Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer's disease.

BACKGROUND: Proteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies. METHODS: We coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimer's disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies. RESULTS: Quantitative multiplexed proteomics by TMT-MS of CD11b + MACS-enriched (N = 5 mice) and FACS-isolated (N = 5 mice), from adult wild-type mice, identified 1791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround Aß plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased Aß phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction. CONCLUSIONS: Using FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

Simulation-based Training in Ectopic Pregnancy and Salpingostomy.

Objective Ectopic pregnancy leads to approximately 3% of deaths in pregnancy. Surgical management is indicated when patients are hemodynamically unstable or have signs of a ruptured ectopic pregnancy. Salpingectomy is more commonly performed, but salpingostomy is preferred in a patient with prior salpingectomy with a desire for future pregnancy. Due to the lack of exposure, salpingostomy is not frequently performed and most residents do not feel adequately trained. Our goal was to provide a hands-on simulation about ectopic pregnancy and salpingostomy in hopes that the simulation will improve the resident's confidence and knowledge in recognizing an ectopic pregnancy, identifying an appropriate candidate for surgical management, and performing a salpingostomy. Methods The educational initiative was aimed towards postgraduate year (PGY) 1-4 OB/GYN residents (n=11). Knowledge and confidence questionnaires were given to participants prior to and post-simulation. A gynecologic mannequin was modified by taking the existing pelvic organs and creating a tubal pregnancy. In the first part of the simulation, a hemodynamically unstable patient presented with lab and imaging findings consistent with an ectopic pregnancy. Once recognized and the decision made for surgical intervention, participants were transferred to a simulated operating room where they performed salpingostomy or salpingectomy on the mannequin. The simulation was followed by a debriefing session to discuss the actions and thought processes of participants, provide reflection, and incorporate improvement opportunities for future cases. Finally, participants engaged in a didactic lecture where they were educated about the incidence, presentation, and management of tubal ectopic pregnancy. Results Analysis of the knowledge questionnaires showed the median score pre- and post-intervention was 9 and 12, respectively, with a median change of 3 (p=0.001). The median confidence value pre- and post-intervention were 28 and 42, respectively, with a median value change of 12 (p<0.001). Conclusion Our intervention improved residents' confidence and knowledge in recognizing an ectopic pregnancy, identifying an appropriate candidate for surgical management, and performing a salpingostomy.

Oxidation of glutathione and cysteine in human plasma associated with smoking.

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years (n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (-64 +/- 16 mV) was more oxidized than nonsmokers (- 76 +/- 11 mV; p <.001), with decreased Cys in smokers (9 +/- 5 microM) compared to nonsmokers (13 +/- 6 microM; p <.001). The GSH/GSSG redox was also more oxidized in smokers (-128 +/- 18 mV) than in nonsmokers (-137 +/- 17 mV; p =.01) and GSH was lower in smokers (1.8 +/- 1.3 microM) than in nonsmokers (2.4 +/- 1.0; p <.005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.