Mónica Roldán

Protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (R9) peptide

AIMS Arginine(R)-rich cationic peptides are powerful tools in drug delivery since, alone or when associated with polyplexes, proteins or chemicals, they confer DNA condensation, membrane translocation and blood-brain barrier crossing abilities. The unusual stability and high in vivo performance of their associated drugs suggest a particulate organization or R(n) complexes, which this study aimed to explore. MATERIALS & METHODS We have analyzed the particulate organization and biological performance in DNA delivery of a model, R9-containing green fluorescent protein by dynamic light scattering, transmission electron microscopy, atomic force microscopy, single cell confocal microscopy and flow cytometry. RESULTS A deep nanoscale examination of R9-powered constructs reveals a novel and promising feature of R9, that when fused to a scaffold green fluorescent protein, promote its efficient self-assembling as highly stable, regular disk-shaped nanoparticles of 20 x 3 nm. These constructs are efficiently internalized in mammalian cells and rapidly migrate through the cytoplasm towards the nucleus in a fully bioactive form. Besides, such particulate platforms accommodate, condense and deliver plasmid DNA to the nucleus and promote plasmid-driven transgene expression. CONCLUSION The architectonic properties of arginine-rich peptides at the nanoscale reveal a new category of protein nanoparticles, namely nanodisks, and provide novel strategic concepts and architectonic tools for the tailored construction of new-generation artificial viruses for gene therapy and drug delivery.

Exploring the secrets of the three-dimensional architecture of phototrophic biofilms in caves

INTRODUCTION Biofilms are collectives of one or more species of microorganisms. They provide protection for growth, enabling microorganisms to survive hostile environments (Prakash et al., 2003) and are significant in sediment stabilization and construction (Golubic & Schneider, 2003). Biofilms comprise sessile microorganisms in different stages of growth; hence, they respond quickly to variable conditions (Costerton et al., 1987). When microorganisms adhere to a surface, their immobilised cells grow, replicate and secrete extracellular polymeric substances (EPS) that engulf them in a gelatinous matrix (Brading et al., 1995). The development of complex, adhered or aggregated communities plays a key role in the survival and reproductive success of the microorganisms involved. Biofilms can provide refuge for species that face

Noninvasive pigment identification in single cells from living phototrophic biofilms by confocal imaging spectrofluorometry.

ABSTRACT A new imaging technique for the analysis of fluorescent pigments from a single cell is reported. It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods. The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages.

Why there is such luxurious growth in the hypogean environments

Organisms building biofilms are of considerable interest in the context of degradation of cultural heritage. Particularly, hypogean environments exposed to artificial light are colonized by microbial communities, which damage walls and frescoes. In order to ascertain the mechanisms by which phototophic biofilms thrive under the particular conditions of hypogea, the organism composition and three-dimensional structure of biofilms from the Roman catacombs St. Callistus and Domitilla were studied. The main phototrophic organisms forming the biofilms were filamentous sheathed cyanobacteria and mosses. Biofilms were spatially very heterogeneous in thickness, density and organism composition but could be classified as regards their main organisms. There was a trend of decreasing diversity in the phototrophic composition of the biofilms under lower irradiances, the one at the lowest irradiance being uniquely built by erected filaments of Leptolynghya sp. Except for this biofilm, the main organism composition was not clearly related with decreasing irradiance. However, biofilms from dim light samples were porous and the filamentous cyanobacteria in them were erected. Leplolyngbya sp., the most ubiquitous species, displayed a high number of phycobilisomes and its hormogonia a gliding movement that allowed colonization of substrata. Such mechanisms may have an important role for thriving under the low light conditions of the catacombs.

QDs versus Alexa: reality of promising tools for immunocytochemistry

BackgroundThe unique photonic properties of the recently developed fluorescent semiconductor nanocrystals (QDs) have made them a potential tool in biological research. However, QDs are not yet a part of routine laboratory techniques. Double and triple immunocytochemistries were performed in HeLa cell cultures with commercial CdSe QDs conjugated to antibodies. The optical characteristics, due to which QDs can be used as immunolabels, were evaluated in terms of emission spectra, photostability and specificity.ResultsQDs were used as secondary and tertiary antibodies to detect β-tubulin (microtubule network), GM130 (Golgi complex) and EEA1 (endosomal system). The data obtained were compared to homologous Alexa Fluor 594 organic dyes. It was found that QDs are excellent fluorochromes with higher intensity, narrower bandwidth values and higher photostability than Alexa dyes in an immunocytochemical process. In terms of specificity, QDs showed high specificity against GM130 and EEA1 primary antibodies, but poor specificity against β-tubulin. Alexa dyes showed good specificity for all the targets tested.ConclusionThis study demonstrates the great potential of QDs, as they are shown to have superior properties to Alexa dyes. Although their specificity still needs to be improved in some cases, QDs conjugated to antibodies can be used instead of organic molecules in routine immunocytochemistry.

Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles.

Fluorescent fingerprints of endolithic phototrophic cyanobacteria living within halite rocks in the Atacama Desert

ABSTRACT Halite deposits from the hyperarid zone of the Atacama Desert reveal the presence of endolithic microbial colonization dominated by cyanobacteria associated with heterotrophic bacteria and archaea. Using the λ-scan confocal laser scanning microscopy (CLSM) option, this study examines the autofluorescence emission spectra produced by single cyanobacterial cells found inside halite rocks and by their photosynthetic pigments. Photosynthetic pigments could be identified according to the shapes of the emission spectra and wavelengths of fluorescence peaks. According to their fluorescence fingerprints, three groups of cyanobacterial cells were identified within this natural extreme microhabitat: (i) cells producing a single fluorescence peak corresponding to the emission range of phycobiliproteins and chlorophyll a, (ii) cells producing two fluorescence peaks within the red and green signal ranges, and (iii) cells emitting only low-intensity fluorescence within the nonspecific green fluorescence signal range. Photosynthetic pigment fingerprints emerged as indicators of the preservation state or viability of the cells. These observations were supported by a cell plasma membrane integrity test based on Sytox Green DNA staining and by transmission electron microscopy ultrastructural observations of cyanobacterial cells.

Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria

Biofabrication is attracting interest as a means to produce nanostructured functional materials because of its operational versatility and full scalability. Materials based on proteins are especially appealing, as the structure and functionality of proteins can be adapted by genetic engineering. Furthermore, strategies and tools for protein production have been developed and refined steadily for more than 30 years. However, protein conformation and therefore activity might be sensitive to production conditions. Here, we have explored whether the downstream strategy influences the structure and biological activities, in vitro and in vivo, of a self-assembling, CD44-targeted protein-only nanoparticle produced in Escherichia coli. This has been performed through the comparative analysis of particles built from soluble protein species or protein versions obtained by in vitro protein extraction from inclusion bodies, through mild, non-denaturing procedures. These methods have been developed recently as a convenient alternative to the use of toxic chaotropic agents for protein resolubilization from protein aggregates. The results indicate that the resulting material shows substantial differences in its physicochemical properties and its biological performance at the systems level, and that its building blocks are sensitive to the particular protein source.

CXCR4+-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis

AIM Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. MATERIALS & METHODS Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. RESULTS Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. CONCLUSION In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.

Functional inclusion bodies produced in the yeast Pichia pastoris.

BackgroundBacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria.ResultsTo overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity.ConclusionsStructurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.