Bioadhesiveness and efficient mechanotransduction stimuli synergistically provided by bacterial inclusion bodies as scaffolds for tissue engineering.

Background: Bacterial inclusion bodies (IBs), mechanically stable, submicron protein particles of 50-500 nm dramatically favor mammalian cell spread when used for substrate surface decoration. The mechanisms supporting fast colonization of IB-modified surfaces have not yet been identified.

Results: This study provides evidence of mechanotransduction-mediated stimulation of mammalian cell proliferation on IB-decorated surfaces, as observed by the enhanced phosphorylation of the signal-regulated protein kinase and by the dramatic emission of filopodia in the presence of IBs.

Biological activities of histidine-rich peptides; merging biotechnology and nanomedicine.

Histidine-rich peptides are commonly used in recombinant protein production as purification tags, allowing the one-step affinity separation of the His-tagged proteins from the extracellular media or cell extracts. Genetic engineering makes feasible the post-purification His-tag removal by inserting, between the tag and the main protein body, a target site for trans-acting proteases or a self-proteolytic peptide with regulatable activities. However, for technical ease, His tags are often not removed and the fusion proteins eventually used in this form.

Bacterial inclusion bodies: making gold from waste.

Many protein species produced in recombinant bacteria aggregate as insoluble protein clusters named inclusion bodies (IBs). IBs are discarded from further processing or are eventually used as a pure protein source for in vitro refolding. Although usually considered as waste byproducts of protein production, recent insights into the physiology of recombinant bacteria and the molecular architecture of IBs have revealed that these protein particles are unexpected functional materials.

Co-production of GroELS discriminates between intrinsic and thermally-induced recombinant protein aggregation during substrate quality control.

Background: The effects and effectiveness of the chaperone pair GroELS on the yield and quality of recombinant polypeptides produced in Escherichia coli are matter of controversy, as the reported activities of this complex are not always consistent and eventually indicate undesired side effects. The divergence in the reported data could be due, at least partially, to different experimental conditions in independent research approaches.

Results: We have then selected two structurally different model proteins (namely GFP and E.

Overexpression of the immunoreceptor CD300f has a neuroprotective role in a model of acute brain injury.

It is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). We have analyzed the function of cluster of differentiation (CD)300f immunoreceptor in a model of excitotoxic rat brain damage. First, to explore the presence of endogenous ligand(s) for this receptor we used a human CD300f-Ig soluble protein and confocal microscopy, showing specific staining mainly in CNS white matter and on the surface of oligodendrocytes and certain astrocytes.

Interleukin-10 overexpression does not synergize with the neuroprotective action of RGD-containing vectors after postnatal brain excitotoxicity but modulates the main inflammatory cell responses.

Antiinflammatory cytokines such as interleukin-10 (IL-10) have been used to modulate and terminate inflammation and provide neuroprotection. Recently, we reported that the modular recombinant transfection vector NLSCt is an efficient tool for transgene overexpression in vivo, which induces neuroprotection as a result of its RGD-mediated integrin-interacting capacity. We here sought to evaluate the putative synergic neuroprotective action exerted by IL-10 overexpression using NLSCt as a transfection vector after an excitotoxic injury to the postnatal rat brain.

How to break recombinant bacteria: does it matter?

Recombinant proteins and other materials of industrial interest produced in Escherichia coli are usually retained within the bacterial cell, in the cytoplasmic space, where they have been produced. Different protocols for cell disruption have been implemented as an initial downstream step, which keeps the biological and mechanical properties of the process products. Being necessarily mild, these approaches often result in 95-99% cell disruption, what is more than acceptable from the yield point of view.

Post-production protein stability: trouble beyond the cell factory.

Being protein function a conformation-dependent issue, avoiding aggregation during production is a major challenge in biotechnological processes, what is often successfully addressed by convenient upstream, midstream or downstream approaches. Even when obtained in soluble forms, proteins tend to aggregate, especially if stored and manipulated at high concentrations, as is the case of protein drugs for human therapy. Post-production protein aggregation is then a major concern in the pharmaceutical industry, as protein stability, pharmacokinetics, bioavailability, immunogenicity and side effects are largely dependent on the extent of aggregates formation.

Use of a generalized fisher equation for global optimization in chemical kinetics.

A new approach for parameter estimation in chemical kinetics has been recently proposed (Ross et al. Proc. Natl.

Dual Effect of low-level laser therapy (LLLT) on the acute lung inflammation induced by intestinal ischemia and reperfusion: Action on anti- and pro-inflammatory cytokines.

Background And Objective: It is unknown if pro- and anti-inflammatory mediators in acute lung inflammation induced by intestinal ischemia and reperfusion (i-I/R) can be modulated by low-level laser therapy (LLLT).

