In silico specificity determination of neoantigen-reactive T-lymphocytes

Effective personalized immunotherapies of the future will need to capture not only the peculiarities of the patient’s tumor but also of his immune response to it. In this study, using results of in vitro high-throughput specificity assays, and combining comparative models of pMHCs and TCRs using molecular docking, we have constructed all-atom models for the putative complexes of all their possible pairwise TCR-pMHC combinations. For the models obtained we have calculated a dataset of physics-based scores and have trained binary classifiers that perform better compared to their solely sequence-based counterparts. These structure-based classifiers pinpoint the most prominent energetic terms and structural features characterizing the type of protein-protein interactions that underlies the immune recognition of tumors by T cells.

Identification of the side population associated with ATP-binding cassette transporters activity using imaging flow cytometry

DyeCycle Violet efflux, caused by ATP-binding cassette transporters activity, was analyzed in human colorectal adenocarcinoma cell lines SW480, HT-29, Caco-2 by neans of FACSAria III flow cytometer and ImageStreamX Mk II imaging flow cytometer. Along with similarity of cytometry data obtained on the two instruments, the use of imaging flow cytometry made it possible to characterize the morphology of side population cells, as well as morphology of other cell populations differing in the degree of dye accumulation. The population of cells, which are smaller than the side population cells and practically do not take the dye, is of the special interest. Probably, this population may contribute to the tumor resistance to chemotherapy.

Atmospheric reactive oxygen species and some aspects of the antiviral protection of the respiratory epithelium

The review focuses on molecular and biochemical mechanisms of nonspecific protection of respiratory epithelium. The authors provide a comprehensive analysis of up-to-date data on the activity of the lactoperoxidase system expressed on the surface of the respiratory epithelium which provides the generation of hypothiocyanate and hypoiodite in the presence of locally produced or inhaled hydrogen peroxide. Molecular mechanisms of production of active compounds with antiviral and antibacterial effects, expression profiles of enzymes, transporters and ion channels involved in the generation of hypothiocyanite and hypoiodate in the mucous membrane of the respiratory system in physiological and pathological conditions (inflammation) are discussed. In the context of antibacterial and antiviral defense special attention is paid to recent data confirming the effects of atmospheric air composition on the efficiency of hypothiocyanite and hypoiodate synthesis in the respiratory epithelium. The causes and outcomes of lactoperoxidase system impairment due to the action of atmospheric factors are discussed in the context of controlling the sensitivity of the epithelium to the action of bacterial agents and viruses. Restoration of the lactoperoxidase system activity can be achieved by application of pharmacological agents aimed to compensate for the lack of halides in tissues, and by the control of chemical composition of the inhaled air.

Methylation profiles of genes in breast cancer luminal HER2-negative primary tumor during regional lymphnode metastasis

Aberrant methylation is strongly associated with development of cancer, but limited data exist on correlation between methylation and regional lymph node metastasis (RLNM). The aim of this research was to study using of methylation levels of WIF1, RASSF1A, CDO1 and MEST aberrant methylated genes in a primary breast cancer for prediction of regional lymph node metastases. We used MS-HRM (Methylation Sensitive High Resolution Melting) to assess methylation levels. The results were confirmed by pyrosequencing. The study included 66 women with LumA and 46 women with HER2- (LumB-), 22 and 26 of them had metastasis in at least one lymph node respectively. It was found that methylation levels between LumA and LumB subtypes differed significantly in genes: WIF1 (p<0.001), CDO1 (p=0.002) and MEST (p=0.033). In the Lum A subtype statistically significant differences in level of methylation of WIF1 gene between patients with metastases in RLNM and patients without metastases were found (p=0.03). Analysis of tumors longer than 2 cm in the LumA subtype, revealed an increase of statistical significance of WIF1 gene — p=0.009 (AUC (95%CI) = 0.76 (0.59−0.93)). In LumB- subtype RASSF1A, CDO1 and MEST had statistically significant differences in methylation level between groups (p=0.03, p=0.048 and p=0.045 respectively). ROC analysis showed that combining of three genes by logistic regression, AUC (95%CI) was 0.74 (0.6−0.88). Analysis of tumors longer than 2 cm, did not increase statistical significance for these genes (p=0.046; p=0.089 and p=0.076, respectively). Thus, the study of methylation in primary tumors may be useful for prediction of lymph node metastasis, as well as for better understanding of biological process inside breast cancer.

Features of the in vitro expression profile of hippocampal neurogenic niche cells during optogenetic stimulation

In the central nervous system of mammals, there are specialized areas in which neurogenesis — neurogenic niches — is observed in the postnatal period. It is believed that astrocytes in the composition of neurogenic niches play a significant role in the regulation of neurogenesis, and therefore they are considered as a promising “target” for the possible control of neurogenesis, including the use of optogenetics. In the framework of this work, we formed an in vitro model of a neurogenic niche, consisting of cerebral endothelial cells, astrocytes and neurospheres. Astrocytes in the neurogenic niche model expressed canalorodopsin ChR2 and underwent photoactivation. The effect of photoactivated astrocytes on the expression profile of neurogenic niche cells was evaluated using immunocytochemical analysis methods. It was found that intact astrocytes in the composition of the neurogenic niche contribute to neuronal differentiation of stem cells, as well as the activation of astroglia expressing photosensitive proteins, changes the expression of molecules characterized by intercellular interactions of pools of resting and proliferating cells in the composition of the neurogenic niche with the participation of NAD+ (Cx43, CD38, CD157), lactate (MCT1). In particular, the registered changes reflect a violation of the paracrine intercellular interactions of two subpopulations of cells, one of which acts as a source of NAD+, and the second as a consumer of NAD+ to ensure the processes of intracellular signal transduction; a change in the mechanisms of lactate transport due to aberrant expression of the lactate transporter MCT1 in cells forming a pool of cells developing along the neuronal path of differentiation. In general, with photostimulation of niche astrocytes, the total proliferative activity increases mainly due to neural progenitor cells, but not neural stem cells. Thus, optogenetic activation of astrocytes can become a promising tool for controlling the activity of neurogenesis processes and the formation of a local proneurogenic microenvironment in an in vitro model of a neurogenic niche.

Changes in the mitochondrial subproteome of mouse brain Rpn13-binding proteins induced by the neurotoxin MPTP and the neuroprotector isatin

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.

The effect of L-carnitine depletion induced by long-term therapy of mice with meldonium on brain mitochondrial balance

Meldonium is a metabolic drug used for treatment of coronary heart disease. The effect of the drug lies in its ability to inhibit synthesis and transport of L-carnitine. At the same time, a long-term deficiency of L-carnitine can theoretically negatively affect the activity of the transcription factor Nrf2, which is extremely important for maintaining mitochondrial balance in cells. We have shown that meldonium therapy for 3 months at a dose of 100 mg/kg in mice causes a decrease in the expression of the Nrf2 gene in the brain. A decrease in the Nrf2 level causes suppression of mitochondrial biogenesis, which is manifested in a decrease in the level of mtDNA and the level of Cox1 expression. However, no negative effect of meldonium on the bioenergetics parameters of mitochondria was found, as evidenced by the maintenance of a stable mitochondrial potential and the level of production of reactive oxygen species. Jne mohth after the end of the meldonium therapy, expression of the genes responsible for mitochondrial biogenesis and mitophagy (p62, Pink1, Tfam) was observed and the expression level of genes responsible for mitochondrial fusion returned to control values. These changes may be associated with the normalization of the level of L-carnitine in brain cells.

The effect of lipid derivative of anti-tumor drug sarcolysin embedded in phospholipid nanoparticles in the experiments in vivo

To improve the therapeutic properties of the antitumor agent Sarcolysin, we have previously developed and characterized a dosage form representing its ester conjugate with decanol embedded in ultra-small phospholipid nanoparticles less than 30 nm in size (“Sarcolysin-NP”). The effect of the resulting composition was investigated in vivo in comparison with the free substance of sarcolysin. The composition intravenous administration to mice showed an improvement in the pharmacokinetic parameters of sarcolysin associated with its initial higher (by 22%) level in the blood and prolonged circulation, which was also observed in mice with P388 tumor. In mice with three types of tumors — lymphocytic leukemia P388, lymphocytic leukemia L1210, and adenocarcinoma of the mammary gland Ca755 — administration of two doses of sarcolysin over a period of 7 days showed its predominant antitumor effect. The maximum tumor growth inhibition was noted for lymphocytic leukemia L1210 and adenocarcinoma of the mouse mammary gland Ca755 (at a dose of Sarcolysin-NP — 8,4 mg/kg), which was higher in comparison with free substance by more than 24% and 17%, respectively. Differences in the life span of the treated animals were revealed significantly at a dose of 10 mg/kg and amounted to 25% and 17,4% for lymphocytic leukemia P388 and L1210, respectively, and 11% for adenocarcinoma Ca755. In an experiment on rats, acute toxicity of Sarcolysin-NP administered intravenously showed that an average LD50 value 2-3 times exceeded a similar parameter for commercial preparations of free sarcolysin (Melphalan and Alkeran), which indicates its lower toxicity.

Antibacterial effects of peptides synthesized based on the sequence of ribosome protein S1

Antibiotic resistance of bacteria is a topical problem on a global scale. Sometimes vigorous human activity leads to an increase in the number of bacteria carrying resistance genes in the environment. Antimicrobial peptides (AMPs) and similar compounds are potential candidates for combating antibiotic-resistant bacteria. Previously, we proposed and successfully tested on Thermus thermophilus a new mechanism of AMP action. This mechanism of directed coaggregation is based on the interaction of a peptide capable of forming fibrils with a target protein. In this work, we discuss the criteria for choosing a target for the targeted action of AMP, describe the features of the “parental” S1 ribosomal proteins T. thermophilus and Escherichia coli and the studied peptides using bioinformatic analysis methods, assess the antimicrobial effect of the synthesized peptides on a model organism of E. coli and cytotoxicity on cells of human fibroblasts. The obtained results will be important for the creation of new AMPs for pathogenic organisms.

Molecular mechanism of chromogenic substrate hydrolysis in the active site of human carboxylesterase-1

Human carboxylesterases are involved in the protective processes of detoxification during the hydrolytic metabolism of xenobiotics. Knowledge of the molecular mechanisms of substrates hydrolysis in the enzymes active site is necessary for the rational drug design. In this work, the molecular mechanism of the hydrolysis reaction of para-nitrophenyl acetate in the active site of human carboxylesterase was determined using modern methods of molecular modeling. According to the combined method of quantum mechanics/molecular mechanics calculations, the chemical reaction occurs within four elementary steps, including two steps of the acylation stage, and two steps of the deacylation stage. All elementary steps have low energy barriers, with the gradual lowering of the intermediate energies that stimulates reaction in the forward direction. The molecular docking was used to estimate the binding constants of the enzyme-substrate complex and the dissociation constant of enzyme-product complexes. The effective kinetic parameters of the enzymatic hydrolysis in the active site of carboxylesterase are determined by numerical solution of the differential kinetic equations.