Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.

Acclimation of photosynthetic processes and metabolic responses to elevated temperatures in cereals.

The aim of the present work was to better understand the molecular mechanisms of heat acclimation processes in cereals. A large number of winter and spring wheat, barley and oat varieties were grown under either control conditions (22/20°C) or under a mild heat stress (30°C) that induce the acclimation processes. The temperature dependence of chlorophyll a fluorescence induction and gas exchange parameters showed that heat acclimation increased the thermotolerance of the photosynthetic apparatus, but these changes did not differ sharply in the winter-spring type cereals. Similarly, to wheat, elevated temperature also led to increasing transpiration rate and reduced water use efficiency in barley and oat plants. A non-targeted metabolomic analysis focusing on polar metabolites in two selected barley (winter type Mv Initium and spring type Conchita) and in two oat varieties (winter type Mv Hópehely and spring type Mv Pehely) revealed substantial differences between both the two species and between the acclimated and non-acclimated plants. Several compounds, including sugars, organic acids, amino acids and alcohols could be separated and detected. The expression level of the CYP707, HSP90, galactinol synthase, raffinose synthase and α-galactosidase genes showed genotype-dependent changes after 1 day; however, the CYP707 was the only one, which was still upregulated in at least some of the genotypes. Results suggest that heat acclimation itself does not require general induction of primary metabolites. However, induction of specific routes, e.g. the induction of the raffinose family oligosaccharides, especially the synthesis of galactinol, may also contribute the improved heat tolerance in cereals.

Modulated Light Dependence of Growth, Flowering, and the Accumulation of Secondary Metabolites in Chilli.

Chili is widely used as a food additive and a flavouring and colouring agent and also has great importance in health preservation and therapy due to the abundant presence of many bioactive compounds, such as polyphenols, flavonoids, carotenoids, and capsaicinoids. Most of these secondary metabolites are strong antioxidants. In the present study, the effect of light intensity and spectral composition was studied on the growth, flowering, and yield of chilli together with the accumulation of secondary metabolites in the fruit. Two light intensities (300 and 500 µmol m-2 s-1) were applied in different spectral compositions. A broad white LED spectrum with and without FR application and with blue LED supplement was compared to blue and red LED lightings in different (80/20 and 95/5%) blue/red ratios. High light intensity increased the harvest index (fruit yield vs. biomass production) and reduced the flowering time of the plants. The amount of secondary metabolites in the fruit varied both by light intensity and spectral compositions; phenolic content and the radical scavenging activity were stimulated, whereas capsaicin accumulation was suppressed by blue light. The red colour of the fruit (provided by carotenoids) was inversely correlated with the absolute amount of blue, green, and far-red light. Based on the results, a schematic model was created, representing light-dependent metabolic changes in chilli. The results indicated that the accumulation of secondary metabolites could be modified by the adjustment of light intensity and spectral composition; however, different types of metabolites required different light environments.

Stressed tadpoles mount more efficient glucocorticoid negative feedback in anthropogenic habitats due to phenotypic plasticity.

Coping with anthropogenic environmental change is among the greatest challenges faced by wildlife, and endocrine flexibility is a potentially crucial coping mechanism. Animals may adapt to anthropogenic environments by dampening their glucocorticoid stress response, but empirical tests of this hypothesis have provided mixed evidence. An alternative hypothesis is that a non-attenuated stress response and efficient negative feedback are favored in anthropogenic habitats. To test this idea, we non-invasively sampled corticosterone release rates of common toad (Bufo bufo) tadpoles in agricultural, urban, and natural habitats, and quantified their stress response and negative feedback by a standardized stress-and-recovery protocol. We repeated the same sampling with tadpoles raised from eggs from the same ponds in a common-garden experiment to infer if the differences observed between populations in different habitats were due to individual phenotypic plasticity rather than microevolution or transgenerational effects. We found that, compared to tadpoles in natural ponds, urban tadpoles had higher baseline and stressed corticosterone release rates, and tadpoles in agricultural ponds had similar corticosterone release rates but greater stress-induced change, indicating stronger stress responses in both types of anthropogenic habitats. As predicted, tadpoles in both agricultural and urban ponds showed more efficient negative feedback than did tadpoles in natural ponds. Water pollution levels, as indicated by the concentrations of carbamazepine and corticoid-disrupting compounds in pond water, contributed to elevating the stress response regardless of land use. Infection by neither Batrachochytrium dendrobatidis nor Ranavirus was detected in free-living tadpoles. No habitat-related glucocorticoid differences persisted in the common-garden experiment. These results suggest that toad tadpoles in anthropogenic habitats increased their glucocorticoid flexibility via phenotypic plasticity. The coupling of stronger stress response and stronger negative feedback in these habitats supports the importance of rapidly "turning on and off" the stress response as a mechanism for coping with anthropogenic environmental change.

Contribution of Sample Processing to Variability and Accuracy of the Results of Pesticide Residue Analysis in Plant Commodities.

Significant reduction of concentration of some pesticide residues and substantial increase of the uncertainty of the results derived from the homogenization of sample materials have been reported in scientific papers long ago. Nevertheless, performance of methods is frequently evaluated on the basis of only recovery tests, which exclude sample processing. We studied the effect of sample processing on accuracy and uncertainty of the measured residue values with lettuce, tomato, and maize grain samples applying mixtures of selected pesticides. The results indicate that the method is simple and robust and applicable in any pesticide residue laboratory. The analytes remaining in the final extract are influenced by their physical-chemical properties, the nature of the sample material, the temperature of comminution of sample, and the mass of test portion extracted. Consequently, validation protocols should include testing the effect of sample processing, and the performance of the complete method should be regularly checked within internal quality control.