An ancient haplotype containing antimicrobial peptide gene variants is associated with severe fungal skin disease in Persian cats

Dermatophytosis, also known as ringworm, is a contagious fungal skin disease affecting humans and animals worldwide. Persian cats exhibit severe forms of the disease more commonly than other breeds of cat, including other long-haired breeds. Certain types of severe dermatophytosis in humans are reportedly caused by monogenic inborn errors of immunity. The goal of this study was to identify genetic variants in Persian cats contributing to the phenotype of severe dermatophytosis. Whole-genome sequencing of case and control Persian cats followed by a genome-wide association study identified a highly divergent, disease-associated haplotype on chromosome F1 containing the S100 family of genes. S100 calcium binding protein A9 (S100A9), which encodes a subunit of the antimicrobial heterodimer known as calprotectin, contained 13 nonsynonymous variants between cases and controls. Evolutionary analysis of S100A9 haplotypes comparing cases, controls, and wild felids suggested the divergent disease-associated haplotype was likely introgressed into the domestic cat lineage and maintained via balancing selection. We demonstrated marked upregulation of calprotectin expression in the feline epidermis during dermatophytosis, suggesting involvement in disease pathogenesis. Given this divergent allele has been maintained in domestic cat and wildcat populations, this haplotype may have beneficial effects against other pathogens. The pathogen specificity of this altered protein should be investigated before attempting to reduce the allele frequency in the Persian cat breed. Further work is needed to clarify if severe Persian dermatophytosis is a monogenic disease or if hidden disease-susceptibility loci remain to be discovered. Consideration should be given to engineering antimicrobial peptides such as calprotectin for topical treatment of dermatophytosis in humans and animals.     

Subfreezing volumetric behavior and stochastic modeling of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopic observations were made of the volumetric behavior and kinetics of intracellular ice formation (IIF) in Drosophila melanogaster embryos in a modified cell culture medium (BD.20) or BD.20 + 2 M ethylene glycol. After rapid cooling to a given temperature, transient volumetric contraction of the embryos during the isothermal period was quantified by computerized video image analysis. Fitting these data to the numerical solution of the volume flux equation yielded estimates of the hydraulic permeability coefficient (Lp) for individual embryos at various subfreezing temperatures. Lp approximately followed an Arrhenius relation between -2 and -9 degrees C, with a value of 0.168 microns/(min-atm) extrapolated to 0 degrees C and an apparent activation energy delta E of 38.9 kcal/mol. IIF during an isothermal period occurred at random times whose characteristic temperature range and kinetics were affected by the presence of ethylene glycol. A stochastic process model developed to fit these data indicated the influence of both time-dependent and instantaneous components of IIF, presumed to be the result of seeding and heterogeneous nucleation, respectively. The presence of 2 M ethylene glycol depressed the characteristic temperature of instantaneous IIF by about 12 degrees C and reduced the rate constant for time-dependent IIF. Comparison with observed incidences of IIF yielded an estimate of the supercooling tolerance of 3 to 5 degrees C.     

Estimation of breast burn size

It is the authors' opinion that the size of chest burns on large-breasted women can be significantly underestimated, especially if the methods of calculation rely on burn charts, such as the Lund and Browder burns chart. This latter chart is based on data derived from only three women and eight men. The surface area of the torsos of 60 volunteers (20 men, 20 small-breasted women, and 20 large-breasted women) was measured using two well-established techniques. The torso surface area was divided into two parts: the anterior trunk and the posterior trunk (i.e., torso surface area = posterior trunk + anterior trunk). The anterior trunk was subdivided and the area above the costal margins defined as the pectoral region. These areas were measured separately for each individual. The volunteers' total body surface area was calculated using normograms, based on their weight and height. The area of each torso section was recorded as a percentage of the total body surface area and torso surface area. Whereas the torso surface area/total body surface area ratio did not vary significantly between the groups, the proportion of anterior to posterior trunk size did depend on the sex and on breast size. There was a direct correlation between the woman's bra cup size and the ratio of anterior-to-posterior trunk surface area. A simple chart was therefore derived that estimates the relative size of a woman's torso surface area once her bra cup size is known. Such a chart can be used to improve accuracy in adult female chest burn estimation, when used in conjunction with a burns chart. Breast burns in larger breasted women are underestimated when calculated using current burn charts. We recommend that a correction be made when estimating chest burns in women to account for the increased surface area of the breasts. A chart, such as the one we have developed, could be used in conjunction with a burn chart (e.g., Lund and Browder) to make this correction.     

Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87 degrees C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10(2) V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.     

Accurate detection of aneuploidies in array CGH and gene expression microarray data

MOTIVATION: Chromosomal copy number changes (aneuploidies) are common in cell populations that undergo multiple cell divisions including yeast strains, cell lines and tumor cells. Identification of aneuploidies is critical in evolutionary studies, where changes in copy number serve an adaptive purpose, as well as in cancer studies, where amplifications and deletions of chromosomal regions have been identified as a major pathogenetic mechanism. Aneuploidies can be studied on whole-genome level using array CGH (a microarray-based method that measures the DNA content), but their presence also affects gene expression. In gene expression microarray analysis, identification of copy number changes is especially important in preventing aberrant biological conclusions based on spurious gene expression correlation or masked phenotypes that arise due to aneuploidies. Previously suggested approaches for aneuploidy detection from microarray data mostly focus on array CGH, address only whole-chromosome or whole-arm copy number changes, and rely on thresholds or other heuristics, making them unsuitable for fully automated general application to gene expression datasets. There is a need for a general and robust method for identification of aneuploidies of any size from both array CGH and gene expression microarray data. RESULTS: We present ChARM (Chromosomal Aberration Region Miner), a robust and accurate expectation-maximization based method for identification of segmental aneuploidies (partial chromosome changes) from gene expression and array CGH microarray data. Systematic evaluation of the algorithm on synthetic and biological data shows that the method is robust to noise, aneuploidal segment size and P-value cutoff. Using our approach, we identify known chromosomal changes and predict novel potential segmental aneuploidies in commonly used yeast deletion strains and in breast cancer. ChARM can be routinely used to identify aneuploidies in array CGH datasets and to screen gene expression data for aneuploidies or array biases. Our methodology is sensitive enough to detect statistically significant and biologically relevant aneuploidies even when expression or DNA content changes are subtle as in mixed populations of cells. AVAILABILITY: Code available by request from the authors and on Web supplement at http://function.cs.princeton.edu/ChARM/     

HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.