Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure.

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

[Plasma Exosomes in Inherited Forms of Parkinson's Disease].

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Alpha-synuclein misfolding and aggregation resulting in neurototoxicity is a hallmark of PD. The prion properties of alpha-synuclein are still under discussion. Exosomes (extrcellular vesicles 40-100 nm in size) can play a key role in the transport of pathogenic forms of alpha-synuclein. The most frequent inherited forms of the disease are PD associated with mutation in the leucine-rich repeat kinase 2 (LRRK2-PD) and glucocerebrosidase (GBA-PD) genes. The aim of our work is to evaluate the concentration and size of exosomes derived from blood plasma of patients with GBA-PD, asymptomatic GBA mutation carriers, and the effect of GBA and LRRK2 mutations on alpha-synuclein level in exosomes derived from peripheral blood plasma. Plasma extracellular vesicles were isolated via chemical precipitation and sequential ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis (NTA), and flow cytometry. Total alpha-synuclein level in plasma exosomes was estimated by enzyme-linked immunosorbent assay. Patients with sporadic PD, PD with dementia, patients with inherited PD (GBA-PD, LRRK2-PD), and GBA mutation carriers were included in the study. The concentration on plasma exosomes was higher in GBA-PD patients that in sporadic PD patients, asymptomatic carriers of mutations on GBA gene, and control (p = 0.004, 0.019 and 0.0001 respectively). The size of plasma exosomes was higher in GBA-PD patients compared to asymptomatic carriers of GBA mutations and control (p = 0.009 and 0.0001, respectively). No significant difference was found for exosomal alpha-synuclein levels in the studied groups. Our results allowed us to suggest that a decrease in GBA activity may affect the pool of plasma exosomes, and mutations in the LRRK2 and GBA genes do not influence the level of plasma exosomal alpha-synuclein.

[Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods]. / Sravnitel'nyi analiz metodov vydeleniia vnekletochnykh mikrovezikul iz kul'tural'noi sredy.

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by "horizontal" exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional features of the EV is being commonly studies in in vitro condition. Several methods of EV isolation from cell culture medium are established, however selection of method might influence on obtained results. The choice of the optimal method depends usually from the amount of medium and the aims of the research while is still challenging issue. We performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with a 30% sucrose/D2O "cushion", precipitation with plant proteins and immune-affinity capturing. EV isolated by different approaches were compared in terms of following parameters: size, concentration, morphology of EV, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, RNA, and several glioma-associated miRNAs. Applied methods included nano-patricle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. On the base of obtained results, we developed practical recommendations that may help researchers to make a best choice of EV isolation method.

AGGREGATION BY LECTIN-METHODICAL APPROACH FOR EFFECTIVE ISOLATION OF EXOSOMES FROM CELL CULTURE SUPERNATANT FOR PROTEOME PROFILING.

Exosomes are small membrane vesicles secreted by most cell types in vivo and in vitro. Exosomes are found in cell culture media, blood, urine, amniotic fluid, malignant ascite fluids and contain distinct subsets of microRNAs and proteins depending upon the tissue from which they are secreted. Thus exosomes constitute potential biomarkers of human diseases, such as cancer. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. Isolation of exosomes can be a tedious, non-specific, and difficult process. Here, we provide a preparative technique for isolation of exosomes based on their ability to aggregate in the presence of lectins. The new method for lectin-based isolation of exosomes from cell culture media was developed as a sample preparation step for exosome-based protein biomarker research.

[Histone deacetylase inhibitors cause the TP53-dependent induction of p21/Waf1 in tumor cells carrying mutations in TP53].

p21/Waf1 protein is one of the main cell cycle arrest regulators and one of the most well-known transcriptional targets of TP53 protein. Here, we demonstrated the activation of expression of the p21/Waf1 gene when the cells were treated to sodium butyrate (NaBu)--one of the natural inhibitors of deacetylase, and investigated whether this phenomenon depends on the presence of functionally active TP53 protein. We compared the effect of the NaBu treatment on the human cell line with different TP53 mutation profile, including: wild-type TP53, single nucleotide substitutions, and the complete absence of TP53 gene. NaBu activated the TP53 protein via hyper acetylation at lysine residue K382, without significant changes in the level of protein expression. Western blotting demonstrated that the addition of NaBu triggers a significant increase in the p21/Waf1 protein level in both the TP53 wild-type cells and in the cells with single nucleotide substitutions in the domain responsible for the binding of TP53 protein to DNA. At the same time, no the p21/Waf1 protein induction was observed in the cells with complete deletion of the TP53 gene. However, NaBu was not able to induce the p2 1/Waf1 production when the expression of TP53 was transiently knocked down by the p53 siRNA. Overall, our results suggest that the NaBu-dependent induction of p21/Waf1 does require the presence of TP53 protein but unexpectedly it can occur regardless of mutational changes in the domain responsible for the TP53 binding to DNA. One of the hypothetical explanations is that NaBu increases the level of TP53 acetylation, and the modified protein is able to establish a new network of protein-protein interactions or trigger some conformational changes affecting the TP53-dependent transcriptional machinery even when its DNA binding ability is impaired.

