Metodologias emergentes de larga escala na era pós-genómica: benefícios e questões éticas

ABSTRACT: The fields of molecular cell biology and human genetics have recently experienced several revolutions that have brought new knowledge and new technologies. Large scale sequencing and global functional characterization studies allow simultaneously monitoring thousands of sequences and molecules, thus generating a large amount of data giving a real-time molecular details snapshot of an individual. In this review, we briefly refer to some of these novel techniques, including next-generation sequencing methods and CRISPR/Cas9 editing gene systems. The major applications and implications of these novel technologies in research and molecular diagnostics are presented. Ethical issues raised by the implementation of these methods in healthcare systems in the near future are discussed.RESUMO: As áreas da biologia celular e molecular e da genética humana sofreram recentemente uma grande revolução que trouxe novo conhecimento e novas tecnologias. Os estudos de sequenciação em larga escala e de caracterização funcional permitem a monitorização simultânea de milhares de sequências e moléculas, gerando uma grande quantidade de dados e fornecendo detalhes moleculares em tempo real e de modo instantâneo de um indivíduo. Nesta revisão faz-se uma referência breve a algumas destas novas técnicas, incluindo os métodos de sequenciação de next-generation e os métodos de edição de genes baseados no sistema CRISPR/Cas9. São apresentadas as potenciais aplicações e implicações destas novas tecnologias nas áreas de investigação e diagnóstico molecular. São também discutidas as questões éticas levantadas pela implementação destas metodologias num futuro próximo.info:eu-repo/semantics/publishedVersio

New high-throughput technologies of post genome era: benefits and ethical issues

ABSTRACT: The fields of molecular cell biology and human genetics have recently experienced several revolutions that have brought new knowledge and new technologies. Large scale sequencing and global functional characterization studies allow simultaneously monitoring thousands of sequences and molecules, thus generating a large amount of data giving a real-time molecular details snapshot of an individual. In this review we briefly refer to some of these novel techniques, including next-generation sequencing methods and CRISPR/Cas9 editing gene systems. The major applications and implications of these novel technologies in research and molecular diagnostics are presented. Ethical issues raised by the implementation of these methods in healthcare systems in the near future are discussed

Prevalence of Aspergillus section Fumigati in portuguese slaughterhouses: a fungal and mycotoxin concern

Within the Aspergillus genus, Aspergillus fumigatus species is one of the most ubiquitous saprophytic fungi and is considered the section of species with higher clinical relevance. This section is the most common cause of invasive aspergillosis and a major source of infection-related mortality in immunocompromised patients. One of the most abundant metabolites produced by this fungus is the metabolite gliotoxin, which exhibits a diverse array of biologic effects on the immune system. The aim of the present work was to determine the prevalence of Aspergillus section Fumigati by cultural and molecular methods in poultry; swine/bovine; and large animal (bovine and horses) slaughterhouses. Air samples were collected through an impaction method, while surface samples were collected by the swabbing method and subject to further macro and microscopic observations. In addition, we collected air samples using the impinger method in order to perform real-time quantitative PCR (qPCR) amplification of genes from A. fumigatus complex. Aspergillus section Fumigati was present only in Stacker, Bleeding and Evisceration air collected from the Poultry Slaughterhouse (10 – 30 CFU.m3), and in air from the Gut Room collected in the Bovine Slaughterhouse (10 CFU.m3). Molecular tools amplified successfully DNA from the A. fumigatus complex in six sampling sites where the presence of this fungal species was not identified by conventional methods. Besides suggesting A. fumigatus complex as an indicator of harmful fungal contamination in this occupational setting, this study also indicates that conventional and molecular tools should be used as a combined strategy to ensure a proper characterization of fungal occupational exposure. Moreover, in the considered slaughterhouses, fungal contamination results pinpoint to co-exposure to other mycotoxins. In fact, occupational exposure to aflatoxin B1 has already been detected in Poultry Slaughterhouse. Therefore, there is a need for considering possible interactions between mycotoxins and fungi and this should be taken into account in the risk assessment process.N/

Poultry production chain: where is the highest occupational threat?

