DFM synthesis approach based on product-process interface modelling. Application to the peen forming process.

Engineering design approach are curently CAD-centred design process. Manufacturing information is selected and assessed very late in the design process and above all as a reactive task instead of being proactive to lead the design choices. DFM appraoches are therefore assesment methods that compare several design alternatives and not real design approaches at all. Main added value of this research work concerns the use of a product-process interface model to jointly manage both the product and the manufacturing data in a proactive DFM way. The DFM synthesis approach and the interface model are presented via the description of the DFM software platform

Impact of Actinobacillus pleuropneumoniae biofilm mode of growth on the lipid A structures and stimulation of immune cells

Actinobacillus pleuropneumoniae (APP), the etiologic agent of porcine pleuropneumonia, forms biofilms on biotic and abiotic surfaces. APP biofilms confers resistance to antibiotics. To our knowledge, no studies have examined the role of APP biofilm in immune evasion and infection persistence. This study was undertaken to (i) investigate biofilm-associated LPS modifications occurring during the switch to biofilm mode of growth; and (ii) characterize pro-inflammatory cytokines expression in porcine pulmonary alveolar macrophages (PAMs) and proliferation in porcine PBMCs challenged with planktonic or biofilm APP cells. Extracted lipid A samples from biofilm and planktonic cultures were analyzed by HPLC high-resolution, accurate mass spectrometry. Biofilm cells displayed significant changes in lipid A profiles when compared with their planktonic counterparts. Furthermore, in vitro experiments were conducted to examine the inflammatory response of PAMs exposed to UV-inactivated APP grown in biofilm or in suspension. Relative mRNA expression of pro-inflammatory genes IL1, IL6, IL8 and MCP1 decreased in PAMs when exposed to biofilm cells compared to planktonic cells. Additionally, the biofilm state reduced PBMCs proliferation. Taken together, APP biofilm cells show a weaker ability to stimulate innate immune cells, which could be due, in part, to lipid A structure modifications

Baseline urinary metabolites predict albuminuria response to spironolactone in type 2 diabetes

The mineralocorticoid receptor antagonist spironolactone significantly reduces albuminuria in subjects with diabetic kidney disease, albeit with a large variability between individuals. Identifying novel biomarkers that predict response to therapy may help to tailor spironolactone therapy. We aimed to identify a set of metabolites for prediction of albuminuria response to spironolactone in subjects with type 2 diabetes. Systems biology molecular process analysis was performed a priori to identify metabolites linked to molecular disease processes and drug mechanism of action. Individual subject data and urine samples were used from 2 randomized placebo controlled double blind clinical trials (NCT01062763, NCT00381134). A urinary metabolite score was developed to predict albuminuria response to spironolactone therapy using penalized ridge regression with leave-one-out cross validation. Bioinformatic analysis identified a set of 18 metabolites linked to a diabetic kidney disease molecular model and potentially affected by spironolactone mechanism of action. Spironolactone reduced UACR relative to placebo by median -42% (25th to 75% percentile -65 to 6) and -29% (25th to 75% percentile -37 to -1) in the test and replication cohorts, respectively. In the test cohort, UACR reduction was higher in the lowest tertile of the baseline urinary metabolite score compared with middle and upper tertiles -58% (25th to 75% percentile -78 to 33), -28% (25th to 75% percentile -46 to 8), -40% (25th to 75% percentile -52% to 31), respectively, P= 0.001 for trend). In the replication cohort, UACR reduction was -54% (25th to 75% percentile -65 to -50), -41 (25th to 75% percentile -46% to 30), and -17% (25th to 75% percentile -36 to 5), respectively, P= 0.010 for trend). We identified a set of 18 urinary metabolites through systems biology to predict albuminuria response to spironolactone in type 2 diabetes. These data suggest that urinary metabolites may be used as a tool to tailor optimal therapy and move in the direction of personalized medicine

DFM synthesis approach based on product-process interface modelling. Application to the peen forming process.

International audienceEngineering design approach are curently CAD-centred design process. Manufacturing information is selected and assessed very late in the design process and above all as a reactive task instead of being proactive to lead the design choices. DFM appraoches are therefore assesment methods that compare several design alternatives and not real design approaches at all. Main added value of this research work concerns the use of a product-process interface model to jointly manage both the product and the manufacturing data in a proactive DFM way. The DFM synthesis approach and the interface model are presented via the description of the DFM software platform

Flexibility of a biotinylated ligand in artificial metalloenzymes based on streptavidin—an insight from molecular dynamics simulations with classical and ab initio force fields

In the field of enzymatic catalysis, creating activity from a non catalytic scaffold is a daunting task. Introduction of a catalytically active moiety within a protein scaffold offers an attractive means for the creation of artificial metalloenzymes. With this goal in mind, introduction of a biotinylated d6-piano-stool complex within streptavidin (SAV) affords enantioselective artificial transfer-hydrogenases for the reduction of prochiral ketones. Based on an X-ray crystal structure of a highly selective hybrid catalyst, displaying significant disorder around the biotinylated catalyst [η6-(p-cymene)Ru(Biot-p-L)Cl], we report on molecular dynamics simulations to shed light on the protein–cofactor interactions and contacts. The results of these simulations with classical force field indicate that the SAV-biotin and SAV-catalyst complexes are more stable than ligand-free SAV. The point mutations introduced did not affect significantly the overall behavior of SAV and, unexpectedly, the P64G substitution did not provide additional flexibility to the protein scaffold. The metal-cofactor proved to be conformationally flexible, and the S112K or P64G mutants proved to enhance this effect in the most pronounced way. The network of intermolecular hydrogen bonds is efficient at stabilizing the position of biotin, but much less at fixing the conformation of an extended biotinylated ligand. This leads to a relative conformational freedom of the metal-cofactor, and a poorly localized catalytic metal moiety. MD calculations with ab initio potential function suggest that the hydrogen bonds alone are not sufficient factors for full stabilization of the biotin. The hydrophobic biotin-binding pocket (and generally protein scaffold) maintains the hydrogen bonds between biotin and protein

β-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora

Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO3−) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into α-, β-, γ-, δ- and ζ-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of β-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding β-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Δcas1, Δcas2, and Δcas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Δcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Δcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions