RESUMO
Weeds are one of the main causes of the decrease in crop yields, with Johnsongrass (Sorghum halepense L.) being one of the most significant. Weeds can be controlled by herbicides, but some have developed resistance. Quantitative PCR is the technique of choice for studying gene expression related to herbicide resistance because of its high sensitivity and specificity, although its quantitative accuracy is highly dependent on the stability of the reference genes. Thus, in this study we evaluated the stability of different reference genes of glyphosate-resistant S. halepense. Nine genes frequently used as reference genes were selected: MDH, ADP, PP2A, EIF4α, ACT, ARI8, DnaJ, Hsp70, and ALS1, and their expression analyzed in susceptible and resistant biotypes at 0, 24 and 72 h post-application of glyphosate. The stability was analyzed with the geNorm, NormFinder, and BestKeeper software programs and using the ΔCt method. RefFinder was used to generate a comprehensive stability ranking. The results showed that PP2A and ARI8 were the most stable genes under the test conditions. EPSPS expression was also verified against the best two and the worst two reference genes. This study provides useful information for gene expression analysis under glyphosate stress and will facilitate resistance mechanism studies in this weed species.
RESUMO
Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0-24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function.
