1,076 research outputs found
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)
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 3Â % of total lipids in uninfected to 27Â % 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)
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
Enteroviruses in Respiratory Samples from Paediatric Patients of a Tertiary Care Hospital in Germany
Enteroviruses are associated with various diseases accompanied by rare but severe complications. In recent years, outbreaks of enterovirus D68 and enterovirus A71 associated with severe respiratory infections and neurological complications have been reported worldwide. Since information on molecular epidemiology in respiratory samples is still limited, the genetic diversity of enteroviruses was retrospectively analysed over a 4-year period (2013–2016) in respiratory samples from paediatric patients. Partial viral major capsid protein gene (VP1) sequences were determined for genotyping. Enteroviruses were detected in 255 (6.1%) of 4187 specimens. Phylogenetic analyses of 233 (91.4%) strains revealed 25 different genotypes distributed to Enterovirus A (39.1%), Enterovirus B (34.3%), and Enterovirus D (26.6%). The most frequently detected genotypes were enterovirus D68 (26.6%), coxsackievirus A6 (15.9%), and enterovirus A71 (7.3%). Enterovirus D68 detections were associated with lower respiratory tract infections and increased oxygen demand. Meningitis/encephalitis and other neurological symptoms were related to enterovirus A71, while coxsackievirus A6 was associated with upper respiratory diseases. Prematurity turned out as a potential risk factor for increased oxygen demand during enterovirus infections. The detailed analysis of epidemiological and clinical data contributes to the non-polio enterovirus surveillance in Europe and showed high and rapidly changing genetic diversity of circulating enteroviruses, including different enterovirus D68 variants
Sex-specific Separation of Plasmodium falciparum Gametocyte Populations
Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence- activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.We are
grateful to the Australian Red Cross for providing human red blood cells and serum. Funding was
provided by the Australian Research Council (DP180103212). M.C.R. is supported by the Australian
Government Research Training Program Scholarship and The Australian National University
Novel Method for the Separation of Male and Female Gametocytes of the Malaria Parasite Plasmodium falciparum That Enables Biological and Drug Discovery
We developed a flow-cytometry-based method to separate and collect cocultured male and female Plasmodium falciparum gametocytes responsible for malaria transmission. The purity of the collected cells was estimated at >97% using flow cytometry, and sorted cells were observed by Giemsa-stained thin-smear and live-cell fluorescence microscopy. The expression of validated sex-specific markers corroborated the sorting strategy. Collected male and female gametocytes were used to confirm three novel sex-specific markers by quantitative real-time PCR that were more enriched in sorted male and female gametocyte populations than existing sex-specific markers. We also applied the method as a proof-of-principle drug screen that allows the identification of drugs that kill gametocytes in a sex-specific manner. Since the developed method allowed for the separation of male and female parasites from the same culture, we observed for the first time a difference in development time between the sexes: females developed faster than males. Hence, the ability to separate male and female gametocytes opens the door to a new field of sex-specific P. falciparum gametocyte biology to further our understanding of malaria transmission.Funding was provided by the Australian Research Council (DP180103212). M.C.R. is
supported by the Australian Government Research Training Program Scholarship and
The Australian National Universit
Dialogue acts in Verbmobil 2
This report describes the dialogue phases and the second edition dialogue acts which are used in the VERBMOBIL 2 project [...]. While in the first project phase the scenario was restricted to appointment scheduling dialogues, it has been extended to travel planning in the second phase with appointment scheduling being only a part of the new scenario
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