Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3β (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading.     

Long Term Immune Response Produced by the SputnikV Vaccine

SputnikV is a vaccine against SARS-CoV-2 developed by the Gamaleya National Research Centre for Epidemiology and Microbiology. The vaccine has been shown to induce both humoral and cellular immune responses, yet the mechanisms remain largely unknown. Forty SputnikV vaccinated individuals were included in this study which aimed to demonstrate the location of immunogenic domains of the SARS-CoV-2 S protein using an overlapping peptide library. Additionally, cytokines in the serum of vaccinated and convalescent COVID-19 patients were analyzed. We have found antibodies from both vaccinated and convalescent sera bind to immunogenic regions located in multiple domains of SARS-CoV-2 S protein, including Receptor Binding Domain (RBD), N-terminal Domain (NTD), Fusion Protein (FP) and Heptad Repeats (HRs). Interestingly, many peptides were recognized by immunized and convalescent serum antibodies and correspond to conserved regions in circulating variants of SARS-CoV-2. This breadth of reactivity was still evident 90 days after the first dose of the vaccine, showing that the vaccine has induced a prolonged response. As evidenced by the activation of T cells, cellular immunity strongly suggests the high potency of the SputnikV vaccine against SARS-CoV-2 infection.     

Myelodysplastic Syndromes and Metabolism

Myelodysplastic syndromes (MDS) are acquired clonal stem cell disorders exhibiting ineffective hematopoiesis, dysplastic cell morphology in the bone marrow, and peripheral cytopenia at early stages; while advanced stages carry a high risk for transformation into acute myeloid leukemia (AML). Genetic alterations are integral to the pathogenesis of MDS. However, it remains unclear how these genetic changes in hematopoietic stem and progenitor cells (HSPCs) occur, and how they confer an expansion advantage to the clones carrying them. Recently, inflammatory processes and changes in cellular metabolism of HSPCs and the surrounding bone marrow microenvironment have been associated with an age-related dysfunction of HSPCs and the emergence of genetic aberrations related to clonal hematopoiesis of indeterminate potential (CHIP). The present review highlights the involvement of metabolic and inflammatory pathways in the regulation of HSPC and niche cell function in MDS in comparison to healthy state and discusses how such pathways may be amenable to therapeutic interventions.     

Electrostatic Map of the SARS-CoV-2 Virion Specifies Binding Sites of the Antiviral Cationic Photosensitizer

Electrostatics is an important part of virus life. Understanding the detailed distribution of charges over the surface of a virus is important to predict its interactions with host cells, antibodies, drugs, and different materials. Using a coarse-grained model of the entire viral envelope developed by D. Korkin and S.-J. Marrink’s scientific groups, we created an electrostatic map of the external surface of SARS-CoV-2 and found a highly heterogeneous distribution of the electrostatic potential field of the viral envelope. Numerous negative patches originate mainly from negatively charged lipid domains in the viral membrane and negatively charged areas on the “stalks” of the spike (S) proteins. Membrane (M) and envelope (E) proteins with the total positive charge tend to colocalize with the negatively charged lipids. In the E protein pentamer exposed to the outer surface, negatively charged glutamate residues and surrounding lipids form a negative electrostatic potential ring around the channel entrance. We simulated the interaction of the antiviral octacationic photosensitizer octakis(cholinyl)zinc phthalocyanine with the surface structures of the entire model virion using the Brownian dynamics computational method implemented in ProKSim software (version r661). All mentioned negatively charged envelope components attracted the photosensitizer molecules and are thus potential targets for reactive oxygen generated in photosensitized reactions.     

Neural network (ANN) approach to biodiesel analysis: Analysis of biodiesel density, kinematic viscosity, methanol and water contents using near infrared (NIR) spectroscopy

