Ekaterina Dadachova

Melanin, radiation, and energy transduction in fungi

Melanin pigments are found in many diverse fungal species, where they serve a variety of functions that promote fitness and cell survival. Melanotic fungi inhabit some of the most extreme habitats on earth such as the damaged nuclear reactor at Chernobyl and the highlands of Antarctica, both of which are high-radiation environments. Melanotic fungi migrate toward radioactive sources, which appear to enhance their growth. This phenomenon, combined with the known capacities of melanin to absorb a broad spectrum of electromagnetic radiation and transduce this radiation into other forms of energy, raises the possibility that melanin also functions in harvesting such energy for biological usage. The ability of melanotic fungi to harness electromagnetic radiation for physiological processes has enormous implications for biological energy flows in the biosphere and for exobiology, since it provides new mechanisms for survival in extraterrestrial conditions. Whereas some features of the way melanin-related energy transduction works can be discerned by linking various observations and circumstantial data, the mechanistic details remain to be discovered.

Resistance of an Antarctic cryptoendolithic black fungus to radiation gives new insights of astrobiological relevance

The Antarctic black meristematic fungus Cryomyces antarcticus CCFEE 515 occurs endolithically in the McMurdo Dry Valleys of Antarctica, one of the best analogue for Mars environment on Earth. To date, this fungus is considered one of the best eukaryotic models for astrobiological studies and has been repeatedly selected for space experiments in the last decade. The obtained results are reviewed here, with special focus on responses to space relevant irradiation, UV radiation, and both sparsely and densely ionizing radiation, which represent the major injuries for a putative space-traveller. The remarkable resistance of this model organism to space stress, its radioresistance in particular, and mechanisms involved, significantly contributed to expanding our concept of limits for life and provided new insights on the origin and evolution of life in planetary systems, habitability, and biosignatures for life detection as well as on human protection during space missions.

Radioimmunotherapy of infection with 213Bi-labeled antibodies

Bismuth-213 ((213)Bi) (physical half-life 46 min) is a beta-emitter (97%) and an alpha-emitter (3%) which decays to short lived alpha-emitter Polonium-213 and could therefore be used as an in vivo generator of alpha particles with the energy of around 8 MeV. (213)Bi has been successfully used during the last decade in both clinical and pre-clinical work for radioimmunotherapy (RIT) of cancer with (213)Bi-labeled monoclonal antibodies (mAbs). RIT has been proposed as a novel techonology for treatment of infectious diseases. (213)Bi-labeled mAbs have been successfully used for treatment of experimental fungal, bacterial and viral infections with transient or none hematologic toxicity. The mechanisms of RIT of infection with (213)Bi-labeled mAbs include direct killing of cells and induction of apoptosis. In vivo RIT results in decrease of inflammation in infected organs. Among the delivery vehicles for RIT of infection whole IgG1 mAbs seem to be the most suitable in terms of the highest uptake in the target organs and the lowest - in normal tissues. RIT with alpha-emitter (213)Bi involves the application of established technology developed for the treatment of malignancies to infectious diseases. The development of RIT for infectious diseases is potentially easier than its application to tumor therapy given antigenic and tissue perfusion differences between sites of microbial infection and tumor infiltration. Nevertheless, considerable pre-clinical and clinical development work is likely to be required to learn how to use RIT for infection optimally.

The second International Symposium on Fungal Stress: ISFUS

The topic of fungal stress is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.

Spectroelectrochemical Reverse Engineering DemonstratesThat Melanin’s Redox and Radical Scavenging Activities Are Linked

Melanins are ubiquitous in nature but their biological activities and functions have been difficult to discern. Conventional approaches to determine material function start by resolving structure and then characterize relevant properties. These approaches have been less successful for melanins because of their complex structure and insolubility, and because their relevant properties are not readily characterized by conventional methods. Here, we report a novel spectroelectrochemical reverse engineering approach that focuses on redox and radical scavenging activities. In this method, the melanin is immobilized in a permeable hydrogel film adjacent to an electrode and this immobilized melanin is probed using diffusible mediators and complex electrical inputs. Response characteristics are measured using two modalities, electrochemical currents associated with the reaction of diffusible mediators, and optical absorbance associated with the presence of diffusible free radicals. Using this method, we observed that both Sepia and fungal melanins are redox active and can repeatedly exchange electrons to be switched between oxidized and reduced states. Further, we observed that these melanins can quench radicals either by donating or accepting electrons. Finally, we demonstrate that the melanins radical scavenging activities are dependent on their redox state such that a melanin must be reduced to have donatable electrons to quench oxidative free radicals, or must be oxidized to accept electrons from reductive free radicals. While the observation that melanin is redox-active is consistent with their well-accepted beneficial (radical-scavenging) and detrimental (pro-oxidant) activities, these observations may also support less well-accepted proposed functions for melanin in energy harvesting and redox communication.

