Profile of a serial killer: cellular and molecular approaches to study individual cytotoxic T-cells following therapeutic vaccination.

T-cell vaccination may prevent or treat cancer and infectious diseases, but further progress is required to increase clinical efficacy. Step-by-step improvements of T-cell vaccination in phase I/II clinical studies combined with very detailed analysis of T-cell responses at the single cell level are the strategy of choice for the identification of the most promising vaccine candidates for testing in subsequent large-scale phase III clinical trials. Major aims are to fully identify the most efficient T-cells in anticancer therapy, to characterize their TCRs, and to pinpoint the mechanisms of T-cell recruitment and function in well-defined clinical situations. Here we discuss novel strategies for the assessment of human T-cell responses, revealing in part unprecedented insight into T-cell biology and novel structural principles that govern TCR-pMHC recognition. Together, the described approaches advance our knowledge of T-cell mediated-protection from human diseases.

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita.

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.

Design and analysis of ChIP-Seq experiments for nuclear factor I DNA-binding proteins

SUMMARY : Eukaryotic DNA interacts with the nuclear proteins using non-covalent ionic interactions. Proteins can recognize specific nucleotide sequences based on the sterical interactions with the DNA and these specific protein-DNA interactions are the basis for many nuclear processes, e.g. gene transcription, chromosomal replication, and recombination. New technology termed ChIP-Seq has been recently developed for the analysis of protein-DNA interactions on a whole genome scale and it is based on immunoprecipitation of chromatin and high-throughput DNA sequencing procedure. ChIP-Seq is a novel technique with a great potential to replace older techniques for mapping of protein-DNA interactions. In this thesis, we bring some new insights into the ChIP-Seq data analysis. First, we point out to some common and so far unknown artifacts of the method. Sequence tag distribution in the genome does not follow uniform distribution and we have found extreme hot-spots of tag accumulation over specific loci in the human and mouse genomes. These artifactual sequence tags accumulations will create false peaks in every ChIP-Seq dataset and we propose different filtering methods to reduce the number of false positives. Next, we propose random sampling as a powerful analytical tool in the ChIP-Seq data analysis that could be used to infer biological knowledge from the massive ChIP-Seq datasets. We created unbiased random sampling algorithm and we used this methodology to reveal some of the important biological properties of Nuclear Factor I DNA binding proteins. Finally, by analyzing the ChIP-Seq data in detail, we revealed that Nuclear Factor I transcription factors mainly act as activators of transcription, and that they are associated with specific chromatin modifications that are markers of open chromatin. We speculate that NFI factors only interact with the DNA wrapped around the nucleosome. We also found multiple loci that indicate possible chromatin barrier activity of NFI proteins, which could suggest the use of NFI binding sequences as chromatin insulators in biotechnology applications. RESUME : L'ADN des eucaryotes interagit avec les prot��ines nucl��aires par des interactions noncovalentes ioniques. Les prot��ines peuvent reconna��tre les s��quences nucl��otidiques sp��cifiques bas��es sur l'interaction st��rique avec l'ADN, et des interactions sp��cifiques contr��lent de nombreux processus nucl��aire, p.ex. transcription du g��ne, la r��plication chromosomique, et la recombinaison. Une nouvelle technologie appel��e ChIP-Seq a ��t�� r��cemment d��velopp��e pour l'analyse des interactions prot��ine-ADN �� l'��chelle du g��nome entier et cette approche est bas��e sur l'immuno-pr��cipitation de la chromatine et sur la proc��dure de s��quen��age de l'ADN �� haut d��bit. La nouvelle approche ChIP-Seq a donc un fort potentiel pour remplacer les anciennes techniques de cartographie des interactions prot��ine-ADN. Dans cette th��se, nous apportons de nouvelles perspectives dans l'analyse des donn��es ChIP-Seq. Tout d'abord, nous avons identifi�� des artefacts tr��s communs associ��s �� cette m��thode qui ��taient jusqu'�� pr��sent insoup��onn��s. La distribution des s��quences dans le g��nome ne suit pas une distribution uniforme et nous avons constat�� des positions extr��mes d'accumulation de s��quence �� des r��gions sp��cifiques, des g��nomes humains et de la souris. Ces accumulations des s��quences art��factuelles cr��era de faux pics dans toutes les donn��es ChIP-Seq, et nous proposons diff��rentes m��thodes de filtrage pour r��duire le nombre de faux positifs. Ensuite, nous proposons un nouvel ��chantillonnage al��atoire comme un outil puissant d'analyse des donn��es ChIP-Seq, ce qui pourraient augmenter l'acquisition de connaissances biologiques �� partir des donn��es ChIP-Seq. Nous avons cr���� un algorithme d'��chantillonnage al��atoire et nous avons utilis�� cette m��thode pour r��v��ler certaines des propri��t��s biologiques importantes de prot��ines liant �� l'ADN nomm��s Facteur Nucl��aire I (NFI). Enfin, en analysant en d��tail les donn��es de ChIP-Seq pour la famille de facteurs de transcription nomm��s Facteur Nucl��aire I, nous avons r��v��l�� que ces prot��ines agissent principalement comme des activateurs de transcription, et qu'elles sont associ��es �� des modifications de la chromatine sp��cifiques qui sont des marqueurs de la chromatine ouverte. Nous pensons que l��s facteurs NFI interagir uniquement avec l'ADN enroul�� autour du nucl��osome. Nous avons ��galement constat�� plusieurs r��gions g��nomiques qui indiquent une ��ventuelle activit�� de barri��re chromatinienne des prot��ines NFI, ce qui pourrait sugg��rer l'utilisation de s��quences de liaison NFI comme s��quences isolatrices dans des applications de la biotechnologie.

Analysing the innovation process in the EU Integrated Programme, ISAFRUIT

ISAFRUIT is an integrated European Union Project focussed on increasing fruit consumption as a means to improve human health, through evaluating the fruit chain and addressing bottlenecks therein.The innovations which are being developed throughout the ISAFRUIT Project have been analysed to determine both the success factors and the obstacles in reaching the commercialisation stage. Only 9.58% of the deliverables planned within the Project were focussed on developing technological innovations.There is evidence, however, of successes in the development of new innovations arising from the ISAFRUIT Project, with several other potential innovations in the pipeline. Of the technologies identified, 67% are still at the ���invention stage���; that is, the stage prior to bridging the so-called ���valley of death���, the stage between an invention and an innovation. Those which are considered to have moved over the ���valley of death��� either had industry partners included in the Project, or had consulted with industry to ensure that the technology was relevant, or met a recognised industry need. Many of the technologies which made less progress did not have the same interactions with industry. A number of other issues were identified which prevented further progress towards innovation. The need for scientists to publish scientific papers, both for their career pathways and to increase their chances of future funding, was identified as one issue, although the filing of patents is now becoming more accepted and recognised. The patenting system is considered complex by many scientists and is not well-understood. Finally, agreements between partners on the sharing of intellectual property rights can cause a delay in the innovation process.

RNA-driven cyclin-dependent kinase regulation: when CDK9/cyclin T subunits of P-TEFb meet their ribonucleoprotein partners.

The positive transcription elongation factor (P-TEFb) consists of CDK9, a cyclin-dependent kinase and its cyclin T partner. It is required for transcription of most class II genes. Its activity is regulated by non-coding RNAs. The 7SK cellular RNA turns the HEXIM cellular protein into a P-TEFb inhibitor that binds its cyclin T subunit. Thus, P-TEFb activity responds to variations in global cellular transcriptional activity and to physiological conditions linked to cell differentiation, proliferation or cardiac hypertrophy. In contrast, the Tat activation region RNA plays an activating role. This feature at the 5' end of the human immunodeficiency (HIV) viral transcript associates with the viral protein Tat that in turn binds cyclin T1 and recruits active P-TEFb to the HIV promoter. This results in enhanced P-TEFb activity, which is critical for an efficient production of viral transcripts. Although discovered recently, the regulation of P-TEFb becomes a paradigm for non-coding RNAs that regulate transcription factors. It is also a unique example of RNA-driven regulation of a cyclindependent kinase.

