Potential Original Drug for Aspergillosis: In Vitro and In Vivo Effects of 1-N,N-Dimethylamino-5-Isocyanonaphthalene (DIMICAN) on Aspergillus fumigatus.

As the recent outbreak of coronavirus disease 2019 (COVID-19) has shown, viral infections are prone to secondary complications like invasive aspergillosis with a high mortality rate, and therefore the development of novel, effective antifungals is of paramount importance. We have previously demonstrated that 1-amino-5-isocyanonaphthalene (ICAN) derivatives are promising original drug candidates against Candida strains (Patent pending), even against fluconazole resistant C. albicans. Consequently, in this study ICANs were tested on Aspergillus fumigatus, an opportunistic pathogen, which is the leading cause of invasive and systematic pulmonary aspergillosis in immunosuppressed, transplanted and cancer- or COVID-19 treated patients. We have tested several N-alkylated ICANs, a well as 1,5-naphthalene-diisocyanide (DIN) with the microdilution method against Aspergillus fumigatus strains. The results revealed that the diisocyanide (DIN) was the most effective with a minimum inhibitory concentration (MIC) value as low as 0.6 µg mL-1 (3.4 µM); however, its practical applicability is limited by its poor water solubility, which needs to be overcome by proper formulation. The other alkylated derivatives also have in vitro and in vivo anti-Aspergillus fumigatus effects. For animal experiments the second most effective derivative 1-N, N-dimethylamino-5-isocyanonaphthalene (DIMICAN, MIC: 7-8 µg mL-1, 36-41 µM) was selected, toxicity tests were made with mice, and then the antifungal effect of DIMICAN was tested in a neutropenic aspergillosis murine model. Compared to amphotericin B (AMB), a well-known antifungal, the antifungal effect of DIMICAN in vivo turned out to be much better (40% vs. 90% survival after eight days), indicating its potential as a clinical drug candidate.

The Camouflage Machine: Optimizing protective coloration using deep learning with genetic algorithms.

Evolutionary biologists frequently wish to measure the fitness of alternative phenotypes using behavioral experiments. However, many phenotypes are complex. One example is coloration: camouflage aims to make detection harder, while conspicuous signals (e.g., for warning or mate attraction) require the opposite. Identifying the hardest and easiest to find patterns is essential for understanding the evolutionary forces that shape protective coloration, but the parameter space of potential patterns (colored visual textures) is vast, limiting previous empirical studies to a narrow range of phenotypes. Here, we demonstrate how deep learning combined with genetic algorithms can be used to augment behavioral experiments, identifying both the best camouflage and the most conspicuous signal(s) from an arbitrarily vast array of patterns. To show the generality of our approach, we do so for both trichromatic (e.g., human) and dichromatic (e.g., typical mammalian) visual systems, in two different habitats. The patterns identified were validated using human participants; those identified as the best for camouflage were significantly harder to find than a tried-and-tested military design, while those identified as most conspicuous were significantly easier to find than other patterns. More generally, our method, dubbed the "Camouflage Machine," will be a useful tool for identifying the optimal phenotype in high dimensional state spaces.

MTT Test and Time-lapse Microscopy to Evaluate the Antitumor Potential of Nucleoside Analogues.

BACKGROUND/AIM: Conventional viability tests, help to screen the cellular effects of candidate molecules, but the endpoint of these measurements lacks sufficient information regarding the molecular aspects. A non-invasive, easy-to-setup live-cell microscopic method served to in-depth analysis of mechanisms of potential anticancer drugs. MATERIALS AND METHODS: The proposed method combining the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test with time-lapse scanning microscopy (TLS), provided additional data related to the cell-cycle and the dynamic properties of cell morphology. Apoptotic and necrotic events became detectable with these methods. RESULTS: Quantification of the results was assisted by image analysis of the acquired image sequences. After demonstrating the potential of the TLS method, a series of experiments compared the in vitro effect of a known and a newly synthesized nucleoside analogue. CONCLUSION: The proposed approach provided a more in-depth insight into the cellular processes that can be affected by known chemotherapeutic agents including nucleoside analogues rather than applying repeated individual treatments.

Antifungal Activity of an Original Amino-Isocyanonaphthalene (ICAN) Compound Family: Promising Broad Spectrum Antifungals.

