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Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report thatput1-/-andput2-/- strains, carrying defectiveProlineUTilization genes, display remarkable proline sensitivity withput2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate P5C, which inhibits mitochondrial respiration. Theput1-/- andput2-/-mutations attenuate virulence inDrosophilaand murine candidemia models. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages ofC. albicanscells colonizing a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not ofput2-/-cells. Multiple members of theCandidaspecies complex, includingC. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

Neutrophils are evolutionarily conserved innate defense cells implicated in diverse pathological processes. Zebrafish models have contributed substantially to our understanding of neutrophil functions, but similarities to human neutrophil maturation have not been characterized limiting applicability to study human disease.We generated transgenic zebrafish strains to distinguish neutrophil maturation gradesin vivoand established a high-resolution transcriptional profile of neutrophil maturation. We linked gene expression at each stage to characteristic transcription factors, including C/ebpβ, important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human confirmed high molecular similarity in immature stages and discriminated zebrafish-specific from pan-species gene signatures. Applying pan-species neutrophil maturation signatures in RNA-seq data from neuroblastoma patients revealed an association of metastatic tumor cell infiltration in the bone marrow with an increase in mature neutrophils.Our detailed neutrophil maturation atlas provides a valuable resource for studying neutrophil function at different stages across species in health and disease.Graphical abstract

Echinocandins such as caspofungin are frontline antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by upregulation of chitin synthesis, thereby sustaining cell wall integrity (CWI). When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a subpopulation of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable supra-high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway. This article has an associated First Person interview with the first author of the paper., Summary: The pathogenic fungus Candida can limit the overproduction of chitin in its cell wall to prevent loss of fitness via a calcineurin-dependent mechanism.

Polymicrobial infections are of paramount importance because of the potential severity of clinical manifestations, often associated with increased resistance to antimicrobial treatment. The intricate interplay with the host and the immune system, and the impact on microbiome imbalance, are of importance in this context. The equilibrium of microbiota in the human host is critical for preventing potential dysbiosis and the ensuing development of disease. Bacteria and fungi can communicate via signaling molecules, and produce metabolites and toxins capable of modulating the immune response or altering the efficacy of treatment. Most of the bacterial−fungal interactions described to date focus on the human fungal pathogen Candida albicans and different bacteria. In this review, we discuss more than twenty different bacterial−fungal interactions involving several clinically important human pathogens. The interactions, which can be synergistic or antagonistic, both in vitro and in vivo, are addressed with a focus on the quorum-sensing molecules produced, the response of the immune system, and the impact on clinical outcome.

Immunodetection is described in this chapter as a technique for producing specific antibodies for antigen detection of the major human fungal pathogens. In the case of Candida spp., heat-killed cells are used to immunize mice over a couple of weeks and then splenocytes are isolated and further fused with myelomas to easily propagate the antibodies produced in the mice. The resulting antibodies follow a purification process where antibody levels and concentrations are determined. Fungal cells are also lysed to obtain whole cell extracts as a prior step for identification of antigens using immunoprecipitation. Finally, this method permits the production of specific antibodies against fungi and the identification of the respective antigens in an in vivo model.

Immunodetection is described in this chapter as a technique for producing specific antibodies for antigen detection of the major human fungal pathogens. In the case of Candida spp., heat-killed cells are used to immunize mice over a couple of weeks and then splenocytes are isolated and further fused with myelomas to easily propagate the antibodies produced in the mice. The resulting antibodies follow a purification process where antibody levels and concentrations are determined. Fungal cells are also lysed to obtain whole cell extracts as a prior step for identification of antigens using immunoprecipitation. Finally, this method permits the production of specific antibodies against fungi and the identification of the respective antigens in an in vivo model.

Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays.

O presente estudo tem como fundamento a crescente importância das competências transversais e a alteração do mercado de trabalho quanto à sua procura e necessidades. Atualmente, as competências específicas estão a perder lugar para as competências transversais, sendo que nos processos de recrutamento e seleção estas são cada vez mais valorizadas. Assim, o estudo pretende compreender se os estudantes e os empregadores estão alinhados com o tipo de competências transversais consideradas importantes, entender o valor atribuído pelos formandos às competências transversais e perceber se consideram que os estabelecimentos de ensino superior desenvolvem este tipo de competências. A amostra do presente estudo foi constituída por 295 estudantes do ensino superior, aos quais foi aplicado um inquérito por questionário via online, de modo a obter o anonimato das respostas e chegar a um número alargado de pessoas. Os formandos inquiridos consideram que têm um bom desenvolvimento das competências transversais e que o ensino superior teve uma contribuição razoável para esse domínio. Além disso, tanto os empregadores como os estudantes consideram as competências transversais uma dimensão importante para o bom desempenho profissional, sendo que os formandos revelam como as mais essenciais a autonomia, a persistência e a resolução de problemas. The following study bases itself around the increase in relevance given to soft skills, and the shifts in the demand and supply of the labor market. Currently hard skills are giving way to soft skills, making them each time more relevant in a recruitment and selection scenarios. The study aims to have a better understanding to which soft skills two separate groups consider to be the most important, understand the value given to soft skills by students, and their opinion on whether or not these soft skills are developed in their higher learning facility. The sample consisted in the opinion of 295 college students, the inquiries were all filled online in order to protect the subjects anonymity and to reach a broad number of students. Based on the inquires students believe to have well developed soft skills, and that their college had a reasonable contribution on their domain of the same. Both employers and students believe that soft skills are important to increase their job performance, students valued autonomy, perseverance and problem solving to be the most.

Uma série de diretrizes foram impostas ao Sistema de Ensino S uperior Português resultantes do Processo de Bolonha. A execução das linhas mestras de uma nova agenda reformista pluralizou inúmeros desafios às Instituições Portuguesas de Ensino Superior. A presente investigação explora a perspetiva dos diplomados da Business School do ISCTE- IUL sobre o desenvolvimento de competências transversais. Para o efeito, delinearam - se como principais objetivos do estudo apurar o nível de desenvolvimento das competências transversais que os diplomados da Business School do ISCTE - IUL consideram ter, saber qual a importância atribuída pelos diplomados da Business School do ISCTE- IUL às competências e i dentificar a importância atribuída pelos diplomados da Business School ao ISCTE- IUL no desenvolvimento de competências transversais. A amostra foi constituída por 107 diplomados da Business School do ISCTE- IUL que terminaram as licenciaturas entre os anos le tivos 2009/2010 e 2012/2013, aos quais foi aplicado um inquérito por questionário. Os principais resultados da investigação indicam que os diplomados consideram ter um nível de desenvolvimento das competências transversais bastante razoável, assim como as consideram bastante importantes para o seu desempenho profissional. Relativamente ao ISCTE - IUL, na ótica dos diplomados, é de sua responsabilidade desenvolver competências transversais nos seus alunos e, de forma geral, existe reconhecimento de que isso efetivamente acontece. Sugere- se que o ISCTE- IUL tome medidas ao nível da alteração das metodologias pedagógicas dos docentes e que futuramente se jam revistos modelos mistos, baseados no trabalho ou inseridos/integrados para implementação. From the Bologna Process, a lot of guidelines were imposed to the Portuguese Higher Educational System. Executing the orientation lines for the new calendar created several challenges to the Higher Superior Institutions in Portugal. This investigation explores the perspectives of graduates from ISCTE - IUL Business School regarding the development of transferable competencies. Th e main goals of this study were get a grasp on the level of development the graduates of ISCTE - IUL Business School believe they possess on transferable competencies, to know the importance given by these sam e graduates to the competencies and to identify the importance given by these same graduates to the ISCTE- IUL in develop transferable competencies. The sample constitutes 107 graduate s from ISCTE - IUL Business School that finished their degrees between 2009/2010 and 2012/2013, to whom was given a survey. The investigation main results indicate that graduates believe they have pretty reasonable level of transferable competencies, as well as consider these as very important to their professional performance. Regarding ISCTE- IUL, from the graduates perspective, it is its responsibility to develop transferable competencies in its students and, in a gross mode, there is the acknowledgment tha t that does happen. It is suggested that ISCTE- IUL should adopt different strategies regarding the teaching metho ds of their professors and that in the future they can look into mixed models, based on work or integrating for implementation.