Game theoretical modelling of survival strategies of Candida albicans inside macrophages.

The polymorphic fungus Candida albicans can live as an aggressive pathogen that causes a wide variety of diseases in humans. Host resistance against these infections is mediated predominantly by phagocytes, namely neutrophils and macrophages. This report provides two game theoretical models of ingested C. albicans cells in macrophages. Two strategies are available for each pathogenic yeast cell: avoiding lysis transiently (called silencing) or forming hyphae and escaping (called piercing because the macrophage is pierced from inside). In dependence on parameter values, two different outcomes can be derived from the model: when the difference of the costs of the two strategies is low, all fungal cells inside a macrophage will play the piercing strategy, while in the high-cost case, a mixed population of piercing and silencing cells is the only stable solution. Further, the role of the SAP gene family encoding secreted proteinases and the Sap proteins is investigated with the help of known studies and is put in relation to the costs of the strategies, the most important parameter of this model. Our results are in agreement with wet-lab results presented by other groups and the model parameters can be estimated from experimental data.

Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples.

A reliable, sensitive and selective method was developed to determine different Fusarium mycotoxins (trichothecenes Type A and B, zearalenone) simultaneously in cereals and cereal-based samples using liquid chromatography with tandem mass spectrometry (LC-ESI-MS/MS). Sample preparation is based on a standard solvent extraction step followed by two different kinds of solid-phase clean-up procedures: using a multifunctional MycoSep material for trichothecenes and zearalenone. The average recoveries for trichothecenes ranged from 65% for nivalenol (NIV) up to 96% for deoxynivalenol (DON) and 89% for zearalenone (ZON). The limit of quantification varied between 0.02 and 10 ppb for each substance. In addition, a screening survey with 685 samples was carried out to compare contents of T-2 toxin and deoxynivalenol and to investigate potential coherence in contamination pattern.

Overview of key phytoplankton toxins and their recent occurrence in the North and Baltic Seas.

The frequency and intensity of harmful algal blooms (HABs) appear to be on the rise globally. There is also evidence of the geographic spreading of toxic strains of these algae. Consequently, methods had to be established and new ones are still needed for the evaluation of possible hazards caused by increased algal toxin production in the marine food chain. Different clinical effects of algae-related poisoning have attracted scientific attention; paralytic shellfish poisoning, diarrhetic shellfish poisoning, and amnesic shellfish poisoning are among the most common. Additionally, cyanobacteria (blue-green algae) in brackish waters often produce neurotoxic and hepatotoxic substances. Bioassays with mice or rats are common methods to determine algal and cyanobacterial toxins. However, biological tests are not really satisfactory because of their low sensitivity. In addition, there is growing public opposition to animal testing. Therefore, there has been increasing effort to determine algal toxins by chemical methods. Plankton samples from different European marine and brackish waters were taken during research cruises and analyzed on board directly. The ship routes covered marine areas in the northwest Atlantic, Orkney Islands, east coast of Scotland, and the North and Baltic seas. The first results on the occurrence and frequency of harmful algal species were obtained in 1997 and 1998. During the 2000 cruise an HPLC/MS coupling was established on board, and algal toxins were measured directly after extraction of the plankton samples. In contrast to earlier cruises, the sampling areas were changed in 2000 to focusing on coastal zones. The occurrence of toxic algae in these areas was compared to toxin formation during HABs in the open sea. It was found that the toxicity of the algal blooms depended on the prevailing local conditions. This observation was also confirmed by monitoring cyanobacterial blooms in the Baltic Sea. Optimal weather conditions, for example, during the summers of 1997 and 2003, favored blooms of cyanobacteria in all regions of the Baltic. The dominant species regarding the HABs in the Baltic was Nodularia spumigena. However, in addition to high concentrations of Nodularia spumigena in coastal zones, other blue-green algae are involved in bloom formation, with changes in plankton communities influencing both toxin profiles and toxicity.

A multicomponent method for Fusarium toxins in cereal based food and feed samples using HPLC-MS/MS.

