Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry最新文献

When developing vaccines and immunotherapeutic and enzymatic drugs, it is usually necessary to obtain a functional protein preparation, which can later be used for immunization or analytical studies in the process of creating a drug. Production in E. coli bacterial cells is the fastest and cheapest way to obtain such proteins. However, recombinant proteins, when produced in E. coli, often form insoluble and functionally inactive aggregates. The purpose of this work is to create a set of expression vectors for rapid screening of optimal conditions for the production of recombinant proteins prone to aggregation in E. coli in soluble form. The work used modern genetic engineering methods, including optimization and de novo synthesis of nucleotide sequences encoding helper polypeptides. For the production and chromatographic purification of proteins, the standard strain BL21(DE3) and the affinity chromatography method using a metal chelate sorbent were used. As a result, a set of nine vectors with helper polypeptides was obtained to increase solubility and increase product yield during the production of recombinant proteins in E. coli cells. The efficiency of obtaining protein drugs prone to molecular aggregation using this set of vectors has been demonstrated by the exampe of three cytokines: interleukin-31 (IL-31), interleukin-33 (IL-33), and transforming growth factor beta (TGF-beta1). The resulting set allows for rapid selection of a suitable helper polypeptide as well as optimization of conditions for obtaining a soluble form of recombinant proteins in E. coli during the pharmaceutical development of therapeutic drugs.

The development of effective delivery systems for antitumor drugs with increased specificity and controlled release is one of the challenges for biomedical chemistry. This paper presents studies of non-covalent interactions of human serum albumin, which may be a promising carrier for drug delivery, with antitumor antibiotic doxorubicin and folic acid, which have the potential of a guide ligand. Intermolecular interactions of doxorubicin and folic acid with human serum albumin were studied by spectroscopic methods at various pH levels and temperatures. The binding strength of doxorubicin and folic acid responded differently to pH changes. The affinity of the drug to protein increased with the transition from acidic to alkaline conditions, while pH 7.4 was optimal for binding for folic acid. In the case of the triple system, it was found that there was no significant effect of albumin complexation with folic acid on non-covalent interaction with doxorubicin. As expected, binding to these active compounds altered the conformation of the protein. At the same time, this change was minimal in physiological pH for folic acid and in alkaline for doxorubicin. Additionally, the therapeutic properties of doxorubicin, non-covalently bound to human serum albumin, were shown to be preserved in vitro.

Melanoma is one of the most aggressive types of cancer. Melanoma morbidity is increasing every year and mortality rate is high besides target therapy. Calorie restriction is lifestyle approach that can alter the activity of the pathways regulating key processes of melanomagenesis. The signaling cascades modulated by fasting includes JAK/STAT. Female С57Bl/6 mice were used for the investigation of the calorie restriction effect on the tumor growth and development. Control group is the mice on ad libitum diet, CR group is the mice on 30% calorie restricted diet. B16 melanoma cell transplantation has been carried out in the mice of the both groups after 3 months of ad libitum and CR regimes. During 15 days after melanoma cell implantation the formation of solid tumor has been occurred. Then the animals were euthanized and average mouse weight and tumor mass in both groups were evaluated. JAK/STAT gene expression was assessed by means of real-time PCR. The activity of glutathione peroxidase and glutathione-S-transferase and glutathione level were registered spectrophotometrically. 30% calorie restriction in mice with B16 melanoma decreased of tumor mass by 1.8 times (р = 0.049) as compared with the tumor mass of control group animals. STAT1 expression increased by 1.9 times, STAT3, STAT5b, STAT6, JAK1 and JAK2 expression decreased by 2.8, 3.8, 2.6, 3 and 4.5 times accordingly. Activity of glutathione peroxidase, glutathione-S-transferase and glutathione level decreased by 1.6, 3 and 1.5 times accordingly compare with control group. The received data can become pathogenical basis for the creation of new adjuvant method of anticancer therapy.

Many disorders can be accurately diagnosed using ribonucleic acids (RNAs). A panel of RNAs specific to colorectal cancer (CRC), generated in silico, was used in this study. This panel is composed of Nucleosome Assembly LINC00654 (Long Intergenic Non-Protein Coding RNA 654) long nucleolar RNA, SRY-box transcription factor 5 (sox5 mRNA) mRNA, small nucleolar RNA host gene (Sox5), and homo sapiens microRNA-133a (miR-133a) from the genetic and epigenetic database based on in silico data analysis. Validation and characterization of the proposed RNA network were done by qPCR in sera samples from 130 cases. These cases included 70 CRC patients with a malignant tumour, 40 patients with a benign tumour, and 20 healthy controls. Moreover, the panel expression was verified in a representative CRC, HT29, cell line. Our data revealed that the expression of LINC00654 and Sox5 RNAs was higher in the sera from CRC compared with the control group, while miR-133a showed the opposite expression pattern. These data may, at least in part, validate the in-silico relationship and enhance the possibility that miR-133a might be sponged by LINC00654 and thus leave the chance for Sox5 upregulation in CRC patients. Taken together, our findings may introduce a novel molecular network. Therefore, this RNA panel could be recommended as a potential diagnostic marker for CRC patients.

