Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

High diversity of skin-associated bacterial communities of marine fishes is promoted by their high variability among body parts, individuals and species

Animal-associated microbiotas form complex communities, which are suspected to play crucial functions for their host fitness. However, the biodiversity of these communities, including their differences between host species and individuals, has been scarcely studied, especially in case of skin-associated communities. In addition, the intraindividual variability (i.e. between body parts) has never been assessed to date. The objective of this study was to characterize skin bacterial communities of two teleostean fish species, namely the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), using a high-throughput DNA sequencing method. In order to focus on intrinsic factors of host-associated bacterial community variability, individuals of the two species were raised in controlled conditions. Bacterial diversity was assessed using a set of four complementary indices, describing the taxonomic and phylogenetic facets of biodiversity and their respective composition (based on presence/absence data) and structure (based on species relative abundances) components. Variability of bacterial diversity was quantified at the interspecific, interindividual and intraindividual scales. We demonstrated that fish surfaces host highly diverse bacterial communities, whose composition was very different from that of surrounding bacterioplankton. This high total biodiversity of skin-associated communities was supported by the important variability, between host species, individuals and the different body parts (dorsal, anal, pectoral and caudal fins).

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.

Investigating novel approaches to the detection of virus neutralising antibodies to rabies and Rift Valley fever virus

Serosurveillance is a powerful tool fundamental to understanding infectious disease dynamics. The presence of virus neutralising antibody (VNAb) in sera is considered the best evidence of infection, or indeed vaccination, and the gold standard serological assay for their detection is the virus neutralisation test (VNT). However, VNTs are labour intensive, costly and time consuming. In addition, VNTs for the detection of antibodies to highly pathogenic viruses require the use of high containment facilities, restricting the application of these assays to the few laboratories with adequate facilities. As a result, robust serological data on such viruses are limited. In this thesis I develop novel VNTs for the detection of VNAb to two important, highly pathogenic, zoonotic viruses; rabies and Rift Valley fever virus (RVFV). The pseudotype-based neutralisation test developed in this study allows for the detection of rabies VNAb without the requirement for high containment facilities. This assay was utilised to investigate the presence of rabies VNAb in animals from a variety of ecological settings. In this thesis I present evidence of natural rabies infection in both domestic dogs and lions from rabies endemic settings. The assay was further used to investigate the kinetics of VNAb response to rabies vaccination in a cohort of free-roaming dogs. The RVFV neutralisation assay developed herein utilises a recombinant luciferase expressing RVFV, which allows for rapid, high-throughput serosurveillance of this important neglected pathogen. In this thesis I present evidence of RVFV infection in a variety of domestic and wildlife species from Northern Tanzania, in addition to the detection of low-level transmission of RVFV during interepidemic periods. Additionally, the investigation of a longitudinal cohort of domestic livestock also provided evidence of rapid waning of RVF VNAb following natural infection. Collectively, the serological data presented in this thesis are consistent with existing data in the literature generated using the gold standard VNTs. Increasing the availability of serological assays will allow the generation of robust serological data, which are imperative to enhancing our understanding of the complex, multi-host ecology of these two viruses.

Epigenetics and alternative splicing

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Effect of cinnamon tea on postprandial glucose concentration

"Glycaemic control, in particular at postprandial period, has a key role in prevention of different diseases, including diabetes and cardiovascular events. Previous studies suggest that postprandial high blood glucose levels (BGL) can lead to an oxidative stress status, which is associated with metabolic alterations. Cinnamon powder has demonstrated a beneficial effect on postprandial glucose homeostasis in animals and human models. The purpose of this study is to investigate the effect of cinnamon tea (C. burmannii) on postprandial capillary blood glucose level on nondiabetic adults. Participants were given oral glucose tolerance test either with or without cinnamon tea in a randomized clinical trial. The data revealed that cinnamon tea administration slightly decreased postprandial BGL. Cinnamon tea ingestion also results in a significantly lower postprandial maximum glucose concentration and variation ofmaximum glucose concentration (p < 0.05). Chemical analysis showed that cinnamon tea has a high antioxidant capacity, whichmay be due to its polyphenol content. The present study provides evidence that cinnamon tea, obtained from C. burmannii, could be beneficial for controlling glucose metabolism in nondiabetic adults during postprandial period."

