Sensitive and specific detection of viable Mycobacterium avium subsp. paratuberculosis in raw milk by the Peptide-mediated magnetic separation (PMS)-phage assay

This study describes further validation of a previously described Peptide-mediated magnetic separation (PMS)-Phage assay, and its application to test raw cows’ milk for presence of viable Mycobacterium avium subsp. paratuberculosis (MAP). The inclusivity and exclusivity of the PMS-phage assay were initially assessed, before the 50% limit of detection (LOD50) was determined and compared with those of PMS-qPCR (targeting both IS900 and f57) and PMS-culture. These methods were then applied in parallel to test 146 individual milk samples and 22 bulk tank milk samples from Johne’s affected herds. Viable MAP were detected by the PMS-phage assay in 31 (21.2%) of 146 individual milk samples (mean plaque count of 228.1 PFU/50 ml, range 6-948 PFU/50 ml), and 13 (59.1%) of 22 bulk tank milks (mean plaque count of 136.83 PFU/50 ml, range 18-695 PFU/50 ml). In contrast, only 7 (9.1%) of 77 individual milks and 10 (45.4%) of 22 bulk tank milks tested PMS-qPCR positive, and 17 (11.6%) of 146 individual milks and 11 (50%) of 22 bulk tank milks tested PMS-culture positive. The mean 50% limits of detection (LOD50) of the PMS-phage, PMS-IS900 qPCR and PMS-f57 qPCR assays, determined by testing MAP-spiked milk, were 0.93, 135.63 and 297.35 MAP CFU/50 ml milk, respectively. Collectively, these results demonstrate that, in our laboratory, the PMS-phage assay is a sensitive and specific method to quickly detect the presence of viable MAP cells in milk. However, due to its complicated, multi-step nature, the method would not be a suitable MAP screening method for the dairy industry.

Development of A Flow Cytometry Assay for Measurement of CD4+ T Cell Proliferation

Thesis (Master's)--University of Washington, 2016-06

Field Evaluation of a Competitive Lateral-flow Assay for Detection of Alternaria brassicae in Vegetable Brassica Crops

On-site detection of inoculum of polycyclic plant pathogens could potentially contribute to management of disease outbreaks. A 6-min, in-field competitive immunochromatographic lateral flow device (CLFD) assay was developed for detection of Alternaria brassicae (the cause of dark leaf spot in brassica crops) in air sampled above the crop canopy. Visual recording of the test result by eye provides a detection threshold of approximately 50 dark leaf spot conidia. Assessment using a portable reader improved test sensitivity. In combination with a weather-driven infection model, CLFD assays were evaluated as part of an in-field risk assessment to identify periods when brassica crops were at risk from A. brassicae infection. The weather-driven model overpredicted A. brassicae infection. An automated 7-day multivial cyclone air sampler combined with a daily in-field CLFD assay detected A. brassicae conidia air samples from above the crops. Integration of information from an in-field detection system (CLFD) with weather-driven mathematical models predicting pathogen infection have the potential for use within disease management systems.

Relation between DNA damage measured by comet assay and OGG1 Ser326Cys polymorphism in antineoplastic drugs biomonitoring

Antineoplastic drugs are hazardous chemical agents used mostly in the treatment of patients with cancer, however health professionals that handle and administer these drugs can become exposed and develop DNA damage. Comet assay is a standard method for assessing DNA damage in human biomonitoring and, combined with formamidopyrimidine DNA glycosylase (FPG) enzyme, it specifically detects DNA oxidative damage. The aim of this study was to investigate genotoxic effects in workers occupationally exposed to cytostatics (n = 46), as compared to a control group with no exposure (n = 46) at two Portuguese hospitals, by means of the alkaline comet assay. The potential of the OGG1 Ser326Cys polymorphism as a susceptibility biomarker was also investigated. Exposure was evaluated by investigating the contamination of surfaces and genotoxic assessment was done by alkaline comet assay in peripheral blood lymphocytes. OGG1 Ser326Cys (rs1052133) polymorphism was studied by Real Time PCR. As for exposure assessment, there were 121 (37%) positive samples out of a total of 327 samples analysed from both hospitals. No statistically significant differences (Mann-Whitney test, p > 0.05) were found between subjects with and without exposure, regarding DNA damage and oxidative DNA damage, nevertheless the exposed group exhibited higher values. Moreover, there was no consistent trend regarding the variation of both biomarkers as assessed by comet assay with OGG1 polymorphism. Our study was not statistically significant regarding occupational exposure to antineoplastic drugs and genetic damage assessed by comet assay. However, health professionals should be monitored for risk behaviour, in order to ensure that safety measures are applied and protection devices are used correctly.

Comet assay as a human biomonitoring tool: application in occupational exposure to antineoplastic drugs

Antineoplastic drugs are a heterogeneous group of chemicals used in the treatment of cancer, and have been proved by IARC to be mutagens, carcinogens and teratogens agents. In general, chemicals that interact directly with DNA by biding covalently or by intercalating, or indirectly by interfering with DNA synthesis, were among the first chemotherapeutics developed. Also, these drugs can induce reactive oxygen species that can lead to DNA damage and, consequently, mutations. These drugs are often used in combination to achieve synergistic effects on tumour cells resulting from their differing modes of action. However, most if not all of these chemical agents are generally nonselective and, along with tumour cells, normal cells may undergo cytotoxic/genotoxic damage. The in vivo exposure to antineoplastic drugs has been shown to induce different types of lesions in DNA, depending on the particular stage of cell cycle at the time of treatment. Besides the patients that use these drugs as a treatment, workers that handle and/or administer these drugs can be exposed to these substances; namely pharmacy, and nursing personnel in hospital context.

