Evaluation of multiplex real-time PCR for identifying dermatophytes in clinical samples-A multicentre study.

The gold-standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real-time polymerase chain reaction (RT-PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT-PCR was further evaluated in 200 community-based samples obtained from a health maintenance organisation and 103 military-personnel-based samples analysed at a central laboratory. In hospital-based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non-concordance, sequencing was consistent with RT-PCR results. RT-PCR correctly identified all smear- and culture-positive cases in community and military-personnel samples. The results were available within 4 hours. The multiplex RT-PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.

Peptides mimicking the unique ARTS-XIAP binding site promote apoptotic cell death in cultured cancer cells.

PURPOSE: XIAP [X-linked inhibitor of apoptosis (IAP) protein] is the best characterized mammalian caspase inhibitor. XIAP is frequently overexpressed in a variety of human tumors, and genetic inactivation of XIAP in mice protects against lymphoma. Therefore, XIAP is an attractive target for anticancer therapy. IAP antagonists based on a conserved IAP-binding motif (IBM), often referred to as "Smac-mimetics," are currently being evaluated for cancer therapy in the clinic. ARTS (Sept4_i2) is a mitochondrial proapoptotic protein which promotes apoptosis by directly binding and inhibiting XIAP via a mechanism that is distinct from all other known IAP antagonists. Here, we investigated the ability of peptides derived from ARTS to antagonize XIAP and promote apoptosis in cancer cell lines. EXPERIMENTAL DESIGN: The ability of synthetic peptides, derived from the C-terminus of ARTS, to bind to XIAP, stimulate XIAP degradation, and induce apoptosis was examined. We compared the response of several cancer cell lines to different ARTS-derived peptides. Pull-down assays were used to examine binding to XIAP, and apoptosis was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, caspase activation, and Western blot analyses of caspase substrates. RESULTS: The C-terminus of ARTS contains a unique sequence, termed ARTS-IBM (AIBM), which is important for binding to XIAP and cell killing. AIBM peptides can bind to XIAP-BIR3, penetrate cancer cells, reduce XIAP levels, and promote apoptosis. CONCLUSIONS: Short synthetic peptides derived from the C-terminus of ARTS are sufficient for binding to XIAP and can induce apoptosis in cancer cells. These results provide proof-of-concept for the feasibility of developing ARTS-based anticancer therapeutics.

Proteomics of the response of Arabidopsis thaliana to infection with Alternaria brassicicola.

We have studied the proteome of the model plant Arabidopsis thaliana infected with a necrotrophic fungal pathogen, Alternaria brassicicola. The Arabidopsis-A. brassicicola host-pathogen pair is being developed as a model genetic system for incompatible plant-fungal interactions, in which the spread of disease is limited by plant defense responses. After confirming that a defense response was induced at the transcriptional level, we identified proteins whose abundance on 2-DE gels increased or decreased in infected leaves. At least 11 protein spots showed reproducible differences in abundance, increasing or decreasing during the progress of the infection. The pathogenesis-related protein PR4, a glycosyl hydrolase, and the antifungal protein osmotin are strongly up-regulated. Two members of the Arabidopsis glutathione S-transferase (GST) family increased in abundance in infected leaves. The spots in which these GST proteins were identified contain additional members of the GST family. Representation of GST family members in several protein spots migrating at similar molecular weight suggests post-translational modifications. The signature of GST regulation may be specific for the type of plant-pathogen interaction. The proteomic view of the defense response to A. brassicicola can be compared with other types of plant-pathogen interactions, and to leaf senescence, identifying unique regulatory patterns.

Large-scale identification of leaf senescence-associated genes.

Leaf senescence is a form of programmed cell death, and is believed to involve preferential expression of a specific set of "senescence-associated genes" (SAGs). To decipher the molecular mechanisms and the predicted complex network of regulatory pathways involved in the senescence program, we have carried out a large-scale gene identification study in a reference plant, Arabidopsis thaliana. Using suppression subtractive hybridization, we isolated approximately 800 cDNA clones representing SAGs expressed in senescing leaves. Differential expression was confirmed by Northern blot analysis for 130 non-redundant genes. Over 70 of the identified genes have not previously been shown to participate in the senescence process. SAG-encoded proteins are likely to participate in macromolecule degradation, detoxification of oxidative metabolites, induction of defense mechanisms, and signaling and regulatory events. Temporal expression profiles of selected genes displayed several distinct patterns, from expression at a very early stage, to the terminal phase of the senescence syndrome. Expression of some of the novel SAGs, in response to age, leaf detachment, darkness, and ethylene and cytokinin treatment was compared. The large repertoire of SAGs identified here provides global insights about regulatory, biochemical and cellular events occurring during leaf senescence.