Synthetic genistein derivatives as modulators of glycosaminoglycan synthesis

Background: Mucopolysaccharidoses (MPS) are severe metabolic disorders caused by 26 accumulation of undegraded glycosaminoglycans (GAGs) in lysosomes due to defects in certain 27 lysosomal hydrolases. Substrate reduction therapy (SRT) has been proposed as one of potential 28 treatment procedures of MPS. Importantly, small molecules used in such a therapy might 29 potentially cross the blood-brain barrier (BBB) and improve neurological status of patients, as 30 reported for a natural isoflavone, 5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one, 31 also known as genistein. Although genistein is able to cross BBB to some extent, its delivery to 32 the central nervous system is still relatively poor (below 10% efficiency). Thus, we aimed to 33 develop a set of synthetically modified genistein molecules and characterize physicochemical as 34 well as biological properties of these compounds. Methods: Following parameters were 35 determined for the tested synthetic derivatives of genistein: cytotoxicity, effects on cell 36 proliferation, kinetics of GAG synthesis, effects on epidermal growth factor (EGF) receptor’s 37 tyrosine kinase activity, effects on lysosomal storage, potential ability to cross BBB. Results: We 38 observed that some synthetic derivatives inhibited GAG synthesis similarly to, or more 39 efficiently than, genistein and were able to reduce lysosomal storage in MPS III fibroblasts. The 40 tested compounds were generally of low cytotoxicity and had minor effects on cell proliferation. 41 Moreover, synthetic derivatives of genistein revealed higher lipophilicity (assessed in silico) than 42 the natural isoflavone. Conclusion: Some compounds tested in this study might be promising 43 candidates for further studies on therapeutic agents in MPS types with neurological symptoms

Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage

A large scale analysis presented in this article focuses on biological and physiological variety of bacteriophages. A collection of 83 bacteriophages, isolated from urban sewage and able to propagate in cells of different bacterial hosts, has been obtained (60 infecting Escherichia coli, 10 infecting Pseudomonas aeruginosa, 4 infecting Salmonella enterica, 3 infecting Staphylococcus sciuri, and 6 infecting Enterococcus faecalis). High biological diversity of the collection is indicated by its characteristics, both morphological (electron microscopic analyses) and biological (host range, plaque size and morphology, growth at various temperatures, thermal inactivation, sensitivity to low and high pH, sensitivity to osmotic stress, survivability upon treatment with organic solvents and detergents), and further supported by hierarchical cluster analysis. By the end of the research no larger collection of phages from a single environmental source investigated by these means had been found. The finding was confirmed by whole genome analysis of 7 selected bacteriophages. Moreover, particular bacteriophages revealed unusual biological features, like the ability to form plaques at low temperature (4 °C), resist high temperature (62 °C or 95 °C) or survive in the presence of an organic solvents (ethanol, acetone, DMSO, chloroform) or detergent (SDS, CTAB, sarkosyl) making them potentially interesting in the context of biotechnological applications

Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor-dependent pathway

<p>Abstract</p> <p>Background</p> <p>Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs). Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone) inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome).</p> <p>Methods</p> <p>To learn on details of the molecular mechanism of genistein-mediated inhibition of GAG synthesis, efficiency of this process was studied by measuring of incorporation of labeled sulfate, storage of GAGs in lysosomes was estimated by using electron microscopic techniques, and efficiency of phosphorylation of epidermal growth factor (EGF) receptor was determined by using an ELISA-based assay with fluorogenic substrates.</p> <p>Results</p> <p>Effects of genistein on inhibition of GAG synthesis and accumulation in fibroblasts from patients suffering from various MPS types were abolished in the presence of an excess of EGF, and were partially reversed by an increased concentration of genistein. No such effects were observed when an excess of 17β-estradiol was used instead of EGF. Moreover, EGF-mediated stimulation of phsophorylation of the EGF receptor was impaired in the presence of genistein in both wild-type and MPS fibroblasts.</p> <p>Conclusion</p> <p>The results presented in this report indicate that the mechanism of genistein-mediated inhibition of GAG synthesis operates through epidermal growth factor (EGF)-dependent pathway.</p

CRP-binding bacteriophage as a new element of layer-by-layer assembly carbon nanofiber modified electrodes

Recently, bacteriophage particles have started to be applied as a new biomaterial for developing sensing platforms. They can be used as both a recognition element or/and as building blocks, template/scaffold. In this paper, we studied a bacteriophage selected through phage-display technology. The chosen bacteriophage acted as a building block for creating a carbon nanofiber-based electrode and as a new receptor/binding element that recognizes C-reactive protein (CRP) – one of the markers of inflammatory processes in the human body. The binding efficiency of the selected phage towards CRP is two orders of magnitude higher than in the wild type. We demonstrate that the phage-based sensor is selective against other proteins. Finally, we show that layer-by-layer methods are suitable for deposition of negatively charged phages (wild or CRP-binding) with positively charged carbon nanofibers for electrode surface modification. A three-layered electrode was successfully used for molecular recognition of CRP, and the molecular interactions were studied using electrochemical, biological, and optical methods, including microscopic and spectroscopic analyses

Heat Treatment Effect on Eu 3+

Glass systems of 73TeO2-4BaO-3Bi2O3-2Eu2O3-xAg (in molar ratio where x = 0, 1, 2, and 3) compositions have been successfully synthesized. Silver nanoparticles were obtained with the employment of heat treatment (HT) procedure executed at 350°C. Glass transition temperatures of different compositions have been determined through DSC measurements. XRD results presented characteristic amorphous halo indicating lack of long range order in the samples. FTIR structural studies revealed that glass matrix is mainly composed of TeO3 and TeO4 species and is stable after different applied heat treatment times. X-ray photoelectron spectroscopy (XPS) measurements confirmed that in selected samples part of Ag ions changed oxidation state to form Ag0 species. TEM measurements revealed nanoparticles of size in the range of 20–40 nm. UV-vis absorption results demonstrated characteristic transitions of Eu3+ ions. Additionally, UV-vis spectra of samples heat-treated for 6, 12, 24, and 48 hours presented bands related to silver nanoparticles. Photoluminescence (PL) studies have been performed with excitation wavelength of λexc=395 nm. Obtained spectra exhibited peaks due to 5D0-7FJ (where J=2,3,4) and 5D1-7FJ (where J=1,2,3) transitions of Eu3+. Moreover, luminescence measurement indicated enhancement of rare earth ions emissions in several of the annealed samples. Increase of emission intensity of about 35% has been observed

Avian pathogenic Escherichia coli-targeting phages for biofilm biocontrol in the poultry industry

Avian pathogenic Escherichia coli (APEC) is a principal etiologic agent of avian colibacillosis, responsible for significant economic losses in the poultry industry due to high mortality and disease treatment with antibiotics. APEC and its ability to form biofilms on food and processing surfaces contributes to its persistence within farms. Bacteriophages are promising antibacterial agents for combating APEC. This study focused on characterization of the newly isolated phages UPWr_E1, UPWr_E2, and UPWr_E4 as well as the UPWr_E124 phage cocktail containing these three phages. Methods included efficiency of plating assay, transmission electron microscopy, and characterization of their resistance to different pH values and temperatures. Moreover, phage genomes were sequenced, annotated and analyzed, and were compared with previously sequenced E. coli phages. All three phages are virulent and devoid of undesirable genes for therapy. Phage UPWr_E1 belongs to the genus Krischvirus within the order Straboviridae and both UPWr_E2 and UPWr_E4 belong to the genus Tequatrovirus within the subfamily Tevenvirinae, sharing over 95 % nucleotide identity between them. For their use on poultry farms, UPWr_E phages and the UPWr_E124 phage cocktail were tested for their anti-biofilm activity on two E. coli strains – 158B (APEC) and the strong biofilm producer NCTC 17848 – on two abiotic surfaces: a 96-well microplate, a stainless steel surface, and one biotic surface, represented by lettuce leaves. The reduction of biofilm formed by both strains in the 96-well microplate, on the stainless steel and lettuce leaf surface for bacteriophage treatment was very efficient, reducing biofilms by ranges of 50.2–83.6, 58.2–88.4 and 53–99.4 %, respectively. Therefore, we conclude that UPWr_E phages and the UPWr_E124 phage cocktail are promising candidates for APEC biocontrol

Coupling of transcription and replication machineries in λ DNA replication initiation: evidence for direct interaction of Escherichia coli RNA polymerase and the λO protein

Transcription proceeding downstream of the λ phage replication origin was previously shown to support initial steps of the λ primosome assembly in vitro and to regulate frequency and directionality of λ DNA replication in vivo. In this report, the data are presented indicating that the RNA polymerase β subunit makes a direct contact with the λO protein, a replication initiator of λ phage. These results suggest that the role of RNA polymerase during the initiation of λ phage DNA replication may be more complex than solely influencing DNA topology. Results demonstrated in this study also show that gyrase supercoiling activity stimulates the formation of a complex between λO and RNA polymerase, suggesting that the introduction of negative supercoils by DNA gyrase, besides lowering the energy required for DNA strand separation, may play an additional role in modeling protein–protein interactions at early steps of DNA replication initiation

Investigation of Peptides for Molecular Recognition of C-Reactive Protein–Theoretical and Experimental Studies

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0–100 μg mL–1) with a detection limit (LOD = 3σ/S) of 0.34 μg mL–1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 μg mL–1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.Polish National Science Centre, 2017/26/D/ST5/0098

The Substantial Role of Cell and Nanoparticle Surface Properties in the Antibacterial Potential of Spherical Silver Nanoparticles [Response to Letter]

