Decreased sialylation elicits complement-related microglia response and bipolar cell loss in the mouse retina.

Sialylation plays an important role in self-recognition, as well as keeping the complement and innate immune systems in check. It is unclear whether the reduced sialylation seen during aging and in mice heterozygous for the null mutant of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (Gne+/-), an essential enzyme for sialic acid biosynthesis, contributes to retinal inflammation and degeneration. We found a reduction of polysialic acid and trisialic acid expression in several retinal layers in Gne+/- mice at 9 months of age compared to Gne+/+ wildtype (WT) mice, which was associated with a higher microglial expression of the lysosomal marker CD68. Furthermore, the total number of rod bipolar cells was reduced in 12 months old Gne+/- mice in comparison to WT mice, demonstrating loss of these retinal interneurons. Transcriptome analysis showed up-regulation of complement, inflammation, and apoptosis-related pathways in the retinas of Gne+/- mice. Particularly, increased gene transcript levels of the complement factors C3 and C4 and the pro-inflammatory cytokine Il-1ß were observed by semi-quantitative real-time polymerase chain reaction (sqRT-PCR) in 9 months old Gne+/- mice compared to WT mice. The increased expression of CD68, loss of rod bipolar cells, and increased gene transcription of complement factor C4, were all prevented after crossing Gne+/- mice with complement factor C3-deficient animals. In conclusion, our data show that retinal hyposialylation in 9 and 12 months old Gne+/- mice was associated with complement-related inflammation and lysosomal microglia response, as well as rod bipolar cells loss, which was absent after genetic deletion of complement factor C3.

Expression of sPD-L1 levels in an ex vivo liver perfusion model.

The programmed cell death protein 1 (PD-1) acts as a central inhibitory immune checkpoint receptor. The soluble form of its primary ligand, sPD-L1, was found to be elevated in serum of patients with cancer, infectious diseases and chronic inflammation. So far, the hepatic origin of sPD-L1 has received relatively little attention and is therefore subject of this study in the context of normothermic machine perfusion (NMP) of liver grafts. sPD-L1 concentrations as well as several well-established clinically relevant laboratory parameters were determined in the perfusate of 16 donor liver grafts undergoing normothermic machine perfusion (NMP) up to 30 hours (h). sPD-L1 levels continuously increased during NMP and significantly correlated with markers of hepatic synthesis (cholinesterase), acute-phase proteins (von Willebrand factor, procalcitonin, antithrombin, interleukin-6, fibrinogen) and liver decay markers (gamma-glutamyltransferase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase). Perfusate leukocytes were in the lower reference range, and decreased after 12 h. Mean sPD-L1 levels in the perfusate correlated with donor levels of gamma-glutamyltransferase, alanine aminotransferase, creatinine and blood urea nitrogen. Our study reveals a significant increase of concentration of sPD-L1 following ischemia-reperfusion injury in a hepatic ex vivo model. sPD-L1 concentrations during NMP correlate with established acute-phase proteins and liver cell decay markers suggesting that hepatic sPD-L1 synthesis or shedding increases during the acute phase and cell decay. Furthermore, sPD-L1 correlates with established liver function and synthesis parameters as well as with donor laboratory values and might therefore be a potential biomarker for hepatic function of liver grafts.

Predictive value of early postoperative lactate (<6†h) during normothermic machine perfusion and outcome after liver transplantation: results from a multicentre study.

