Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes

Many bacterial species contain intracellular nano- and micro-compartments consisting of self-assembling proteins that form protein-only shells. These structures are built up by combinations of a reduced number of repeated elements, from 60 repeated copies of one unique structural element self-assembled in encapsulins of 24 nm to 10,000-20,000 copies of a few protein species assembled in a organelle of around 100-150 nm in cross-section. However, this apparent simplicity does not correspond to the structural and functional sophistication of some of these organelles. They package, by not yet definitely solved mechanisms, one or more enzymes involved in specific metabolic pathways, confining such reactions and sequestering or increasing the inner concentration of unstable, toxics or volatile intermediate metabolites. From a biotechnological point of view, we can use the self assembling properties of these particles for directing shell assembling and enzyme packaging, mimicking nature to design new applications in biotechnology. Upon appropriate engineering of the building blocks, they could act as a new family of self-assembled, protein-based vehicles in Nanomedicine to encapsulate, target and deliver therapeutic cargoes to specific cell types and/or tissues. This would provide a new, intriguing platform of microbial origin for drug delivery

Boosting the chances to improve stroke treatment

Background and Purpose—There is a lack of agreement regarding measuring the effects of stroke treatment in clinical trials, which often relies on the dichotomized value of 1 outcome scale. Alternative analyses consist mainly of 2 strategies: use all the information from an ordinal scale and combine information from several outcome scales in a single estimate. Methods—We reanalyzed 3 outcome scales that assessed patient recovery (modified Rankin Scale, National Institutes of Health Stroke Scale, and Barthel Index). With data collected from the 1652 patients in the Citicoline pooling data analysis, we used 2 standard techniques of exploratory multivariate analysis to analyze the distances among ranks and to isolate the common and the unique information provided by each of the 3 scales. Results—The different scale values correspond to gradually different patient status, confirming that information is lost when a scale is collapsed to just 2 values, whether recovered or not. The scales shared 90.7% (95% CI, 84.5–96.9) of their information, with no individual scale contributing unique information. Conclusions—Salient stroke outcome information is lost when an ordinal scale is collapsed into fewer categories. In contrast, the full scales provide a comprehensive patient outcome estimate. Furthermore, in the context of stroke clinical trials, those scales are highly correlated, providing the rationale to pool them into a single estimate. These insights may be used to optimize the analysis of stroke trials to increase study power to detect efficacious interventions.Postprint (published version

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories : production of human alpha-galactosidase A

This work was supported by ERANET-IB08-007 project from the European Union and its linked national project EUI2008- 03610 to AV. We also appreciate the support from EME2007-08 to NFM from Universitat Autonoma de Barcelona, from Antartide 2010 to MLT and EP, from MIUR Azioni Integrate Italia-Spagna 2010 Prot. IT10LECLM9 to MLT, from MINECO (IT2009-0021) to AV and LT, from AGAUR (2009SGR-108) to AV. AV is also supported by The Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN, Spain), an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. PS has received predoctoral fellowship from ISCIII, and AV has been distinguished with an ICREA ACADEMIA award (Catalonia, Spain).Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems

Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders

Highly versatile polyelectrolyte complexes for improving the enzyme replacement therapy of lysosomal storage disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically crosslinked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and -galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity and hemocompatibility of RGD-targeted and un-targeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freezedrying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows keeping intact the activity of the therapeutic protein. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders

A polynomial-time algorithm for deciding bisimulation equivalence of normed Basic Parallel Processes

SIGLEAvailable from British Library Document Supply Centre- DSC:5186.0913(EU-ECS-LFCS--94-288) / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Boosting the chances to imporve stroke treatment

Background and Purpose— There is a lack of agreement regarding measuring the effects of stroke treatment in clinical trials, which often relies on the dichotomized value of 1 outcome scale. Alternative analyses consist mainly of 2 strategies: use all the information from an ordinal scale and combine information from several outcome scales in a single estimate. Methods— We reanalyzed 3 outcome scales that assessed patient recovery (modified Rankin Scale, National Institutes of Health Stroke Scale, and Barthel Index). With data collected from the 1652 patients in the Citicoline pooling data analysis, we used 2 standard techniques of exploratory multivariate analysis to analyze the distances among ranks and to isolate the common and the unique information provided by each of the 3 scales. Results— The different scale values correspond to gradually different patient status, confirming that information is lost when a scale is collapsed to just 2 values, whether recovered or not. The scales shared 90.7% (95% CI, 84.5–96.9) of their information, with no individual scale contributing unique information. Conclusions— Salient stroke outcome information is lost when an ordinal scale is collapsed into fewer categories. In contrast, the full scales provide a comprehensive patient outcome estimate. Furthermore, in the context of stroke clinical trials, those scales are highly correlated, providing the rationale to pool them into a single estimate. These insights may be used to optimize the analysis of stroke trials to increase study power to detect efficacious interventionsPeer Reviewe

Boosting the chances to improve stroke treatment

Background and Purpose—There is a lack of agreement regarding measuring the effects of stroke treatment in clinical trials, which often relies on the dichotomized value of 1 outcome scale. Alternative analyses consist mainly of 2 strategies: use all the information from an ordinal scale and combine information from several outcome scales in a single estimate. Methods—We reanalyzed 3 outcome scales that assessed patient recovery (modified Rankin Scale, National Institutes of Health Stroke Scale, and Barthel Index). With data collected from the 1652 patients in the Citicoline pooling data analysis, we used 2 standard techniques of exploratory multivariate analysis to analyze the distances among ranks and to isolate the common and the unique information provided by each of the 3 scales. Results—The different scale values correspond to gradually different patient status, confirming that information is lost when a scale is collapsed to just 2 values, whether recovered or not. The scales shared 90.7% (95% CI, 84.5–96.9) of their information, with no individual scale contributing unique information. Conclusions—Salient stroke outcome information is lost when an ordinal scale is collapsed into fewer categories. In contrast, the full scales provide a comprehensive patient outcome estimate. Furthermore, in the context of stroke clinical trials, those scales are highly correlated, providing the rationale to pool them into a single estimate. These insights may be used to optimize the analysis of stroke trials to increase study power to detect efficacious interventions