The Protective Role of 1,8-Dihydroxynaphthalene-Melanin on Conidia of the Opportunistic Human Pathogen Aspergillus fumigatus Revisited: No Role in Protection against Hydrogen Peroxide and Superoxides.

Previously, 1,8-dihydroxynaphthalene (DHN)-melanin was described to protect Aspergillus fumigatus against hydrogen peroxide (H2O2), thereby protecting this opportunistic human pathogen from reactive oxygen species generated by the immune system. This was based on the finding that the ATCC 46645 mutant with mutations in the pksP gene of the DHN-melanin synthesis pathway showed increased sensitivity to reactive oxygen species compared to the wild type. Here, it is shown that deletion of the pksP gene in A. fumigatus strain CEA10 did not affect sensitivity for H2O2 and superoxide in a plate stress assay. In addition, direct exposure of the dormant white conidia of the pksP deletion strains to H2O2 did not result in increased sensitivity. Moreover, complementation of the ATCC 46645 pksP mutant strain with the wild-type pksP gene did result in pigmented conidia but did not rescue the H2O2-sensitive phenotype observed in the plate stress assay. Genome sequencing of the ATCC 46645 pksP mutant strain and its complemented strain revealed a mutation in the cat1 gene, likely due to the UV mutagenesis procedure used previously, which could explain the increased sensitivity toward H2O2. In summary, DHN-melanin is not involved in protection against H2O2 or superoxide and, thus, has no role in survival of conidia when attacked by these reactive oxygen species. IMPORTANCE Opportunistic pathogens like Aspergillus fumigatus have strategies to protect themselves against reactive oxygen species like hydrogen peroxides and superoxides that are produced by immune cells. DHN-melanin is the green pigment on conidia of Aspergillus fumigatus and more than 2 decades ago was reported to protect conidia against hydrogen peroxide. Here, we correct this misinterpretation by showing that DHN-melanin actually is not involved in protection of conidia against hydrogen peroxide. We show that UV mutagenesis that was previously used to select a pksP mutant generated many more genome-wide mutations. We discovered that a mutation in the mycelial catalase gene cat1 could explain the observed phenotype of increased hydrogen peroxide sensitivity. Our work shows that UV mutagenesis is not the preferred methodology to be used for generating mutants. It requires genome sequencing with single-nucleotide polymorphism analysis as well as additional validations to discard unwanted and confirm correct phenotypes.

Correction: Variation of virulence of five Aspergillus fumigatus isolates in four different infection models.

[This corrects the article DOI: 10.1371/journal.pone.0252948.].

Variation of virulence of five Aspergillus fumigatus isolates in four different infection models.

Conidia of Aspergillus fumigatus are inhaled by humans on daily basis. As a consequence, these conidia can cause infections that differ in severity ranging from allergic bronchopulmonary aspergillosis to invasive aspergillosis. In this study we compared virulence of five A. fumigatus isolates in four different infection models to address the predictive value of different model systems. Two of the A. fumigatus strains were isolated from dogs with a non-invasive sino-nasal aspergillosis (DTO271-B5 and DTO303-F3), while three strains were isolated from human patients with invasive aspergillosis (Af293, ATCC46645 and CEA10). Infection models used encompassed cultured type II A549 lung epithelial cells, Protostelium aurantium amoeba, Galleria melonella larvae and zebrafish embryos. No major differences in virulence between these five strains were observed in the lung epithelial cell model. In contrast, strain ATCC46645 was most virulent in the amoeba and zebrafish model, whereas it was much less virulent in the Galleria infection model. DTO303-F3 was most virulent in the latter model. In general, reference strain Af293 was less virulent as compared to the other strains. Genome sequence analysis showed that this latter strain differed from the other four strains in 136 SNPs in virulence-related genes. Together, our results show that virulence of individual A. fumigatus strains show significant differences between infection models. We conclude that the predictive value of different model systems varies since the relative virulence across fungal strains does not hold up across different infection model systems.

Nurse telephone triage: good quality associated with appropriate decisions.

BACKGROUND: Triage at out-of-hours GP cooperatives (GPCs) is aimed at determining medical urgency and guiding decisions. Both medical knowledge and communication skills are required for this complex task. OBJECTIVE: To explore the impact of quality of consultation and estimated urgency on the appropriateness of decisions. METHODS: We performed a secondary analysis of telephone triage consultations by nurses at 29 Dutch GPCs. Consultations were taped and assessed by trained observers, using a validated instrument. Measures concerned quality of consultation, nurse-estimated urgency (four levels) and appropriateness of decisions (urgency, follow-up advice and timing). Bivariate analyses and logistic multilevel regression analyses were used to explore the impact of quality of consultation (controlling for urgency) on appropriateness of decisions. RESULTS: The sample included 6739 telephone contacts, most of which (90%) were non-urgent. The majority of decisions were appropriate (91% for urgency, 96% for follow-up advice and 95% for timing). In bivariate analyses, appropriateness of decisions was positively related to higher quality of consultation. Estimated urgency was negatively associated with quality of consultation. Logistic multilevel analysis showed that higher quality of consultation was related to a more appropriate estimation of urgency [odds ratio (OR) = 1.82; 95% confidence interval (CI): 1.69-1.95], follow-up advice (OR = 2.69; 95% CI: 2.41-3.01) and timing of decisions (OR = 2.41; 95% CI: 2.20-2.63). CONCLUSIONS: High quality of consultation has a positive, but small, impact on the appropriateness of decisions. Quality of consultation needs to be targeted in training and support of triage nurses, especially when it concerns highly urgent contacts.

Non-tryptophan fluorescence of crystallins from normal and cataractous human lenses.

The soluble proteins of a newborn human lens exhibit almost no non-tryptophan fluorescence. On aging, an increase in the fluorescence of all native crystallins is found except for gamma 2-crystallin. With formation of nuclear cataract, a further increase is seen for gamma 1-crystallin. The fluorophore, excitation 355 nm/emission 420 nm, is mainly associated with one species of the gamma 1-crystallin population. It is also present as such in the soluble fraction and increases significantly with nuclear cataract formation. At least one of the gamma 1-crystallins seems to play an important role in the cataractogenic process.

First-trimester diagnosis of Lesch-Nyhan syndrome.

Chorionic villi were sampled at 8-9 weeks' gestation in four women whose fetuses were at risk for Lesch-Nyhan syndrome. Radiochemical assay of hypoxanthine phosphoribosyl transferase activity and fetal sexing (in two fetuses by means of a Y chromosome specific cDNA probe) showed that three fetuses were affected. The biochemical findings were confirmed after therapeutic abortion in two cases and spontaneous abortion in the third. Chorion biopsy and ultramicroscale enzymology may be a suitable alternative to recombinant-DNA-based methods for first-trimester diagnosis in selected diseases where the abnormal gene product is an enzyme expressed in the chorion by the 8th week of pregnancy.