The Plasmodium Export Element Revisited

We performed a bioinformatical analysis of protein export elements (PEXEL) in the putative proteome of the malaria parasite Plasmodium falciparum. A protein family-specific conservation of physicochemical residue profiles was found for PEXEL-flanking sequence regions. We demonstrate that the family members can be clustered based on the flanking regions only and display characteristic hydrophobicity patterns. This raises the possibility that the flanking regions may contain additional information for a family-specific role of PEXEL. We further show that signal peptide cleavage results in a positional alignment of PEXEL from both proteins with, and without, a signal peptide

Human in vitro reporter model of neuronal development and early differentiation processes

<p>Abstract</p> <p>Background</p> <p>During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop <it>in vitro </it>reporter models using doublecortin promoter sequences.</p> <p>Results</p> <p>Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, βIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression.</p> <p>Conclusion</p> <p>Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.</p

The Plasmodium falciparum STEVOR Multigene Family Mediates Antigenic Variation of the Infected Erythrocyte

Modifications of the Plasmodium falciparum–infected red blood cell (iRBC) surface have been linked to parasite-associated pathology. Such modifications enable the parasite to establish long-lasting chronic infection by evading antibody mediate immune recognition and splenic clearance. With the exception of the well-demonstrated roles of var-encoded PfEMP1 in virulence and immune evasion, the biological significance of other variant surface antigens (rif and stevor) is largely unknown. While PfEMP1 and RIFIN have been located on the iRBC surface, recent studies have located STEVOR at the iRBC membrane where it may be exposed on the erythrocyte surface. To investigate the role of STEVOR in more detail, we have developed antibodies against two putative STEVOR proteins and used a combination of indirect immunofluorescence assays (IFA), live IFA, flow cytometry, as well as agglutination assays, which enable us to demonstrate that STEVOR is clonally variant at the surface of schizont stage parasites. Crucially, expression of different STEVOR on the surface of the iRBC changes the antigenic property of the parasite. Taken together, our data for the first time demonstrate that STEVOR plays a role in creating antigenic diversity of schizont stage parasites, thereby adding additional complexity to the immunogenic properties of the iRBC. Furthermore, it clearly demonstrates that to obtain a complete understanding of how parasite-induced pathology is linked to variation on the surface of the iRBC, focusing the interactions of multiple multigene families needs to be considered

Compartmentation of Redox Metabolism in Malaria Parasites

Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito – a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes – glutathione reductase and thioredoxin reductase – Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention

The Malaria Secretome: From Algorithms to Essential Function in Blood Stage Infection

The malaria agent Plasmodium falciparum is predicted to export a “secretome” of several hundred proteins to remodel the host erythrocyte. Prediction of protein export is based on the presence of an ER-type signal sequence and a downstream Host-Targeting (HT) motif (which is similar to, but distinct from, the closely related Plasmodium Export Element [PEXEL]). Previous attempts to determine the entire secretome, using either the HT-motif or the PEXEL, have yielded large sets of proteins, which have not been comprehensively tested. We present here an expanded secretome that is optimized for both P. falciparum signal sequences and the HT-motif. From the most conservative of these three secretome predictions, we identify 11 proteins that are preserved across human- and rodent-infecting Plasmodium species. The conservation of these proteins likely indicates that they perform important functions in the interaction with and remodeling of the host erythrocyte important for all Plasmodium parasites. Using the piggyBac transposition system, we validate their export and find a positive prediction rate of ∼70%. Even for proteins identified by all secretomes, the positive prediction rate is not likely to exceed ∼75%. Attempted deletions of the genes encoding the conserved exported proteins were not successful, but additional functional analyses revealed the first conserved secretome function. This gave new insight into mechanisms for the assembly of the parasite-induced tubovesicular network needed for import of nutrients into the infected erythrocyte. Thus, genomic screens combined with functional assays provide unexpected and fundamental insights into host remodeling by this major human pathogen

Biophysics of Malarial Parasite Exit from Infected Erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.Singapore. Agency for Science, Technology and ResearchSingapore-MIT AllianceGlobal Enterprise for Micro-Mechanics and Molecular MedicineNational University of SingaporeNational Institutes of Health (U.S.) (Grant R01 HL094270-01A1)National Institutes of Health (U.S.) (Grant 1-R01-GM076689-01)National Institutes of Health (U.S.) (P41-RR02594-18-24

