Calcium sequestration by fungal melanin inhibits calcium-calmodulin signalling to prevent LC3-associated phagocytosis.

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca2+ signalling pathway that depends on intracellular Ca2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca2+-CaM signalling in aspergillosis. Finally, we demonstrate that Ca2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca2+-CaM signalling to inhibit LAP. These findings reveal the important role of Ca2+-CaM signalling in antifungal immunity and identify an immunological function of Ca2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.

Analytical speciation of mercury in fish tissues by reversed phase liquid chromatography-inductively coupled plasma mass spectrometry with Bi(3+) as internal standard.

In this work, the quantification of two mercury species (Hg(2+) and CH(3)Hg(+)) in fish tissues has been revisited. The originality of our approach relies on the use of Bi(3+) as internal standard (IS) and on the modification of typical extraction conditions. The IS (125 microl, 1000 microg l(-1) Bi(3+)) was added to the aliquot of fresh fish tissue (400-500 mg). A high-speed blender and ultrasound-assisted homogenization/extraction was carried out in the presence of perchloric acid (1.5 ml, 0.6 mol l(-1)), l-cysteine (500 microl, 0.75 mol l(-1)) and 500 microl toluene:methanol (1:1). Perchloric acid was used for protein denaturation and precipitation, toluene helped to destroy lipid structures potentially sequestering CH(3)Hg(+), L-cysteine was used to form water-soluble complexes with Bi(3+), Hg(2+) and CH(3)Hg(+). The excess of perchloric acid was eliminated by addition of potassium hydroxide (pH 5 with acetic acid). The obtained extract, was diluted with the mobile phase (1:1) and introduced (20 microl) to the reversed phase HPLC-ICP-MS system. The separation was achieved by isocratic elution (2.5 mmol l(-1) cysteine, 12.5 mmol l(-1) (NH(4))(2)HPO(4), 0.05% triethylamine, pH 7.0:methanol (96:4)) at a flow rate 0.6 ml min(-1). Column effluent was on-line introduced to ICP-MS for specific detection of (202)Hg, (200)Hg and (209)Bi. Analytical signal was defined as the ratio between (202)Hg/(209)Bi peak areas. The detection limits evaluated for Hg(2+) and CH(3)Hg(+) were 0.8 and 0.7 microg l(-1). Recovery of the procedure, calculated as the sum of species concentrations found in the sample with respect to total ICP-MS-determined Hg was 91.9% for king mackerel muscle and 89.5% for red snapper liver. In the standard addition experiments, the recovery results were 98.9% for Hg(2+) and 100.6% for CH(3)Hg(+). It should be stressed that the use of Bi(3+) as IS enabled to improve analytical performance by compensating for incomplete extraction and for imprecision of sample handling during relatively non-rigorous protocol.

Effect of some heavy metals and soil humic substances on the phytochelatin production in wild plants from silver mine areas of Guanajuato, Mexico.

Phytochelatins (PCs) were determined in the wild plants, focusing on their relationship with the levels of heavy metals and humic substances (HS) in soil. Ricinus communis and Tithonia diversifolia were collected from several sites in Guanajuato city (Mexico), which had long been the silver and gold mining center. The analysis of PCs in root extracts was carried out by liquid chromatography (derivatization with monobromobimane). Total Ag, Cd, Cu and Pb in plant roots and in soil samples, as well as soil HS were determined. The association of metals with HS in soils was evaluated by size exclusion chromatography (SEC) with UV and ICP-MS detection. The results obtained revealed the induction of PCs in R. communis but not in T. diversifolia. The levels of Cd and Pb in plant roots presented strong positive correlation with PC-2 (r=0.9395, p=0.005; r=0.9573, p=0.003, respectively), indicating that these two metals promote PCs induction in R. communis. On the other hand, the inverse correlation was found between soil HS and metal levels in roots of R. communis (Cu>Pb>Cd>Ag), in agreement with the decreasing affinity of these metals to HS. Importantly, the inverse correlation between soil HS and plant PC-2 was observed (r=-0.7825, p=0.066). These results suggest that metals strongly bound to HS could be less bioavailable to plants, which in turn would limit their role in the induction of PCs. Indeed, the SEC elution profiles showed Pb but not Cd association with HS and the correlation between metal in soil and PC-2 in plant was statistically significant only for Cd (r=0.7857, p=0.064). Based on these results it is proposed that the role of heavy metals in PCs induction would depend on their uptake by R. communis, which apparently is controlled by the association of metals with soil HS. This work provides further evidence on the role of environmental conditions in the accumulation of heavy metals and phytochelatin production in plants.