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Alicja Hinz,1 Sylwia Stankiewicz,1,2 Jacek Jakub Litewka,2,3 Paweł E Ferdek,3 Maja Sochalska,4 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Doctoral School of Exact and Natural Sciences Jagiellonian University, Kraków, Poland; 3Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 4Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, PolandCorrespondence: Monika Bzowska, Email monika.bzowska@uj.edu.plPurpose: Given the increased use of polyethylene glycol (PEG) in refining the therapeutic activity of medicines, our research focuses on explaining the potential mechanism of immune reactions associated with this polymer. We aim to investigate the interaction of different types of PEG with mouse and human immune cells, thereby contributing to understanding PEG interactions with the immune system and verifying the proinflammatory activity of the tested polymers.Patients and Methods: Mouse macrophage and neutrophil cell lines, human peripheral blood mononuclear cells, and polymorphonuclear cells isolated from healthy donors were exposed to various PEGs. ROS, NO, and cytokine production were analyzed using fluorescence intensity, absorbance, or cytometric measurements. Toll-like receptor (TLR) signaling was verified using HEK-blue-reporter cell lines. Finally, neutrophil trap formation was studied using immunofluorescence labeling, and calcium imaging was performed using a Ca2+-sensitive indicator and fluorescence microscope.Results: Our findings show that specific PEG and mPEG are not toxic to tested mouse and human cells. However, they exert proinflammatory activity against human immune cells, as evidenced by the increased secretion of proinflammatory cytokines, such as IFN-a2, IFN-γ, TNF-α, MCP-1, IL-8, IL-17A, and IL-23. This phenomenon is independent of PEG signaling via TLR. Additionally, mPEG10 induced the formation of neutrophil extracellular traps and intracellular calcium signaling.Conclusion: Our finding suggests that some PEG types have proinflammatory activity against human immune cells, manifesting in the upregulated production of cytokines and neutrophils trap releasing.Keywords: drug delivery, immunomodulation, in vitro nanotoxicity, nanoparticle functionalization, PEGylated therapeutics

The current therapies against gastric pathogen Helicobacter pylori are ineffective in over 20% of patients. Enzymes belonging to the purine salvage pathway are considered as novel drug targets in this pathogen. Therefore, the main aim of the current study was to determine the antibacterial activity of pyridoxal 5’-phosphate (PLP), an active form of vitamin B6, against reference and clinical strains of H. pylori. Using a broad set of microbiological, physicochemical (UV absorption, LC-MS, X-ray analysis) and in silico experiments, we were able to prove that PLP inhibits adenylosuccinate synthetase (AdSS) from H. pylori by the competition with GTP (IC50eq ∼30 nM). This behaviour was attributed to formation of a Schiff base with a lysine residue (a covalent bond with Lys322 in the GTP binding site of AdSS) and was potentiated by the presence of vitamin C. This antibacterial activity of PLP gives hope for its future use against H. pylori.

Introduction Suicidality is a common concern in psychiatric patients and one of the leading causes of death in adolescents and young adults. (Adolescent health. (2019, November 26) WHO). Some mental health professionals engage in a no-suicide contract with their patients. In this type of intervention, the patient usually agrees to not harm or kill himself/herself. There is an increasing body of evidence to support brief interventions, such as group of safety planning-type interventions (SPTIs) (McCabe et al. MC Psychiatry, 2018, May 3; 18(1)). Safety planning is derived from cognitive therapy and cognitive behavioral therapy used for suicide prevention. Objectives Our objective was to summarize and critically analyze current evidence of effectiveness of SPTIs and no-suicide contracts in suicide prevention. Methods We conducted a literature review to compare no-suicide contract to safety-planning interventions in suicide prevention. Results Although no-suicide contracts may work for some individuals, there is not enough quantitative evidence to support such contracts as clinically effective tools. A recent meta-analysis has shown that SPTIs were associated with reductions in suicidal behaviors although no effect was identified with frequency of suicidal thoughts (Nuij et al. (2021, April 30). The British Journal of Psychiatry, 219 (2), 419–426). Conclusions Based on the evidence and straightforward implementation of SPTIs in different clinical settings it may to be a more effective alternative to no-suicide contracts. Disclosure of Interest None Declared

Macrophages are highly plastic cells ubiquitous in various tissues, where they perform diverse functions. They participate in the response to pathogen invasion and inflammation resolution following the immune response, as well as the maintenance of homeostasis and proper tissue functions. Macrophages are generally considered long-lived cells with relatively strong resistance to numerous cytotoxic factors. On the other hand, their death seems to be one of the principal mechanisms by which macrophages perform their physiological functions or can contribute to the development of certain diseases. In this review, we scrutinize three distinct pro-inflammatory programmed cell death pathways – pyroptosis, necroptosis, and ferroptosis – occurring in macrophages under specific circumstances, and explain how these cells appear to undergo dynamic yet not always final changes before ultimately dying. We achieve that by examining the interconnectivity of these cell death types, which in macrophages seem to create a coordinated and flexible system responding to the microenvironment. Finally, we discuss the complexity and consequences of pyroptotic, necroptotic, and ferroptotic pathway induction in macrophages under two pathological conditions – atherosclerosis and cancer. We summarize damage-associated molecular patterns (DAMPs) along with other microenvironmental factors, macrophage polarization states, associated mechanisms as well as general outcomes, as such a comprehensive look at these correlations may point out the proper methodologies and potential therapeutic approaches.

Ectosomes are carriers of proangiogenic factors during cancer progression. This study investigated whether the proangiogenic effect exerted by melanoma-derived ectosomes on recipient endothelial cells is mediated by ectosomal αvβ3 and αvβ5 integrins. Ectosomes were isolated from the conditioned culture media of four melanoma cell lines and melanocytes. Changes in gene and protein expression of αvβ3 and αvβ5 integrins, as well as VEGF and TNF-α were assessed in ectosome-treated endothelial cells. To confirm the functional involvement of ectosomal integrins in functional tests (Alamar Blue, wound healing and tube formation assays), ectosomes were also pretreated with anti-integrin antibodies and integrin-blocking peptides echistatin and cilengitide. Melanoma-derived ectosomes induced changes in the expression of αvβ3 and αvβ5 integrins in recipient endothelial cells, leading to increased viability, migratory properties, and tube formation potential. The extent of proangiogenic stimulation varied depending on the types of cells releasing ectosomes and the recipient cells. The use of anti-integrin antibodies and integrin-blocking peptides revealed a more significant role for the αvβ5 integrin/VEGF than the αvβ3 integrin/TNF-α pathway in the interactions between ectosomes and endothelial cells. The study demonstrated the functional role of ectosomal αvβ3 and αvβ5 integrins. It also provided a baseline understanding of ectosome-mediated αvβ3 integrin/TNF-α and αvβ5 integrin/VEGF signaling in angiogenesis.

Helicobacter pylori represents a global health threat with around 50% of the world population infected. Due to the increasing number of antibiotic-resistant strains, new strategies for eradication of H. pylori are needed. In this study, we suggest purine nucleoside phosphorylase (PNP) as a possible new drug target, by characterising its interactions with 2- and/or 6-substituted purines as well as the effect of these compounds on bacterial growth. Inhibition constants are in the micromolar range, the lowest being that of 6-benzylthio-2-chloropurine. This compound also inhibits H. pylori 26695 growth at the lowest concentration. X-ray structures of the complexes of PNP with the investigated compounds allowed the identification of interactions of inhibitors in the enzyme’s base-binding site and the suggestion of structures that could bind to the enzyme more tightly. Our findings prove the potential of PNP inhibitors in the design of drugs against H. pylori.

