Apoptotic Janus-faced mycotoxins against thoracal and breast metastases.

Abdominal organs (liver, kidney, spleen) are frequent targets of cancer cell invasion but their primary tumours are less known for their metastatic potential to other organs e.g. to the breast. Despite the known connection of the pathogenesis from breast cancer to liver metastasis, the study of the spread in the opposite direction has been neglected. The notion that breast cancer could be a metastasis besides being a primary tumour is based on rodents' tumour models upon implantation of tumour cells under the capsule of the kidney or under the Glisson's capsule of the liver of rats and mice. Tumour cells develop into a primary tumour at the site of subcutaneous implantation. The metastatic process starts with peripheral disruptions of blood vessels near the surface of primary tumours. Tumour cells released into the abdomen cross the apertures of the diaphragm, enter the thoracal lymph nodes and accumulate in parathymic lymph nodes. Abdominal colloidal carbon particles injected into the abdomen faithfully mimicked the migration of tumour cells and deposited in parathymic lymph nodes (PTNs). An explanation is provided why the connection between abdominal tumours and mammary tumours escaped attention, notably, parathymic lymph nodes in humans were referred to as internal mammary or parasternal lymph nodes. The apoptotic effect of Janus-faced cytotoxins is suggested to provide a new approach against the spread of abdominal primary tumours, and metastatic development.

Macromolecular Structure of Linearly Arranged Eukaryotic Chromosomes.

Eukaryotic chromosomes have not been visualized during the interphase. The fact that chromosomes cannot be seen during the interphase of the cell cycle does not mean that there are no means to make them visible. This work provides visual evidence that reversible permeabilization of the cell membrane followed by the regeneration of cell membranes allows getting a glimpse behind the nuclear curtain. Reversibly permeable eukaryotic cells have been used to synthesize nascent DNA, analyze the 5'-end of RNA primers, view individual replicons and visualize interphase chromosomes. Dextran T-150 in a slightly hypotonic buffer prevented cells from disruption. Upon reversal of permeabilization, the nucleus could be opened at any time during the interphase. A broad spectrum of a flexible chromatin folding pattern was revealed through a series of transient geometric forms of chromosomes. Linear attachment of chromosomes was visualized in several mammalian and lower eukaryotic cells. The linear connection of chromosomes is maintained throughout the cell cycle showing that rather than individual chromosomes, a linear array of chromosomes is the functional giant macromolecule. This study proves that not only the prokaryotic genome but also linearly attached eukaryotic chromosomes form a giant macromolecular unit.

Janus-Faced Molecules against Plant Pathogenic Fungi.

The high cytotoxicity of the secondary metabolites of mycotoxins is capable of killing microbes and tumour cells alike, similarly to the genotoxic effect characteristic of Janus-faced molecules. The "double-edged sword" effect of several cytotoxins is known, and these agents have, therefore, been utilized only reluctantly against fungal infections. In this review, consideration was given to (a) toxins that could be used against plant and human pathogens, (b) animal models that measure the effect of antifungal agents, (c) known antifungal agents that have been described and efficiently prevent the growth of fungal cells, and (d) the chemical interactions that are characteristic of antifungal agents. The utilization of apoptotic effects against tumour growth by agents that, at the same time, induce mutations may raise ethical issues. Nevertheless, it deserves consideration despite the mutagenic impact of Janus-faced molecules for those patients who suffer from plant pathogenic fungal infections and are older than their fertility age, in the same way that the short-term cytotoxicity of cancer treatment is favoured over the long-term mutagenic effect.

Prebiotic Pathway from Ribose to RNA Formation.

At the focus of abiotic chemical reactions is the synthesis of ribose. No satisfactory explanation was provided as to the missing link between the prebiotic synthesis of ribose and prebiotic RNA (preRNA). Hydrogen cyanide (HCN) is assumed to have been the principal precursor in the prebiotic formation of aldopentoses in the formose reaction and in the synthesis of ribose. Ribose as the best fitting aldopentose became the exclusive sugar component of RNA. The elevated yield of ribose synthesis at higher temperatures and its protection from decomposition could have driven the polymerization of the ribose-phosphate backbone and the coupling of nucleobases to the backbone. RNA could have come into being without the involvement of nucleotide precursors. The first nucleoside monophosphate is likely to have appeared upon the hydrolysis of preRNA contributed by the presence of reactive 2'-OH moieties in the preRNA chain. As a result of phosphorylation, nucleoside monophosphates became nucleoside triphosphates, substrates for the selective synthesis of genRNA.

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 Gentamicin B1 Against Systemic Plant Mycoses.

BACKGROUND: Gentamicin is a broad-spectrum aminoglycoside antibiotic produced by Micromonospora purpurea bacteria, effective against Gram-negative bacterial infections. Major fractions of the gentamicin complex (C1, C1a, C2, C2a) possess weak antifungal activity and one of the minor components (A, A1-A4, B, B1, X), gentamicin B1 was found to be a strong antifungal agent. METHODS: This work uses in vitro and in vivo dilution methods to compare the antifusarial, antiaspergillic and anticryptococcal effects of gentamicin derivatives and structurally-related congeners. RESULTS: The in vitro antifusarial activity of gentamicin B1 (minimum inhibitory concentration (MIC) 0.4 µg/mL) and structurally-related compounds (MIC 0.8-12.5 µg/mL) suggests that the purpuroseamine ring substituents are responsible for the specific antimycotic effect. The functional groups of the garoseamine and 2-deoxystreptamine rings of gentamicin derivatives are identical in gentamicin compounds and are unlikely to exert a significant antifungal effect. Among soil dermatophytes, Microsporum gypseum was more susceptible to gentamicin B1 (MIC 3.1 µg/mL) than Trichophyton gypseum (MIC 25 µg/mL). The in vitro antifungal effect of gentamicin B1 against plant pathogenic fungi was comparable to primary antifungal agents. CONCLUSION: Gentamicin is already in medical use. In vitro and preclinical in vivo synergisms of gentamicin B1 with amphotericin B suggest immediate clinical trials starting with subtoxic doses.

Ribose Selected as Precursor to Life.

The chemical or prebiotic evolution referred also to as pre-Darwinian evolution describes chemical reactions up to the origin of a self-replicating system that was capable of Darwinian evolution. These chemical processes took place on Earth between about 3.7 and 4.5 billion years ago when cellular life came into being. The pre-Darwinian chemical evolution usually assumes hereditary elements, but does not regard them as self-organizing processes. Physical and chemical self-organization led to uninterrupted pre-Darwinian and Darwinian evolution. Thus, it is not justified to distinguish between different types of evolution. From the many possible solutions, evolution selected among those reactions that generated catalytic networks incorporating chemical sequence information and under gradually changing circumstances produced a reproducible and stable living system that adapted to these conditions. Major issues in this review involve prebiotic reactions leading to genetic evolution involving (1) abiotic sources of components of ribonucleotides and xenobiotic nucleotides, (2) formation of prebiotic RNA, (3) development of genetic RNA from random-sequence noncoding RNA, (4) transition from RNA World to DNA Empire, (5) the role of oxygenic photosynthesis in genetic transitions, and (6) hierarchical arrangement of processes involved in the optimized genetic system.

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.

Origin of Coding RNA from Random-Sequence RNA.

D-ribose and D-arabinose differ only by the steric orientation of their C2-OH groups. The initial reactions and emergence of RNA depended on the position, reactivity, and flexibility of the C2-OH moiety in the ribose molecule. The steric relationship of the C2- and C3-OH groups favored the selection of ribose, ribonucleotide, and RNA synthesis and excluded the possibility of xenonucleic acid-based life on Earth. This brief review provides a hypothesis based on the absence of nucleotides and enzymes under prebiotic conditions and on the polymerization of ribose 5-phosphate units leading to the polarized formation of the ribose-phosphate backbone. The strong covalent bond formation in the sugar-phosphate backbone was followed by the somewhat less reactive interaction between ribose and nucleobase and supplemented by even weaker hydrogen-bonded and stacking interactions. This hypothesis proposes a scheme how prebiotic random-sequence RNA was formed under abiotic conditions and hydrolyzed to oligomers and nucleotides. The term random-sequence prebiotic RNA refers to nucleobases attached randomly to the ribose-phosphate backbone and not to cellular RNA sequences as proteins and cells did not probably exist at the time of abiotic RNA formation. It is hypothesized that RNA generated under abiotic conditions containing random nucleobases was hydrolyzed to nucleotides that served as a pool for the selected synthesis of genetic RNA.

Controlled release of methotrexate from functionalized silica-gelatin aerogel microparticles applied against tumor cell growth.

Methotrexate functionalized silica-gelatin hybrid aerogel (SGM) was synthesized by the sol-gel method and co-gelation. The drug methotrexate (MTX) is covalently linked to the collagen molecules of the hybrid aerogel backbone by amide-bond. The characteristic MTX content of the functionalized hybrid aerogel is ca. 6 wt% by the dry weight. The micronization of SGM aerogel in water yields cell sized (d = 10-20 µm) particles. The cytotoxicity of these microparticles against tumor cell lines (SCC VII and HL-60) is unprecedentedly high, it is approximately equivalent to that of an equal dose of free (dissolved) MTX, as proved by in vitro experiments. Thus, the activity of MTX is intact after aerogel functionalization, and the mass specific cytotoxicity of SGM is high enough for medical applications. Drug release studies verified that MTX cannot be liberated from this drug delivery system solely by chemical hydrolysis, however, collagenase enzymatic activity releases MTX from the functionalized hybrid aerogel. The cytotoxicity of SGM towards various cancerous and non-cancerous cell lines correlates with the collagenase activities of cells. Therefore, conjugation with the hybrid aerogel provides a controlled release system for the antineoplastic agent MTX. The morphology of the delivery vehicle was chosen to adapt the size of cancer cells; thus the metastatic pathways of the tumor cells can get flooded.

Metastatic Spread from Abdominal Tumor Cells to Parathymic Lymph Nodes.

Metastatic studies on rats showed that after subrenal implantation of tumor cells under the capsule of the kidney or subhepatic implantation under Glisson's capsule of the liver generated primary tumors in these organs. It was assumed that tumor cells that escaped through the disrupted peripheral blood vessels of primary tumors entered the peritoneal cavity, crossed the diaphragm, and appeared in the thoracal, primarily in the parathymic lymph nodes. This explanation did not answer the question whether distant lymph nodes were reached via the blood stream from the primary tumor or through the thoracal lymphatic vessels. In this work, we investigated the metastatic pathway in C3H/HeJ mice, after direct intraperitoneal administration of murine SCC VII cells bypassing the hematogenic spread of tumor cells. The direct pathway was also mimicked by intraperitoneal injection of Pelican Ink colloidal particles, which appeared in the parathymic lymph nodes, similarly to the tumor cells that caused metastasis in the parathymic lymph nodes and in the thymic tissue. The murine peritoneal-parathymic lymph node route indicates a general mechanism of tumor progression from the abdominal effusion. This pathway starts with the growth of abdominal tumors, continues as thoracal metastasis in parathymic lymph nodes and may proceed as mammary lymph node metastasis.

Retrospective evaluation of in vitro effect of gentamicin B1 against Fusarium species.

