Identification of putative markers of triclabendazole resistance by a genome-wide analysis of genetically recombinant Fasciola hepatica

Despite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.

Transposable Elements Are a Significant Contributor to Tandem Repeats in the Human Genome

Sequence repeats are an important phenomenon in the human genome, playing important roles in genomic alteration often with phenotypic consequences. The two major types of repeat elements in the human genome are tandem repeats (TRs) including microsatellites, minisatellites, and satellites and transposable elements (TEs). So far, very little has been known about the relationship between these two types of repeats. In this study, we identified TRs that are derived from TEs either based on sequence similarity or overlapping genomic positions. We then analyzed the distribution of these TRs among TE families/subfamilies. Our study shows that at least 7,276 TRs or 23% of all minisatellites/satellites is derived from TEs, contributing ∼0.32% of the human genome. TRs seem to be generated more likely from younger/more active TEs, and once initiated they are expanded with time via local duplication of the repeat units. The currently postulated mechanisms for origin of TRs can explain only 6% of all TE-derived TRs, indicating the presence of one or more yet to be identified mechanisms for the initiation of such repeats. Our result suggests that TEs are contributing to genome expansion and alteration not only by transposition but also by generating tandem repeats.

Identification des réseaux transcriptionnnels de résistance aux antifongiques chez Candida albicans

Plusieurs souches cliniques de Candida albicans résistantes aux médicaments antifongiques azolés surexpriment des gènes encodant des effecteurs de la résistance appartenant à deux classes fonctionnelles : i) des transporteurs expulsant les azoles, CDR1, CDR2 et MDR1 et ii) la cible des azoles 14-lanostérol déméthylase encodée par ERG11. La surexpression de ces gènes est due à la sélection de mutations activatrices dans des facteurs de transcription à doigts de zinc de la famille zinc cluster (Zn2Cys6) qui contrôlent leur expression : Tac1p (Transcriptional activator of CDR genes 1) contrôlant l’expression de CDR1 et CDR2, Mrr1p (Multidrug resistance regulator 1), régulant celle de MDR1 et Upc2p (Uptake control 2), contrôlant celle d’ERG11. Un autre effecteur de la résistance clinique aux azoles est PDR16, encodant une transférase de phospholipides, dont la surexpression accompagne souvent celle de CDR1 et CDR2, suggérant que les trois gènes appartiennent au même régulon, potentiellement celui de Tac1p. De plus, la régulation transcriptionnelle du gène MDR1 ne dépend pas seulement de Mrr1p, mais aussi du facteur de transcription de la famille basic-leucine zipper Cap1p (Candida activator protein 1), un régulateur majeur de la réponse au stress oxydatif chez C. albicans qui, lorsque muté, induit une surexpression constitutive de MDR1 conférant la résistance aux azoles. Ces observations suggèrent qu’un réseau de régulation transcriptionnelle complexe contrôle le processus de résistance aux antifongiques azolés chez C. albicans. L’objectif de mon projet au doctorat était d’identifier les cibles transcriptionnelles directes des facteurs de transcription Tac1p, Upc2p et Cap1p, en me servant d’approches génétiques et de génomique fonctionnelle, afin de i) caractériser leur réseau transcriptionnel et les modules transcriptionnels qui sont sous leur contrôle direct, et ii) d’inférer leurs fonctions biologiques et ainsi mieux comprendre leur rôle dans la résistance aux azoles. Dans un premier volet, j’ai démontré, par des expériences de génétique, que Tac1p contrôle non seulement la surexpression de CDR1 et CDR2 mais aussi celle de PDR16. Mes résultats ont identifié une nouvelle mutation activatrice de Tac1p (N972D) et ont révélé la participation d’un autre régulateur dans le contrôle transcriptionnel de CDR1 et PDR16 dont l’identité est encore inconnue. Une combinaison d’expériences de transcriptomique et d’immunoprécipitation de la chromatine couplée à l’hybridation sur des biopuces à ADN (ChIP-chip) m’a permis d’identifier plusieurs gènes dont l’expression est contrôlée in vivo et directement par Tac1p (PDR16, CDR1, CDR2, ERG2, autres), Upc2p (ERG11, ERG2, MDR1, CDR1, autres) et Cap1p (MDR1, GCY1, GLR1, autres). Ces expériences ont révélé qu’Upc2p ne contrôle pas seulement l’expression d’ERG11, mais aussi celle de MDR1 et CDR1. Plusieurs nouvelles propriétés fonctionnelles de ces régulateurs ont été caractérisées, notamment la liaison in vivo de Tac1p aux promoteurs de ses cibles de façon constitutive et indépendamment de son état d’activation, et la liaison de Cap1p non seulement à la région du promoteur de ses cibles, mais aussi celle couvrant le cadre de lecture ouvert et le terminateur transcriptionnel putatif, suggérant une interaction physique avec la machinerie de la transcription. La caractérisation du réseau transcriptionnel a révélé une interaction fonctionnnelle entre ces différents facteurs, notamment Cap1p et Mrr1p, et a permis d’inférer des fonctions biologiques potentielles pour Tac1p (trafic et la mobilisation des lipides, réponse au stress oxydatif et osmotique) et confirmer ou proposer d’autres fonctions pour Upc2p (métabolisme des stérols) et Cap1p (réponse au stress oxydatif, métabolisme des sources d’azote, transport des phospholipides). Mes études suggèrent que la résistance aux antifongiques azolés chez C. albicans est intimement liée au métabolisme des lipides membranaires et à la réponse au stress oxydatif.

