Design and Applications of In-Situ Differential Scanning Electron Microscopy

Contrast enhancement in the scanning electron microscope (SEM) is usually achieved by either of the two techniques of black level suppression, and differential imaging. This paper is mainly concerned with the latter method. Differential imaging of SEM images is commonly accomplished by either using a selective electronic filtering circuit (time sensitive) on the video signals, or the post processing of a collected digitized image (application of special kernel operatives). A technique is described that is capable of generating true in-situ SEM differential video signals of local sample features. Characteristics of this method are enhanced sample feature-boundary sensitivity, suppression of large background signals, and the ability to perform critical pattern alignments prior to feature measurements. Results are presented on the application of the technique to the general field of electron microscopy, as well as to integrated circuit micro-metrology

Loss of locus coeruleus noradrenergic neurons alters the inflammatory response to LPS in substantia nigra but does not affect nigral cell loss

This is the accepted version of the following article: Mahmoud M. Iravani, Mona Sadeghian, Sarah Rose and Peter Jenner, “Loss of locus coeruleus noradrenergic neurons alters the inflammatory response to LPS in substantia nigra but does not affect nigral cell loss”, Journal of Neural Transmission, Vol. 121(12): 1493-1505, first published online 30 April 2014. The version of record is available online via doi: 10.1007/s00702-014-1223-1. © Springer-Verlag Wien 2014In Parkinson's disease (PD), destruction of noradrenergic neurons in the locus coeruleus (LC) may precede damage to nigral cells and subsequently exaggerate dopaminergic cell loss. We examine if destruction of the locus coeruleus with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) alters dopaminergic cell loss in substantia nigra (SN) initiated by lipopolysaccharide (LPS) in the rat through an effect on glial cell activation. In rats, a single intraperitoneal dose of DSP-4 administered 8 days previously, caused a marked loss of tyrosine hydroxylase positive neurons in LC but no change in dopaminergic cell number in SN. Unilateral nigral LPS administration resulted in marked dopaminergic cell death with reactive microgliosis associated with enhanced p47 phox in OX-6 and OX-42 positive microglia. There was proliferation of inducible nitric oxide synthase (iNOS)-positive cells, formation of 3-nitrotyrosine (3-NT) and proliferation of astrocytes that expressed glial cell line-derived neurotrophic factor (GDNF). Following combined DSP-4 treatment and subsequent administration of LPS, unexpectedly, no further loss of tyrosine hydroxylase (TH)-immunoreactivity (-ir) occurred in the SN compared to the effects of LPS alone. However, there was a marked alteration in the morphology of microglial cell and a reduction of 3-NT- and iNOS-ir was evident. Expression of p47 phox was downregulated in microglia but up-regulated in TH-ir neurons. No further change in GFAP-ir was observed compared to that produced by DSP-4 alone or LPS alone, but the expression of GDNF was markedly reduced. This study suggests that in contrast to previous reports, prior LC damage does not influence subsequent nigral dopaminergic cell degeneration induced by LPS. Rather it appears to attenuate the microglial response thought to contribute to disease progression in PD.Peer reviewedFinal Accepted Versio

Bird Community Responses to Rest-Rotation Grazing in Western Canada\u27s Grasslands

Western Canada’s native grasslands support high levels of avian diversity including both resident and migrant species. Many grassland specialist bird populations, however, are in serious decline due to widespread habitat loss resulting from agricultural conversion and adverse land management. As the primary use on remaining grasslands, cattle grazing largely determines the availability and quality of bird species’ habitat, depending on the timing, intensity, and frequency of livestock use. While adaptive multi-paddock grazing (AMP, a short duration, high-intensity grazing system that prioritises plant recovery between grazing events) is growing in popularity, comprehensive assessments of bird diversity in relation to AMP grazing practices are largely lacking. As part of a larger grazing management study, we examined how AMP grazing practices influence the taxonomic and phylogenetic diversity of bird species, compared to neighbouring (n-AMP) properties managed with more conventional grazing practices. In addition to the AMP/n-AMP contrast, we used rancher survey information to test for the influence of specific grazing practices over and above biophysical effects. Bird communities were surveyed at 309 point count locations across 38 ranches (set up as matched pairs) using visual and acoustic detection. Overall, we identified 96 bird species, of which 81 species were recorded on AMP-grazed ranches compared to 84 species on grasslands under n-AMP grazing, ranging from 10-32 species per ranch. We observed a considerable grazing management signal on species abundance and diversity including significant associations between some threatened species and n-AMP grazing. Moreover, AMP grazing, and specifically the use of higher rest-to-grazing ratios early in the growing season (prior to August 1), was associated with phylogenetically more clustered bird communities. Overall, this study highlights the potential of specialized rotational grazing systems to alter the composition and phylogenetic diversity of grassland bird communities. In conclusion, we stress the importance for prioritisation of strategic management plans to safeguard and restore North America’s grassland bird communities