Development of a lab-on-a-chip device for rapid nanotoxicity assessment in vitro

Increasing useof nanomaterials in consumer products and biomedical applications creates the possibilities of intentional/unintentional exposure to humans and the environment. Beyond the physiological limit, the nanomaterialexposure to humans can induce toxicity. It is difficult to define toxicity of nanoparticles on humans as it varies by nanomaterialcomposition, size, surface properties and the target organ/cell line. Traditional tests for nanomaterialtoxicity assessment are mostly based on bulk-colorimetric assays. In many studies, nanomaterials have found to interfere with assay-dye to produce false results and usually require several hours or days to collect results. Therefore, there is a clear need for alternative tools that can provide accurate, rapid, and sensitive measure of initial nanomaterialscreening. Recent advancement in single cell studies has suggested discovering cell properties not found earlier in traditional bulk assays. A complex phenomenon, like nanotoxicity, may become clearer when studied at the single cell level, including with small colonies of cells. Advances in lab-on-a-chip techniques have played a significant role in drug discoveries and biosensor applications, however, rarely explored for nanomaterialtoxicity assessment. We presented such cell-integrated chip-based approach that provided quantitative and rapid response of cellhealth, through electrochemical measurements. Moreover, the novel design of the device presented in this study was capable of capturing and analyzing the cells at a single cell and small cell-population level. We examined the change in exocytosis (i.e. neurotransmitterrelease) properties of a single PC12 cell, when exposed to CuOand TiO2 nanoparticles. We found both nanomaterials to interfere with the cell exocytosis function. We also studied the whole-cell response of a single-cell and a small cell-population simultaneously in real-time for the first time. The presented study can be a reference to the future research in the direction of nanotoxicity assessment to develop miniature, simple, and cost-effective tool for fast, quantitative measurements at high throughput level. The designed lab-on-a-chip device and measurement techniques utilized in the present work can be applied for the assessment of othernanoparticles' toxicity, as well.^

The impact of whey protein consumption and exercise on the composition and diversity of the gut microbiota: a high through-put DNA sequencing approach

Advances in culture independent technologies over the last decade have highlighted the pivotal role which the gut microbiota plays in maintaining human health. Conversely, perturbations to the composition or actions of the ‘normal/functioning’ microbiota have been frequently associated with the pathogenesis of several disease states. Therefore the selective modulation of enteric microbial communities represents a viable target for the development of novel treatments for such diseases. Notably, while bovine whey proteins and exercise have been shown to positively influence several physiological processes, such as energy balance, their effect on the composition or functionality of the gut microbiota remains largely unknown. In this thesis, a variety of ex vivo, murine and human models are used in conjunction with high-throughput DNA sequencing-based analysis to provide valuable and novel insights into the impact of both whey proteins and exercise on enteric microbial communities. Overall the results presented in this thesis highlight that the consumption both whey protein isolate (WPI), and individual component proteins of whey such as bovine serum albumin (BSA) and lactoferrin, reduce high fat diet associated body weight gain and are associated with beneficial alterations within the murine gut microbiota. Although the impact of exercise on enteric microbial communities remains less clear, it may be that longer term investigations are required for the true effect of exercise on the gut microbiota to be fully elucidated.

Changes In The Bacterial Community Of Soil From A Neutral Mine Drainage Channel.

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.

Iis--integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools.

High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. IIS is freely available online at: http://www.lge.ibi.unicamp.br/lnbio/IIS/.

Association Of Single Nucleotide Polymorphisms In The Gene Encoding Glut1 And Diabetic Nephropathy In Brazilian Patients With Type 1 Diabetes Mellitus.