BACKGROUND: Biomarkers with strong predictive capacity towards transplantation outcome for livers undergoing normothermic machine perfusion (NMP) are needed. We investigated lactate clearing capacity as a basic function of liver viability during the first 6 h of NMP. METHODS: A trial conducted in 6 high-volume transplant centres in Europe. All centres applied a back-to-base NMP approach with the OrganOx metra system. Perfusate lactate levels at start, 1, 2, 4 and 6 h of NMP were assessed individually and as area under the curve (AUC) and correlated with EAD (early allograft dysfunction), MEAF (model for early allograft function) and modified L-GrAFT (liver graft assessment following transplantation) scores. RESULTS: A total of 509 livers underwent ≥6 h of NMP before transplantation in 6 centres in the UK, Germany and Austria. The donor age was 53 (40-63) years (median, i.q.r.).The total NMP time was 10.8 (7.9-15.7) h. EAD occurred in 26%, MEAF was 4.72 (3.54-6.05) and L-GrAFT10 -0.96 (-1.52--0.32). Lactate at 1, 2 and 6 h correlated with increasing robustness with MEAF. Rather than a binary assessment with a cut-off value at 2 h, the actual 2 h lactate level correlated with the MEAF (P = 0.0306 versus P = 0.0002, Pearson r = 0.01087 versus r = 0.1734). The absolute lactate concentration at 6 h, the AUC of 0-6 h and 1-6 h (P < 0.0001, r = 0.3176) were the strongest predictors of MEAF. CONCLUSION: Lactate measured 1-6 h and lactate levels at 6 h correlate strongly with risk of liver allograft dysfunction upon transplantation. The robustness of predicting MEAF by lactate increases with perfusion duration. Monitoring lactate levels should be extended to at least 6 h of NMP routinely to improve clinical outcome.

Assessment of Mitochondrial Respiration During Hypothermic Storage of Liver Biopsies Following Normothermic Machine Perfusion.

Organ quality can be assessed prior to transplantation, during normothermic machine perfusion (NMP) of the liver. Evaluation of mitochondrial function by high-resolution respirometry (HRR) may serve as a viability assessment concept in this setting. Freshly collected tissue is considered as optimal sample for HRR, but due to technical and personnel requirements, more flexible and schedulable measurements are needed. However, the impact of cold storage following NMP before processing biopsy samples for mitochondrial analysis remains unknown. We aimed at establishing an appropriate storage protocol of liver biopsies for HRR. Wedge biopsies of 5 human livers during NMP were obtained and assessed by HRR. Analysis was performed after 0, 4, 8, and 12 h of hypothermic storage (HTS) in HTK organ preservation solution at 4°C. With HTS up to 4 h, mitochondrial performance did not decrease in HTS samples compared with 0 h (OXPHOS, 44.62 [34.75-60.15] pmol·s-1·mg wet mass-1 vs. 43.73 [40.69-57.71], median [IQR], p > 0.999). However, at HTS beyond 4 h, mitochondrial respiration decreased. We conclude that HTS can be safely applied for extending the biopsy measurement window for up to 4 h to determine organ quality, but also that human liver respiration degrades beyond 4 h HTS following NMP.

The Predictive Value of Graft Viability and Bioenergetics Testing Towards the Outcome in Liver Transplantation.

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (-0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation.

BRENDA, the ELIXIR core data resource in 2021: new developments and updates.

The BRENDA enzyme database (https://www.brenda-enzymes.org), established in 1987, has evolved into the main collection of functional enzyme and metabolism data. In 2018, BRENDA was selected as an ELIXIR Core Data Resource. BRENDA provides reliable data, continuous curation and updates of classified enzymes, and the integration of newly discovered enzymes. The main part contains >5 million data for ∼90 000 enzymes from ∼13 000 organisms, manually extracted from ∼157 000 primary literature references, combined with information of text and data mining, data integration, and prediction algorithms. Supplements comprise disease-related data, protein sequences, 3D structures, genome annotations, ligand information, taxonomic, bibliographic, and kinetic data. BRENDA offers an easy access to enzyme information from quick to advanced searches, text- and structured-based queries for enzyme-ligand interactions, word maps, and visualization of enzyme data. The BRENDA Pathway Maps are completely revised and updated for an enhanced interactive and intuitive usability. The new design of the Enzyme Summary Page provides an improved access to each individual enzyme. A new protein structure 3D viewer was integrated. The prediction of the intracellular localization of eukaryotic enzymes has been implemented. The new EnzymeDetector combines BRENDA enzyme annotations with protein and genome databases for the detection of eukaryotic and prokaryotic enzymes.

Bioenergetic and Cytokine Profiling May Help to Rescue More DCD Livers for Transplantation.

The majority of organs used for liver transplantation come from brain-dead donors (DBD). In order to overcome the organ shortage, increasingly donation after circulatory death (DCD) organs are also considered. Since normothermic machine perfusion (NMP) restores metabolic activity and allows for in-depth assessment of organ quality and function prior to transplantation, such organs may benefit from NMP. We herein compare the bioenergetic performance through a comprehensive evaluation of mitochondria by high-resolution respirometry in tissue biopsies and the inflammatory response in DBD and DCD livers during NMP. While livers were indistinguishable by perfusate biomarker assessment and histology, our findings revealed a greater impairment of mitochondrial function in DCD livers after static cold storage compared to DBD livers. During subsequent NMPs, DCD organs recovered and eventually showed a similar performance as DBD livers. Cytokine expression analysis showed no differences in the early phase of NMP, while towards the end of NMP, significantly elevated levels of IL-1ß, IL-5 and IL-6 were found in the perfusate of DCD livers. Based on our results, we find it worthwhile to reconsider more DCD organs for transplantation to further extend the donor pool. Therefore, donor organ quality criteria must be developed, which may include an assessment of bioenergetic function and cytokine quantification.

Hyperspectral Imaging as a Tool for Viability Assessment During Normothermic Machine Perfusion of Human Livers: A Proof of Concept Pilot Study.

Normothermic machine perfusion (NMP) allows for ex vivo viability and functional assessment prior to liver transplantation (LT). Hyperspectral imaging represents a suitable, non-invasive method to evaluate tissue morphology and organ perfusion during NMP. Liver allografts were subjected to NMP prior to LT. Serial image acquisition of oxygen saturation levels (StO2), organ hemoglobin (THI), near-infrared perfusion (NIR) and tissue water indices (TWI) through hyperspectral imaging was performed during static cold storage, at 1h, 6h, 12h and at the end of NMP. The readouts were correlated with perfusate parameters at equivalent time points. Twenty-one deceased donor livers were included in the study. Seven (33.0%) were discarded due to poor organ function during NMP. StO2 (p < 0.001), THI (p < 0.001) and NIR (p = 0.002) significantly augmented, from static cold storage (pre-NMP) to NMP end, while TWI dropped (p = 0.005) during the observational period. At 12-24h, a significantly higher hemoglobin concentration (THI) in the superficial tissue layers was seen in discarded, compared to transplanted livers (p = 0.036). Lactate values at 12h NMP correlated negatively with NIR perfusion index between 12 and 24h NMP and with the delta NIR perfusion index between 1 and 24h (rs = -0.883, p = 0.008 for both). Furthermore, NIR and TWI correlated with lactate clearance and pH. This study provides first evidence of feasibility of hyperspectral imaging as a potentially helpful contact-free organ viability assessment tool during liver NMP.

Assessment of binocular fixational eye movements including cyclotorsion with split-field binocular scanning laser ophthalmoscopy.

Fixational eye movements are a hallmark of human gaze behavior, yet little is known about how they interact between fellow eyes. Here, we designed, built and validated a split-field binocular scanning laser ophthalmoscope to record high-resolution eye motion traces from both eyes of six observers during fixation in different binocular vergence conditions. In addition to microsaccades and drift, torsional eye motion could be extracted, with a spatial measurement error of less than 1 arcmin. Microsaccades were strongly coupled between fellow eyes under all conditions. No monocular microsaccade occurred and no significant delay between microsaccade onsets across fellow eyes could be detected. Cyclotorsion was also firmly coupled between both eyes, occurring typically in conjugacy, with gradual changes during drift and abrupt changes during saccades.

Straightness metric for measurement of turbulence-induced distortion in long-range imaging.

Algorithms used for mitigation of the effects of atmospheric turbulence on video sequences often rely on a process for creating a reference image to register all of the frames. Because such a pristine image is generally not available, no-reference image quality metrics can be used to identify frames in a sequence that have minimum distortion. Here, we propose a metric that quantifies image warping by measuring image straightness based on line detection. The average length of straight lines in a frame is used to select best frames in a sequence and to generate a reference frame for a subsequent dewarping algorithm. We perform tests with this metric on simulated data that exhibits varying degrees of distortion and blur and spans normalized turbulence strengths between 0.75 and 4.5. We show, through these simulations, that the metric can differentiate between weak and moderate turbulence effects. We also show in simulations that the optical flow that uses a reference frame generated by this metric produces consistently improved image quality. This improvement is even higher when we employ the metric to guide optical flow that is applied to three real video sequences taken over a 7 km path.

Cell-Based Regeneration and Treatment of Liver Diseases.