The use of ethanol and biodiesel, which are alternative fuels or biofuels, has increased in the last few years. Modern official standards list 25 parameters that must be determined to certify biodiesel quality, and these analyses are expensive and time-consuming. Near infrared (NIR/NIRS) spectroscopy (4000-12,820 cm⁻¹) is a cheap and fast alternative to analyse biodiesel quality, when compared with infrared, Raman, or NMR methods, and quality control can be done in realtime (on-line).We compared the performance of linear and non-linear calibration techniques - namely, multiple linear regression (MLR), principal component regression (PCR), partial least squares regression (PLS), polynomial and Spline-PLS versions, and artificial neural networks (ANN) - for prediction of biodiesel properties from near infrared spectra. The model was created for four important biodiesel properties: density (at 15 °C), kinematic viscosity (at 40 °C), water content, and methanol content. We also investigated the influence of different pre-processing methods (Savitzky-Golay derivatives, orthogonal signal correction) on the model prediction capability. The lowest root mean squared errors of prediction (RMSEP) of ANN for density, viscosity, water percentage, and methanol content were 0.42 kg m⁻³, 0.068 mm² s⁻¹, 45 ppm, and 51 ppm, respectively. The artificial neural network (ANN) approach was superior to the linear (MLR, PCR, PLS) and “quasi”-non-linear (Poly-PLS, Spline-PLS) calibration methods.     

Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters,MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018–2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.     

Cytoprotective Activity of Polyamines Is Associated with the Alternative Splicing of RAD51A Pre-mRNA in Normal Human CD4+ T Lymphocytes

Physiological polyamines are ubiquitous polycations with pleiotropic biochemical activities, including regulation of gene expression and cell proliferation as well as modulation of cell signaling. They can also decrease DNA damage and promote cell survival. In the present study, we demonstrated that polyamines have cytoprotective effects on normal human CD4⁺ T lymphocytes but not on cancer Jurkat or K562 cells. Pretreatment of lymphocytes with polyamines resulted in a significant reduction in cells with DNA damage induced by doxorubicin, cisplatin, or irinotecan, leading to an increase in cell survival and viability. The induction of RAD51A expression was in response to DNA damage in both cancer and normal cells. However, in normal cells, putrescin pretreatment resulted in alternative splicing of RAD51A and the switch of the predominant expression from the splice variant with the deletion of exon 4 to the full-length variant. Induction of RAD51A alternative splicing by splice-switching oligonucleotides resulted in a decrease in DNA damage and cell protection against cisplatin-induced apoptosis. The results of this study suggest that the cytoprotective activity of polyamines is associated with the alternative splicing of RAD51A pre-mRNA in normal human CD4⁺ T lymphocytes. The difference in the sensitivity of normal and cancer cells to polyamines may become the basis for the use of these compounds to protect normal lymphocytes during lymphoblastic chemotherapy.     

Self-Assembled Particles Combining SARS-CoV-2 RBD Protein and RBD DNA Vaccine Induce Synergistic Enhancement of the Humoral Response in Mice

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.     

Comparative Analysis of Tumor-Associated microRNAs and Tetraspanines from Exosomes of Plasma and Ascitic Fluids of Ovarian Cancer Patients

Ovarian cancer (OC) is one of the most common and fatal types of gynecological cancer. In the early phase of OC detection, the current treatment and diagnostic methods are not efficient and sensitive enough. Therefore, it is crucial to explore the mechanisms of OC metastasis and discover valuable factors for early diagnosis of female cancers and novel therapeutic strategies for metastasis. Exosomes are known to be involved in the development, migration, and invasion of cancer cells, and their cargo could be useful for the non-invasive biopsy development. CD151- and Tspan8-positive exosomes are known to support the degradation of the extracellular matrix, and are involved in stroma remodeling, angiogenesis and cell motility, as well as the association of miR-24 and miR-101 with these processes. The objective of this study was to explore the relationship of these components of exosomal cargo, in patients with OC, to clarify the clinical significance of these markers in liquid biopsies. The levels of tetraspanins Tspan8+ and CD151+ exosomes were significantly higher in plasma exosomes of OC patients compared with healthy females (HFs). The relative levels of miR-24 and miR-101 in plasma exosomes of HFs were significantly higher than in plasma exosomes of OC patients, while the levels of these microRNAs in exosomes from plasma and ascites of ill females showed no difference. Our study revealed a strong direct correlation between the change in the ascites exosomes CD151+Tspan8+ subpopulation level and the expression levels of the ascites (R = 0.81, p < 0.05) and plasma exosomal miR-24 (R = 0.74, p < 0.05) in OC patients, which confirms the assumption that exosomal cargo act synergistically to increase cellular motility, affecting cellular processes and signaling. Bioinformatics analysis confirmed the involvement of CD151 and Tspan8 tetraspanins and genes controlled by miR-24-3p and miR-101 in signaling pathways, which are crucial for carcinogenesis, demonstrating that these tetraspanins and microRNAs are potential biomarkers for OC screening, and predictors of poor clinicopathological behavior in tumors.