Développement de procédés efficaces pour la construction et la production de vecteurs adénoviraux

L’adénovirus possède plusieurs caractéristiques faisant de ce virus un candidat de choix pour la construction de vecteurs utiles dans les études de génomique fonctionnelle. Dans la majorité de ces applications, on a recours à un vecteur adénoviral de première génération délété de sa région E1. L’utilisation de vecteurs adénoviraux comprend deux maillons faibles : la construction du vecteur et la production subséquente de ce dernier. Le développement de méthodes alternatives est donc nécessaire pour renforcer ces deux maillons, permettant ainsi une utilisation étendue de ces vecteurs. Ce développement va s’articuler sur deux axes : l’ingénierie du vecteur de transfert pour la construction de l’adénovirus recombinant et l’ingénierie d’une lignée cellulaire pour la production du vecteur. En utilisant un vecteur de transfert adénoviral co-exprimant, à partir d’un promoteur régulable à la tétracycline, la protéase de l’adénovirus et une protéine de fluorescence verte (GFP) par l’intermédiaire d’un site d’entrée ribosomal interne (IRES), notre groupe a établi que la sélection positive, via l’expression ectopique de la protéase, est un processus efficace pour la création de librairie d’adénovirus recombinants. Par contre, la diversité atteinte dans ce premier système est relativement faible, environ 1 adénovirus recombinant par 1 000 cellules. Le travail effectué dans le cadre de cette thèse vise à construire un nouveau transfert de vecteur dans lequel l’expression de la protéase sera indépendante de celle du transgène permettant ainsi d’optimiser l’expression de la protéase. Ce travail d’optimisation a permis de réduire le phénomène de transcomplémentation du virus parental ce qui a fait grimper la diversité à 1 virus recombinant par 75 cellules. Ce système a été mis à l’épreuve en générerant une librairie adénovirale antisens dirigée contre la GFP. La diversité de cette librairie a été suffisante pour sélectionner un antisens réduisant de 75% l’expression de la GFP. L’amplification de ce vecteur adénoviral de première génération doit se faire dans une lignée cellulaire exprimant la région E1 telle que les cellules 293. Par contre, un adénovirus de première génération se répliquant dans les cellules 293 peut échanger, par recombinaison homologue, son transgène avec la région E1 de la cellule créant ainsi un adénovirus recombinant réplicatif (RCA), compromettant ainsi la pureté des stocks. Notre groupe a déjà breveté une lignée cellulaire A549 (BMAdE1) exprimant la région E1, mais qui ne peut pas recombiner avec le transgène du virus. Par contre, le niveau de réplication de l’adénovirus dans les BMAdE1 est sous-optimal, à peine 15-30% du niveau obtenu dans les cellules 293. Le travail fait dans le cadre de cette thèse a permis de mettre en évidence qu’une expression insuffisante d’E1B-55K était responsable de la mauvaise réplication du virus dans les BMAdE1. Nous avons produit de nouveaux clones à partir de la lignée parentale via une transduction avec un vecteur lentiviral exprimant E1B-55K. Nous avons confirmé que certains clones exprimaient une plus grande quantité d’E1B-55K et que ces clones amplifiaient de manière plus efficace un vecteur adénoviral de première génération. Ce clone a par la suite été adapté à la culture en suspension sans sérum.