Mesangial cells subject to high extracellular glucose concentrations, as occur in hyperglycaemic states, are unable to down regulate glucose influx, resulting in intracellular activation of deleterious biochemical pathways. A high expression of GLUT1 participates in the development of diabetic glomerulopathy. Variants in the gene encoding GLUT1 (SLC2A1) have been associated to this diabetic complication. The aim of this study was to test whether polymorphisms in SLC2A1 confer susceptibility to diabetic nephropathy (DN) in Brazilian type 1 diabetes patients. Four polymorphisms (rs3820589, rs1385129, rs841847 and rs841848) were genotyped in a Brazilian cohort comprised of 452 patients. A prospective analysis was performed in 155 patients. Mean duration of follow-up was 5.6±2.4years and the incidence of renal events was 18.0%. The rs3820589 presented an inverse association with the prevalence of incipient DN (OR: 0.36, 95% CI: 0.16 - 0.80, p=0.01) and with progression to renal events (HR: 0.20; 95% CI: 0.03 - 0.70; p=0.009). AGGT and AGAC haplotypes were associated with the prevalence of incipient DN and the AGAC haplotype was also associated with the prevalence of established/advanced DN. In conclusion, rs3820589 in the SLC2A1 gene modulates the risk to DN in Brazilian patients with inadequate type 1 diabetes control.

Evaluation of the performance of different plastics used to seal nylon cDNA arrays

cDNA arrays are a powerful tool for discovering gene expression patterns. Nylon arrays have the advantage that they can be re-used several times. A key issue in high throughput gene expression analysis is sensitivity. In the case of nylon arrays, signal detection can be affected by the plastic bags used to keep membranes humid. In this study, we evaluated the effect of five types of plastics on the radioactive transmittance, number of genes with a signal above the background, and data variability. A polyethylene plastic bag 69 μm thick had a strong shielding effect that blocked 68.7% of the radioactive signal. The shielding effect on transmittance decreased the number of detected genes and increased the data variability. Other plastics which were thinner gave better results. Although plastics made from polyvinylidene chloride, polyvinyl chloride (both 13 μm thick) and polyethylene (29 and 7 μm thick) showed different levels of transmittance, they all gave similarly good performances. Polyvinylidene chloride and polyethylene 29 mm thick were the plastics of choice because of their easy handling. For other types of plastics, it is advisable to run a simple check on their performance in order to obtain the maximum information from nylon cDNA arrays.

The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system

Background: The cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps. Results: Here we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 mu M 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 mu M 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization. Conclusions: The genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox

Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 A, beta = 96.03 degrees. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V (M) of 2.12 A3 Da-1, corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques.

Integration and visualization of systems biology data in context of the genome

Background: High-density tiling arrays and new sequencing technologies are generating rapidly increasing volumes of transcriptome and protein-DNA interaction data. Visualization and exploration of this data is critical to understanding the regulatory logic encoded in the genome by which the cell dynamically affects its physiology and interacts with its environment. Results: The Gaggle Genome Browser is a cross-platform desktop program for interactively visualizing high-throughput data in the context of the genome. Important features include dynamic panning and zooming, keyword search and open interoperability through the Gaggle framework. Users may bookmark locations on the genome with descriptive annotations and share these bookmarks with other users. The program handles large sets of user-generated data using an in-process database and leverages the facilities of SQL and the R environment for importing and manipulating data. A key aspect of the Gaggle Genome Browser is interoperability. By connecting to the Gaggle framework, the genome browser joins a suite of interconnected bioinformatics tools for analysis and visualization with connectivity to major public repositories of sequences, interactions and pathways. To this flexible environment for exploring and combining data, the Gaggle Genome Browser adds the ability to visualize diverse types of data in relation to its coordinates on the genome. Conclusions: Genomic coordinates function as a common key by which disparate biological data types can be related to one another. In the Gaggle Genome Browser, heterogeneous data are joined by their location on the genome to create information-rich visualizations yielding insight into genome organization, transcription and its regulation and, ultimately, a better understanding of the mechanisms that enable the cell to dynamically respond to its environment.