Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes.

Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection

Comparative analysis of the alpha-like globin clusters in mouse, rat, and human chromosomes indicates a mechanism underlying breaks in conserved synteny.

We have sequenced and fully annotated a 65,871-bp region of mouse Chromosome 17 including the Hba-ps4 alpha-globin pseudogene. Comparative sequence analysis with the functional alpha-globin loci at human Chromosome 16p13.3 and mouse Chromosome 11 shows that this segment of mouse Chromosome 17 contains a group of three alpha-like pseudogenes (Hba-psm-Hba-ps4-Hba-q3), similar to the duplicated sets found at the functional mouse cluster on Chromosome 11. In addition, exons 7 to 12 of the mLuc7L gene are present just downstream from the pseudogene cluster, indicating that this clone contains the region in which human 16p13.3 switches in synteny between mouse Chromosomes 11 and 17. Comparison of the sequences around the alpha-like clusters on the two mouse chromosomes reveals the presence of conserved tandem repeats. We propose that these repetitive elements have played a role in the fragmentation of the mouse alpha cluster during evolution

Validation of ASH Optical Depth and Layer Height from IASI using Earlinet Lidar Data

The 2010 eruptions of the Icelandic volcano Eyjafjallajökull attracted the attention of the public and the scientific community to the vulnerability of the European airspace to volcanic eruptions. The European Space Agency project “Satellite Monitoring of Ash and Sulphur Dioxide for the mitigation of Aviation Hazards”, called for the creation of an optimal End-to-End System for Volcanic Ash Plume Monitoring and Prediction. This system is based on improved and dedicated satellite-derived ash plume and sulphur dioxide level assessments, as well as an extensive validation, using among others ground-based measurements (Koukouli et al., 2014). The validation of volcanic ash levels and height extracted from IASI/MetopA is presented in this work with emphasis on the ash plume height and ash optical depth levels. European Aerosol Research Lidar Network [EARLINET] lidar measurements are compared to different satellite estimates for two eruptive episodes. The validation results are extremely promising within the estimated uncertainties of each of the comparative datasets

Blood Cells Mol. Dis.

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing - and -globin genes from the pufferfish Fugu rubripes. By DNA FISH analysis we show that Fugu contains two distinct hemoglobin loci situated on separate chromosomes. One locus contains only -globin genes ( -locus) while the other also contains a -globin gene ( -locus). This is the first poikilothermic species analysed where the physical linkage of the - and -globin genes has been uncoupled, supporting a model in which the separation of the - and -globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu -locus suggest that a strong distal Locus Control Region regulating the activity of the globin genes, as found in mammalian -globin clusters, may not be present in the Fugu -locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are in close proximity to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the five human globin loci known to date, each encoding proteins with specific functions in specific cell types

Peptides Interact with Keratinocytes.

<p>(A) 800 peptide or 800SCR tagged with tetramethylrhodamine (TMR) were added to primary keratinocytes at a concentration of 200nM for 30 minutes, then fixed and viewed by confocal microscopy. Scale bars: 25μM. (B) Cells were treated with no peptide (control), 50nM untagged peptide 800, the TMR-tagged form or its scrambled peptide for 30 minutes, then infected with bacteria for 1 hour. They were then fixed and bacteria quantified by fluorescence microscopy. Data are presented as % of an untreated control, transformed by Y = log₁₀(Y) before analysis with one-way ANOVA. **p≤0.01 ***p˂0.0001.</p

Wounding and Infection Affect Cytokine Production by TEskin.

<p>(A) TEskin was wounded by burning, or left intact. (B) TEskin was burned, and either infected with S235 strain Staphylococcus aureus or left uninfected. Cytokines were taken at 24 hours, and measured using a cytometric bead array. Data analysed by unpaired T test, * p≤0.05 **p≤0.01. n = 3, performed in triplicate.</p

Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences.

An important step toward improving the annotation of the human genome is to identify cis-acting regulatory elements from primary DNA sequence. One approach is to compare sequences from multiple, divergent species. This approach distinguishes multispecies conserved sequences (MCS) in noncoding regions from more rapidly evolving neutral DNA. Here, we have analyzed a region of approximately 238kb containing the human alpha globin cluster that was sequenced and/or annotated across the syntenic region in 22 species spanning 500 million years of evolution. Using a variety of bioinformatic approaches and correlating the results with many aspects of chromosome structure and function in this region, we were able to identify and evaluate the importance of 24 individual MCSs. This approach sensitively and accurately identified previously characterized regulatory elements but also discovered unidentified promoters, exons, splicing, and transcriptional regulatory elements. Together, these studies demonstrate an integrated approach by which to identify, subclassify, and predict the potential importance of MCSs

Tetraspanin Peptides Do Not Affect Bacterial Viability of Cell Migration.

<p>(A) HaCaT cells were seeded into a 24 well plate then treated with MTT stain for 1 hour. They were then permeabilised and the supernatant analysed using a plate reader at 562nm n = 3, duplicate. (B) HaCat cells were seeded into a 24 well plate to form a confluent monolayer. A scratch was made using a pipette tip, and the healing rate quantified using ImageJ, n = 5, performed in duplicate, Analysis by one-way ANOVA, ****p˂0.0001. (C) A TEskin model was set up to measure the migration of the epidermis across bare dermis over 10 days, with constant peptide treatment. Measurements were taken at 0 and 10 days using a Resazurin blue stain and ImageJ area measurements tools, n = 3 (D) S235 Bacteria were grown for 48 hours in the presence of 200nM peptide, and CFU measurements taken. n = 3, performed in duplicate.</p

Dose- and Time-Dependent Effects of Tetraspanin Peptide.

<p>(A and B) Varying dosages of 800 peptide were applied to HaCaT cells for 30 minutes then infected with bacteria S235 strain <i>S</i>. <i>aureus</i> for 1 hour before fixing and analysis. (C and D) 50nM peptide was applied to HaCaT cells at various time points then washed away after 1 hour. S235 Bacteria were then added at an MOI of 30 for one hour. Best fit was obtained when data were modelled using plateau followed by one-phase dissociation. A and C represent the proportion of infected cells, B and D represent the number of bacteria per 100 cells.</p

Tetraspanin Expression on Skin Cells.

<p>The expression levels of CD9, CD63, CD81 and CD151 on HaCaT cells, fibroblasts and keratinocytes were quantified by flow cytometry. The expression of each tetraspanin was compared for differentiated primary keratinocytes grown in Green’s medium and undifferentiated keratinocytes from the same donor grown in MCDB15132 low calcium medium. Data are from 3 separate HaCaT cultures, 10 individual fibroblast donors and 2 individual keratinocyte donors. * p≤0.05, unpaired t test.</p