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Neurofilament light chain (NfL), released during central nervous injury, has evolved as a powerful serum marker of disease severity in many neurological disorders, including infectious diseases. So far NfL has not been assessed in cerebral malaria in human or its rodent model experimental cerebral malaria (ECM), a disease that can lead to fatal brain edema or reversible brain edema. In this study we assessed if NfL serum levels can also grade disease severity in an ECM mouse model with reversible (n = 11) and irreversible edema (n = 10). Blood–brain-barrier disruption and brain volume were determined by magnetic resonance imaging. Neurofilament density volume as well as structural integrity were examined by electron microscopy in regions of most severe brain damage (olfactory bulb (OB), cortex and brainstem). NfL plasma levels in mice with irreversible edema (317.0 ± 45.01 pg/ml) or reversible edema (528.3 ± 125.4 pg/ml) were significantly increased compared to controls (103.4 ± 25.78 pg/ml) by three to five fold, but did not differ significantly in mice with reversible or irreversible edema. In both reversible and irreversible edema, the brain region most affected was the OB with highest level of blood–brain-barrier disruption and most pronounced decrease in neurofilament density volume, which correlated with NfL plasma levels (r = − 0.68, p = 0.045). In cortical and brainstem regions neurofilament density was only decreased in mice with irreversible edema and strongest in the brainstem. In reversible edema NfL plasma levels, MRI findings and neurofilament volume density normalized at 3 months' follow-up. In conclusion, NfL plasma levels are elevated during ECM confirming brain damage. However, NfL plasma levels fail short on reliably indicating on the final outcomes in the acute disease stage that could be either fatal or reversible. Increased levels of plasma NfL during the acute disease stage are thus likely driven by the anatomical location of brain damage, the olfactory bulb, a region that serves as cerebral draining pathway into the nasal lymphatics. [ABSTRACT FROM AUTHOR]

The vaccinia virus (VACV) precursor membrane, the crescent, consists of an open membrane sheet and is formed by rupture of a cellular compartment. Here, we asked whether A17, a viral membrane protein, plays a role in membrane rupture. Without A17 synthesis, crescents are not formed, and instead, tubular and vesicular membranes accumulate (Rodriguez et al. in J Virol 69:4640-4648, ). We used electron tomography (ET) to analyze whether the viral membranes lacking A17 consist of open membrane sheets. Tubular, vesicular and so far not described onion-shaped membranes, which consisted of open membrane sheets, were seen. Thus, the data show that membrane rupture occurs independently of the A17 protein. [ABSTRACT FROM AUTHOR]

The outer membranes of mitochondria and chloroplasts are distinguished by the presence of ß-barrel membrane proteins. The outer membrane of Gram-negative bacteria also harbours ß-barrel proteins. In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10). These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers. Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with a-helical segments, very little is known about how ß-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures. Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane ß-barrel proteins (TOB). We present evidence that important elements of the topogenesis of ß-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors. [ABSTRACT FROM AUTHOR]

The glms ribozyme system has been used as an amenable tool to conditionally control expression of genes of interest. It is generally assumed that insertion of the ribozyme sequence does not affect expression of the targeted gene in the absence of the inducer glucosamine-6-phosphate, although experimental support for this assumption is scarce. Here, we report the unexpected finding that integration of the glms ribozyme sequence in the 3′ untranslated region of a gene encoding a HECT E3 ubiquitin ligase, termed Plasmodium falciparum ubiquitin transferase (PfUT), increased steady state RNA and protein levels 2.5-fold in the human malaria parasite P. falciparum. Overexpression of pfut resulted in an S/M phase-associated lengthening of the parasite's intraerythrocytic developmental cycle and a reduced merozoite invasion efficiency. The addition of glucosamine partially restored the wild type phenotype. Our study suggests a role of PfUT in controlling cell cycle progression and merozoite invasion. Our study further raises awareness regarding unexpected effects on gene expression when inserting the glms ribozyme sequence into a gene locus. [ABSTRACT FROM AUTHOR]

During intraerythrocytic development, the human malaria parasite Plasmodium falciparum alters the mechanical deformability of its host cell. The underpinning biological processes involve gain in parasite mass, changes in the membrane protein compositions, reorganization of the cytoskeletons and its coupling to the plasma membrane, and formation of membrane protrusions, termed knobs. The hemoglobinopathies S and C are known to partially protect carriers from severe malaria, possibly through additional changes in the erythrocyte biomechanics, but a detailed quantification of cell mechanics is still missing. Here, we combined flicker spectroscopy and a mathematical model and demonstrated that knob formation strongly suppresses membrane fluctuations by increasing membrane-cytoskeleton coupling. We found that the confinement increased with hemoglobin S but decreases with hemoglobin C in spite of comparable knob densities and diameters. We further found that the membrane bending modulus strongly depends on the hemoglobinopathetic variant, suggesting increased amounts of irreversibly oxidized hemichromes bound to membranes. Fröhlich et al. show that membrane protrusions of malaria parasite-infected blood cells reduce their membrane fluctuation due to the enforced coupling of membrane and cytoskeleton. Differential cellular mechanics of blood cells possessing variant hemoglobins that protect people from malaria suggests that oxidative stress may explain the selective advantage of certain hemoglobin variants. [ABSTRACT FROM AUTHOR]

PfEMP1 (erythrocyte membrane protein 1) adhesins play a pivotal role in the pathophysiology of falciparum malaria, by mediating sequestration of Plasmodium falciparum-infected erythrocytes in the microvasculature. PfEMP1 variants are expressed by var genes and are presented on membrane elevations, termed knobs. However, the organization of PfEMP1 on knobs is largely unclear. Here, we use super-resolution microscopy and genetically altered parasites expressing a modified var2csa gene in which the coding sequence of the photoactivatable mEOS2 was inserted to determine the number and distribution of PfEMP1 on single knobs. The data were verified by quantitative fluorescence-activated cell sorting analysis and immuno-electron microscopy together with stereology methods. We show that knobs contain 3.3 ± 1.7 and 4.3 ± 2.5 PfEMP1 molecules, predominantly placed on the knob tip, in parasitized erythrocytes containing wild type and sickle haemoglobin, respectively. The ramifications of our findings for cytoadhesion and immune evasion are discussed. Cecilia Sanchez et al. investigate the organization of immune-variant adhesins on Plasmodium falciparum-infected erythrocytes by super-resolution microscopy. Using a fluorescently tagged protein, they obtain quantitative and spatial information about the distribution of adhesin molecules on knobs, which are structures used by the malaria parasite to anchor to the host cells. [ABSTRACT FROM AUTHOR]