Cytogenetic effects of cyclophosphamide on human lymphocytes in vivo and in vitro

A comparative study of cytogenetic effects in human lymphocytes caused in vivo by cyclophosphamide (CP) after intravenous injection and in vitro by exposure of plasma of the same patients was carried out. It was found that the frequency of induced chromosome aberrations (CA) and sister-chromatid exchanges (SCE) increased linearly for SCE and exponentially for CA within the 'dose' of alkylating activity of CP metabolites. Parameters of 'cytogenetic effect-dose' in vivo and in vitro coincided. The intensity of cytogenetic effects varied between individuals.     

An Exposure to the Oxidized DNA Enhances Both Instability of Genome and Survival in Cancer Cells

Background

Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases and acts upon the cells. Response to cfDNA depends on concentrations and levels of the damage within cfDNA. Oxidized extracellular DNA acts as a stress signal and elicits an adaptive response.

Principal Findings

Here we show that oxidized extracellular DNA stimulates the survival of MCF-7 tumor cells. Importantly, in cells exposed to oxidized DNA, the suppression of cell death is accompanied by an increase in the markers of genome instability. Short-term exposure to oxidized DNA results in both single- and double strand DNA breaks. Longer treatments evoke a compensatory response that leads to a decrease in the levels of chromatin fragmentations across cell populations. Exposure to oxidized DNA leads to a decrease in the activity of NRF2 and an increase in the activity of NF-kB and STAT3. A model that describes the role of oxidized DNA released from apoptotic cells in tumor biology is proposed.

Conclusions/Significance

Survival of cells with an unstable genome may substantially augment progression of malignancy. Further studies of the effects of extracellular DNA on malignant and normal cells are warranted.     

Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems‐level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14‐protein core network critical to the viability of multiple EGFR‐mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR‐mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.     

Transport of endogenous nucleosides in guinea pig heart

The purpose of this study was to investigate the characteristics of transport of endogenous nucleosides into cardiac tissue from coronary circulation. The study was performed on the isolated perfused guinea pig heart, using the rapid paired tracers single-pass technique. The maximal cellular uptake (U(max)) and total cellular uptake (U(tot)) of adenosine, deoxyadenosine, thymidine, uridine, and cytidine were determined. The cellular uptake of adenosine was significantly higher than the cellular uptake of other studied nucleosides. To elucidate the mechanisms of nucleoside transport, competition studies were performed and the influence of S-(p-nitrobenzyl)-6-thioinosine (NBTI) and sodium ion absence on U(max) and U(tot) was investigated. Self- and cross-inhibition studies indicated the saturable mechanism of nucleosides transport into cardiac tissue and the involvement of different transport mechanisms for purine and pyrimidine nucleosides. The study also showed that both equilibrative-sensitive (es) and sodium-dependent transport were responsible for adenosine and thymidine cellular uptake.     

Ratio of the frequency of chromosome aberrations and sister chromatid exchanges to the dose of the mutagenic exposure to cyclophosphamide in vivo and in vitro

Cyclophosphamide (CP) and its metabolites were used to compare the rate of chromosomal aberrations (CA) and sister chromatid exchange (SCE) in the rabbit lymphocytes in vivo and in vitro. The dose-dependent increase of cytogenetic effects rate appeared to be of linear and exponential dependence for SCE and CA, respectively, both in vivo and in vitro. The regression equation coefficients coincided in in vivo and in vitro experiments.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Assessment of radiation exposure level for hydrobionts in some special industrial ponds at the "Mayak" PA

Evaluation of the radionuclide content in the ecosystem components (water, sediments, aquatic organisms) of industrial reservoirs-storages of liquid radioactive waste of the "Mayak" PA (reservoirs R-4, R-10, R-11, R-17, R-9) and the estimation of the absorbed dose rate in aquatic organisms of these reservoirs using the software package ERICA Assessment Tool 1.0 May 2009 have been performed. Gradient of the absorbed dose rate for the detected taxonomic groups of hydrobionts in the series of the studied reservoirs R-11 --> R-10 --> R-4 --> R-17 --> R-9 was almost equal to one order of magnitude. The estimated absorbed dose rate for phytoplankton ranged from 5.4 x 10(0) mGy/day (R-11) to 4.0 x 10(4) mGy/day (R-9), for zooplankton--from 6.4 x 10(-1) mGy/day (R-11) to 3.8 x 10(3) mGy/day (R-9), for zoobenthos (chironomids)--from 5.6 x 10(0) mGy/day (R-11) to 1.1 x 10(3) mGy/day (R-17), for fish (roach)--from 8.0 x 10(-1) mGy/day (R-11) to 1.9 x 10(1) mGy/day (R-4).     

Ecotoxicological study of bottom sediments from reservoir 11 of the Techa River cascade

The ecotoxicological properties of sediments from reservoir R-11 of the Techa River cascade are studied in comparison to the Shershni reservoir. Radiochemical analysis of sediment and water samples from R-11 show that the radioactivity of the sediments ranges from 240 to 360 kBq/dm³ dry weight for ⁹⁰Sr and from 10 to 161 kBq/dm³ dry weight for ¹³⁷Cs. The absorbed doses for Oligochaeta from R-11 are calculated on the basis of radiochemical data. The hydrobiological studies include (1) study of the population density and diversity of Oligochaeta species in the reservoirs and (2) laboratory bioassay of sediments with Tubifex tubifex as a biomonitor species. The results indicate that the Oligochaeta population density in R-11 is less than in the Shershni reservoir. No significant effects of the absorbed dose rate on population density, survival rate, or fertility are found in a laboratory Tubificidae bioassay of R-11 sediments. However, a decrease in fertility is noted in experiments at a higher absorbed dose.     

Comparison of the frequency of chromosome aberrations induced by thiophosphamide in rabbit lymphocytes in vitro and in vivo

Rabbit lymphocytes were treated with thiophosphamide in vivo and in vitro. In-vitro doses were calculated by multiplying thiophosphamide concentrations by the time of treatment (the doses ranged within 0-1500 mg/min/ml). In-vivo doses were calculated as integral of thiophosphamide concentration function from the time of administration till the time of blood sample collection (the doses ranged within 0-1900 mg/min/ml). It was shown that with the dose increase the rate of chromosome aberrations and the number of disruptions per cell rise exponentially in vivo and in vitro. At the same time the parameters of regression equations coincide. This evidences that thiophosphamide produces the same effect in vivo and in vitro.     

Use of Atomic Force Microscopy and Transmission Electron Microscopy for Correlative Studies of Bacterial Capsules

Bacteria can possess an outermost assembly of polysaccharide molecules, a capsule, which is attached to their cell wall. We have used two complementary, high-resolution microscopy techniques, atomic force microscopy (AFM) and transmission electron microscopy (TEM), to study bacterial capsules of four different gram-negative bacterial strains: Escherichia coli K30, Pseudomonas aeruginosa FRD1, Shewanella oneidensis MR-4, and Geobacter sulfurreducens PCA. TEM analysis of bacterial cells using different preparative techniques (whole-cell mounts, conventional embeddings, and freeze-substitution) revealed capsules for some but not all of the strains. In contrast, the use of AFM allowed the unambiguous identification of the presence of capsules on all strains used in the present study, including those that were shown by TEM to be not encapsulated. In addition, the use of AFM phase imaging allowed the visualization of the bacterial cell within the capsule, with a depth sensitivity that decreased with increasing tapping frequency.