Fungal protoplasts as a genetic tool

For the last three decades, protoplasts have been shown to be an effective tool in studying the biochemistry and genetics of fungi, as the thick cell wall is no more a barrier. In this work we describe and discuss this versatile tool in mycology in two ways, preparation level and utilisation level. At the preparation level we describe the optimal conditions to obtain yeast protoplasts, including buffers employed, incubation time, regeneration of protoplasts, and compare the methodology to obtain the protoplasts from yeast and filamentous fungi. At the utilisation level we analyse the methodology to introduce foreign DNA into host genomes via protoplast transformation, probably the most universal method of gene transfer, and via protoplast fusion, a tool to "illegitimaly" transfer nuclear and/or organellar genetic information beyond phylogenetic boundaries. Finally, we conclude about the importance and actuality of fungal protoplasts in mycological research

In vitro impact of tenebrio molitor insect flour on human gut microbiota

A farinha de inseto de Tenebrio molitor (TMIF) é considerada um ingrediente alimentar nutritivo que ainda carece de avaliação do seu potencial nutricional na dieta humana. Um dos maiores indicadores do efeito da dieta na saúde humana é a composição da microbiota intestinal, principalmente a presença de grupos bacterianos benéficos, como as bactérias probióticas. Desta forma, foram elaborados dois modelos in vitro da microbiota intestinal para avaliar o efeito da TMIF na microbiota intestinal. Um dos modelos usou culturas puras de estirpes de Lactobacillus e Bifidobacterium em monoculturas e co-culturas (pares e consórcio) para avaliar o efeito da TMIF e a atividade metabólica destas bactérias. Adicionalmente, o efeito direto da farinha nas células bacterianas quando estas se encontram em stress nutritivo também foi avaliado. Posteriormente, a interação de todos os grupos presentes da microbiota intestinal bacteriana foi avaliada a partir de fezes de voluntários humanos, utilizando neste caso TMIF sujeitas ou não a uma pré-digestão. A avaliação da viabilidade celular e atividade metabólica foi realizada e comparada em ambos modelos. No primeiro modelo com culturas puras, não foram observados efeitos negativos da TMIF na viabilidade e no crescimento das bactérias probióticas, ocorrendo um aumento do crescimento e da produção de ácidos gordos de cadeia curta (SCFA) e lactato. Durante o tempo de incubação em stress nutritivo, o número de células bacterianas viáveis foi mantido mostrando que a farinha não apresenta qualquer efeito direto tóxico nas células. No segundo modelo, o modelo in vitro fecal, a TMIF digerida e não digerida demonstrou ter efeitos positivos no crescimento dos grupos bacterianos considerados benéficos (ex. Bacteroidaceae e Prevotellaceae) sem promover o crescimento significativo nos grupos com impacto negativo na saúde humana (ex. Clostridium histolyticum, Desulfovibrionales e Desulfuromonales). A TMIF promoveu uma produção mais elevada de ácidos orgânicos como o acetato e o propionato. Na presença de TMIF a produção de amoníaco foi na gama de concentrações consideradas não citotóxicas. Em relação ao conteúdo de aminoácidos das amostras de TMIF, a forma não digerida apresentou ter maior concentração de aminoácidos totais enquanto que a amostra digerida a maior concentração em aminoácidos livres. Como conclusão, a TMIF pode ser um potencial substituto de carne graças ao seu conteúdo nutricional e ao impacto na microbiota intestinal.Tenebrio molitor insect flour (TMIF) is considered a nutritious food ingredient but still needs assessment of its nutritional potential in the human diet. One of the major indicators of the effect of diet on human health is the composition of the gut microbiota, especially the presence of beneficial bacterial groups, such as probiotic bacteria. In this way, two in vitro models of the gut microbiota were elaborated to evaluate the effect of TMIF on the gut microbiota. One of the models used pure cultures of Lactobacillus and Bifidobacterium strains in monocultures and co-cultures (pairs and consortium) to evaluate the effect of TMIF and the metabolic activity of these bacteria. Additionally, the direct effect of the flour on bacterial cells when they are in nutritive stress was also evaluated. Subsequently, the interaction of all groups present in the bacterial gut microbiota was evaluated from human volunteer faeces, using in this case TMIF subjects with or without pre-digestion. The evaluation of cell viability and metabolic activity was performed and compared in both models. In the first model with pure cultures, no negative effects of TMIF on the viability and growth of probiotic bacteria were observed, with an increase in the growth and production of short chain fatty acids (SCFA) and lactate. During incubation time under nutritional stress, the number of viable bacterial cells was maintained showing that the flour does not have any direct toxic effect on the cells. In the second model, the in vitro faecal model, digested and undigested TMIF have been shown to have positive effects on the growth of bacterial groups considered beneficial (e.g. Bacteroidaceae and Prevotellaceae) without promoting significant growth in groups with a negative impact on human health (e.g. Clostridium histolyticum, Desulfovibrionales and Desulfuromonales). TMIF promoted a higher production of organic acids such as acetate and propionate. In the presence of TMIF the production of ammonia was in the range of concentrations considered non-cytotoxic. Regarding the amino acid content of the TMIF samples, the undigested form presented to have higher concentration of total amino acids while the digested sample the highest concentration of free amino acids. As a conclusion, TMIF may be a potential meat substitute because of its nutritional content and the impact on the intestinal microbiota

Physiological responses of Tetrahymena pyriformis to copper, zinc, cycloheximide and Triton X-100

Protozoa, and particularly ciliates, are essential in aerobic purification processes of wastewaters and have proved to be very sensitive to environmental changes. The physiological response of the ciliate Tetrahymena pyriformis was assessed in terms of mortality, growth and grazing capacity after exposure to four toxicants: copper, zinc, cycloheximide and Triton X-100. In the ranges of concentrations used, mortality, inhibition of growth and inhibition of grazing were observed with all toxicants employed, but in different ways. Copper and zinc showed lower toxicity than observed in other studies with protozoa, though some of the present results are in accordance with those reported by other authors. This supports the importance of the organism tested and the experimental conditions of the bioassays.Instituto de Biotecnologia e Química Fina (IBQF).Fundação para a Ciência e a Tecnologia (FCT) - Praxis XXI - 2/2.1/BIO/1118/95, Praxis XXI - grant BD/5080/95

Mutagenesis and transformation of Aspergillus terreus based on the nitrate reductase pathway

Aspergillus terreus is an efficient producer of different extracellular enzymes which are used in food industries. Thus, we are interested in developing a genetic transformation system based on the nitrate structural gene niaD. In order to develop this system the nitrate assimilation pathway in A. terreus was studied and spontaneous mutants defective in the genes required for nitrate assimilation were obtained on the basis of chlorate resistance. Of particular interest were niaD mutants which failed to grow on nitrate but grew as wild type on other sole nitrogen sources. Afterwards the transformation system was developed for mutants defective in nitrate reductase of A. terreus using pSTA10 vector containing niaD gene from A. niger. The transformation frequency obtained was c.a. 0.5 per ug DNA. The vector appeared to be mitotically stable and Southern hybridization analysis of transformants showed that transformation events occurred by integration into to the recipient genome. To assess the amount of product made by the transformants, nitrate reductase assays were carried out under inducing conditions. Experiments to improved transformation frequency and to characterize the integration are now in progress

Participatory Workshops as a Tool for Building Inclusivity in New Towns in Africa

Africa is currently the fastest-urbanizing continent in the world. As part of this rapid growth, New Towns are increasingly employed by private developers as a means of providing well-serviced urban environments to middle- and upper-income groups. These comprehensively-planned New Towns are often seen in contrast to the perceived ‘chaos’ and ‘congestion’ of large African cities. As a result, two urban forms, the highly controlled New Towns and the unregulated settlements at their edges, engage in complex social and economic exchanges, but remain spatially segregated and socially exclusive. Current research points to the need for an alternative approach to top-down New Town planning in Africa. Participatory workshops are one alternative that can offer planners access to local knowledge that is otherwise difficult to access. This paper explores the potential of short-term reflective, design, and serious gaming workshops by reflecting on the experiences of the authors in four recent workshops. The paper evaluates the effectiveness of these workshops as useful tools to increase inclusivity in African New Towns by bringing together stakeholders with competing agendas and supporting open discussion, negotiation, and informed decision-making. The paper concludes that participation from stakeholder groups that would normally be marginalized from the planning process (such as current residents, temporary users, and residents of adjacent unregulated communities), can offer new insights to planning bodies and inform more inclusive New Towns across the continent

Utilization of mycelial growth to study the tolerance of some white rot fungi to phenolic compounds

Phanerochaete chrysosporium, Aureobasidium pu/lulans, Coriolus versicolor, Pleurotus ostreatus and Dichomitus sqoo/ens were some white-rot fungi selected to study the effect of increasing concentrations of phenol, catechol and resorcinol on the mycelial growth on solid media. The increasing concentrations of the phenolic compounds added to a mineral Czapec-Dox agar medium were progressively inhibitory up to a certain maximum value beyond which growth became impossible. P. ostreatu.s was the most affected fungus by the high concentrations used, following by C. versicolor, while P. chrysosporium, A. pul/ulans, and D. squalens tolerated better the presence of the phenolic compounds. It was observed that the toxicity of these compounds towards the microorganisms progressed in accordance to the following order: resorcinol< phenol<cathecol. Furthermore, the capability of growing at the expense of agar, agarose and gelatine as sole carbon and energy source was demonstrated by all the fungi