Proteomic analysis of aquatic microbial responses to nanoparticulate and ionic silver

Environmental biomarkers are the most promising next generation risk assessment tools, augmenting measurements of direct and highly sensitive responses at the cellular and sub-cellular levels. Silver nanoparticles (AgNPs) are among the mostly used nanoparticles and likely to be released in significant amounts to aquatic environments. Due to their antimicrobial properties, it is relevant to examine whether AgNPs can pose a risk to aquatic microbes in natural ecosystems. We used a bacterial strain, Pseudomonas sp. M1 (PsM1), isolated from sediments in a metal-polluted stream, to gain insights into the molecular mechanisms underlying its ability to deal with the toxic effects of AgNPs using a proteomic approach. We identified changes in the protein expression at AgNP concentrations inhibiting biomass production in 20% (EC20). After SDS-PAGE, the LC-MS/MS identified almost 200 proteins, about 50% of which increased its abundance under stress induced by AgNPs and Ag. Silver is known to react with proteins by combining with the thiol groups of enzymes, leading to protein inactivation. After AgNPs exposure, some of the upregulated proteins were associated with the degradation of transiently denatured and unfolded proteins, accumulated in the periplasm under stress conditions (e.g. periplasmic serine endoprotease). Exposure to AgNPs also induced proteins related to stress response, in particular, antioxidant enzymes, such as catalase-peroxidase and superoxide dismutase. The antioxidant response was consistent with our previous work suggesting that the ability to initiate an efficient antioxidant response is essential for the bacterium to cope with AgNP toxicity. We also found an increase in the proteins involved in amino acid (e.g. ornithine carbamoyltransferase) and energy metabolism (e.g. fructose-bisphosphate aldolase), which may reveal an AgNP-induced reorganization of the metabolic fluxes, that is compatible with an increased need of the bacterial cells to generate energy to support the defense mechanisms against AgNPs toxicity. An increased amount of chaperones (e.g. chaperone protein ClpB) was also found. These proteins play an essential role in the cell by assisting the correct folding of nascent and stress accumulated misfolded proteins and preventing their aggregation. AgNPs can likewise interact with elements of bacterial membranes, causing structural changes, dissipation of the proton motive + force, which is consistent with the increase in a specific porin with serine protease activity. Overall, PsM1’s response to the stress induced by AgNPs involved, among others, stress response proteins, proteins of the energy metabolism and transport proteins. Since the risk of the appearance of bacterial strains with augmented silver resistance is growing, it is highly recommended that the knowledge obtained from PsM1’s response to AgNPs be considered in future studies

Study of the dimorphic fungus Paracoccidioides brasiliensis: development of molecular tools and morphology evaluation