The processes of progressive decrease in muscle mass and weakening of mitochondrial function that occur during aging in skeletal muscles remain poorly understood as does, above all, the cause-and-effect relationship between the ultrastructure of mitochondria and atrophic processes in skeletal muscles. An ultrastructural study of the features of the internal structural organization of mitochondria during aging of skeletal muscle was carried out on representatives of rapidly aging mammalian species (Wistar rats, OXYS, mice) and representatives of long-lived species: the naked mole rat (Heterocephalus glaber) and human. Previously unknown, age-related structural changes in the internal organization of skeletal muscle mitochondria in OXYS rats at the age of 24 months are shown: the appearance in each mitochondria of local areas of altered arrangement of cristae in the form of stellate structures, as well as the presence of extremely large structural formations, apparently the result of destructive processes in mitochondria, as well as the appearance of mitochondria that are abnormal in size and internal ultrastructure. It was shown that structural changes in mice at the age of 10 months and naked mole rats at the age of 11 years were multidirectional. If disturbances in the normal ultrastructure in mice affected not only mitochondria but also muscle fibers and the sarcoplasmic reticulum, then not only no pathological changes are observed in mole rats but also, on the contrary, the powerfully developed structure of mitochondria indicates the functional activity of these organelles. For the first time, the ultrastructure of mitochondria in human skeletal muscle at the age of 68–81 and 25–28 years was compared using biopsy material. In elderly patients, the phenomenon of mitochondrial proliferation is shown: a compensatory structural response to mitochondrial dysfunction. Mitochondria are small, with a small number of cristae. In young people, the ultrastructure of mitochondria corresponded to classical ideas about the features of the structural organization of skeletal muscle mitochondria. Literary ideas about the possible role of autophagy in the development of aging processes are considered.

Mast cells (MCs) are an important population of connective tissue cells that play a key role in the development of allergic diseases. The main pathway of MC activation in allergies is associated with the interaction of antigen complexes with immunoglobulin E and their subsequent binding to the FcεRI receptor. This leads to rapid release of secretory granules and cytokine production. In recent years, there has been increasing evidence for the involvement of autophagy in many processes, including MC function. Therefore, autophagy regulators can be used as potential inhibitors of MC activity and as therapeutic agents for the treatment of allergic diseases. In the present study, we investigated the effects of two autophagy inhibitors SBI-0206965 and LY294002, and one autophagy activator rapamycin on FcεRI-dependent activation of RBL-2H3, which usually used as model of MCs and basophils. Cell activation was assessed by determining the content of the secretory granule marker β-hexosaminidase and the levels of the cytokines TNF, IL-4 and IL-13. Treatment of cells with SBI-0206965 and LY294002 was shown to reduce FcεRI-dependent degranulation and IL-4 cytokine secretion by RBL-2H3 cells, while the use of rapamycin resulted in reduction of the level of IL-13 cytokine. This indicates the prospect of potential application of these autophagy regulators in the therapy of allergic diseases.

Background: This study assessed the association of vitamin D status with the frequency of incidence of metabolic health risk factors, including obesity, hypo-alpha cholesterol, hypertriglyceridemia, carbohydrate metabolism disorders (hyperglycemia and insulin resistance), and cardiovascular system stress conditions in men of different ages. Aim: The research aimed at identifying cardiometabolic disorders in young, mature, and elderly men with suboptimal and optimal concentrations of 25(OH) vitamin D. Materials and methods: Two hundred and thirty-four Magadan oblast residents at early adulthood, maturity, and extreme old age participated in the survey. Each age group was divided into two samples: with optimal or suboptimal vitamin D level. We used photometric, immunochemiluminescent research methods, as well as standard methods for assessing body mass index and cardiovascular system. Results: The results showed rather high incidence of suboptimal 25(OH) vitamin D levels among the Magadan oblast population. Optimal vitamin D concentration was associated with a significant fall in the percentage of people with a hypertensive character of cardiovascular performance, with a lower incidence of hypoalphacholesterolemia, hyperglycemia, hypertriglyceridemia, and insulin resistance throughout all age groups, and was more common in elderly men. Conclusions: The suboptimal concentration of vitamin D can be considered as an additional nonstandard driving factor for age-associated cardiometabolic disorders. Metabolic healthcare needs preventive measures to level these disorders to contribute to active longevity and life expectancy.

Different efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) has been observed between lung cancer patients with 19 exon deletion (19Del) and with L858R mutation. We investigate the multi-omics information from the TCGA Lung adenocarcinoma (LUAD) dataset and validate it using the GEO (GSE190139, GSE147377) and MSK datasets. Somatic loss-of-function alteration of RBM10 and altered Immune infiltration profile correlated with L858R decreased survival. Meanwhile, 9p21.3 loss, CDKN2B methylation, and increased cell cycle-related gene expression are differential characteristics in the L858R mutation group. Comprehensive genomic and phenotypic analysis of the EGFR-mutated lung cancer subtypes reveals distinctive features of each subtype, laying the groundwork for subtype-specific treatment and care options for lung cancer patients.

The E3 ubiquitin ligase Cullin 5 (CUL5) has been linked to a variety of cell biological functions, such as developmental process regulation, DNA repair, and cell cycle control, but the role of CUL5 in the unfolded protein response (UPR) remains unclear. We found that the knocked-down of the CUL5 gene results in the upregulated levels of binding immunoglobulin protein (BiP) protein, a major chaperone protein in unfolded protein response (UPR), whereas the over-expression of CUL5 downregulated BiP protein levels in breast cancer cells. Further investigation revealed that CUL5 binds with BiP, leading to the ubiquitination of BiP. Our findings suggest that CUL5 is involved critically in the proteasome-degradation of BiP, leading to weaker UPR.

This work is an overview of deep machine learning methods aimed at predicting binding sites in protein structures. Several classes of methods are selected: prediction of binding sites for small molecules, proteins, and nucleic acids. For each class, various approaches to prediction are considered (prediction of binding atoms, residues, surfaces, pockets). Specifics of feature selection and neural network architectures inherent to each class and approach are highlighted, and an attempt is made to explain these specifics and foresee the further direction of their development.