Lipophosphoramidates as lipidic part of lipospermines for gene delivery

The DNA compacting properties of polyamines (especially spermine) are well-known, hence the use of spermine as the cationic part in several synthetic DNA carriers. Here, we describe the synthesis of modified spermines, with a "lipophosphoramidate" as the lipidic part, and their use for efficient in vitro transfection. Physicochemical measurements (particle size, zeta potentials, pKa determination) and gel retardation assays were also performed. Theoretical membrane-disrupting ability was established by FRET. Taken together, our results indicate that lipophosphoramidates constitute an interesting alternative to "classical" lipidic parts of cationic lipids used as DNA carriers.     

Reprint of "Structural and mechanistic aspects of Amt/Rh proteins" [J. Struct. Biol. 158 (2007) 472-481

Amt/Rh proteins, which mediate movement of ammonium across cell membranes, are spread throughout the three kingdoms of life. Most functional studies on various members of the family have been performed using cellular assays in heterologous expression systems, which are, however, not very well suited for detailed mechanistic studies. Although now generally considered to be ammonia conducting channels, based on a number of experimental studies and structural insights, the possibility remains that some plant Amts facilitate net ammonium ion transport. The Escherichia coli channel AmtB has become the model system of choice for analysis of the mechanism of ammonia conductance, increasingly also through molecular dynamics simulations. Further progress in a more detailed mechanistic understanding of these proteins requires a reliable in vitro assay using purified protein, allowing quantitative kinetic measurements under a variety of experimental conditions for different Amt/Rh proteins, including mutants. Here, we critically review the existing functional data in the context of the most interesting and unresolved mechanistic questions and we present our results, obtained using an in vitro assay set up with the purified E. coli channel AmtB.     

How NOD2 mutations predispose to Crohn's disease?

NOD2 mutations are associated with the development of granulomatous inflammatory diseases, such as early-onset sarcoidosis (EOS), Blau syndrome (BS) and Crohn's disease (CD). As a pathogen-recognition molecule for muramyl dipeptide (MDP), NOD2 controls both innate and adaptive immune responses, through the regulation of cytokines, chemokines and antimicrobial peptides production. Notably, Nod2-deficient mice experienced increased susceptibility to enteric infection and to antigen-specific colitis. Furthermore, mutant mice bearing the orthologue of the major CD-associated NOD2^3020ins allele showed increased susceptibility to DSS-induced colitis. However, many questions remain open. (i) Is antimicrobial function deficiency sufficient to initiate the development of CD? (ii) How impaired and mutant NOD2 might lead to increased adaptive immune response? (iii) How do the other disease-associated NOD2 mutations contribute to the development of chronic intestinal inflammation? Whatever the relevant mechanism(s), it provides a casual link between abnormal bacterial sensing and development of inflammatory disorders. Further work should now focus on restoring abnormal NOD2 function by modulating antimicrobial function and regulatory mechanisms of the adaptive immune system.     

Natural antibody--complement dependent neutralization of bovine herpesvirus 4 by human serum

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are sensitive or even permissive to BoHV-4. These observations led to the hypothesis that cross-species transmission of BoHV-4 could lead to human infections. In the present study, we investigate the sensitivity of BoHV-4 to neutralization by na?ve human sera in order to determine if humans exhibit innate anti-viral activities against this virus. Our results demonstrate that human sera from na?ve individuals, in contrast to the sera of na?ve subjects from various animal species, neutralize BoHV-4 efficiently. A series of complementary experiments were performed to unravel the mechanism(s) of this neutralization. The data obtained in this study demonstrates that human serum neutralizes BoHV-4 in a complement dependent manner activated by natural antibodies raised against the Galalpha1-3Galbeta1-4GlcNAc-R epitope expressed by bovine cells.     

Epigenetic heredity in mice: involvement of RNA and miRNas.