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.

Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities.

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings.

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Els cultius in vitro i el desenvolupament vegetal

Biotecnologia Vegetal de materials de treball que incorporin les TICs. El material elaborat ha estat un llibre electr��nic utilitzable tant on-line (web) com off-line (CD). Els materials generats com a resultes d���aquest projecte han estat situats en un servidor de la UdL accessible des de l���exterior de la Universitat. Aquest llibre electr��nic ��s d���acc��s obert i es pot consultar on-line a l���URL (http://sakai.udl.es/cursos/76304/indexC.htm). El llibre electr��nic cont�� un total de 98 Mb d���informaci�� hiperm��dia i hipertexual distribu��da en m��s de 380 arxius dels quals 50 s��n p��gines html, 10 arxius doc, 10 arxius pdf, 258 imatges fixes i 8 arxius de v��deo en format flash (swf). L���acc��s al llibre electr��nic es realitza a trav��s d���una pantalla inicial que d��na pas a un men�� que distribueix els materials en 7 apartats. Els textos estan acompanyats de gran quantitat d���imatges fotogr��fiques, gr��fics, esquemes, imatges infogr��fiques i videoclips generats de novo per aquest projecte. En dissenyar la web s���han tingut en compte criteris de confiabilitat, accessibilitat i usabilitat. El primer disseny de la web ha estat validat per un panel d���usuaris i utilitzat posteriorment amb alumnes durant el curs 2007-2008. Les observacions i suggeriments fets per aquests ja han estat incorporats en aquest document final. Una primera enquesta de satisfacci�� realitzada amb aquest alumnat permet concloure, a t��tol provisional donat lo redu��t de la poblaci�� enquestada, que l���alumnat mostra un grau de coneixement de les eines TIC suficient i que consideren positiva la incorporaci�� de materials multim��dia com a recurs educatiu. A m��s a m��s, la realitzaci�� d���aquest projecte i la seva aplicaci�� a l���aula ha estat presentada en dues pon��ncies al ���Congreso Internacional de Docencia y Innovaci��n Universitaria���.

Dermatologie. 2009: quoi de neuf en dermatologie [Dermatology]

Many significant advances in dermatology were published during 2009, focussing on infectious diseases, inflammatory disorders and oncology. Molecular medicine, as a result of the human genome project, also modifies the field of dermatology. Bioinformatics and biotechnology revolutionize the daily clinical practice in dermatology. A change of paradigm occurs notably in infectious diseases.

