Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

Antibiotic prescribing in the emergency department versus primary care: Implications for stewardship.

OBJECTIVE: This study sought to compare the appropriateness of antibiotic prescribing by drug, dose, duration, and indication between the emergency department (ED) and primary care (PC) within the Veterans Affairs Western New York Healthcare System (VAWNYHCS) to aid in focusing antimicrobial stewardship efforts. DESIGN: In this prospective observational cohort study, patients were identified using electronic alerts at the time of antibiotic prescribing. Prescriptions were retrospectively analyzed for appropriateness of antibiotic indication, drug choice, dose, and duration on the basis of current guideline recommendations. Data were compared between the ED and PC to determine the impact of visit location on prescribing habits. Baseline characteristics were compared using descriptive statistics, and a multivariable analysis was performed to identify statistically significant risk factors for inappropriate prescribing. SETTING AND PARTICIPANTS: Patients prescribed outpatient antibiotics at the VAWNYHCS ED and PC settings between June 2017 and February 2018. OUTCOME MEASURES: Appropriateness of antibiotic prescribing by drug, dose, duration, and indication between the ED and PC settings. RESULTS: The cohort included 1566 antibiotic prescriptions (ED = 488, PC = 1078). The appropriate drug, dose, and duration for antibiotics prescribed in the ED versus PC were 63.1% versus 43.4% (P < 0.001), 88.1% versus 88.2% (P = 0.953), and 86.1% versus 71.1% (P < 0.001), respectively. Azithromycin was the most inappropriately prescribed antibiotic in both the ED (37.8%) and PC (49.0%). Two factors predicted whether patients received the correct antibiotic empirically: location of the visit and antibiotic allergy. Overall, 56.6% of ED prescriptions and 82% of PC prescriptions were inappropriate with respect to at least 1 component. CONCLUSION: Stewardship is needed in both the ED and PC settings. However, initial efforts should be focused on PC because this setting had a statistically significant amount of inappropriate antibiotic prescribing. Pharmacist-led education and interventions should focus on the determination of drug, duration, and indication for the use of antibiotics.

Advancing Eosinophilic Esophagitis Diagnosis and Phenotype Assessment with Deep Learning Computer Vision.

Eosinophilic Esophagitis (EoE) is an inflammatory esophageal disease which is increasing in prevalence. The diagnostic gold-standard involves manual review of a patient's biopsy tissue sample by a clinical pathologist for the presence of 15 or greater eosinophils within a single high-power field (400× magnification). Diagnosing EoE can be a cumbersome process with added difficulty for assessing the severity and progression of disease. We propose an automated approach for quantifying eosinophils using deep image segmentation. A U-Net model and post-processing system are applied to generate eosinophil-based statistics that can diagnose EoE as well as describe disease severity and progression. These statistics are captured in biopsies at the initial EoE diagnosis and are then compared with patient metadata: clinical and treatment phenotypes. The goal is to find linkages that could potentially guide treatment plans for new patients at their initial disease diagnosis. A deep image classification model is further applied to discover features other than eosinophils that can be used to diagnose EoE. This is the first study to utilize a deep learning computer vision approach for EoE diagnosis and to provide an automated process for tracking disease severity and progression.