A Flow Cytometric Assay for the Study of E3 Ubiquitin Ligase Activityb

This is the peer reviewed version of the following article: Hilliard, J. G., Cooper, A. L., Slusser, J. G. and Davido, D. J. (2009), A flow cytometric assay for the study of E3 ubiquitin ligase activity. Cytometry, 75A: 634–641. doi:10.1002/cyto.a.20738, which has been published in final form at http://doi.org/10.1002/cyto.a.20738. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.BACKGROUND: Current methods for monitoring E3 ubiquitin ligase activity in cell culture or in vivo are limited. As a result, the degradation of cellular targets by many E3 ubiquitin ligases in live cells has not yet been examined. METHODS: A target of an E3 ubiquitin ligase was expressed as a fluorescently labeled protein in cell culture. If the E3 ubiquitin ligase mediates the degradation of a target protein in cell culture, it is expected that the target will show a reduced fluorescence signal by FCM analysis. We initially used the E3 ubiquitin ligase, herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) and one of its targets, promyelocytic leukemia (PML) protein, to determine the feasibility of our approach. Cells expressing a PML-GFP fusion protein were selected by cell sorting and infected with an adenoviral vector expressing ICP0. RESULTS: In contrast to mock-infected cells, only PML-GFP-expressing cells infected with the ICP0 adenoviral vector led to a significant decrease in the fluorescence signal of PML-GFP when examined by fluorescence microscopy and FCM analysis. CONCLUSIONS: Using HSV-1 ICP0 as a paradigm, it is possible to examine the live activity of an E3 ubiquitin ligase (via one of its targets) in cell culture with FCM analysis

Identification of genes involved in alcohol consumption and cigarettes smoking

We compared the results of quantitative linkage analysis using single-nucleotide polymorphisms and microsatellite markers and introduced a new screening test for multivariate quantitative linkage analysis using the Collaborative Study on the Genetics of Alcoholism data. We analyzed 115 extended non-Hispanic White families and tested for linkage using two phenotypes: the maximum number of drinks in a 24-hour period and the number of packs smoked per day for one year. Our results showed that the linkage signal increased using single-nucleotide polymorphisms compared with microsatellite markers and that the screening test gave similar results to that of the bivariate analysis, suggesting its potential use in reducing overall analysis time

Receipt of Corrective Lenses and Academic Performance of Low-Income Students

ObjectiveUntreated vision problems are associated with poor school performance. Whether providing glasses alone improves performance, however, remains unknown. We sought to test whether receiving glasses was associated with improved school performance for low-income minority students in Los Angeles.MethodsFrom 2017 to 2018, we analyzed achievement marks in mathematics and language arts from 406 first to fifth grade students attending 24 public elementary schools who received glasses through a free school-based vision program between February and May 2014, and 23,393 of their nonparticipating same-school, same-grade peers. We calculated students' percentile rank during each grading period in 1 year before and 2 years since they received glasses. Multilevel linear regressions tested whether percentile rank differed from baseline at each subsequent grading period. Models accounted for clustering at the school level and controlled for gender, grade level, and baseline class rank. Interaction terms tested whether associations differed by gender and class rank.ResultsStudents increased 4.5 percentile points (P = .02) in language arts in the second year after receiving glasses. There was no change in math achievement overall; however, those with baseline performance in the bottom tercile had an immediate and sustained improvement of 10 to 24 percentile points from baseline (interaction term P < .001). Class rank for behavior marks decreased during the fourth grading period after receiving glasses but subsequently returned to baseline. There were no significant changes in work habits and no variation in results by gender.ConclusionsEnsuring access to vision care may be a simple, scalable strategy to improve language arts performance for low-income minority children

The Vascular-Renal Connection in Patients Hospitalized With Hypertensive Crisis: A Population-Based Study

Objective: To determine the risks of acute kidney injury development and long-term clinical outcomes of patients with hypertensive crisis. Patients and Methods: This was a population study of Olmsted County residents with hypertensive crisis between January 1, 2000, and December 31, 2008, with follow-up until June 30, 2016. Results: The results demonstrated that those with underlying chronic kidney disease upon admission for hypertensive crisis, defined as a systolic blood pressure above 180 mm Hg or diastolic blood pressure above 120 mm Hg, were more likely to develop acute kidney injury during hospitalization (odds ratio, 6.04; 95% CI, 1-26; P=.02). Hospitalization length of stay was increased when patients developed acute kidney injury during hypertensive crisis hospitalization (7.6±9 vs 3.4±4 days; P=.04). Furthermore, those who developed acute kidney injury had increased cardiac rehospitalization frequency over 10 years (87% vs 46%; P=.009). These results suggest that those with poor renal reserve are more likely to have further acute kidney damage in the setting of hypertensive crisis, likely due to decreased renal perfusion and neurohormonal dysregulation. Conclusion: In patients hospitalized for hypertensive crisis, chronic renal insufficiency was a risk factor associated with acute kidney injury development during hospitalization. Those who developed acute kidney injury had longer hospitalizations with increased rehospitalization frequency. Future studies are warranted to further investigate whether the preservation of renal function will improve clinical outcomes in hospitalized patients with hypertensive crisis