Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome

Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.     

A combined hydraulic and toxicological approach to assess re-suspended sediments during simulated flood events—part II: an interdisciplinary experimental methodology

Purpose

Flood events are expected to increase both in intensity and frequency due to climate change in the near future. From an environmental toxicology perspective, there is concern that such flood events could lead to the remobilization of contaminated sediment layers in rivers. The aim of this pilot study was to establish a novel and interdisciplinary framework combining methods of hydrodynamic engineering and ecotoxicological assessment to enable investigation of the potential risks associated with such remobilization events.

Materials and methods

Formulated sediment was prepared according to OECD guideline 218 and spiked with a mixture of four polycyclic aromatic hydrocarbons (phenanthrene, chrysene, pyrene, benzo[a]pyrene) at concentrations of 3.3?C8.3?mg?kg^?1 dry weight. Rainbow trout (Oncorhynchus mykiss) were exposed as test animals to re-suspended sediments in three out of five experiments. The experiments were carried out in an annular flume designed to investigate transport behaviour of fine-grained sediments. Several physicochemical (e.g. pH) and sedimentological (e.g. turbidity) parameters were measured to characterise environmental conditions and erosion behaviour of sediments. Furthermore, exposure concentrations were measured by means of an in vitro assay (7-ethoxyresorufin-o-deethylase (EROD), RTL-W1 cell assay) and chemical analysis.

Results and discussion

Preparation and spiking of large amounts of formulated sediments were feasible but not practical. Successful spiking could be confirmed by the bioanalytical methods with the spiked sediments showing significantly elevated EROD induction compared to control sediments. Conditions within the annular flume remained stable throughout all experiments and were adequate to support rainbow trout. Flood events were successfully simulated, resulting in re-suspension of formulated sediment. Different erosion behaviours of sediments during the simulated flood events were observed and could be associated with changes in microbial composition of sediments due to differences in storage conditions. Therefore, maintaining constant storage conditions of formulated sediments is crucial to enable consistency and comparability among erosion experiments.

Conclusions

This study clearly demonstrated the feasibility of a combined hydro-toxicological approach in support of the investigation of the potential ecotoxicological relevance of sediment re-suspension events. However, based on the results presented here, it is recommended to include additional physicochemical parameters, such as redox potential and conductivity, and to extend the experimental setup to natural sediments and different aquatic organisms. Future studies will use natural sediments containing representative microbial communities and extracellular polymeric substances to enable extrapolation from the annular flume to conditions in natural flowing waters.     

First morphological characterization of 'Candidatus Mycoplasma turicensis' using electron microscopy

At least three haemotropic mycoplasmas have been recognized in cats: Mycoplasma haemofelis (Mhf), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus M. turicensis' (CMt). The latter was originally identified in a Swiss pet cat with haemolytic anaemia and shown to be prevalent in domestic cats and wild felids worldwide using molecular methods. So far, there has been no confirmatory morphological evidence of the existence of CMt presumably due to low blood loads during infection while CMhm has only been characterized by light microscopy with discrepant results. This study aimed to provide for the first time electron microscopic characteristics of CMt and CMhm and to compare them to Mhf. Blood samples from cats experimentally infected with CMt, CMhm and Mhf were used to determine copy numbers in blood by real-time PCR and for transmission and scanning electron microscopy. High resolution scanning electron microscopy revealed CMt and CMhm to be discoid-shaped organisms of 0.3 μm in diameter attached to red blood cells (RBCs). In transmission electron microscopy of CMt, an oval organism of about 0.25 μm with several intracellular electron dense structures was identified close to the surface of a RBC. CMhm and CMt exhibited similar morphology to Mhf but had a smaller diameter. This is the first study to provide morphological evidence of CMt thereby confirming its status as a distinct haemoplasma species, and to present electron microscopic features of CMhm.     

A combined hydraulic and toxicological approach to assess re-suspended sediments during simulated flood events. Part I–multiple biomarkers in rainbow trout

Purpose

One of the central issues related to global changes in weather is the increasing occurrence of flood events that can result in the re-suspension of contaminated sediments in rivers. Here, we report on a proof-of-concept study combining hydraulic engineering and ecotoxicology in a new interdisciplinary approach to assess the toxicity of re-suspended polluted sediments after a simulated flood event.

Materials and methods

Rainbow trout (Oncorhynchus mykiss) were exposed for 5 days under simulated flood conditions in an annular flume with artificial sediments that were spiked with a mixture of polycyclic aromatic hydrocarbons (PAH) at environmentally relevant concentrations. Specifically, the objective of this study was to bridge the gap between the physical re-suspension of pollutants and resulting toxicological impacts on aquatic organisms. A suite of different molecular, biochemical and histological markers was used to test the hypothesis that re-suspension of sediments can lead to re-mobilization of PAHs and subsequently to effects on aquatic organisms.

