Cold atmospheric pressure plasma (CAPP) as a new alternative treatment method for onychomycosis caused by Trichophyton verrucosum: in vitro studies.

PURPOSE: Anthropophilic dermatophytes as etiological factors of onychomycoses are more common than zoophilic fungi. In the case of the latter, reverse zoonoses are possible, which poses a threat to the persistence of dermatophytes in the environment. Nevertheless, without treatment, both types of tinea unguium may lead to complete nail plate destruction and secondary mixed infections with fungi and bacteria. One of the zoophilic dermatophytes that cause onychomycosis is Trichophyton verrucosum, whose prevalence has been increasing in recent years. Such infections are usually treated with allylamines and/or azoles, but such a conventional treatment of infections caused by T. verrucosum often fails or is discontinued by patients. METHODS: Herein, we reveal the results of our in vitro studies related to direct application of cold atmospheric pressure plasma (CAPP) on Trichophyton verrucosum growth, germination and adherence to nail as a new alternative treatment method of such types of dermatomycoses. RESULTS: Our in vitro studies showed that, while exposure to CAPP for 10 min delays germination of conidia and clearly impairs the fitness of the fungal structures, 15 min is enough to kill all fungal elements exposed to plasma. Moreover, the SEM images revealed that T. verrucosum cultures exposed to CAPP for 10 and 15 min were not able to invade the nail fragments. CONCLUSION: The results revealed that single exposure to CAPP was able to inhibit T. verrucosum growth and infection capacity. Hence, cold atmospheric pressure plasma should be considered as a promising alternative treatment of onychomycoses.

Therapeutic efficacy of topically used luliconazole vs. terbinafine 1% creams.

OBJECTIVES: Dermatomycoses of zoophilic origin, especially those caused by Trichophyton mentagrophytes, often pose considerable therapeutic problems. This is reflected in the growing number of strains of this species with resistance to terbinafine caused by a mutation in the squalene epoxidase (SQLE) gene. Therefore, it is reasonable to look for alternative therapies to the commonly used terbinafine. The aim of the present study was to assess the in vivo effectiveness of topical therapy with luliconazole or terbinafine 1% cream. METHODS: Therapeutic efficacy was assessed using direct examination in KOH with DMSO, qPCR analysis with pan-dermatophyte primers and culturing. Moreover, in vitro susceptibility tests for luliconazole and terbinafine were performed. RESULTS: The results demonstrated significantly higher antifungal activity of luliconazole than terbinafine against dermatomycoses caused by T. mentagrophytes. The geometric mean of the MIC value for luliconazole against all T. mentagrophytes strains was 0.002 µg/ml, while this value for terbinafine was 0.004 µg/ml. In all studied cases, 28-day local therapy with luliconazole contributed to complete eradication of the aetiological agent of infection. CONCLUSIONS: Given the increasingly frequent reports of difficult-to-treat dermatophytoses caused by zoophilic terbinafine-resistant strains, the 1% luliconazole cream can be alternative solution in topical therapy.

Continuous sheath-free separation of drug-treated human fungal pathogen Cryptococcus neoformans by morphology in biocompatible polymer solutions.

Cryptococcal meningitis caused by Cryptococcus neoformans is the leading cause of fungal central nervous system infections. Current antifungal treatments for cryptococcal infections are inadequate partly due to the occurrence of drug resistance. Recent studies indicate that the treatment of the azole drug fluconazole changes the morphology of C. neoformans to form enlarged "multimeras" that consist of three or more connected cells/buds. To analyze if these multimeric cells are a prerequisite for C. neoformans to acquire drug resistance, a tool capable of separating them from normal cells is critical. We extend our recently demonstrated sheath-free elasto-inertial particle separation technique to fractionate drug-treated C. neoformans cells by morphology in biocompatible polymer solutions. The separation performance is evaluated for both multimeric and normal cells in terms of three dimensionless metrics: efficiency, purity, and enrichment ratio. The effects of flow rate, polymer concentration, and microchannel height on cell separation are studied.

