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Aims: To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism. Methods and Results: Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax ( Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10-μl sample volume onto the sample support was 106 particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 μl, the detection limit could be reduced to 105 particles per ml for spores and to 5 × 104 particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated. Conclusions: The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support. Significance and Impact of the Study: Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique. [ABSTRACT FROM AUTHOR]

Summary: High‐pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high‐pressure freezer. We explored the potential of a 10 μm thin polyester filter membrane (PE‐filter) as a versatile sample substrate for high‐pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE‐filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE‐filter. These microorganism‐loaded PE‐filters were used for high‐pressure freezing, freeze‐substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross‐sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa‐cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism‐loaded PE‐filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze‐substituted PE‐filters samples with scanning electron microscopy, demonstrates the versatility of the PE‐filter as a sample substrate for a wide array of microorganisms. Lay Description: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology‐changes due to drying artefacts or the impact of chemical stabilisers, high‐pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to –196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology‐destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 μm in optimal cases). High‐pressure freezing is regarded as the gold‐standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high‐pressure freezer and their transfer into the high‐pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 μm thin polyester filter membrane (PE‐filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE‐filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface‐adhering higher cells were able to grow directly on the PE‐filter. These microorganism‐loaded PE‐filters could be directly used for high‐pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross‐sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa‐cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism‐loaded PE‐filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze‐substituted PE‐filters samples with scanning‐electron microscopy, demonstrates the versatility of the PE‐filter as a sample substrate for a wide array of microorganisms. [ABSTRACT FROM AUTHOR]

Aims Successive application of negative staining transmission electron microscopy ( TEM) and tip-enhanced Raman spectroscopy ( TERS) is a new correlative approach that could be used to rapidly and specifically detect and identify single pathogens including bioterrorism-relevant viruses in complex samples. Our objective is to evaluate the TERS-compatibility of commonly used electron microscopy ( EM) grids (sample supports), chemicals and negative staining techniques and, if required, to devise appropriate alternatives. Methods and Results While phosphortungstic acid ( PTA) is suitable as a heavy metal stain, uranyl acetate, paraformaldehyde in HEPES buffer and alcian blue are unsuitable due to their relatively high Raman scattering. Moreover, the low thermal stability of the carbon-coated pioloform film on copper grids (pioloform grids) negates their utilization. The silicon in the cantilever of the silver-coated atomic force microscope tip used to record TERS spectra suggested that Si-based grids might be employed as alternatives. From all evaluated Si-based TEM grids, the silicon nitride ( Si N) grid was found to be best suited, with almost no background Raman signals in the relevant spectral range, a low surface roughness and good particle adhesion properties that could be further improved by glow discharge. Conclusions Charged Si N grids have excellent particle adhesion properties. The use of these grids in combination with PTA for contrast in the TEM is suitable for subsequent analysis by TERS. Significance and Impact of the Study The study reports fundamental modifications and optimizations of the negative staining EM method that allows a combination with near-field Raman spectroscopy to acquire a spectroscopic signature from nanoscale biological structures. This should facilitate a more precise diagnosis of single viral particles and other micro-organisms previously localized and visualized in the TEM. [ABSTRACT FROM AUTHOR]

Aims: The objective of this study was to determine the effects of Ca‐dipicolinic acid (CaDPA), cortex‐lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. Methods and Results: Bacillus subtilis spores, wild‐type, CaDPA‐less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine‐treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild‐type and CaDPA‐less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores ± CaDPA, with up to 99% killing with 1 mol l−1 dodecylamine in 4 h at 45°C with spores at ~108 ml−1. Dodecylamine killing of decoated wild type and CLE‐less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA‐less spores, with ≥99% killing in 5 min. Propidium iodide stained intact spores ± CaDPA minimally, decoated CaDPA‐replete spores or dodecylamine‐killed CLE‐less spores peripherally, and cores of decoated CaDPA‐less spores and dodecylamine‐killed intact spores with CLEs. The IM of some decoated CaDPA‐less spores was greatly reorganized. Conclusions: Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. Significance and Impact of the Study: This work gives new information on killing bacterial spores by dodecylamine, and how spore IM's relative impermeability is maintained. [ABSTRACT FROM AUTHOR]

The morphology and structure of biological nanoparticles, such as viruses, can be efficiently analysed by transmission electron microscopy (TEM). To chemically characterise such nanoparticles in heterogeneous samples at the single particle level, we suggest tip-enhanced Raman spectroscopy (TERS) as a correlative method. Here we describe a TERS-compatible staining procedure for TEM which involves sample pre-scanning by TEM imaging, nanoparticle relocalisation by atomic force microscopy (AFM) followed by spectroscopic characterization of the virus nanoparticles using TERS. First successful correlative measurements are demonstrated on tobacco mosaic virus particles deposited on silicon-based TEM sample supports. In addition, the advantages and problems of this methodology are discussed. [ABSTRACT FROM AUTHOR]

Early occurrence of small-fibre neuropathy (SFN) is a common feature of Fabry disease (FD) - an X-linked storage disorder caused by reduced activity of the α-galactosidase A ( α-GAL). Although SFN may result from different disorders, the cause is often unclear. Therefore, we investigated the frequency of FD in patients with SFN of unknown aetiology. Patients with idiopathic SFN, established by sensory quantitative testing and/or skin biopsy, were examined for mutations in the α-GAL gene. Where mutations in the α-GAL gene were identified, levels of globotriaosylceramide (Gb) were measured in urine and blood and the α-GAL activity was evaluated. When new mutations were detected, a diagnostic work-up was performed as well as a Gb accumulation in the skin, lyso-Gb in blood and Gb_24 in urine were proved. Twenty-four of 29 eligible patients were enrolled in the study. Mutations in the α-GAL gene were observed in five patients. A typical mutation for FD (c.424T>C, [C142R]) was detected in one patient. In four patients, a complex intronic haplotype within the α-GAL gene (IVS0-10C>T [rs2071225], IVS4-16A>G [rs2071397], IVS6-22C>T [rs2071228]) was identified. The relevance of this haplotype in the pathogenesis of FD remains unclear until now. However, these patients showed increased concentrations of Gb and/or lyso-Gb, while no further manifestations for FD could be proved. Fabry disease should be considered in patients with SFN of unknown aetiology, and screening for FD should be included in the diagnostic guidelines for SFN. The significance of the intronic haplotype regarding SFN needs further evaluation. [ABSTRACT FROM AUTHOR]

The concept of prebiotics is well known from food products, where several ingredients are used to stimulate the beneficial gut microflora. However, prebiotic substances can in principle be applied to balance almost any microbial community to achieve advantageous effects. Since many cosmetic products target skin bacteria as a cause for undesirable conditions like inflammation or body odour, prebiotic actives can help to cope with these problems in a very effective way. To ensure a focused and successful mode of action we demonstrated that it is essential to analyse the microflora and the interactions between different bacterial species carefully. These analyses were made using a molecular method, fluorescence in situ hybridization (FISH), allowing a precise determination of a bacterial community without the drawbacks of classical culture methods. In a further step it was then possible to identify new actives that inhibit harmful or unwanted bacterial species while protecting the beneficial flora. Using a quick and reliable in vitro screening method we found several plant extracts (e.g. Ginseng or Black currant) which inhibit the inflammation-causing bacterium Propionibacterium acnes, but do not affect beneficial species like coagulase-negative staphylococci. In addition, it could also be demonstrated that a prebiotic product line for treatment of inflamed (or acne prone) skin showed the predicted effects on the microflora composition in a first in vivo trial, i.e. decrease of Propionibacterium acnes at a constant level of total bacteria. After 3 weeks, the microflora of 91% of the volunteers had been rebalanced in this way. Furthermore, these formulations have been shown to exhibit excellent skin compatibility compared to an antibacterial product. Thus, prebiotic substances have the potential to provide a gentle and sustainable alternative to undirected antibacterial ingredients which can cause irritation, and frequently only show short time effects. Paper presented at the IFSCC Conference 2005, Florence, Italy. [ABSTRACT FROM AUTHOR]

IFSCC Magazine, 12 (2009) (2) 101–105 Extracts of grapevine, especially in the fermented form of wine, are reported to exert beneficial effects to human health. Grapevine polyphenols are considered to be the main compounds responsible for this beneficial activity, and of these, resveratrol has been studied in most detail. Because of its antioxidant properties, resveratrol demonstrates cell protective characteristics, but it is also able to reduce cell proliferation and induce apoptosis, especially in cancer cells. Investigations on the effects of resveratrol on UVB irradiated primary keratinocytes and HaCats have demonstrated protective properties also in skin cells. Although grapevine extracts are routinely used as an active ingredient in skincare cosmetics, the activity of these extracts on skin, apart from that of resveratrol, is only poorly understood. Here the effects on skin cells of a grapevine extract enriched with the resveratrol dimer ε-viniferin were analyzed in vitro and in vivo. Keywords: Fibroblast, collagen contraction, confocal laser microscopy, resveratrol, senescence This paper was presented as a poster at the 25th IFSCC Congress 2008, Barcelona, Spain [ABSTRACT FROM AUTHOR]

Plant secondary metabolites such as flavonoids, isoflavones and phytosterols have been proposed as cosmetic ingredients displaying anti-aging effects. On the cellular level, however, the activity profiles of these ingredients are only partially understood. In this study we analyzed the effects of apple seed phytosterols on age-related structural and functional parameters using cell biochemical, molecular biological and bioengineering techniques. The expression of age-related genes was studied using skin equivalents and cDNA microarrays. Incubation of skin equivalents with apple seed phytosterols had significant consequences: (i) differential regulation of a set of genes associated with keratinocyte proliferation and differentiation, (ii) stimulation of hyaluronic acid synthesis, and (iii) increase of epidermal thickness.In vivostudies revealed that apple seed phytosterols improve skin elasticity and decrease skin roughness. In conclusion, apple seed phytosterols display distinct biological effects and significantly improve the structure and function of mature skin. [ABSTRACT FROM AUTHOR]

Synopsis A tissue engineered human skin equivalent is successfully used for the testing of raw materials and cosmetic formulations. This reconstructed skin is supported by a collagen-glycosaminoglycan-chitosan biopolymer in which human keratinocytes and dermal fibroblasts were co-cultured to form a tissue that closely reproduces the in vivo architecture of normal human skin and takes into account the complex interactions between epidermis and dermis. On the other hand, dermal and epidermal responses can be assessed separately in the dermal or skin equivalent. The three-dimensional model has important advantages compared to monolayer cell cultures and epidermis models in efficacy testing: (i) the possibility of long-term cultivation with repeated application of cream formulations containing bioactives and (ii) the similarity to human skin concerning the interaction between dermis and epidermis. These similarities include the expression of keratinocyte differentiation markers such as cytokeratin 10, filaggrin and transglutaminase, as well as proteins of the basal lamina (laminin, collagen type IV) and extracellular matrix proteins such as elastin. The efficacy of selected bioactives was determined using different endpoints, for example, stimulation of collagen synthesis in the dermal and skin equivalents was shown in comparison to vitamin C as a positive control. On skin equivalents using immunofluorescence techniques we also demonstrated stimulation of the differentiation marker filaggrin, which is important for skin moisturization. The results could be used for claim substantiation, e.g. for the treatment of dry and aged skin. [ABSTRACT FROM AUTHOR]