The in vitro susceptibility of gentamicin fractions against Fusarium growth was the subject of this retrospective study. Fusariosis was earlier an exceptionally rare human disease and an unrealistic idea to treat soil saprophytes and plant pathogens with expensive antibiotics such as gentamicins or their minor components. Disseminated fusariosis is now the second most frequent lethal fungal infection after aspergillosis especially in neutropenic patients with hematologic malignancy. Results of this study obtained between May and November 1973 were interesting but not practicable and remained unpublished. Seven Fusarium and 28 other fungal strains were tested for their susceptibility to gentamicin B1. The anti-Fusarium activity of gentamicin B1 was between 0.2 and 3.1 µg/ml minimum inhibitory concentration (MIC) values. The MIC values of clotrimazol and amphotericin B against Fusarium species were significantly higher, 3.1-12.5 µg/ml and 3.1-50 µg/ml, respectively. Gentamicin B1 and its structurally related congeners including hygromycin B, paromomycin, tobramycin (nebramycin factor 5'), nebramycin (nebramycin factor 4), and sisomicin exerted strong in vitro inhibition against Fusarium species between 0.2 and 12.5 µg/ml concentrations. The antibacterial MIC concentration of gentamicin B1 tested on 20 bacterial strains ranged between 0.1 and 50 µg/ml. Gentamicin B1, a minor fraction of the gentamicin complex, inhibited effectively the growth of Gram-positive (Staphylococcus, Streptococcus, Bacillus subtilis) bacteria and Gram-negative (Escherichia coli, Salmonella, Proteus, Pseudomonas) pathogens. Gentamicins and related aminoglycoside antibiotics are used in medical practice. It is proposed that due to the increasing incidence of fusariosis and drug resistance, gentamicin components, particularly minor fraction B1 and related aminoglycoside antibiotics, could be tested for their in vivo activity against fusariosis and aspergillosis either alone or in combination with other antifungal agents.

Improved and adopted murine models to combat pulmonary aspergillosis.

The insufficient basic and clinical knowledge about invasive mold infections necessitated to review aspergillosis rodent models. The scope of this review has two major aspects. (1) It briefly summarizes Aspergillus toxicoses, the adverse effects of Aspergillus mycotoxins, the virulence factors of Aspergillus fumigatus, and how mild Aspergillus infections can turn to immunosuppressive diseases, ultimately to lethal invasive pulmonary aspergillosis. (2) The second major aspect of the review deals with earlier and recent murine models of pulmonary aspergillosis. Particular attention will be paid to the development of unified and generally applicable methods to detect, follow, and combat aspergillosis by medical treatments. Additionally, the review raises the question of responsibility regarding the application of immunosuppressive agents that initiate, contribute, and aggravate aspergillosis. Future studies of immunosuppression by chemical agents impacting aspergillosis deserve more studies.

Antineoplastic potential of mycotoxins.

Fungal toxins are secondary metabolites, in which many of them were mycotoxins, affecting eukaryotic cells with a broad range of structural and functional variety contributing to the multitude of their classification. This refers to the harmful genotoxic (mutagenic, teratogenic, and carcinogenic) effects of mycotoxins on the one hand, and their cytocidic and antineoplastic properties on the other hand. This "double edged sword" effect could be utilized against the spread of tumors in older patients when the survival is much more important than the mutagenic side effects. To decide which fungal toxins could be used as combined cytotoxic and antimetastatic agents, mycotoxins were divided into three categories: (a) highly genotoxic (mutagenic, teratogenic, and carcinogenic), (b) adversely toxic, and (c) antitumorigenic agents. Highly cytotoxic mycotoxins with tolerable side effects, combined with an antineoplastic character, could be potential candidates against metastasis. From the structure-function relationship of antimetastatic mycotoxins, only general conclusions have been drawn. The presence of ring structures containing heteroatoms, functional groups, and the cumulative presence of oxygen atoms contributed to the oxidative stress and cytotoxicity of mycotoxins. The preselection of mycotoxins excluded category (a), and only the categories (b) and

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.

MICAN, a new fluorophore for vital and non-vital staining of human cells.

