Advancing characterisation with statistics from correlative electron diffraction and X-ray spectroscopy, in the scanning electron microscope.

The routine and unique determination of minor phases in microstructures is critical to materials science. In metallurgy alone, applications include alloy and process development and the understanding of degradation in service. We develop a correlative method, exploring superalloy microstructures, which are examined in the scanning electron microscope (SEM) using simultaneous energy dispersive X-ray spectroscopy (EDS) and electron backscatter diffraction (EBSD). This is performed at an appropriate length scale for characterisation of carbide phases' shape, size, location, and distribution. EDS and EBSD data are generated using two different physical processes, but each provide a signature of the material interacting with the incoming electron beam. Recent advances in post-processing, driven by 'big data' approaches, include use of principal component analysis (PCA). Components are subsequently characterised to assign labels to a mapped region. To provide physically meaningful signals, the principal components may be rotated to control the distribution of variance. In this work, we develop this method further through a weighted PCA approach. We use the EDS and EBSD signals concurrently, thereby labelling each region using both EDS (chemistry) and EBSD (crystal structure) information. This provides a new method of amplifying signal-to-noise for very small phases in mapped regions, especially where the EDS or EBSD signal is not unique enough alone for classification.

HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus.

Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1-4), which confer distinct biophysical properties on the channel. Despite understanding the structure-function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shown that HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PV-expressing cells in the hippocampal CA1 subfield (93.5%; ±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory, and sleep as well as the pathogenesis of several neurological diseases, these findings provide important insights into the identity and neurochemical status of cells that could underlie such conditions.

The leukocyte integrin p150,95 (CD11c/CD18) as a receptor for iC3b. Activation by a heterologous beta subunit and localization of a ligand recognition site to the I domain.

p150,95 is a member of the leukocyte integrin family of adhesion proteins. Compared with LFA-1 and Mac-1, p150,95 is less well functionally characterized. Although p150,95 has complement receptor activity for iC3b and has been designated complement receptor type 4, transfected cells expressing p150,95 do not bind iC3b-sensitized cells. We report that cells cotransfected with a human p150,95 alpha subunit and a chicken, but not human, beta subunit bind IgM-iC3b-coated erythrocytes, suggesting that interactions between the alpha and beta subunits can regulate p150,95 adhesiveness. Furthermore, purified human p150,95 binds to cell-bound iC3b-coated erythrocytes. Because binding to iC3b by cellular and purified p150,95 is specifically abolished by mAbs that localize to the I domain of p150,95, we suggest that the I domain of the p150,95 alpha subunit is an important ligand recognition site for iC3b.

Cloning and expression of the chicken CD18 cDNA.

The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the chicken beta 2 (CD18) subunit, common to the leukocyte integrin family. The deduced 748-amino-acid sequence reveals a transmembrane protein with 65% and 64% identity with its human and murine homologues, respectively. The chicken beta 2 can associate on the cell surface with the human alpha subunit of LFA-1 and yields a hybrid molecule capable of binding to purified ICAM-1 and ICAM-3.

The identification of the beta 2-microglobulin binding antigen encoded by the human CD1D gene.

Human cluster of differentiation (CD1) is a family of cell surface glycoproteins composed of a 43-49-kDa heavy chain non-covalently associated with beta 2-microglobulin. Five human CD1 genes have been detected and cloned. Three genes (CD1A, -B and -C) encode the serologically defined CD1a, -b and -c antigens. Thus two genes remain, CD1D and CD1E, whose protein products have not been characterized so far. This report describes how a beta-galactosidase-CD1D fusion protein was used to raise specific antisera and a monoclonal antibody against the CD1D gene product. The monoclonal antibody defines a cell surface molecule expressed on a cortical thymocyte cell line and is composed of a 49-kDa heavy chain associated with beta 2-microglobulin, which is serologically distinct from CD1a.

Structure and expression of the human thymocyte antigens CD1a, CD1b, and CD1c.

The CD1 human antigens are a family of at least three components, CD1a, CD1b, and CD1c, that are characteristic of the cortical stage of thymocyte maturation. CD1a was originally named HTA1 or T6 and thought to be the human equivalent of mouse Tla. The genes coding for all three have now been identified by transfection into mouse cells. The transfectants express the surface antigens that can then be recognized by the corresponding cluster of monoclonal antibodies used to define the three members of CD1. The full sequence of the genomic DNA is described for all three. The intron-exon structure of CD1a is deduced by comparison with a near-full-length cDNA clone. Similar structures are proposed for the other two, largely based on sequence homology. An unusually long 5'-untranslated exon (280 bases long) is highly conserved between the three genes, suggesting an important but unknown function. CD1c has a duplicated form of this exon that is thought to be spliced out. The major homology between the three antigens is in the beta 2-microglobulin-binding domain. The general relatedness to major histocompatibility complex class I and class II molecules is significant but low, with no section of higher homology to mouse Tla.