A comparative molecular and 3-dimensional structural investigation into cross-continental and novel avian Trypanosoma spp. in Australia.

BACKGROUND: Molecular and structural information on avian Trypanosoma spp. throughout Australia is limited despite their intrinsic value in understanding trypanosomatid evolution, diversity, and structural biology. In Western Australia tissue samples (n = 429) extracted from 93 birds in 25 bird species were screened using generic PCR primers to investigate the diversity of Trypanosoma spp. To investigate avian trypanosome structural biology the first 3-dimensional ultrastructural models of a Trypanosoma spp. (Trypanosoma sp. AAT) isolated from a bird (currawong, Strepera spp.) were generated using focussed ion beam milling combined with scanning electron microscopy (FIB-SEM). RESULTS: Here, we confirm four intercontinental species of avian trypanosomes in native Australian birds, and identify a new avian Trypanosoma. Trypanosome infection was identified in 18 birds from 13 different bird species (19%). A single new genotype was isolated and found to be closely related to T. culicavium (Trypanosoma sp. CC2016 B002). Other Trypanosoma spp. identified include T. avium, T. culicavium, T. thomasbancrofti, Trypanosoma sp. TL.AQ.22, Trypanosoma sp. AAT, and an uncharacterised Trypanosoma sp. (group C-III sensu Zidková et al. (Infect Genet Evol 12:102-112, 2012)), all previously identified in Australia or other continents. Serially-sectioning Trypanosoma sp. AAT epimastigotes using FIB-SEM revealed the disc-shaped kinetoplast pocket attached perpendicular to the branching mitochondrion. Additionally, the universal minicircle sequence within the kinetoplast DNA and the associated binding protein were determined in Trypanosoma sp. AAT. CONCLUSIONS: These results indicate that bird trypanosomes are relatively conserved across continents, while being locally diverse, which supports the hypothesis that bird trypanosomes exist as fewer species than described in the literature. Evidence exists that avian Trypanosoma spp. are infecting mammals and could be transmitted by haemadipsid leeches. Trypanosoma sp. AAT is most likely a separate species currently found only in Australia and the first 3-dimentional ultrastructural analysis of an avian trypanosome provides interesting information on their morphology and organelle arrangement.

Accumulation and precipitation of magnesium, calcium, and sulfur in two Acacia (Leguminosae; Mimosoideae) species grown in different substrates proposed for mine-site rehabilitation.

PREMISE OF THE STUDY: Few studies have investigated the effects of substrates on the accumulation and precipitation of magnesium, calcium, and sulfur in plants. Acacia stipuligera and A. robeorum growing in their natural habitats with different substrates show different accumulation and precipitation patterns of these elements. Here, we compared the accumulation and precipitation of magnesium, calcium, and sulfur in A. stipuligera and A. robeorum grown in different substrates proposed for mine-site rehabilitation and expected the differences in substrates to have significant effects on the accumulation and precipitation of these elements in the two species. METHODS: Saplings were grown in sandy topsoil or in a topsoil-siltstone mixture in a glasshouse. Phyllode magnesium, calcium, and sulfur concentrations of 25-wk-old plants were measured. Precipitation of these elements in phyllodes and branchlets was investigated by means of scanning electron microscopy and energy-dispersive x-ray spectroscopy. KEY RESULTS: Phyllode magnesium, calcium, and sulfur concentrations were generally significantly greater in A. robeorum than in A. stipuligera. The two species responded in unique ways to the substrate, with A. stipuligera having similar phyllode magnesium and calcium concentrations in both substrates, but greater sulfur concentration in the topsoil-siltstone mixture, while A. robeorum showed lower phyllode magnesium, calcium, and sulfur concentrations in the topsoil-siltstone mixture. For both substrates, mineral precipitates were observed in both species, with A. robeorum having more mineral precipitates containing magnesium, calcium, and sulfur in its phyllodes than A. stipuligera did. CONCLUSIONS: The accumulation and precipitation patterns of magnesium, calcium, and sulfur are more species-specific than substrate-affected.

