Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor

<p>Abstract</p> <p>Background</p> <p>Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in <it>Staphylococcus aureus </it>spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, <it>Lactococcus lactis</it>, and purified from the culture medium.</p> <p>Results</p> <p>The gene segment corresponding to the <it>S. aureus </it>nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An <it>L. lactis </it>subsp <it>cremoris </it>model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure.</p> <p>Conclusions</p> <p>In <it>L. lactis</it>, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD₆₀₀of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg.</p

Regulation of FATTY ACID ELONGATION1 expression in embryonic and vascular tissues of Brassica napus

International audienceThe expression of the FATTY ACID ELONGATION1 genes was characterised to provide insight into the regulation of very long chain fatty acid (VLCFA) biosynthesis in Brassica napus embryos. Each of the two rapeseed homoeologous genes (Bn-FAE1.1 and Bn-FAE1.2) encoding isozymes of 3-keto-acylCoA synthase, a subunit of the cytoplasmic acyl-CoA elongase complex that controls the production of elongated fatty acids, are expressed predominantly in developing seeds. The proximal regions of the Bn-FAE1.1 and Bn-FAE1.2 promoters possess strong sequence identity suggesting that transcriptional control of expression is mediated by this region which contains putative cis-elements characteristic of those found in the promoters of genes expressed in embryo and endosperm. Histochemical staining of rapeseed lines expressing Bn-FAE1.1 promoter:reporter gene fusions revealed a strong expression in the embryo cotyledon and axis throughout the maturation phase. Quantitative analyses revealed the region, −331 to −149, exerts a major control on cotyledon specific expression and the level of expression. A second region, −640 to −475, acts positively to enhance expression levels and extends expression of Bn-FAE1.1 into the axis and hypocotyl but also acts negatively to repress expression in the root meristem. The expression of the Bn-FAE1.1 gene was not restricted to the seed but was also detected in the vascular tissues of germinating seedlings and mature plants in the fascicular cambium tissue present in roots, stem and leaf petiole. We propose that Bn-FAE1.1 expression in vascular tissue may contribute VLCFA for barrier lipid synthesis and reflects the ancestral function of FAE1 encoded 3-keto-acylCoA synthase

Proteomic Analysis of Saliva from Patients with Oral Chronic Graft-Versus-Host Disease

AbstractChronic graft-versus-host disease (cGVHD) is an immune-mediated disorder and is the major long-term complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The oral mucosa, including the salivary glands, is affected in the majority of patients with cGVHD; however, at present there is only a limited understanding of disease pathobiology. In this study, we performed a quantitative proteomic analysis of saliva pooled from patients with and without oral cGVHD—cGVHD(+) and cGVHD(−), respectively—using isobaric tags for relative and absolute quantification labeling, followed by tandem mass spectrometry. Among 249 salivary proteins identified by tandem mass spectrometry, 82 exhibited altered expression in the oral cGVHD(+) group compared with the cGVHD(−) group. Many of the identified proteins function in innate or acquired immunity, or are associated with tissue maintenance functions, such as proteolysis or the cytoskeleton. Using ELISA immunoassays, we further confirmed that 2 of these proteins, IL-1 receptor antagonist and cystatin B, showed decreased expression in patients with active oral cGVHD (P < .003). Receiver operating curve characteristic analysis revealed that these 2 markers were able to distinguish oral cGVHD with a sensitivity of 85% and specificity of 60%, and showed slightly better discrimination in newly diagnosed patients evaluated within 12 months of allo-HSCT (sensitivity, 92%; specificity 73%). In addition to identifying novel potential salivary cGVHD biomarkers, our study demonstrates that there is coordinated regulation of protein families involved in inflammation, antimicrobial defense, and tissue protection in oral cGVHD that also may reflect changes in salivary gland function and damage to the oral mucosa

MIL-91(Ti), a small pore metal-organic framework which fulfils several criteria : an upscaled green synthesis, excellent water stability, high CO2 selectivity and fast CO2 transport