[Aberrant expression of sox2 gene in malignant gliomas].

Both genetic and epigenetic changes underlite the mechanisms of tumor initiation and progression. In the present study we analyze sox2 gene expression and its epigenetic regulation in primary cultures of malignant gliomas. The sox2 expression was detected in the vast majority (74%) of the investigated gliomas and absent in morphologically normal brain tissue. This indicates the process of glioma malignant transformation. We have also shown that the association of different areas of the sox2 gene with important epigenetic markers, posttranslational modifications of H3 histone H3K4ac and H3K9met3, does not correlate with the sox2 expression. However, this may indicate the stochastic nature of the regulation of sox2 gene expression in malignant gliomas.

[Human RAD51 recombinase: the role in the cell cycle checkpoint and cellular survival].

The RAD51 protein, an eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous recombination. Here, we demonstrate that short-term silencing of Rad51 gene by specific small interfering (si) RNA induces cell death of the most part of investigated cancer cell lines and normal fibroblasts. Disruption of the Rad51 gene in these cells results in S or (and) G2 cell cycle arrest leading to apoptosis. But some human cancer cell lines demonstrate abolishment of pre-mitotic checkpoint and are not sensitive to siRNA silencing of RAD51 recombinase. Recent experiments show that normal functioning of the recombination repair system is essential for maintenance of genome stability, proliferation of vertebrate cells and, finally, for prevention of dramatic cell death.

[Response to tamoxifen in estrogen receptor-positive cell line MCF-7 is independent of p53 expression].

The study was concerned with identification of predictive value of p53 expression on sensitivity to tamoxifen in breast cancer management. Estrogen receptor-positive cell line MCF-7 was used to establish p53 expression influence on the rate of cell proliferation after tamoxifen. The investigation demonstrated the absence of that effect when p53 was silenced.

[Microsatellite instability of genome in cells of sporadic and hereditary carcinoma of the gastrointestinal tract]. / Mikrosatellitnaia nestabil'nost' genoma v kletkakh sporadicheskikh i nasledstvennykh kartsinom zheludochno-kishechnogo trakta.

Microsatellite instability (MIN) of human genome, i.e. instability of very short (1-5 nt) DNA tandem repeats, points to a deficiency in the mismatch repair system (MMR). To investigate the role of MMR in sporadic and hereditary carcinogenesis in the gastrointestinal tract, four types of carcinomas were compared: sporadic (GC), familial (FGC) gastric carcinoma, sporadic colorectal (CC) and hereditary nonpolyposis colorectal (HNPCC) carcinoma. No significant difference in MIN frequency was found between GC (9 out of 27) (33%) and CC (7 out of 29) (24%). In hereditary carcinoma group, MIN occurrence appeared 2-3 times as high: FGC in 7 out of 10 (70%) and HNPCC in 6 out of 8 patients (75%). No significant differences were recorded in MIN occurrence at early and later stages of the disease in all groups. Therefore, it can be suggested that disorders in the MMR develop at earlier stages of carcinogenesis and may be responsible for tumor progression.

[Damage to the transforming growth factor TGF-beta type II receptor gene and microsatellite instability in carcinoma cells of the gastrointestinal tract]. / Povrezhdeniia gena retseptora tipa II transformiruiushchego faktora rosta TGF-beta i mikrosatellitnaia nestabil'nost' genoma v kletkakh kartsinom zheludochno-kishechnogo trakta.

In order to compare the frequency of damage to the transforming growth factor TGF-beta receptor type II gene (RII gene) and microsatellite instability (MIN) in oncogenesis of sporadic and hereditary cancer of gastrointestinal tract (GIT), 4 groups of carcinomas were analyzed. They included sporadic gastric (GC), family gastric (FGC), sporadic colorectal (CC) and hereditary nonpolyposis colorectal (HNPCC) carcinomas having appropriate clinical and pathological characteristics. Each group consisted of two types of carcinomas, one of them showing MIN. The RII gene damage occurred in 89% of GC (8 cases out of 9), 86% of CC (6 out of 7), 71% of FGC (5 out of 7), 50% of HNPCC (3 out of 6) for carcinomas coupled with MIN, whereas only in 6% (1 out of 18) of GC and 5% (1 out of 22) of CC for carcinomas without MIN. No damage to RII gene was found in the cases of hereditary carcinomas which did not show MIN though the number of cases analyzed was not sufficient for final conclusions (3 cases of FGC and 3 HNPCC). The data revealed a correlation between the MIN phenotype and mutations in RII gene both for sporadic (p < 0.001) and for hereditary (p < 0.02) cases. For all 4 groups the frequency of RII gene damage was found for early and advanced carcinomas. This suggests that the deficiency of TGF-beta receptor complex in both sporadic and hereditary carcinomas of GIT is revealed at early stages of tumor development and consequently may be responsible for tumor progression. The correlation between RII gene damages and MIN in GIT carcinoma cells suggests that genetic change predetermined the neoplasia of colorectal and gastric epithelium and partially overlapped for both sporadic and hereditary cases.