Andreas Kiesow

Melanin Is Not Required for Turgor Generation but Enhances Cell-Wall Rigidity in Appressoria of the Corn Pathogen Colletotrichum graminicola

The ascomycete and causative agent of maize anthracnose and stem rot, Colletotrichum graminicola, differentiates melanized infection cells called appressoria that are indispensable for breaching the plant cell wall. High concentrations of osmolytes accumulate within the appressorium, and the internal turgor pressure of up to 5.4 MPa provides sufficient force to penetrate the leaf epidermis directly. In order to assess the function of melanin in C. graminicola appressoria, we identified and characterized the polyketide synthase 1 (CgPKS1) gene which displayed high similarity to fungal polyketide synthases (PKS) involved in synthesis of 1,3,6,8-tetrahydronaphthalene, the first intermediate in melanin biosynthesis. Cgpks1 albino mutants created by targeted gene disruption were unable to penetrate intact leaves and ruptured frequently but, surprisingly, were able to penetrate ultrathin polytetrafluoroethylene membranes mimicking the plant surface. Nonmelanized Cgpks1 appressoria were sensitive to externally applied cell-wall-degrading enzymes whereas melanized appressoria were not affected. Expression studies using a truncated CgPKS1 fused to green fluorescent protein revealed fluorescence in immature appressoria and in setae, which is in agreement with transcript data obtained by RNA-Seq and quantitative polymerase chain reaction. Unexpectedly, surface scans of mutant and wild-type appressoria revealed considerable differences in cell-wall morphology. Melanization of appressoria is indispensable for successful infection of intact leaves. However, cell collapse experiments and analysis of the appressorial osmolyte content by Mach-Zehnder interferometry convincingly showed that melanin is not required for solute accumulation and turgor generation, thus questioning the role of melanin as a barrier for osmolytes in appressoria of C. graminicola.

Formation of metal particle nanowires induced by ultrashort laser pulses

We report a simple method to generate metallic nanowire arrays. By irradiating thin organic films with embedded silver or gold nanoparticles with series of ultrashort laser pulses, wire arrays containing more than 30 nearly parallel wires several microns in length are formed. The individual nanowires are between 100 and 200 nm wide with equal line space ratios, significantly smaller than the laser wavelength used. The wire direction correlates to the linear polarization of the laser pulses. This structural dependence on the laser polarization is mirrored in corresponding anisotropic optical and electrical film properties.

Generation of wavelength-dependent, periodic line pattern in metal nanoparticle-containing polymer films by femtosecond laser irradiation

Thin polymer films containing metal nanoparticles were irradiated with ultrashort, linearly polarized laser pulses. As result of irradiation, nanostructural changes occur in a type of periodically arranged, line-like areas with modified particle size and shape distribution. The periodic formation in this nanocomposite material is observed only for a small filling factor range, which can be attributed to the percolation region or nearby. Transmission (also in cross section) and scanning electron microscopy were applied to investigate the obtained structure modifications. A linear dependency between the period Λ of the line structures and the laser wavelength λ (800, 528, 400, and 266nm) used with Λ∕λ≈0.70 is assumed. The structural changes and the physical mechanism of the periodic formation are discussed.

Controlled nail delivery of a novel lipophilic antifungal agent using various modern drug carrier systems as well as in vitro and ex vivo model systems

The penetration behavior into human nails and animal hoof membranes of a novel antifungal agent (EV-086K) for the treatment of onychomycosis was investigated in this study. The new drug provides a high lipophilicity which is adverse for penetration into nails. Therefore, four different formulations were developed, with particular focus on a colloidal carrier system (CCS) due to its penetration enhancing properties. On the one hand, ex vivo penetration experiments on human nails were performed. Afterwards the human nail plates were cut by cryomicrotome in order to quantify the drug concentration in the dorsal, intermediate and ventral nail layer using high-performance liquid chromatography (HPLC) with UV detection. On the other hand, equine and bovine hoof membranes were used to determine the in vitro penetration of the drug into the acceptor compartment of an online diffusion cell coupled with Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy. In combination, both results should exhibit a correlation between the EV-086K penetration behavior in human nail plates and animal hoof membranes. The investigations showed that the developed CCS could increase drug delivery through the human nail most compared to other formulations (nail lacquer, solution and hydrogel). Using animal hooves in the online diffusion cell, we were able to calculate pharmacokinetic data of the penetration process, especially diffusion and permeability coefficients. Finally, a qualitative correlation between the penetration results of human nails and equine hooves was established.

Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

Interaction of water with different cellulose ethers: a Raman spectroscopy and environmental scanning electron microscopy study.

Different non‐ionic cellulose ethers like methyl cellulose (MC), hydroxypropyl cellulose (HPC) and hydroxypropylmethyl cellulose (HPMC) were investigated. The characterization of the cellulose ethers was carried out by thermogravimetry and sorption/desorption isotherms. Differences in the properties of the cellulose ether films were described by time‐dependent contact angle measurements. Changes in molecular structure of the raw materials, gels and films caused by water contact were studied using Raman spectroscopy. Differences between the substitution types and changes due to the gel or film formation were observed. An environmental scanning electron microscopy (ESEM) technique was used to distinguish the morphological behaviour of the cellulose ether films in contact with water. Based on in‐situ ESEM experiments, the swelling and drying behaviour of the various stages of cellulose ether films (film‐hydrated film‐dried film) were quantified by using image analysis.

