New evidence of unexpectedly high animal density and diet diversity will benefit the conservation of the Critically Endangered western ringtail possum

A comprehensive and contemporary understanding of habitat and resource requirements has been critical to the conservation of multiple taxa and ecosystems globally. Until recently, much of the ecological knowledge that contributes to conservation priorities and strategies for the Critically Endangered western ringtail possum (Pseudocheirus occidentalis) was largely derived from decades‐old observations in peppermint (Agonis flexuosa) and marri‐jarrah (Corymbia calophylla and Eucalyptus marginanta) woodlands in the northern parts of the species range. These observations do not account for more recent evidence of their flexible use of habitat resources in other regions of its range. This may represent a significant conservation opportunity for the species through the identification of additional habitats that warrant protection. In a region where knowledge of their ecology is scarce, we used scat analysis and quantitative spotlighting to determine the diet and density of western ringtail possums in three vegetation types: peppermint, sheoak (Allocasuarina fraseriana) and marri‐eucalypt (C. calophylla, E. marginanta and Eucalyptus staerii) woodlands. Given the species’ reported dependence on peppermint woodlands and dominant canopy species for food sources, we hypothesised that western ringtail possums would be most abundant in this habitat type and that their diet would comprise the foliage of few (≤2 species) canopy species. We found western ringtail possums consumed a higher diversity of plant species than expected (8–14), exhibited dietary preference for non‐dominant canopy species and were present in all sampled vegetation types at substantially higher densities than previously recorded for the region (as high as 17 possums ha^?1). Our results confirm (i) the western ringtail possum is flexible in its use of habitat resources and (ii) the significant conservation value of sheoak and marri‐eucalypt woodlands in the southernmost portion of its distribution.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Sheoak woodlands: a newly identified habitat for western ringtail possums

Although critically endangered western ringtail possums (Pseudocheirus occidentalis) have been recorded from a variety of vegetation types in southwestern Australia, the extent to which many of these vegetation types are occupied by the species remains unknown. We conducted spotlight surveys for western ringtail possums between March and April 2018 in Albany, southwestern Australia, to determine the species’ occupancy in 2 vegetation types. Using occupancy models, we demonstrated that sheoak (Allocasuarina fraseriana) woodlands, previously unrecognized as providing western ringtail possum habitat, support the species, although the median probability of their occupancy was lower than in marri (Corymbia calophylla) and eucalypt (Eucalyptus marginata and E. staeri) woodlands. Use of trees and other habitat components by western ringtail possums varied within and between vegetation types. Sheoak woodlands are likely critical for western ringtail possums and given the apparent flexibility in vegetation types used by the species, investigation of the potential value of other vegetation types for this species has conservation merit. © 2019 The Wildlife Society.     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.