The research leading to these results has received funding from the European Community Seventh Framework Program (FP7/2007-2013) [grant agreement number 608490] (project M4CO2) and from the ANR ‘CHESDENS’ (ANR-13-SEED-0001-01).A multidisciplinary approach combining advanced experimental and modelling tools was undertaken to characterize the promises of a small-pore type Ti-based metal-organic framework, MIL-91(Ti) for CO2 capture. This material was prepared using two synthesis strategies, i.e. under hydrothermal conditions and under reflux, and its single component adsorption behaviour with respect to CO2, CH4 and N2 was first revealed by gravimetry measurements. This hydrophilic and highly water stable MOF is characterized by a relatively high CO2 adsorption enthalpy. Molecular simulations combined with in situ powder X-ray diffraction evidenced that this is due to the combined interaction of this probe with N-H and P-O groups in the phosphonate linker. High CO2 selectivities in the presence of either N2 or CH4 were also predicted and confirmed by co-adsorption measurements. The possibility to prepare this sample under reflux represents an environmentally friendly route which can easily be upscaled. This green synthesis route, excellent water stability, high selectivities and relatively fast transport kinetics of CO2 are significant points rendering this sample of utmost interest for CO2 capture.PostprintPostprintPeer reviewe

Functional Characterization of Aquaporin-4 Specific T Cells: Towards a Model for Neuromyelitis Optica

Antibodies to the water channel protein aquaporin-4 (AQP4), which is expressed in astrocytic endfeet at the blood brain barrier, have been identified in the serum of Neuromyelitis optica (NMO) patients and are believed to induce damage to astrocytes. However, AQP4 specific T helper cell responses that are required for the generation of anti-AQP4 antibodies and most likely also for the formation of intraparenchymal CNS lesions have not been characterized. specific T cells were present in the natural T cell repertoire of wild type C57BL/6 mice and T cell lines were raised. However, active immunization with these AQP4 peptides did not induce signs of spinal cord disease. Rather, sensitization with AQP4 peptides resulted in production of IFN-γ, but also IL-5 and IL-10 by antigen-specific T cells. Consistent with this cytokine profile, the AQP4 specific antibody response upon immunization with full length AQP4 included IgG1 and IgG2, which are associated with a mixed Th2/Th1 T cell response. restricted AQP4 specific T cell epitopes will allow us to investigate how AQP4 specific autoimmune reactions are regulated and to establish faithful mouse models of NMO that include both cellular and humoral responses against AQP4

Extra-Intestinal Manifestations of Familial Adenomatous Polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disorder, which results from a germ line mutation in the APC (adenomatous polyposis coli) gene. FAP is characterized by the formation of hundreds to thousands of colorectal adenomatous polyps. Although the development of colorectal cancer stands out as the most prevalent complication, FAP is a multisystem disorder of growth. This means, it is comparable to other diseases such as the MEN syndromes, Von Hippel-Lindau disease and neurofibromatosis. However, the incidence of many of its clinical features is much lower. Therefore, a specialized multidisciplinary approach to optimize health care—common for other disorders—is not usually taken for FAP patients. Thus, clinicians that care for and counsel members of high-risk families should have familiarity with all the extra-intestinal manifestations of this syndrome. FAP-related complications, for which medical attention is essential, are not rare and their estimated lifetime risk presumably exceeds 30%. Affected individuals can develop thyroid and pancreatic cancer, hepatoblastomas, CNS tumors (especially medulloblastomas), and various benign tumors such as adrenal adenomas, osteomas, desmoid tumors and dental abnormalities. Due to improved longevity, as a result of better prevention of colorectal cancer, the risk of these clinical problems will further increase

Seed maturation: Simplification of control networks in plants

International audienceNetworks controlling developmental or metabolic processes in plants are often complex as a consequence of the duplication and specialisation of the regulatory genes as well as the numerous levels of transcriptional and post-transcriptional controls added during evolution. Networks serve to accommodate multicellular complexity and increase robustness to environmental changes. Mathematical simplification by regrouping genes or pathways in a limited number of hubs has facilitated the construction of models for complex traits. In a complementary approach, a biological simplification can be achieved by using genetic modification to understand the core and singular ancestral function of the network, which is likely to be more prevalent within the plant kingdom rather than specific to a species. With this viewpoint, we review examples of simplification successfully undertaken in yeast and other organisms. A strategy of progressive complementation of single, double and triple mutants of seed maturation confirmed the fundamental role of the AFL sub-family of B3 transcription factors as master regulators of seed maturation, illustrating that biological simplification of complex networks could be more widely applied in plants. Defining minimal control networks will facilitate evolutionary comparisons of regulatory processes and the identification of an essential gene set for synthetic biology

