RNA sequencing (RNAseq) is currently a method of choice for the high-throughput RNA-level analysis of gene expression. Furthermore, RNAseq data can be used for the prediction of numerous cancer biomarkers e.g. microsatellite instability, tumor mutational burden, gene signatures, and immunohistochemical markers expression. In this analysis, central step is comparison with the pre-existing pool of normal/healthy control tissue profiles. However, technically different RNAseq platforms and protocols usually provide poorly compatible gene expression outputs that can be difficult to pool together and analyze in a direct comparison due to platform/protocol-specific bias. We recently published Oncobox RNA sample preparation and sequencing protocol for Illumina platform that can be used for the analysis of gene expression in cancer molecular diagnostics to personalize treatments, as validated in preclinical and clinical studies. Here we report adaptation of this protocol for DNBSEQ-G50 engine of a competitor MGI sequencing platform. We demonstrate common clustering and similar gene expression portraits for the RNAseq profiles obtained for the same 16 formalin-fixed, paraffin-embedded model experimental cancer biosamples using both Illumina and MGI sequencing platforms. The adopted Oncobox protocol enables retention of the case-to-normal ratios, calculated values of molecular pathway activation, and also of predicted cancer drug efficiency scores. Our findings suggest clinical applicability of Oncobox molecular diagnostics with both Illumina and MGI sequencing platforms. This also evidence that no specific data harmonization is needed to compare the molecular profiles obtained with either platform when using the Oncobox protocol, e.g. with the previously published ANTE experimental panel of normal tissues.

Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) was applied to monitor simultaneously apoptosis using the genetically encoded sensor of caspase-3, mKate2-DEVD-iRFP, and the autofluorescence of redox cofactors in colorectal cancer cells upon stimulation of apoptosis with staurosporine, cisplatin or hydrogen peroxide. We found that, irrespective of the apoptotic stimulus used, ROS accumulation correlated well with both the elevated pool of mitochondrial, enzyme-bound NADH and caspase-3 activation. Meanwhile, a shift in the contribution of bound NADH could develop independently of the apoptosis, and this was observed in the case of cisplatin. An increase in the proportion of bound NADPH was detected only in staurosporine-treated cells, this likely being associated with a high level of ROS production and their resulting detoxification. The results of the study favor the discovery of new therapeutic strategies based on manipulation of the cellular redox balance, which could help improve the anti-tumor activity of drugs and overcome apoptotic resistance.

Cytostatic chemotherapeutics provide a classical means to treat cancer, but conventional treatments have not increased in efficacy in the past years, warranting a search for new approaches to therapy. The aim of the study was, therefore, to obtain methacrylic acid (MAA) (co)polymers and to study their immunopharmacological properties. 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CDSPA) and 2-cyano-2-propyl dodecyl trithiocarbonate (CPDT) were used as reversible chain transfer agents. Experiments were carried out in Wistar rats. The MTT assay was used to evaluate the cytotoxic effect of the polymeric systems on peritoneal macrophages. An experimental tumor model was obtained by grafting RMK-1 breast cancer cells. Serum cytokine levels of tumor-bearing rats were analyzed. The chain transfer agents employed in classical radical polymerization substantially reduced the molecular weight of the resulting polymers, but a narrow molecular weight distribution was achieved only with CDSPA and high CPDT concentrations. Toxicity was not observed when incubating peritoneal macrophages with polymeric systems. In tumor-bearing rats, the IL-10 concentration was 1.7 times higher and the IL-17 concentration was less than half that of intact rats. Polymeric systems decreased the IL-10 concentration and normalized the IL-17 concentration in tumor-bearing rats. The maximum effect was observed for a MAA homopolymer with a high molecular weight. The anion-active polymers proposed as carrier constituents are promising for further studies and designs of carrier constituents of drug derivatives.

The phase of the cell cycle determines numerous aspects of cancer cell behaviour including invasiveness, ability to migrate and responsiveness to cytotoxic drugs. To non-invasively monitor progression of cell cycle in vivo, a family of genetically encoded fluorescent indicators, FUCCI (fluorescent ubiquitination-based cell cycle indicator), has been developed. Existing versions of FUCCI are based on fluorescent proteins of two or more different colors fused to cell-cycle-dependent degradation motifs. Thus, FUCCI-expressing cells emit light of different colors in different phases providing a robust way to monitor cell cycle progression by fluorescence microscopy and flow cytometry but limiting the possibility to simultaneously visualize other markers. To overcome this limitation, we developed a single-color variant of FUCCI, called FUCCI-Red, which utilizes two red fluorescent proteins with distinct fluorescence lifetimes, mCherry and mKate2. Similarly to FUCCI, these proteins carry cell cycle-dependent degradation motifs to resolve G1 and S/G2/M phases. We showed utility of FUCCI-Red by visualizing cell cycle progression of cancer cells in 2D and 3D cultures and monitoring development of tumors in vivo by confocal and fluorescence lifetime imaging microscopy (FLIM). Single-channel registration and red-shifted spectra make FUCCI-Red sensor a promising instrument for multiparameter in vivo imaging applications, which was demonstrated by simultaneous detection of cellular metabolic state using endogenous fluorescence in the blue range.

