Pesticides are important chemicals or biological agents that deter or kill pests. The use of pesticides has continued to increase as it is still considered the most effective method to reduce pests and increase crop growth. However, pesticides have other consequences, including potential toxicity to humans and wildlife. Pesticides have been associated with increased risk of cardiovascular disease, cancer, and birth defects. Labels on pesticides also suggest limiting exposure to these hazardous chemicals. Based on experimental evidence, various types of pesticides all seem to have a common effect, the induction of oxidative stress in different cell types and animal models. Pesticide-induced oxidative stress is caused by both reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are associated with several diseases including cancer, inflammation, and cardiovascular and neurodegenerative diseases. ROS and RNS can activate at least five independent signaling pathways including mitochondrial-induced apoptosis. Limited in vitro studies also suggest that exogenous antioxidants can reduce or prevent the deleterious effects of pesticides.

The tumor suppressor p53 is thought to play a key role in the maintenance of cell size and homeostasis, but relatively little is known about its role in skeletal muscle. Based on its ability to suppress cell growth, we hypothesized that inhibiting the function of wild-type p53 through the overexpression of a dominant-negative p53 mutant (DDp53) could result in muscle fiber hypertrophy. To test this hypothesis, we electroporated adult rat tibialis anterior muscles with DDp53 and collected the tissue three weeks later. We confirmed successful overexpression of DDp53 on a histological and biochemical level and found pronounced changes to muscle architecture, metabolism, and molecular signaling. Muscle mass, fiber cross-sectional area, and fiber diameter significantly decreased with DDp53 overexpression. We found histopathological changes in DDp53 transfected muscle which were accompanied by increased levels of proteins that are associated with membrane damage and repair. In addition, DDp53 decreased oxidative phosphorylation complex I and V protein levels, and despite its negative effects on muscle mass and fiber size, caused an increase in muscle protein synthesis as assessed via the SUnSET technique. Interestingly, the increase in muscle protein synthesis was concomitant with a decrease in phospho-S6K1 (Thr389). Furthermore, the muscle wasting in the DDp53 electroporated leg was accompanied by a decrease in global protein ubiquitination and an increase in proteasome activity. In conclusion, overexpression of a dominant-negative p53 mutant in skeletal muscle results in decreased muscle mass, myofiber size, histological muscle damage, a metabolic phenotype, and perturbed homeostasis between muscle protein synthesis and degradation.

Western blotting (immunoblotting) is a powerful and commonly used technique that is capable of detecting or semiquantifying an individual protein from complex mixtures of proteins extracted from cells or tissues. The history surrounding the origin of western blotting, the theory behind the western blotting technique, a comprehensive protocol and the uses of western blotting are presented. Lesser known and significant problems in the western blotting field and troubleshooting of common problems are highlighted and discussed. This work is a comprehensive primer and guide for new western blotting researchers and those interested in a better understanding of the technique or getting better results.

Background Pesticides are important biological agents that deter or kill pests. Globally, about 2 million tons of pesticides are being utilized each year which highlights their importance. Previous studies have suggested that high levels of pesticide exposure are associated with increased risk of chronic diseases, including cancers, cardiotoxicity, Parkinson’s disease, diabetes mellitus, neurological deficits, birth defects, and reproductive disorders. Prometryn is a commonly used selective triazine herbicide, which is currently used as an alternative to atrazine‐based products. It is widely applied to agriculture as it has lower acute toxicity than atrazine herbicides, stable chemical properties, and a long period of effectiveness. Although past studies have shown that triazine herbicides have carcinogenic potential in humans, the cytotoxic effects of prometryn on cardiac and hepatic systems are not well investigated or understood. The aim of this study was to determine the effects of prometryn on the heart and liver. Methods H9c2 cardiac and HepG2 liver carcinoma cells were used to study the in‐vitro effects of prometryn. Cells were treated with varying concentrations of prometryn for 24 h. Cell viability, mitochondrial membrane potential, and reactive oxygen species (ROS) levels were assessed. For animal studies, male mice (10 weeks old) (n=24) were randomized to two treatment groups (control and prometryn). The control group received corn oil while the prometryn treated group were exposed to 185mg/kg b.w. prometryn dissolved in corn oil every 48 hours for 7 days. After a total of 4 doses, mice were euthanized, and the heart and liver tissues were collected. Proteasome and immunoproteasome activity were measured in the heart and liver. Protein expression levels of markers for oxidative stress, apoptosis, and proteasomal degradation such as GSTT2, GSTA1/2, Hsp70, Hsp27, NADPH oxidase, PARP‐1, p53, PSMD12, LMP2, DNP, VU‐1 were detected using immunoblotting. Protein oxidation and polyubiquitination were detected as an indicator of oxidative stress. A paired t‐test was used to assess group differences for protein levels and p values <0.05 were deemed statistically significant. Result We found that prometryn had a concentration‐dependent effects on H9c2 and HepG2 cell lines when we assessed cell viability, mitochondria function, and total intracellular reactive oxygen species formation. Increasing the concentration of prometryn increased the ROS formation, decreased cell viability and decreased mitochondrial membrane potential in these cell lines. Furthermore, in animal studies we observed changes in the proteasome and immunoproteasome activities in the liver but not in the heart. Changes in protein expression levels of oxidative stress and proteasomal degradation markers were only detected in the liver. Conclusion These results indicate that H9c2 and HepG2 cells are sensitive to prometryn in‐vitro. However, prometryn effects on the heart and liver tissue in male mice is significantly different. Overall, our data s...

Cancer-associated cachexia (CAC) is a critical contributor to pancreatic ductal adenocarcinoma (PDAC) mortality. Thus, there is an urgent need for new strategies to mitigate PDAC-associated cachexia; and the exploration of dietary interventions is a critical component. We previously observed that a ketogenic diet (KD) combined with gemcitabine enhances overall survival in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. In this study, we investigated the effect and cellular mechanisms of a KD in combination with gemcitabine on the maintenance of skeletal muscle mass in KPC mice. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD), a KD, a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. We observed that a KD or a KG-mitigated muscle strength declined over time and presented higher gastrocnemius weights compared CD-fed mice. Mechanistically, we observed sex-dependent effects of KG treatment, including the inhibition of autophagy, and increased phosphorylation levels of eIF2α in KG-treated KPC mice when compared to CG-treated mice. Our data suggest that a KG results in preservation of skeletal muscle mass. Additional research is warranted to explore whether this diet-treatment combination can be clinically effective in combating CAC in PDAC patients.