The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans

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Multianalyte analysis of volatile compounds in virgin olive oils using SPME-GC with FID or MS detection: results of an international interlaboratory validation

The organoleptic assessment (Panel test) is the only procedure within the official methods for determining the quality of virgin olive oils that involves an expert panel. There is an urgent need for analytical methodology that can reliably measure volatile compounds in virgin olive oils that is capable of supporting and anticipating the official Panel test. For this reason, a new method based on solid-phase microextraction–gas chromatography with the choice of two possible detectors (FID or MS) was subjected to a large international interlaboratory validation study. The study involved a two-stage process: first, a pretrial phase in which 7 participants were exposed to the method for the first time to identify any initial problems with the methodology; then, a formal validation stage (trial proper), which involved 20 laboratories from Europe, USA, Japan and China. The performance of the different detectors was investigated. While both methods have advantages, the method using FID provided better results for 11 compounds, in terms of reproducibility, compared to MS. This information will allow to implement the method with accurate information of the method performance depending on the detector used. Practical applications: This study provides information from an interlaboratory validation of a method for measuring volatile compounds in virgin olive oils conducted with laboratories (from industry and academia) working in the olive oil sector. The information on the expected analytical errors in the determination of each volatile compound is necessary to apply this method for supporting the official Panel test (sensory analysis). The SPME-GC-MS/FID methods proposed in this work can be used for the internal quality control of a company/distributor/quality control laboratory and could also be used in cases of difficult/contradictory organoleptic assessment, or to confirm results from sensory panels in cases of disputes/disagreement (Reg. EU 2022/2105)

Participation of Candida albicans transcription factor Rlm1 in cell wall biogenesis and virulence

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.This work was supported by CBMA (Centre of Molecular and Environmental Biology) through the FCT (Fundacao para a Ciencia e Tecnologia) project PEst-C/BIA/UI4050/2011. Yolanda Delgado-Silva was supported by an ALbAN scholarship (No E07D400922PE), and Alexandra Correia by SFRH/BD/31354/2006 fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Remarkable Reduction of MAP2 in the Brains of Scrapie-Infected Rodents and Human Prion Disease Possibly Correlated with the Increase of Calpain

Microtubule-associated protein 2 (MAP2) belongs to the family of heat stable MAPs, which takes part in neuronal morphogenesis, maintenance of cellular architecture and internal organization, cell division and cellular processes. To obtain insight into the possible alteration and the role of MAP2 in transmissible spongiform encephalopathies (TSEs), the MAP2 levels in the brain tissues of agent 263K-infected hamsters and human prion diseases were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, MAP2 levels in the brain tissues of scrapie infected hamsters, a patient with genetic Creutzfeldt-Jakob disease (G114V gCJD) and a patient with fatal familial insomnia (FFI) were almost undetectable. The decline of MAP2 was closely related with prolonged incubation time. Exposure of SK-N-SH neuroblastoma cell line to cytotoxic PrP106-126 peptide significantly down-regulated the cellular MAP2 level and remarkably disrupted the microtubule structure, but did not alter the level of tubulin. Moreover, the levels of calpain, which mediated the degradation of a broad of cytoskeletal proteins, were significantly increased in both PrP106-126 treated SK-N-SH cells and brain tissues of 263K prion-infected hamsters. Our data indicate that the decline of MAP2 is a common phenomenon in TSEs, which seems to occur at an early stage of incubation period. Markedly increased calpain level might contribute to the reduction of MAP2

Prediction of Phenotype-Associated Genes via a Cellular Network Approach: A Candida albicans Infection Case Study

Candida albicans is the most prevalent opportunistic fungal pathogen in humans causing superficial and serious systemic infections. The infection process can be divided into three stages: adhesion, invasion, and host cell damage. To enhance our understanding of these C. albicans infection stages, this study aimed to predict phenotype-associated genes involved during these three infection stages and their roles in C. albicans–host interactions. In light of the principles that proteins that lie closer to one another in a protein interaction network are more likely to have similar functions, and that genes regulated by the same transcription factors tend to have similar functions, a cellular network approach was proposed to predict the phenotype-associated genes in this study. A total of 4, 12, and 3 genes were predicted as adhesion-, invasion-, and damage-associated genes during C. albicans infection, respectively. These predicted genes highlight the facts that cell surface components are critical for cell adhesion, and that morphogenesis is crucial for cell invasion. In addition, they provide targets for further investigations into the mechanisms of the three C. albicans infection stages. These results give insights into the responses elicited in C. albicans during interaction with the host, possibly instrumental in identifying novel therapies to treat C. albicans infection

THE CHLOROPEROXIDASE-CATALYZED OXIDATION OF PHENOLS - MECHANISM, SELECTIVITY, AND CHARACTERIZATION OF ENZYME-SUBSTRATE COMPLEXES

The reactivity of a series of para-substituted phenolic compounds in the peroxidation catalyzed by chloroperoxidase was investigated, and the results were interpreted on the basis of the binding characteristics of the substrates to the active site of the enzyme. Marked selectivity effects are observed. These operate through charge, preventing phenolic compounds carrying amino groups on the substituent chain to act as substrates for the enzyme, and through size, excluding potential substrates containing bulky substituents to the phenol nucleus. Also, chiral recognition is exhibited by chloroperoxidase in the oxidation of N-acetyltyrosine, where only the L isomer is oxidized. Kinetic measurements show that, in general, the efficiency of chloroperoxidase in the oxidation of phenols is lower than that of horseradish peroxidase. Paramagnetic NMR spectra and relaxation rate measurements of chloroperoxidase-phenol complexes are consistent with binding of the substrates close to the heme, in the distal pocket, with the phenol group pointing toward the iron atom. On the other hand, phenolic compounds which are not substrates for chloroperoxidase bind to the enzyme with a much different disposition, with the phenol group very distant from the iron and probably actually outside the active-site cavity

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast-hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast-hypha morphogenesis in C. albicans