Justyna Śmiałek,1,* Monika Bzowska,2,* Alicja Hinz,2 Renata Mężyk-Kopeć,2 Kamilla Sołtys,2 Paweł Mak1 1Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland*These authors contributed equally to this workCorrespondence: Paweł Mak, Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 St., Kraków, 30-387, Poland, Tel +48 12 664 6511, Fax +48 12 664 6902, Email pawel.mak@uj.edu.plPurpose: The zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 – an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules.Methods: The study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin – nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides.Results: BacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide.Conclusion: All naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.Keywords: cytokines, neutrophils, NF-kappa-B activation, nisin, nitric oxide, posttranslational modifications, Staphylococcus pseudintermedius 222

The current therapies against gastric pathogen Helicobacter pylori are ineffective in over 20% of patients. Enzymes belonging to the purine salvage pathway are considered as novel drug targets in this pathogen. Therefore, the main aim of the current study was to determine the antibacterial activity of pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, against reference and clinical strains of H. pylori. Using a broad set of microbiological, physicochemical (UV absorption, LC-MS, X-ray analysis) and in silico experiments, we were able to prove that PLP inhibits adenylosuccinate synthetase (AdSS) from H. pylori by the competition with GTP (IC50eq ∼30 nM). This behaviour was attributed to formation of a Schiff base with a lysine residue (a covalent bond with Lys322 in the GTP binding site of AdSS) and was potentiated by the presence of vitamin C. This antibacterial activity of PLP gives hope for its future use against H. pylori. [ABSTRACT FROM AUTHOR]

Alicja Hinz,1 Marta Szczęch,2 Krzysztof Szczepanowicz,2 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Kraków, PolandCorrespondence: Monika BzowskaDepartment of Cell Biochemistry, Faculty of Biochemistry, Jagiellonian University in Kraków, Biophysics and Biotechnology, 7 Gronostajowa Street, Kraków, 30-387, Poland, Tel/Fax +48 12 664 63 88, Email monika.bzowska@uj.edu.plKrzysztof SzczepanowiczJerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8 Street, Kraków, 30-239, Poland, Tel/Fax +48 12 639 51 21, Email krzysztof.szczepanowicz@ikifp.edu.plIntroduction: Biodistribution of nanocarriers with a structure consisting of core and shell is most often analyzed using methods based on labeling subsequent compartments of nanocarriers. This approach may have serious limitations due to the instability of such complex systems under in vivo conditions.Methods: The core-shell polyelectrolyte nanocarriers were intravenously administered to healthy BALB/c mice with breast cancer. Next, biodistribution profiles and elimination routes were determined post mortem based on fluorescence measurements performed for isolated blood, tissue homogenates, collected urine, and feces.Results: Despite the surface PEGylation with PLL-g-PEG, multilayer polyelectrolyte nanocarriers undergo rapid degradation after intravenous administration. This process releases the shell components but not free Rhodamine B. Elements of polyelectrolyte shells are removed by hepatobiliary and renal clearance.Conclusion: Multilayer polyelectrolyte nanocarriers are prone to rapid degradation after intravenous administration. Fluorophore localization determines the obtained results of biodistribution and elimination routes of core-shell nanomaterials. Therefore, precise and reliable analysis of in vivo stability and biodistribution of nanomaterials composed of several compartments requires nanomaterials labeled within each compartment.Keywords: in vivo studies, fluorescently labeled nanomaterials, stealth polymers, pharmacokinetics, routes of elimination, nanocarriers stability and degradation

Fluorescent markers play important roles in spectroscopic and microscopic research techniques and are broadly used in basic and applied sciences. We have obtained markers with fluorescent properties, two etheno derivatives of 2-aminopurine, as follows: 1,N2-etheno-2-aminopurine (1,N2-ε2APu, I) and N2,3-etheno-2-aminopurine (N2,3-ε2APu, II). In the present paper, we investigate their interaction with two key enzymes of purine metabolism, purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO), using diffraction of X-rays on protein crystals, isothermal titration calorimetry, and fluorescence spectroscopy. Crystals were obtained and structures were solved for WT PNP and D204N-PNP mutant in a complex with N2,3-ε2APu (II). In the case of WT PNP—1,N2-ε2APu (I) complex, the electron density corresponding to the ligand could not be identified in the active site. Small electron density bobbles may indicate that the ligand binds to the active site of a small number of molecules. On the basis of spectroscopic studies in solution, we found that, in contrast to PNP, 1,N2-ε2APu (I) is the ligand with better affinity to XO. Enzymatic oxidation of (I) leads to a marked increase in fluorescence near 400 nm. Hence, we have developed a new method to determine XO activity in biological material, particularly suitable for milk analysis. [ABSTRACT FROM AUTHOR]

Abstract The binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells. Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells.