Searching for novel gene functions in yeast : identification of thousands of novel molecular interactions by protein-fragment complementation assay followed by automated gene function prediction and high-throughput lipidomics

La compréhension de processus biologiques complexes requiert des approches expérimentales et informatiques sophistiquées. Les récents progrès dans le domaine des stratégies génomiques fonctionnelles mettent dorénavant à notre disposition de puissants outils de collecte de données sur l’interconnectivité des gènes, des protéines et des petites molécules, dans le but d’étudier les principes organisationnels de leurs réseaux cellulaires. L’intégration de ces connaissances au sein d’un cadre de référence en biologie systémique permettrait la prédiction de nouvelles fonctions de gènes qui demeurent non caractérisées à ce jour. Afin de réaliser de telles prédictions à l’échelle génomique chez la levure Saccharomyces cerevisiae, nous avons développé une stratégie innovatrice qui combine le criblage interactomique à haut débit des interactions protéines-protéines, la prédiction de la fonction des gènes in silico ainsi que la validation de ces prédictions avec la lipidomique à haut débit. D’abord, nous avons exécuté un dépistage à grande échelle des interactions protéines-protéines à l’aide de la complémentation de fragments protéiques. Cette méthode a permis de déceler des interactions in vivo entre les protéines exprimées par leurs promoteurs naturels. De plus, aucun biais lié aux interactions des membranes n’a pu être mis en évidence avec cette méthode, comparativement aux autres techniques existantes qui décèlent les interactions protéines-protéines. Conséquemment, nous avons découvert plusieurs nouvelles interactions et nous avons augmenté la couverture d’un interactome d’homéostasie lipidique dont la compréhension demeure encore incomplète à ce jour. Par la suite, nous avons appliqué un algorithme d’apprentissage afin d’identifier huit gènes non caractérisés ayant un rôle potentiel dans le métabolisme des lipides. Finalement, nous avons étudié si ces gènes et un groupe de régulateurs transcriptionnels distincts, non préalablement impliqués avec les lipides, avaient un rôle dans l’homéostasie des lipides. Dans ce but, nous avons analysé les lipidomes des délétions mutantes de gènes sélectionnés. Afin d’examiner une grande quantité de souches, nous avons développé une plateforme à haut débit pour le criblage lipidomique à contenu élevé des bibliothèques de levures mutantes. Cette plateforme consiste en la spectrométrie de masse à haute resolution Orbitrap et en un cadre de traitement des données dédié et supportant le phénotypage des lipides de centaines de mutations de Saccharomyces cerevisiae. Les méthodes expérimentales en lipidomiques ont confirmé les prédictions fonctionnelles en démontrant certaines différences au sein des phénotypes métaboliques lipidiques des délétions mutantes ayant une absence des gènes YBR141C et YJR015W, connus pour leur implication dans le métabolisme des lipides. Une altération du phénotype lipidique a également été observé pour une délétion mutante du facteur de transcription KAR4 qui n’avait pas été auparavant lié au métabolisme lipidique. Tous ces résultats démontrent qu’un processus qui intègre l’acquisition de nouvelles interactions moléculaires, la prédiction informatique des fonctions des gènes et une plateforme lipidomique innovatrice à haut débit , constitue un ajout important aux méthodologies existantes en biologie systémique. Les développements en méthodologies génomiques fonctionnelles et en technologies lipidomiques fournissent donc de nouveaux moyens pour étudier les réseaux biologiques des eucaryotes supérieurs, incluant les mammifères. Par conséquent, le stratégie présenté ici détient un potentiel d’application au sein d’organismes plus complexes.

Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps

The constitutive production of AMPs in shrimps ensures that animals are able to protect themselves from low-level assaults by pathogens present in the environment. As these molecules play important roles in the shrimp immune defense system, the expression level of these AMPs are possible indicators of the immune state of shrimps. The present study also indicates the antiviral property of AMPs, especially ALF, stressing the importance of their up-regulation through the application of immunostimulants/probiotics as a prophylactic strategy in aquaculture. The present study shows that shrimp defense system is equipped enough to evade WSSV infection to a certain extent, when the animals were maintained on marine yeast and probiotic diet, whereas the control diet fed group succumbed to WSSV infection. This study reveals that marine yeast and probiotic supplemented diet can delay the process of WSSV infection and confer greater protection to the animals. Particularly, the protection conferred by marine yeast, C. haemulonii S27 and Bacillus MCCB101 were highly promising imparting greater hope to the aquaculture community to overcome the prevailing disease problems in aquaculture. It may be inferred from the present study that up-regulation of AMP genes could be effected by the application of immunostimulants and probiotics. Also, AMP expression profile could be used as an effective tool for screening immunostimulants and probiotics for application in shrimp culture. Ultimately, it is likely that no single compound or strategy will provide a solution to the problem of disease within aquaculture and that, in reality, a suite of techniques will be required including the manipulation of the rearing environment, addition of probionts as a matter of routine during culture, and the use of immunostimulants and other supplements during vulnerable growth phases. Finally, the development of good management practices, the control of environmental variables, genetic improvement in the penaeid species, understanding of host-virus interaction, modulation of the shrimp immune system, supported by functional genomics and proteomics of this crustacean, as a whole suggests that the control of WSSV is not far.

El perfil criminal del asesino en serie colombiano desde la perspectiva psicodinámica. Una revisión de literatura

El objetivo de la presente investigación consiste en describir las características de un asesino en serie colombiano desde la perspectiva psicodinámica. En este sentido, el abordaje teórico realizado en este trabajo se compone inicialmente de una concepción de asesinos en serie, posteriormente se hace una revisión acerca de las bases biológicas y los factores sociales del homicida serial, igualmente, se explican tres teorías psicodinámicas a trabajar (Sigmund Freud y Erick Erickson). Finalmente, se hace mención dentro de la investigación a la comparación casuística de los asesinos en serie, teniendo en cuenta a cuatro asesinos en serie mediante el abordaje psicodinámico. Por otra parte, a nivel metodológico, el tipo de estudio realizado es descriptivo con un corte cualitativo y un diseño no experimental, basado en la revisión de fuentes bibliográficas. Como producto se pretende hacer una aproximación al perfil correspondiente de la personalidad de un asesino en serie colombiano mediante las teorías psicodinámicas.

Major advances in fundamental dairy cattle nutrition

Fundamental nutrition seeks to describe the complex biochemical reactions involved in assimilation and processing of nutrients by various tissues and organs, and to quantify nutrient movement (flux) through those processes. Over the last 25 yr, considerable progress has been made in increasing our understanding of metabolism in dairy cattle. Major advances have been made at all levels of biological organization, including the whole animal, organ systems, tissues, cells, and molecules. At the whole-animal level, progress has been made in delineating metabolism during late pregnancy and the transition to lactation, as well as in whole-body use of energy-yielding substrates and amino acids for growth in young calves. An explosion of research using multicatheterization techniques has led to better quantitative descriptions of nutrient use by tissues of the portal-drained viscera (digestive tract, pancreas, and associated adipose tissues) and liver. Isolated tissue preparations have provided important information on the interrelationships among glucose, fatty acid, and amino acid metabolism in liver, adipose tissue, and mammary gland, as well as the regulation of these pathways during different physiological states. Finally, the last 25 yr has witnessed the birth of "molecular biology" approaches to understanding fundamental nutrition. Although measurements of mRNA abundance for proteins of interest already have provided new insights into regulation of metabolism, the next 25 yr will likely see remarkable advances as these techniques continue to be applied to problems of dairy cattle biology. Integration of the "omics" technologies (functional genomics, proteomics, and metabolomics) with measurements of tissue metabolism obtained by other methods is a particularly exciting prospect for the future. The result should be improved animal health and well being, more efficient dairy production, and better models to predict nutritional requirements and provide rations to meet those requirements.