Study Design/material And Methods: A controlled ex vivo study was developed in which rats were irradiated (660 nm, 30 mW, 0.08 cm² of spot size) on the skin over the right upper bronchus 1 hour post-mesenteric artery occlusion and euthanized 4 hours later.

Nanoparticulate architecture of protein-based artificial viruses is supported by protein-DNA interactions.

Unlabelled: AIM & METHODS: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine-glycine-aspartic acid cell-binding domains and promote significant transgene expression levels.

Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells.

Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested.

Biological role of bacterial inclusion bodies: a model for amyloid aggregation.

Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry.

Analytical approaches for assessing aggregation of protein biopharmaceuticals.

Production of protein-based pharmaceuticals is a major issue in conventional pharmacology, biomedicine and nanomedicine. Being mostly obtained by genetic engineering, the quality and activity of protein drugs is a steady matter of concern. Although the physiology of the host recombinant cells, mostly mammalian and microbial, is progressively understood, the complexity of the cellular quality control systems escapes rational protein and process engineering, and recombinant proteins are often unstable, aggregate and/or do not reach the fully native conformation compatible with proper biological activity.

Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production.

Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield.

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis.

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance.

Engineering building blocks for self-assembling protein nanoparticles.

Like natural viruses, manmade protein cages for drug delivery are to be ideally formed by repetitive subunits with self-assembling properties, mimicking viral functions and molecular organization. Naturally formed nanostructures (such as viruses, flagella or simpler protein oligomers) can be engineered to acquire specific traits of interest in biomedicine, for instance through the addition of cell targeting agents for desired biodistribution and specific delivery of associated drugs. However, fully artificial constructs would be highly desirable regarding finest tuning and adaptation to precise therapeutic purposes.

Low-level laser therapy (LLLT) acts as cAMP-elevating agent in acute respiratory distress syndrome.

The aim of this work was to investigate if the low-level laser therapy (LLLT) on acute lung inflammation (ALI) induced by lipopolysaccharide (LPS) is linked to tumor necrosis factor (TNF) in alveolar macrophages (AM) from bronchoalveolar lavage fluid (BALF) of mice. LLLT has been reported to actuate positively for relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased TNF mRNA expression and dysfunction of cAMP generation observed in ALI can be influenced by LLLT.

Low-level laser therapy associated to N-acetylcysteine lowers macrophage inflammatory protein-2 (MIP-2) mRNA expression and generation of intracellular reactive oxygen species in alveolar macrophages.

Objective: The aim of this work was to investigate the low-level laser therapy (LLLT) effect on alveolar macrophages (AM) activated by oxidative stress and lipopolysaccharide (LPS).

Background Data: LLLT has been reported to actuate positively relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased MIP-2 mRNA expression and intracellular reactive oxygen species (ROS) generation observed in acute lung inflammation (ALI) can be influenced by LLLT.

Environmental quality of mussel farms in the Vigo estuary: pollution by PAHs, origin and effects on reproduction.

This work analyzes the influence of environmental and physiological parameters on PAHs accumulation in cultured mussels. Lipid content and reproductive stage are directly related with PAHs accumulation pattern. We observed a rapid accumulation and depuration of PAHs, mainly during periods of nutrients accumulation, spawns and gonadic restorations.

Internalization and kinetics of nuclear migration of protein-only, arginine-rich nanoparticles.

Understanding the intracellular trafficking of nanoparticles internalized by mammalian cells is a critical issue in nanomedicine, intimately linked to therapeutic applications but also to toxicity concerns. While the uptake mechanisms of carbon nanotubes and polymeric particles have been investigated fairly extensively, there are few studies on the migration and fate of protein-only nanoparticles other than natural viruses. Interestingly, protein nanoparticles are emerging as tools in personalized medicines because of their biocompatibility and functional tuneability, and are particularly promising for gene therapy and also conventional drug delivery.

Isolation of cell-free bacterial inclusion bodies.

Background: Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces.

Side effects of chaperone gene co-expression in recombinant protein production.

Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield.

Comparative study of the topical application of Aloe vera gel, therapeutic ultrasound and phonophoresis on the tissue repair in collagenase-induced rat tendinitis.

The aim of our study was to compare topical use of Aloe vera gel, pulsed mode ultrasound (US) and Aloe vera phonophoresis on rat paw with collagenase-induced tendinitis. Edema size, tensile tendon strength, tendon elasticity, number of inflammatory cells and tissue histology were studied at 7 and 14 days after tendinitis induction. Pulse mode US parameters were: 1 MHz frequency, 100 Hz repetition rate, 10% duty cycle, and 0.