to the type of clotting factor replacement. Twenty patients had received only cryoprecipitate during the two years preceding the evaluation (group I); 11 additional patients were treated predominantly with cryoprecipitate but had also received up to nine bottles of factor VIII concentrate (group II); six patients received factor VIII concentrate (group III); six patients received factor IX concentrate (group IV). There was no clinical or laboratory evidence of immunodeficiency among the 43 patients. The mean absolute number of Th cells was normal in all patient groups, but the mean absolute number of Ts cells was increased compared with controls, both in patients treated with cryoprecipitate and in patients treated with factor VIII or factor IX concentrate. There was no correlation between the Th/Ts ratio and patient age, alanine aminotransferase level, hepatitis serology, in vitro lymphocyte function, or amount of clotting factor administered. Our observations demonstrate that the volunteer or commercial origin of clotting factor replacement cannot fully explain the alterations in lymphocyte subset distribution previously described in patients with hemophilia A.     

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein’s folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein’s functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.All proteins are biosynthesized as linear chains, and most need to fold into 3D structures to function. Studies on protein folding in buffers have revealed that a kinetic competition typically exists between protein folding, misfolding, and aggregation. It is the role of the protein homeostasis or proteostasis network in each subcellular compartment to regulate this competition and keep the folded and functional proteome within the physiological concentration range, while minimizing misfolding and aggregation in the face of stresses (1–4). It remains a challenge to discern how the proteostasis network affects the folding of proteins into biologically active conformations required for function in vivo (5).Current methodologies allow for quantification of the partitioning of a protein of interest (POI) between soluble and aggregated states but cannot determine the proportion of the soluble population that is properly folded and functional. Published folding probes have the potential to report on the folded fraction in cells or cell lysate (6–9); however, the extent to which they shift folding equilibria and quantify the folded and functional fraction faithfully has not been studied. Herein, we create POI folding probes by adapting the principle of activity-based protein profiling (10) to quantify the soluble folded and functional fraction of a particular protein in a cell lysate. We seek folding probes that bind to and selectively react with only the folded and functional state of a POI in a cell, leaving the nonfunctional states and other cellular proteins unmodified (Fig. 1A).Open in a separate windowFig. 1.A small molecule folding probe strategy to quantify the soluble folded and functional fraction of a POI in a cell lysate. (A) Overview of the general strategy to selectively covalently label a folded and functional POI without labeling its nonfunctional conformations and other cellular proteins. (B) The experimental scheme to quantify the ratio of the soluble POI that is functional (R_f).Fluorescent folding probes for the de novo-designed enzyme, retroaldolase (RA) (11), and fluorogenic folding probes (12) for the nonenzyme protein, transthyretin (TTR), were developed and scrutinized. We show that destabilized mutant RA and TTR proteins partition into folded and functional as well as misfolded soluble conformations and that this partitioning is sensitive to proteostasis network perturbations. Experiments show that a snapshot of the distribution between folded and functional vs. soluble and misfolded conformational states can be preserved during the small molecule folding probe labeling period, provided that the cellular chaperone holdase activity is sufficient, achieved by rapid ATP depletion in parallel with cell lysis. Sufficient chaperone/chaperonin holdase activity minimizes changes in the folded and functional concentration associated with probe binding and reaction with the POI and renders the relative folding and conjugation rates much less influential.