Characterisation of the intracellular-glutamate decarboxylase system (GAD): analysis of its function, transcription and role in the acid resistance of various strains of Listeria monocytogenes

The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABA(i)) as a standard response against acid in any medium, and this occurs in all strains tested. Since these systems can occur independently of one another, we refer to them as the extracellular (GAD(e)) and intracellular (GAD(i)) systems. We show here that GAD(i) contributes to acid resistance since in a ΔgadD1D2 mutant, reduced GABA(i) accumulation coincided with a 3.2-log-unit reduction in survival at pH 3.0 compared to that of wild-type strain LO28. Among 20 different strains, the GAD(i) system was found to remove 23.11% ± 18.87% of the protons removed by the overall GAD system. Furthermore, the GAD(i) system is activated at milder pH values (4.5 to 5.0) than the GAD(e) system (pH 4.0 to 4.5), suggesting that GAD(i) is the more responsive of the two and the first line of defense against acid. Through functional genomics, we found a major role for GadD2 in the function of GAD(i), while that of GadD1 was minor. Furthermore, the transcription of the gad genes in three common reference strains (10403S, LO28, and EGD-e) during an acid challenge correlated well with their relative acid sensitivity. No transcriptional upregulation of the gadT2D2 operon, which is the most important component of the GAD system, was observed, while gadD3 transcription was the highest among all gad genes in all strains. In this study, we present a revised model for the function of the GAD system and highlight the important role of GAD(i) in the acid resistance of L. monocytogenes.

Progress in the genetics of common obesity and type 2 diabetes

The prevalence of obesity and diabetes, which are heritable traits that arise from the interactions of multiple genes and lifestyle factors, continues to rise worldwide, causing serious health problems and imposing a substantial economic burden on societies. For the past 15 years, candidate gene and genome-wide linkage studies have been the main genetic epidemiological approaches to identify genetic loci for obesity and diabetes, yet progress has been slow and success limited. The genome-wide association approach, which has become available in recent years, has dramatically changed the pace of gene discoveries. Genome-wide association is a hypothesis-generating approach that aims to identify new loci associated with the disease or trait of interest. So far, three waves of large-scale genome-wide association studies have identified 19 loci for common obesity and 18 for common type 2 diabetes. Although the combined contribution of these loci to the variation in obesity and diabetes risk is small and their predictive value is typically low, these recently identified loci are set to substantially improve our insights into the pathophysiology of obesity and diabetes. This will require integration of genetic epidemiological methods with functional genomics and proteomics. However, the use of these novel insights for genetic screening and personalised treatment lies some way off in the future.

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.

Interaction of cationic bilayer fragments with a model oligonucleotide

The interaction between cationic bilayer fragments and a model oligonucleotide was investigated by differential scanning calorimetry, turbidimetry, determination of excimer to monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidyl-choline in bilayer fragment dispersions and dynamic light scattering for sizing and zeta-potential analysis. Salt (Na(2)HPO(4)), mononucleotide (2`-deoxyadenosine-5`-monophosphate) or poly (dA) oligonucleotide (3`-AAA AAA AAA A-5`) affected structure and stability of dioctadecyldimethylammonium bromide bilayer fragments. Oligonucleotide and salt increased bilayer packing due to bilayer fragment fusion. Mononucleotide did not reduce colloid stability or did not cause bilayer fragment fusion. Charge neutralization of bilayer fragments by poly (dA) at 1:10 poly (dA):dioctadecyldimethylammonium bromide molar ratio caused extensive aggregation, maximal size and zero of zeta-potential for the assemblies. Above charge neutralization, assemblies recovered colloid stability due to charge overcompensation. For bilayer fragments/poly (dA), the nonmonotonic behavior of colloid stability as a function of poly (dA) concentration was unique for the oligonucleotide and was not observed for Na(2)HPO(4) or 2`-deoxyadenosine-5`-monophosphate. For the first time, such interactions between cationic bilayer fragments and mono- or oligonucleotide were described in the literature. Bilayer fragments/oligonucleotide assemblies may find interesting applications in drug delivery. (c) 2010 Elsevier B.V. All rights reserved.

Heterologous microarray experiments used to identify the early gene response to heat stress in a coral reef fish

Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. In this study, we tested the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Results from a comparative genomic hybridization experiment and direct sequence comparisons indicate that for most genes there is considerable sequence similarity between the two species, suggesting that the D. rerio array is useful for genomic studies of P. moluccensis. We employed this heterologous microarray approach to characterize the early transcriptional response to heat stress in P. moluccensis. A total of 111 gene loci, many of which are involved in protein processing, transcription, and cell growth, showed significant changes in transcript abundance following exposure to elevated temperatures. Changes in transcript abundance were validated for a selection of candidate genes using quantitative real-time polymerase chain reaction. This study demonstrates that heterologous microarrays can be successfully employed to study species for which specific microarrays have not yet been developed, and so have the potential to greatly enhance the utility of microarray technology to the field of environmental and functional genomics.