The liver, in combination with a functional biliary system, is responsible for maintaining a great number of vital body functions. However, acute and chronic liver diseases may lead to irreversible liver damage and, ultimately, liver failure. At the moment, the best curative option for patients suffering from end-stage liver disease is liver transplantation. However, the number of donor livers required by far surpasses the supply, leading to a significant organ shortage. Cellular therapies play an increasing role in the restoration of organ function and can be integrated into organ transplantation protocols. Different types and sources of stem cells are considered for this purpose, but highly specific immune cells are also the focus of attention when developing individualized therapies. In-depth knowledge of the underlying mechanisms governing cell differentiation and engraftment is crucial for clinical implementation. Additionally, novel technologies such as ex vivo machine perfusion and recent developments in tissue engineering may hold promising potential for the implementation of cell-based therapies to restore proper organ function.

Ex Vivo Mesenchymal Stem Cell Therapy to Regenerate Machine Perfused Organs.

Transplantation represents the treatment of choice for many end-stage diseases but is limited by the shortage of healthy donor organs. Ex situ normothermic machine perfusion (NMP) has the potential to extend the donor pool by facilitating the use of marginal quality organs such as those from donors after cardiac death (DCD) and extended criteria donors (ECD). NMP provides a platform for organ quality assessment but also offers the opportunity to treat and eventually regenerate organs during the perfusion process prior to transplantation. Due to their anti-inflammatory, immunomodulatory and regenerative capacity, mesenchymal stem cells (MSCs) are considered as an interesting tool in this model system. Only a limited number of studies have reported on the use of MSCs during ex situ machine perfusion so far with a focus on feasibility and safety aspects. At this point, no clinical benefits have been conclusively demonstrated, and studies with controlled transplantation set-ups are urgently warranted to elucidate favorable effects of MSCs in order to improve organs during ex situ machine perfusion.

Restoring Mitochondrial Function While Avoiding Redox Stress: The Key to Preventing Ischemia/Reperfusion Injury in Machine Perfused Liver Grafts?

Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion.

Tracking gene expression and oxidative damage of O2-stressed Clostridioides difficile by a multi-omics approach.

Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15 min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O2 is based on a complex and far-reaching adjustment of global gene expression which leads to only a slight change in phenotype.

Consumer Willingness to Pay for Vehicle Attributes: What Do We Know?

As standards for vehicle greenhouse gas emissions and fuel economy have become more stringent, concerns have arisen that the incorporation of fuel-saving technologies may entail tradeoffs with other vehicle attributes important to consumers such as acceleration performance. Assessing the effects of these tradeoffs on consumer welfare requires estimates of both the degree of the tradeoffs, and consumer willingness to pay (WTP) for the foregone benefits. This paper has two objectives. The first is to review recent literature that presents, or can be used to calculate, marginal WTP (MWTP) for vehicle attributes to describe the attributes that have been studied and the estimated MWTP values. We found 52 U.S.-focused papers with sufficient data to calculate WTP values for 142 different vehicle attributes, which we organized into 15 general groups of comfort, fuel availability, fuel costs, fuel type, incentives, model availability, non-fuel operating costs, performance, pollution, prestige, range, reliability, safety, size, and vehicle type. Measures of dispersion around central MWTP values typically show large variation in MWTP values for attributes. We explore factors that may contribute to this large variation via analysis of variance (ANOVA) and find that, although most have statistically significant effects, they account for only about one third of the observed variation. Case studies of papers that provide estimates from a variety of model formulations and estimation methods suggest that decisions made by researchers can strongly influence MWTP estimates. The paper's second objective is to seek consensus estimates for WTP for fuel cost reduction and increased acceleration performance. Meta-analysis of MWTP for reduced fuel cost indicates that estimates based on revealed vs. stated preference data differ, as do estimates from models that account for endogeneity and those that do not. We find greater consistency in estimates of MWTP for acceleration despite substantial uncertainty about the overall mean. We conclude with recommendations for improving the understanding of consumers' MWTP for vehicle attributes.

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates.

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δerm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut.

Plant resistance against the parasitic nematode Heterodera schachtii is mediated by MPK3 and MPK6 kinases, which are controlled by the MAPK phosphatase AP2C1 in Arabidopsis.

