The Ascomycete Verticillium longisporum Is a Hybrid and a Plant Pathogen with an Expanded Host Range

Hybridization plays a central role in plant evolution, but its overall importance in fungi is unknown. New plant pathogens are thought to arise by hybridization between formerly separated fungal species. Evolution of hybrid plant pathogens from non-pathogenic ancestors in the fungal-like protist Phytophthora has been demonstrated, but in fungi, the most important group of plant pathogens, there are few well-characterized examples of hybrids. We focused our attention on the hybrid and plant pathogen Verticillium longisporum, the causal agent of the Verticillium wilt disease in crucifer crops. In order to address questions related to the evolutionary origin of V. longisporum, we used phylogenetic analyses of seven nuclear loci and a dataset of 203 isolates of V. longisporum, V. dahliae and related species. We confirmed that V. longisporum was diploid, and originated three different times, involving four different lineages and three different parental species. All hybrids shared a common parent, species A1, that hybridized respectively with species D1, V. dahliae lineage D2 and V. dahliae lineage D3, to give rise to three different lineages of V. longisporum. Species A1 and species D1 constituted as yet unknown taxa. Verticillium longisporum likely originated recently, as each V. longisporum lineage was genetically homogenous, and comprised species A1 alleles that were identical across lineages

Deciphering the secret message within bone microstructure

Bones are subjected to a variety of loads during a lifetime. Already in the 19th century it was suggested that the microstructure of bones is adapted to these loads and that this is achieved by continuous modelling and remodelling in which cells add tissue where needed and resorb it where not needed. This process ultimately leads to a mechanically optimised structure with a uniform tissue loading distribution and is nowadays commonly accepted and referred to as ‘Wolff’s law’. Assuming that bone strives for a uniform tissue loading, we here hypothesise that it might be possible to derive a bone’s loading history from its microstructure by finding a set of external forces that results in the most uniform tissue loading. This would be the inverse of ‘Wolff’s law’. If deriving loading histories from bone microstructure would be indeed possible, this would enable estimations of bone loading where it is difficult or impossible to measure them in any other way, such as for fossil bones or bones in vivo. The former would enable, for example, making inferences about the locomotion of extinct animals from the derived loading histories. The latter would enable to determine in vivo loading conditions that are required for patient-specific bone remodelling studies. In such remodelling studies, load-driven bone remodelling simulations could be used to study the effects of diseases and treatments on the bone microstructure of patients. However, in vivo loading is usually not known and it has not yet been shown that load-driven remodelling can be assumed in human bones. In my doctoral thesis, I therefore explored the concept of deriving loading histories from bone microstructure and investigated the clinically important application of estimating realistic in vivo loading conditions for patient-specific bone remodelling simulations. To do so, a novel approach to estimate bone loading histories from bone microstructure was first developed. The method can be summarized in three steps: First, unit load cases are defined and the resultant tissue loading is calculated using micro-finite element analysis. Second, the predefined load cases are scaled until the most uniform tissue loading is achieved when the results of each load case are added. Third, the final loading history is determined from the scaling factors of the unit load cases. Using this approach, validation and feasibility studies were carried out to investigate the accuracy and robustness of the algorithm. In a first study, compressive forces that were applied to murine caudal vertebrae during an animal experiment were estimated from the developed bone microstructures measured in vivo by micro-CT. The algorithm successfully derived the magnitude of this simple load case. In order to further validate the load estimation algorithm for more complex loading situations and to control the state of mechanical adaptation, we conducted a study in which the loading history from a set of synthetic bone structures was derived. These structures were generated by performing bone remodelling simulations where the loading history as well as the degree of mechanical adaptation can be controlled exactly. It was found that differences between the estimates based on the adapted structures and the actually applied loading in the simulations were less than 4.4%. To challenge the load estimation algorithm even more and to validate it with human bones, forces working in the human distal radius were estimated based on high-resolution peripheral CT images and compared to literature data in a third study. It was found that these estimates well compared to values reported in experimental studies. Finally, as a proof-of-concept of this approach to decipher realistic more complex loading patterns as they occur at the hip joint, forces acting at the human and canine femoral head were estimated from the bone microstructure as obtained from micro-CT scans. Here also, the estimated forces were in good agreement with direct in vivo measurements reported in the literature and reflected the loading conditions during walking and thus a realistic loading pattern. To investigate the clinically important application of the load estimation algorithm – to determine in vivo loading conditions needed for patient-specific bone remodelling studies – two more studies were performed. In a first study, the load estimation algorithm was used to derive patient-specific loading conditions from bone biopsies based on which bone remodelling simulations of hypoparathyroidism were performed. It was found that the changes in the bone structure predicted by the simulation compared successfully to those seen in the patients. In a second study, bone loading was estimated from high-resolution in vivo peripheral CT images of the human distal tibia. Additionally, bone remodelling sites were quantified by comparing baseline and follow-up scans. It was found that the local bone tissue loading conditions were good predictors for bone loss and gain as quantified from the images. This result thus confirms the assumption of load-driven bone remodelling. Furthermore, we developed a prototype for clinical bone remodelling simulations. Healthy remodelling was simulated based on high-resolution in vivo peripheral CT images of the distal radius and resultant microstructures were compared to follow-up measurements. Morphometric parameters of baseline did not differ much from 6-months follow-up and simulated bone microstructures, indicating a realistic prediction of bone remodelling. It also confirms the estimation of realistic in vivo loading conditions for such simulations because otherwise the bone microstructure would have changed due to mechanical adaptation. These results are in agreement with our hypothesis that there is a strong correlation between bone microstructure and functional use and that this form-function relationship allows deriving loading histories from bone microstructure, which can be used for patient-specific bone remodelling simulations. We conclude that the results obtained so far support the idea that deciphering the secret message within bone microstructure is possible