Material compatibility and antimicrobial activity of consumer products commonly used to clean dentures

STATEMENT OF PROBLEM Regular denture cleaning is essential to good oral health, but only limited evidence is available regarding the effects of common cleaning routines. PURPOSE The purpose of this in vitro study was to determine the compatibility of denture materials with and the antimicrobial effects of typical cleaning regimens. MATERIAL AND METHODS The evaluated treatments were derived from a study of dental professional recommendations and consumer habits, including denture cleanser tablets, toothpaste, mouthwash, isopropyl alcohol (IPA), household bleach, soap, and vinegar. The material integrity of denture materials, including polymethyl methacrylate (PMMA) and metals, was evaluated by scanning electron microscopy and profilometry after treatment with laboratory regimens simulating 2 years of typical consumer use. Treatments were also evaluated in a microbial kill time assay against a range of oral microorganisms with typical treatment regimens. RESULTS Alcohol-based mouthwash and IPA damaged the surface of PMMA, and brushing with toothpaste caused scratching and surface material loss. Bleach caused limited damage to PMMA, but corroded CoCr alloy (pitting) and solder (layer formation). Denture tablets caused little damage to any materials apart from the layer formation on silver solder. Vinegar and soap were compatible with all materials. In antimicrobial assays, bleach gave excellent results, and IPA and mouthwash required concentrated dilutions to be effective. Cleanser tablets were effective at 5 minutes treatment time against all organisms. Toothpaste was effective against bacteria but not Candida albicans. Vinegar, soaps, salt, and sodium bicarbonate were microbially ineffective. CONCLUSIONS Bleach was highly antimicrobial but incompatible with metal dental prosthesis components. IPA and mouthwash were antimicrobial but damaged PMMA. Specialist denture cleanser tablets gave a good combination of microbial efficacy and reasonable material compatibility.

EMMPRIN/CD147 deficiency disturbs ameloblast–odontoblast cross-talk and delays enamel mineralization

Tooth development is regulated by a series of reciprocal inductive signaling between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissue formation. We report a delay in enamel deposition and formation that is clearly distinguishable in the growing incisor and associated with a significant reduction of MMP-3 and MMP-20 expression in tooth germs of KO mice. Insufficient basement membrane degradation is evidenced by a persistent laminin immunostaining, resulting in a delay of both odontoblast and ameloblast differentiation. Consequently, enamel volume and thickness are decreased in adult mutant teeth but enamel maturation and tooth morphology are normal, as shown by micro-computed tomographic (micro-CT), nanoindentation, and scanning electron microscope analyses. In addition, the dentino-enamel junction appears as a rough calcified layer of approximately 10±5μm thick (mean±SD) in both molars and growing incisors of KO adult mice. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. The mild tooth phenotype observed in EMMPRIN KO mice suggests that the direct effect of EMMPRIN may be limited to a short time window, comprised between basement membrane degradation allowing direct cell contact and calcified matrix deposition.

Thin PTFE-like membranes allow characterizing germination and mechanical penetration competence of pathogenic fungi.

Investigating the penetration behavior of pathogenic fungi often fails because natural substrata vary significantly with respect to morphological and microstructural properties. To establish in vitro penetration assays, reproducible production of thin membranes with defined properties such as thickness, mechanical and chemical stability, roughness and hydrophobicity is essential. In this paper we describe the fabrication and characterization of membranes mimicking plant surfaces with respect to hydrophobicity and report on penetration assays with plant pathogenic fungi known to exert enormous force during the infection process. In order to reach high hydrophobicity, polytetrafluoroethylene-like membranes were used. By varying membrane thickness, the penetration competence of different pathogens could be evaluated and quantified. In addition, a relationship between surface roughness in the nanometer scale and the germination rate has been observed.

A Descriptive in vitro Electron Microscopic Study of Acidic Fluoride-Treated Enamel: Potential Anti-Erosion Effects

This study aimed to investigate the surface zones of acidic fluoride-treated enamel. Human teeth were each divided into three or four enamel specimens that were treated for 10 min with solutions of 0.2 and 0.4% HF (pH 3.09 and 2.94), 1.74% SnF2 (pH 2.9), 0.68% TiF4 (pH 1.6) and 0.84% NaF (pH 4.5). Untreated specimens functioned as negative controls. The microstructure and elemental composition of the surface zones were studied by scanning electron microscopy/energy-dispersive X-ray (EDX) analysis, transmission electron microscopy (TEM) and nanospot-EDX following cross-sectional preparation using focused ion beam technology. TEM/EDX analyses of NaF-treated specimens showed a 500-nm-thick closed surface film containing 20-40 at% (atomic percent) F. HF-treated specimens had a distinct surface film 200-600 nm thick (dense, not globular) containing 45-47 at% F. TiF4-treated specimens had a surface film of 200-300 nm in thickness containing 8-11 at% Ti but no detectable fluoride. SnF2-treated specimens had a modified surface enamel layer varying in thickness from 200 to 800 nm with an inhomogeneous distribution of Sn. Local spots were detected with as high as 8 at% Sn (30 wt%, weight percent). The results suggest that the reaction mechanisms of SnF2 and TiF4 solutions with dental enamel differ from those occurring after enamel exposure to acidulated NaF and HF solutions. While the HF and NaF treatments resulted in the formation of CaF2-like material as shown by EDX, no significant surface fluoridation was found for SnF2 and TiF4 solutions within the TEM/EDX detection limits. These results suggest that the erosion-protective mechanisms of these latter compounds probably relate more to the formation of hardly soluble and acid-resistant reaction surface films and less to surface fluoride incorporation.