Isolation of Talaromyces marneffei from the Skin of an Egyptian Mongoose ( Herpestes ichneumon) in Portugal.

We report a case of Talaromyces marneffei skin infection in an Egyptian mongoose ( Herpestes ichneumon) in Portugal. The isolated fungus was identified through its mycologic characteristics, morphology, and PCR amplification.

The combination of Cl-IB-MECA with paclitaxel: a new anti-metastatic therapeutic strategy for melanoma.

PURPOSE: Metastatic melanoma is considered one of the most aggressive malignant tumours, representing the deadliest form of skin cancer. Melanoma progression is associated with the abrogation of normal controls that limit cell proliferation, migration, and invasion, eventually leading to metastasis. Based on the variety of cellular mechanisms involved in metastatic progression, we aimed to evaluate the effect of inosine (50 µM) and of the combination of Cl-IB-MECA (10 µM) with paclitaxel (10 ng/mL) on several stages of melanoma progression. METHODS: Proliferation, migration, adhesion, invasion, and colony formation assays were performed on human C32 and A375 metastatic melanoma cells. Levels of ERK1/2 were also determined using an ELISA kit. Moreover, mouse aortic rings were treated with vascular endothelial growth factor in order to assess the microvessel sprouting (an indicator of angiogenesis) in the presence of the referred compounds. RESULTS: We demonstrate that inosine induced, through A3 adenosine receptor activation, proliferation, migration, adhesion, and invasion on C32 and A375 melanoma cells, although with dissimilar importance on the two melanoma cell lines. Inosine also increased colony formation on A375 cells. Levels of ERK1/2 were increased after inosine exposure and that increase was dependent on A3 adenosine receptor activation in both cell lines. Moreover, microvessel sprouting stimulated by inosine was decreased by the combination of Cl-IB-MECA with paclitaxel. CONCLUSIONS: Cl-IB-MECA combined with paclitaxel was able to impair almost all of the referred metastatic related mechanisms induced by inosine, making this approach a valuable tool for combinatory therapy against metastatic melanoma.

Combination of Cl­IB­MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR­dependent autophagy and mitotic catastrophe on human melanoma cells.

PURPOSE: Metastatic melanoma is the deadliest form of skin cancer. It is highly resistant to conventional therapies,particularly to drugs that cause apoptosis as the main anticancer mechanism. Recently, induction of autophagic cell death is emerging as a novel therapeutic target for apoptotic-resistant cancers. We aimed to investigate the underlying mechanisms elicited by the cytotoxic combination of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide(Cl-IB-MECA, a selective A(3) adenosine receptor agonist; 10 µM) and paclitaxel (10 ng/mL) on human C32 and A375 melanoma cell lines. METHODS: Cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase leakage assays, after 48-h incubation. Autophagosome and autolysosome formation was detected by fluorescence through monodansylcadaverine-staining and CellLight(®) Lysosomes-RFP-labelling, respectively. Cell nuclei were visualized by Hoechst staining, while levels of p62 were determined by an ELISA kit. Levels of mammalian target of rapamycin (mTOR) and the alterations of microtubule networks were evaluated by immunofluorescence. RESULTS: We demonstrated, for the first time, that the combination of Cl-IB-MECA with paclitaxel significantly increases cytotoxicity, with apoptosis and autophagy the major mechanisms involved in cell death. Induction of autophagy, using clinically relevant doses,was confirmed by visualization of autophagosome and autolysosome formation, and downregulation of mTOR and p62 levels. Caspase-dependent and caspase-independent mitotic catastrophe evidencing micro- and multinucleation was also observed in cells exposed to our combination. CONCLUSIONS: The combination of Cl-IB-MECA and paclitaxel causes significant cytotoxicity on two melanoma cell lines through multiple mechanisms of cell death. This multifactorial hit makes this therapy very promising as it will help to avoid melanoma multiresistance to chemotherapy and therefore potentially improve its treatment.

Potentiation of cytotoxicity of paclitaxel in combination with Cl-IB-MECA in human C32 metastatic melanoma cells: A new possible therapeutic strategy for melanoma.

Metastatic melanoma monotherapies with drugs such as dacarbazine, cisplatin or paclitaxel (PXT) are associated with significant toxicity and low efficacy rates. These facts reinforce the need for development of novel agents or combinatory strategies. Cl-IB-MECA is a small molecule, orally bioavailable, well tolerated and currently under clinical trials as an anticancer agent. Our aim was to investigate a possible combinatory therapeutic strategy using PXT and Cl-IB-MECA on human C32 melanoma cells and its underlying mechanisms. Cytotoxicity was evaluated using MTT reduction, lactate dehydrogenase leakage and neutral red uptake assays, for different concentrations and combinations of both agents, at 24 and 48 h. Apoptosis was also assessed using fluorescence microscopy and through the evaluation of caspases 8, 9, and 3 activities. We demonstrated, for the first time, that combination of PXT and Cl-IB-MECA significantly increases cytotoxicity for clinically relevant concentrations. This combination seems to act synergistically in disrupting membrane integrity, but also causing lysosomal and mitochondrial dysfunction. When using the lowest PTX concentration (10 ng/mL), co-incubation with CI-IB-MECA (micromolar concentrations) potentiated overall cytotoxic effects and morphological signs of apoptosis. All combinations studied enhanced caspase 8, 9, and 3 activities, suggesting the involvement of both intrinsic and extrinsic apoptotic pathways. The possibility that cytotoxicity elicited by Cl-IB-MECA, alone or in combination with PXT, involves adenosine receptor activation was discarded and results confirmed that oxidative stress is only involved in cytotoxicity after treatment with PXT, alone. Being melanoma a very apoptosis-resistance cancer, this combination seems to hold promise as a new therapeutic strategy for melanoma.

Photosynthetic responses of three C4 grasses of different metabolic subtypes to water deficit.

C4 plants are considered to be less sensitive to drought than C3 plants because of their CO2 concentrating mechanism. The C4 grasses, Paspalum dilatatum Poiret (NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK) were compared in their response to water deficit imposed by the addition of polyethylene glycol to the nutrient solution in which they were grown. The effects of drought on leaf relative water content (RWC), net photosynthesis, stomatal conductance, carboxylating enzyme activities and chlorophyll a fluorescence were investigated. In C. dactylon the RWC was more sensitive, but the photosynthetic activity was less sensitive, to water deficit than in P. dilatatum and Z. japonica. The decrease of photosynthesis in P. dilatatum under water deficit was not closely related to the activities of the carboxylating enzymes or to chlorophyll a fluorescence. However, decreased activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase, in addition to decreased stomatal conductance, may have contributed to the decrease of photosynthesis with drought in C. dactylon and Z. japonica. The different responses to water deficit are discussed in relation to the natural habitats of C4 grasses.