A Combined Molecular, Cell and Structural Biology Approach Towards Characterising Malaria Alveolins

Intermediate filament (IF)-­‐based cytoskeletal networks in metazoans have key roles in cell architecture and plasticity, and as mechanical stress absorbers. Much less is known about IFs in protozoans. Alveolins are a family of putative IF proteins found exclusively in apicomplexan parasites (causative agents of diseases such as malaria, toxoplasmosis, cryptosporidiosis), dinoflagellate algae and ciliates. All alveolins share functional domains that are characterized by possessing 12 amino-­‐acid tandem repeats. These ‘alveolin’ modules resemble conserved domains found in other protozoan cytoskeletal proteins like articulins. The demonstrated essential nature of alveolins in malaria parasite development, their expression throughout the life cycle, and their absence in vertebrates makes them potentially attractive drug targets for malaria treatment, prophylaxis and transmission control. Moreover, such drugs could be active against a broad range of other apicomplexan parasites, as well as against related pathogenic protozoans. In this context, a better understanding of the core architecture of the Plasmodium alveolins and their assembly mechanisms is important. This project set out to study the structural requirements of the alveolins and their conserved domains for assembly of the protein into the IF network, and for their functional contribution to cell shape, tensile strength and motility in live malaria parasites, using the Plasmodium berghei mouse malaria model. The results reveal, based on the ookinete and sporozoite-­‐ expressed alveolin IMC1h, that the ‘alveolin’ module is required for recruitment into the cortical cytoskeleton, consistent with the notion that it holds the properties for IF formation. In addition, the carboxy-­‐terminal conserved domain of IMC1h, structurally unrelated to the ‘alveolin’ module, is implicated in facilitating parasite motility through direct or indirect interactions with the motility apparatus. In addition, a structural biology approach was undertaken, aimed at determining the core atomic structure of the alveolins, with various techniques at hand to try and determine both tertiary and secondary structures formed by these proteins. Bioinformatic-­‐based analyses indicated that the ‘alveolin’ module is structurally ordered, and adopts a predominantly β-­‐ strand architecture. High level expression, in soluble form, of various P. berghei alveolin domains in bacteria was achieved as amino-­‐terminal fusions with the protein tag NusA. However, further purification of these recombinant alveolins was severely hampered by problems with solubility after cleavage of the NusA tag, or after concentration, resulting in protein precipitation. Whilst these problems have thus far precluded structural analyses by biophysical means, the observations could reflect actual physical properties of the alveolins and the way by which these molecules assemble in the cell into the insoluble IF network structure, possibly via the intermittent formation of shorter oligomers (protofilaments). Work is ongoing to optimise purification protocols

Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months

Pharmacological inhibition of Akt and downstream pathways modulates the expression of COX-2 and mPGES-1 in activated microglia

<p>Abstract</p> <p>Background</p> <p>Microglia are considered a major target for modulating neuroinflammatory and neurodegenerative disease processes. Upon activation, microglia secrete inflammatory mediators that contribute to the resolution or to further enhancement of damage in the central nervous system (CNS). Therefore, it is important to study the intracellular pathways that are involved in the expression of the inflammatory mediators. Particularly, the role of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 (GSK-3) pathways in activated microglia is unclear. Thus, in the present study we investigated the role of Akt and its downstream pathways, GSK-3 and mTOR, in lipopolysaccharide (LPS)-activated primary rat microglia by pharmacological inhibition of these pathways in regard to the expression of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1) and to the production of prostaglandin (PG) E₂and PGD₂.</p> <p>Findings</p> <p>We show that inhibition of Akt by the Akt inhibitor X enhanced the production of PGE₂and PGD₂without affecting the expression of COX-2, mPGES-1, mPGES-2 and cytosolic prostaglandin E synthase (cPGES). Moreover, inhibition of GSK-3 reduced the expression of both COX-2 and mPGES-1. In contrast, the mTOR inhibitor rapamycin enhanced both COX-2 and mPGES-1 immunoreactivity and the release of PGE₂and PGD₂. Interestingly, NVP-BEZ235, a dual PI3K/mTOR inhibitor, enhanced COX-2 and reduced mPGES-1 immunoreactivity, albeit PGE₂and PGD₂levels were enhanced in LPS-stimulated microglia. However, this compound also increased PGE₂in non-stimulated microglia.</p> <p>Conclusion</p> <p>Taken together, we demonstrate that blockade of mTOR and/or PI3K/Akt enhances prostanoid production and that PI3K/Akt, GSK-3 and mTOR differently regulate the expression of mPGES-1 and COX-2 in activated primary microglia. Therefore, these pathways are potential targets for the development of novel strategies to modulate neuroinflammation.</p

