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.     

Hawksbill turtle terra incognita: conservation genetics of?eastern Pacific rookeries

Prior to 2008 and the discovery of several important hawksbill turtle (Eretmochelys imbricata) nesting colonies in the EP (Eastern Pacific), the species was considered virtually absent from the region. Research since that time has yielded new insights into EP hawksbills, salient among them being the use of mangrove estuaries for nesting. These recent revelations have raised interest in the genetic characterization of hawksbills in the EP, studies of which have remained lacking to date. Between 2008 and 2014, we collected tissue samples from 269 nesting hawksbills at nine rookeries across the EP and used mitochondrial DNA sequences (766 bp) to generate the first genetic characterization of rookeries in the region. Our results inform genetic diversity, population differentiation, and phylogeography of the species. Hawksbills in the EP demonstrate low genetic diversity: We identified a total of only seven haplotypes across the region, including five new and two previously identified nesting haplotypes (pooled frequencies of 58.4% and 41.6%, respectively), the former only evident in Central American rookeries. Despite low genetic diversity, we found strong stock structure between the four principal rookeries, suggesting the existence of multiple populations and warranting their recognition as distinct management units. Furthermore, haplotypes EiIP106 and EiIP108 are unique to hawksbills that nest in mangrove estuaries, a behavior found only in hawksbills along Pacific Central America. The detected genetic differentiation supports the existence of a novel mangrove estuary “reproductive ecotype” that may warrant additional conservation attention. From a phylogeographic perspective, our research indicates hawksbills colonized the EP via the Indo‐Pacific, and do not represent relict populations isolated from the Atlantic by the rising of the Panama Isthmus. Low overall genetic diversity in the EP is likely the combined result of few rookeries, extremely small reproductive populations and evolutionarily recent colonization events. Additional research with larger sample sizes and variable markers will help further genetic understanding of hawksbill turtles in the EP.     

Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.     

Is COPD a Progressive Disease? A Long Term Bode Cohort Observation

BackgroundThe Global Initiative for Obstructive Lung Diseases (GOLD) defines COPD as a disease that is usually progressive. GOLD also provides a spirometric classification of airflow limitation. However, little is known about the long-term changes of patients in different GOLD grades.ObjectiveExplore the proportion and characteristics of COPD patients that change their spirometric GOLD grade over long-term follow-up.MethodsPatients alive for at least 8 years since recruitment and those who died with at least 4 years of repeated spirometric measurements were selected from the BODE cohort database. We purposely included the group of non survivors to avoid a “survival selection” bias. The proportion of patients that had a change (improvement or worsening) in their spirometric GOLD grading was calculated and their characteristics compared with those that remained in the same grade.ResultsA total of 318 patients were included in the survivor and 217 in the non-survivor groups. Nine percent of survivors and 11% of non survivors had an improvement of at least one GOLD grade. Seventy one percent of survivors and non-survivors remained in the same GOLD grade. Those that improved had a greater degree of airway obstruction at baseline.ConclusionsIn this selected population of COPD patients, a high proportion of patients remained in the same spirometric GOLD grade or improved in a long-term follow-up. These findings suggest that once diagnosed, COPD is usually a non-progressive disease.     

Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats

We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg^-1d^-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg^-1d^-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt–treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt–treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation.     

The European antibody network's practical guide to finding and validating suitable antibodies for research

Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.     

Influence of Obesity and Metabolic Disease on Carotid Atherosclerosis in Patients with Coronary Artery Disease (CordioPrev Study)

BackgroundRecent data suggest that the presence of associated metabolic abnormalities may be important modifiers of the association of obesity with a poorer prognosis in coronary heart disease. We determined the influence of isolated overweight and obesity on carotid intima media thickness (IMT-CC), and also assessed whether this influence was determined by the presence of metabolic abnormalities.Methods1002 participants from the CordioPrev study were studied at entry. We determined their metabolic phenotypes and performed carotid ultrasound assessment. We evaluated the influence of obesity, overweight and metabolic phenotypes on the IMT-CC.ResultsMetabolically sick participants (defined by the presence of two or more metabolic abnormalities) showed a greater IMT-CC than metabolically healthy individuals (p = 4 * 10⁻⁶). Overweight and normal weight patients who were metabolically healthy showed a lower IMT-CC than the metabolically abnormal groups (all p<0.05). When we evaluated only body weight (without considering metabolic phenotypes), overweight or obese patients did not differ significantly from normal-weight patients in their IMT-CC (p = 0.077). However, obesity was a determinant of IMT-CC when compared to the composite group of normal weight and overweight patients (all not obese).ConclusionsIn coronary patients, a metabolically abnormal phenotype is associated with a greater IMT-CC, and may be linked to a higher risk of suffering new cardiovascular events. The protection conferred in the IMT-CC by the absence of metabolic abnormality may be blunted by the presence of obesity.

Trial Registration

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