Interface organique-inorganique et coordination dans des solides cristallins construits à partir de tétrathiafulvalènes fonctionnalisés par des groupements iodo, amidopyridine, amido-2,2'-bipyridine

Ce travail décrit la synthèse et la caractérisation de solides cristallins contenant des dérivés du TétraThiaFulvalène ou TTF. Ces molécules électroactives sont susceptibles d'être oxydées en radicaux cations stables, qui peuvent être associés à divers anions inorganiques. Dans ces sels d'ions radicaux, l'arrangement structural gouverne les propriétés physiques macroscopiques (conduction électrique, supraconduction). Un dérivé halogéné du TTF, EDT-TTF-I2, a été associé à des anions iodoplombates par électrocristallisation. Un premier sel présentant une partie anionique polymérique bidimensionnelle a pu être isolé. Il peut être structuralement décrit comme un composé d'intercalation de radicaux cations entre des plans PbI2 lacunaires en métal. Ces lacunes peuvent être comblées par des monocations. Les propriétés physiques de ces matériaux sont présentées et reliées à leurs structures électroniques. Un autre sel présentant un anion unidimensionnel a également été synthétisé, son mode de synthèse, sa structure cristalline et ses propriétés physiques sont discutés. La seconde partie de ce travail décrit la synthèse de dérivés du TTFs porteurs de groupements amidopyridines. L'organisation de ces molécules à l'état solide peut être corrélée aux différentes interactions intermoléculaires directionnelles mises en jeu. Les descriptions structurales d'un de ces dérivés dans différents états (neutre non coordiné, oxydé non coordiné, neutre coordiné) sont présentées. La synthèse est ensuite étendue à des dérivés amido-2,2'-bipyridine. L'un d'entre eux, 5,5'-bis(DMT-TTF-CONH)-2,2'-biPy, est engagé comme chélate face à des ions cuivre (I) et (II). Les propriétés électrochimiques de ces complexes sont discutées.This work describes the synthesis and physical characterization of molecular crystalline solids containing TetraThiaFulvalene (TTF) derivatives. These molecules can be oxydized to stable cation radicals which are associated with various anions to afford cation radicals salts. The physical properties of these salts are highly dependent on the relative position of the electroactive cores in the solid state. A halogenated TTF derivative has been engaged by electrocristallization with iodoplumbates anions. Some salts containing polymeric anions have been synthesized, their physical properties have been correlated with their crystallographic and electronic structures. The synthesis of amidopyridine functionalized TTF is described. Structural arrangement of these molecules is correlated with directional intermolecular interactions. The synthesis has been applied to 2,2'-bipyridine derivatives. Preparation of copper (I) and (II) complexes is described and their redox properties discussed.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

Porphyrin and phthalocyanine-based metal organic frameworks beyond metal-carboxylates

International audienceGiven the ubiquitous role of porphyrins in natural systems, these molecules and related derivatives such as phthalocyanines are fascinating building units to achieve functional porous materials. Porphyrin-based MOFs have been developed since the past three decades, yet chemically robust frameworks, necessary for applications, have been achieved much more recently and this field is expanding. This progress is partially driven by the development of porphyrins and phthalocyanines bearing alternative coordinating groups (phosphonate, azolates, phenolates…) that allowed to move the related MOFs beyond metalcarboxylates and achieve new topologies and properties. In this perspective article we first give a brief outline of the synthetic pathways towards simple porphyrins and phthalocyanines bearing these complexing groups. The related MOF compounds are then described; their structural and textural properties are discussed, as well as their stability and physical properties. An overview of the resulting nets and topologies is proposed, showing both the similarities with metal-carboxylate phases and the peculiarities related to the alternative coordinating groups. Eventually, the opportunities offered by this recent research topic, both in term of synthesis pathways, modulation of pore size and shape, stability and physical properties, are discusse