Fluorescence time-lapse microscopy is in connection with the invasive properties of fluorochrome applied, and with the toxicity of the excitation energy and wavelength of the dye itself. Experiments with the newly synthesized fluorescent dye 1-N-methylamino-5-isocyanonaphthalene (MICAN) served to test its cytotoxicity on human HaCaT keratinocyte cell cultures. Experiments related to staining capability were performed with paraformaldehyde (PFA) fixed cells and observed with fluorescence microscope. It was assumed that the fluorophore 1-amino-5-isocyanonaphthalene (ICAN) and especially its N-methylamino derivative MICAN, containing condensed aromatic rings could serve as a nonselective fluorescent dye capable to stain cellular structures of fixed, living, damaged and dead cells. This notion was confirmed by the MICAN staining of cytoplasmic proteins primarily rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SEM) and less efficiently nuclear proteins suggesting the involvement of staining of subcellular structures involved in protein synthesis. MICAN was not only well tolerated by living cells but turned out to be a strong heterochromatin and RER staining agent. This led to the development of a MICAN staining protocol for native and living samples. Relative to other fluorescent dyes, MICAN is not only useful but also cost-effective. Toxicology tests were performed using 30, 10, 5, 0.5 µg/ml MICAN concentrations. Time-lapse videomicroscopy at near-infrared (NIR) illumination has been used for the examination of MICAN effect on cell division. It was found that MICAN as a vital stain had no significant harmful effect on HaCaT cells. MICAN turned out to be a non-toxic, highly quantum-efficient vital stain with minimal, or no photobleaching, and can be applied to co-stain with propidium-iodide due the strong spectral separation.

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.

Methods to detect apoptotic cell death.

In concert with the increased understanding that there are many ways for cells to die, several methods have been developed to detect cell death. The classification of cell death posed some difficulties that were overcome by implementing strict selection criteria that should also apply to the detection methods. The selection of assays is based on morphological criteria and distinguishable marks of apoptotic patways. The detection of apoptosis includes methods related to membrane alterations, DNA fragmentation, cytotoxicity and cell proliferation, mitochondrial damage, immunological detection and mechanism based assays. Other less frequently used detections of apoptosis are: (a) light-scattering flow cytometry to avoid underestimating the extent and timing of apoptosis, (b) time-lapse microscopy perfusion platform to support the temporal aspects of detection, to measure cell surface area and cellular adhesion, and (c) genotoxicity specific chromatin changes. Attention is called to the advantages and limitations of various methods.

Mille modis morimur: We die in a thousand ways.

Dying cells subjected to apoptotic programs are engulfed by neighboring cells or by professional phagocytes, without inflammation or immunological reactions in the tissue where apoptosis takes place. Apoptotic cells release danger-associated project signals to their neighbours, through different molecular patterns, stimulate antigen production and immune responses. Microenvironmental effects with several functional consequences indicate that cell death is a complex process and may take place in several ways. This idea is expressed by the title of the Special Issue and by the title of the guest editorial "Mille modis morimur" meaning that not only multicellular organisms, but also single cells may die in a thousand ways. This idea is demonstrated by the papers serving as examples for cell death. Apoptosis was induced by clary sage oil in Candida cells. Heavy metal (Gd) induced cell motility and apoptosis was found in mammalian cells. RNA oxidation enhanced the reversion frequency of apoptosis in yeast mutants. The frequency of apoptotic micronucleus formation increased in a concentration-dependent manner by methotrexate. The antioxidant coenzyme Q10 protected renal proximal tubule cells against nicotine-induced apoptosis. The synergy of 2-deoxy-D-glucose combined with berberine induced lysosome/autophagy. The mitochondrial apoptotic pathway could be regulated by glucocorticoid receptor in collaboration with Bcl-2 family proteins in developing T cells. Cylindrospermopsin induced biochemical changes led to apoptosis in plants. Mechanisms of stress seriously impacted the risk of apoptosis. Transcriptional control of apoptotic cell clearance was achieved by macrophage nuclear receptors. Finally, the clinical aspects of apoptosis-induced lymphopenia were reviewed in sepsis and other severe injuries. These examples not only support the view of many ways of cell death, but predict further potential ways to induce or reduce the risk of cell death.