Clonality and α-a recombination in the Australian Cryptococcus gattii VGII population--an emerging outbreak in Australia

Background

Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure.

Methodology/Principal Findings

A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MAT a VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MAT a populations. Our results also support the hypothesis of a “smouldering” outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion.

Conclusions/Significance

The detection of sexual recombination in MATα-MAT a population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.     

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.     

Cellular associations of pituitary gonadotrophs in a rodent (Lagostomus maximus maximus) with photoperiod-dependent reproduction

The morphological characteristics and percentage of the cellular associations between gonadotrophs (LH- and FSH-secreting cells) and other cellular types were studied in pituitary pars distalis of adult male viscachas (Lagostomus maximus maximus) by double immunohistochemistry using specific antibodies to LH, FSH, PRL, GH, ACTH, TSH and S-100 protein (by folliculostellate cells; FSC), during long and short photoperiods. Bihormonal gonadotrophs were observed in ventro-medial and dorsal regions, interspersed between monohormonal gonadotrophs, and their number increased in short photoperiod. LH- and FSH-gonadotrophs were found around lactotrophs, enclosed by somatotrophs in the dorsal region, and associated with irregular corticotrophs. Gonadotrophs and thyrotrophs were associated along blood vessels and follicular structures. The cytoplasmic prolongations of FSC were in contact with both gonadotrophs. The percentage of LH–FSH, LH–ACTH, LH–FSC, FSH–LH, FSH–PRL, FSH–GH, FSH–ACTH, FSH–TSH and FSH–FSC associations decreased, whereas LH–PRL increased in short as compared to long photoperiod. The most abundant associations were LH–GH and LH–TSH during long photoperiod, but LH–GH and LH–PRL during short photoperiod. FSH–GH and FSH–PRL were the most numerous associations, and LH–FSC and FSH–FSC were the less abundant ones in both photoperiods. These results provide the morphological evidence for specific cellular associations between gonadotrophs and other cellular types of viscacha pituitary.     

Synopsis of the CASIROZ case study: carbon sink strength of Fagus sylvatica L. in a changing environment--experimental risk assessment of mitigation by chronic ozone impact

Databases are needed for the ozone (O(3)) risk assessment on adult forest trees under stand conditions, as mostly juvenile trees have been studied in chamber experiments. A synopsis is presented here from an integrated case study which was conducted on adult FAGUS SYLVATICA trees at a Central-European forest site. Employed was a novel free-air canopy O(3) fumigation methodology which ensured a whole-plant assessment of O(3) sensitivity of the about 30 m tall and 60 years old trees, comparing responses to an experimental 2 x ambient O(3) regime (2 x O(3), max. 150 nl O(3) l (-1)) with those to the unchanged 1 x ambient O(3) regime (1 x O(3)=control) prevailing at the site. Additional experimentation on individual branches and juvenile beech trees exposed within the forest canopy allowed for evaluating the representativeness of young-tree and branch-bag approaches relative to the O(3) sensitivity of the adult trees. The 2 x O(3) regime did not substantially weaken the carbon sink strength of the adult beech trees, given the absence of a statistically significant decline in annual stem growth; a 3 % reduction across five years was demonstrated, however, through modelling upon parameterization with the elaborated database. 2 x O(3) did induce a number of statistically significant tree responses at the cell and leaf level, although the O(3) responsiveness varied between years. Shade leaves displayed an O(3) sensitivity similar to that of sun leaves, while indirect belowground O(3) effects, apparently mediated through hormonal relationships, were reflected by stimulated fine-root and ectomycorrhizal development. Juvenile trees were not reliable surrogates of adult ones in view of O(3) risk assessment. Branch sections enclosed in (climatized) cuvettes, however, turned out to represent the O(3) sensitivity of entire tree crowns. Drought-induced stomatal closure decoupled O(3) intake from O(3) exposure, as in addition, also the "physiologically effective O(3) dose" was subject to change. No evidence emerged for a need to lower the "Critical Level for Ozone" in risk assessment of forest trees, although sensitive tree parameters did not necessarily reflect a linear relationship to O(3) stress. Exposure-based concepts tended to overestimate O(3) risk under drought, which is in support of current efforts to establish flux-related concepts of O(3) intake in risk assessment.     

Evidence of a landlocked reproducing population of the marine pejerrey Odontesthes argentinensis (Actinopterygii; Atherinopsidae)

In South America, the order Atheriniformes includes the monophyletic genus Odontesthes with 20 species that inhabit freshwater, estuarine and coastal environments. Pejerrey Odontesthes argentinensis is widely distributed in coastal and estuarine areas of the Atlantic Ocean and is known to foray into estuaries of river systems, particularly in conditions of elevated salinity. However, to our knowledge, a landlocked self-sustaining population has never been recorded. In this study, we examined the pejerrey population of Salada de Pedro Luro Lake (south-east of Buenos Aires Province, Argentina) to clarify its taxonomic identity. An integrative taxonomic analysis based on traditional meristic, landmark-based morphometrics and genetic techniques suggests that the Salada de Pedro Luro pejerrey population represents a novel case of physiological and morphological adaptation of a marine pejerrey species to a landlocked environment and emphasises the environmental plasticity of this group of fishes.     

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K_d value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R⁴⁹ that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.     

Comparative effects of desiccation,heat shock and high temperatures on seed germination of savanna and forest tree species

Although forest and savanna biomes predominate in tropics regions, the factors that control their distribution remain unclear. South American savannas occur in regions that are considered warm and humid enough to support forests, indicating that agents other than climate determine the occurrence of one or the other physiognomy. Herbivory, fire and water deficit have been considered environmental filters that limit the forest species encroachment in savanna physiognomies, but the effects of these filters on the capability of these species to recruit from seeds remain poorly understood. In this study we investigated how stress factors characteristic of savanna environments, such as soil desiccation, heat shocks and high temperatures affect the survival and germination of seeds from savanna and forest tree species. We found that desiccation (to 5%) reduced the germination percentage of forest seeds, but had no effect on the germination of savanna seeds. Forest seeds were less tolerant to heat shocks of 140°C and 200°C, and showed lower germination percentage at temperatures of 35 and 40°C, when compared with savanna seeds. Savanna seeds presented longer germination times and higher germination variance than forest seeds, indicating a risk‐spreading germination strategy among savanna species. The low tolerance of forest seeds to desiccation, heat shock and high temperatures may explain the low recruitment of forest trees into savanna physiognomies. Climate change models predict lower soil moisture, higher temperatures and higher fires frequency for South America biomes. Our results suggest that savanna species are likely to be more capable of withstanding the effects of these changes than forest species.