Introduction: Poultry farmers are occupationally exposed to many respiratory hazards, being the two most reported the fungal burden and particles. Besides poultries, we also must consider poultry slaughterhouses, since it may confine the same exposure risks. Objective: In this study, we aimed to access fungal and particles’ exposure in seven poultries with floor-housed operations and in one poultry slaughterhouse to assess and compare occupational exposure to both risk factors in the two occupational settings.info:eu-repo/semantics/publishedVersio

Toxigenic fungi in coffee samples: a menace to public health

Introduction - Mycotoxin contamination was reported to occur in some food and commodities, such as coffee, particularly due to the presence of toxigenic fungi such as Aspergillus, Penicillium and Fusarium spp. Aspergilli are known to produce high levels of mycotoxins, such as ochratoxin and aflatoxin. Aspergillus ochraceus has been proposed as the major cause of ochratoxin A contamination in coffee beans. Aim of the study - The aim of this work was to evaluate the prevalence of Aspergillus sections Circumdati, Flavi and Fumigati in 28 green coffee samples to be used by Portuguese coffee industry, from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) species from different origins

Fungi distribution in poultry feed

Feed can easily be contaminated and colonized by fungi that use up the nutrients for their own metabolism and growth, producing secondary metabolites such as mycotoxins that are not eliminated throughout the feed processing. The major problems associated with mycotoxin contaminated animal feed are metabolic disturbances resulting in poor animal productivity. In addition, handling contaminated animal feed can also raise health issues regarding workers exposure to fungi and mycotoxins. The scope of this work was to characterize fungal distribution in 11 poultry feed samples. Twenty grams of feed were suspended in 180 mL of distilled water and homogenized during 20 minutes at 200 rpm. The washed supernatant was plated in malt extract agar (MEA) and dichloran glycerol agar base (DG18) media for morphological identification of the mycobiota present. Using macro- and microscopic analysis of the colonies, fungal contamination was evident in 72.7% of the analyzed poultry feed samples. Fungal load ranged from 0 to 13140 CFU/g, and the most prevalent species/genera were F. graminearum complex (71.1%), Penicillium sp. (11.6%), Cladosporium sp. (8.8%), and Fusarium poae (3.6%). In addition to these species, we also isolated Aspergillus sections Circumdati, Nigri and Aspergilli, and Mucor and Rhizopus genus albeit at a lower abundance. The data obtained showed that, besides high fungal contamination, mycotoxins contamination is probably a reality, particularly in the final product since mycotoxins resist to all the processing operations including thermal treatment. Additionally, data claimed attention for the probable co-exposure to fungi and mycotoxins of the workers in feed industries

Analysis of fungal burden by conventional and molecular methods in different settings and matrices: implications for public and occupational health

Fungal burden has traditionally been detected by conventional culture analysis. This method allows the identification and quantification of organisms posing high health/occupational risk and is widely used by the scientific community. The fungal burden determined by culture analysis can, in most studies, be compared with legal and scientific guidelines allowing an estimation of the degree of severity of the exposure. However, this method is limited by several factors, including incubation conditions such as the incubation time, which can be very long for some species, thus preventing a quick assessment of fungal burden. Another limiting factor is competition between species: clinically relevant species might possess lower growth rates than other non-toxic fast growing fungi thus hampering their detection. These limitations can be overcome by the use of quantitative real-time PCR (qPCR). This method, based on the amplification of genomic regions specific to certain fungal species, increases sensitivity, allowing the specific detection of a given species and removing interference by other species present in the sample. qPCR also allows the detection of dormant forms of fungi, such as spores. Thus the ideal scenario is to use these the two methods in parallel, as they complement each other to provide useful information for the assessment of exposure to fungi. We briefly describe several studies where both methods were used to detect the presence of toxigenic fungi, namely Aspergillus, particularly from the Fumigati, Flavi and Circumdati sections. These include fungal analysis from different matrices such as air, feed and coffee and within different settings, including wastewater treatment plants, slaughterhouses, feed industries, poultry and swine pavilions. The results obtained with both conventional and molecular methods are compared and discussed as well as its implications for the exposed workers' health.info:eu-repo/semantics/draf

Bioburden characterization by active and passive methods in Portuguese dwellings

Projeto IPL/2017/B2CF_ESTeSL, Projeto IPL/2017/B2 Hemo_ESTeSLThe living environment can be a source for bioburden and azole-resistant mycobiota that depending on the occupant’s health status can be a serious public health problem. The objective of this study was to assess bioburden in Portuguese dwellings located in the Lisbon Region through active methods (air sampling) coupled with passive methods (Electrostatic dust cloth - EDC).info:eu-repo/semantics/publishedVersio

Occupational exposure to fungi and mycotoxins in cork industry: an exploratory study

Cork oak is the second most dominant forest species in Portugal and makes this country the work leader in cork export. This type of industry has already been associated with occupational exposure to several fungal species, most commonly with Penicillium globrum (belonging to Penicillium section Aspergilloides) and Chrysonilia sitophila. However, occupational exposure to mycotoxins has never been studied. Here we describe an exploratory work developed in a cork industry located in Portugal aiming at assessing exposure to fungi and mycotoxins.info:eu-repo/semantics/publishedVersio