By contrast with a wide definition of the 'epigenetic variation', including all changes in gene expression that do not result from alteration of the gene structure, a more restricted class had been defined, initially in plants, under the name 'paramutation'. It corresponds to epigenetic modifications distinct from the regulatory interactions of the cell differentiation pathways, mitotically stable and sexually transmitted with non-Mendelian patterns. This class of epigenetic changes appeared for some time restricted to the plant world, but examples progressively accumulated of epigenetic inheritance in organisms ranging from mice to humans. Occurrence of paramutation in the mouse and possible mechanisms were then established in the paradigmatic case of a mutant phenotype maintained and hereditarily transmitted by wild type homozygotes. Together with recent findings in plants indicative of a necessary step of RNA amplification in the reference maize paramutation, the mouse studies point to a new role of RNA, as an inducer and hereditary determinant of epigenetic variation. Given the known presence of a wide range of RNAs in human spermatozoa, as well as a number of unexplained cases of familial disease predisposition and transgenerational maintenance, speculations can be extended to possible roles of RNA-mediated inheritance in human biology and pathology.La paramutation est une modification épigénétique héréditaire, découverte chez des plantes et récemment, chez la souris. C'est un changement héréditaire du phénotype associé avec un allèle sauvage à la suite de son passage dans une structure hétérozygote avec un allèle mutant (phénomène quelquefois appelé "conversation interchromosomique"). Souvent il est considéré comme une exception à la base de lois de Mendel, "les allèles sont retrouvés inchangés lors des ségrégations au cours des croisements". Au contraire la paramutation observée chez la souris résulte d'une modification de l'allèle sauvage du gène Kit après transmission à partir d'un hétérozygote avec un allèle mutant "insertion". Le phénotype des taches blanches visibles aisément par la couleur du pelage est transmis en absence de l'allèle inducteur sur plusieurs générations. Il est corrélé avec une diminution du niveau d'ARNm de Kit et une accumulation d'ARN de taille variable de Kit dans les spermatozo?des des souris paramutantes. La micro-injection de l'ARN de l'hétérozygote, ou de l'ARN et des microARN spécifiques de Kit dans l'oeuf fécondé induit le phénotype "taches blanches". Le r?le de l'ARN dans l'établissement et le maintien d'un état épigénétique héréditaire est proposé et discuté.     

Minimizing the overlap problem in protein NMR: a computational framework for precision amino acid labeling

MOTIVATION: Recent advances in cell-free protein expression systems allow specific labeling of proteins with amino acids containing stable isotopes ((15)N, (13) C and (2)H), an important feature for protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. Given this labeling ability, we present a mathematical optimization framework for designing a set of protein isotopomers, or labeling schedules, to reduce the congestion in the NMR spectra. The labeling schedules, which are derived by the optimization of a cost function, are tailored to a specific protein and NMR experiment. RESULTS: For 2D (15)N-(1)H HSQC experiments, we can produce an exact solution using a dynamic programming algorithm in under 2 h on a standard desktop machine. Applying the method to a standard benchmark protein, calmodulin, we are able to reduce the number of overlaps in the 500 MHz HSQC spectrum from 10 to 1 using four samples with a true cost function, and 10 to 4 if the cost function is derived from statistical estimates. On a set of 448 curated proteins from the BMRB database, we are able to reduce the relative percent congestion by 84.9% in their HSQC spectra using only four samples. Our method can be applied in a high-throughput manner on a proteomic scale using the server we developed. On a 100-node cluster, optimal schedules can be computed for every protein coded for in the human genome in less than a month. AVAILABILITY: A server for creating labeling schedules for (15)N-(1)H HSQC experiments as well as results for each of the individual 448 proteins used in the test set is available at http://nmr.proteomics.ics.uci.edu.     

Differentiating megakaryocytes in myelodysplastic syndromes succumb to mitochondrial derangement without caspase activation

Myelodysplastic syndromes (MDS) constitute a preneoplastic condition in which potentially malignant cancer stem cells continuously die during differentiation. This MDS-associated cell death often involves caspase-3 activation, yet can also occur without caspase activation, for instance in differentiating megakaryocytes (MK). We investigated, the mechanisms through which MK from MDS patients undergo premature cell death. While polyploid, mature MK from healthy subjects or MDS patients manifested caspase-3 activation during terminal differentiation, freshly isolated, immature MK from MDS died without caspase-3 activation. Similarly, purified bone marrow CD34⁺ cells from MDS patients that were driven into MK differentiation in vitro died without caspase-3 activation at an immature stage, before polyploidization. The premature death of MDS MK was accompanied by the mitochondrial release of cytochrome c, Smac/DIABLO and endonuclease G, a caspase-independent death effector, as well loss of the mitochondrial membrane potential and plasma membrane phosphatidylserine exposure before definitive loss of viability. Thus, a stereotyped pattern of mitochondrial alterations accompanies differentiation-associated MK death in MDS. T. Braun and G. Carvalho contributed equally to this paper.     

Genome ecosystem and transposable elements species

Transposable elements are known to be “selfish DNA” sequences able to spread and be maintained in all genomes analyzed so far. Their evolution depends on the interaction they have with the other components of the genome, including genes and other transposable elements. These relationships are complex and have often been compared to those of species living and competing in an ecosystem. The aim of this current work is a proposition to fill the conceptual gap existing between genome biology and ecology, assuming that genomic components, such as transposable elements families, can be compared to species interacting in an ecosystem. Using this framework, some of the main models defined in the population genetics of transposable elements can then been reformulated, and some new kinds of realistic relationships, such as symbiosis between different genomic components, can then be modelled and explored.