Vol 15, No 4 (2023)

Mitochondrial diseases (MDs) associated with nuclear gene mutations are part of a large group of inherited diseases caused by the suppression of energy metabolism. These diseases are of particular interest, because nuclear genes encode not only most of the structural proteins of the oxidative phosphorylation system (OXPHOS), but also all the proteins involved in the OXPHOS protein import from the cytoplasm and their assembly in mitochondria. Defects in any of these proteins can lead to functional impairment of the respiratory chain, including dysfunction of complex I that plays a central role in cellular respiration and oxidative phosphorylation, which is the most common cause of mitopathologies. Mitochondrial diseases are characterized by an early age of onset and a progressive course and affect primarily energy-consuming tissues and organs. The treatment of MDs should be initiated as soon as possible, but the diagnosis of mitopathologies is extremely difficult because of their heterogeneity and overlapping clinical features. The molecular pathogenesis of mitochondrial diseases is investigated using animal models: i.e. animals carrying mutations causing MD symptoms in humans. The use of mutant animal models opens new opportunities in the study of genes encoding mitochondrial proteins, as well as the molecular mechanisms of mitopathology development, which is necessary for improving diagnosis and developing approaches to drug therapy. In this review, we present the most recent information on mitochondrial diseases associated with nuclear gene mutations and animal models developed to investigate them.

The recent advances achieved in microscopy technology have led to a significant breakthrough in biological research. Super-resolution fluorescent microscopy now allows us to visualize subcellular structures down to the pin-pointing of the single molecules in them, while modern electron microscopy has opened new possibilities in the study of protein complexes in their native, intracellular environment at near-atomic resolution. Nonetheless, both fluorescent and electron microscopy have remained beset by their principal shortcomings: the reliance on labeling procedures and severe sample volume limitations, respectively. Soft X-ray microscopy is a candidate method that can compensate for the shortcomings of both technologies by making possible observation of the entirety of the cellular interior without chemical fixation and labeling with an isotropic resolution of 40–70 nm. This will thus bridge the resolution gap between light and electron microscopy (although this gap is being narrowed, it still exists) and resolve the issue of compatibility with the former, and possibly in the near future, the latter methods. This review aims to assess the current state of soft X-ray microscopy and its impact on our understanding of the subcellular organization. It also attempts to look into the future of X-ray microscopy, particularly as relates to its seamless integration into the cell biology toolkit.

This review focuses on in vitro modeling of diseases and the development of therapeutic strategies using iPSCs for the two most common types of optical pathologies: hereditary neuropathies and retinopathies. Degeneration of retinal ganglion cells and the subsequent optic nerve atrophy leads to various types of neuropathies. Damage to photoreceptor cells or retinal pigment epithelium cells causes various retinopathies. Human iPSCs can be used as a model for studying the pathological foundations of diseases and for developing therapies to restore visual function. In recent years, significant progress has also been made in creating ganglionic and retinal organoids from iPSCs. Different research groups have published data pertaining to the potential of using iPSCs for the modeling of optic neuropathies such as glaucoma, Leber hereditary optic neuropathy, etc., including in the development of therapeutic approaches using gene editing tools.

One of the major problems of regenerative medicine is the development of hypertrophic scars and keloids. The protein kinase RIPK3 is involved in necroptosis; however, recent evidence indicates that it also has non-canonical functions, including its involvement in the development of renal fibrosis. The aim of our work was to study the expression of RIPK3 in mouse and human skin models of fibrotic processes. A subpopulation of RIPK3+Vim+ cells was found in both human keloid and a mouse wound, with the cell number being significantly greater in the mouse wound bed compared to healthy skin. Real-time polymerase chain reaction (RT-PCR) detected expression of the Ripk3 and fibroblast biomarkers Acta2, Fap, Col1a1, and Fn1 in the cells isolated from the wound bed, indicating that RIPK3 can be expressed by wound bed fibroblasts. An analysis of the human fibroblasts stained with anti-RIPK3 antibodies demonstrated an increase in the fluorescence intensity in the presence of lipopolysaccharide (LPS) at concentrations of 5, 10, 25, 50, and 100 ng/ml and TGF-β at concentrations of 0.1, 1, 2, and 5 ng/ml compared to the control. At the same time, the expression levels of RIPK3 and fibroblast activation markers in the presence of TGF-β and LPS did not differ significantly from the control. It is possible that RIPK3 expression in wound fibroblasts is not directly associated with fibrotic processes, and that kinase plays a different, yet unknown role in wound healing.

The coronavirus disease (COVID-19) pandemic has brought into sharp relief the threat posed by coronaviruses and laid the foundation for a fundamental analysis of this viral family, as well as a search for effective anti-COVID drugs. Work is underway to update existent vaccines against COVID-19, and screening for low-molecular-weight anti-COVID drug candidates for outpatient medicine continues. The opportunities and ways to accelerate the development of antiviral drugs against other pathogens are being discussed in the context of preparing for the next pandemic. In 2012–2015, Tsyshkova et al. synthesized a group of water-soluble low-molecular-weight compounds exhibiting an antiviral activity, whose chemical structure was similar to that of arbidol. Among those, there were a number of water-soluble compounds based on 5-methoxyindole-3-carboxylic acid aminoalkyl esters. Only one member of this rather extensive group of compounds, dihydrochloride of 6-bromo-5-methoxy-1-methyl-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole, exhibited a reliable antiviral effect against SARS-CoV-2 in vitro. At a concentration of 52.0 μM, this compound completely inhibited the replication of the SARS-CoV-2 virus with an infectious activity of 10⁶ TCID₅₀/mL. The concentration curves of the analyzed compound indicate the specificity of its action. Interferon-inducing activity, as well as suppression of syncytium formation induced by the spike protein (S-glycoprotein) of SARS-CoV-2 by 89%, were also revealed. In view of its synthetic accessibility − high activity (IC₅₀ = 1.06 µg/mL) and high selectivity index (SI = 78.6) − this compound appears to meets the requirements for the development of antiviral drugs for COVID-19 prevention and treatment.

Despite the significant potential of photodynamic therapy (PDT) as a minimally invasive treatment modality, the use of this method in oncology has remained limited due to two serious problems: 1) limited penetration of the excitation light in tissues, which makes it impossible to affect deep-seated tumors and 2) use of chemical photosensitizers that slowly degrade in the body and cause photodermatoses and hyperthermia in patients. To solve these problems, we propose a fully biocompatible targeted system for PDT that does not require an external light source. The proposed system is based on bioluminescent resonance energy transfer (BRET) from the oxidized form of the luciferase substrate to the photosensitizing protein SOPP3. The BRET-activated system is composed of the multimodal protein DARP-NanoLuc-SOPP3, which contains a BRET pair NanoLuc-SOPP3 and a targeting module DARPin. The latter provides the interaction of the multimodal protein with tumors overexpressing tumor-associated antigen HER2 (human epidermal growth factor receptor type II). In vitro experiments in a 2D monolayer cell culture and a 3D spheroid model have confirmed HER2-specific photo-induced cytotoxicity of the system without the use of an external light source; in addition, experiments in animals with subcutaneous HER2-positive tumors have shown selective accumulation of DARP-NanoLuc-SOPP3 on the tumor site. The fully biocompatible system for targeted BRET-induced therapy proposed in this work makes it possible to overcome the following limitations: 1) the need to use an external light source and 2) the side phototoxic effect from aberrant accumulation of chemical photosensitizers. The obtained results demonstrate that the fully protein-based self-excited BRET system has a high potential for targeted PDT.