MD3F: Multivariate Distance Drift Diffusion Framework for High-Dimensional Datasets.

High-dimensional biomedical datasets have become easier to collect in the last two decades with the advent of multi-omic and single-cell experiments. These can generate over 1000 measurements per sample or per cell. More recently, focus has been drawn toward the need for longitudinal datasets, with the appreciation that important dynamic changes occur along transitions between health and disease. Analysis of longitudinal omics data comes with many challenges, including type I error inflation and corresponding loss in power when thousands of hypothesis tests are needed. Multivariate analysis can yield approaches with higher statistical power; however, multivariate methods for longitudinal data are currently limited. We propose a multivariate distance-based drift-diffusion framework (MD3F) to tackle the need for a multivariate approach to longitudinal, high-throughput datasets. We show that MD3F can result in surprisingly simple yet valid and powerful hypothesis testing and estimation approaches using generalized linear models. Through simulation and application studies, we show that MD3F is robust and can offer a broadly applicable method for assessing multivariate dynamics in omics data.

Update on Stachybotrys chartarum-Black Mold Perceived as Toxigenic and Potentially Pathogenic to Humans.

In nature, there are many species of fungi known to produce various mycotoxins, allergens and volatile organic compounds (VOCs), as well as the commonly known etiological agents of various types of mycoses. So far, none of them have provoked so much emotion among homeowners, builders, conservators, mycologists and clinicians as Stachybotrys chartarum. This species compared to fungi of the genera Fusarium and Aspergillus is not as frequently described to be a micromycete that is toxigenic and hazardous to human and animal health, but interest in it has been growing consistently for three decades. Depending on the authors of any given review article, attention is focused either on the clinical aspects alongside the role of this fungus in deterioration of biomaterials, or aspects related to its biology, ecology and taxonomic position. On the one hand, it is well established that inhalation of conidia, containing the highest concentrations of toxic metabolites, may cause serious damage to the mammalian lung, particularly with repeated exposure. On the other hand, we can find articles in which authors demonstrate that S. chartarum conidia can germinate and form hyphae in lungs but are not able to establish an effective infection. Finally, we can find case reports that suggest that S. chartarum infection is linked with acute pulmonary hemorrhage, based on fungal structures recovered from patient lung tissue. New scientific reports have verified the current state of knowledge and note that clinical significance of this fungus is exceedingly controversial. For these reasons, understanding S. chartarum requires reviewing the well-known toxigenic features and harmful factors associated with this fungus, by gathering the newest ones into a coherent whole. The research problem related to this fungus seems to be not overly publicized, and there is still a demand to truthfully define the real threats of S. chartarum and phylogenetically related species. The most important problem, which should be fully elucidated as soon as possible, remains the clarification of the pathogenicity of S. chartarum and related species. Maybe it is urgent time to ask a critical question, namely what exactly do we know 28 years after the outbreak of pulmonary hemorrhage in infants in Cleveland, Ohio, USA most likely caused by S. chartarum?

In vitro evaluation of photodynamic activity of methylene blue against Trichophyton verrucosum azole-susceptible and -resistant strains.

The intense search for the "Holy Grail" of antifungal therapy can be observed today. The searches are not limited only to discovery of potential antifungal drugs, but also new therapeutic strategies involving the use of chemosensitizers to achieve synergistic effect or physicochemical factors inducing stress conditions in fungal cells. In this study was examined in vitro effectiveness of photodynamic antifungal strategy with methylene blue using a light beam with a wavelength equal to 635 nm toward the Trichophyton verrucosum susceptible and itraconazole- and/or fluconazole-resistant strains. Methylene blue used at concentration equal to 5 µg/mL and in the presence of 40 J/cm2 of light energy showed fungicidal effect toward the susceptible strains. However, for azole-resistant isolates, only the energy dose equal to 60 J/cm2 at 5 µg/mL of methylene blue allowed to kill the pathogen. This study confirms that methylene blue induced by red light has a definite inhibitory effect on zoophilic dermatophytes.

