Emil Kryukov

Geographic atrophy or late-stage dry age-related macular degeneration (AMD) is characterized by drusen deposition and progressive retinal pigment epithelium (RPE) degeneration, leading to irreversible vision loss. The formation of drusen leads to dyshomeostasis, oxidative stress, and irreversible damage to the RPE. In this study, we used an in vitro model of oxidized low-density lipoprotein (ox-LDL)-induced human RPE damage/death to investigate the mechanism through which a sterically hindered phenol antioxidant compound, PMC (2,2,5,7,8-pentamethyl-6-chromanol), protects the RPE against ox-LDL-induced damage. We show that PMC exerts its protective effect by preventing the upregulation of stress-responsive heme oxygenase-1 (HMOX1/HO-1) and NAD(P)H: quinone oxidoreductase (NQO1) at the mRNA and protein levels. This effect was due to PMC’s blockade of ROS generation, which in turn blocked nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, ultimately preventing the upregulation of antioxidant response elements (AREs), including HMOX1 and NQO1. The key role of HO-1 was demonstrated when the protective effect of PMC was inhibited by the knockdown of HMOX1. Additionally, PMC treatment under different experimental conditions and at different time points revealed that the continuous presence of PMC is required for the optimal protection against ox-LDL-induced cytotoxicity, defining the cellular pharmacokinetics of this molecule. Our data demonstrate the involvement of a key antioxidant pathway through which PMC mitigates the oxidative stress induced by ox-LDL and provides a potential therapeutic strategy for suppressing RPE degeneration/damage during AMD progression.

Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor “bridge” cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3A^high human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in “bridge” progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.

The aim was to study the volume of blood loss and the duration of a surgical session in patients with destructive pulmonary tuberculosis using threedimensional anatomical models of the lungs. Methods. The study was conducted in 2020 – 2021 in the State Budgetary Healthcare Institution of the Nizhniy Novgorod region “Nizhny Novgorod Regional Clinical Tuberculosis Dispensary” as part of an internal grant from the Federal State Budgetary Educational Institution of Higher Education “Privolzhsky Research Medical University” of the Ministry of Health of the Russian Federation. The prospective clinical study included 80 patients. All patients were divided into two groups. The main group included 40 patients. Preoperative lung reconstruction was performed for these patients. Thoracic surgeons used the three-dimensional anatomical models of the lungs to study the syntopy of healthy and diseased tissue (visual assessment) and prepare for the surgical session (cutting the model with a scalpel). Threedimensional preoperative lung reconstruction was not performed for the other 40 patients in the control group. In this group, the thoracic surgeons did not use 3D anatomical models of the lungs. Comparison of the results in the control and the main group was carried out using the Mann – Whitney U-test and Spearman and Kendall correlation tests. The obtained data are described with absolute and relative values, boxplots reflecting median (Me) and interquartile interval (IQI), scatter plots with trend lines, and probability density plots. The level of statistical significance of differences for testing the hypotheses was chosen at p < 0.05. Results. It was shown that the volume of blood loss (U = 590; p = 0.042) and the duration of surgery (U = 587; p = 0.041) in patients of the main group are smaller than in patients in the control group. A moderate correlation was established between the volume of blood loss and the duration of the surgical session (ρ = 0.54; p < 0.001) in the main group using the Spearman’s test. Kendall’s test also showed a positive correlation (τ = 0.42; p < 0.001). Conclusion. The established differences between the main group and control group in the volume of blood loss and the duration of surgery due to the inclusion of polymer models of the lungs in preoperative preparation of thoracic surgeons confirm our hypothesis about the effect of additive technologies on the above parameters and indicate the significance of the applied reconstruction method.

The present study focused on anaerobic digestion (AD) performance, microbial community, as well as morphological and topological features of biofilms on various materials used to promote direct interspecies electron transfer (DIET) during AD of low-concentration swine manure. The best AD kinetics were observed in stainless steel mesh/digested cow manure (CM) and polyester felt/digested sewage sludge (SS) combinations used as material/inoculum, respectively. Microbial analysis of biofilms revealed the dominance of putatively syntrophic bacteria of the MBA03 group of the Limnochordia class in CM inoculated reactors, and syntrophic proteolytic bacteria of the genus Coprothermobacter and acetogenic Clostridium sensu stricto 1 , known for their ability to carry out DIET, in SS inoculated reactors. Biofilms on non-conductive materials contained pili-like structures, which were observed only in SS inoculated reactors. Polyester felt had denser biofilms than carbon felt, regardless of the inoculum used. These results provide new insights into the different types of DIET that can occur in low-loaded AD systems with attached growth.

Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects.

SPIE Digital Library Proceedings

The changes in cell metabolism can affect the epigenome-modifying enzymes activity during iPSCs differentiation and thus control the functional potential of the final cell. Therefore, for therapeutic applications, the restoration of a fully functional mitochondrial network specific for the cell types derived from iPSCs will be required to support the energy and other mitochondrial factors. Recently, FLIM method allows to study the metabolic changes that accompanying cell differentiation noninvasively and without additional labels. In this study, we investigated the metabolic changes in iPSCs during neural differentiation using two-photon fluorescence microscopy and FLIM. Cellular metabolism was examined by monitoring the optical redox ratio (FAD/NAD(P)H), the fluorescence lifetime contributions of the free and bound forms of NADH and NADPH. Given that neural differentiation is also accompanied by synthetic processes and oxidative stress, this process was included in the scope of this work. We demonstrated an increased contribution of protein-bound NADH and NADPH in neuron associated with metabolic switch to oxidative phosphorylation and the biosynthetic processes or oxidative stress, respectively. We also found that the optical redox ratio FAD/NAD(P)H decreased during neural differentiation, and this was likely to be explained by the intensive lipid membrane synthesis or ROS generating and the enhanced NADPH production associated with them. The biochemical analysis was carried out to verify the metabolic status of iPSCs and their neural derivatives. Based on the data on glucose consumption, lactate and ATP amount we registered the trend to the metabolic pathways redistribution towards the oxidative phosphorylation in neuron.

The differentiation of endothelial cells from human iPSC has incontestable advantages in diseases research and therapeutic applications. However, the safe use of iPSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Shifts in cellular metabolism associated with intracellular pH changes affect the enzymes that control epigenetic configuration, which impact chromatin reorganization and gene expression changes during reprogramming and differentiation. FLIM-based metabolic imaging of NADH and FAD is a powerful tool for measuring mitochondrial metabolic state and widely used diagnostic method for identification of neoplastic diseases, skin diseases, ocular pathologies and stem cells differentiation. Therefore, in this study, we used the potential of FLIM-based metabolic imaging and fluorescence microscopy of NADH and FAD to study the metabolic changes during iPSC differentiation in endothelial cells. The evaluation of the intracellular pH was carried out with the fluorescent pH-sensor SypHer-2 and fluorescence microscopy to obtain complete information about metabolic status of iPSC and their endothelial derivatives. Based on the FAD/NAD(P)H optical redox ratios increase and the contributions rise of the NAD(P)H fluorescence lifetime in iPSC during endothelial differentiation, we demonstrated an contribution increase of OXPHOS to cellular metabolism. Based on the shift toward more acidic intracellular pH in endothelial cell derived from iPSCs we verified their oxidative state.