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.

Survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) often experience chronic symptoms that include fatigue, shortness of breath, and brain fog. The collection of ongoing post-COVID-19 symptoms have been classified as Post-Acute Sequela of SARS-CoV-2 (PASC). Older adult patients are especially susceptible to experiencing PASC related complications and have a high risk for long-term cognitive impairment symptoms. Definitions for categorizing PASC- associated cognitive impairment and neuropsychological assessments used to evaluate cognitive impairment are inconsistent between studies examining older adults. This systematic review aims to identify which neuropsychological tests best identify cognitive impairments associated with PASC and suggest a guide to standardize the measurement of PASC-related cognitive impairments. Through a literature search using PubMed, we included within this review 14 studies that fulfilled our inclusion and exclusion criteria evaluating middle-aged and older adult populations affected by PASC-associated cognitive impairments. The majority of the studies used tests designed to screen for general cognitive function to test for the prevalence of cognitive impairment, with the most common one being Montreal Cognitive Assessment (MoCA), followed by MMSE and TICS. MoCA reported the highest prevalence of the general cognitive screeners which suggests higher sensitivity and specificity. Telephone Interview for Cognitive Status (TICS) demonstrated similar scores as MoCA despite administration being remote while MMSE identified the lowest prevalence. Four studies also used domain-specific cognitive evaluations and reported instances of cognitive impairment in individuals who had previously tested healthy. Furthermore, the results gathered in this review were stratified based on disease symptom severity. This review identifies MoCA to be better suited for evaluating general cognitive impairment in older adults. TICS has the added utility in being able to access a wider range of older adults through remote screening. Disease severity must be clearly defined to allow better comparisons between studies and allow for standardization. Early identification of PASC-associated cognitive impairment in middle-aged and older adults can be performed using general cognitive function evaluations and administering a baseline cognitive evaluation one month after infection is suggested.