Characterization of the effects of IL-17F on CHO cell line generation

CHO is the most commonly used mammalian host for the generation of cell lines allowing for the production of high quality therapeutic proteins. The generation of such cell lines is a lengthy and resource-intensive process requiring extensive screening in order to isolate candidates with optimal characteristics, such as growth, stability and productivity. For this reason, the biotechnology industry invests much effort in attempts to optimize CHO expression systems in order to streamline and shorten the cell line selection process. Based on preliminary observations of a facilitated selection of CHO-GS cell lines expressing members of the IL-17 cytokine family, this study investigates the use of IL-17F as a novel enhancing factor for CHO cell line generation. Using two different CHO expression systems (exploiting GS and DHFR-based selection), we demonstrated that IL-17F expression caused a significant increase in the occurrence of colonies during the selection process. All colonies selected produced substantial amounts of IL-17F, suggesting that benefits were conferred, during selection, to those cells expressing the cytokine. Furthermore, transgene expression levels were significantly increased when the selection pressure was raised to a level that would not normally be permissive for colony selection (i.e. 100 |o.M MSX for the CHO-GS expression system or 1000 nM MTX for the CHO-DHFR system). Finally, IL-17F expression was also found to enhance the rate of appearance of clones during single cell subcloning in the absence of selection pressure. Overall, these benefits have the potential to allow a substantial reduction in the length of cell line generation while significantly increasing cell line productivity. Nevertheless, we found that the high IL-17F expression levels required to convey enhancing effects was a limitation when attempting to co-express IL-17F and a recombinant soluble protein of therapeutic interest from independent CMV promoters within the same expression vector. In order to understand and overcome this limitation, studies were designed to characterize the IL-17F enhancing effect at the molecular and cellular level. Regular supplementation of recombinant biologically-active IL-17F into the culture medium during cell line selection was not able to reproduce the enhancing effects of endogenous IL-17F expression. In addition, increased IL-17F expression correlated with increased CHO-GS selection transgene expression at the single cell level. This data suggested a possible effect of IL-17F on viral promoter activity or transgene mRNA stability. It also provided direct evidence that the cells expressing the highest amounts of IL-17F obtained the most benefit. Overall data obtained from these study implied that IL-17F may act through an intracellular mechanism, possibly exerted during secretion. We therefore initiated experiments designed to determine the specific compartment(s) within which IL-17F triggers its effect. This work has identified IL-17F as a potentially powerful tool to optimize the CHO cell line generation process. The characterization of this enhancing effect at the molecular level has given us several insights into overcoming the current limitations, thus paving the way for the development of a viable technology that can be exploited within the biotechnology industry. - La CHO est la cellule h��te de mammifere la plus couramment utilis��e dans la cr��ation de lign��e cellulaire produisant des prot��ines th��rapeutiques de haute qualit��. La g��n��ration de ces lign��es cellulaires est un processus long et exigeant l'utilisation de techniques de s��lection robustes afin d'isoler des candidats poss��dants les caract��ristiques optimales de croissance, de productivit�� et de stabilit�� d'expression. Les industries biopharmaceutiques ont investi beaucoup d'efforts afin d'optimiser les syst��mes d'expression CHO dans le but raccourcir la longueur du proc��d�� de s��lection de lign��es cellulaires et aussi d'en augmenter l'efficacit��. A partir d'observations pr��liminaires obtenues lors de la g��n��ration de lign��es cellulaires CHO- GS exprimant une cytokine appartenant �� la famille des IL-17, nous avons r��alis�� une ��tude portant sur l'utilisation de l'IL-17F humaine (IL-17F) comme nouveau facteur d'optimisation pour la g��n��ration de lign��es cellulaires CHO. Nous avons d��montr��, en utilisant les deux syst��mes de s��lection et d'expression CHO couramment utilis��s (le premier exploitant la GS et l'autre bas��e sur la DHFR), que l'expression de l'IL-17F permet une augmentation significative de la fr��quence d'apparition de colonies durant le processus de s��lection de lign��es cellulaires. Les diff��rentes colonies s��lectionn��es expriment des quantit��s substantielles d'IL-17F, sugg��rant un effet b��n��fique lors de la s��lection qui serait exclusivement conf��r�� aux cellules exprimant la cytokine. En outre, le niveau d'expression du transgene se trouve significativement augment�� lorsque la pression de s��lection est port��e �� un niveau habituellement trop ��lev�� pour permettre la s��lection de colonies (soit 100 |JM MSX pour le syst��me d'expression CHO-GS ou 1000 nM MTX pour le syst��me CHO- DHFR). Enfin, l'expression d'IL-17F permet ��galement d'am��liorer la vitesse d'apparition de clones pendant une ��tape de sous-clonage en l'absence de pression de s��lection. L'ensemble de ces effets b��n��fiques permettent une r��duction substantielle de la dur��e de g��n��ration de lign��es cellulaires tout en augmentant consid��rablement la productivit�� des lign��es obtenues. N��anmoins, nous avons constat�� que la n��cessit�� d'exprimer des niveaux ��lev��s d'IL-17F afin obtenir l'ensemble de ses effets b��n��fiques devient une contrainte lors de l'utilisation d'un vecteur d'expression compos�� de deux promoteurs CMV ind��pendants pour la co-expression de la cytokine et d'une prot��ine soluble pr��sentant un int��r��t th��rapeutique. Afin de mieux comprendre et de surmonter cette limitation, plusieurs ��tudes ont ��t�� effectu��es dans le but de mieux caract��riser l'effet de IL-17F au niveau subcellulaire. L'apport r��gulier en IL-17F recombinante et biologiquement active dans le milieu de culture lors de la s��lection de lign��es cellulaires ne permet pas de reproduire les effets b��n��fiques observ��s par l'expression endog��ne d'IL-17F. En outre, nous avons constat�� que, lors de l'utilisation du syst��me CHO- GS, l'augmentation d'expression de 1TL-17F est corr��l��e �� un accroissement de l'expression du marqueur de s��lection au niveau cellulaire. Ces r��sultats sugg��rent un possible effet d'IL- 17F sur l'activit�� des promoteurs viraux et ainsi fournissent une preuve directe que les cellules exprimant de haut niveau d'IL-17F sont celles qui en profitent le plus. L'ensemble de ces observations mettrait en avant que l'effet d'IL-17F se ferait selon un m��canisme intracellulaire. Nous avons donc ��tudi�� le(s) compartiment(s) sp��cifique(s) dans lequel IL-17F pourrait exercer son effet. Ce travail a permis de d��finir IL-17F comme un puissant outil pour l'optimisation des proc��d��s de g��n��ration de lign��es cellulaires CHO. La caract��risation de cette am��lioration de l'effet au niveau mol��culaire nous a donn�� plusieurs indications sur la mani��re de d��passer les limitations actuelles, ouvrant ainsi la voie au d��veloppement d'une technologie viable qui peut ��tre exploit��e pars l'industrie biotechnologique.

Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases.

Defensins and cathelicidins are anti-microbial peptides (AMPs) that act as natural antibiotics and are part of the innate immune defence in many species. We consider human defensins and LL37, the only human member of the cathelicidin family. In particular, we refer to the human alpha-defensins called human neutrophil peptides (HNP1 through 4), which are produced by neutrophils, HD5 and HD6, mainly expressed in Paneth cells of intestine, the human beta-defensins HBD1, HBD2 and HBD3, synthesized by epithelial cells and LL37, which is located in granulocytes, but is also produced by epithelial cells of the skin, lungs, and gut. In the last years, the study of AMPs activity and regulation has allowed to understand the important role of these peptides not only in the innate defence mechanisms against bacteria, viruses, fungi, but also in the regulation of immune cell activation and migration. Complementary studies have disclosed a role for AMPs in modulating many physiological processes that involve non-immune cells, such as activation of wound healing, angiogenesis, cartilage remodeling. Due to the pleiotropic tasks of these peptides, many of them are now being discovered to contribute to immune pathology of chronic diseases that affect skin, gut, joints; this is supported by many examples of immune-mediated pathologies in which their expression is disregulated. In this article we review the current literature that suggests a role for human defensins and LL37 in pathogenic mechanisms of several chronic diseases that are considered of auto-immune or auto-inflammatory origin.

Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation.

Root diseases caused by fungal pathogens can be suppressed by certain rhizobacteria that effectively colonize the roots and produce extracellular antifungal compounds. To be effective, biocontrol bacteria need to be present at sufficiently high cell densities. These conditions favor the operation of positive feedback mechanisms that control the production of antifungal compounds in biocontrol strains of fluorescent pseudomonads, via both transcriptional and post-transcriptional mechanisms.