Abstract E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.

Alicja Karabasz,1 Monika Bzowska,1 Krzysztof Szczepanowicz2 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków, PolandCorrespondence: Monika BzowskaDepartment of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 7 Gronostajowa Street, Kraków 30-387, PolandTel/Fax +48 12 664 63 88Email monika.bzowska@uj.edu.plKrzysztof SzczepanowiczJerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, 8 Niezapominajek Street, Kraków 30-239, PolandTel/Fax +48 12 639 51 21Email ncszczep@cyf-kr.edu.plAbstract: Polymeric nanomaterials have become a prominent area of research in the field of drug delivery. Their application in nanomedicine can improve bioavailability, pharmacokinetics, and, therefore, the effectiveness of various therapeutics or contrast agents. There are many studies for developing new polymeric nanocarriers; however, their clinical application is somewhat limited. In this review, we present new complex and multifunctional polymeric nanocarriers as promising and innovative diagnostic or therapeutic systems. Their multifunctionality, resulting from the unique chemical and biological properties of the polymers used, ensures better delivery, and a controlled, sequential release of many different therapeutics to the diseased tissue. We present a brief introduction of the classical formulation techniques and describe examples of multifunctional nanocarriers, whose biological assessment has been carried out at least in vitro. Most of them, however, also underwent evaluation in vivo on animal models. Selected polymeric nanocarriers were grouped depending on their medical application: anti-cancer drug nanocarriers, nanomaterials delivering compounds for cancer immunotherapy or regenerative medicine, components of vaccines nanomaterials used for topical application, and lifestyle diseases, ie, diabetes.Keywords: polymeric nanocarriers, biomedical application, anti-cancer, cancer immunotherapy, modern vaccines, regenerative medicine, lifestyle diseases – diabetes

Ectosomes are carriers of proangiogenic factors during cancer progression. This study investigated whether the proangiogenic effect exerted by melanoma-derived ectosomes on recipient endothelial cells is mediated by ectosomal αvβ3 and αvβ5 integrins. Ectosomes were isolated from the conditioned culture media of four melanoma cell lines and melanocytes. Changes in gene and protein expression of αvβ3 and αvβ5 integrins, as well as VEGF and TNF-α were assessed in ectosome-treated endothelial cells. To confirm the functional involvement of ectosomal integrins in functional tests (Alamar Blue, wound healing and tube formation assays), ectosomes were also pretreated with anti-integrin antibodies and integrin-blocking peptides echistatin and cilengitide. Melanoma-derived ectosomes induced changes in the expression of αvβ3 and αvβ5 integrins in recipient endothelial cells, leading to increased viability, migratory properties, and tube formation potential. The extent of proangiogenic stimulation varied depending on the types of cells releasing ectosomes and the recipient cells. The use of anti-integrin antibodies and integrin-blocking peptides revealed a more significant role for the αvβ5 integrin/VEGF than the αvβ3 integrin/TNF-α pathway in the interactions between ectosomes and endothelial cells. The study demonstrated the functional role of ectosomal αvβ3 and αvβ5 integrins. It also provided a baseline understanding of ectosome-mediated αvβ3 integrin/TNF-α and αvβ5 integrin/VEGF signaling in angiogenesis. [ABSTRACT FROM AUTHOR]

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of the research is to examine the existing literature on the economic foundations of agricultural crop production in Ukraine, focusing on the evolving management strategies in light of climate change challenges during the war. Design/methodology/approach: The systemic method applied to research involves an integrated approach focusing on interconnected elements within agricultural systems. The analysis was based on the official data of crops growing in Ukraine in 1991-2022, constituting a list of scientific publications. The systemic method applied to research involves an integrated approach focusing on interconnected elements within agricultural systems. Combining multiple methods provided a more comprehensive and nuanced understanding of the complex interactions between crops growing and analyzing the structure of the information about new plant cultures. Findings: The study embraced an adaptive approach, recognizing that the agricultural system is dynamic and subject to constant change. It aimed to adapt strategies based on evolving insights and feedback from stakeholders involved in the system. Research limitations/implications: The implications of this study underscore the importance of stakeholder engagement and adaptive management for effective integration. Practical implications suggest the need for policy coordination, capacity building, and innovative incentive mechanisms to foster harmonious coexistence between economic development and agricultural crops production. Originality/value: Before the war, 45 agricultural enterprises controlled a total of about 4.1 million hectares of agricultural land. Their total income exceeded 10.8 billion US dollars. The average farm in Ukraine occupies an area of 1000 hectares, while in the EU - only 16 hectares, and in Poland – 11 hectares. It is not difficult to calculate that one Ukrainian tycoon owns the area of approximately 46,000 Polish farmers. This might be a new direction in correction of modern agricultural policy. These results could be especially interesting for researchers whose studies are interdisciplinary. [ABSTRACT FROM AUTHOR]

Alicja Karabasz,1 Krzysztof Szczepanowicz,2 Agnieszka Cierniak,3,4 Renata Mezyk-Kopec,1 Grzegorz Dyduch,5 Marta Szczęch,2 Joanna Bereta,1 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków, Poland; 3Department of General Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 4Department of Biochemistry, Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski Krakow University, Kraków, Poland; 5Department of Pathomorphology, Jagiellonian University Medical College, Kraków, PolandCorrespondence: Monika BzowskaDepartment of Cell Biochemistry, Faculty of Biochemistry, Jagiellonian University in Kraków, Biophysics and Biotechnology, 7 Gronostajowa Street, Kraków 30-387, PolandTel/Fax +48 12 664 63 88Email monika.bzowska@uj.edu.plBackground: The functionalization of a nanoparticle surface with PEG (polyethylene glycol) is an approach most often used for extending nanomaterial circulation time, enhancing its delivery and retention in the target tissues, and decreasing systemic toxicity of nanocarriers and their cargos. However, because PEGylated nanomedicines were reported to induce immune response including production of anti-PEG antibodies, activation of the complement system as well as hypersensitivity reactions, hydrophilic polymers other than PEG are gaining interest as its replacement in nanomaterial functionalization. Here, we present the results of in vivo evaluation of polyelectrolyte nanocapsules with biodegradable, polyelectrolyte multilayer shells consisting of poly-l-lysine (PLL) and poly-l-glutamic (PGA) acid as a potential drug delivery system. We compared the effects of nanocapsules functionalized with two different “stealth” polymers as the external layer of tested nanocapsules was composed of PGA (PGA-terminated nanocapsules, NC-PGA) or the copolymer of poly-l-lysine and polyethylene glycol (PEG-terminated nanocapsules, NC-PEG).Methods: Nanocapsules pharmacokinetics, biodistribution and routes of eliminations were analysed postmortem by fluorescence intensity measurement. Toxicity of intravenously injected nanocapsules was evaluated with analyses of blood morphology and biochemistry and by histological tissue analysis. DNA integrity was determined by comet assay, cytokine profiling was performed using flow cytometer and detection of antibodies specific to PEG was performed by ELISA assay.Results: We found that NC-PGA and NC-PEG had similar pharmacokinetic and biodistribution profiles and both were eliminated by hepatobiliary and renal clearance. Biochemical and histopathological evaluation of long-term toxicity performed after a single as well as repeated intravenous injections of nanomaterials demonstrated that neither NC-PGA nor NC-PEG had any acute or chronic hemato-, hepato- or nephrotoxic effects. In contrast to NC-PGA, repeated administration of NC-PEG resulted in prolonged increased serum levels of a number of cytokines.Conclusion: Our results indicate that NC-PEG may cause undesirable activation of the immune system. Therefore, PGA compares favorably with PEG in equipping nanomaterials with stealth properties. Our research points to the importance of a thorough assessment of the potential influence of nanomaterials on the immune system.Keywords: polyelectrolyte nanocapsules, stealth polymers, animal studies