Cancer-Associated Fibroblasts Suppress CD8⁺ T-cell Infiltration and Confer Resistance to Immune-Checkpoint Blockade

\ua92022 The Authors. Immune-checkpoint blockade (ICB) promotes antitumor immune responses and can result in durable patient benefit. However, response rates in breast cancer patients remain modest, stimulating efforts to discover novel treatment options. Cancer-associated fibroblasts (CAF) represent a major component of the breast tumor microenvironment and have known immunosuppressive functions in addition to their well-established roles in directly promoting tumor growth and metastasis. Here we utilized paired syngeneic mouse mammary carcinoma models to show that CAF abundance is associated with insensitivity to combination aCTLA4 and aPD-L1 ICB. CAF-rich tumors exhibited an immunologically cold tumor microenvironment, with transcriptomic, flow cytometric, and quantitative histopathologic analyses demonstrating a relationship between CAF density and a CD8+ T-cell–excluded tumor phenotype. The CAF receptor Endo180 (Mrc2) is predominantly expressed on myofibroblastic CAFs, and its genetic deletion depleted a subset of aSMA-expressing CAFs and impaired tumor progression in vivo. The addition of wild-type, but not Endo180-deficient, CAFs in coimplantation studies restricted CD8+ T-cell intratumoral infiltration, and tumors in Endo180 knockout mice exhibited increased CD8+ T-cell infiltration and enhanced sensitivity to ICB compared with tumors in wild-type mice. Clinically, in a trial of melanoma patients, high MRC2 mRNA levels in tumors were associated with a poor response to aPD-1 therapy, highlighting the potential benefits of therapeutically targeting a specific CAF subpopulation in breast and other CAF-rich cancers to improve clinical responses to immunotherapy

A Role for Fibrillar Collagen Deposition and the Collagen Internalization Receptor Endo180 in Glioma Invasion

Glioblastoma multiforme (GBM, WHO grade IV) is the most common and most malignant of astrocytic brain tumors, and is associated with rapid invasion into neighboring tissue. In other tumor types it is well established that such invasion involves a complex interaction between tumor cells and locally produced extracellular matrix. In GBMs, surprisingly little is known about the associated matrix components, in particular the fibrillar proteins such as collagens that are known to play a key role in the invasion of other tumor types.In this study we have used both the Masson's trichrome staining and a high resolution multiple immunofluorescence labeling method to demonstrate that intratumoral fibrillar collagens are an integral part of the extracellular matrix in a subset of GBMs. Correlated with this collagen deposition we observed high level expression of the collagen-binding receptor Endo180 (CD280) in the tumor cells. Further, interrogation of multiple expression array datasets identified Endo180 as one of the most highly upregulated transcripts in grade IV GBMs compared to grade III gliomas. Using promoter analysis studies we show that this increased expression is, in part, mediated via TGF-β signaling. Functionally, we demonstrate that Endo180 serves as the major collagen internalization receptor in GBM cell lines and provide the first evidence that this activity is critical for the invasion of GBM cells through fibrillar collagen matrices.This study demonstrates, for the first time, that fibrillar collagens are extensively deposited in GBMs and that the collagen internalization receptor Endo180 is both highly expressed in these tumors and that it serves to mediate the invasion of tumor cells through collagen-containing matrices. Together these data provide important insights into the mechanism of GBM invasion and identify Endo180 as a potential target to limit matrix turnover by glioma cells and thereby restrict tumor progression