Wnt and Hedgehog Are Critical Mediators of Cigarette Smoke-Induced Lung Cancer

BACKGROUND: Lung cancer is the leading cause of cancer death in the world, and greater than 90% of lung cancers are cigarette smoke-related. Current treatment options are inadequate, because the molecular basis of cigarette-induced lung cancer is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that human primary or immortalized bronchial epithelial cells exposed to cigarette smoke for eight days in culture rapidly proliferate, show anchorage-independent growth, and form tumors in nude mice. Using this model of the early stages of smoke-induced tumorigenesis, we examined the molecular changes leading to lung cancer. We observed that the embryonic signaling pathways mediated by Hedgehog and Wnt are activated by smoke. Pharmacological inhibition of these pathways blocked the transformed phenotype. CONCLUSIONS/SIGNIFICANCE: These experiments provide a model in which the early stages of smoke-induced tumorigenesis can be elicited, and should permit us to identify molecular changes driving this process. Results obtained so far indicate that smoke-induced lung tumors are driven by activation of two embryonic regulatory pathways, Hedgehog (Hh) and Wnt. Based on the current and emerging availability of drugs to inhibit Hh and Wnt signaling, it is possible that an understanding of the role of Hh and Wnt in lung cancer pathogenesis will lead to the development of new therapies

Sulindac Sulfide Reverses Aberrant Self-Renewal of Progenitor Cells Induced by the AML-Associated Fusion Proteins PML/RARα and PLZF/RARα

Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings

Involvement of the Glycogen Synthase Kinase-3 Signaling Pathway in TBI Pathology and Neurocognitive Outcome

BACKGROUND: Traumatic brain injury (TBI) sets in motion cascades of biochemical changes that result in delayed cell death and altered neuronal architecture. Studies have demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) effectively reduces apoptosis following a number of stimuli. The Wnt family of proteins, and growth factors are two major factors that regulate GSK-3 activity. In the absence of stimuli, GSK-3 is constitutively active and is complexed with Axin, adenomatous polyposis coli (APC), and casein kinase Iα (CK1α) and phosphorylates ß-Catenin leading to its degradation. Binding of Wnt to Frizzled receptors causes the translocation of GSK-3 to the plasma membrane, where it phosphorylates and inactivates the Frizzled co-receptor lipoprotein-related protein 6 (LRP6). Furthermore, the translocation of GSK-3 reduces ß-Catenin phosphorylation and degradation, leading to ß-Catenin accumulation and gene expression. Growth factors activate Akt, which in turn inhibits GSK-3 activity by direct phosphorylation, leading to a reduction in apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Using a rodent model, we found that TBI caused a rapid, but transient, increase in LRP6 phosphorylation that is followed by a modest decrease in ß-Catenin phosphorylation. Phospho-GSK-3β immunoreactivity was found to increase three days post injury, a time point at which increased Akt activity following TBI has been observed. Lithium influences several neurochemical cascades, including inhibiting GSK-3. When the efficacy of daily lithium was assessed, reduced hippocampal neuronal cell loss and learning and memory improvements were observed. These influences were partially mimicked by administration of the GSK-3-selective inhibitor SB-216763, as this drug resulted in improved motor function, but only a modest improvement in memory retention and no overt neuroprotection. CONCLUSION/SIGNIFICANCE: Taken together, our findings suggest that selective inhibition of GSK-3 may offer partial cognitive improvement. As a broad spectrum inhibitor of GSK-3, lithium offers neuroprotection and robust cognitive improvement, supporting its clinical testing as a treatment for TBI

