Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

The implications of oxidative stress in the pathogenesis of many chronic human diseases has led to the widely accepted view that low molecular weight antioxidants could be beneficial and postpone or even prevent these diseases. Small molecules of either plant or synthetic origins, which contain Michael acceptor functionalities (olefins or acetylenes conjugated to electron-withdrawing groups) protect against the toxicity of oxidants and electrophiles indirectly, i.e., by inducing phase 2 cytoprotective enzymes. Some of these molecules, e.g., flavonoid and curcuminoid analogues that have phenolic hydroxyl groups in addition to Michael acceptor centers, are also potent direct antioxidants, and may therefore be appropriately designated: bifunctional antioxidants. By use of spectroscopic methods we identified phenolic chalcone and bis(benzylidene)acetone analogues containing one or two Michael acceptor groups, respectively, as very efficient scavengers of two different types of radicals: (a) the nitrogen-centered 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS.+) radical cation, and (b) the oxygen-centered galvinoxyl (phenoxyl) radical. The most potent scavengers are those also bearing hydroxyl substituents on the aromatic ring(s) at the ortho-position(s). The initial reaction velocities are very rapid and concentration-dependent. In the human keratinocyte cell line HaCaT, the same compounds coordinately increase the intracellular levels of glutathione, glutathione reductase, and thioredoxin reductase. Thus, such bifunctional antioxidants could exert synergistic protective effects against oxidants and electrophiles which represent the principal biological hazards by: (i) scavenging hazardous oxidants directly and immediately; and (ii) inducing the phase 2 response to prevent and resolve the consequences of hazardous processes that are already in progress, i.e., acting indirectly, but with much more diverse and long-lasting effects.

Myosin light chain 2 (MYL2) gene encodes the myosin regulatory light chain (RLC) simultaneously in heart ventricles and in slow‐twitch skeletal muscle. Using transgenic mice with cardiac‐specific expression of the human R58Q‐RLC mutant, we sought to determine whether the hypertrophic cardiomyopathy phenotype observed in papillary muscles (PMs) of R58Q mice is also manifested in slow‐twitch soleus (SOL) muscles. Skinned SOL muscles and ventricular PMs of R58Q animals exhibited lower contractile force that was not observed in the fast‐twitch extensor digitorum longus muscles of R58Q vs. wild‐type‐RLC mice, but mutant animals did not display gross muscle weakness in vivo. Consistent with SOL muscle abnormalities in R58Q vs. wild‐type mice, myosin ATPase staining revealed a decreased proportion of fiber type I/type II only in SOL muscles but not in the extensor digitorum longus muscles. The similarities between SOL muscles and PMs of R58Q mice were further supported by quantitative proteomics. Differential regulation of proteins involved in energy metabolism, cell‐cell interactions, and protein‐protein signaling was concurrently observed in the hearts and SOL muscles of R58Q mice. In summary, even though R58Q expression was restricted to the heart of mice, functional similarities were clearly observed between the hearts and slow‐twitch skeletal muscle, suggesting that MYL2 mutated models of hypertrophic cardiomyopathy may be useful research tools to study the molecular, structural, and energetic mechanisms of cardioskeletal myopathy associated with myosin RLC.—Kazmierczak, K., Liang, J., Yuan, C.‐C, Yadav, S., Sitbon, Y. H., Walz, K., Ma, W., Irving, T. C., Cheah, J. X., Gomes, A. V., Szczesna‐Cordary, D. Slow‐twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain. FASEB J. 33, 3152–3166 (2019). http://www.fasebj.org

In this study we aimed to provide an in-depth proteomic analysis of differentially expressed proteins in the hearts of transgenic mouse models of pathological and physiological cardiac hypertrophy using tandem mass tag labeling and liquid chromatography tandem mass spectrometry. The Δ43 mouse model, expressing the 43-amino-acid N-terminally truncated myosin essential light chain (ELC) served as a tool to study the mechanisms of physiological cardiac remodeling, while the pathological hypertrophy was investigated in A57G (Alanine 57 → Glycine) ELC mice. The results showed that 30 proteins were differentially expressed in Δ43 versus A57G hearts as determined by multiple pair comparisons of the mutant versus wild-type (WT) samples with P < 0.05. The A57G hearts showed differential expression of nine mitochondrial proteins involved in metabolic processes compared to four proteins for Δ43 hearts when both mutants were compared to WT hearts. Comparisons between Δ43 and A57G hearts showed an upregulation of three metabolically important mitochondrial proteins but downregulation of nine proteins in Δ43 hearts. The physiological model of cardiac hypertrophy (Δ43) showed no changes in the levels of Ca2+-binding proteins relative to WT, while the pathologic model (A57G) showed the upregulation of three Ca2+-binding proteins, including sarcalumenin. Unique differences in chaperone and fatty acid metabolism proteins were also observed in Δ43 versus A57G hearts. The proteomics data support the results from functional studies performed previously on both animal models of cardiac hypertrophy and suggest that the A57G- and not Δ43- mediated alterations in fatty acid metabolism and Ca2+ homeostasis may contribute to pathological cardiac remodeling in A57G hearts.

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Gastrointestinal symptoms affect the great majority of patients with systemic sclerosis. Management of these complications is often challenging as any region of the gastrointestinal tract may be involved, and significant heterogeneity exists in clinical presentation, kinetics, and outcomes. Here, we highlight new findings relevant to the management of systemic sclerosis-related gastrointestinal disease (lights) and consider areas that we have yet to elucidate (shadows).

Objective. Up to 50% of patients with systemic sclerosis (SSc) experience slow colonic transit, which may be associated with severe outcomes. Our objective, therefore, was to identify specific clinical features associated with slow colonic transit in SSc.Methods. SSc patients with gastrointestinal symptoms were prospectively enrolled and completed a scintigraphy-based whole gut transit study. Clinical features were compared between patients with and without slow colonic transit in univariate and multivariable logistic regression analyses.Results. Forty-eight of 100 patients (48%) in our cohort had slow colonic transit. In the univariate analyses, slow colonic transit was positively associated with female sex (odds ratio [OR] 12.61 [95% confidence interval (95% CI) 1.56-101.90]), telangiectasia (OR 4.00 [95% CI 1.32-12.10]), anticentromere antibodies (OR 3.25 [95% CI 1.25-8.44]), prior or current smoking (OR 2.56 [95% CI 1.06-6.21]), and a Medsger gastrointestinal severity score of ≥3 (OR 3.94 [95% CI 1.16-13.36]). Patients were less likely to have significant restriction on pulmonary function tests (OR 0.23 [95% CI 0.09-0.63]). In our multivariable model, the association between slow colonic transit and telangiectasia ) and less restrictive lung disease on pulmonary function tests (OR 0.28 [95% CI 0.09-0.86]) remained statistically significant, though a trend with smoking remained ). Interestingly, there were no significant associations between slow colonic transit and delayed transit in other regions of the gastrointestinal tract.Conclusion. Distinct clinical features are associated with slow colonic transit in SSc. Such features may provide insight in risk stratification and the study of disease mechanism in more homogeneous subgroups.

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