Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs

EU funds as an important source of financing for environmental protection at the local level on the example of Sadecczyzna region

Tematem niniejszej pracy jest przedstawienie istotnej roli funduszy europejskich w finansowaniu działań prośrodowiskowych na szczeblu lokalnym na przykładzie Sądecczyzny. Praca składa się z dwóch rozdziałów teoretycznych oraz jednego empirycznego. Celem pracy było przybliżenie działań Unii Europejskiej w zakresie środowiska przyrodniczego i podkreślenie istotnej roli środków pieniężnych z funduszy europejskich na ochronę środowiska.The subject of this paper is to present the important role of European funds in financing pro-environmental activities at the local level, on the example of the Sądecczyzna region. The work consists of two theoretical and one empirical chapters. The aim of the study was to present the activities of the European Union in the field of the natural environment and to emphasize the important role of funds from European funds for environmental protection

Neurotoksyczność ołowiu. Hipotetyczny molekularny mechanizm zaburzeń funkcji synaptycznych

Lead (Pb) toxicity is still a major health problem associated with both environmental and occupational exposure. Special attention is given to the neurotoxic effect of lead. Along with the newly emerging data, the Pb concentration in the body that can be considered safe is declining. Numerous studies on the neurotoxicity of Pb have shown multiple cellular ‘molecular targets’ of this metal at the biochemical and molecular levels, and differences in sensitivity to its toxic action among various neural cells. One possible target of the neurotoxic effect of Pb (at the synapse level) is N-methyl-D-aspartic acid (NMDA) receptors. This review presents the hypothetical molecular mechanism by which Pb disrupts synapse formation and plasticity in developing hippocampal neurons and the role of the NMDA receptor-dependent signaling pathway and brain-derived neurotrophic factor (BDNF) as a mechanism of Pb neurotoxicity at the synapse level.Toksyczność ołowiu (Pb) stanowi problem zdrowotny wynikający z narażenia środowiskowego i zawodowego. W centrum zainteresowania jest neurotoksyczne działanie Pb. W badaniach nad neurotoksycznością Pb obserwuje się tendencję do obniżania progu “bezpiecznego stężenia” Pb. Na poziomie biochemicznym i molekularnym wykazano wiele “punktów uchwytu” Pb w procesach komórkowych oraz niejednakową wrażliwość poszczególnych rodzajów komórek nerwowych na jego toksyczne działanie. Jednym z celów działania Pb na poziomie synapsy są glutaminianergiczne receptory jonotropowe dla kwasu A-metylo-ö-asparaginowego (NMDA). W pracy przedstawiono przypuszczalny mechanizm molekularny, poprzez który Pb zakłóca formowanie i plastyczność synapsy w rozwijających się neuronach hipokampa, oraz omówiono rolę zaburzenia szlaku sygnałowego zależnego od receptora NMDA i czynnika wzrostu pochodzenia mózgowego (BDNF) jako mechanizmu neurotoksycznego działania Pb na poziomie synapsy

DataSheet_1_Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.docx

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.</p