Work of morphogenesis in the Department of Embryology of Moscow State University: toward understanding the dynamic architecture of development

This is a review of studies on morphogenesis carried out at the Department of Embryology, Moscow State University, over the past 30 years. The main direction of studies has been to reveal and describe the properties of self-organizing fields of mechanical stresses in developing embryos.     

Correlation of fine structures of distributions of amplitudes of a photomultiplier dark current fluctuations with the Earth rotations about its axis

The fine structures of distributions of photomultiplier dark current fluctuations measured in two laboratories 2000 km distant from other: in the international Institute of Biophysics (Neuss, Germany) and in the Moscow State University (Moscow, Russia) were compared. It is shown that similar forms of appropriate histograms are apparently more often realized at both locations at the same local time. This confirms the previous conclusion that the fine structure of distributions correlates with rotation of the Earth about its axis.     

Flavopiridol inhibits the growth of GL261 gliomas in vivo: implications for malignant glioma therapy

The mechanism of action of many chemotherapeutic agents targets the cell cycle. Recently, we demonstrated cytotoxic and other anti-tumor effects of flavopiridol, the first synthetic cyclin dependent kinase (CDK) inhibitor to enter clinical trials, on the murine GL261 glioma cell line in vitro (Newcomb et al., Cell Cycle 2003; 2:243). Given that flavopiridol has demonstrated anti-tumor activity in several human xenograft models, we wanted to evaluate it for anti-glioma activity in vivo in our established subcutaneous and intracranial GL261 experimental tumor models. In particular, the intracranial animal model recapitulates many of the histopathological and biological features of human high-grade glioma including both necrosis with pseudopalisading and invasion of the brain adjacent to tumor. Here we tested the activity of flavopiridol against tumors formed by GL261 cells, first as subcutaneous implants, and then in the intracranial model. We demonstrate efficacy of flavopiridol as a single modality treatment in delaying tumor growth in both animal models. We hypothesize that flavopiridol treatment induced tumor growth delay by two possible mechanisms involving growth arrest combined with recruitment of tumor cells to S-phase. Based on our findings, flavopiridol should be considered as a treatment approach for patients with high-grade glioma.     

Statistical study of rapid mechanodependent cell movements in deformed explants of african clawed frog <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryonic tissues

Computer analysis of artificially deformed (stretched or compressed) double explants (sandwiches) of the blastocoel roof (BRs) and suprablastoporal region (SBRs) of African clawed frog Xenopus laevis early gastrula has been performed using frames of time-lapse microfilming. During the first 14 min after cutting off, the velocities and displacement angles of several hundreds of cells relative to one another, as well as to fixed points and the extension axis, were measured in the control and deformed samples. It has been found that the deformation of samples leads to a rapid reorientation of large cell masses and increase in the velocities of movements along the extension axes or perpendicularly to the compression axes. In addition, an increase in the velocities of mutual cell displacements in the stretched BRs and cell convergence to the extension axes have been observed. Comparison of different angular sectors demonstrates a statistically significant positive correlation between the mean velocities of cell movements and the number of cells moving within an individual sector. This suggests cooperativity of mechanodependent cell movements. In general, these results demonstrate an important role of mechanical factors in regulation of collective cell movements.     

In vivo cleavage of transgene donors promotes nuclease‐mediated targeted integration

Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology‐based and homology‐independent processes. Both pathways start the integration process with a site‐specific break in the chromosome, typically from a zinc‐finger nuclease (ZFN). We previously described an efficient homology‐independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs. We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in efficient integration of large, transgene‐sized DNA molecules into the chromosomal double‐strand break. Successful targeted integration via in vivo donor linearization is demonstrated at five distinct loci in two mammalian cell types, highlighting the generality of the approach. Finally, we show that CHO cells, a cell type recalcitrant to homology‐based integration, are proficient at capture of in vivo‐linearized transgene donors. Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN‐ or TALE nuclease‐mediated integration of an antibody cassette. Our results enable efficient targeted transgene addition to cells and organisms that fare poorly with traditional homology‐driven approaches. Biotechnol. Bioeng. 2013; 110: 871–880. © 2012 Wiley Periodicals, Inc.     

A Family of Indoles Regulate Virulence and Shiga Toxin Production in Pathogenic E. coli

Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) are intestinal pathogens that cause food and water-borne disease in humans. Using biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA), and indole-3-acetic acid (IAA), as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE), which encodes virulence factors that cause “attaching and effacing” (A/E) lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling.     

Statistical study of rapid mechanodependent cell movements in deformed explants of African clawed frog Xenopus laevis embryonic tissues

Computer analysis of artificially deformed (stretched or compressed) double explants (sandwiches) of the blastocoel roof (BRs) and suprablastoporal region (SBRs) of African clawed frog Xenopus laevis early gastrula has been performed using frames of time-lapse microfilming. During the first 14 min after cutting off, the velocities and displacement angles of several hundreds of cells relative to one another, as well as to fixed points and the extension axis, were measured in the control and deformed samples. It has been found that the deformation of samples leads to a rapid reorientation of large cell masses and increase in the velocities of movements along the extension axes or perpendicularly to the compression axes. In addition, an increase in the velocities of mutual cell displacements in the stretched BRs and cell convergence to the extension axes have been observed. Comparison of different angular sectors demonstrates a statistically significant positive correlation between the mean velocities of cell movements and the number of cells moving within an individual sector. This suggests cooperativity of mechanodependent cell movements. In general, these results demonstrate an important role of mechanical factors in regulation of collective cell movements.