Spectrophotometric and Smartphone-Assisted Determination of Phenolic Compounds Using Crude Eggplant Extract.

In order to develop a simple, reliable and low cost enzymatic method for the determination of phenolic compounds we studied polyphenol oxidase activity of crude eggplant (S. melongena) extract using 13 phenolic compounds. Catechol, caffeic and chlorogenic acids, and L-DOPA have been rapidly oxidized with the formation of colored products. Monophenolic compounds have been oxidized at a much slower speed. Ferulic acid, quercetin, rutin, and dihydroquercetin have been found to inhibit polyphenol oxidase activity of crude eggplant extract. The influence of pH, temperature, crude eggplant extract amount, and 3-methyl-2-benzothiazolinone hydrazone (MBTH) concentration on the oxidation of catechol, caffeic acid, chlorogenic acid, and L-DOPA has been investigated spectrophotometrically. Michaelis constants values decrease by a factor of 2 to 3 in the presence of MBTH. Spectrophotometric (cuvette and microplate variants) and smartphone-assisted procedures for phenolic compounds determination have been proposed. Average saturation values (HSV color model) of the images of the microplate wells have been chosen as the analytical signal for smartphone-assisted procedure. LOD values for catechol, caffeic acid, chlorogenic acid, and L-DOPA equaled 5.1, 6.3, 5.8 and 30.0 µM (cuvette procedure), 12.2, 13.2, 13.2 and 80.4 µM (microplate procedure), and 23.5, 26.4, 20.8 and 120.6 µM (smartphone procedure). All the variants have been successfully applied for fast (4-5 min) and simple TPC determination in plant derived products and L-DOPA determination in model biological fluids. The values found with smartphone procedure are in good agreement with both spectrophotometric procedures values and reference values. Using crude eggplant extract- mediated reactions combined with smartphone camera detection has allowed creating low-cost, reliable and environmentally friendly analytical method for the determination of phenolic compounds.

Mechanisms of Inflammatory Injury of Renal Tubular Cells in a Cellular Model of Pyelonephritis.

Previously, we have assembled a cellular model of pyelonephritis which contains a primary culture of renal tubular epithelial cells, mononuclear leukocytes, and bacterial lysate or lipopolysaccharide. After cocultivation of renal cells with leukocytes and bacterial lysate, proinflammatory changes were observed in the renal cells, followed by nitrosative and oxidative stress and cell death. The interaction of bacterial antigens not only with leukocytes, but also with epithelial cells of the renal tubules, was partially mediated by signaling pathways involving Toll-like receptors (TLR2 and TLR4). Activation of these receptors led to increased levels of oxidative stress and synthesis of proinflammatory cytokines (TNF, IL-6, IL-1α) in the renal epithelium, while TLR4 blockade decreased the severity of these processes. Apart from the fact that activation of inflammatory signaling in response to bacterial antigens is observed directly in the renal cells, the presence of leukocytes significantly amplifies the inflammatory response as measured by the level of cytokines generated in the ensemble. In the presence of activated leukocytes, higher expression of TLR2 on the surface of renal cells was observed in response to exposure to bacterial components, which might explain the increased inflammatory response in the presence of leukocytes. The synthesis of IL-1α in the epithelial cells of the renal tubules in this inflammatory model leads to its accumulation in the nuclei, which has been reduced by the TLR4 antagonist polymyxin. TLR2 agonists also led to increased levels of IL-1α. The elevation in the content of IL-1α in nuclei was accompanied by increased acetylation of nuclear proteins, which has been reduced to control values after exposure to protective agents (Trolox, mitochondria-targeted antioxidant SkQR1 or LiCl). The high level of acetylation of histones is probably regulated by proinflammatory cytokines, and to some extent it is a marker of inflammation, which can indirectly be reduced by protective agents.

Perspectives of mitochondrial medicine.

Mitochondrial medicine was established more than 50 years ago after discovery of the very first pathology caused by impaired mitochondria. Since then, more than 100 mitochondrial pathologies have been discovered. However, the number may be significantly higher if we interpret the term "mitochondrial medicine" more widely and include in these pathologies not only those determined by the genetic apparatus of the nucleus and mitochondria, but also acquired mitochondrial defects of non-genetic nature. Now the main problems of mitochondriology arise from methodology, this being due to studies of mitochondrial activities under different models and conditions that are far from the functioning of mitochondria in a cell, organ, or organism. Controversial behavior of mitochondria ("friends and foes") to some extent might be explained by their bacterial origin with possible preservation of "egoistic" features peculiar to bacteria. Apparently, for normal mitochondrial functioning it is essential to maintain homeostasis of a number of mitochondrial elements such as mitochondrial DNA structure, membrane potential, and the system of mitochondrial quality control. Abrogation of these elements can cause a number of pathologies that have become subjects of mitochondrial medicine. Some approaches to therapy of mitochondrial pathologies are discussed.

Mitochondria-targeted antioxidant SkQR1 ameliorates gentamycin-induced renal failure and hearing loss.

The influence of the mitochondria-targeted antioxidant SkQR1 on gentamycin-induced nephrotoxicity and ototoxicity has been analyzed. SkQR1 reduces the death of kidney epithelium cells and decreases the severity of renal failure caused by gentamycin application and also lowers the animals' mortality. Treatment with SkQR1 also decreases gentamycin-induced hearing loss. Mitochondria-targeted antioxidants, such as SkQR1, are new promising agents for preventing negative consequences of therapy with antibiotics.

The phenoptosis problem: what is causing the death of an organism? Lessons from acute kidney injury.

Programmed execution of various cells and intracellular structures is hypothesized to be not the only example of elimination of biological systems - the general mechanism can also involve programmed execution of organs and organisms. Modern rating of programmed cell death mechanisms includes 13 mechanistic types. As for some types, the mechanism of actuation and manifestation of cell execution has been basically elucidated, while the causes and intermediate steps of the process of fatal failure of organs and organisms remain unknown. The analysis of deaths resulting from a sudden heart arrest or multiple organ failure and other acute and chronic pathologies leads to the conclusion of a special role of mitochondria and oxidative stress activating the immune system. Possible mechanisms of mitochondria-mediated induction of the signaling cascades involved in organ failure and death of the organism are discussed. These mechanisms include generation of reactive oxygen species and damage-associated molecular patterns in mitochondria. Some examples of renal failure-induced deaths are presented with mechanisms and settings determined by some hypothetical super system rather than by the kidneys themselves. This system plays the key role in the process of physiological senescence and termination of an organism. The facts presented suggest that it is the immune system involved in mitochondrial signaling that can act as the system responsible for the organism's death.

New-generation Skulachev ions exhibiting nephroprotective and neuroprotective properties.

A mitochondria-targeted chimeric compound consisting of a rhodamine derivative linked to a plastoquinone molecule (10-(6'-plastoquinonyl)decylrhodamine, SkQR1) was studied under conditions of acute brain or kidney damage. A protective effect of this compound was demonstrated in a model of focal brain ischemia, rat kidney ischemia/reperfusion, myoglobinuria (rhabdomyolysis, or crush syndrome), and pyelonephritis. We found that a single intraperitoneal injection of SkQR1 diminishes the size of the ischemic zone in the brain and improves performance of a test characterizing neurological deficit in ischemic animals. Control substance not containing plastoquinone appeared to be not neuroprotective. The data show that SkQR1 is a nephroprotectant and neuroprotectant, which can be due to the antioxidative action of this Skulachev cation.