Keratin 13 deficiency causes white sponge nevus in mice.

White sponge nevus (WSN) is a benign autosomal dominant disorder characterized by the formation of white spongy plaques in the oral mucosa. Keratin (KRT) 13 is highly expressed in the mucosa, and mutations in this gene have been commonly associated with WSN patients. However, it remains unknown whether there is a causal relationship between KRT13 mutations and WSN and what the underlying mechanisms might be. Here, we use mouse genetic models to demonstrate that Krt13 is crucial for the maintenance of epithelial integrity. Krt13 knockout mice show a WSN-like phenotype in several tissues, including the tongue, buccal mucosa, and esophagus. Transcriptome analyses uncover that Krt13 regulates a cohort of gene networks in tongue epithelial cells, including epithelial differentiation, immune responses, stress-activated kinase signaling, and metabolic processes. We also provide evidence that epithelial cells without Krt13 are susceptible to mechanical stresses experienced during postnatal life, resulting in unbalanced cell proliferation and differentiation. These data demonstrate that Krt13 is essential for maintaining epithelial homeostasis and loss of Krt13 causes the WSN-like phenotype in mice.

Three-dimensional bright-field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms.

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.

Chitosan-based nanoformulated (-)-epigallocatechin-3-gallate (EGCG) modulates human keratinocyte-induced responses and alleviates imiquimod-induced murine psoriasiform dermatitis.

BACKGROUND: Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation, and inflammation, leading to disrupted skin barrier function. The use of natural agents that can abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) mitigated inflammation and increased the expression of caspase-14 while promoting epidermal differentiation and cornification. However, bioavailability issues have restricted the development of EGCG for the treatment of psoriasis. MATERIALS AND METHODS: To overcome these limitations, we employed a chitosan-based polymeric nanoparticle formulation of EGCG (CHI-EGCG-NPs, hereafter termed nanoEGCG) suitable for topical delivery for treating psoriasis. We investigated and compared the efficacy of nanoEGCG versus native or free EGCG in vitro and in an in vivo imiquimod (IMQ)-induced murine psoriasis-like dermatitis model. The in vivo relevance and efficacy of nanoEGCG formulation (48 µg/mouse) were assessed in an IMQ-induced mouse psoriasis-like skin lesion model compared to free EGCG (1 mg/mouse). RESULTS: Like free EGCG, nanoEGCG treatment induced differentiation, and decreased proliferation and inflammatory responses in cultured keratinocytes, but with a 4-fold dose advantage. Topically applied nanoEGCG elicited a significant (p<0.01) amelioration of psoriasiform pathological markers in IMQ-induced mouse skin lesions, including reductions in ear and skin thickness, erythema and scales, proliferation (Ki-67), infiltratory immune cells (mast cells, neutrophils, macrophages, and CD4+ T cells), and angiogenesis (CD31). We also observed increases in the protein expression of caspase-14, early (keratin-10) and late (filaggrin and loricrin) markers of differentiation, and the activator protein-1 factor (JunB). Importantly, a significant modulation of several psoriasis-related inflammatory cytokines and chemokines was observed compared to the high dose of free EGCG (p<0.05). Taken together, topically applied nanoEGCG displayed a >20-fold dose advantage over free EGCG. CONCLUSION: Based on these observations, our nanoEGCG formulation represents a promising drug-delivery strategy for treating psoriasis and possibly other inflammatory skin diseases.

Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor.

Fibroblasts produce extracellular matrix and play an important role in wound healing and scarring. Hepatocyte growth factor (HGF) has strong antifibrotic activity, and has been suggested to have therapeutic potential for treatment of fibrotic diseases. In the present in vitro study, morphological and functional changes of human vocal fold fibroblasts with HGF were examined by transmission electron microscopy and enzyme-linked immunosorbent assay to help clarify the potential use of HGF in the prevention or treatment of vocal fold scarring. The HGF stimulated the production of hyaluronic acid (HA) and decreased the production of collagen type I from the fibroblasts in Reinke's space (FbRS), whereas fibronectin production was not affected. The HGF also changed the shape of the FbRS from an oval shape toward a spindle and stellate shape, and developed Golgi apparatus (GA) and rough endoplasmic reticulum (rER) in the FbRS. The fibroblasts in the macula flava (FbMF) presented with much more production of HA and collagen type I than did FbRS, and were more frequently formed in a stellate shape with well-developed GA and rER. The HGF decreased the production of collagen type I from the FbMF, but barely affected the FbMF in terms of the shape of the cells, the development of GA and rER, or the production of HA. These results were interpreted to suggest that the FbMF are not as susceptible to HGF as are FbRS. On the contrary, HGF appeared to activate the FbRS and modify the function. The increased HA and decreased collagen type I production from the FbRS suggest that HGF may be useful in the prevention or treatment of fibrotic vocal fold scarring.

Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.

Subcellular distribution of Aedes aegypti sterol carrier protein-2 (AeSCP-2) and AeSCP-x was studied using electron microscopy. In both cultured A. aegypti cells and in the larval midgut, AeSCP-2 was detected mostly in the cytosol, with some labeling mitochondria and nucleus, but not in membranous vesicles. The widespread distribution of AeSCP-2 in the midgut epithelium is consistent with its potential lipid transfer function in all phases of cholesterol absorption. In contrast, AeSCP-x was found mostly in the peroxisome. Differences in the subcellular distribution of AeSCP-2 and AeSCP-x suggest that these two members of the SCP-2 gene family are functionally distinct. Overexpression of AeSCP-2 in A. aegypti cells showed increased localization of AeSCP-2 to cytosol, mitochondria, and nucleus. This is the first report on the nuclear distribution of an SCP. Overexpression of AeSCP-2 resulted in increased cholesterol incorporation in cells, suggesting that AeSCP-2 enhances cholesterol uptake.

Transposon-5 mutagenesis transforms Corynebacterium matruchotii to synthesize novel hybrid fatty acids that functionally replace corynomycolic acid.

Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery.