Reducing the amount of error and introduced data variability increases the accuracy of Western blot results. In this study, different methods of normalization for loading differences and data alignment were explored with respect to their impact on Western blot results. GAPDH was compared to the LI-COR Revert total protein stain as a loading control. The impact of normalizing data to a control condition, which is commonly done to align Western blot data distributed over several immunoblots, was also investigated. Specifically, this study addressed whether normalization to a small subset of distinct controls on each immunoblot increases pooled data variability compared to a larger set of controls. Protein expression data for NOX-2 and SOD-2 from a study investigating the protective role of the bradykinin type 1 receptor in angiotensin-II induced left ventricle remodeling were used to address these questions but are also discussed in the context of the original study. The comparison of GAPDH and Revert total protein stain as a loading control was done by assessing their correlation and comparing how they affected protein expression results. Additionally, the impact of treatment on GAPDH was investigated. To assess how normalization to different combinations of controls influences data variability, protein data were normalized to the average of 5 controls, the average of 2 controls, or an average vehicle and the results by treatment were compared. The results of this study demonstrated that GAPDH expression is not affected by angiotensin-II or bradykinin type 1 receptor antagonist R-954 and is a less sensitive loading control compared to Revert total protein stain. Normalization to the average of 5 controls tended to reduce pooled data variability compared to 2 controls. Lastly, the results of this study provided preliminary evidence that R-954 does not alter the expression of NOX-2 or SOD-2 to an expression profile that would be expected to explain the protection it confers against Ang-II induced left ventricle remodeling.

Depression is a worldwide public health problem that affects millions of people every year. Due to recent reports that depressed individuals have an altered gut microbiome composition, there is speculation that treatments that influence microorganisms in the gut could potentially lead to alleviation of depressive symptoms. Apple cider vinegar has been studied extensively for its health-promoting properties and benefits. Apple cider vinegar’s main ingredient is the short chain fatty acid, acetic acid. Short chain fatty acids have been shown to improve mood state and depressive symptoms, as well as amplify the effect of prebiotics in restoring the gut microbiome. This experimental design study examined the effects of ingesting 2 tbsp. apple cider vinegar (1 g acetic acid) twice daily with a meal on the levels of urinary metabolites in 14 college students compared to a control group of 11 college students that took one vinegar supplement tablet (0.015 g of acetic acid) daily for 28 days. All participants were healthy, normal to underactive (< 300 minutes of moderate exercise a week), and free of chronic or acute illnesses. Urinary metabolite analysis revealed a significant production of enzymes involved in the hexosamine pathway in the liquid vinegar group compared to baseline levels. However, anticipation of an alteration in tryptophan metabolites, a possible consequence of altered metabolism of gut microflora, was not observed. These data suggest that apple cider vinegar might be a potential treatment for depression through the production of hexosamine pathway enzymes.