Mechanisms of institutional continuity in neoliberal "success stories" : developmental regimes in Chile and Estonia

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Several mycoplasmas, such as the emergent human pathogen Mycoplasma genitalium, developed a complex polar structure, known as the terminal organelle (TO), responsible for a new type of cellular motility, which is involved in a variety of cell functions: cell division, adherence to host cells, and virulence. The TO cytoskeleton is organized as a multisubunit dynamic motor, including three main ultrastructures: the terminal button, the electrodense core, and the wheel complex. Here, we describe the interaction between MG200 and MG491, two of the main components of the TO wheel complex that connects the TO with the cell body and the cell membrane. The interaction between MG200 and MG491 has a KD in the 80 nM range, as determined by surface plasmon resonance. The interface between the two partners was confined to the >enriched in aromatic and glycine residues> (EAGR) box of MG200, previously described as a protein-protein interaction domain, and to a 25-residue-long peptide from the C-terminal region of MG491 by surface plasmon resonance and NMR spectroscopy studies. An atomic description of the MG200 EAGR box binding surface was also provided by solution NMR. An M. genitalium mutant lacking the MG491 segment corresponding to the peptide reveals specific alterations in cell motility and cell morphology indicating that the MG200-MG491 interaction plays a key role in the stability and functioning of the TO.This work was supported by Ministerio de Ciencia e Innovacion Grants BFU2012-36827 (to I. F.) and BFU2010-22209-C02-01 (to E. Q.), a grant from the Centre de Referencia de R+D de Biotecnologia (Generalitat de Catalunya, Spain) (to E. Q.), and by FEDER funds through the Operational Competitiveness Programme-COMPETE and by Portuguese national funds through FCT-Fundação para a Ciência e a Tecnologia under Project FCOMP-01-0124-FEDER-027581 (EXPL/BBB-BQB/0546/2012) (to B. C.). The NMR characterization was conducted through the FP7 Access to Research Infrastructures (Bio-NMR Contract 261863) and by Instruct, which is part of the European Strategy Forum on Research Infrastructures (ESFRI) and supported by national member subscriptionsPeer Reviewe

Functional protein-based nanomaterial produced in GRAS microorganism : a new platform for biotechnology

Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest

In situ photoelectron spectroscopy study of water adsorption on model biomaterial surfaces

Using in situ photoelectron spectroscopy at near ambient conditions, we compare the interaction of water with four different model biomaterial surfaces: self-assembled thiol monolayers on Au(111) that are functionalized with methyl, hydroxyl, and carboxyl groups, and phosphatidylcholine (POPC) lipid films on Silicon. We show that the interaction of water with biomaterial surfaces is mediated by polar functional groups that interact strongly with water molecules through hydrogen bonding, resulting in adsorption of 0.2-0.3 ML water on the polar thiol films in 700 mTorr water pressure and resulting in characteristic N1s and P2p shifts for the POPC films. Provided that beam damage is carefully controlled, in situ electron spectroscopy can give valuable information about water adsorption which is not accessible under ultra-high vacuum conditions

Integrating mechanical and biological control of cell proliferation through bioinspired multi-effector materials

In nature, cells respond to complex mechanical and biological stimuli whose understanding is required for tissue construction in regenerative medicine. However, the full replication of such bimodal effector networks is far to be reached. Engineering substrate roughness and architecture allows regulating cell adhesion, positioning, proliferation, differentiation and survival, and the external supply of soluble protein factors (mainly growth factors and hormones) has been long applied to promote growth and differentiation. Further, bioinspired scaffolds are progressively engineered as reservoirs for the in situ sustained release of soluble protein factors from functional topographies. We review here how research progresses toward the design of integrative, holistic scaffold platforms based on the exploration of individual mechanical and biological effectors and their further combination

Rapid quantification of 4-ethylphenol in wine using high-performance liquid chromatography with a fluorimetric detector

A versatile evaporation-assisted methodology based on the coffee-drop effect is described to deposit nanoparticles on surfaces, obtaining for the first time patterned gradients of protein nanoparticles (pNPs) by using a simple custom-made device. Fully controllable patterns with specific periodicities consisting of stripes with different widths and distinct nanoparticle concentration as well as gradients can be produced over large areas (∼10 cm²) in a fast (up to 10 mm²/min), reproducible, and cost-effective manner using an operational protocol optimized by an evolutionary algorithm. The developed method opens the possibility to decorate surfaces "a-la-carte" with pNPs enabling different categories of high-throughput studies on cell motility

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions

Stable nanovesicles formed by intrinsically planar bilayers

Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation.This work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 712949 (TECNIOspring PLUS) and from the Agency for Business Competitiveness of the Government of Catalonia. The production of quatsomes and part of their characterization has been performed by the ICTS “NANBIOSIS”, more specifically by the Biomaterial Processing and Nanostructuring Unit (U6), Unit of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). Authors acknowledge financial support from the Spanish Ministry of Science and Innovation through grants “MOL4BIO” (PID2019-105622RB-I00), “SimBioSoft” (PID2021-124297NB-C33) and the FUNFUTURE-FIP-2020 Severo Ochoa project, from Generalitat de Catalunya through grant 2017-SGR-918, from CSIC through grant 2019AEP133, and from the European Commission through the H2020 PHOENIX project (contract no. 953110). We acknowledge the support of the Israel scienceIsrael science Foundation, grant 1117/2016, and thank Dr. Inbal Ionita for her professional assistance in the cryo-TEM analysis. We thank Jannik Nedergaard Pedersen and Beatrice Plazzotta for help with the SAXS measurements. The simulations reported here were performed using the Cori Supercomputing facility of the National Energy Research Scientific Computing Center (NERSC), a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-05CH11231.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

In vivo architectonic stability of fully de novo designed protein-only nanoparticles

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids

Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces. Although the surface density and spacing of the RGD peptide at the nanoscale have already shown a significant influence on cell adhesion, the impact of its hierarchical nanostructure is still rather unexplored. Accordingly, a versatile colloidal system named quatsomes, based on fluid nanovesicles formed by the self-assembling of cholesterol and surfactant molecules, has been devised as a novel template to achieve hierarchical nanostructures of the RGD peptide. To this end, RGD was anchored on the vesicle's fluid membrane of quatsomes, and the RGD-functionalized nanovesicles were covalently anchored to planar gold surfaces, forming a state of quasi-suspension, through a long poly(ethylene glycol) (PEG) chain with a thiol termination. An underlying self-assembled monolayer (SAM) of a shorter PEG was introduced for vesicle stabilization and to avoid unspecific cell adhesion. In comparison with substrates featuring a homogeneous distribution of RGD peptides, the resulting hierarchical nanoarchitectonic dramatically enhanced cell adhesion, despite lower overall RGD molecules on the surface. The new versatile platform was thoroughly characterized using a multitechnique approach, proving its enhanced performance. These findings open new methods for the hierarchical immobilization of biomolecules on surfaces using quatsomes as a robust and novel tissue engineering strategy.This work was supported by MICINN (PID2019-105622RBI00, MAT2016-80826-R, PID2019-111682RB-I00, PID2020-115296RA-I00, CTQ2015-66194-R; SAF2014-60138-R, RTI2018-093831-B-I00, and PDC2021-121481-I00); Instituto de Salud Carlos III (ISCIII) through the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN (FlexQS-skin, FlexCAB, BBN18PI01, BBN20PIV02, and CB/06/0074); Generalitat de Catalunya (grants 2017-SGR-918, 2017-SGR-229, 2017-SGR-1442, 2017-SGR-1439); the Fundació Marató de TV3 (Nr. 201812); the COST Action CA15126 Between Atom and Cell, and “ERDF A way of making Europe”. J.G. acknowledges financial support from the Ramón y Cajal Program (RYC-2017-22614) from MICINN and the Max Planck Society through the Max Planck Partner Group “Dynamic Biomimetics for Cancer Immunotherapy” in collaboration with the Max Planck Institute for Medical Research (Heidelberg, Germany). This work has received funding from the European Union’s Horizon 2020 research and innovation program through grant agreements 953110 (PHOENIX), 720942 (Smart4Fabry), 101007804 (MICRO4NANO), and 801342 (granted to the Agency for Business Competitiveness ACCIÓ through a Tecniospring Industry fellowship (TECSPR19-1-0065)). ICMAB acknowledges support from MICINN through the “‘Severo Ochoa”’ Programme for Centres of Excellence in R&D (CEX2019-000917-S). J.M. acknowledges a “Juan de la Cierva” fellowship from MICINN. J.T-M. acknowledges an FI-AGAUR grant (2020FI_B2 00137) from Generalitat de Catalunya and the European Social Fund. We also acknowledge the ICTS “NANBIOSIS for the support of the Synthesis of Peptides Unit (U3) at IQAC–CSIC (https://www.nanbiosis.es/portfolio/u3-synthesis-of-peptides-unit/) and the Biomaterial Processing and Nanostructuring Unit (U6) at ICMAB-CSIC (https://www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit/). We are grateful to the SMP unit of the Scientific and Technological Centers of University of Barcelona (CCiTUB). This work has been developed under the “Biochemistry, Molecular Biology and Biomedicine” and “Materials Science” Ph.D. programs of Universitat Autònoma de Barcelona (UAB).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe