Development and assessment of Diversity Arrays Technology for high-throughput DNA analyses in <Emphasis Type="Italic">Musa</Emphasis>

Diversity Arrays Technology (DArT) is a DNA hybridisation-based molecular marker technique that can detect simultaneously variation at numerous genomic loci without sequence information. This efficiency makes it a potential tool for a quick and powerful assessment of the structure of germplasm collections. This article demonstrates the usefulness of DArT markers for genetic diversity analyses of Musa spp. genotypes. We developed four complexity reduction methods to generate DArT genomic representations and we tested their performance using 48 reference Musa genotypes. For these four complexity reduction methods, DArT markers displayed high polymorphism information content. We selected the two methods which generated the most polymorphic genomic representations (PstI/BstNI 16.8%, PstI/TaqI 16.1%) to analyze a panel of 168 Musa genotypes from two of the most important field collections of Musa in the world: Cirad (Neufchateau, Guadeloupe), and IITA (Ibadan, Nigeria). Since most edible cultivars are derived from two wild species, Musa acuminata (A genome) and Musa balbisiana (B genome), the study is restricted mostly to accessions of these two species and those derived from them. The genomic origin of the markers can help resolving the pedigree of valuable genotypes of unknown origin. A total of 836 markers were identified and used for genotyping. Ten percent of them were specific to the A genome and enabled targeting this genome portion in relatedness analysis among diverse ploidy constitutions. DArT markers revealed genetic relationships among Musa genotype consistent with those provided by the other markers technologies, but at a significantly higher resolution and speed and reduced cost.     

Maximizing power production in a stack of microbial fuel cells using multiunit optimization method

This study demonstrates real‐time maximization of power production in a stack of two continuous flow microbial fuel cells (MFCs). To maximize power output, external resistances of two air–cathode membraneless MFCs were controlled by a multiunit optimization algorithm. Multiunit optimization is a recently proposed method that uses multiple similar units to optimize process performance. The experiment demonstrated fast convergence toward optimal external resistance and algorithm stability during external perturbations (e.g., temperature variations). Rate of the algorithm convergence was much faster than in traditional maximum power point tracking algorithms (MPPT), which are based on temporal perturbations. A power output of 81–84 mW/L_A (A = anode volume) was achieved in each MFC. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

Aldosterone regulation of T-type calcium channels

Voltage-operated calcium channels play a crucial role in signal transduction in many excitable and non-excitable cell types. While a rapid modulation of their activity by hormone-activated kinases and/or G proteins has been recognized for a long time, a sustained control of their expression level has been only recently demonstrated. In adrenal H295R cells, for example, aldosterone treatment selectively increased low threshold T-type calcium current density without affecting L-type currents. Antagonizing the mineralocorticoid receptor (MR) with spironolactone prevented aldosterone action on T-type currents. By RT-PCR, we detected in these cells the presence of two different isoforms of L-type channels, alpha(1)C and alpha(1)D, and one isoform of T channel, alpha(1)H. A second T channel isoform (alpha(1)G) was also observed under particular culture conditions. Quantification of the specific messenger RNA by real time RT-PCR allowed us to show a 40% increase of the alpha1H messenger levels upon aldosterone treatment (alpha(1)G was insensitive), a response that was also completely prevented by spironolactone. Because T-type, but not L-type channel activity is linked to steroidogenesis, this modulation represents a positive, intracrine feed back mechanism exerted by aldosterone on its own production.Aldosterone has been also implicated in the pathogenesis and progression of ventricular hypertrophy and heart failure independently of its action on arterial blood pressure. We have observed that, in rat neonatal cardiomyocytes, aldosterone increases (by two-fold) L-type calcium current amplitude in ventricular but not in atrial cells. No significant effect of aldosterone could be detected on T-type currents, that were much smaller than L-type currents in these cells. However, aldosterone exerted opposite effects on T channel isoform expression, increasing alpha(1)H and decreasing alpha(1)G. Although the functional role of T channels is still poorly defined in ventricular cardiomyocytes, an overexpression of alpha(1)H could be partially responsible for the arrhythmias linked to hyperaldosteronism.Finally, T channels also appear to be involved in the neuroendocrine differentiation of prostate epithelial cells, a poor prognosis in prostate cancer. We have shown that the only calcium channel expressed in the prostatic LNCaP cells is the alpha(1)H isoform and that induction of cell differentiation with cAMP leads to a concomitant increase in both T-type current and alpha(1)H mRNA. In spite of the presence of MR in these cells, aldosterone only modestly increased alpha(1)H mRNA levels. A functional role for these channels was suggested by the observation that low nickel concentrations prevent neuritic process outgrowth.In conclusion, it appears that T-type calcium channel expression vary in different patho-physiological conditions and that aldosterone, in several cell types, is able to modulate this expression.     

Development and validation of experimental protocols for use of cardinal models for prediction of microorganism growth in food products

An experimental protocol to validate secondary-model application to foods was suggested. Escherichia coli, Listeria monocytogenes, Bacillus cereus, Clostridium perfringens, and Salmonella were observed in various food categories, such as meat, dairy, egg, or seafood products. The secondary model validated in this study was based on the gamma concept, in which the environmental factors temperature, pH, and water activity (aw) were introduced as individual terms with microbe-dependent parameters, and the effect of foodstuffs on the growth rates of these species was described with a food- and microbe-dependent parameter. This food-oriented approach was carried out by challenge testing, generally at 15 and 10 degrees C for L. monocytogenes, E. coli, B. cereus, and Salmonella and at 25 and 20 degrees C for C. perfringens. About 222 kinetics in foods were generated. The results were compared to simulations generated by existing software, such as PMP. The bias factor was also calculated. The methodology to obtain a food-dependent parameter (fitting step) and therefore to compare results given by models with new independent data (validation step) is discussed in regard to its food safety application. The proposed methods were used within the French national program of predictive microbiology, Sym'Previus, to include challenge test results in the database and to obtain predictive models designed for microbial growth in food products.     

Making and repairing the mammalian brain--in vitro production of dopaminergic neurons

Midbrain dopamine (DA) neurons play an essential role in modulating motor control, and their degeneration is the hallmark feature of Parkinson's disease (PD). In vitro production of DA neurons provides insight into the mechanisms that control cell fate choice, and offers an alternative to the use of fetal tissue for experimental cell replacement in PD. Here we will review the advantages and disadvantages of the various renewable cell sources and protocols tested, and discuss their relevance for basic studies and for cell therapy.