Activity of CD101, a long-acting echinocandin,against clinical isolates of Candida auris

CD101 is a new echinocandin with a prolonged half-life. CD101 was tested by broth microdilution against 100 isolates of the emerging yeast Candida auris. It showed activity against most isolates, including some that were resistant to other echinocandins.     

Parallel analysis of genetic selections using whole genome oligonucleotide arrays

Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein–protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.     

Release of a potent polymorphonuclear leukocyte chemoattractant is regulated by white-opaque switching in Candida albicans

Previous studies employing transmembrane assays suggested that Candida albicans and related species, as well as Saccharomyces cerevisiae, release chemoattractants for human polymorphonuclear leukocytes (PMNs). Because transmembrane assays do not definitively distinguish between chemokinesis and chemotaxis, single-cell chemotaxis assays were used to confirm these findings and test whether mating-type or white-opaque switching affects the release of attractant. Our results demonstrate that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata release bona fide chemoattractants for PMNs. S. cerevisiae, however, releases a chemokinetic factor but not a chemoattractant. Characterization of the C. albicans chemoattractant revealed that it is a peptide of approximately 1 kDa. Whereas the mating type of C. albicans did not affect the release of chemoattractant, switching did. White-phase cells released chemoattractant, but opaque-phase cells did not. Since the opaque phase of C. albicans represents the mating-competent phenotype, it may be that opaque-phase cells selectively suppress the release of chemoattractant to facilitate mating.     

Suicide in women with gynecologic cancer

Objective(s)

To characterize the suicide rates among patients with gynecologic cancer in the Unites States and to identify factors associated with high suicide rates.

Method(s)

Subjects with a diagnosis of gynecologic cancer were identified from the Surveillance, Epidemiology, and End Results (SEER) program for the period 1988-2007. Comparison with women in the general US population was based on WHO data 2005, matched for age in 10-year categories. Cox regression models were used to perform multivariate modeling for factors associated with suicide.

Result(s)

Among 252,235 patients followed for 1,207,278 person-years, the suicide rate was 8.3 per 100,000 person-years, with a standardized mortality ratio (SMR) of 1.4 (95% CI 1.2-1.7, p < 0.001). The highest suicide rates were observed in patients with ovarian cancer and within the first year following diagnosis. Suicide risk was associated with younger age at diagnosis, high grade disease and absence of surgical intervention.

Conclusion(s)

Patients with gynecologic cancer have an increased suicide risk when compared to the general population. Suicide rates vary by cancer site and time since diagnosis. Effective screening and appropriate treatment of psychosocial stress among women with gynecologic cancer are warranted.     

Complete nucleotide sequence of rose yellow leaf virus,a new member of the family Tombusviridae

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.     

Molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia

Trauma intensive care unit (TICU) patients requiring mechanical respiratory support frequently develop ventilator-associated pneumonia (VAP). Oral and oropharyngeal bacteria are believed to be responsible for many cases of VAP, but definitive evidence of this relationship is lacking. Earlier studies used conventional culture-based methods for identification of bacterial pathogens, but these methods are insufficient, as some bacteria may be uncultivable or difficult to grow. The purpose of this study was to use a culture-independent molecular approach to analyze and compare the bacterial species colonizing the oral cavity and the lungs of TICU patients who developed VAP. Bacterial samples were acquired from the dorsal tongue and bronchoalveolar lavage fluid of 16 patients. Bacterial DNA was extracted, and the 16S rRNA genes were PCR amplified, cloned into Escherichia coli, and sequenced. The sequencing data revealed the following: (i) a wide diversity of bacterial species in both the oral and pulmonary sites, some of them novel; (ii) known and putative respiratory pathogens colonizing both the oral cavity and lungs of 14 patients; and (iii) a number of bacterial pathogens (e.g., Dialister pneumosintes, Haemophilus segnis, Gemella morbillorum, and Pseudomonas fluorescens) in lung samples that had not been reported previously at this site when culture-based methods were used. Our data indicate that the dorsal surface of the tongue serves as a potential reservoir for bacterial species involved in VAP. Furthermore, it is clear that the diversity of bacterial pathogens for VAP is far more complex than the current literature suggests.     

Chromosome 13q neocentromeres: molecular cytogenetic characterization of three additional cases and clinical spectrum

We report three new cases of chromosome 13 derived marker chromosomes, found in unrelated patients with dysmorphisms and/or developmental delay. Molecular cytogenetic analysis was performed using fluorescence in situ hybridization (FISH) with chromosome-specific painting probes, alpha satellite probes, and physically mapped probes from chromosome 13q, as well as comparative genomic hybridization (CGH). This analysis demonstrated that these markers consisted of inversion duplications of distal portions of chromosome 13q that have separated from the endogenous chromosome 13 centromere and contain no detectable alpha satellite DNA. The presence of a functional neocentromere on these marker chromosomes was confirmed by immunofluorescence with antibodies to centromere protein-C (CENP-C). The cytogenetic location of a neocentromere in band 13q32 was confirmed by simultaneous FISH with physically mapped YACs from 13q32 and immunofluorescence with anti-CENP-C. The addition of these three new cases brings the total number of described inv dup 13q neocentic chromosomes to 11, representing 21% (11/52) of the current overall total of 52 described cases of human neocentric chromosomes. This higher than expected frequency suggests that chromosome 13q may have an increased propensity for neocentromere formation. The clinical spectrum of all 11 cases is presented, representing a unique collection of polysomy for different portions of chromosome 13q without aneuploidies for additional chromosomal regions. The complexity and variability of the phenotypes seen in these patients does not support a simple reductionist view of phenotype/genotype correlation with polysomy for certain chromosomal regions.     

Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans.

Randomly amplified polymorphic DNA (RAPD) analysis, multilocus enzyme electrophoresis (MLEE), and Southern blot hybridization with moderately repetitive DNA probes have emerged as effective fingerprinting methods for the infectious fungus Candida albicans. The three methods have been compared for their capacities to identify identical or highly related isolates, to cluster weakly related isolates, to discriminate between unrelated isolates, and to assess microevolution within a strain. By computing similarity coefficients between 29 isolates from three cities within the continental United States, strong concordance of the results is demonstrated for RAPD analysis, MLEE, and Southern blot hybridization with the moderately repetitive probe Ca3, and weaker concordance of the results is demonstrated for these three fingerprinting methods and Southern blot hybridization with the moderately repetitive probe CARE2. All methods were also demonstrated to be able to resolve microevolution within a strain, with the Ca3 probe exhibiting the greatest resolving power. The strong correlations demonstrated between polymorphic markers assessed by the four independent fingerprinting methods and the nonrandom association between loci demonstrated by RAPD analysis and MLEE provide evidence for strong linkage disequilibrium and a clonal population structure for C. albicans. In addition, a synapomorphic allele, Pep-3A, was found to be present in all members of one of the three clusters discriminated by RAPD analysis, MLEE, and Ca3 fingerprinting, supporting the concordance of the clustering capacities of the three methods, the robustness of the clusters, and the clonal nature of the clusters.     

Antimicrobial activity of truncated alpha-defensin (human neutrophil peptide (HNP)-1) analogues without disulphide bridges

Antimicrobial peptides play an important role in host defence, particularly in the oral cavity where there is constant challenge by microorganisms. The alpha-defensin antimicrobial peptides comprise 30-50% of the total protein in the azurophilic granules of human neutrophils, the most abundant of which is human neutrophil peptide 1 (HNP-1). Despite its antimicrobial activity, a limiting factor in the potential therapeutic use of HNP-1 is its chemical synthesis with the correct disulphide topology. In the present study, we synthesised a range of truncated defensin analogues lacking disulphide bridges. All the analogues were modelled on the C-terminal region of HNP-1 and their antimicrobial activity was tested against a range of microorganisms, including oral pathogens. Although there was variability in the antimicrobial activity of the truncated analogues synthesised, a truncated peptide named 2Abz(23)S(29) displayed a broad spectrum of antibacterial activity, effectively killing all the bacterial strains tested. The finding that truncated peptides, modelled on the C-terminal beta-hairpin region of HNP-1 but lacking disulphide bridges, display antimicrobial activity could aid their potential use in therapeutic interventions.