Untitled

Funder

Organizational Unit

Publications

Evaluation of methanol preservation for molecular and morphological studies in cyanobacteria using Planktothrix agardhii

Publication . Churro, Catarina; Valério, Elisabete; Vieira, Luís; Pereira, Paulo; Vasconcelos, Vitor

Molecular studies on cyanobacteria often involve filtering and freezing of samples leading to loss of cell morphological features. Methanol is often used in preservation of biological materials in association with other fixatives. This study evaluates the application of methanol in the preservation of DNA for molecular studies as well as for the preservation of cell morphology for morphometric analysis in filamentous cyanobacteria. In the present study, both culture and environmental bloom samples were preserved using a cold methanol dehydration series (50, 70, and 100 %) and stored at −20 °C for up to 2 years. The DNA quantity and quality, nucleotide sequence retrieval, and real-time PCR quantification were analyzed over time. Morphometric cell analysis was performed on preserved samples. Results show that the DNA extracted from samples preserved up to 6 months was successfully quantified by real-time PCR. After that period, the DNA quantity decreased with the preservation time. Nevertheless, we were able to detect/amplify the target fragment in samples preserved up to 2 years. The DNA sequence and cell morphology were also maintained during the preservation time. Thus, methanol preservation is an adequate method to preserve molecular information and morphological features after long storage periods.

2015-10-29Journal article

Restricted access

Proteomic and Real-Time PCR analyses of Saccharomyces cerevisiae VL3 exposed to microcystin-LR reveals a set of protein alterations transversal to several eukaryotic models

Publication . Valério, Elisabete; Campos, Alexandre; Osório, Hugo; Vasconcelos, Vitor

Some of the most common toxins present in freshwater, in particular microcystins (MCs), are produced by cyanobacteria. These toxins have a negative impact on human health, being associated with episodes of acute hepatotoxicity and being considered potentially carcinogenic to humans. To date the exact mechanisms of MC-induced toxicity and tumor promotion were not completely elucidated. To get new insights underlying microcystin-LR (MCLR) molecular mechanisms of toxicity we have performed the proteomic profiling using two-dimensional electrophoresis and MALDI-TOF/TOF of Saccharomyces cerevisiae cells exposed for 4 h-1 nM and 1 μM of MCLR, and compared them to the control (cells not exposed to MCLR). We identified 14 differentially expressed proteins. The identified proteins are involved in metabolism, genotoxicity, cytotoxicity and stress response. Furthermore, we evaluated the relative expression of yeast's PP1 and PP2A genes and also of genes from the Base Excision Repair (BER) DNA-repair system, and observed that three out of the five genes analyzed displayed dose-dependent responses. Overall, the different proteins and genes affected are related to oxidative stress and apoptosis, thus reinforcing that it is probably the main mechanism of MCLR toxicity transversal to several organisms, especially at lower doses. Notwithstanding these MCLR responsive proteins could be object of further studies to evaluate their suitability as biomarkers of exposure to the toxin.

2016-03-15Journal article

Restricted access