The aim Was to study the role of post-translational modifications of cofilin in the regulation of respiration and autophagy in murine brain mitochondria.MATERIALS AND METHODS:The experiments were performed with C57BL/6 mice. To obtain cytoplasmic and mitochondrial fractions of the brain tissue, differential centrifugation was used. Expressions of cofilin, phospho-cofilin, K48- and K63-associated chains of ubiquitin, and the autophagy marker LC3B were determined using electrophoresis, immunoprecipitation and Western blot methods. To study the processes of ubiquitination, we used PR619 - the inhibitor of deubiquitinating enzymes. Respiratory activity of brain mitochondria was evaluated using high-resolution fluorespirometry.RESULTS:Modification of cofilin by non-canonical K63 multiubiquitin chains in the cytoplasm and mitochondria from murine brain was demonstrated. Different levels of phospho-cofilin, cofilin, and its ubiquitinated proteoforms were found. PR619, the inhibitor of deubiquitinating enzymes, affects the expression of phosphorylated and ubiquitinated forms of cofilin in the mitochondria and cytoplasm, at the same time it changes the activity of tissue respiration and mitophagy.CONCLUSION:The sensitivity of cofilin to the inhibitor of deubiquitinating enzymes indicates the existence of a new non-catabolic mechanism of cofilin modification, which may be involved in the regulation of mitochondrial functions, specifically, the mitochondrial respiration and autophagy. The data help understand the molecular mechanisms of mitochondrial function in normal and pathological conditions, which may be useful in developing novel methods for the treatment of diseases of the nervous system.

A series of methacrylic acid co(polymers) were obtained using various reversible chain transfer agents. The molecular weight characteristics of the synthesized (co)polymers were determined using gel permeation chromatography. In vitro studies showed the absence of cytotoxicity of the obtained compounds against immune cells (peritoneal macrophages) and the absence of an effect on the functional activity of the cells.

The aim of the present study was to evaluate the current body of knowledge regarding tumor-associated macrophages (TAMs) and their potential use in antitumor therapy, based on their role in the pathological process of tumorigenesis. For this purpose, a critical analysis of published data and summarization of the findings available from original studies, focusing on the role of TAMs in the pathological process, and their potential therapeutic application was performed. Promising key avenues of research were identified in this field. The following issues seem the most promising and thus worth further investigation: i) The process of M1/M2 macrophage polarization, macrophage characteristics at intermediate polarization steps and their role in the tumor process; ii) determining the conditions necessary for transitions between the M1 and M2 macrophage phenotypes and the role of signals from the microenvironment in this process; iii) cause-and-effect associations between the quantity and quality of macrophages, and the prognosis and outcome of the pathological process; iv) modulation of macrophages and stimulation of their phagocytic activity with drugs; v) targeted vector-based systems for drug delivery to macrophages; and vi) targeted drug delivery systems with macrophages as carriers, thus potentially combining chemotherapy and immunotherapy.

The ubiquitin-proteasome system is a major protein degradative pathway involved in the maintenance of cellular structure and function. Actin cytoskeleton plays a critical role in the morphology and structural changes of the nerve cells. Cofilin is a main regulator of actin filament assembly/disassembly. Many diseases of the nerve system are associated with actin-cofilin remodeling. Nevertheless, the role of ubiquitylation in the regulation of cofilin activity and actin cytoskeleton structure in the nerve cells is little investigated. Confocal fluorescence microscopy-based methods are valuable tools for the precise imaging and quantitative evaluation of actin cytoskeleton changes. The aim of this research was to analyze the effect of a proteasome inhibitor MG132 on actin cytoskeleton remodeling in the nerve cells using fluorescence imaging. Cofilin was shown to be colocalized with actin filaments and ubiquitin in hippocampal cells. Analysis of cofilin proteoforms revealed the presence of cofilin modified by K63-linked multiubiquitin chains. The treatment of hippocampal cells with MG132 led to the changes in the structure of actin cytoskeleton, expression of cofilin, and activation of autophagy. Therefore, the results suggest the relationship between cofilin ubiquitylation and actin cytoskeleton remodeling in the nerve cells. It may be important for the development of new methods of diagnosis and treatment of the nerve system disorders.

Actin-binding proteins and, in particular, members of the actin-depolymerization factor (ADF)/cofilin family, are involved in the regulation of the actin cytoskeleton in response to various intracellular and extracellular signals. Recent studies point to the exceptional role of this group of proteins in the development and functioning of the nervous system. This review presents the latest data on the functions of cofilin in the cell and the signaling pathways involved in its regulation. Special attention is paid to studies of the relationship between cofilin and actin dynamics in such processes as the control of synaptic plasticity, apoptosis of neurons, and neuroinflammation. We show the molecular mechanisms of cofilin activation-inactivation and the specific structure of actin in nerve cells during neurodegeneration in the in vitro and in vivo models. We review new directions in the study of cofilin and related proteins as prognostic markers and therapeutic targets in the diagnosis and treatment of diseases of the nervous system.

Cysteine proteases, caspases, play an important role in the process of programmed cell death. In apoptosis, the cell signaling pathways overlap at executioner caspases regardless of the initial stimulus. Caspase-3 is the key one. This review considers the sensors for activity of caspase-3 and some other caspases. Over the past decade, many such sensors utilizing a variety of detection principles have been created; fluorescence and luminescence are the most common detection methods. The intracellular sensors are of particular interest; they allow visualization of the activation of caspases in living systems. Herein, we briefly describe sensors of various designs, the advantages and disadvantages of which must be taken into account when choosing a sensor for a particular experimental system.