Sexual violence in the protracted conflict of DRC programming for rape survivors in South Kivu

BACKGROUND: Despite international acknowledgement of the linkages between sexual violence and conflict, reliable data on its prevalence, the circumstances, characteristics of perpetrators, and physical or mental health impacts is rare. Among the conflicts that have been associated with widespread sexual violence has been the one in the Democratic Republic of the Congo (DRC). METHODS: From 2003 till to date Malteser International has run a medico-social support programme for rape survivors in South Kivu province, DRC. In the context of this programme, a host of data was collected. We present these data and discuss the findings within the frame of available literature. RESULTS: Malteser International registered 20,517 female rape survivors in the three year period 2005-2007. Women of all ages have been targeted by sexual violence and only few of those - and many of them only after several years - sought medical care and psychological help. Sexual violence in the DRC frequently led to social, especially familial, exclusion. Members of military and paramilitary groups were identified as the main perpetrators of sexual violence. CONCLUSION: We have documented that in the DRC conflict sexual violence has been - and continues to be - highly prevalent in a wide area in the East of the country. Humanitarian programming in this field is challenging due to the multiple needs of rape survivors. The easily accessible, integrated medical and psycho-social care that the programme offered apparently responded to the needs of many rape survivors in this area

Comprehensive Gene-Expression Survey Identifies Wif1 as a Modulator of Cardiomyocyte Differentiation

During chicken cardiac development the proepicardium (PE) forms the epicardium (Epi), which contributes to several non-myocardial lineages within the heart. In contrast to Epi-explant cultures, PE explants can differentiate into a cardiomyocyte phenotype. By temporal microarray expression profiles of PE-explant cultures and maturing Epi cells, we identified genes specifically associated with differentiation towards either of these lineages and genes that are associated with the Epi-lineage restriction. We found a central role for Wnt signaling in the determination of the different cell lineages. Immunofluorescent staining after recombinant-protein incubation in PE-explant cultures indicated that the early upregulated Wnt inhibitory factor-1 (Wif1), stimulates cardiomyocyte differentiation in a similar manner as Wnt stimulation. Concordingly, in the mouse pluripotent embryogenic carcinoma cell line p19cl6, early and late Wif1 exposure enhances and attenuates differentiation, respectively. In ovo exposure of the HH12 chicken embryonic heart to Wif1 increases the Tbx18-positive cardiac progenitor pool. These data indicate that Wif1 enhances cardiomyogenesis

Sexual violence in the protracted conflict of DRC programming for rape survivors in South Kivu

BACKGROUND: Despite international acknowledgement of the linkages between sexual violence and conflict, reliable data on its prevalence, the circumstances, characteristics of perpetrators, and physical or mental health impacts is rare. Among the conflicts that have been associated with widespread sexual violence has been the one in the Democratic Republic of the Congo (DRC). METHODS: From 2003 till to date Malteser International has run a medico-social support programme for rape survivors in South Kivu province, DRC. In the context of this programme, a host of data was collected. We present these data and discuss the findings within the frame of available literature. RESULTS: Malteser International registered 20,517 female rape survivors in the three year period 2005-2007. Women of all ages have been targeted by sexual violence and only few of those - and many of them only after several years - sought medical care and psychological help. Sexual violence in the DRC frequently led to social, especially familial, exclusion. Members of military and paramilitary groups were identified as the main perpetrators of sexual violence. CONCLUSION: We have documented that in the DRC conflict sexual violence has been - and continues to be - highly prevalent in a wide area in the East of the country. Humanitarian programming in this field is challenging due to the multiple needs of rape survivors. The easily accessible, integrated medical and psycho-social care that the programme offered apparently responded to the needs of many rape survivors in this area