Validation of a novel assay for cardiology drug screening using hESC-derived cardiomyocyte spheroids - “mini-hearts”

This is an abstract of a presented talk at the European Biotechnology Conference held in Latvia during 05–07 May 2016

Elaboração de paté de lapa, Patella spp. com adição de medronho, Arbutus unedo: caraterização físico-química, microbiológica e sensorial e avaliação da estabilidade oxidativa

As lapas do género Patella são moluscos gastrópodes em crescente utilização na alimentação humana, sendo um petisco muito apreciado em várias regiões costeiras de Portugal. De acordo com a fileira do pescado, o consumo de produtos da pesca ricos em ácidos gordos polinsaturados têm vindo a substituir os produtos de origem animal terrestre através dos produtos filetados, reestruturados e pré-cozinhados. A oxidação dos alimentos é a causa mais importante na deterioração da qualidade nutricional e sensorial dos produtos da pesca. Assim nos últimos anos, tem-se verificado um interesse crescente na utilização de antioxidantes naturais de origem vegetal (frutas e vegetais) em detrimento de antioxidantes sintéticos, aquando da elaboração de produtos reestruturados de origem animal (hambúrgueres, salsichas e patés). Para além deste efeito antioxidante, existe uma relação benéfica entre o consumo de frutas e vegetais, ricos em compostos fenólicos e a prevenção de certas doenças. Este trabalho pretende produzir e caracterizar ao nível físico-químico, sensorial e microbiológico um paté elaborado a partir de lapas, com adição do fruto silvestre medronho, bem como a valorização destas matérias-primas ao proporcionar uma mais-valia nutricional/funcional e económica com o desenvolvimento de um produto inovador (paté). Por outro lado, avaliou-se a estabilidade oxidativa do paté em armazenamento refrigerado durante 90 dias bem como a aceitabilidade do mesmo. Sendo assim, produziram-se três tipos de patés inovadores: i) paté de lapa com BHT (PCON), ii) paté de lapa com 3% de medronho (PAU3) e iii) paté de lapa com 6% de medronho (PAU6). Os resultados da avaliação da estabilidade oxidativa (PAU6>PAU3>PCON) confirmam a elevada capacidade do extrato de medronho rico em compostos fenólicos como inibidor eficaz na oxidação lipídica do paté de lapa e com boa aceitação sensorial.

Optimizing PAT data transmission: assessing the accuracy of temperature summary data to estimate environmental conditions

Pop-up archival tags (PAT) provide summary and high-resolution time series data at predefined temporal intervals. The limited battery capabilities of PATs often restrict the transmission success and thus temporal coverage of both data products. While summary data are usually less affected by this problem, as a result of its lower size, it might be less informative. We here investigate the accuracy and feasibility of using temperature at depth summary data provided by PATs to describe encountered oceanographic conditions. Interpolated temperature at depth summary data was found to provide accurate estimates of three major thermal water column structure indicators: thermocline depth, stratification and ocean heat content. Such indicators are useful for the interpretation of the tagged animal's horizontal and vertical behaviour. The accuracy of these indicators was found to be particularly sensitive to the number of data points available in the first 100 m, which in turn depends on the vertical behaviour of the tagged animal. Based on our results, we recommend the use of temperature at depth summary data as opposed to temperature time series data for PAT studies; doing so during the tag programming will help to maximize the amount of transmitted time series data for other key data types such as light levels and depth.

The cytokinesis-block micronucleus (CBMN) assay in human populations exposed to styrene: a systematic review and meta-analysis

Styrene is a building-block of several compounds used in a wide array of materials and products. The most important human exposure to this substance occurs in industrial settings, especially among reinforced-plastics industry workers. The effect of occupational exposure to styrene on cytogenetics biomarkers has been previously reviewed with positive association observed for chromosomal aberrations, and inconclusive data for the micronucleus assay. Some limitations were noted in those studies, including inadequate exposure assessment and poor epidemiological design. Furthermore, in earlier studies micronuclei frequency was measured with protocols not as reliable as cytokinesis-block micronucleus (CBMN) assay. Aim of the present systematic review and meta-analysis is to investigate genomic instability and DNA damage as measured by the CBMN assay in lymphocytes of subjects exposed to styrene. A total of 11 studies published between 2004 and 2012 were included in the meta-analysis encompassing 479 styrene-exposed workers and 510 controls. The quality of each study was estimated by a quality scoring system which ranked studies according to the consideration of major confounders, exposure characterization, and technical parameters. An overall increase of micronuclei frequencies was found in styrene-exposure workers when compared to referents (meta-MR 1.34; 95% CI 1.18–1.52), with significant increases achieved in six individual studies. The consistency of results in individual studies, the independence of this result from major confounding factors and from the quality of the study strengthens the reliability of risk estimates and supports the use of the CBMN assay in monitoring genetic risk in styrene workers.

A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis

Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.

The RGBarrier assay: the parallel study of gene regulatory element performance at defined chromosomal locations

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase seminested PCR and restriction fragment length polymorphism assay

Rotavirus double-stranded RNA was detected directly in sewage treatment plant samples over a 1-year period by reverse transcription followed by PCR amplification of the VP7 gene and Southern blot hybridization. The presence of naturally occurring rotaviruses was demonstrated in 42% of raw sewage samples and in 67% of treated effluent samples, Amplified viral sequences were analyzed bg restriction enzymes. Ten different restriction profiles were characterized, most of which were found in treated effluent samples. A mixture of restriction profiles was observed in 75% of contaminated effluent samples, The profiles were compared with those obtained from human rotavirus isolates involved in infections in children from the same area (six different profiles were detected), Five identical viral sequences were detected in both environmental and clinical samples, Restriction profiles sere also compared io profiles from known genomic sequences of human and animal viruses. Both human and animal origins of rotavirus contamination of water seemed likely.