: Multiple drug resistant fungi pose a serious threat to human health, therefore the development of completely new antimycotics is of paramount importance. The in vitro antifungal activity of the original, 1-amino-5-isocyanonaphthalenes (ICANs) was evaluated against reference strains of clinically important Candida species. Structure-activity studies revealed that the naphthalene core and the isocyano- together with the amino moieties are all necessary to exert antifungal activity. 1,1-N-dimethylamino-5-isocyanonaphthalene (DIMICAN), the most promising candidate, was tested further in vitro against clinical isolates of Candida species, yielding a minimum inhibitory concentration (MIC) of 0.04-1.25 µg/mL. DIMICAN was found to be effective against intrinsically fluconazole resistant Candida krusei isolates, too. In vivo experiments were performed in a severly neutropenic murine model inoculated with a clinical strain of Candida albicans. Daily administration of 5 mg/kg DIMICAN intraperitoneally resulted in 80% survival even at day 13, whereas 100% of the control group died within six days. Based on these results, ICANs may become an effective clinical lead compound family against fungal pathogens.

Iridescence as Camouflage.

Iridescence is a striking and taxonomically widespread form of animal coloration [1], but that its intense and varying hues could function as concealment [2] rather than signaling seems completely counterintuitive. Here, we show that the color changeability of biological iridescence, produced by multilayer cuticle reflectors in jewel beetle (Sternocera aequisignata) wing cases, provides effective protection against predation by birds. Importantly, we also show that the most likely mechanism to explain this increase in survival is camouflage and not some other protective function, such as aposematism. In two field experiments using wild birds and humans, we measured both the "survival" and direct detectability of iridescent and non-iridescent beetle models and demonstrated that the iridescent treatment fared best in both experiments. We also show that an increased level of specular reflection (gloss) of the leaf background leads to an increase in the survival of all targets and, for detectability by humans, enhances the camouflage effect of iridescence. The latter suggests that some prey, particularly iridescent ones, can increase their chance of survival against visually hunting predators even further by choosing glossier backgrounds. Our study is the first to present direct empirical evidence that biological iridescence can work as a form of camouflage, providing an adaptive explanation for its taxonomically widespread occurrence. VIDEO ABSTRACT.

Effect of amphotericin B and voriconazole on the outgrowth of conidia of Aspergillus fumigatus followed by time-lapse microscopy.

Studies of morphological measurements from the outgrowth of cells to a network of hyphae have been extended from Candida albicans (Nagy et al. in Appl Microbiol Biotechnol 98(11):5185-5194. https://doi.org/10.1007/s00253-014-5696-5 , 2014) to invasive conidiospores of Aspergillus fumigatus upon treatment with antifungal agents. The understanding of mycelial processes is important to optimize industrial processes such as fermentation and contributes to the fight against pathogenic fungi. This brief study combines TLS with digital image analysis. The TLS system was adapted to get information related to the adherence and growth dynamics of filamentous fungi. This approach was used earlier to distinguish among subphases of bacterial and fungal infections of mammal cells by detecting Mycoplasma infection in cell cultures causing serious damages in cell cultures. We describe changes in adherence, germination of spores, and hyphal growth of A. fumigatus, taking place in the absence and presence of amphotericin B (AMB) and voriconazole (VRC). These growth parameters were measured by TLS in CO2 incubator under physiological Photomicrography by TLS and extended for a longer period of time up to several weeks combined with image analysis represents a comfortable and reliable means to characterize the growth dynamism of A. fumigatus. The most important observation of medical importance related to the pathomechanism of VRC was that it did not adhere to conidiospores, i.e. that it did not contribute to the attachment of spores to the growth surface, and did not prevent germination but delayed hypha protrusion and elongation. In contrast AMB adhered to conidia, inhibited germination, hypha elongation and branching. It was concluded that AMB was efficient against the therapy of growth but not against the prevention of fungal infection.

Murine model to follow hyphal development in invasive pulmonary aspergillosis.

Aspergillus fumigatus is an opportunistic pathogen, the leading cause of invasive and disseminated aspergillosis in systemic immunocompromised patients, and an important cause of mortality. The aim of the present study was to adapt a pulmonary aspergillosis murine model, to determine pathodynamical parameters quantitatively, and to follow the progression of fungal infection in vivo. The nasal inoculation of Aspergillus conidia in mice previously subjected to immunosuppression with cyclophosphamide (CP) turned out to be a more suitable model than that of immunosuppressed with hydrocortisone (HC). The following parameters were found to correlate quantitatively with the progress of the infection: (i) survival rate, (ii) weight loss of mice, (iii) infected focal plaque size, (iv) hyphal density, (v) hyphal length distribution of A. fumigatus, and the (vi) the histopathological status and scores. These parameters will be essential elements for the development of antifungal drugs and therapies, and important for the investigation of the pathogenicity in different strains of A. fumigatus.

Mycoplasma infection followed by time-lapse microscopy.

Early detection of mycoplasma infection is crucial for saving precious often irreplaceable data from the tissues of patients. Mycoplasma infections cause diseases in the upper and lower respiratory tracts, urethritis in men resulting in painful dysuria, urgency and urethral discharge. Cough, fever, headache, urethritis may persist for several weeks and convalescence is slow. The symptoms of these diseases are aggravated by the detection of mycoplasma infections, that takes either a long time, besides being expensive or is specific and restricted to only a limited number of contaminant strains. Mycoplasmas are hard to detect visually but could be seen and followed by time-lapse microscopy. Our hypothesis is that one can detect mycoplasma infection irrespective of its origin and type of mycoplasma. Main lines of supporting evidence are provided by the time-lapse microscopy showing dynamic morphological alterations caused by mycoplasmas before changes in human cell cultures become visible. Morphometric measurements of mycoplasma infections revealed four subphases: i) detachment of infected cells, ii) aggregation, iii) biofilm formation and iv) shrinkage of infected cells. The applicability of time-lapse microscopy for the detection of mycoplasma infection was validated by a mycoplasma test Kit. Most important implications related to morphometric parameters include the observation of mycoplasma infected cultures for an extended period of time instead of applying static snap-shot microscopy. A reliable method is offered to estimate the time of mycoplasma exposure that elapsed during the cell growth. This microphotometric approach served a more economical detection of mycoplasma contamination at its early stage of cell growth and spread, irrespective of the origin of contaminated serum, without defining the type of mycoplasma.

The biology of color.

Coloration mediates the relationship between an organism and its environment in important ways, including social signaling, antipredator defenses, parasitic exploitation, thermoregulation, and protection from ultraviolet light, microbes, and abrasion. Methodological breakthroughs are accelerating knowledge of the processes underlying both the production of animal coloration and its perception, experiments are advancing understanding of mechanism and function, and measurements of color collected noninvasively and at a global scale are opening windows to evolutionary dynamics more generally. Here we provide a roadmap of these advances and identify hitherto unrecognized challenges for this multi- and interdisciplinary field.

Cultural evolution of military camouflage.

While one has evolved and the other been consciously created, animal and military camouflage are expected to show many similar design principles. Using a unique database of calibrated photographs of camouflage uniform patterns, processed using texture and colour analysis methods from computer vision, we show that the parallels with biology are deeper than design for effective concealment. Using two case studies we show that, like many animal colour patterns, military camouflage can serve multiple functions. Following the dissolution of the Warsaw Pact, countries that became more Western-facing in political terms converged on NATO patterns in camouflage texture and colour. Following the break-up of the former Yugoslavia, the resulting states diverged in design, becoming more similar to neighbouring countries than the ancestral design. None of these insights would have been obtained using extant military approaches to camouflage design, which focus solely on concealment. Moreover, our computational techniques for quantifying pattern offer new tools for comparative biologists studying animal coloration.This article is part of the themed issue 'Animal coloration: production, perception, function and application'.

Chemically Induced Cell Cycle Arrest in Perfusion Cell Culture.

In contrast to most present methods, continuous imaging of live cells would require full automation in each processing step. As an integrated system that would meet all requirements does not exist, we have established a long-term scanning-perfusion platform that: (a) replaces old medium with fresh one, (b) bypasses physical contact with the cell culture during continuous cell growth, (c) provides uninterrupted photomicrography of single cells, and (d) secures near physiological conditions and sterility up to several weeks. The system was validated by synchronizing cells using serum starvation and butyrate-induced cell cycle arrest of HaCaT cells.

Antibiotics delay in vitro human stem cell regrowth.

Stem cell line from human limbal area was established to study in vitro cell growth and response to the toxic effects of antibiotics used in ophthalmology in terms of cell migration rates and structure of interphase chromatin. Recovery from cellular damages caused by ophthalmologic antibiotics was mimicked by an in vitro scratch model and followed by time-lapse microscopy, scanning electronmicroscopy and chromatin image analysis. Experiments revealed that broad spectrum antibiotics, chloramphenicol (0.5-1.0mg/ml) and rifampicin (0.1-0.2mg/ml), corresponding to concentrations in common clinical practice, slowed down the regeneration process. Results show that nuclei of naturally occurring limbal cells contain the same intermediates of chromatin condensation as seen in mammalian tumor cells and follow the common pathway of chromosome condensation. These intermediates included decondensed veil-like chromatin, fibrillary chromatin, supercoiled ribbon, chromatin bodies, early linear forms and metaphase chromosomes. Upon chloramphenicol and rifampicin treatment characteristic distorsions took place in the intermediates of chromosome condensation. Damaging effects in limbal stem cells in the presence of chloramphenicol or rifampicin indicate that ophthalmologic treatment with antibiotics should be used cautiously.