High Throughput Multi-Omics Approaches for Clinical Trial Evaluation and Drug Discovery.

High throughput single cell multi-omics platforms, such as mass cytometry (cytometry by time-of-flight; CyTOF), high dimensional imaging (>6 marker; Hyperion, MIBIscope, CODEX, MACSima) and the recently evolved genomic cytometry (Citeseq or REAPseq) have enabled unprecedented insights into many biological and clinical questions, such as hematopoiesis, transplantation, cancer, and autoimmunity. In synergy with constantly adapting new single-cell analysis approaches and subsequent accumulating big data collections from these platforms, whole atlases of cell types and cellular and sub-cellular interaction networks are created. These atlases build an ideal scientific discovery environment for reference and data mining approaches, which often times reveals new cellular disease networks. In this review we will discuss how combinations and fusions of different -omic workflows on a single cell level can be used to examine cellular phenotypes, immune effector functions, and even dynamic changes, such as metabolomic state of different cells in a sample or even in a defined tissue location. We will touch on how pre-print platforms help in optimization and reproducibility of workflows, as well as community outreach. We will also shortly discuss how leveraging single cell multi-omic approaches can be used to accelerate cellular biomarker discovery during clinical trials to predict response to therapy, follow responsive cell types, and define novel druggable target pathways. Single cell proteome approaches already have changed how we explore cellular mechanism in disease and during therapy. Current challenges in the field are how we share these disruptive technologies to the scientific communities while still including new approaches, such as genomic cytometry and single cell metabolomics.

Genetic contribution to high temperature tolerance in Cryptococcus neoformans.

The human fungal pathogen Cryptococcus neoformans relies on a complex signaling network for the adaptation and survival at the host temperature. Protein phosphatase calcineurin is central to proliferation at 37°C but its exact contributions remain ill-defined. To better define genetic contributions to the C. neoformans temperature tolerance, 4031 gene knockouts were screened for genes essential at 37°C and under conditions that keep calcineurin inactive. Identified 83 candidate strains, potentially sensitive to 37°C, were subsequently subject to technologically simple yet robust assay, in which cells are exposed to a temperature gradient. This has resulted in identification of 46 genes contributing to the maximum temperature at which C. neoformans can proliferate (Tmax). The 46 mutants, characterized by a range of Tmax on drug-free media, were further assessed for Tmax under conditions that inhibit calcineurin, which led to identification of several previously uncharacterized knockouts exhibiting synthetic interaction with the inhibition of calcineurin. A mutant that lacked septin Cdc11 was among those with the lowest Tmax and failed to proliferate in the absence of calcineurin activity. To further define connections with calcineurin and the role for septins in high temperature growth, the 46 mutants were tested for cell morphology at 37°C and growth in the presence of agents disrupting cell wall and cell membrane. Mutants sensitive to calcineurin inhibition were tested for synthetic lethal interaction with deletion of the septin-encoding CDC12 and the localization of the septin Cdc3-mCherry. The analysis described here pointed to previously uncharacterized genes that were missed in standard growth assays indicating that the temperature gradient assay is a valuable complementary tool for elucidating the genetic basis of temperature range at which microorganisms proliferate.

Yeast cell fractionation by morphology in dilute ferrofluids.

Morphology is an important particle (both biological and synthetic) property and potentially a useful marker for label-free particle separation. We present in this work a continuous-flow morphology-based fractionation of a heterogeneous mixture of drug-treated yeast cells in dilute ferrofluids. Such a diamagnetic cell separation technique utilizes the negative magnetophoretic motion to direct pre-focused yeast cells to morphology-dependent streamlines in a laminar flow. The separation performance is evaluated by comparing the exiting positions of the four classified groups of yeast cells: Singles, Doubles, Triples, and Others. We also develop a three-dimensional numerical model to simulate the separation process by the use of the experimentally determined correction factor for each group of non-spherical cells. The determining factors in this separation are studied both experimentally and numerically, the results of which show a reasonable agreement.