Antimicrobial stewardship during a time of rapid antimicrobial development: Potential impact on industry for future investment.

In response to the antimicrobial resistance crisis, pharmaceutical industry reinvested in and produced new antibiotics. Antimicrobial stewardship programs influence optimal antimicrobial use, which often places them at the cross-roads of resistance and treatments. We surveyed a clinical administration database of US medical centers between 2014 and 2018 for index antimicrobial utilization date of six Qualified Infectious Diseases Products (QIDP). Among 132 hospitals identified, the median time to use any agent was 398 days (range 13 to >1478 days). QIDP antibiotic use was more likely among academic medical centers (range 34%-88%) and hospitals >400 beds (range 39%-86%) compared to non-academic medical center (3-51%) and smaller and hospitals (range 0-61%). The South was quickest to use all QIDP (median 733 days), while the Northeast was longest at 1370 days. New antimicrobials have limited clinical use, which impacts manufacturers' ability to stay in the antimicrobial market and further risking a depleted antimicrobial pipeline.

Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment.

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst-matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.

Issues associated with assessing nuclear localization of N-terminally unphosphorylated beta-catenin with monoclonal antibody 8E7.

BACKGROUND: Beta-catenin is a dual function adhesion/transcriptional co-activator protein, and both functions are critical for normal tissue homeostasis. Since the transcriptional functions of beta-catenin are more often implicated in various disease processes, there is much interest in the development and use of reagents to interrogate spatial and temporal evidence of beta-catenin nuclear signaling in cells and tissues. An important study demonstrated that the signaling form of beta-catenin is specifically unphosphorylated at residues S37 and T41, and suggested that this form exhibits a propensity for cytosolic/nuclear accumulation relative to the total pool of beta-catenin. RESULTS: We show that monoclonal antibody, 8E7, which recognizes the signaling form of beta-catenin specifically unphosphorylated at S37 and T41 (Active B-Catenin, ABC), also cross-reacts with a widely expressed, variably accessible nuclear antigen that is not beta-catenin. In cell types commonly used to study Wnt activation, this non-specific nuclear staining can be robust, obscuring the ABC signal. Definitive detection of nuclear localized ABC can be confirmed through an ability of classical cadherins to sequester ABC to cell junctions. In tissues, milder antigen retrieval methods can reduce the accessibility of mAb 8E7 to this cross-reacting nuclear antigen. CONCLUSION: These findings reveal that interpretation of nuclear, signaling active beta-catenin using monoclonal antibody 8E7 should be considered judiciously, and in conjunction with independent methods.