Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices

Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.

Laser ablation-based one-step generation and bio-functionalization of gold nanoparticles conjugated with aptamers

Background: Bio-conjugated nanoparticles are important analytical tools with emerging biological and medical applications. In this context, in situ conjugation of nanoparticles with biomolecules via laser ablation in an aqueous media is a highly promising one-step method for the production of functional nanoparticles resulting in highly efficient conjugation. Increased yields are required, particularly considering the conjugation of cost-intensive biomolecules like RNA aptamers. Results: Using a DNA aptamer directed against streptavidin, in situ conjugation results in nanoparticles with diameters of approximately 9 nm exhibiting a high aptamer surface density (98 aptamers per nanoparticle) and a maximal conjugation efficiency of 40.3%. We have demonstrated the functionality of the aptamer-conjugated nanoparticles using three independent analytical methods, including an agglomeration-based colorimetric assay, and solid-phase assays proving high aptamer activity. To demonstrate the general applicability of the in situ conjugation of gold nanoparticles with aptamers, we have transferred the method to an RNA aptamer directed against prostate-specific membrane antigen (PSMA). Successful detection of PSMA in human prostate cancer tissue was achieved utilizing tissue microarrays. Conclusions: In comparison to the conventional generation of bio-conjugated gold nanoparticles using chemical synthesis and subsequent bio-functionalization, the laser-ablation-based in situ conjugation is a rapid, one-step production method. Due to high conjugation efficiency and productivity, in situ conjugation can be easily used for high throughput generation of gold nanoparticles conjugated with valuable biomolecules like aptamers.

EMMA 2--a MAGE-compliant system for the collaborative analysis and integration of microarray data

Background: Understanding transcriptional regulation by genome-wide microarray studies can contribute to unravel complex relationships between genes. Attempts to standardize the annotation of microarray data include the Minimum Information About a Microarray Experiment (MIAME) recommendations, the MAGE-ML format for data interchange, and the use of controlled vocabularies or ontologies. The existing software systems for microarray data analysis implement the mentioned standards only partially and are often hard to use and extend. Integration of genomic annotation data and other sources of external knowledge using open standards is therefore a key requirement for future integrated analysis systems. Results: The EMMA 2 software has been designed to resolve shortcomings with respect to full MAGE-ML and ontology support and makes use of modern data integration techniques. We present a software system that features comprehensive data analysis functions for spotted arrays, and for the most common synthesized oligo arrays such as Agilent, Affymetrix and NimbleGen. The system is based on the full MAGE object model. Analysis functionality is based on R and Bioconductor packages and can make use of a compute cluster for distributed services. Conclusion: Our model-driven approach for automatically implementing a full MAGE object model provides high flexibility and compatibility. Data integration via SOAP-based web-services is advantageous in a distributed client-server environment as the collaborative analysis of microarray data is gaining more and more relevance in international research consortia. The adequacy of the EMMA 2 software design and implementation has been proven by its application in many distributed functional genomics projects. Its scalability makes the current architecture suited for extensions towards future transcriptomics methods based on high-throughput sequencing approaches which have much higher computational requirements than microarrays.

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

Background The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. Conclusions This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

Identification of regulatory polymorphisms associated with breast cancer risk

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Functionalized Carbon Micro/Nanostructures for Biomolecular Detection

Advancements in the micro-and nano-scale fabrication techniques have opened up new avenues for the development of portable, scalable and easier-to-use biosensors. Over the last few years, electrodes made of carbon have been widely used as sensing units in biosensors due to their attractive physiochemical properties. The aim of this research is to investigate different strategies to develop functionalized high surface carbon micro/nano-structures for electrochemical and biosensing devices. High aspect ratio three-dimensional carbon microarrays were fabricated via carbon microelectromechanical systems (C-MEMS) technique, which is based on pyrolyzing pre-patterned organic photoresist polymers. To further increase the surface area of the carbon microstructures, surface porosity was introduced by two strategies, i.e. (i) using F127 as porogen and (ii) oxygen reactive ion etch (RIE) treatment. Electrochemical characterization showed that porous carbon thin film electrodes prepared by using F127 as porogen had an effective surface area (Aeff 185%) compared to the conventional carbon electrode. To achieve enhanced electrochemical sensitivity for C-MEMS based functional devices, graphene was conformally coated onto high aspect ratio three-dimensional (3D) carbon micropillar arrays using electrostatic spray deposition (ESD) technique. The amperometric response of graphene/carbon micropillar electrode arrays exhibited higher electrochemical activity, improved charge transfer and a linear response towards H2O2 detection between 250μM to 5.5mM. Furthermore, carbon structures with dimensions from 50 nano-to micrometer level have been fabricated by pyrolyzing photo-nanoimprint lithography patterned organic resist polymer. Microstructure, elemental composition and resistivity characterization of the carbon nanostructures produced by this process were very similar to conventional photoresist derived carbon. Surface functionalization of the carbon nanostructures was performed using direct amination technique. Considering the need for requisite functional groups to covalently attach bioreceptors on the carbon surface for biomolecule detection, different oxidation techniques were compared to study the types of carbon–oxygen groups formed on the surface and their percentages with respect to different oxidation pretreatment times. Finally, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor oncoprotein detection on functionalized three-dimensional carbon microarrays platform was demonstrated. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5 pmol.

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Utility of a panviral microarray for detection of swine respiratory viruses in clinical samples

Several factors have recently converged, elevating the need for highly parallel diagnostic platforms that have the ability to detect many known, novel, and emerging pathogenic agents simultaneously. Panviral DNA microarrays represent the most robust approach for massively parallel viral surveillance and detection. The Virochip is a panviral DNA microarray that is capable of detecting all known viruses, as well as novel viruses related to known viral families, in a single assay and has been used to successfully identify known and novel viral agents in clinical human specimens. However, the usefulness and the sensitivity of the Virochip platform have not been tested on a set of clinical veterinary specimens with the high degree of genetic variance that is frequently observed with swine virus field isolates. In this report, we investigate the utility and sensitivity of the Virochip to positively detect swine viruses in both cell culture-derived samples and clinical swine samples. The Virochip successfully detected porcine reproductive and respiratory syndrome virus (PRRSV) in serum containing 6.10 × 10(2) viral copies per microliter and influenza A virus in lung lavage fluid containing 2.08 × 10(6) viral copies per microliter. The Virochip also successfully detected porcine circovirus type 2 (PCV2) in serum containing 2.50 × 10(8) viral copies per microliter and porcine respiratory coronavirus (PRCV) in turbinate tissue homogenate. Collectively, the data in this report demonstrate that the Virochip can successfully detect pathogenic viruses frequently found in swine in a variety of solid and liquid specimens, such as turbinate tissue homogenate and lung lavage fluid, as well as antemortem samples, such as serum.

Transcriptional analysis of PRRSV-infected porcine dendritic cell response to Streptococcus suis infection reveals up-regulation of inflammatory-related genes expression

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.

Heat and water stress induce unique transcriptional signatures of heat-shock proteins and transcription factors in grapevine

Grapevine is an extremely important crop worldwide.In southern Europe, post-flowering phases of the growth cycle can occur under high temperatures, excessive light, and drought conditions at soil and/or atmospheric level. In this study, we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress(WDS), and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular net works involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays, we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high-throughput transcriptomic grapevine array, the 23K custom-made Affymetrix Vitis GeneChip. We identified 1,594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat-shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However, across-talk between the response pathways to both stresses was observed at the level of AP2/ERF transcription factors.

Transcriptional analysis of PRRSV-infected porcine dendritic cell response to Streptococcus suis infection reveals up-regulation of inflammatory-related genes expression

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.

Genomics to elucidate the molecular basis of calcific aortic valve disease

Le rétrécissement valvulaire aortique (RVA) est causé par une calcification et une fibrose progressive de la valve aortique. Le risque de développer la maladie augmente avec l’âge. À cause de l’augmentation de l’espérance de vie, le RVA est devenu un problème de santé publique. Le RVA est fatal en absence de traitement médical. Actuellement, la chirurgie est le seul traitement pour le stade sévère de la maladie, mais près de 50% des individus avec RVA n’y sont pas éligibles, principalement due à la présence de comorbidités. Plusieurs processus biologiques ont été associés à la maladie, mais les voies moléculaires spécifiques et les gènes impliqués dans le développement et la progression du RVA ne sont pas connus. Il est donc urgent de découvrir les gènes de susceptibilité pour le RVA afin d’identifier les personnes à risque ainsi que les biomarqueurs et les cibles thérapeutiques pouvant mener au développement de médicaments pour inverser ou limiter la progression de la maladie. L’objectif de cette thèse de doctorat était d’identifier la base moléculaire du RVA. Des approches modernes en génomique, incluant l’étude de gènes candidats et le criblage génomique par association (GWAS), ont été réalisées à l’aide de collections d’ADN provenant d’un grand nombre de patients bien caractérisés pour le RVA. Des études complémentaires en transciptomique ont comparé le profil d’expression global des gènes entre des valves calcifiées et non-calcifiées à l’aide de biopuces à ADN et de séquençage de l’ARN. Une première étude a identifié des variations dans le gène NOTCH1 et suggère pour la première fois la présence d’un polymorphisme commun dans ce gène conférant une susceptibilité au RVA. La deuxième étude a combiné par méta-analyse deux GWAS de patients provenant de la ville de Québec et Paris (France) aux données transcriptomiques. Cette étude de génomique intégrative a confirmé le rôle de RUNX2 dans le RVA et a permis l’identification d’un nouveau gène de susceptibilité, CACNA1C. Les troisième et quatrième études sur l’expression des gènes ont permis de mieux comprendre les bases moléculaires de la calcification des valves aortiques bicuspides et ainsi d’identifier de nouvelles cibles thérapeutiques pour le RVA. Les données générées par ce projet sont la base de futures découvertes importantes qui permettront d’améliorer les options de traitement et la qualité de vie des patients atteints du RVA.

Global transcriptional response to salt shock of the plant microsymbiont Mesorhizobium loti MAFF303099

Soil salinity affects rhizobia both as free-living bacteria and in symbiosis with the host. The aim of this study was to examine the transcriptional response of the Lotus microsymbiont Mesorhizobium loti MAFF303099 to salt shock. Changes in the transcriptome of bacterial cells subjected to a salt shock of 10% NaCl for 30 min were analyzed. From a total of 7231 protein-coding genes, 385 were found to be differentially expressed upon salt shock, among which 272 were overexpressed. Although a large number of overexpressed genes encode hypothetical proteins, the two most frequently represented COG categories are "defense mechanisms" and "nucleotide transport and metabolism". A significant number of transcriptional regulators and ABC transporters genes were upregulated. Chemotaxis and motility genes were not differentially expressed. Moreover, most genes previously reported to be involved in salt tolerance were not differentially expressed. The transcriptional response to salt shock of a rhizobium with low ability to grow under salinity conditions, but enduring a salinity shock, may enlighten us concerning salinity stress response mechanisms.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

ABSTRACT: BACKGROUND: Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. RESULTS: Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total ß-carotene, containing all-E-, 9-Z-, and 13-Z-ß-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no ?-carotene was observed, variable amounts of a ?-ring derived xanthophyll, lutein, was detected; with greater accumulation of ?-ring xanthophylls than of ß-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total ß-carotene accumulation. CONCLUSIONS: Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total ß-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.