Results and discussion

The micronucleus frequency was significantly 4.3-fold elevated after exposure. There was no significant indication of Aryl hydrocarbon receptor signaling (no EROD induction or increased CYP1A protein content, only slight induction of CYP1A gene expression). Biliary metabolite concentration was the most sensitive marker of PAH exposure. Results for other biomarkers (glutathione-S-transferase, catalase and lipid peroxidation) were inconclusive.

Conclusions

In combination with chemical analyses of suspended matter, the presented approach will be used to improve understanding of the re-mobilization of pollutants from sediments in support of environmental risk assessment.     

In search for the ecological and toxicological relevance of sediment re-mobilisation and transport during flood events

In response to increasing concerns about the potential toxicological impacts of (extreme) flood events, scientists from several disciplines have joined to form the interdisciplinary research project named FLOODSEARCH. FLOODSEARCH is one of the recent Pathfinder Projects supported by the German Excellence Initiative via the Exploratory Research Space at RWTH Aachen (ERS). FLOODSEARCH aims to combine methodologies of hydraulic engineering and ecotoxicology in a new interdisciplinary approach to assess the risks associated with the re-mobilisation of particulate bound contaminants often observed after severe flood events. Impacts of extreme flood events and aspects of re-mobilisation of sediment-bound toxic compounds will be characterised and evaluated in controlled experiments fusing flood simulation technologies with biological effects assessment. The overall goal is to establish a novel and more realistic approach towards flood event testing that can be applied to a number of different questions and species. Specifically, model aquatic species such as rainbow trout (Onchorhynchus mykiss) will be exposed to particle-bound contaminants in flood-like conditions in a specifically designed annular flume that permits monitoring of both physical/chemical and biological parameter. Ultimately, this approach will assist to further our understanding of the potential biological risks associated with increasingly frequent extreme flood events, e.g., as a consequence of climate change, by bridging the gap between the physical (re-)mobilisation of contaminants and resulting toxicological impacts on aquatic organisms. Thus, it is the objective of the project to derive relationships between the hydrodynamic parameters such as velocities and turbulences, the parameters associated to sediment transport such as sediment concentration and grain sizes and the biological parameters.     

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3⁺CD5⁺CD6⁺ T-cells (−40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8 and CD8β lymphocytes, but was not expressed in γδ T-cells or monocyte/macrophages. The perforin positive CD3⁻ subset was phenotypically homogeneous and defined as CD5⁻CD6⁻CD8β⁻CD16⁺CD11b⁺. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3⁺) could be divided into at least three subsets. The first subset was CD4⁻CD5⁺CD6⁺CD11b⁻CD16⁻ most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8β. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4⁺CD5⁺CD6⁺CD8β⁺CD16⁻CD11b⁻ and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3⁺CD4⁻CD5⁺CD6⁻CD8β^±CD11b⁺CD16⁺, and possessed MHC-unrestricted cytotoxicity and LAK activity.     

Clinical outcomes in cats with renal carcinoma undergoing nephrectomy: A retrospective study

Renal carcinomas (RC) are uncommonly encountered in feline medicine. Limited information regarding clinical presentation and postoperative outcomes is available. The purpose of this multi-institutional, retrospective study was to describe the presenting features and clinical outcomes of cats with RC undergoing nephrectomy. Thirty-six client-owned cats were included. Medical records from participating institutions were searched to identify cats that had a histopathologic diagnosis of RC and underwent nephrectomy from January 2001 to October 2021. The most common presenting complaints were weight loss (36.1%) and hyporexia (30.6%). Based on preoperative imaging and intraoperative findings, eight cats had suspected metastasis at the time of surgery (22.2%). Twenty-eight cats survived to discharge (77.8%). Median progression free interval (PFI) could not be determined, as only six cats developed suspected recurrence (16.7%) and seven cats developed suspected metastasis (19.4%). The all-cause median survival time (MST) was 203 days (95% confidence interval [CI]: 84, 1379 days). When cases that died prior to discharge were excluded, MST increased to 1217 days (95% CI: 127, 1641 days). One-year, two-year, and three-year survival rates were all 40.4%. Neither renal tumour histologic subtype nor the presence of preoperative azotemia, anaemia, erythrocytosis, haematuria, or suspected metastasis at diagnosis were found to influence survival. For cats surviving to discharge, prolonged survival times were possible. Further studies are necessary to elucidate other potential prognostic factors, the utility of postoperative adjuvant treatment, and to identify cats at-risk of mortality in the perioperative period.