Conformational plasticity of the Gerstmann-Sträussler-Scheinker disease peptide as indicated by its multiple aggregation pathways

The existence of several prion strains and their capacity of overcoming species barriers seem to point to a high conformational adaptability of the prion protein. To investigate this structural plasticity, we studied here the aggregation pathways of the human prion peptide PrP82-146, a major component of the Gerstmann-Sträussler-Scheinker amyloid disease.By Fourier transform infrared (FT-IR) spectroscopy, electron microscopy, and atomic force microscopy (AFM), we monitored the time course of PrP82-146 fibril formation. After incubation at 37 °C, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands. At a critical oligomer concentration, the emergence of a new FT-IR band allowed to detect fibril formation. A different intermolecular β-sheet interaction of the peptides in oligomers and in fibrils is, therefore, detected by FT-IR spectroscopy, which, in addition, suggests a parallel orientation of the cross β-sheet structures of PrP82-146 fibrils. By AFM, a wide distribution of PrP82-146 oligomer volumes—the smallest ones containing from 5 to 30 peptides—was observed. Interestingly, the statistical analysis of AFM data enabled us to detect a quantization in the oligomer height values differing by steps of ∼ 0.5 nm that could reflect an orientation of oligomer β-strands parallel with the sample surface. Different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly.Thermal aggregation of PrP82-146 was also investigated by FT-IR spectroscopy, which indicated for these aggregates an intermolecular β-sheet interaction different from that observed for oligomers and fibrils. Unexpectedly, random aggregates, induced by solvent evaporation, were found to display a significant α-helical structure as well as several β-sheet components.All these results clearly point to a high plasticity of the PrP82-146 peptide, which was found to be capable of undergoing several aggregation pathways, with end products displaying different secondary structures and intermolecular interactions.     

Medium chain fatty acids in intrauterine growth restricted and small for gestational age pregnancies

Introduction

Low birth weight is associated with an increased risk of heart disease, high blood pressure and diabetes in adult life. Fetal growth is determined by nutrient availability, which is related to placenta nutrient transport. Medium chain fatty acids (MCFAs) are a particular class of nutrients, known to be a readily available energy source. Until now no data are reported on these MCFAs in low birth weight fetus.

Aim

This is a prospective study conducted in a tertiary center of prenatal diagnosis to investigate the maternal and fetal MCFAs levels in appropriate for gestational age (AGA), intrauterine growth restricted (IUGR), and small for gestational age (SGA) pregnancies.

Method

The plasmatic levels of MCFAs in AGA, IUGR and SGA mother–infant pairs were quantified by gas chromatography–mass spectrometry. The analytical method had a linearity range of 0.1–50 mg/L and a limit of quantification of 0.03 mg/L. Reduced fetal growth was defined as an estimated fetal weight below the 3rd–10th percentile for gestational age, with (IUGR) or without (SGA) fetal Doppler abnormalities.

Result

Maternal and fetal MCFAs plasma levels were significantly different among SGA, IUGR and AGA groups. Additionally, the observed MCFAs fetal to maternal ratio is >1 for IUGR group, whilst for SGA and AGA the fetal to maternal ratio is less than one.

Conclusion

Changes in MCFAs levels in fetal and maternal plasma are not related to placental functionality or nutrients availability, suggesting the presence of a de novo biosynthesis.

     

Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages

Chitosan is a linear polymer of N-acetyl-D-glucosamine and deacetylated glucosamine widely used as a wound-healing accelerator in clinical and veterinary medicine. Chitosan enhances the functions of inflammatory cells such as macrophages (Mphi), inducing the production of cytokines as well as the expression of activation markers, Fc receptors and mannose receptor. In this work we studied the effects of chitosan on the arginine metabolic pathways of both resident and inflammatory (proteose-peptone elicited) rat Mphi. Our results show that low molecular weight (LMW) chitosan activated moderately both the inducible nitric oxide synthase (iNOS) and arginase pathways in resident Mphi. In inflammatory Mphi treated with chitosan instead, the arginase activity was strongly enhanced. Supernatants of chitosan-stimulated Mphi enhanced the proliferation of the rat cell line C6. These findings suggest that the healing activity of chitosan could rely on the enhanced arginase activity observed in a wound-associated inflammatory milieu.     

Employment of 16S rDNA gene sequencing techniques for improved identification of difficult-to-identify bacterial veterinary pathogens

To employ 16S rDNA PCR and automated sequencing techniques to identify a collection of bacterial veterinary pathogens from avian, equine, canine and ovine sources, that have proven difficult to identify, employing conventional cultural techniques. Universal or “broad-range” eubacterial PCR was performed on a collection of 46 difficult-to-identify bacterial isolates originating from clinical veterinary specimens. 16S rDNA PCR was performed using two sets of universal primers to successfully generate a composite amplicon of 1,068 bp, which was sequenced to obtain each isolate’s identity. Sequence analysis was able to identify all isolates examined with relative ease. Where the use of molecular identification methods is justified, such as in outbreak control or bioterrorism in animal health, employment of partial 16S rDNA PCR and sequencing employing universal or “broad-range” 16S rDNA, provides a valuable and reliable method of identification of such pathogens.     

Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.     

Environmentally Dependent Morphological Variability in Seven Apomictic Microspecies from Alchemilla L. (Rosaceae)

Apomictic microspecies, for example those of the genus Alchemilla, are often difficult to distinguish. Still, their differences are thought to be persistent due to apomixis. Apomicts are argued to have general-purpose genotypes. The present study aims to assess the variation of morphological characters of Alchemilla depending on environmental conditions both in nature and experimentally; to evaluate the efficacy of characters for identifying the different microspecies; to assess the similarity of different microspecies; and to determine if Alchemilla microspecies have general-purpose genotypes that are not dependent on environmental conditions. The variability of selected characters in seven microspecies of Alchemilla was studied in nature and five microspecies were grown in a common garden experiment. The growing conditions of the latter were subjected to various manipulations (fertilization, shading and irrigation). Typical natural habitats of Alchemilla exhibited only small differences. Environmental conditions therefore had little effect on morphological characters. Neither cultivation in the common garden nor manipulation of conditions therein had a significant impact on the discrimination of microspecies. However, the metric characters were larger in the garden relative to those observed in nature, particularly under fertilization. Fertilization affected most characters, whereas shading and irrigation did not. The most effective characters for discriminating between microspecies were the ratios of the metric characters. Two species pairs: A. vulgaris and A. micans, as well as A. glaucescens and A. hirsuticaulis were morphologically close; however, the species within these pairs could still be distinguished. The morphological characters of Alchemilla microspecies were only slightly dependent on the environmental conditions.     

A malaria parasite-encoded vacuolar H(+)-ATPase is targeted to the host erythrocyte

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H(+)-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H(+)-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pH(i)) of the infected erythrocyte. Our results also indicate that although the pH(i) maintained by the V-H(+)-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.     

Relics of the Europe's warm past: phylogeography of the Aesculapian snake

Understanding how species responded to past climate change can provide information about how they may respond to the current global warming. Here we show how a European reptile species responded to the last natural global warming event at the Pleistocene-Holocene transition that led to the Holocene climatic optimum approximately 5000-8000 years ago. The Aesculapian snake, Zamenis longissimus, is a thermophilous species whose present-day distribution in the southern half of Europe is a remnant of much wider range during the Holocene climatic optimum when populations occurred as far north as Denmark. These northern populations went extinct as the climate cooled, and presently the species is extinct from all central Europe, except few relic populations in locally suitable microhabitats in Germany and the Czech Republic. Our phylogenetic and demographic analyses identified two major clades that expanded from their respective western and eastern refugia after the last glacial maximum (18,000-23,000 years ago) and contributed approximately equally to the present range. Snakes from the relic northern populations carried the Eastern clade, showing that it was primarily the snakes from the eastern, probably Balkan, refugium that occupied the central and northern Europe during the Holocene climatic optimum. Two small, deep-branching clades were identified in near the Black Sea and in Greece. These clades provide evidence for two additional refugia, which did not successfully contribute to the colonization of Europe. If, as our results suggest, some populations responded to the mid-Holocene global warming by shifting their ranges further north than other populations of the same species, knowing what populations were able to expand in different species may provide information about what populations will be important for the species' ability to cope with the current global warming.     

Migration of periodontal ligament fibroblasts on nanometric topographical patterns: influence of filopodia and focal adhesions on contact guidance

Considered to be the "holy grail" of dentistry, regeneration of the periodontal ligament in humans remains a major clinical problem. Removal of bacterial biofilms is commonly achieved using EDTA gels or lasers. One side effect of these treatment regimens is the etching of nanotopographies on the surface of the tooth. However, the response of periodontal ligament fibroblasts to such features has received very little attention. Using laser interference lithography, we fabricated precisely defined topographies with continuous or discontinuous nanogrooves to assess the adhesion, spreading and migration of PDL fibroblasts. PDL fibroblasts adhered to and spread on all tested surfaces, with initial spreading and focal adhesion formation slower on discontinuous nanogrooves. Cells had a significantly smaller planar area on both continuous and discontinuous nanogrooves in comparison with cells on non-patterned controls. At 24 h post seeding, cells on both types of nanogrooves were highly elongated parallel to the groove long axis. Time-lapse video microscopy revealed that PDL fibroblast movement was guided on both types of grooves, but migration velocity was not significantly different from cells cultured on non-patterned controls. Analysis of filopodia formation using time-lapse video microscopy and labeling of vinculin and F-actin revealed that on nanogrooves, filopodia were highly aligned at both ends of the cell, but with increasing time filopodia and membrane protrusions developed at the side of the cell perpendicular to the cell long axis. We conclude that periodontal ligament fibroblasts are sensitive to nanotopographical depths of 85-100 μm, which could be utilized in regeneration of the periodontal ligament.