eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a KD of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [14C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin.

A synthetic approach for the preparation and linkage of functionalized plasticizer molecules to PVC is described. The synthesis of this four-step procedure is economically and ecologically viable because it is based on trichlorotriazine as inexpensive starting material; the reactions can be carried out one-pot with quantitative yields and without need of final purification of the products. The approach is furthermore highly versatile and allows for the preparation of a large number of different plasticizers with properties that may be adjusted to a broad range of applications from highly flexible to semirigid.

(1) IQFR-CSIC; (2) EM-CSIC Polymer nanospheres with different size distributions are prepared by two different methods: with and without the aid of an anionic surfactant. Calorimetric traces of these nanospheres show a different glass transition temperature, with respect to that of the bulk, which has been discussed in terms of an entropy model. The total confinement, imposed by the spherical geometry, leads to a limiting number of repeating polymer units in the sphere and thus to a reduction of the possible configuration states of the polymer chains. This is ultimately related to variations in the bulk value of the glass transition temperature. The model is evaluated against our calorimetric measurements as well as with the data available in the literature. Good agreement between data and model is found for many cases, proving that confinement can be related to reductions in the entropy of these nanosystems.

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Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca2+ influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn2+. Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity.

Integrin ␣IIb␤3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole ␣IIb␤3 in Ca 2؉ /detergent solutions. Model projections after 90°rotation along its long axis show an elongated and "arched" form (135°) not observed before and a "handgun" form. This 20-nm-long structure is well defined, despite ␣IIb␤3 multidomain nature and expected segmental flexibility, with the largest region at the top, followed by two narrower and smaller regions at the bottom. Docking of this SANS envelope into the high resolution structure of ␣IIb␤3, reconstructed from crystallographic and NMR data, shows that the solution structure is less constrained, allows tentative assignment of the disposition of the ␣IIb and ␤3 subunits and their domains within the model, and points out the structural analogies and differences of the SANS model with the crystallographic models of the recombinant ectodomains of ␣IIb␤3 and ␣V␤3 and with the cryo-electron microscopy model of whole ␣IIb␤3. The ectodomain is in the bent configuration at the top of the model, where ␣IIb and ␤3 occupy the concave and convex sides, respectively, at the arched projection, with their bent knees at its apex. It follows the narrower transmembrane region and the cytoplasmic domains at the bottom end. ␣IIb␤3 aggregated in Mn 2؉ /detergent solutions, which impeded to get its SANS model.Blood platelets have distinct mechanisms to respond to the different agonists of platelet activation, all of which end up with the acquisition by the integrin ␣IIb␤3 or glycoprotein IIb/IIIa of the capacity to recognize and bind fibrinogen and other adhesive proteins. Binding of fibrinogen, the major adhesive protein in plasma, to activated ␣IIb␤3 and the binding of fibronectin, the major adhesive protein in the extracellular matrix of subendothelium, to resting or activated ␣IIb␤3 lead to the formation of interplatelet cross-linking and to platelet adhesion to the subendothelium, respectively, and, eventually, to the formation of the platelet plug. Thus, knowledge of the fibrinogen receptor and the mechanisms of induction and modulation of its activity is determinant to understand hemostasis and the pathophysiological paths leading to thrombosis, as well as to develop new ways to prevent, detect, and treat thrombosis effectively, securely, and selectively. After 20 years of molecular genetics and 10 years of high resolution structural biology of integrins, the disposition of an integrin in the membrane is unknown, the modifications in the cytoplasmic domains and the transmembrane segments related with the receptor activation are still not definitively defined, and the molecular mechanism of activation and molecular properties of the activated receptor are still under discussion (1-4). Structural and cell biological informat...

We report on the fabrication of 3D printed pH-responsive and antimicrobial hydrogels with a micrometer-scale resolution achieved by stereolithography (SLA) 3D printing. The preparation of the hydrogels was optimized by selecting the most appropriate difunctional polyethylene glycol dimethacrylates (testing cross-linking agents with chain lengths ranging from 2 up to 14 units ethylene glycol) and introducing acrylic acid (AA) as a monofunctional monomer. As a result of the incorporation of AA, the hydrogels described are able to reversibly swell and shrink upon environmental changes on the pH, and the swelling extent is directly related to the amount of AA and can be thus finely tuned. More interestingly, upon optimization of the UV penetration depth employing a photoabsorber (Sudan I), a reliable procedure for the fabrication of 3D objects with a high model accuracy is shown. Finally, the antimicrobial properties of all of the hydrogels were demonstrated using Staphylococcus aureus as a bacterial model. We found that even those hydrogels with a low amount of AA monomeric units presented excellent antimicrobial properties against S. aureus.