A reliable, sensitive and selective multicomponent method has been developed to determine 12 differentFusarium mycotoxins (trichothecenes type A and B, zearalenone) simultaneously in cereal and grain samples using liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation based on a standard extraction step followed by two different kinds of solid phase clean-up (multifunctional MycoSep(®) material) for trichothecenes, and an immuno-affinity purification which combined antibodies for aflatoxins, ochratoxin A and zearalenone (AOZ-IAC). For quantification of zearalenone (ZON) an internal standard (zearalanone, ZAN) was used, whereas for trichothecenes a recovery standard (verrucarol, VOL) was applied. The average recoveries for the trichothecenes ranged from 65% for nivalenol (NIV) up to 96% for deoxynivalenol (DON) and 89% for zearalenone. The limit of quantification is different for each of the individual trichothecenes and in the range of 1 ppb to 10 ppb.

Determination of paralytic shellfish poisoning toxins by high-performance ion-exchange chromatography.

An efficient LC method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins based on ion-exchange chromatographic separation of the toxins followed by electrochemical post-column oxidation and fluorescence detection as well as mass spectrometric (MS) detection. The method can be applied to the determination of PSP toxins in phytoplankton and to control seafood for PSP content.

Identification of microcystins in cyanobacteria from the Bleiloch former drinking-water reservoir (Thuringia, Germany).

The presence of microcystins in cyanobacterial samples collected from the Bleiloch reservoir, formerly an important drinking-water supply in Thuringia, Germany, was proven by application of a combination of recently developed analytical methods. The raw extracts were cleaned by size-exclusion chromatography (SEC) or solid-phase extraction (SPE). The determination of microcystins was achieved by different HPLC separation followed by the application of alternative detection methods (UV, diode array detection (DAD), and mass spectrometry (MS), respectively). Furthermore, the different results of clean-up by SPE and SEC are demonstrated. The identity of microcystins was verified by MS/MS measurements. In the cyanobacterial sample from 1998, microcystin-RR, -LR and -YR were found, whereas in 1999 only microcystin-LR and -YR were detectable. In addition to detection of cell-bound microcystins, in 1999 traces of dissolved microcystins in water from the Bleiloch reservoir were detected. It can be assumed that not only the Bleiloch reservoir is contaminated with hepatotoxins but also many similar lakes still used for drinking water supply.

Different methods for toxin analysis in the cyanobacterium Nodularia spumigena (Cyanophyceae).

The brackish water cyanobacterium Nodularia spumigena produce the hepatotoxic cyclic pentapeptide nodularin. Intoxications for both human as well as animal may arise when water reservoirs are contaminated with potentially toxic Nodularia species. Here, results of three independent methods for the determination of nodularin in different strains of N. spumigena are presented. The results obtained with a protein phosphatase assay and a HPLC/UV/MS method are compared with the results obtained with a bioluminescence assay, which is successfully introduced here for nodularin determination. Statistical evaluation of the three applied methods revealed a good comparability towards the detected toxin content. The methods were evaluated taking into consideration the parameters: handling, efficiency, sensitivity and selectivity. The detection limit in the protein phosphatase assay is highest (0.05ng nodularin) and lowest (250ng nodularin) in the bioluminescence assay- it was determined with 5ng (MS) and 25ng (UV) for the HPLC/UV/MS methods. The different selectivities and sensitivities are critically discussed and an analytical pathway for the determination of the biotoxin nodularin from Nodularia samples is proposed.

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters.

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.

Automatic high-performance liquid chromatographic method for the determination of diarrhetic shellfish poison.

A rapid HPLC method with fluorescence detection for the determination of okadaic acid (OA) and dinophysistoxin-1 (DTX-1) in mussels and mussel products is presented. For fluorescence labelling of OA and DTX-1, 9-anthryldiazomethane (ADAM) is used. HPLC with a column-switching system is proposed to avoid time-consuming clean-up procedures after derivatization of sample extracts with ADAM. The column-switching system as well as the chromatographic conditions and detection are described.

Determination of chloramphenicol in animal tissue using high-performance liquid chromatography with a column-switching system and ultraviolet detection.

A rapid and universally applicable method for determination of chloramphenicol (CAP) residues in animal tissues using high-performance liquid chromatography with a column-switching system is presented. The clean-up procedure as well as the chromatographic conditions and detection are described. The linearity and repeatability of the data obtained by this method as well as the recovery rates of CAP in several farm animals are presented.