Role of polysaccharide and lipid in lipopolysaccharide induced prion protein conversion

Conversion of native cellular prion protein (PrP^c) from an α-helical structure to a toxic and infectious β-sheet structure (PrP^Sc) is a critical step in the development of prion disease. There are some indications that the formation of PrP^Sc is preceded by a β-sheet rich PrP (PrP^β) form which is non-infectious, but is an intermediate in the formation of infectious PrP^Sc. Furthermore the presence of lipid cofactors is thought to be critical in the formation of both intermediate-PrP^β and lethal, infectious PrP^Sc. We previously discovered that the endotoxin, lipopolysaccharide (LPS), interacts with recombinant PrP^c and induces rapid conformational change to a β-sheet rich structure. This LPS induced PrP^β structure exhibits PrP^Sc-like features including proteinase K (PK) resistance and the capacity to form large oligomers and rod-like fibrils. LPS is a large, complex molecule with lipid, polysaccharide, 2-keto-3-deoxyoctonate (Kdo) and glucosamine components. To learn more about which LPS chemical constituents are critical for binding PrP^c and inducing β-sheet conversion we systematically investigated which chemical components of LPS either bind or induce PrP conversion to PrP^β. We analyzed this PrP conversion using resolution enhanced native acidic gel electrophoresis (RENAGE), tryptophan fluorescence, circular dichroism, electron microscopy and PK resistance. Our results indicate that a minimal version of LPS (called detoxified and partially de-acylated LPS or dLPS) containing a portion of the polysaccharide and a portion of the lipid component is sufficient for PrP conversion. Lipid components, alone, and saccharide components, alone, are insufficient for conversion.     

Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.     

High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species

Background

Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.

Methods/Principal Findings

Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.

Conclusions/Significance

HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.     

Fluorescent substrates for ADAM15 useful for assaying and high throughput screening

A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs −2, –9, and −13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M⁻¹ s⁻¹ and 4300 M⁻¹ s⁻¹, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (k_cat/K_m) of 5200 M⁻¹ s⁻¹. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 μM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.     

Geometric and traditional morphometrics for the assessment of character state identity: multivariate statistical analyses of character variation in the genus Arrenurus (Acari,Hydrachnidia, Arrenuridae)

Geometric and traditional morphometric approaches are tested to describe and reveal taxonomic characters and character states in variation of shape and size of idiosoma morphology in the Arrenuridae. Patterns of variation of idiosoma and glandularia features of males from 11 Mexican species of Arrenurus (Megaluracarus) and two species of subgenus Dadayella were explored with five landmark configurations and three sets of interlandmark distances. Separate principal component analyses (PCA) and canonical variate analyses (CVA) were performed for each data set. The eight multivariate analyses of variance among 13 a priori groups (species) detected significantly different morphometric variants, which were interpreted as different taxonomic character states. Patterns of character state similarity among species were examined with unweighted‐pair grouping method using averages (UPGMA) cluster analyses on Mahalanobis distances. Analyses of five landmark configurations revealed important taxonomical variation in the anterior idiosoma outline (10 character states), the outline of the posterior region or cauda (13 states), the distribution of postocularia, and the second and third pairs of dorsoglandularia (nine states), the fourth pair of dorsoglandularia (three states), and ventroglandularia on the posterior side of idiosoma (nine character states). Multivariate analyses of three sets of distance measurements also resulted in the detection of potential taxonomic characters related to idiosoma size (12 character states), postocularia and dorsoglandularia (13 states), and ventroglandularia (nine character states). Morphometric analyses of distances and shapes provide a formal basis for the interpretation of taxonomic characters, and for the discovery of character states. These characters should be investigated further in a wider sample of species for the phylogenetic systematics of these water mites. In the meantime, idiosoma regions and structures were tested for congruence in a phylogenetic analysis, and were proposed as homologous among the species sampled.     

The response of meiofauna and microphytobenthos to engineering effects of fiddler crabs on a subtropical intertidal sandflat

Fiddler crabs are key bioturbators on tidal flats. During their intense bioturbation process, they manipulate large amounts of sediment, altering the physical state of existing materials. We investigated whether different types of sediment bioturbation produced by fiddler crabs modulate meiofaunal assemblages and microphytobenthic content. We hypothesized that sedimentary structures produced by burrowing (the burrow itself and the excavation pellets) and feeding (feeding pellets) generate different microenvironments compared with areas without apparent signs of fiddler crab disturbance, affecting both meiofauna and microphytobenthos, independent of the sampling period. Our results indicate that the engineering effects of burrow construction and maintenance and the engineering effects of fiddler crab foraging modulate meiofaunal assemblages in different ways. Overall, meiofauna from burrows and excavation pellets was more abundant and diverse than at control sites, whereas feeding pellets contained poor meiofaunal assemblages. By contrast, only foraging effects were detected on microphytobenthos; independent of the sampling period, Chl a and phaeopigment content were higher in the feeding pellets, but similar among burrows, excavation pellets and control sites. The present study demonstrates that the different engineering effects of fiddler crabs are an important source of habitat heterogeneity and a structuring agent of meiofaunal assemblages on subtropical tidal flats.