Determination of physiological external loading conditions based on bone morphology

No abstract

Challenges in longitudinal measurements with HR-pQCT: evaluation of a 3D registration method to improve bone microarchitecture and strength measurement reproducibility

Definition of identical regions between repeated computed tomography (CT) scans is a key factor to monitor changes in bone microarchitecture. In longitudinal studies, accurate determination of the volume of interest (VOI), using three dimensional (3D) registration may improve precision. Therefore, the aim of our study was to investigate the short-term reproducibility of bone geometry, density, microstructure and biomechanical parameters assessed by HR-pQCT and micro-finite element (µFE) derived analyses, using the cross-sectional area (CSA) registration method in comparison with the use of 3D registration, to find overlapping regions between scans. Fifteen healthy individuals (aged 21–47 years) underwent 3 separate scans at the distal radius and tibia, within a one-month interval. Reproducibility was assessed after double contouring the cortical compartment and after applying three different methods to determine the common region between repeated scans: (i) the VOI was determined with no registration, i.e., on 110 slices, (ii) the VOI was determined after CSA-based registration, and (iii) the VOI was determined after 3D registration. Both pre- and post-registration short-term reproducibility for each subject was determined. With no registration, CVrms of geometry parameters ranged from 0.5 to 3.7%, showing a slight variation in the CSA between scans. When the CSA registration method was employed, the variability of geometry (CVrms <1.8%) and density parameters (CVrms <1.8%), was better than that obtained without registration. By removing the effect of repositioning, the 3D registration further improved the reproducibility of cortical bone measurements compared to other methods. Indeed, significant improvements were found for cortical geometry and microstructure measurements (CVrms ranged from 0.4% to 10.7% at both sites; p <0.05), whereas the impact on trabecular bone measurements was restricted to its geometry parameter. The repositioning error was significantly reduced, most markedly at the radius compared to the tibia. For µFE measures, the impact of 3D registration on whole bone stiffness was negligible, indicating adequate assessment of longitudinal changes in estimated biomechanical properties, even without registration. In conclusion, we have shown that the 3D registration improved the identification of the common region retained for longitudinal analysis, contributing to improve the reproducibility of cortical bone parameter measurements. We also quantified the minimally detectable bone changes to help designing future studies with HR-pQCT

Subject-specific bone loading estimation in the human distal radius

High-resolution in vivo bone micro-architecture assessment, as possible now for the distal forearm, in combination with bone remodelling simulation algorithms could, eventually, predict patient-specific bone morphology changes. To simulate load-adaptive bone remodelling, however, physiological loading conditions must be defined. In this paper we test a previously developed algorithm to estimate such physiological loading conditions from the bone micro-architecture. The aims of this study were to investigate if realistic boundary forces and moments are predicted for the scanned distal radius section and how these predicted forces and moments should be distributed to the scanned section in order to obtain a load transfer similar to that in situ. Images at in vivo resolution were generated for the clinically measured section of nine distal radius cadaver bones, converted to micro-finite element models and used for load estimation. Models of the full distal radius were created to analyse tissue loading distributions of the sections in situ. It was found that predicted forces and moments at the boundaries of the scanned region varied considerably but, when translated to equivalent radiocarpal joint forces, agreed well with values reported in the literature. Bone tissue loading distribution was in best agreement with in situ distributions when loading was applied to an extra layer of material at both ends of the clinical scan region. The agreement of the predicted loading to previous studies and the wide range of predicted loading values indicate that subject-specific bone loading estimation is possible and necessary

Bone remodelling in humans is load-driven but not lazy

During bone remodelling, bone cells are thought to add and remove tissue at sites with high and low loading, respectively. To predict remodelling, it was proposed that bone is removed below and added above certain thresholds of tissue loading and within these thresholds, called a ‘lazy zone’, no net change in bone mass occurs. Animal experiments linking mechanical loading with changes in bone density or microstructure support load-adaptive bone remodelling, while in humans the evidence for this relationship at the micro-scale is still lacking. Using new high-resolution CT imaging techniques and computational methods, we quantify microstructural changes and physiological tissue loading in humans. Here, we show that bone remodelling sites in healthy postmenopausal women strongly correlate with tissue loading following a linear relationship without a ‘lazy zone’ providing unbiased evidence for load-driven remodelling in humans. This suggests that human and animal bones both react to loading induced remodelling in a similar fashion

Large-scale microstructural simulation of load-adaptive bone remodeling in whole human vertebrae

\u3cp\u3eIdentification of individuals at risk of bone fractures remains challenging despite recent advances in bone strength assessment. In particular, the future degradation of the microstructure and load adaptation has been disregarded. Bone remodeling simulations have so far been restricted to small-volume samples. Here, we present a large-scale framework for predicting microstructural adaptation in whole human vertebrae. The load-adaptive bone remodeling simulations include estimations of appropriate bone loading of three load cases as boundary conditions with microfinite element analysis. Homeostatic adaptation of whole human vertebrae over a simulated period of 10 years is achieved with changes in bone volume fraction (BV/TV) of less than 5 %. Evaluation on subvolumes shows that simplifying boundary conditions reduces the ability of the system to maintain trabecular structures when keeping remodeling parameters unchanged. By rotating the loading direction, adaptation toward new loading conditions could be induced. This framework shows the possibility of using large-scale bone remodeling simulations toward a more accurate prediction of microstructural changes in whole human bones.\u3c/p\u3