Marta Krychowiak-Ma&sacute;nicka,1,&ast; Weronika Paulina Wojciechowska,1,&ast; Karolina Bogaj,1 Aleksandra Bielicka-Gie&lstrok;do&nacute;,2 Ewa Czechowska,3 Magdalena Zi&aogon;bka,4 Magdalena Narajczyk,5 Anna Kawiak,6 Tomasz Mazur,7 Beata Szafranek,2 Aleksandra Kr&oacute;licka1 1University of Gdansk, Intercollegiate Faculty of Biotechnology, Laboratory of Biologically Active Compounds, Gdansk, Poland; 2University of Gdansk, Faculty of Chemistry, Gdansk, Poland; 3University of Gdansk, Intercollegiate Faculty of Biotechnology, Laboratory of Experimental and Translational Immunology, Gdansk, Poland; 4AGH University of Krakow, Faculty of Materials Science and Ceramics, Department of Ceramics and Refractories, Krakow, Poland; 5University of Gdansk, Faculty of Biology, Bioimaging Laboratory, Gdansk, Poland; 6University of Gdansk, Intercollegiate Faculty of Biotechnology, Laboratory of Plant Protection and Biotechnology, Gdansk, Poland; 7AGH University of Krakow, Academic Centre for Materials and Nanotechnology, Krakow, Poland&ast;These authors contributed equally to this workCorrespondence: Marta Krychowiak-Ma&sacute;nicka, Laboratory of Biologically Active Compounds, Intercollegiate Faculty of Biotechnology of the University of Gdansk and the Medical University of Gdansk, University of Gdansk, Abrahama 58, Gdansk, 80-307, Poland, Email [email protected]

The Substantial Role of Cell and Nanoparticle Surface Properties in the Antibacterial Potential of Spherical Silver Nanoparticles

Marta Krychowiak-Ma&sacute;nicka,1,&ast; Weronika Paulina Wojciechowska,1,&ast; Karolina Bogaj,1 Aleksandra Bielicka-Gie&lstrok;do&nacute;,2 Ewa Czechowska,3 Magdalena Zi&aogon;bka,4 Magdalena Narajczyk,5 Anna Kawiak,6 Tomasz Mazur,7 Beata Szafranek,2 Aleksandra Królicka1 1University of Gdansk, Intercollegiate Faculty of Biotechnology, Laboratory of Biologically Active Compounds, Gdansk, Poland; 2University of Gdansk, Faculty of Chemistry, Gdansk, Poland; 3University of Gdansk, Intercollegiate Faculty of Biotechnology, Laboratory of Experimental and Translational Immunology, Gdansk, Poland; 4AGH University of Krakow, Faculty of Materials Science and Ceramics, Department of Ceramics and Refractories, Krakow, Poland; 5University of Gdansk, Faculty of Biology, Bioimaging Laboratory, Gdansk, Poland; 6University of Gdansk, Intercollegiate Faculty of Biotechnology, Laboratory of Plant Protection and Biotechnology, Gdansk, Poland; 7AGH University of Krakow, Academic Centre for Materials and Nanotechnology, Krakow, Poland&ast;These authors contributed equally to this workCorrespondence: Marta Krychowiak-Ma&sacute;nicka, Laboratory of Biologically Active Compounds, Intercollegiate Faculty of Biotechnology of the University of Gdansk and the Medical University of Gdansk, University of Gdansk, Abrahama 58, Gdansk, 80-307, Poland, Email [email protected]: Although it is well known that the size, shape, and surface chemistry affect the biological potential of silver nanoparticles (AgNPs), the published studies that have considered the influence of AgNP surface on antibacterial activity have not provided conclusive results. This is the first study whose objective was to determine the significance of the surface net charge of AgNPs on their antibacterial potential, attraction to bacterial cells, and cell envelope disruption, considering differences in bacterial surface properties.Methods: We evaluated five commercial AgNP colloids with identical size and shape but different surface ligands. We thoroughly characterized their physicochemical properties, including the zeta potential, hydrodynamic diameter, and polydispersity index, and determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), along with silver absorption into bacterial cells. Moreover, we investigated structural changes in bacteria treated with AgNPs by using a crystal violet assay and electron microscopy.Results: The zeta potential of AgNPs ranged from − 47.6 to +68.5 mV, with a hydrodynamic diameter of 29– 87 nm and a polydispersity index of 0.349– 0.863. Bacterial susceptibility varied significantly (0.5 ≤ MIC ≤ 256 μg Ag/mL; 1 ≤ MBC ≤ 256 μg Ag/mL); we found the lowest susceptibility in bacteria with a cell wall or a polysaccharide capsule. The most active AgNPs (0.5 ≤ MIC ≤ 32 μg Ag/mL; 2 ≤ MBC ≤ 64 μg Ag/mL) had a moderate surface charge (− 21.5 and +14.9 mV). The antibacterial potential was unrelated to ion dissolution or cell envelope disruption, and bacterial cells absorbed less of the most active AgNPs (1.75– 7.65%).Conclusion: Contrary to previous reports, we found that a moderate surface charge is crucial for the antibacterial activity of AgNPs, and that a significant attraction of the nanoparticle to the cell surface reduces the antibacterial potential of AgNPs. These findings challenge the existing views on AgNP antibacterial mechanisms and interactions with bacterial cells. Keywords: human pathogen, ESKAPE, Klebsiella pneumoniae, cell envelope, ICP-OES, cell–nanoparticle interactio