Utility of polymerase chain reaction for assessment of onychomycosis during topical therapy.

BACKGROUND: Increased detection of fungi including non-dermatophyte molds (NDMs) using polymerase chain reaction (PCR) methods is well-established. However, the use of PCR to evaluate ongoing onychomycosis treatment outcome has not been investigated. METHODS: Nail samples from 28 patients receiving topical efinaconazole were evaluated by both KOH/culture and PCR methods across the study period. Detection of microorganisms by PCR was compared to the culture at baseline and end of study at month 24 (M24). Fungal detection by both methods was evaluated with respect to clinical cure observed as 100% visual clearance of the target toenail. RESULTS: By culture, all 28 subjects were dermatophyte-positive at pre-treatment; only 4/28 also exhibited an NDM microorganism. According to PCR, 24/28 subjects were dermatophyte-positive pre-treatment, with 25/28 also exhibiting NDMs. At M24, 18/28 (64.3%) participants had negative KOH/culture results, in contrast to 4/28 (14.3%) negative PCR results. PCR showed higher rates of NDM detection than the culture at baseline as well as M24. Calculations to compare the diagnostic utility of KOH/culture versus PCR found that positive tests with both methods reliably indicate the presence of onychomycosis, but negative PCR correlated better with onychomycosis cure than did KOH/culture. DISCUSSION: PCR confirmed a high presence of NDMs pre-treatment, and continued presence of NDMs to M24, with unknown significance requiring further investigation. Though both KOH/culture and PCR have diagnostic limitations, PCR showed better overall utility than culture in predicting onychomycosis topical treatment outcome and should be more strongly considered for evaluation of topical therapies.

High prevalence of mixed infections in global onychomycosis.

Onychomycosis is estimated at a prevalence of 10% worldwide with the infecting organism most commonly Trichophyton rubrum (T. rubrum). Traditional culture identification of causative organisms has inherent risks of overestimating dermatophytes, like T. rubrum, by inhibiting the growth of possible nondermatophyte mould (NDM) environmental contaminants which could be causative agents. Recently, molecular methods have revealed that a proportion of onychomycosis cases in North America may be caused by mixed infections of T. rubrum as an agent co-infecting with one or more NDM. Determining the global burden of mixed infections is a necessary step to evaluating the best therapies for this difficult-to-treat disease. To determine the prevalence of mixed infections in a global population, nail samples from onychomycosis patients in Brazil, Canada, and Israel (n = 216) were analyzed by molecular methods for the presence of dermatophytes and five NDMs. If an NDM was detected, repeat sampling was performed to confirm the NDM. T. rubrum was detected in 98% (211/216) of infections with 39% mixed (84/216). The infection type was more likely to be mixed in samples from Brazil, but more likely to be a dermatophyte in samples from Canada and Israel (Χ2 = 16.92, df = 2, P<0.001). The most common cause of onychomycosis was T. rubrum. In all countries (Brazil, Canada and Israel combined) the prevalence of dermatophyte (Χ2 = 211.15, df = 3, P<0.001) and mixed (dermatophyte and NDM; Χ2 = 166.38, df = 3, P<0.001) infection increased with patient age. Our data suggest that mixed infection onychomycosis is more prevalent than previously reported with the aging population being at increased risk for mixed infections.

Trichophyton rubrum DNA Strains in Patients with Onychomycosis with Persistent Mixed Infections Involving a Nondermatophyte Mold.

BACKGROUND: Onychomycosis is estimated to occur in approximately 10% of the global population, with most cases caused by Trichophyton rubrum. Some persistent onychomycosis is caused by mixed infections of T rubrum and one or more co-infecting nondermatophyte molds (NDMs). In onychomycosis, T rubrum strain types may naturally switch and may also be triggered to switch in response to antifungal therapy. T rubrum strain types in mixed infections of onychomycosis have not been characterized. METHODS: T rubrum DNA strains in mixed infections of onychomycosis containing co-infecting NDMs were compared with a baseline North American population through polymerase chain reaction amplification of ribosomal DNA tandemly repetitive subelements (TRSs) 1 and 2. The baseline DNA strain types were determined from 102 clinical isolates of T rubrum. The T rubrum DNA strain types from mixed infections were determined from 63 repeated toenail samples from 15 patients. RESULTS: Two unique TRS-2 types among the clinical isolates contributed to four unique TRS-1 and TRS-2 strain types. Six TRS-1 and TRS-2 strain types represented 92% of the clinical isolates of T rubrum. Four TRS-1 and TRS-2 strain types accounted for 100% of the T rubrum within mixed infections. CONCLUSIONS: Four unique North American T rubrum strains were identified. In support of a shared ancestry, the T rubrum DNA strain types found in mixed infections with NDMs were among the most abundant types. A population of T rubrum strains in mixed infections of onychomycosis has been characterized, with more than one strain detected in some nails. The presence of a co-infecting NDM in mixed infections may contribute to failed therapy by stabilizing the T rubrum strain type, possibly preventing the antifungal therapy-induced strain type switching observed with infections caused by T rubrum alone.

Onychomycosis InfectionsDo Polymerase Chain Reaction and Culture Reports Agree?

BACKGROUND: Mycological culture is the traditional method for identifying infecting agents of onychomycosis despite high false-negative results, slower processing, and complications surrounding nondermatophyte mold (NDM) infections. Molecular polymerase chain reaction (PCR) methods are faster and suited for ascertaining NDM infections. METHODS: To measure agreement between culture and PCR methods for identification of infecting species of suspected onychomycosis, single toenail samples from 167 patients and repeated serial samples from 43 patients with suspected onychomycosis were processed by culture and PCR for identification of 16 dermatophytes and five NDMs. Agreement between methods was quantified using the kappa statistic (κ). RESULTS: The methods exhibited fair agreement for the identification of all infecting organisms (single samples: κ = 0.32; repeated samples: κ = 0.38). For dermatophytes, agreement was moderate (single samples: κ = 0.44; repeated samples: κ = 0.42). For NDMs, agreement was poor with single samples (κ = 0.16) but fair with repeated samples (κ = 0.25). Excluding false-negative reports from analyses improved agreement between methods in all cases except the identification of NDMs from single samples. CONCLUSIONS: Culture was three or four times more likely to report a false-negative result compared with PCR. The increased agreement between methods observed by excluding false-negative reports statistically clarifies and highlights the major discord caused by false-negative cultures. The increased agreement of NDM identification from poor to fair with repeated sampling along with their poor agreement in the single samples, with and without false-negatives, affirms the complications of NDM identification and supports the recommendation that serial samples help confirm the diagnosis of NDM infections.

Molecular determination of mixed infections of dermatophytes and nondermatophyte molds in individuals with onychomycosis.

BACKGROUND: Reports of mixed infections with nondermatophyte molds (NDMs) and dermatophytes in onychomycosis are rare, possibly owing to the inhibition of NDM growth during traditional culture. We sought to determine the prevalence of mixed infections in onychomycosis using molecular identification. METHODS: Molecular analyses were used to identify infecting organisms directly from at least two serial great toenail samples from each of the 44 patients. RESULTS: Mixed infections were present in 41% of the patients (18 of 44). A single coinfecting NDM was the most common mixed infection and was detected in 34% of patients with onychomycosis (15 of 44), with Fusarium oxysporum present in 14% (6 of 44), Scopulariopsis brevicaulis in 9% (4 of 44), Acremonium spp in 2% (1 of 44), Aspergillus spp in 4.5% (2 of 44), and Scytalidium spp in 4.5% (2 of 44). Mixed infections with two NDMs were found in 7% of patients (3 of 44). CONCLUSIONS: Mixed onychomycosis infections may be more prevalent than previously reported.

Molecular Determination of Mixed Infections of Dermatophytes and Nondermatophyte Moulds in individuals with Onychomycosis.

Abstract Background: Reports of mixed infections with NDMs and dermatophytes in onychomycosis are rare, possibly due to the inhibition of NDM growth during traditional culture. Objective: To determine the prevalence of mixed infections in onychomycosis using molecular identification. Methods: Molecular analyses were utilised to identify infecting organisms directly from at least two serial great toenail samples from each of the 44 subjects. Results: Mixed infections were present in 41% (18/44) of the subjects. A single co-infecting NDM was the most common mixed infection and was detected in 34% (15/44) of onychomycosis patients, with F. oxysporum present in 14% (6/44), S. brevicaulis in 9% (4/44), Acremonium spp in 2% (1/44), Aspergillus spp. in 4.5% (2/44) and Scytalidium spp. in 4.5% (2/44) of patients. Mixed infections with two NDMs were found in 7% (3/44) of the subjects. Conclusions: Mixed dermatophyte/NDM onychomycosis infections may be more prevalent than previously reported.

Isolation of hair follicle bulge stem cells from YFP-expressing reporter mice.

In this article we provide a method to isolate hair follicle stem cells that have undergone targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26-yellow fluorescent protein (YFP) reporter background, which results in YFP expression in the targeted stem cell population. These cells are isolated and purified by fluorescence-activated cell sorting, using epidermal stem cell-specific markers in conjunction with YFP fluorescence. The purified cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as viability and capacity for directional migration.

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury.

Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15--expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury.

Analysis of nuclear export using photoactivatable GFP fusion proteins and interspecies heterokaryons.

In this chapter, we review protocols for the analysis of nucleocytoplasmic shuttling of transcription factors and nuclear proteins, using two different approaches. The first involves the use of photoactivatable forms of the protein of interest by fusion to photoactivatable green fluorescent protein to follow its movement out of the nucleus by live-cell confocal microscopy. This methodology allows for the kinetic characterization of protein movements as well as measurement of steady-state levels. In a second procedure to assess the ability of a nuclear protein to move into and out of the nucleus, we describe the use of interspecies heterokaryon assays, which provide a measurement of steady-state distribution. These technologies are directly applicable to the analysis of nucleocytoplasmic movements not only of transcription factors, but also other nuclear proteins.

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis.

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Restoration of peroxisomal catalase import in a model of human cellular aging.

Peroxisomes play an important role in human cellular metabolism by housing enzymes involved in a number of essential biochemical pathways. Many of these enzymes are oxidases that transfer hydrogen atoms to molecular oxygen forming hydrogen peroxide. The organelle also contains catalase, which readily decomposes the hydrogen peroxide, a potentially damaging oxidant. Previous work has demonstrated that aging compromises peroxisomal protein import with catalase being particularly affected. The resultant imbalance in the relative ratio of oxidases to catalase was seen as a potential contributor to cellular oxidative stress and aging. Here we report that altering the peroxisomal targeting signal of catalase to the more effective serine-lysine-leucine (SKL) sequence results in a catalase molecule that more strongly interacts with its receptor and is more efficiently imported in both in vitro and in vivo assays. Furthermore, catalase-SKL monomers expressed in cells interact with endogenous catalase subunits resulting in altered trafficking of the latter molecules. A dramatic reduction in cellular hydrogen peroxide levels accompanies this increased peroxisomal import of catalase. Finally, we show that catalase-SKL stably expressed in cells by retroviral-mediated transduction repolarizes mitochondria and reduces the number of senescent cells in a population. These results demonstrate the utility of a catalase-SKL therapy for the restoration of a normal oxidative state in aging cells.

Rhamm-/- fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair.

Rhamm (receptor for hyaluronan-mediated motility) is an hyaluronan binding protein with limited expression in normal tissues and high expression in advanced cancers. To understand its physiological functions and identify the molecular mechanisms underlying these functions, we created mice with a genetic deletion of Rhamm. We show that Rhamm(-/-) fibroblasts fail to resurface scratch wounds >3 mm or invade hyaluronan-supplemented collagen gels in culture. We identify a requirement for Rhamm in the localization of CD44 to the cell surface, formation of CD44-ERK1,2 (extracellular-regulated kinase 1,2) complexes, and activation/subcellular targeting of ERK1,2 to the cell nucleus. We also show that cell surface Rhamm, restricted to the extracellular compartment by linking recombinant protein to beads, and expression of mutant active mitogen-activated kinase kinase 1 (Mek1) are sufficient to rescue aberrant signaling through CD44-ERK1,2 complexes in Rh(-/-) fibroblasts. ERK1,2 activation and fibroblast migration/differentiation is also defective during repair of Rh(-/-) excisional skin wounds and results in aberrant granulation tissue in vivo. These results identify Rhamm as an essential regulator of CD44-ERK1,2 fibroblast motogenic signaling required for wound repair.

Identification and characterization of the terminal enzyme of siroheme biosynthesis from Arabidopsis thaliana: a plastid-located sirohydrochlorin ferrochelatase containing a 2FE-2S center.

Higher plant sulfite and nitrite reductases contain siroheme as a prosthetic group. Siroheme is synthesized from the tetrapyrrole primogenitor uroporphyrinogen III in three steps involving methylation, oxidation, and ferrochelation reactions. In this paper we report on the Arabidopsis thaliana sirohydrochlorin ferrochelatase At-SirB. The complete precursor protein of 225 amino acids and shorter constructs in which the first 46 or 79 residues had been removed were shown to complement a defined Escherichia coli sirohydrochlorin ferrochelatase mutant. The mature form of the protein appeared to consist of only 150 amino acids, making it much smaller than previously characterized ferrochelatases. Green fluorescent protein tagging revealed that it is located in the chloroplast. The enzyme was easily produced in E. coli as a recombinant protein, and the isolated enzyme was found to have a specific activity of 48.5 nmol/min/mg. Significantly, the protein purified as a brown-colored solution with a UV-visible spectrum containing maxima at 415 and 455 nm, suggestive of an Fe-S center. EPR analysis of the recombinant protein produced a rhombic spectrum with G-values of 2.04, 1.94, and 1.90 and with temperature dependence consistent with a 2Fe-2S center. Redox titration demonstrated that the Fe-S center is highly unstable, with an apparent midpoint reduction potential of about -370 mV. This is the first Fe-S center to be reported in a higher plant ferrochelatase. The implications of the Fe-S center in an enzyme that is so closely associated with the metabolism of sulfur and iron are discussed.

Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide.

Phosphorylation of the transit peptide of several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast protein import pathway in vitro[May, T. & Soll, J. (2000) Plant Cell 12, 53-63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carboxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phosphorylation of the transit peptide is not responsible for the specificity of chloroplast import.

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.