Plant-parasitic cyst nematodes infect plants and form highly sophisticated feeding sites in roots. It is not known which plant cell signalling mechanisms trigger plant defence during the early stages of nematode parasitism. Mitogen-activated protein kinases (MAPKs) are central components of protein phosphorylation cascades transducing extracellular signals to plant defence responses. MAPK phosphatases control kinase activities and the signalling outcome. The involvement and the role of MPK3 and MPK6, as well as the MAPK phosphatase AP2C1, is demonstrated during parasitism of the beet cyst nematode Heterodera schachtii in Arabidopsis. Our data reveal notable activation patterns of plant MAPKs and the induction of AP2C1 suggesting the attenuation of defence signalling in plant cells during early nematode infection. It is demonstrated that the ap2c1 mutant that is lacking AP2C1 is more attractive but less susceptible to nematodes compared with the AP2C1-overexpressing line. This implies that the function of AP2C1 is a negative regulator of nematode-induced defence. By contrast, the enhanced susceptibility of mpk3 and mpk6 plants indicates a positive role of stress-activated MAPKs in plant immunity against nematodes. Evidence is provided that phosphatase AP2C1, as well as AP2C1-targeted MPK3 and MPK6, are important regulators of plant-nematode interaction, where the co-ordinated action of these signalling components ensures the timely activation of plant defence.

Cell-to-cell propagation of infectious cytosolic protein aggregates.

Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals.

Time-resolved amino acid uptake of Clostridium difficile 630Δerm and concomitant fermentation product and toxin formation.

BACKGROUND: Clostridium difficile is one of the major nosocomial threats causing severe gastrointestinal infections. Compared to the well documented clinical symptoms, little is known about the processes in the bacterial cell like the regulation and activity of metabolic pathways. In this study, we present time-resolved and global data of extracellular substrates and products. In a second part, we focus on the correlation of fermentation products and substrate uptake with toxin production. RESULTS: Formation of different fermentation products during growth in a comparison between the two different media in a global approach was studied using non-targeted gas chromatography-mass spectrometry (GC-MS) based analysis. During cultivation in a casamino acids medium and minimal medium, the clinical isolate C. difficile 630Δerm showed major differences in amino acid utilization: In casamino acids medium, C. difficile preferred proline, leucine and cysteine as carbon and energy sources while glutamate and lysine were not or hardly used. In contrast, proline and leucine were consumed at a significantly later stage in minimal medium. Due to the more complex substrate mixture more fermentation products were detectable in the casamino acids medium, accompanied by major changes in the ratios between oxidative and reductive Stickland products. Different glucose consumption dynamics were observed in presence of either casamino acids or the minimal set of amino acids, accompanied by major changes in butanoate formation. This was associated with a variation in both the toxin yield and a change in the ratio of toxin A to toxin B. CONCLUSIONS: Since in all media compositions, more than one substrate was available as a suitable carbon source, availability of different carbon sources and their metabolic fate appears to be the key factor for toxin formation.

Role of stress-related hormones in plant defence during early infection of the cyst nematode Heterodera schachtii in Arabidopsis.

Heterodera schachtii, a plant-parasitic cyst nematode, invades host roots and induces a specific syncytial feeding structure, from which it withdraws all required nutrients, causing severe yield losses. The system H. schachtii-Arabidopsis is an excellent research model for investigating plant defence mechanisms. Such responses are suppressed in well-established syncytia, whereas they are induced during early parasitism. However, the mechanisms by which the defence responses are modulated and the role of phytohormones are largely unknown. The aim of this study was to elucidate the role of hormone-based defence responses at the onset of nematode infection. First, concentrations of main phytohormones were quantified and the expression of several hormone-related genes was analysed using quantitative real-time (qRT)-PCR or GeneChip. Further, the effects of individual hormones were evaluated via nematode attraction and infection assays using plants with altered endogenous hormone concentrations. Our results suggest a pivotal and positive role for ethylene during nematode attraction, whereas jasmonic acid triggers early defence responses against H. schachtii. Salicylic acid seems to be a negative regulator during later syncytium and female development. We conclude that nematodes are able to impose specific changes in hormone pools, thus modulating hormone-based defence and signal transduction in strict dependence on their parasitism stage.