Yarrowia lipolytica lipase production enhanced by increased air pressure

Aims: To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l^?1 at 1 bar to 533·5 U l^?1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high‐density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.     

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.     

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo

Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.     

Binding of N-terminal fragments of anthrax edema factor (EF(N)) and lethal factor (LF(N)) to the protective antigen pore

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA(63), forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA(63)-channels. Here, in experiments with artificial lipid bilayer membranes, EF(N) and LF(N) show block of PA(63)-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF(N), increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA(63)-channel.     

Increased mortality of Acanthoscelides obtectus by alkane-grown Beauveria bassiana

The effect of alkane-growth induction of theentomopathogenic fungus Beauveria bassiana(Balsamo) Vuillemin (Deuteromycotina:Hyphomycetes), on the ability to kill the beanweevil Acanthoscelides obtectus Say(Coleoptera: Bruchidae), was tested. Adultinsects were sprayed with an 0.01% Tween 20aqueous suspension of 4 × 10⁶ conidia/ml.The performance of fungi grown in complete agarmedium containing glucose as carbon source(FS₀) was compared to that of alkane-grownfungi (FS₁) with n-hexadecane as the onlycarbon source. Mortality increased (p< 0.05) from 22 ± 4.5% to 44 ±11.4% at day 7, and from 26 ± 5.5% to 60± 7.1% 14 days after treatment withFS₀ or FS₁ respectively. The insectepicuticular hydrocarbons were analysed bycapillary gas chromatography (CGC); majorcomponents were saturated hydrocarbons, 27 to29 carbons in length. A variety ofmethyl-branched isomers of C27 were theprevailing structures, and nC27 was the majorstraight chain component. Whole insecthydrocarbons were qualitatively identical tothose of the epicuticular surface. Oleic,linoleic and palmitic acids accounted foralmost 88% of the fungal fatty acids,irrespective of the carbon source used forgrowth; however, the unsaturated/saturatedratio diminished markedly from 4.32(FS₀) to 2.47 (FS₁). These resultsindicate that alkane supplementation of culturemedia might be a tool to improve the virulenceof some mycoinsecticides.     

Système pour la stimulation cardiaque du ventricule gauche sur de multiples électrodes reconfigurables avec une électronique encapsulée hermétique et biocompatible

MULTICARDE is a Tecsan ANR exploratory project that was launched in 2007 and ended up in 2009. ELA Medical (SORIN Group) and the CEA-Leti were associated for this development. The project aims at enhancing the therapy for congestive heart failure based on biventricular electrical resynchronization therapy. Several studies proved the efficiency of this therapy, which remains subjected to the good positioning of the left pacing lead. The MULTICARDE project develops a programmable multi-electrodes pacing to improve the hemodynamic status of the patient. Multiple constraints must be taken into account for this kind of project, such as the power consumption, the size and the interoperability towards leads’ standards. To address these requirements an innovative collective packaging process was developed to integrate electronics inside the lead.     

Lack of coupling of D-2 receptors to adenylate cyclase in GH-3 cells exposed to epidermal growth factor. Possible role of a differential expression of Gi protein subtypes.

Exposure of GH-3 cells to epidermal growth factor for 4 consecutive days induced the expression of both D-2(415) and D-2(444) dopamine-receptor isoforms. Epidermal growth factor also promoted a remarkable increase in the content of Gi3 protein, which is responsible for receptor-induced activation of potassium channels in GH-3 cells. D-2 receptors in this model apparently activate a specific transducing pathway, leading to opening of potassium channels and inhibition of prolactin release by cAMP-independent mechanisms. This is shown by: 1) the selective D-2 agonist quinpirole, while inactive on vasoactive intestinal peptide-induced prolactin release, strongly inhibited the hormone secretion induced by neurotensin; 2) quinpirole, up to 100 microM, did not inhibit cAMP production evoked by vasoactive intestinal peptide both in intact cells and in broken cell membrane preparations; and 3) quinpirole and other D-2 agonists strongly potentiated Rb+ efflux when measured in a nominally calcium-free reaction solution containing 100 mM potassium (voltage-dependent component), but did not modify Rb+ efflux if measured in a reaction solution containing 1 mM calcium and 5 mM potassium (calcium-activated, cAMP-dependent component).