Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

Automatic object segmentation from calibrated images

This paper addresses the problem of automatically obtaining the object/background segmentation of a rigid 3D object observed in a set of images that have been calibrated for camera pose and intrinsics. Such segmentations can be used to obtain a shape representation of a potentially texture-less object by computing a visual hull. We propose an automatic approach where the object to be segmented is identified by the pose of the cameras instead of user input such as 2D bounding rectangles or brush-strokes. The key behind our method is a pairwise MRF framework that combines (a) foreground/background appearance models, (b) epipolar constraints and (c) weak stereo correspondence into a single segmentation cost function that can be efficiently solved by Graph-cuts. The segmentation thus obtained is further improved using silhouette coherency and then used to update the foreground/background appearance models which are fed into the next Graph-cut computation. These two steps are iterated until segmentation convergences. Our method can automatically provide a 3D surface representation even in texture-less scenes where MVS methods might fail. Furthermore, it confers improved performance in images where the object is not readily separable from the background in colour space, an area that previous segmentation approaches have found challenging. © 2011 IEEE.

Enhanced dispersibility and deposition of spray-dried powders for pulmonary gene therapy

Spray-drying represents a viable alternative to freeze-drying for preparing dry powder dispersions for delivering macromolecules to the lung. The dispersibility of spray-dried powders is limited however, and needs to be enhanced to improve lung deposition and subsequent biological activity. In this study, we investigate the utility of leucine as a dry powder dispersibility enhancer when added prior to spray-drying a model non-viral gene therapy formulation (lipid:polycation:pDNA, LPD). Freeze-dried lactose-LPD, spray-dried lactose-LPD and spray-dried leucine-lactose-LPD powders were prepared. Scanning electron microscopy showed that leucine, increased the surface roughness of spray-dried lactose particles. Particle size analysis revealed that leucine-containing spray-dried powders were unimodally dispersed with a mean particle diameter of 3.12 μm. Both gel electrophoresis and in vitro cell (A549) transfection showed that leucine may compromise the integrity and biological functionality of the gene therapy vector. The deposition of the leucine containing powder was however significantly enhanced as evidenced by an increase in gene expression mediated by dry powder collected at lower stages of a multistage liquid impinger (MSLI). Further studies are required to determine the potential of leucine as a ubiquitous dispersibility enhancer for a variety of pulmonary formulations. © 2003 Taylor & Francis Ltd.

McKinney, Mark. Redrawing French Empire Comics. Columbus, The Ohio State University Press, 2013. 304 pages. 9780814212202

The history of France and its empires is one that has been well trodden, particularly the French occupation, and subsequent war, in Algeria. In this companion to his earlier work, 2011’s The Colonial Heritage of French Comics,McKinney attempts to examine the reconstruction of French national identity in the wake of decolonisation through the medium of Francophonecomics. He endeavours to study the colonial affrontier (3), the space in which France and its colonies are connected and divided, where they seek to confront each other, or to seek peace and the removal of the division. McKinney argues this affrontier can be found most strongly in the Francophone comics produced dealing with the French colonial experience in Algeria, as well as that of Indochina,and does so from both sides of each conflict. McKinney examines in detail the French colonisation of Algeria (1830sonwards), the French war in Indochina (1946–54) and the Algerian war (1954–62), and his work is the first to approach these well-covered areas of research through the medium of comics. The resulting work takes the form of an investigation into the five forms of genealogical inquiry utilised in comics regarding these conflicts. His approach investigates the familial, ethnic, national, artistic and critical forms of genealogy relating to colonialism and imperialism from a variety of viewpoints, including the previously overlooked perspective of the pieds noirs. He aims to highlight both those cartoonists that critique the colonial ideology, as well as those cartoonists who to some extent attempt to gloss over or even romanticise the French empire, strengthening the affrontier. He positions himself alongside Foucault in seeing genealogy as a useful means of establishing ‘historical knowledge of struggles’ (Foucault1980, 85), but McKinney looks at the colonial representation in a popular medium,including the recent increase in comics produced which consider the French colonial experience. He argues that this consideration of the present, as well as European imperialism, is absent in the work of Foucault. The text is accompanied by a number of black and white facsimiles of pages from the comics he analyses to illustrate the different and often conflicting positions of cartoonists on these issues. Overall, McKinney’s work is a welcome addition to the study of the French colonial experience, which separates its elf from the rest by using Francophonecomics as lenses through which to look at these already well-trodden areas of study. He succeeds in determining if and how cartoonists critique colonial ideology and representations on both sides of the conflicts, a task in which he is unarguably successful. McKinney’s work, however, is unfortunately let down by typo graphicerrors, which occur throughout the text.Nevertheless, McKinney’s work is another important work in the field of Bande Dessine ́e scholarship, and useful for anyone interested in the representations of colonialism and imperialism in French comics, accompanied by anencyclopaedic bibliography of comics produced on this topic.

Application and use of isothermal calorimetry in pharmaceutical development

There are many steps involved in developing a drug candidate into a formulated medicine and many involve analysis of chemical interaction or physical change. Calorimetry is particularly suited to such analyses as it offers the capacity to observe and quantify both chemical and physical changes in virtually any sample. Differential scanning calorimetry (DSC) is ubiquitous in pharmaceutical development, but the related technique of isothermal calorimetry (IC) is complementary and can be used to investigate a range of processes not amenable to analysis by DSC. Typically, IC is used for longer-term stability indicating or excipient compatibility assays because both the temperature and relative humidity (RH) in the sample ampoule can be controlled. However, instrument design and configuration, such as titration, gas perfusion or ampoule-breaking (solution) calorimetry, allow quantification of more specific values, such as binding enthalpies, heats of solution and quantification of amorphous content. As ever, instrument selection, experiment design and sample preparation are critical to ensuring the relevance of any data recorded. This article reviews the use of isothermal, titration, gas-perfusion and solution calorimetry in the context of pharmaceutical development, with a focus on instrument and experimental design factors, highlighted with examples from the recent literature. © 2011 Elsevier B.V.

Quantitative analysis of solid-state processes studied with isothermal microcalorimetry

Quantitative analysis of solid-state processes from isothermal microcalorimetric data is straightforward if data for the total process have been recorded and problematic (in the more likely case) when they have not. Data are usually plotted as a function of fraction reacted (α); for calorimetric data, this requires knowledge of the total heat change (Q) upon completion of the process. Determination of Q is difficult in cases where the process is fast (initial data missing) or slow (final data missing). Here we introduce several mathematical methods that allow the direct calculation of Q by selection of data points when only partial data are present, based on analysis with the Pérez-Maqueda model. All methods in addition allow direct determination of the reaction mechanism descriptors m and n and from this the rate constant, k. The validity of the methods is tested with the use of simulated calorimetric data, and we introduce a graphical method for generating solid-state power-time data. The methods are then applied to the crystallization of indomethacin from a glass. All methods correctly recovered the total reaction enthalpy (16.6 J) and suggested that the crystallization followed an Avrami model. The rate constants for crystallization were determined to be 3.98 × 10-6, 4.13 × 10-6, and 3.98 × 10 -6 s-1 with methods 1, 2, and 3, respectively. © 2010 American Chemical Society.

High-sensitivity differential scanning calorimetry for measurement of steroid entrapment in nebulised liposomes generated from proliposomes

A novel approach to the determination of steroid entrapment in the bilayers of aerosolised liposomes has been introduced using high-sensitivity differential scanning calorimetry (DSC). Proliposomes were dispersed in water within an air-jet nebuliser and the energy produced during atomisation was used to hydrate the proliposomes and generate liposome aerosols. Proliposomes that included the steroid beclometasone dipropionate (BDP) produced lower aerosol and lipid outputs than steroid-free proliposomes. Size analysis and transmission electron microscopy showed an evidence of liposome formation within the nebuliser, which was followed by deaggregation and size reduction of multilamellar liposomes on nebulisation to a two-stage impinger. For each formulation, no difference in thermal transitions was observed between delivered liposomes and those remaining in the nebuliser. However, steroid (5 mole%) lowered the onset temperature and the enthalpy of the pretransition, and produced a similar onset temperature and larger enthalpy of the main transition, with broadened pretransition and main transitions. This indicates that BDP was entrapped and exhibited an interaction with the liposome phospholipid membranes. Since the pretransition was depressed but not completely removed and no phase separation occurred, it is suggested that the bilayers of the multilamellar liposomes can entrap more than 5 mole% BDP. Overall, liposomes were generated from proliposomes and DSC investigations indicated that the steroid was entrapped in the bilayers of aerosolised multilamellar vesicles.