The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

BACKGROUND: Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen. RESULTS: We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence. CONCLUSIONS: Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

Euphresco Sendo: An international laboratory comparison study of molecular tests for Synchytrium endobioticum detection and identification.

An international test performance study (TPS) was organised to generate validation data for three molecular Synchytrium endobioticum tests: van den Boogert et al. (European Journal of Plant Pathology 113, 47-57, 2005), and van Gent-Pelzer et al. (European Journal of Plant Pathology, 126, 129-133, 2010) for the detection of S. endobioticum, and the pathotype 1(D1) identification test described by Bonants et al. (European Journal of Plant Pathology, 143, 495-506, 2015). Two TPS rounds were organised focussing on different test matrices, i.e. round 1: warted potato tissue, and round 2: resting spore suspensions. When using the tests for detection and identification of S. endobioticum in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. When using the tests for detection and identification of S. endobioticum in resting spore suspensions, the van den Boogert and van Gent-Pelzer tests significantly outperform the Bonants test for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the van Gent-Pelzer significantly outperforms the van den Boogert and Bonants tests and is regarded as the test of choice when identifying S. endobioticum from resting spores. Tests regarded fit for purpose for routine testing of wart material and resting spore suspensions are proposed for the update of EPPO standard PM7/28(1) Synchytrium endobioticum.

Effects of temperature and doxorubicin exposure on keratinocyte damage in vitro.

Cancer chemotherapy treatment often leads to hair loss, which may be prevented by cooling the scalp during drug administration. The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow (perfusion) and chemical reaction rates. Reduced perfusion leads to less drugs available for uptake, whereas the reduced temperature decreases uptake of and damage by chemotherapy. Altogether, less damage is exerted to the hair cells, and the hair is preserved. However, the two mechanisms in the hypothesis have not been quantified yet. To quantify the effect of reduced drug damage caused by falling temperatures, we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes. Cells were exposed for 4 h to a wide range of doxorubicin concentrations. During exposure, cells were kept at different temperatures. Cell viability was determined after 3 d using a viability test. Control samples were used to establish a concentration-viability curve. Results show that cell survival is significantly higher in cooled cells (T < 22 degrees C) than in non-cooled cells (T = 37 degrees C), but no significant differences are visible between T = 10 degrees C and T = 22 degrees C. Based on this result and previous work, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided.

Effect of forced-air heaters on perfusion and temperature distribution during and after open-heart surgery.

OBJECTIVES: After cardiopulmonary bypass, patients often show redistribution hypothermia, also called afterdrop. Forced-air blankets help to reduce afterdrop. This study explores the effect of forced-air blankets on temperature distribution and peripheral perfusion. The blood perfusion data is used to explain the observed temperature effects and the reduction of the afterdrop. METHODS: Fifteen patients were enrolled in a randomised study. In the test group (n=8), forced-air warmers were used. In the control group (n=7), only passive insulation was used. Core and skin temperatures and thigh temperatures at 0, 8, 18 and 38 mm depth were measured. Laser Doppler flowmetry (LDF) was used to record skin perfusion from the big toe. Blood flow through the femoral artery was determined with ultrasound. RESULTS: Afterdrop in the test group was smaller than in the control group (1.2+/-0.2 degrees C vs 1.8+/-0.7 degrees C: P=0.04) whilst no significant difference in mean tissue thigh temperature was found between the groups. Local skin temperature was 2.5-3.0 degrees C higher when using forced-air heaters. However, skin perfusion was unaffected. Ultrasound measurements revealed that leg blood flow during the first hours after surgery was reduced to approximately 70% of pre- and peri-operative values. CONCLUSIONS: Forced-air blankets reduce afterdrop. However, they do not lead to clinical relevant changes in deep thigh temperature. LDF measurements show that forced-air heating does not improve toe perfusion. The extra heat especially favours core temperature. This is underlined by the decrease in postoperative leg blood flow, suggesting that the majority of the warmed blood leaving the heart flows to core organs and not to the periphery.

The relationship between local scalp skin temperature and cutaneous perfusion during scalp cooling.

Cooling the scalp during administration of chemotherapy can prevent hair loss. It reduces both skin blood flow and hair follicle temperature, thus affecting drug supply and drug effect in the hair follicle. The extent to which these mechanisms contribute to the hair preservative effect of scalp cooling remains unknown. The purpose of this study was to establish a relationship between local scalp skin temperature and cutaneous blood flow during scalp cooling. We measured skin temperature and cutaneous perfusion during a cooling and re-warming experiment. Experiments on a single subject showed that the measurements were reproducible and that the response was identical for the two positions that were measured. Inter-subject variability was investigated on nine subjects. We found that for the first 10 degrees C of cooling, perfusion of the scalp skin decreases to below 40%. Perfusion can be further reduced to below 30% by a few degrees more cooling, but a plateau is reached after that. We found that a generally accepted relation in thermal physiology between temperature and perfusion (i.e. Q(10) relation) does not describe the data well, but we found an alternative relation that describes the average behavior significantly better.

Discrete vessel heat transfer in perfused tissue--model comparison.

The aim of this paper is to compare two methods of calculating heat transfer in perfused biological tissue using a discrete vessel description. The methods differ in two important aspects: the representation of the vascular system and the algorithm for calculating the heat flux between tissue and blood vessels. The first method was developed at the University of Utrecht between 1994 and 1998 and has been used in several clinical applications. The second method has been proposed by the first author. The methods are briefly described, their assumptions and limitations are discussed. Finally, the test simulation is introduced and the results produced by both methods are compared. The test indicates that the simpler, and less computationally intensive method proposed by the present author for calculating 2D problems containing countercurrent blood vessel systems can reproduce quite well some features of the solution obtained by the more complex 3D method. The observed discrepancies could be explained on physical grounds.