Dissertação de mestrado em Genética MolecularParacoccidioides brasiliensis, a thermal dimorphic fungus, is the etiological agent of paracoccidioidomycosis, the most common systemic mycosis affecting the rural population of Latin America. During the last decade, molecular approaches have allowed a broader insight into P. brasiliensis. However, knowledge regarding fundamental biology of this fungus has been greatly hampered by the absence of appropriate genetic tools. We present results concerning the development of a strategy for gene targeted mutagenesis that could increase the probability of homologous recombination (HR) in P. brasiliensis, using antisense RNA (aRNA) technology to down-regulate the KU80 gene, a key component of the nonhomologous end joining (NHEJ) pathway. However, the aRNAs developed within this work were not able to downregulate PbKU80’s expression, thus other targeted sequences should be employed. The present work also focuses on the analysis of different morphological features (mother cell size and form; bud number, size and form) and possible correlations that might exist among them, and between the expression of the Rholike GTPase Cdc42 (Pbcdc42p), a pivotal molecule in cellular division and shape. We used real-time PCR (RT-PCR) to determine PbCDC42 transcript levels and stereomicroscopy (by contour measurements of 150 mother cells and buds) for morphological analysis of exponentially growing batch culture yeast cells from the 3 described cryptic species (S1, PS2, and PS3), using both clinical and environmental isolates. We show that cell size and cell form of both the mother and bud cells are very heterogeneous both within the same strain and among the strains. Regarding bud number per mother cell, most strains showed no predominance or budding pattern. Moreover, no associations were detected between mother cell and bud cell size and form, suggesting that P. brasiliensis yeast cells do not obey the critical mass rule between mother and daughter cells conversely to other yeast cells like Saccharomyces cerevisiae. Interestingly, expression of PbCDC42, although heterogeneous among the groups of the 3 cryptic species, seems to show a negative correlation with bud number in the tested strains, but not with any of the other analyzed morphological features. Altogether, this study provides a quantitative assessment of morphological traits of P. brasiliensis yeast cells, supporting the hypothesis that P. brasiliensis does not follow standard rules of cell growth.Paracoccidioides brasiliensis, um fungo termo-dimórfico, é o agente etiológico da paracoccidioidomicose, a micose sistémica mais comum que afecta a população rural da América Latina. Durante a última década, abordagens moleculares têm permitido uma visão mais ampla sobre P. brasiliensis. No entanto, o conhecimento acerca da biologia fundamental deste fungo tem sido dificultado pela ausência de ferramentas moleculares adequadas. Neste trabalho apresentamos resultados relativos ao desenvolvimento de uma estratégia para mutagénese dirigida que poderia aumentar a probabilidade de recombinação homóloga (RH) em P. brasiliensis, aplicando a tecnologia de RNA antisense (aRNA) para diminuir a expressão do gene KU80, um interveniente chave na via nonhomologous end joining (NHEJ). No entanto, as moléculas antisense desenvolvidas no âmbito deste trabalho não foram capazes de reduzir a expressão de PbKU80, assim, outras sequências antisense deverão ser desenvolvidas. O presente trabalho também se debruçou na análise de diferentes características morfológicas (tamanho e forma da célula mãe; número, tamanho e forma das gémulas) e possível correlação que possa existir entre elas, e entre a expressão da Rho-like GTPase CDC42 (Pbcdc42p), uma molécula crucial na forma e divisão celulares. Usámos a técnica de PCR em tempo real para determinar os níveis de expressão de PbCDC42 e a estereomicroscopia (por medição do contorno de 150 células mãe e suas gémulas) para a análise morfológica de células de levedura em crescimento exponencial pertencentes a 3 espécies crípticas descritas (S1, PS2 e PS3), usando tanto isolados clínicos como ambientais. Mostrámos que tanto o tamanho como a forma de ambas as células mãe e respectivas gémulas são muito heterogéneos, tanto para a mesma estirpe, como entre estirpes diferentes. Quanto ao número de gémulas por célula mãe, na maioria das estirpes não se verificou qualquer predominância ou padrão de gemulação. Além disso, não foram encontradas associações entre o tamanho e a forma das células mãe e respectivas gémulas, sugerindo que as células leveduriformes de P. brasiliensis não obedecem ao modelo ideal de gemulação exibido entre célula mãe e gémula, ao contrário de outras leveduras como Saccharomyces cerevisiae. Curiosamente, a expressão de PbCDC42, embora heterogénea entre as 3 espécies crípticas, parece apresentar uma correlação negativa com o número de gémulas nas estirpes testadas, mas não com qualquer das outras características morfológicas analisadas. Em suma, este estudo faculta uma avaliação quantitativa das características morfológicas das células leveduriformes de P. brasiliensis, apoiando a hipótese de que este fungo dimórfico não segue as regras standard do crescimento celular.O trabalho apresentado foi desenvolvido no âmbito do projecto PTDC/BIA-MIC/108309/2008 - “Unraveling the specific involvement of the small Rho-like GTPase Cdc42 in the highly polymorphic nature of Paracoccidioides brasiliensis yeast cells

Proteomic responses to nanoparticulate and ionic silver in fungi from metal-polluted and non-polluted streams

Enhanced production and usage of silver nanoparticles (AgNPs) raise concerns about their potential impacts in aquatic ecosystems. Effects of AgNPs and Ag+ were assessed based on the variations in the overall proteome and the activities of selected antioxidant enzymes in two fungal strains of Articulospora tetracladia, one isolated from a non-polluted stream (At72) and the other from a metal-polluted stream (At61). For that, fungi were exposed to concentrations of AgNPs and Ag+ affecting 20% of growth (EC20). A total of 432 proteins were identified, of which 172 belonged to At72 and 260 to At61; 71 were expressed in both strains. At72 had 58% and 52% of the proteins induced by AgNPs and Ag+, respectively. For At61, the percentages were higher ( ˜77%). The higher percentages of proteins suppressed by either Ag forms suggest higher stress in At72 than in At61, which is consistent with the background of this fungal strain. Major groups of proteins were related to carbohydrate metabolism, amino-acid and protein biosynthesis. Both Ag forms also induced stress-responsive proteins, including catalase and superoxide dismutase which remained consistent with the profile of their enzymatic activities. These results supported the ability of these fungi in initiating an efficient antioxidant response to cope with Ag-induced toxicity. Overall, the functional proteomic approach can be useful to get a mechanistic insight on the stress induced by AgNPs or Ag+ in aquatic fungi that play a key role in plant litter decomposition in stream

Proteomic responses to nanoparticulate and ionic silver in aquatic fungi

Enhanced use of silver nanoparticles (AgNPs) has inevitably resulted in their release into aquatic environments raising concern about the risk to aquatic biota and related ecological functions. Functional proteomics is an emerging technology that provides high-throughput analyses augmenting measurements of direct and highly sensitive responses at the cellular and sub-cellular levels. The impacts of AgNPs and its ionic precursor (Ag+ in AgNO3) at low exposure concentrations (close to environmental realism) on a fungal strain isolated from a non-polluted stream were assessed based on the variations in the overall proteome as well as in the activity of selected antioxidant enzymes. A total of 352 proteins were identified, but only 151 proteins were responsive (significantly up- or down-regulated relative to control) of which 65% presented matching alterations. Out of these 151 proteins, 62% increased abundance under stress induced by AgNPs and 56% under stress induced by Ag+. Exposure to both forms of silver induced proteins related to stress response, in particular, antioxidant enzymes. The antioxidant enzymatic responses were consistent with the proteomic responses, suggesting that the ability to initiate an efficient antioxidant response is essential for the fungus to cope with Ag-induced toxicity. Moreover, several proteins involved in the metabolism of carbohydrates, amino acids and lipids were altered. This evidence may reflect the need of generating energy to support the cellular defense mechanisms. Some of the significantly altered proteins were associated with the correct folding of nascent and stress accumulated misfolded proteins or degradation of transiently denatured and unfolded proteins preventing their aggregation. Others were related to the regulation of translation suggesting a compromised protein synthesis system. Overall, the functional proteomic approach can be useful to expand the knowledge on silver-induced stress responses in aquatic fung

Proteomic responses to nanoparticulate and ionic silver in freshwater microbes with different background

ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by the Portuguese Foundation for Science and Technology (FCT) I.P. through the strategic funding UID/BIA/04050/2014 (POCI-01-0145-FEDER-007569) and PTDC/AAC-AMB/121650/201

Omics reveal distinct mechanisms of toxicity of nanoparticulate and ionic silver in microbes

The mechanisms of toxicity of silver nanoparticles (AgNPs) are not clear and the role of Ag+ released from the nanoparticulated form in the overall toxicity requires further attention, especially in aquatic ecosystems where NPs will most likely end up. The impacts of AgNPs and Ag+ were assessed based on the variations in the overall proteome in i) two aquatic fungal ecotypes of Articulospora tetracladia, one isolated from a non-polluted stream (At72) and the other from a metal-polluted stream (At61), and ii) the bacterial strain Pseudomonas sp. M1 (PsM1) isolated from a metal-polluted stream. Transcriptomic responses of At72 in the same exposure conditions were also assessed to complement knowledge from proteomic responses. At72 was the most sensitive to AgNPs, whereas PsM1 was the most tolerant one. The toxicity of AgNPs was supported by data from NP characterization, which showed increased particle stability and lesser agglomeration in presence of At72 that the other microbes. Omic responses to equitoxic levels of AgNPs and Ag+ suggested different mechanisms of toxicity since distinct profiles of protein and gene expression were unveiled. Gene Ontology (GO) enrichment analysis further unravelled several biological processes and allowed to differentiate the effects of AgNPs from those of Ag+. Overall, omic approaches revealed different adaptive responses to Ag+ or AgNPs in the metabolic, energetic and stress pathways. Furthermore, the negligible amount of Ag+ released from AgNPs suggested that toxicity of AgNPs was mainly associated with the particle for

Effects of ethanol and phenanthrene on litter decomposition by aquatic fungal assemblages

Fungi, particularly aquatic hyphomycetes, have been recognized as playing a dominant role in microbial decomposition of plant litter in streams. In this study, we used a microcosm experiment with monocultures and combinations of mixed cultures with up to five aquatic hyphomycete species (Articulospora tetracladia, Tricladium splendens, Heliscus submersus, Tetrachaetum elegans and Flagellospora curta) to assess the potential effect of ethanol, phenanthrene and fungal diversity (species number and identity) on three functional measures: litter decomposition, fungal biomass accrual and sporulation. Alder leaf disks were conditioned by fungi for 7 days and then were exposed to ethanol (0,07%) and/or phenanthrene (1 mg/L) for further 24 days. Exposure to ethanol or phenanthrene decreased both leaf mass loss and fungal sporulation, but increased fungal biomass. All aspects of fungal activity increased with increasing number of fungal species. Fungal activity in mixed cultures was generally higher than expected from the weighted performances of participating species in monoculture, suggesting complementarity between species. However, the activity of fungi in mixed cultures did not exceed the activity of the most productive species in the absence or presence of ethanol and/or phenanthrene.Fundação para a Ciência e a Tecnologia (FCT): POCI/MAR/56964/2

A IMPORTÂNCIA DO ENFERMEIRO NOS SERVIÇOS DE HEMODINÂMICA: UMA REVISÃO INTEGRATIVA

No início do século XX as doenças cardiovasculares provocavam 10% dos óbitos no mundo. Com o objetivo de melhorar a estratégia de tratamento dessas doenças surgem procedimentos com resultados favoráveis, os quais estão inclusos na cardiologia intervencionista, a qual é desenvolvida em Unidades de Hemodinâmica, sendo eles: a angioplastia primária e o emprego de stents. A experiência do médico e de toda a equipe de enfermagem e radiologia é um fator essencial para o resultado obtido nos procedimentos empregados na cardiologia intervencionista. O presente artigo consiste em uma revisão integrativa, no qual tem como objetivo discorrer acerca da importância do enfermeiro nos serviços de hemodinâmica, com o intuito de conscientizar os estudantes e profissionais da área acerca da sua importância nesse setor. Trata-se de uma revisão de literatura do tipo integrativa, na qual foi realizada uma pesquisa bibliográfica, nas bases de dados da Biblioteca Virtual da Saúde: Sistema Latino Americano e do Caribe de informação em Ciências da Saúde e na Biblioteca Eletrônica Científica Online, acerca do tema em estudo. A atuação da Enfermagem no serviço de hemodinâmica cardíaca é de suma importância, visto que tem como objetivo um melhor planejamento do cuidado por meio da Sistematização da Assistência de Enfermagem. Em suma, é possível concluir que as Unidades de Hemodinâmica exigem do enfermeiro conhecimentos referentes à administração e à assistência ao indivíduo, estendendo-se à família e à comunidade

MAMMALS IN PORTUGAL : A data set of terrestrial, volant, and marine mammal occurrences in P ortugal

Mammals are threatened worldwide, with 26% of all species being includedin the IUCN threatened categories. This overall pattern is primarily associatedwith habitat loss or degradation, and human persecution for terrestrial mam-mals, and pollution, open net fishing, climate change, and prey depletion formarine mammals. Mammals play a key role in maintaining ecosystems func-tionality and resilience, and therefore information on their distribution is cru-cial to delineate and support conservation actions. MAMMALS INPORTUGAL is a publicly available data set compiling unpublishedgeoreferenced occurrence records of 92 terrestrial, volant, and marine mam-mals in mainland Portugal and archipelagos of the Azores and Madeira thatincludes 105,026 data entries between 1873 and 2021 (72% of the data occur-ring in 2000 and 2021). The methods used to collect the data were: live obser-vations/captures (43%), sign surveys (35%), camera trapping (16%),bioacoustics surveys (4%) and radiotracking, and inquiries that represent lessthan 1% of the records. The data set includes 13 types of records: (1) burrowsjsoil moundsjtunnel, (2) capture, (3) colony, (4) dead animaljhairjskullsjjaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8),observation in shelters, (9) photo trappingjvideo, (10) predators dietjpelletsjpine cones/nuts, (11) scatjtrackjditch, (12) telemetry and (13) vocalizationjecholocation. The spatial uncertainty of most records ranges between 0 and100 m (76%). Rodentia (n=31,573) has the highest number of records followedby Chiroptera (n=18,857), Carnivora (n=18,594), Lagomorpha (n=17,496),Cetartiodactyla (n=11,568) and Eulipotyphla (n=7008). The data setincludes records of species classified by the IUCN as threatened(e.g.,Oryctolagus cuniculus[n=12,159],Monachus monachus[n=1,512],andLynx pardinus[n=197]). We believe that this data set may stimulate thepublication of other European countries data sets that would certainly contrib-ute to ecology and conservation-related research, and therefore assisting onthe development of more accurate and tailored conservation managementstrategies for each species. There are no copyright restrictions; please cite thisdata paper when the data are used in publications.info:eu-repo/semantics/publishedVersio