Use of receptors immobilized on microspheres to identify ligand binding sites in tissue sections: detection of lymph node ligands for L-Selectin

 Particulate microspheres bearing immobilized probes can be used to identify ligands expressed by cells and require only brightfield microscopy for detection. There are distinct advantages to using microspheres to detect low affinity interactions; microspheres require no secondary amplification or detection procedures subsequent to the binding interaction, reducing opportunities for detachment of bound probe, and concentrating probes on microspheres may greatly increase binding avidity. Selectin leukocyte-endothelial adhesion molecules undergo low affinity binding to ligands, and these interactions may be difficult to detect with standard techniques. The aim of this study was to determine if immobilizing recombinant L-Selectin on microspheres would facilitate detection of specific tissue ligands. Microspheres were incubated with sections of rabbit peripheral lymph node in a modified Stamper-Woodruff assay, and binding was assessed by brightfield microscopy. L-Selectin-IgG microspheres bound to high endothelial venules, known to be sites of expression for L-Selectin ligands. Specificity was indicated by the lack of binding of microspheres coated with control protein, and inhibition of binding by antibody to L-Selectin and by competitive antagonists of L-Selectin ligand interactions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42230/1/418-107-1-57_71070057.pd

The acute inflammatory reaction

A major theme in current developments in this field has been the realization that the target tissue, in particular the endothelium, has an active role in initiating and participating in inflammatory reactions and is not a passive target, as was previously thought. Attempts to delineate the mechanisms involved have benefited immeasurably from the application of the modem techniques of molecular and cellular biology, and will continue to do so. It is axiomatic that a fuller understanding of these mechanisms will result in the development of new strategies designed to block the acute inflammatory response more specifically and effectively.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27722/1/0000111.pd

Monokine-induced gene expression of a human endothelial cell-derived neutrophil chemotactic factor

Monokines have been increasingly recognized as communication signals that interact with both immune and non-immune cells during inflammation. Specifically, interleukin-1alpha (IL-lalpha), interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) possess potent effector activities on various cell types. We present novel data demonstrating that human endothelial cells are a major source of a neutrophil chemotactic factor (NCF) synthesized upon stimulation with either IL-1alpha, IL-1beta, or TNF-alpha; but not with interleukin-6 (IL-6). Northern blot analysis demonstrated that 20 ng/ml of either IL-1 or TNF-alpha could induce endothelial cells to express significant levels of NCF mRNA, while IL-6 was not active in this system. These data demonstrate that monokines play an important role in mediating acute inflammation via induction of an endothelial cell-derived NCF.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27070/1/0000060.pd

The ability of magnetic field sensors to monitor feeding in three domestic herbivores

The rate at which animals ingest food is a fundamental part of animal ecology although it is rarely quantified, with recently-developed animal-attached tags providing a potentially viable approach. However, to date, these methods lack clarity in differentiating various eating behaviours, such as ‘chewing’ from ‘biting’. The aims of this study were to examine the use of inter-mandibular angle sensors (IMASENs), to quantify grazing behaviour in herbivores including cattle (Bos taurus), sheep (Ovis aries) and pygmy goats (Capra aegagrus hircus) eating different foodstuffs. Specifically, we aimed to: (1) quantify jaw movements of each species and determine differences between biting and chewing; (2) assess whether different food types can be discerned from jaw movements; and (3) determine whether species-specific differences in jaw movements can be detected. Subjects were filmed while consuming concentrate, hay, grass and browse to allow comparison of observed and IMASEN-recorded jaw movements. This study shows that IMASENs can accurately detect jaw movements of feeding herbivores, and, based on the rate of jaw movements, can classify biting (taking new material into the mouth) from chewing (masticating material already in the mouth). The biting behaviours associated with concentrate pellets could be identified easily as these occurred at the fastest rate for all species. However, the rates of chewing different food items were more difficult to discern from one another. Comparison of chew:bite ratios of the various food types eaten by each species showed no differences. Species differences could be identified using bite and chew rates. Cattle consistently displayed slower bite and chew rates to sheep and pygmy goats when feeding, while sheep and pygmy goats showed similar bite and chew rates when feeding on concentrate pellets. Species-specific differences in chew:bite ratios were not identified. Magnetometry has the potential to record quantitative aspects of foraging such as the feeding duration, food handling time and food type. This is of major importance for researchers interested in both captive (e.g., agricultural productivity) and wild animal foraging dynamics as it can provide quantitative data with minimal observer interference

The α(1,3)fucosyltransferases FucT-IV and FucT-VII Exert Collaborative Control over Selectin-Dependent Leukocyte Recruitment and Lymphocyte Homing

AbstractE-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by α(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV−/−/FucT-VII−/− mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII−/− mice is revoked in FucT-IV−/−/FucT-VII−/− mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing

Decision rules for determining terrestrial movement and the consequences for filtering high-resolution global positioning system tracks: a case study using the African lion ( Panthera leo )

The combined use of global positioning system (GPS) technology and motion sensors within the discipline of movement ecology has increased over recent years. This is particularly the case for instrumented wildlife, with many studies now opting to record parameters at high (infra-second) sampling frequencies. However, the detail with which GPS loggers can elucidate fine-scale movement depends on the precision and accuracy of fixes, with accuracy being affected by signal reception. We hypothesized that animal behaviour was the main factor affecting fix inaccuracy, with inherent GPS positional noise (jitter) being most apparent during GPS fixes for non-moving locations, thereby producing disproportionate error during rest periods. A movement-verified filtering (MVF) protocol was constructed to compare GPS-derived speed data with dynamic body acceleration, to provide a computationally quick method for identifying genuine travelling movement. This method was tested on 11 free-ranging lions (Panthera leo) fitted with collar-mounted GPS units and tri-axial motion sensors recording at 1 and 40 Hz, respectively. The findings support the hypothesis and show that distance moved estimates were, on average, overestimated by greater than 80% prior to GPS screening. We present the conceptual and mathematical protocols for screening fix inaccuracy within high-resolution GPS datasets and demonstrate the importance that MVF has for avoiding inaccurate and biased estimates of movement

Dead-reckoning animal movements in R: a reappraisal using Gundog.Tracks

BackgroundFine-scale data on animal position are increasingly enabling us to understand the details of animal movement ecology and dead-reckoning, a technique integrating motion sensor-derived information on heading and speed, can be used to reconstruct fine-scale movement paths at sub-second resolution, irrespective of the environment. On its own however, the dead-reckoning process is prone to cumulative errors, so that position estimates quickly become uncoupled from true location. Periodic ground-truthing with aligned location data (e.g., from global positioning technology) can correct for this drift between Verified Positions (VPs). We present step-by-step instructions for implementing Verified Position Correction (VPC) dead-reckoning in R using the tilt-compensated compass method, accompanied by the mathematical protocols underlying the code and improvements and extensions of this technique to reduce the trade-off between VPC rate and dead-reckoning accuracy. These protocols are all built into a user-friendly, fully annotated VPC dead-reckoning R function; Gundog.Tracks, with multi-functionality to reconstruct animal movement paths across terrestrial, aquatic, and aerial systems, provided within the Additional file 4 as well as online (GitHub).ResultsThe Gundog.Tracks function is demonstrated on three contrasting model species (the African lion Panthera leo, the Magellanic penguin Spheniscus magellanicus, and the Imperial cormorant Leucocarbo atriceps) moving on land, in water and in air. We show the effect of uncorrected errors in speed estimations, heading inaccuracies and infrequent VPC rate and demonstrate how these issues can be addressed.ConclusionsThe function provided will allow anyone familiar with R to dead-reckon animal tracks readily and accurately, as the key complex issues are dealt with by Gundog.Tracks. This will help the community to consider and implement a valuable, but often overlooked method of reconstructing high-resolution animal movement paths across diverse species and systems without requiring a bespoke application

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Decay-accelerating factor is a component of subendothelial extracellular matrix in vitro , and is augmented by activation of endothelial protein kinase C

The vasculature is protected from complement activation by regulatory molecules expressed on endothelial cells. However, complement fixation also occurs on subendothelial extracellular matrix (ECM) in vitro, and is initiated simply by retraction or removal of overlying cells. To investigate mechanisms controlling vascular complement activation, we examined subendothelial ECM for the presence of complement regulatory proteins. Decay-accelerating factor (DAF) was found on both human umbilical vein endothelial cells (HUVEC) and in their ECM; in contrast, membrane cofactor protein was found only on cells. ECM and HUVEC DAF were distinguishable based on several properties. While HUVEC DAF is anchored to cell membranes by a phospholipase C-sensitive glycosylphosphatidylinositol linkage, DAF was removed from ECM only by proteolytic digestion. Cytokines (TNF-α, IL-1β, IL-4) increased HUVEC DAF expression, but had minimal effect on ECM DAF; in contrast, phorbol 12-myristate 13-acetate (PMA) and wheat germ agglutinin markedly increased DAF on both HUVEC and ECM. The effect of PMA was mediated by activation of protein kinase C. The complement regulatory potential of ECM DAF was assessed by evaluating the effect of DAF-neutralizing antibodies on C3 deposition on HUVEC ECM, as well as on HeLa cell ECM, which had a considerably higher DAF content. DAF blockade enhanced C3 deposition on HeLa ECM, but had no effect on HUVEC ECM. As ECM DAF is likely to be immobile, i.e. able to interact only with C3 convertases forming in the immediate vicinity, its ability to regulate complement activation may be particularly density dependent, and contingent on endothelial-dependent up-regulation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37965/1/1052_ftp.pd