Secreted production of an elastin-like polypeptide by Pichia pastoris

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium

Multi-Layered Films Containing a Biomimetic Stimuli-Responsive Recombinant Protein

Electrostatic self-assembly was used to fabricate new smart multi-layer coatings, using a recombinant elastin-like polymer (ELP) and chitosan as the counterion macromolecule. The ELP was bioproduced, purified and its purity and expected molecular weight were assessed. Aggregate size measurements, obtained by light scattering of dissolved ELP, were performed as a function of temperature and pH to assess the smart properties of the polymer. The build-up of multi-layered films containing ELP and chitosan, using a layer-by-layer methodology, was followed by quartz-crystal microbalance with dissipation monitoring. Atomic force microscopy analysis permitted to demonstrate that the topography of the multi-layered films could respond to temperature. This work opens new possibilities for the use of ELPs in the fabrication of biodegradable smart coatings and films, offering new platforms in biotechnology and in the biomedical area

X-ray Absorption and Reflection in Active Galactic Nuclei

X-ray spectroscopy offers an opportunity to study the complex mixture of emitting and absorbing components in the circumnuclear regions of active galactic nuclei, and to learn about the accretion process that fuels AGN and the feedback of material to their host galaxies. We describe the spectral signatures that may be studied and review the X-ray spectra and spectral variability of active galaxies, concentrating on progress from recent Chandra, XMM-Newton and Suzaku data for local type 1 AGN. We describe the evidence for absorption covering a wide range of column densities, ionization and dynamics, and discuss the growing evidence for partial-covering absorption from data at energies > 10 keV. Such absorption can also explain the observed X-ray spectral curvature and variability in AGN at lower energies and is likely an important factor in shaping the observed properties of this class of source. Consideration of self-consistent models for local AGN indicates that X-ray spectra likely comprise a combination of absorption and reflection effects from material originating within a few light days of the black hole as well as on larger scales. It is likely that AGN X-ray spectra may be strongly affected by the presence of disk-wind outflows that are expected in systems with high accretion rates, and we describe models that attempt to predict the effects of radiative transfer through such winds, and discuss the prospects for new data to test and address these ideas.Comment: Accepted for publication in the Astronomy and Astrophysics Review. 58 pages, 9 figures. V2 has fixed an error in footnote

Hiding Relations

The present vogue of ‘managing for development results’ is an expression of a historically dominant mode of thought in international aid – ‘substantialism’ – which sees the world primarily in terms of ‘entities’ such as ‘poverty’, ‘basic needs’, ‘rights’, ‘women’, or ‘results’. Another important mode of thought, ‘relationalism’ – in association more generally with ideas of process and complexity – appears to be absent in the thinking of aid institutions. Drawing on my own experiences of working with the UK Department for International Development (DFID), I illustrate how despite formally subscribing to the institution’s substantialist view of the world, some staff are ‘closet relationists’, behaving according to one mode of thought while officially framing their action in terms of the other, more orthodox mode. In so doing, they may be unwittingly keeping international aid sufficiently viable - by the apparent proof of the efficacy of results-based management - to enable the institution as a whole to maintain its substantialist imaginary

Biocatalytic Synthesis of Polymers of Precisely Defined Structures

The fabrication of functional nanoscale devices requires the construction of complex architectures at length scales characteristic of atoms and molecules. Currently microlithography and micro-machining of macroscopic objects are the preferred methods for construction of small devices, but these methods are limited to the micron scale. An intriguing approach to nanoscale fabrication involves the association of individual molecular components into the desired architectures by supramolecular assembly. This process requires the precise specification of intermolecular interactions, which in turn requires precise control of molecular structure

Disordered protein-graphene oxide co-assembly and supramolecular biofabrication of functional fluidic devices

Supramolecular chemistry offers an exciting opportunity to assemble materials with molecular precision. However, there remains an unmet need to turn molecular self-assembly into functional materials and devices. Harnessing the inherent properties of both disordered proteins and graphene oxide (GO), we report a disordered protein-GO co-assembling system that through a diffusion-reaction process and disorder-to-order transitions generates hierarchically organized materials that exhibit high stability and access to non-equilibrium on demand. We use experimental approaches and molecular dynamics simulations to describe the underlying molecular mechanism of formation and establish key rules for its design and regulation. Through rapid prototyping techniques, we demonstrate the system's capacity to be controlled with spatio-temporal precision into well-defined capillary-like fluidic microstructures with a high level of biocompatibility and, importantly, the capacity to withstand flow. Our study presents an innovative approach to transform rational supramolecular design into functional engineering with potential widespread use in microfluidic systems and organ-on-a-chip platforms

Calcitriol modulates the CD46 pathway in T cells

The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. In this pilot study, we examined whether active vitamin D (1,25(OH)2D3 or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated CD4+ T cells from patients with MS. In healthy T cells, calcitriol profoundly affects the phenotype of CD46-costimulated CD4+ T cells, by increasing the expression of CD28, CD25, CTLA-4 and Foxp3 while it concomitantly decreased CD46 expression. Similar trends were observed in MS CD4+ T cells except for CD25 for which a striking opposite effect was observed: while CD25 was normally induced on MS T cells by CD46 costimulation, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNc ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Hence, we show that calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS

The interaction of Thrombospondins with extracellular matrix proteins

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias