Production and characterisation of aegerolysin and protein with MACPF domain from entomopathogenic fungus Beauveria bassiana.

Perforini so proteini, ki s tvorjenjem por v membrane predstavljajo velik biotehnološki potencial na biofarmacevtskih, analitičnih in agronomskih področjih. Znanih je več različnih perforinov in proteinskih parov, ki skupaj tvorijo pore, njihov širok spekter možne uporabe pa kliče po novih raziskavah. Pomembno je natančno poznavanje njihovih molekularnih mehanizmov, ki vplivajo na njihovo biološko funkcijo in možne aplikacije. V tej diplomski nalogi smo se odločili preveriti potencial entomopatogene glive Beauveria bassiana, njenega egerolizina in proteina z domeno MACPF, z namenom ugotavljanja njune interakcije pri formiranju por. Izbrali smo dva gena, za katera je obstajala verjetnost, da glivi omogočata entomopatogeno delovanje s tvorbo por v žuželčjo kutikulo. Tekom raziskave smo uspešno klonirali in izrazili oba proteina v rekombinantni bakteriji, egerolizin smo uspešno izolirali v čisti obliki. Na njem smo uspešno izvedli več analitskih metod, ki so pokazale, da smo protein najverjetneje pridobili v pravilnem zvitju. Sedimentacijski test in površinska plazmonska resonanca sta pokazala močno vezavo egerolizina na kombinacijo lipidov ceramid fosfoetanolamin in sfingomielin, kar nakazuje na predvideno biološko funkcijo. Za natančnejšo določitev bioloških funkcij obeh proteinov in dokončno preverbo njunih potencialov kot pesticidnih sredstev in morebitne druge aplikacije pa so zagotovo potrebne nadaljnje analize.Perforins are proteins that have a large biotechnological potential in biopharmaceutical, analytic and agronomical fields because of their pore forming abilities. There is already a wide spectre of known perforins and protein couples that jointly form pores, but their wide spectre of potential use calls for new research. Analysis of molecular mechanisms that effect their biological function as well as their potential applications is crucial. In this thesis we decided to discover potential of entomopathogenic fungi Beauveria bassiana, its aegerolylin and protein with MACPF domain with a goal of discovering their interaction in formulation of pores. We selected two genes, for which there was a plausibility, that they are enabling fungi its entomopathogenic activity, with formation of pores into insect cuticle. During the research we successfully cloned and expressed both proteins from recombinant bacterium and successfully isolated aegerolysin in a pure form. On this protein we than preceded to conduct analytical methods, that confirmed, that we most likely gained it in its native form. Sedimentation test and SPR showed strong bonds of aegerolysin to lipid combination of ceramide phosphoethanolamine and sphingomyelin which matches predicted biological function. For exact determination of biological functions of both proteins and final discovery of their potential use as pesticide and other applications further research is needed

Integrating multi-taxon palaeogenomes and sedimentary ancient DNA to study past ecosystem dynamics

Ancient DNA (aDNA) has played a major role in our understanding of the past. Important advances in the sequencing and analysis of aDNA from a range of organisms have enabled a detailed understanding of processes such as past demography, introgression, domestication, adaptation and speciation. However, to date and with the notable exception of microbiomes and sediments, most aDNA studies have focused on single taxa or taxonomic groups, making the study of changes at the community level challenging. This is rather surprising because current sequencing and analytical approaches allow us to obtain and analyse aDNA from multiple source materials. When combined, these data can enable the simultaneous study of multiple taxa through space and time, and could thus provide a more comprehensive understanding of ecosystem-wide changes. It is therefore timely to develop an integrative approach to aDNA studies by combining data from multiple taxa and substrates. In this review, we discuss the various applications, associated challenges and future prospects of such an approach

Regulation of immune cells in the periphery and central nervous system after X-ray irradiation

