Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

Reduction of elasmobranch by-catch in the hake semipelagic near-bottom longline fishery in the Algarve (Southern Portugal)

Elasmobranch fish, particularly deep-sea sharks, are the most important component of the by-catch of the hake semipelagic near-bottom 'pedra-e-bola' longline fishery in the Algarve (South Portugal) and most of these fish are discarded. The effects of the removal of the lower hooks were evaluated, in terms of target and by-catch reductions, by quantifying the catches of each hook relative to the distance from the bottom. The analysis showed that most European hake (Merluccius merluccius), the target species of this fishery, were caught in the middle range of the hooks, with very few individuals caught near the bottom, whereas for sharks the situation was the opposite, with most hooked near the bottom. The removal of the lower three pairs of hooks would result in a small reduction in the catch of the target species, but a much more significant reduction in elasmobranch by-catch. In the specific case of the blackmouth catshark (Galeus melastomus), discard mortality would be further minimized due to the fact that the lower hooks capture significantly smaller animals that are always discarded compared with hooks that are more distant from the bottom

Children with cerebral malaria or severe malarial anaemia lack immunity to distinct variant surface antigen subsets

Abstract Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or “lacunae”, in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children

Molecular and neurological characterizations of three Saudi families with lipoid proteinosis

<p>Abstract</p> <p>Background</p> <p>Lipoid proteinosis is a rare autosomal recessive disease characterized by cutaneous and mucosal lesions and hoarseness appearing in early childhood. It is caused by homozygous or compound heterozygous mutations in the <it>ECM1 </it>gene. The disease is largely uncharacterized in Arab population and the mutation(s) spectrum in the Arab population is largely unknown. We report the neurologic and neuroradiologic characteristics and <it>ECM1 </it>gene mutations of seven individuals with lipoid proteinosis (LP) from three unrelated consanguineous families.</p> <p>Methods</p> <p>Clinical, neurologic, and neuro-ophthalmologic examinations; skin histopathology; brain CT and MRI; and sequencing of the full<it>ECM1 </it>gene.</p> <p>Results</p> <p>All seven affected individuals had skin scarring and hoarseness from early childhood. The two children in Family 1 had worse skin involvement and worse hoarseness than affected children of Families 2 and 3. Both children in Family 1 were modestly mentally retarded, and one had typical calcifications of the amygdalae on CT scan. Affected individuals in Families 2 and 3 had no grossneurologic, neurodevelopmental, or neuroimaging abnormalities. Skin histopathology was compatible with LP in all three families. Sequencing the full coding region of <it>ECM1 </it>gene revealed two novel mutationsin Family 1 (c.1300-1301delAA) and Family 2 (p.Cys269Tyr) and in Family 3 a previously described 1163 bp deletion starting 34 bp into intron 8.</p> <p>Conclusions</p> <p>These individuals illustrate the neurologic spectrum of LP, including variable mental retardation, personality changes, and mesial temporal calcificationand imply that significant neurologic involvement may be somewhat less common than previously thought. The cause of neurologic abnormalities was not clear from either neuroimaging or from what is known about <it>ECM1 </it>function. The severity of dermatologic abnormalities and hoarseness generally correlated with neurologic abnormalities, with Family 1 being somewhat more affected in all spheres than the other two families. Nevertheless, phenotype-genotype correlation was not obvious, possibly because of difficulty quantifying the neurologic phenotype and because of genetic complexity.</p

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.Fundacao de Amparo Pesquisa do Estado de Sao Paulo-FAPESP[07/07139-3]Coordenaco de Aperfeioamento de Pessoal de Nivel Superior-CAPESInstituto Nacional de Ciencia e Tecnologia de Complexos Fluidos (INCT)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNP

Diagnostic accuracy of cerebrospinal fluid protein markers for sporadic Creutzfeldt-Jakob disease in Canada: a 6-year prospective study

<p>Abstract</p> <p>Background</p> <p>To better characterize the value of cerebrospinal fluid (CSF) proteins as diagnostic markers in a clinical population of subacute encephalopathy patients with relatively low prevalence of sporadic Creutzfeldt-Jakob disease (sCJD), we studied the diagnostic accuracies of several such markers (14-3-3, tau and S100B) in 1000 prospectively and sequentially recruited Canadian patients with clinically suspected sCJD.</p> <p>Methods</p> <p>The study included 127 patients with autopsy-confirmed sCJD (prevalence = 12.7%) and 873 with probable non-CJD diagnoses. Standard statistical measures of diagnostic accuracy were employed, including sensitivity (Se), specificity (Sp), predictive values (PVs), likelihood ratios (LRs), and Receiver Operating Characteristic (ROC) analysis.</p> <p>Results</p> <p>At optimal cutoff thresholds (empirically selected for 14-3-3, assayed by immunoblot; 976 pg/mL for tau and 2.5 ng/mL for S100B, both assayed by ELISA), Se and Sp respectively were 0.88 (95% CI, 0.81-0.93) and 0.72 (0.69-0.75) for 14-3-3; 0.91 (0.84-0.95) and 0.88 (0.85-0.90) for tau; and 0.87 (0.80-0.92) and 0.87 (0.84-0.89) for S100B. The observed differences in Sp between 14-3-3 and either of the other 2 markers were statistically significant. Positive LRs were 3.1 (2.8-3.6) for 14-3-3; 7.4 (6.9-7.8) for tau; and 6.6 (6.1-7.1) for S100B. Negative LRs were 0.16 (0.10-0.26) for 14-3-3; 0.10 (0.06-0.20) for tau; and 0.15 (0.09-0.20) for S100B. Estimates of areas under ROC curves were 0.947 (0.931-0.961) for tau and 0.908 (0.888-0.926) for S100B. Use of interval LRs (iLRs) significantly enhanced accuracy for patient subsets [<it>e.g</it>., 41/120 (34.2%) of tested sCJD patients displayed tau levels > 10,000 pg/mL, with an iLR of 56.4 (22.8-140.0)], as did combining tau and S100B [<it>e.g</it>., for tau > 976 pg/mL and S100B > 2.5 ng/mL, positive LR = 18.0 (12.9-25.0) and negative LR = 0.02 (0.01-0.09)].</p> <p>Conclusions</p> <p>CSF 14-3-3, tau and S100B proteins are useful diagnostic markers of sCJD even in a low-prevalence clinical population. CSF tau showed better overall diagnostic accuracy than 14-3-3 or S100B. Reporting of quantitative assay results and combining tau with S100B could enhance case definitions used in diagnosis and surveillance of sCJD.</p

The Structure of the Chemokine Receptor CXCR1 in Phospholipid Bilayers and Interactions with IL-8

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumor growth(1-3). IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signaling pathways result in neutrophil migration to the site of inflammation(2). CXCR1 is a class-A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors(4-7). Despite its importance, its molecular mechanism is poorly understood due to the limited structural information available. Recently, structure determination of GPCRs has advanced by tailoring the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences(8), as well as addition of stabilizing antibodies and small molecules(9) that facilitate crystallization in cubic phase monoolein mixtures(10). The intracellular loops of GPCRs are critical for G-protein interactions(11) and activation of CXCR1 involves both N-terminal residues and extracellular loops(2,12,13). Our previous NMR studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity(14). Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed