RESUMO
The management of fungal diseases imposes an urgent need for the development of effective antifungal drugs. Among new drug candidates are the antimicrobial peptides, and especially their derivatives. Here, we investigated the molecular mechanism of action of three bioinspired peptides against the opportunistic yeasts Candida tropicalis and Candida albicans. We assessed morphological changes, mitochondrial functionality, chromatin condensation, ROS production, activation of metacaspases, and the occurrence of cell death. Our results indicated that the peptides induced sharply contrasting death kinetics, of 6 h for RR and 3 h for D-RR to C. tropicalis and 1 h for WR to C. albicans. Both peptide-treated yeasts exhibited increased ROS levels, mitochondrial hyperpolarization, cell size reduction, and chromatin condensation. RR and WR induced necrosis in C. tropicalis and C. albicans, but not D-RR in C. tropicalis. The antioxidant ascorbic acid reverted the toxic effect of RR and D-RR, but not WR, suggesting that instead of ROS there is a second signal triggered that leads to yeast death. Our data suggest that RR induced a regulated accidental cell death in C. tropicalis, D-RR induced a programmed cell death metacaspase-independent in C. tropicalis, while WR induced an accidental cell death in C. albicans. Our results were obtained with the LD100 and within the time that the peptides induce the yeast death. Within this temporal frame, our results allow us to gain clarity on the events triggered by the peptide-cell interaction and their temporal order, providing a better understanding of the death process induced by them.
Assuntos
Antifúngicos , Candida albicans , Espécies Reativas de Oxigênio/metabolismo , Candida albicans/metabolismo , Antifúngicos/química , Morte Celular , Peptídeos/farmacologia , Peptídeos/metabolismo , Candida tropicalis/metabolismo , Cromatina/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Purpose: Determining the post-mortem interval (PMI) is one of the challenging tasks in forensic science due to the lack of quick and inexpensive methods. Our objective is to develop innovative and alternative means for PMI evaluation. Methods: The relationship between PMI and enzymatic modifications in mice tissues was described. After being sacrificed, Swiss mice were randomly divided into groups according to the time elapsed since death. The activities of catalase (CAT) and δ-aminolevulinate dehydratase (δ-ALA-D) were determined in hepatic, renal, skeletal muscle and cerebral tissues. Results: CAT activity increased in kidney and brain 6 h after death and this increase remained for up to 24 h in the brain and 48 h in the kidney. δ-ALA-D had its activity decreased in the liver and kidneys in 6 h. In the skeletal muscle, δ-ALA-D activity was reduced only 48 h after death. Conversely, an increase on δ-ALA-D activity was observed in the brain at 6 h, followed by its decrease at 24 and 48 h. Conclusion: With the association of this set of results, it is possible to provide an estimate of PMI. Additionally, these results can be used as an auxiliary parameter associated with other methods to estimate PMI.
Assuntos
Catalase , Sintase do Porfobilinogênio , Mudanças Depois da Morte , Animais , Autopsia , Catalase/metabolismo , Cérebro/enzimologia , Ensaios Enzimáticos , Rim/enzimologia , Fígado/enzimologia , Camundongos , Músculo Esquelético/enzimologia , Sintase do Porfobilinogênio/metabolismo , Fatores de TempoRESUMO
Determining precisely the postmortem interval (PMI) is a key parameter for forensic researches, given that various physical, biochemical and metabolic changes begin to occur in the body after death. In the present study, the Na+/K+-ATPase, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were evaluated. For this, male adult Swiss mice were killed by isoflurane inhalation anesthesia and divided into four groups according to time of death (0, 6, 24 and 48â¯h). The brain, liver, kidney and skeletal muscle tissues were removed. Our results revealed that at the time of 6â¯h, there was a decrease on Na+/K+-ATPase and GST activities in the brain and liver tissues, respectively. In addition, at this time point, an increase on renal GST activity was verified. At the time of 24â¯h, an increase on the cerebral AChE and renal GST activities was observed, while the cerebral Na+/K+-ATPase activity was decreased. Forty-eight hours after death, cerebral Na+/K+-ATPase and renal GST activities remained decreased and increased, respectively. In addition, no alteration was observed on the GST activity in the skeletal muscle and brain (in PMIs evaluated). The present study revealed that the brain and kidney (at the times of 24 and 48â¯h) were the tissues that suffered the most changes in almost all the enzymes evaluated. Our results demonstrated that enzyme activity assessments are reliable, easy-to-perform and low-cost determinations, and could be promising postmortem markers.
Assuntos
Acetilcolinesterase/metabolismo , Biomarcadores/metabolismo , Encéfalo/enzimologia , Medicina Legal/métodos , Glutationa Transferase/metabolismo , Rim/enzimologia , Fígado/enzimologia , Músculo Esquelético/enzimologia , Mudanças Depois da Morte , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Masculino , Camundongos , Fatores de Tempo , Distribuição TecidualRESUMO
This paper describes for the first time the use of paper-based analytical devices at crime scenes to estimate the post-mortem interval (PMI), based on the colorimetric determination of Fe2+ in vitreous humour (VH) samples. Experimental parameters such as the paper substrate, the microzone diameter, the sample volume and the 1,10-phenanthroline (o-phen) concentration were optimised in order to ensure the best analytical performance. Grade 1 CHR paper, microzone with diameter of 5 mm, a sample volume of 4 µL and an o-phen concentration of 0.05 mol/L were chosen as the optimum experimental conditions. A good linear response was observed for a concentration range of Fe2+ between 2 and 10 mg/L and the calculated values for the limit of detection (LOD) and limit of quantification (LOQ) were 0.3 and 0.9 mg/L, respectively. The specificity of the Fe2+ colorimetric response was tested in the presence of the main interfering agents and no significant differences were found. After selecting the ideal experimental conditions, four HV samples were investigated on paper-based devices. The concentration levels of Fe2+ achieved for samples #1, #2, #3 and #4 were 0.5 ± 0.1, 0.7 ± 0.1, 1.2 ± 0.1 and 15.1 ± 0.1 mg/L, respectively. These values are in good agreement with those calculated by ICP-MS. It important to note that the concentration levels measured using both techniques are proportional to the PMI. The limitation of the proposed analytical device is that it is restricted to a PMI greater than 1 day. The capability of providing an immediate answer about the PMI on the crime scene without any sophisticated instrumentation is a great achievement in modern instrumentation for forensic chemistry. The strategy proposed in this study could be helpful in many criminal investigations.