Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCD_max (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPR_min (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.     

Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach

beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with cellular growth but not with lactone production, although Aox activity on short chains was inversely correlated with lactone accumulation. Due to the poor correlation between Aox activity and lactone production, the modeling of this parameter gave no satisfactory results but growth depending on Aox activity was modeled.     

Molecular,quantitative and abiotic variables for the delineation of evolutionary significant units: case of sandalwood (<Emphasis Type="Italic">Santalum austrocaledonicum</Emphasis> Vieillard) in New Caledonia

Various approaches have been developed to define conservation units for plant and animal species. In this study we combined nuclear microsatellites (from a previous published study) and chloroplast microsatellites (assessed in the present study), leaf and seed morphology traits and abiotic variables (climate and soil) to define evolutionary significant units (ESU) of Santalum austrocaledonicum, a tree species growing in New Caledonia. Results for chloroplast microsatellites showed that the total population heterozygosity was␣high, (H _cp = 0.84) but varied between islands. Differentiation was strong in the total population (F _stcp = 0.66) but also within the main island Grande Terre (F _stcp = 0.73) and within Iles Loyauté (F _stcp = 0.52), highlighting a limited gene flow between populations. These results confirmed those obtained with nuclear microsatellites. The cluster analysis on molecular markers discriminated two main groups constituted by the populations of Grande Terre and the populations of Iles Loyauté. A principal component analysis of leaf and seed morphology traits singled out the populations of Iles Loyauté and the western populations of Grande Terre. Quantitative genetic analyses showed that the variation between populations was under genetic control (broad sense heritability close to 80%). A high correlation between rainfall and morphological traits suggested an impact of climate on this variation. The integration of these results allows to define two ESUs, one corresponding to Grande Terre and Ile des Pins and the other the Iles Loyauté archipelago. This study stresses the need to restore some populations of Grande Terre that are currently threatened by their small size.     

Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.     

Personalized pathology test for Cardio-vascular disease: Approximate Bayesian computation with discriminative summary statistics learning

Cardio/cerebrovascular diseases (CVD) have become one of the major health issue in our societies. But recent studies show that the present pathology tests to detect CVD are ineffectual as they do not consider different stages of platelet activation or the molecular dynamics involved in platelet interactions and are incapable to consider inter-individual variability. Here we propose a stochastic platelet deposition model and an inferential scheme to estimate the biologically meaningful model parameters using approximate Bayesian computation with a summary statistic that maximally discriminates between different types of patients. Inferred parameters from data collected on healthy volunteers and different patient types help us to identify specific biological parameters and hence biological reasoning behind the dysfunction for each type of patients. This work opens up an unprecedented opportunity of personalized pathology test for CVD detection and medical treatment.     

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.