Correction to: Analysis of the Fungal Flora in Environmental Dust Samples by PCR-SSCP Method.

The original version of this article contained a mistake in the co-author's conflict of interest statement. Erika von Mutius wishes to add the following disclosures: "E. von Mutius is listed as an inventor on the following patents: publication number EP 1411977, composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases, granted on 18 April 2007; publication number EP1637147, stable dust extract for allergy protection, granted on 10 December 2008; publication number EP 1964570, pharmaceutical compound to protect against allergies and inflammatory diseases, granted on 21 November 2012. E. von Mutius is listed as inventor and has received royalties on the following patent: publication number EP2361632, specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders, granted on 19 March 2014".

Fluorescent Diarylethene Photoswitches-A Universal Tool for Super-Resolution Microscopy in Nanostructured Materials.

Super-resolution fluorescence microscopy allows for unprecedented in situ visualization of biological structures, but its application to materials science has so far been comparatively limited. One of the main reasons is the lack of powerful dyes that allow for labeling and photoswitching in materials science systems. In this study it is shown that appropriate substitution of diarylethenes bearing a fluorescent closed and dark open form paves the way for imaging nanostructured materials with three of the most popular super-resolution fluorescence microscopy methods that are based on different concepts to achieve imaging beyond the diffraction limit of light. The key to obtain optimal resolution lies in a proper control over the photochemistry of the photoswitches and its adaption to the system to be imaged. It is hoped that the present work will provide researchers with a guide to choose the best photoswitch derivative for super-resolution microscopy in materials science, just like the correct choice of a Swiss Army Knife's tool is essential to fulfill a given task.

Identification of fungal candidates for asthma protection in a large population-based study.

BACKGROUND: Exposure to molds has been related to asthma risk both positively and negatively, depending on the environmental setting. The pertinent results are based on generic markers or culturing methods although the majority of present fungi cannot be cultured under laboratory conditions. The aim of the present analysis was to assess environmental dust samples for asthma-protective fungal candidates with a comprehensive molecular technique covering also non-cultivable and non-viable fungi. METHODS: Mattress dust samples of 844 children from the GABRIELA study were analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) of the fungus-specific internal transcribed spacer (ITS) region. Known asthma candidate species were tested for their associations with asthma, and further gel positions were sought to explain the above. As a second, data-driven, analysis, we tested the association of each individual gel position with asthma. RESULTS: In the hypothesis-driven approach, Penicillium chrysogenum emerged with an odds ratio of 0.80 (95% confidence interval 0.66-0.96; p = 0.020). The effect size was changed by 39% toward the null when adjusting for the two bands 683 (DNA of Metschnikowia sp., Aureobasidium spp.) and 978 (DNA of Epicoccum spp., Galactomyces spp., uncultured Penicillium). The data-driven approach yielded an additional band (containing DNA of Pseudotaeniolina globosa) with reduced risk of asthma (OR = 0.80 [0.66-0.96], p = 0.012). CONCLUSIONS: A large population-based study revealed several fungal taxa with inverse associations with childhood asthma. Molds produce a variety of bioactive compounds with detrimental but also beneficial immunoregulatory capacities, which renders them promising targets for further asthma research.

Analysis of the fungal flora in environmental dust samples by PCR-SSCP method.

Conventional microbiological techniques yield only limited information on the composition of fungal communities in dust. The aim of this study was to establish and optimize PCR-single strand conformation polymorphism (PCR-SSCP) analysis for investigation of fungal diversity in rural dust samples. Three different DNA extraction protocols were tested on 38 fungal cultures. A total of six known universal fungal primer pairs were tested targeting the 18S rRNA gene, the 28S rRNA gene and the ITS region, respectively. Objective evaluation was performed with respect to the following parameters: efficiency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCR-SSCP analysis; possibility to classify the amplified gene fragments to species level. Primer ITS1/ITS4 and PowerSoil™ DNA isolation showed the best performance in most cases and were chosen for further analysis. The detection limit of the developed system was 200 CFU/g dust. Moreover, the reproducibility of the system could be demonstrated, leading to average profile similarities of 94.94% [SD = 2.51] within gels, 93.03% [SD = 4.69] between different days and 87.66% [SD = 6.62] between different gels when testing shed and mattress dust samples. Sequencing allowed identification on species level, in detail: Alternaria alternata, Cladosporium sphaerospermum, Cladosporium cladosporioides as well as the yeasts Candida cabralensis and Candida catenulata. This demonstrates the adaptability of the method. In this study, a standardized system for fungal community analysis was developed that provides reproducible results applicable for epidemiological purposes.

PCR-SSCP-based reconstruction of the original fungal flora of heat-processed meat products.

Food processing of spoiled meat is prohibited by law, since it is a deception and does not comply with food safety aspects. In general, spoilage of meat is mostly caused by bacteria. However, a high contamination level of fungi could be also found in some meat or meat products with certain preserving conditions. In case that unhygienic meat is used to produce heat processed products, the microorganisms will be deactivated by heat, so that they cannot be detected by a standard cultivation method. Therefore, this study aimed to develop and apply a molecular biological method--polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP)--to reconstruct the original fungal flora of heat processed meat. Twenty primer pairs were tested for their specificity for fungal DNA. Since none of them fully complied with all study criteria (such as high specificity and sensitivity for fungal DNA; suitability of the products for PCR-SSCP) in the matrix "meat", we designed a new reverse primer, ITS5.8R. The primer pair ITS1/ITS5.8R amplified DNA from all tested fungal species, but not DNA from meat-producing animals or from ingredients of plant origin (spices). For the final test, 32 DNA bands in acrylamide gel from 15 meat products and 1 soy sauce were sequenced-all originating from fungal species, which were, in other studies, reported to contaminate meat e.g. Alternaria alternata, Aureobasidium pullulans, Candida rugosa, C. tropicalis, C. zeylanoides, Eurotium amstelodami and Pichia membranifaciens, and/or spices such as Botrytis aclada, Guignardia mangiferae, Itersonilia perplexans, Lasiodiplodia theobromae, Lewia infectoria, Neofusicoccum parvum and Pleospora herbarum. This confirms the suitability of PCR-SSCP to specifically detect fungal DNA in heat processed meat products, and thus provides an overview of fungal species contaminating raw material such as meat and spices.

A systematic approach for the identification of novel, serologically reactive recombinant Varicella-Zoster Virus (VZV) antigens.

BACKGROUND: Varicella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells. RESULTS: We present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens (ORFs 1, 4, 14, 49, and 68). These were rearranged in line format and validated with pre-characterized sera. CONCLUSIONS: The line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE) proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens.