Staphylococcus aureus clumping factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal colonization

Staphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high-risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surface-expressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal cytokeratins in vitro . By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis , we demonstrated that adherence to epidermal cytokeratins was conferred by ClfB. Adherence of wild-type S. aureus was inhibited by recombinant ClfB protein or anti-ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.     

Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.     

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.     

Animal models of ischemic stroke: balancing experimental aims and animal care

Animal models of ischemic stroke are examples of an induced model that can present challenges from the perspectives of protocol review and animal management. The review presented here will include a brief summary of the current state of knowledge about clinical stroke; a general synopsis of important unanswered research questions that justify use of animal stroke models; an overview of various animal models of ischemic stroke, including strengths and limitations; and a discussion of animal care issues relative to ischemic stroke models. Good communication and interactive education among primary investigators, laboratory animal veterinarians and caretakers, and institutional animal care and use committee members are critical in achieving a balance between research objectives and animal care issues when using animal stroke models.     

Genetic diversity of peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome

Background  

The genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives.     

The significance of p16INK4a in cell defenses against transformation

Human cells, including fibroblast strains that have been immortalized by telomerase, are much more resistant to transformation than rodent cells. Most of the experimental evidence suggests that transformation of human fibroblasts requires inactivation of both the retinoblastoma (pRb) and p53 tumor suppressors as well as the addition of one or more dominant oncogenes. By starting with strains of primary fibroblast (Leiden and Q34 cells) that are genetically deficient for p16INK4a, we have been able to generate anchorage independent colonies simply by addition of telomerase (hTERT) and either Ras or Myc. Importantly, the transformed cells appear to retain pRb and p53 functions and are essentially diploid. Whereas Leiden cells expressing the individual oncogenes did not form tumors in mice, the combination of hTERT, Myc and Ras enabled them to become tumorigenic, albeit at a frequency suggestive of an additional genetic event. Significantly, we have obtained karyotypically stable tumors without the need to use DNA tumor virus oncoproteins and without deliberate ablation of p53.     

Simulated effects of low atmospheric CO2 on structure and composition of North American vegetation at the Last Glacial Maximum

1. Physiological experiments have indicated that the lower CO₂ levels of the last glaciation (200 μmol mol^?1) probably reduced plant water-use efficiency (WUE) and that they combined with increased aridity and colder temperatures to alter vegetation structure and composition at the Last Glacial Maximum (LGM). 2. The effects of low CO₂ on vegetation structure were investigated using BIOME3 simulations of leaf area index (LAI), and a two-by-two factorial experimental design (modern/LGM CO₂, modern/LGM climate).3. Using BIOME3, and a combination of lowered CO₂ and simulated LGM climate (from the NCAR-CCM1 model), results in the introduction of additional xeric vegetation types between open woodland and closed-canopy forest along a latitudinal gradient in eastern North America.4. The simulated LAI of LGM vegetation was 25–60% lower in many regions of central and eastern United States relative to modern climate, indicating that glacial vegetation was much more open than today.5. Comparison of factorial simulations show that low atmospheric CO₂ has the potential to alter vegetation structure (LAI) to a greater extent than LGM climate.6. If the magnitude of LAI reductions simulated for glacial North America were global, then low atmospheric CO₂ may have promoted atmospheric warming and increased aridity, through alteration of rates of water and heat exchange with the atmosphere.