Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN

The use of taxonomic relationships among species in applied ecological research: Baseline, steps forward and future challenges

Taxonomy is more than a mere exercise of nomenclature and classification of biological diversity: it profiles the identity of species by investigating their biological and ecological traits. Taxonomy is intimately related to ecology which, in turn, cannot be a mere exercise in describing ecological patterns, but instead requires deep knowledge of species’ biological structures, roles, interactions and functions. Thus, the study of taxonomic and phylogenetic relatedness of species is of paramount importance in ecological research, enabling insights into potential evolutionary patterns and processes, allowing a more comprehensive view of biodiversity, and providing opportunities to improve the assessment and monitoring of ecological changes in time and space. The work of K. Robert (‘Bob’) Clarke forged new pathways in this direction, providing new ideas and statistical tools to include and exploit taxonomic relationships in applied marine ecological studies and beyond, also inspiring the next generation of ecologists. In this short review, we synthesise the application and development of these tools and concepts in marine biodiversity research over the last three decades and suggest future pathways in this evolving field

Deriving the structure of pre-supernovae and delta Scuti stars using nonradial oscillations

This is the final report of a three-year Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The objective is to learn more about the internal structure of two classes of variable stars, by using the observational data afforded by their pulsation properties. The authors updated the one-dimensional computer codes to calculate the evolution and pulsation frequencies of representative delta Scuti and LBV models. They compared the observed pulsation properties with model predictions in an iterative process to find a model (or models) with interior structures that matched the observational constraints for several delta Scuti stars. They carried out nonlinear hydrodynamic modeling of LBV envelopes and proposed a mechanism for their periodic outbursts. Finally, they began validation of a two-dimensional stellar evolution code that will be used to investigate the effects of rotation and hydrodynamic instabilities on the interior structure of these stars

LVCN treatment shows enhanced tumor apoptosis in the breast MDA-MB-231 xenograft model.

<p>For this experiment, MDA-MB-231 xenografts were allowed to grow to a significantly larger volume (4 weeks after inoculation) before treatments were initiated. The animals received either liposomal VCN (at the dose-equivalent of 100 µg of VCN per injection) or Avastin (400 µg per injection) administered intravenously every other day and compared to a control group that received saline only. All animals were sacrificed after receiving 6 consecutive doses of each treatment. (<b>i</b>) To assess the impact of VCN on cell death, tumor cryostat sections from each group were stained with FITC-TUNEL, and counterstained with Hoechst 33342. Representative confocal images from multiple experiments taken at ×250 magnification are shown above (scale bar, 100 µm; panels A₁-C₁ - TUNEL-Hoechst, panels A₂-C₂ - TUNEL only). (<b>ii</b>) The amount of cell death was quantitated as ‘number of TUNEL⁺ nuclei/total number of nuclei x 100’ by counting all nuclei in ‘hotspot’ areas from multiple fields using a computer-assisted approach (the ‘SimplePCI’ imaging software). The liposomal VCN group shows a significantly increased amount of cell death compared to either Avastin or control. (<b>iii</b>) The impact of VCN treatment on tumor proliferation was assessed by Ki-67 immunoperoxidase staining. Representative Ki-67 images are shown above (scale bar, 200 µm). (<b>iv</b>) Cell proliferation was quantitated using the same approach as for TUNEL staining. The differences in cell proliferation between the treatment groups were much smaller than those observed for cell death. The data was analyzed with ANOVA followed by <i>post-hoc</i> tests (* signifies a P<0.01).</p

Disintegrin-integrin biding kinetics by fluorescence polarization.

<p>The binding kinetics were calculated from the fluorescence anisotropy data generated by the steady state binding of FITC-labeled disintegrins to either purified (αvβ3 and αvβ5) or recombinant (α5β1) functional human integrins. The dissociation constants for interactions of either CN or VCN with soluble integrins were determined by Scatchard analysis using a non-linear curve fit.</p

The expression, purification and initial characterization of VCN as an active disintegrin.

<p><b>Panel A –</b> The production of Trx-VCN was assessed in different <i>E. coli</i> strains that were transformed, grown, induced, and processed under identical conditions. The same amount (5 µl) of cell lysates from each induced strain was loaded on a precast gel and Coomassie stained. Unlike the BL21 (DE3) strain, the lysates from both AD494 (DE3) and Origami B (DE3) strains generate a unique and consistent Trx-VCN band (shown by the arrow). By employing a modified media recipe, the Origami B (DE3) Trx-VCN transformants achieve higher cell densities at the end of the induction time, generating up to 200 mg of soluble VCN per L of bacterial culture after purification. <b>Panel B –</b> Coomassie stained gel showing the migration of Trx-VCN before and after TEV proteolysis (lanes 2 and 3, respectively) versus C18 reverse phase-HPLC purified VCN (lane 4). <b>Panel C </b><b>–</b> VCN and native CN exhibit an almost identical dose-dependent inhibitory effect against ADP-induced platelet aggregation when incubated with human platelet-rich plasma (with a calculated IC₅₀ of ∼60 nM). In contrast, the rCN construct, which is also expressed as a soluble polypeptide in Origami B (DE3), shows no inhibitory activity. <b>Panel D </b><b>–</b> The agonistic activity of VCN (FAK activation) was assessed in serum-starved non-migratory MDA-MB-435 cells kept in suspension and exposed to increasing concentrations of disintegrins for 30 min. Similar to dimeric CN, VCN is also shown to engage integrins agonistically (outside-in signaling).</p