In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

A New Rhesus Macaque Assembly and Annotation for Next-Generation Sequencing Analyses

BACKGROUND: The rhesus macaque (Macaca mulatta) is a key species for advancing biomedical research. Like all draft mammalian genomes, the draft rhesus assembly (rheMac2) has gaps, sequencing errors and misassemblies that have prevented automated annotation pipelines from functioning correctly. Another rhesus macaque assembly, CR_1.0, is also available but is substantially more fragmented than rheMac2 with smaller contigs and scaffolds. Annotations for these two assemblies are limited in completeness and accuracy. High quality assembly and annotation files are required for a wide range of studies including expression, genetic and evolutionary analyses. RESULTS: We report a new de novo assembly of the rhesus macaque genome (MacaM) that incorporates both the original Sanger sequences used to assemble rheMac2 and new Illumina sequences from the same animal. MacaM has a weighted average (N50) contig size of 64 kilobases, more than twice the size of the rheMac2 assembly and almost five times the size of the CR_1.0 assembly. The MacaM chromosome assembly incorporates information from previously unutilized mapping data and preliminary annotation of scaffolds. Independent assessment of the assemblies using Ion Torrent read alignments indicates that MacaM is more complete and accurate than rheMac2 and CR_1.0. We assembled messenger RNA sequences from several rhesus tissues into transcripts which allowed us to identify a total of 11,712 complete proteins representing 9,524 distinct genes. Using a combination of our assembled rhesus macaque transcripts and human transcripts, we annotated 18,757 transcripts and 16,050 genes with complete coding sequences in the MacaM assembly. Further, we demonstrate that the new annotations provide greatly improved accuracy as compared to the current annotations of rheMac2. Finally, we show that the MacaM genome provides an accurate resource for alignment of reads produced by RNA sequence expression studies. CONCLUSIONS: The MacaM assembly and annotation files provide a substantially more complete and accurate representation of the rhesus macaque genome than rheMac2 or CR_1.0 and will serve as an important resource for investigators conducting next-generation sequencing studies with nonhuman primates. REVIEWERS: This article was reviewed by Dr. Lutz Walter, Dr. Soojin Yi and Dr. Kateryna Makova

VERTICO. IV. Environmental effects on the gas distribution and star formation efficiency of Virgo cluster spirals

GalaxiesInterstellar matter and star formatio

The Neutrophil's Eye-View: Inference and Visualisation of the Chemoattractant Field Driving Cell Chemotaxis In Vivo

As we begin to understand the signals that drive chemotaxis in vivo, it is becoming clear that there is a complex interplay of chemotactic factors, which changes over time as the inflammatory response evolves. New animal models such as transgenic lines of zebrafish, which are near transparent and where the neutrophils express a green fluorescent protein, have the potential to greatly increase our understanding of the chemotactic process under conditions of wounding and infection from video microscopy data. Measurement of the chemoattractants over space (and their evolution over time) is a key objective for understanding the signals driving neutrophil chemotaxis. However, it is not possible to measure and visualise the most important contributors to in vivo chemotaxis, and in fact the understanding of the main contributors at any particular time is incomplete. The key insight that we make in this investigation is that the neutrophils themselves are sensing the underlying field that is driving their action and we can use the observations of neutrophil movement to infer the hidden net chemoattractant field by use of a novel computational framework. We apply the methodology to multiple in vivo neutrophil recruitment data sets to demonstrate this new technique and find that the method provides consistent estimates of the chemoattractant field across the majority of experiments. The framework that we derive represents an important new methodology for cell biologists investigating the signalling processes driving cell chemotaxis, which we label the neutrophils eye-view of the chemoattractant field

Decreased number of mast cells infiltrating into needle biopsy specimens leads to a better prognosis of prostate cancer

Mast cell infiltration is often observed around human tumours. Inflammatory cells such as macrophages, neutrophils and mast cells infiltrating around tumours are known to contribute to tumour growth; however, the clinical significance of mast cell invasion in prostate cancer (PCa) has not been investigated. Mast cell infiltration was evaluated in 104 patients (age range, 45–88 years; median, 72 years), who underwent needle biopsy of the prostate and were confirmed to have PCa. Needle biopsy specimens of prostate were sliced into 5-μm-thick sections and immunostained for mast cells with monoclonal antibody against mast cell-specific tryptase. Mast cells were counted systematically under a microscope (× 400 magnification), and the relations between mast cell numbers and clinicopathologic findings were evaluated. The mast cell count was evaluated for prognostic value by multivariate analysis. Mast cells were immunostained around the cancer foci. The median number of mast cells in each case was 16. The mast cell count was higher around cancer foci in patients with higher Gleason scores than in those with low Gleason scores. The mast cell number correlated well with clinical stage (P<0.001). Prostate-specific antigen-free survival of patients with higher mast cell counts was better than that in patients with lower mast cell counts (P<0.001). Multivariate analysis revealed that mast cell count was a significant prognostic factor (P<0.005). The number of mast cells infiltrating around cancer foci in prostate biopsy specimens can be a significant prognostic factor of PCa

Reshaping teacher and student roles in technology-enriched classrooms

This paper draws on data from a three-year longitudblal study of secondary school classrooms to examine pedagogical issues in using tectmology resources in mathematics teaching-in particular, graphics calculators and overhead projection panels that allow screen output to be viewed by the whole class. We theorise four roles for technology in relation to such teaching and learning interactions-master, servant, partner, and extension of self-and illustrate this taxonomy with observational data from five senior secondary mathematics classrooms. Our research shows how technology can facilitate collaborative inquiry during both small group interactions and whole class discussions when students use their calculators and the overhead projection panel to share their mathematical understanding

Effect of ethanol and of noise on reaction time in the monkey: Variation with stimulus level

To determine whether the latency-increasing effects of ethanol were differential with respect to the intensity of the stimulus that initiated the response, three rhesus monkeys were trained on a behavioral task in which the latency of a simple motor response was measured following the onset of a pure tone stimulus. Following training, the animals were tested at a number of different tone intensities and functions relating latency to tone intensity were constructed. When these were stable, the animals were given ethanol in doses of 1.0–2.5 g/kg and the effects on response latencies to different tone intensities were determined. It was found that, for all except the lowest stimulus levels, the effect of ethanol was dose-related, while for a given dose the effect was equal across intensity. These results indicate that the effects of ethanol in this situation are on response execution rather than stimulus detection. The effects of ethanol were compared to those of exposure to high intensity noise. This treatment, which affects primarily the inner ear, resulted in substantial increases in latency to low intensity tones, but little, if any, shift at high intensities.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46413/1/213_2004_Article_BF00426520.pd