We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand-receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically-fixed samples that reduced autonucleation, and a new pre-embedding goldenlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies.

Understanding the pharmacology of microbiome‐based therapeutics is required to support the development of new medicines. Strains of E. coli Nissle (EcN) were genetically modified and administered to cynomolgus monkeys at doses of 1 × 109 and 1 × 1012 colony‐forming units (CFU)/day for 28 days. A clinical study to evaluate the exposure and clearance of EcN in healthy volunteers was also performed. Healthy subjects received oral doses of EcN, 2.5 to 25 × 109 CFU 3 times daily for 28 days or a single day. In cynomolgus monkeys, replicating strains yielded higher fecal concentrations than nonreplicating strains and persisted for longer following cessation of dosing. In the clinical study, all subjects cleared EcN following cessation of dosing with median clearance of 1 week. Quantitative methodology can be applied to microbiome‐based therapeutics, and similar kinetics and clearance were observed for EcN in cynomolgus monkeys and humans.

Aims To examine psychological insulin resistance (PIR), the unwillingness to accept insulin therapy, within a unique U.S. population of patients with diabetes. Methods A cross-sectional survey of PIR among low-income, U.S. Latino and African-American (AA) patients with type 2 diabetes recruited from a diabetes specialty clinic. Results Data from 136 insulin-naïve respondents (57% female, 69% Latino, mean age 51.1 ± 10.3 years; $200–$1,000 median monthly household income; grade 8–12 median education) revealed a 48% prevalence of complete unwillingness to begin insulin. In comparing Latinos to AA, Latino respondents were younger, lived fewer years in the U.S., had less education, were more likely unwilling to use insulin (53% vs. 30%, p = 0.03), and reported a more negative attitude to 8 of 9 PIR domains (p ≤ 0.01 for each). Fewer years in the U.S. predicted greater unwillingness and a more negative attitude on 8 of 9 PIR domains (p ≤ 0.03 for each); and less education predicted greater feelings of unfairness (p = 0.01). Conclusions PIR is highly prevalent among low income, U.S. Latino patients with type 2 diabetes. Our data may help to better guide culturally appropriate counseling regarding insulin use.