Application of SEM and EDX in studying biomineralization in plant tissues.

This chapter describes protocols using formalin-acetic acid-alcohol (FAA) to fix plant tissues for studying biomineralization by means of scanning electron microscopy (SEM) and qualitative energy-dispersive X-ray microanalysis (EDX). Specimen preparation protocols for SEM and EDX mainly include fixation, dehydration, critical point drying (CPD), mounting, and coating. Gold-coated specimens are used for SEM imaging, while gold- and carbon-coated specimens are prepared for qualitative X-ray microanalyses separately to obtain complementary information on the elemental compositions of biominerals. During the specimen preparation procedure for SEM, some biominerals may be dislodged or scattered, making it difficult to determine their accurate locations, and light microscopy is used to complement SEM studies. Specimen preparation protocols for light microscopy generally include fixation, dehydration, infiltration and embedding with resin, microtome sectioning, and staining. In addition, microwave processing methods are adopted here to speed up the specimen preparation process for both SEM and light microscopy.

Fibrin sealant promotes migration and proliferation of human articular chondrocytes: possible involvement of thrombin and protease-activated receptors.

Fibrin sealant (FS), a biological adhesive material, has been recently recommended as an adjunct in autologous chondrocyte implantation (ACI). While FS has been shown to possess osteoinductive potential, little is known about its effects on chondrogenic cells. In this study, we assessed the bioactivity of FS (Tisseel) on the migration and proliferation of human articular chondrocytes in vitro. Using a co-culture assay to mimic matrix-induced ACI (MACI), chondrocytes were found to migrate from collagen membranes towards FS within 12 h of culture, with significant migratory activity evident by 24 h. In addition, 5-bromo-2'-deoxyuridine (BrdU) incorporation experiments revealed that thrombin, the active component of the tissue glue, stimulated chondrocyte proliferation, with maximal efficacy observed at 48 h post-stimulation (1-10 U/ml). In an effort to elucidate the molecular mechanisms underlying these thrombin-induced effects, we examined the expression and activation of protease-activated receptors (PARs), established thrombin receptors. Using a combination of RT-PCR and immunohistochemistry, all four PARs were detected in human chondrocytes, with PAR-1 being the major isoform expressed. Moreover, thrombin and a PAR-1, but not other PAR-isotype-specific peptide agonists, were found to induce rapid intracellular Ca2+ responses in human chondrocytes in calcium mobilization assays. Together, these data demonstrate that FS supports both the migration and proliferation of human chondrocytes. We propose that these effects are mediated, at least in part, via thrombin-induced PAR-1 signalling in human chondrocytes.

In situ characterisation of oligodendrocyte progenitor cells in adult mammalian optic nerve.

Oligodendrocyte progenitors from the adult rodent optic nerve were described in vitro more than a decade ago but their identification in vivo has been more elusive. We have previously traced newly-generated oligodendrocytes remyelinating focal demyelination of adult feline optic nerve back to their origin in surrounding fascicles. Extending the search to normal tissue has now enabled the description of an oligodendrocyte progenitor population in situ. By combining serial 1 microm section immunocytochemistry with electron microscopy of perfusion-fixed tissue, every cell of interest could be characterised. Oligodendrocyte progenitors were readily identifiable in transverse and longitudinal sections dual-stained with antibodies to glutamine synthetase and either S-100 or HNK-1. Oligodendrocyte progenitors of the adult feline optic nerve were mainly located centrally within fascicles, comprised 4% of the total macroglia and had both ultrastructural and immunocytochemical features suggesting roles additional to that of a progenitor cell. The identification and characterisation of oligodendrocyte progenitors in situ in fixed tissue sections should help the understanding of their role in the adult CNS and in disease such as multiple sclerosis.