Combinatorial Binding in Human and Mouse Embryonic Stem Cells Identifies Conserved Enhancers Active in Early Embryonic Development

Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES) cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas “gene regulatory hotspots” which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints

Human pluripotent embryonal carcinoma NTERA2 cl.D1 cells maintain their typical morphology in an angiomyogenic medium

BACKGROUND: Pluripotent embryonal carcinomas are good potential models, to study, "in vitro," the mechanisms that control differentiation during embryogenesis. The NTERA2cl.D1 (NT2/D1) cell line is a well known system of ectodermal differentiation. Retinoic acid (RA) induces a dorsal pattern of differentiation (essentially neurons) and bone morphogenetic protein (BMP) or hexamethylenebisacetamide (HMBA) induces a more ventral (epidermal) pattern of differentiation. However, whether these human cells could give rise to mesoderm derivatives as their counterpart in mouse remained elusive. We analyzed the morphological characteristics and transcriptional activation of genes pertinent in cardiac muscle and endothelium differentiation, during the growth of NT2/D1 cells in an inductive angiomyogenic medium with or without Bone Morphogenetic Protein 2 (BMP2). RESULTS: Our experiments showed that NT2/D1 maintains their typical actin organization in angiomyogenic medium. Although the beta myosin heavy chain gene was never detected, all the other 15 genes analyzed maintained their expression throughout the time course of the experiment. Among them were early and late cardiac, endothelial, neuronal and teratocarcinoma genes. CONCLUSION: Our results suggest that despite the NT2/D1 cells natural tendency to differentiate into neuroectodermal lineages, they can activate genes of mesodermal lineages. Therefore, we believe that these pluripotent cells might still be a good model to study biological development of mesodermal derivatives, provided the right culture conditions are met

The Tumorigenicity of Mouse Embryonic Stem Cells and In Vitro Differentiated Neuronal Cells Is Controlled by the Recipients' Immune Response

Embryonic stem (ES) cells have the potential to differentiate into all cell types and are considered as a valuable source of cells for transplantation therapies. A critical issue, however, is the risk of teratoma formation after transplantation. The effect of the immune response on the tumorigenicity of transplanted cells is poorly understood. We have systematically compared the tumorigenicity of mouse ES cells and in vitro differentiated neuronal cells in various recipients. Subcutaneous injection of 1×106 ES or differentiated cells into syngeneic or allogeneic immunodeficient mice resulted in teratomas in about 95% of the recipients. Both cell types did not give rise to tumors in immunocompetent allogeneic mice or xenogeneic rats. However, in 61% of cyclosporine A-treated rats teratomas developed after injection of differentiated cells. Undifferentiated ES cells did not give rise to tumors in these rats. ES cells turned out to be highly susceptible to killing by rat natural killer (NK) cells due to the expression of ligands of the activating NK receptor NKG2D on ES cells. These ligands were down-regulated on differentiated cells. The activity of NK cells which is not suppressed by cyclosporine A might contribute to the prevention of teratomas after injection of ES cells but not after inoculation of differentiated cells. These findings clearly point to the importance of the immune response in this process. Interestingly, the differentiated cells must contain a tumorigenic cell population that is not present among ES cells and which might be resistant to NK cell-mediated killing

Stripping the Boss : The Powerful Role of Humor in the Egyptian Revolution 2011

The Egyptian Revolution 2011 has shaken the Arab world and stirred up Middle-East politics. Moreover, it caused a rush in political science and the neighboring disciplines, which had not predicted an event like this and now have troubles explaining it. While many things can be learned from the popular uprising, and from the limitations of previous scholarship, our focus will be on a moral resource, which has occasionally been noticed, but not sufficiently explored: the role of humor in keeping up the spirit of the Revolution. For eighteen days, protestors persevered at Liberation Square in Central Cairo, the epicenter of resistance; at times a few dozens, at times hundreds of thousands. What they did was to fight the terror of the regime, which reached absurd peaks during those days, with humor – successfully. We offer a social-functionalist account of the uprising, which includes behavioral as well as cultural levels of analysis, and illuminates how humorous means helped to achieve deadly serious goals. By reconstructing how Egyptians laughed themselves into democracy, we outline a social psychology of resistance, which uses humor both as a sword and a shield.Peer reviewe