Space environment presents great danger for astronauts' health. One of the major risk factors is exposure to ionizing radiation, which has the potential to ionize atoms and molecules. This can have detrimental effects to cellular mechanisms as well as the biological systems as a whole. Due to the overlap of different biological systems it is impossible to study them individually. Two important organ systems severely affected by ionizing radiation are the immune system and the central nervous system (CNS). The role of irradiated immune cells of the CNS in the pathogenesis of cognitive deficits in addition to those caused by radiation exposure of neuronal cells still needs to be elucidated. The aim of this thesis was to understand the response of microglia with an activated or an inactivated phenotype to different doses of X-irradiation. The murine BV-2 cell line was used as model for microglial cells. DNA damage, apoptotic activity and phagocyic rates after irradiation were determined. For comparison, DNA double strand breaks (DSB) were also measured in the monocyte cell line (U937) after irradiation. While X-irradiation caused a dosedependent increase in DSB, no significant induction of apoptosis or reduction of phagocytotic activity was observed. The responses were comparable for inactivated and activated microglia with an exception of significant results observed in the amount of DNA DSB after irradiation with a dose of 4 Gy. A trend of decreased phagocitic activity was observed in both activated and inactivated phenotype 24 hours post irradiation for the dose of 4 Gy, but did not yield a significant result. The radiation-induced effects 24-hours post irradiation decreased in comparison to the 1- hour post irradiation timepoint which entails the efficiency of the repair mechanisms. Further experiments should be done in order to truly understand the implications of the activated and inactivated phenotype as well as the X-irradiation dose effects on the immune cells of the CNS and the resulting neurological damage or protection

Lipid-binding aegerolysin from biocontrol fungus Beauveria bassiana

Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis. We performed an in-depth bioinformatics analysis of aegerolysins in entomopathogenic fungi and selected a candidate aegerolysin, beauveriolysin A (BlyA) from Beauveria bassiana. BlyA was expressed as a recombinant protein in Escherichia coli, and purified to further determine its functional and structural properties, including lipid-binding ability. Aegerolysins were found to be encoded in genomes of entomopathogenic fungi, such as Beauveria, Cordyceps, Metarhizium and Ophiocordyceps. Detailed bioinformatics analysis revealed that they are linked to MACPF-like genes in most genomes. We also show that BlyA interacts with an insect-specific membrane lipid. These results were placed in the context of other fungal and bacterial aegerolysins and their partner proteins. We believe that aegerolysins play a role in promoting the entomopathogenic and antagonistic activity of B. bassiana, which is an active ingredient of bioinsecticides

The Dynamics of Heparin-Binding Protein in Cardiothoracic Surgery—A Pilot Study

Objectives: To explore the preoperative, intraoperative, and postoperative dynamics of heparin-binding protein (HBP) in cardiothoracic surgery. Design: This was a prospective, observational study. Setting: The study was conducted at a single university hospital. Participants: Thirty patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) were included, 15 of whom underwent coronary artery bypass grafting surgery and 15 of whom underwent complex procedures. Ten patients undergoing lung surgery also were included as a conventional surgery reference group. Interventions: No interventions were performed. Measurements and Main Results: HBP was measured at nine different perioperative times. HBP levels increased immediately after heparin administration, further increased during CPB, but decreased rapidly after protamine administration. At arrival to the intensive care unit, median HBP levels were 24.8 (15.6-38.1) ng/mL for coronary artery bypass grafting patients and 51.2 (34.0-117.7) ng/mL for complex surgery patients (p = 0.011). One day after surgery, HBP levels in all three groups were below the proposed cutoff of 30 ng/mL, which previously was found to predict development of organ dysfunction in patients with infection. Conclusions: HBP levels are elevated by the administration of heparin and the use of CPB but reduced by protamine administration. At postoperative day one, HBP levels were less than the threshold for organ dysfunction in patients with infection. The usefulness of HBP for predicting postoperative infections in cardiothoracic surgery should be investigated in future studies