Fungal strategies for dealing with environment- and agriculture-induced stresses

The Fungal Kingdom is responsible for many ecosystem services as well as many industrial and agricultural products. Nevertheless, how these fungal species function and carry out these services is dependent on their capacity to grow under different stress conditions caused by a variety of abiotic factors such as ionizing radiation, UV radiation, extremes of temperature, acidity and alkalinity, and environments of low nutritional status, low water activity, or polluted with, e.g. toxic metals or xenobiotics. This article reviews some natural or synthetic environments where fungi thrive under stress and have important impacts in agriculture and forestry.

The effect of protracted X-ray exposure on cell survival and metabolic activity of fast and slow growing fungi capable of melanogenesis: Fast and slow growing fungi capable of melanogenesis

The aim of this study was to analyse how protracted exposure to X-rays delivered at low dose rates of 0.0032-0.052 kGyxa0h-1 affects the survival and metabolic activity of two microfungi capable of melanogenesis: fast-growing Cryptococcus neoformans (CN) and slow-growing Cryomyces antarcticus (CA). Melanized CN and CA cells survived the protracted exposure better than non-melanized ones, which was consistent with previous reports on the radioprotective role of melanin in these fungi after high dose rate exposures. The survival data were described by the linear quadratic dose response model. The XTT metabolic profiles were practically identical for melanized CN and CA with activity dose-dependent increasing: no changes in the activity of the non-melanized CN and CA were recorded by this assay. In contrast, the MTT assay, which measures the intracellular energy-related processes, recorded an increase in activity of non-melanized CN and CA cells, but not in their melanized counterparts. This could reflect intensive repair processes initiated by the non-melanized cells post exposure. This study suggests that differences in radiation responses between melanized and non-melanized fungal cells occur over a wide range of radiation dose rates.

Morphological changes in melanized and non-melanized Cryptococcus neoformans cells post exposure to sparsely and densely ionizing radiation demonstrate protective effect of melanin

There is a need for novel and effective prophylactic treatments and radioprotective materials to protect civilians and military personnel from ionizing radiation in contaminated environments. Melanin, a naturally occurring, ubiquitous pigment, has been shown to confer radioresistance, acting as a potential radioprotective agent. We have demonstrated that melanized Cryptococcus neoformans (CN) cells had improved survival post ionizing irradiation than non-melanized ones. The goal of this study was to identify morphological changes in melanized and non-melanized CN cells following irradiation with densely-ionizing deuterons and alpha particles relative to sparsely-ionizing gamma radiation. We observed significant differences between the melanized and non-melanized CN cellular ultrastructure following irradiation. Melanized CN cells were relatively resistant to mid and max-dose levels of alpha particles and deuterons irradiation. Following irradiation the capsule was stripped, but the cell wall was intact and structural integrity was maintained. At the maximum dose, cytoplasmic vacuolization, and mitochondrial swelling started to occur. In contrast, the non-melanized CN strain was sensitive to the mid-dose radiation. Non-melanized cells presented two morphologies: small condensed, and swollen, lacking structural integrity. This morphological investigation provides the first direct evidence of the radioprotective properties of melanin in CN cells subjected to high RBE and high LET ionizing radiation.

Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

In vivo Evaluation of Free and Chelated Accelerator-produced Actinium- 225 - Radiation Dosimetry and Toxicity Results

BACKGROUND AND OBJECTIVEnThe demand for the alpha-emitting radionuclide Actinium-225 (225Ac) for use in radionuclide therapy is growing. Producing 225Ac using high energy linear accelerators, cyclotrons or photoinduction could increase its supply. One potential problem with accelerator produced 225Ac using Thorium-232 targets is the presence in final product of 0.1-0.3% by activity of the long-lived 227Ac impurity at the end of irradiation. It is important to comprehensively evaluate the behavior of accelerator-produced 225Ac in vivo before using it in pre-clinical and clinical applications.nnnMETHODSnBiodistribution of accelerator-produced 225Ac in acetate (free) and DOTA complex forms was performed in male and female CD-1 mice. The biodistribution data was used for radiation dosimetry calculations. The toxicity studies of free 225Ac were conducted in CD-1 mice at 1.036 and 2.035 kBq/g body weight. Blood counts, body weight and post-mortem histology were evaluated.nnnRESULTSnIn both genders, there was a pronounced uptake of free 225Ac in the liver when compared to 225Ac-DOTA which resulted in 200 and 50 times higher liver radiation dose for free 225Ac in male and female mice, respectively. 227Ac contribution to radiation dose delivered by 225Ac was calculated to be negligible. Mice given free 225Ac did not lose weight, had only transient effect on their blood counts and showed no histological damage to the liver and bone marrow.nnnCONCLUSIONnOur biodistribution/dosimetry/toxicity study of accelerator-produced 225Ac demonstrated the patterns very similar to 229Th-derived 225Ac. We conclude that accelerator-produced 225Ac is suitable for the developmental work of targeted radionuclide therapy.