Alicja Karabasz1, Dorota Lachowicz2, Anna Karewicz3, Renata Mezyk-Kopec1, Krystyna Stalińska1, Ewa Werner4,5, Agnieszka Cierniak6, Grzegorz Dyduch7, Joanna Bereta1, Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, Kraków, Poland; 3Faculty of Chemistry, Jagiellonian University, Kraków, Poland; 4Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 5Department of Animal Reproduction and Anatomy, Faculty of Animal Science, University of Agriculture, Krakow, Poland; 6Department of General Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 7Department of Pathomorphology, Jagiellonian University Medical College, Kraków, PolandCorrespondence: Monika BzowskaJagiellonian University in Kraków, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biochemistry, 7 Gronostajowa St., Kraków 30-387, PolandTel +48 12 664 6388Fax +48 12 664 6388Email monika.bzowska@uj.edu.plBackground: Curcumin is a natural polyphenol with anti-inflammatory, chemopreventive and anticancer activity. However, its high hydrophobicity and poor bioavailability limit its medical application. The development of nanocarriers for curcumin delivery is an attractive approach to overcome its low bioavailability and fast metabolism in the liver. We synthesized a blood compatible alginate-curcumin conjugate, AA-Cur, which formed colloidally stable micelles of approximately 200 nm and, as previously shown, exerted strong cytotoxicity against mouse cancer cell lines. Here we analyze in vivo toxicity and antitumor activity of AA-Cur in two different mouse tumor models.Method: Potential toxicity of intravenously injected AA-Cur was evaluated by: i) analyses of blood parameters (morphology and biochemistry), ii) histology, iii) DNA integrity (comet assay), and iv) cytokine profiling (flow cytometry). Antitumor activity of AA-Cur was evaluated by measuring the growth of subcutaneously inoculated colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in control mice vs mice treated with AA-Cur.Results: Injections of four doses of AA-Cur did not reveal any toxicity of the conjugate, thus indicating the safety of its use. AA-Cur elicited moderate anti-tumor activity toward colon MC38-CEA or breast 4T1 carcinomas.Conclusion: The tested conjugate of alginate and curcumin, AA-Cur, is non-toxic and safe, but exhibits limited anticancer activity.Keywords: curcumin, alginate conjugates, in vivo toxicity, antitumor activity

Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis

Cholesterol-laden, foam macrophages constitute the most characteristic component of human atherosclerotic plaques. Persistent uptake of oxLDLs results in accumulation of lipid bodies inside the cells and determines their phenotype and subsequent functions. In this work, we describe the phenotype of human monocyte-derived foam cells obtained by differentiation in the constant presence of oxLDLs for 30 days (prolonged-hMDFCs). Although neither the total cellular nor the cell surface expression of Toll-like receptors (TLR) was regulated by oxLDLs, the prolonged-hMDFCs changed dramatically their responsiveness to TLR ligands and inactivated bacteria. Using multiplex technology, we observed an acute decline in cytokine and chemokine production after surface and endosomal TLR stimulation with the exception of TLR2/6 triggering with agonists Pam2CSK4 and MALP-2. We also noted significant reduction of some surface receptors which can have accessory function in recognition of particulate antigens (CD47, CD81, and CD11b). In contrast, the prolonged-hMDFCs responded to inflammasome activation by LPS/nigericin with extensive, necrotic type cell death, which was partially independent of caspase-1. This pyroptosis-like cell death was aggravated by necrostatin-1 and rapamycin. These findings identify a potential contribution of mature foam cells to inflammatory status by increasing the immunogenic cell death burden. The observed cross-talk between foam cell death pathways may lead to recognition of a potential new marker for atherosclerosis disease severity. Overall, our study demonstrates that, in contrast to other cellular models of foam cells, the prolonged-hMDFCs acquire a functional phenotype which may help understanding the role of foam cells in the pathogenesis of atherosclerosis.

Alicja Karabasz,1 Krzysztof Szczepanowicz,2 Agnieszka Cierniak,1,3 Joanna Bereta,1 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków, Poland; 3Department of Biochemistry, Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski Kraków University, Kraków, Poland Background: Toxicity of nanomaterials is one of the most important factors limiting their medical application. Evaluation of in vitro nanotoxicity allows for the identification and elimination of most of the toxic materials prior to animal testing. The current knowledge of the possible side effects of biodegradable nanomaterials, such as liposomes and polymeric organic nanoparticles, is limited. Previously, we developed a potential drug delivery system in the form of nanocapsules with polyelectrolyte, biodegradable shells consisting of poly-L-lysine and poly-L-glutamic acid (PGA), formed by the layer-by-layer adsorption technique. Methods: Hemolysis assay, viability tests, flow cytometry analysis of vascular cell adhesion molecule-1 expression on endothelium, analysis of nitric oxide production, measurement of intracellular reactive oxygen species levels, detection of antioxidant enzyme activity, and analysis of DNA damage with comet assay were performed to study the in vitro toxicity of nanocapsules. Results: In this work, we present the results of an in vitro analysis of toxicity of five-layer positively charged poly-L-lysine–terminated nanocapsules (NC5), six-layer negatively charged PGA-terminated nanocapsules (NC6) and five-layer PEGylated nanocapsules (NC5-PEG). PGA and polyethylene glycol (PEG) were used as two different “stealth” polymers. Of all the polyelectrolyte nanocapsules tested for blood compatibility, only cationic NC5 showed acute toxicity toward blood cells, expressed as hemolysis and aggregation. Neither NC6 nor NC5-PEG had proinflammatory activity evaluated through changes in the expression of NF-κB–dependent genes, iNOS and vascular cell adhesion molecule-1, induced oxidative stress, or promoted DNA damage in various cells. Conclusion: Our studies clearly indicate that PGA-coated (negatively charged) and PEGylated polyelectrolyte nanocapsules do not show in vitro toxicity, and their potential as a drug delivery system may be safely studied in vivo. Keywords: polyelectrolyte nanocapsules, layer-by-layer, nanotoxicity, oxidative stress, genotoxicity

Purpose: This paper reviews the impacts of heavy metals on soil degradation and examines the influence of military equipment on agricultural lands during the war. Design/methodology/approach: The systematic research method involves a comprehensive approach focused on interrelated elements of land pollution by heavy metals during the war. The analysis is based on official data on soil contamination by heavy metals (Lead (Pb), Cadmium (Cd), Mercury (Hg), Chromium (Cr), Arsenic (As)) in Ukraine and other countries of the world during the war period from burned military equipment. Combining multiple methods has provided a more complete and detailed understanding of complex interactions. Findings: This study aims to assess the spatial distribution, concentration levels, and potential sources of heavy metal contamination in Ukrainian soils during the period of war. By employing a multidisciplinary approach encompassing field surveys, soil sampling, laboratory analyses, and geographic information systems mapping, the authors seek to elucidate the impact of warfare activities on soil quality and identify hotspots of contamination across war affected regions. Research limitations/implications: The implications of this study underscore the importance of stakeholder engagement and adaptive management for effective integration. Originality/value: These results could be especially interesting for researchers whose studies are interdisciplinary. [ABSTRACT FROM AUTHOR]

Adenylosuccinate synthetase (AdSS) is an enzyme at regulatory point of purine metabolism. In pathogenic organisms which utilise only the purine salvage pathway, AdSS asserts itself as a promising drug target. One of these organisms is Helicobacter pylori, a wide-spread human pathogen involved in the development of many diseases. The rate of H. pylori antibiotic resistance is on the increase, making the quest for new drugs against this pathogen more important than ever. In this context, we describe here the properties of H. pylori AdSS. This enzyme exists in a dimeric active form independently of the presence of its ligands. Its narrow stability range and pH-neutral optimal working conditions reflect the bacterium’s high level of adaptation to its living environment. Efficient inhibition of H. pylori AdSS with hadacidin and adenylosuccinate gives hope of finding novel drugs that aim at eradicating this dangerous pathogen.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrAHp) belongs to the well conserved family of serine proteases. HtrAHp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrAHp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrAHp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrAHp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrAHp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrAHp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrAHp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.