Virtual Prototyping for validation of functional architectures

International audienceThis paper will present a new approach how to use virtual prototyping to validate functional architectures. The approach points out how functional architecture can be set up and tested in order to get highly validated data before making final architectural/design decisions. The introduction will speak about the current facts, the constrains and the tendencies to create a common and understandable picture in which the proposed solution may be implemented. Then we will give a description and definition of MiLMiL / SiLSiL / HiL, rapid prototyping and virtual prototyping to avoid confusion. The main part will discuss functional and logical architecture, the mapping of both as well a the variants that have to be considered. It will be shown how validation by virtual prototyping can help to define and proof architectural decision. Finally we will give a short outlook on how to migrate from virtual prototyping systems via rapid prototyping via fullpass and bypass to HiL applications. The conclusion summarizes the advantages compared to the current situation

Binding of coatomer by the PEX11 C-terminus is not required for function

AbstractMicrobodies are single membrane-bound organelles found in eukaryotes from trypanosomes to man. Although they have diverse roles in metabolism, the mechanisms and molecules involved in membrane biogenesis and matrix protein import are conserved. Similarly, the basic mechanisms and structures involved in vesicular transport are similar throughout eukaryotic evolution. The PEX11 proteins are required for the division of microbodies in trypanosomes, yeast and mammals, and a role of coatomer in this process has been suggested. We show here that the binding of trypanosome, yeast and bovine coatomers to selected peptides is identical. Coatomer binds to the C-termini of trypanosome PEX11 and rat Pex11α, but not yeast Pex11p or human Pex11β. Mutations of the C-terminus of trypanosome PEX11 that eliminated coatomer binding did not affect function in yeast or trypanosomes. Thus binding of coatomer to the C-terminus of PEX11 is not required for PEX11 function

Plasmodium falciparum exported protein PFE60 influences Maurer's clefts architecture and virulence complex composition

Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature erythrocyte host cell upon invasion for its own survival. Maurer's clefts (MC) are membraneous structures established by the parasite in the cytoplasm of infected cells. These organelles are deemed essential for trafficking of virulence complex proteins. The display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell and the subsequent cytoadhesion of infected cells in the microvasculature of vital organs is the key mechanism that leads to the pathology associated with malaria infection. In a previous study we established that PFE60 (PIESP2) is one of the protein components of this complex. Here we demonstrate that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected red blood cells. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore - unlike all other described resident MC membrane proteins - PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a red blood cell surface antigen.This work was supported by the Australian Research Council (DP1093518 and DP0878953)

Automated Fourier space region-recognition filtering for off-axis digital holographic microscopy

Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding to extracts the optimal shape of frequency components. The region recognition technique offers fully automated adaptive filtering that can operate with a variety of samples and imaging conditions. When imaging through optically scattering biological hydrogel matrix, the technique surpasses previous histogram thresholding techniques without requiring any manual intervention. Finally, we automate the extraction of the statistical difference of optical height between malaria parasite infected and uninfected red blood cells. The method described here pave way to greater autonomy in automated DHM imaging for imaging live cell in thick cell cultures

Teamwork in an honours group writing assignment

Scientific practice is essentially collaborative. Most research publications list multiple authors making collaborative writing a key skill for scientists. This paper reports the student experience of a collaborative writing task for honours students in experimental science. Students were asked to work in groups of five to research and construct a scientific review suitable for publication in a peer-reviewed journal. Students submitted a piece of individual writing as well as the final group review and where also asked to assess the contribution of group members. Students found group work demanding and this appeared to overshadow the experience of collaborative writing. However, at the same time, students strongly agreed that teamwork skills and collaboration were essential for successful research. This dichotomy between the need for collaborative skills and the difficulty of putting this into practice argues for greater development of teamwork skills in the undergraduate curriculum in preparation for research training

The role of cholesterol in invasion and growth of malaria parasites

Malaria parasites are unicellular eukaryotic pathogens that develop through a complex lifecycle involving two hosts, an anopheline mosquito and a vertebrate host. Throughout this lifecycle, the parasite encounters widely differing conditions and survives in distinct ways, from an intracellular lifestyle in the vertebrate host to exclusively extracellular stages in the mosquito. Although the parasite relies on cholesterol for its growth, the parasite has an ambiguous relationship with cholesterol: cholesterol is required for invasion of host cells by the parasite, including hepatocytes and erythrocytes, and for the development of the parasites in those cells. However, the parasite is unable to produce cholesterol itself and appears to remove cholesterol actively from its own plasma membrane, thereby setting up a cholesterol gradient inside the infected host erythrocyte. Overall a picture emerges in which the parasite relies on host cholesterol and carefully controls its transport. Here, we describe the role of cholesterol at the different lifecycle stages of the parasites

Invasion by P. falciparum Merozoites Suggests a Hierarchy of Molecular Interactions

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor–ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines

Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum

Abstract Background The development of differentiated sexual stages (gametocytes) within human red blood cells is essential for the propagation of the malaria parasite, since only mature gametocytes will survive in the mosquito’s midgut. Hence gametocytogenesis is a pre-requisite for transmission of the disease. Physiological changes involved in sexual differentiation are still enigmatic. In particular the lipid metabolism—despite being central to cellular regulation and development—is not well explored. Methods Here the lipid profiles of red blood cells infected with the five different sexual stages of Plasmodium falciparum were analysed by mass spectrometry and compared to those from uninfected and asexual trophozoite infected erythrocytes. Results Fundamental differences between erythrocytes infected with the different parasite stages were revealed. In mature gametocytes many lipids that decrease in the trophozoite and early gametocyte infected red blood cells are regained. In particular, regulators of membrane fluidity, cholesterol and sphingomyelin, increased significantly during gametocyte maturation. Neutral lipids (serving mainly as caloriometric reserves) increased from 3 % of total lipids in uninfected to 27 % in stage V gametocyte infected red blood cells. The major membrane lipid class (phospholipids) decreased during gametocyte development. Conclusions The lipid profiles of infected erythrocytes are characteristic for the particular parasite life cycle and maturity stages of gametocytes. The obtained lipid profiles are crucial in revealing the lipid metabolism of malaria parasites and identifying targets to interfere with this deadly disease.We are grateful to the Australian Red Cross for providing human RBCs and serum. Support of the Australian Research Council is acknowledged. TWM is an Australian Research Council Future Fellow (FT110100249)