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1.
Materials (Basel) ; 14(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199341

RESUMO

The aim of the work was to study the interaction between boron-rich boron carbide nanoparticles and selected tumor and immune phagocytic cells. Experiments were performed to investigate the feasibility of the application of boron carbide nanoparticles as a boron carrier in boron neutron capture therapy. Boron carbide powder was prepared by the direct reaction between boron and soot using the transport of reagents through the gas phase. The powder was ground, and a population of nanoparticles with an average particle size about 80 nm was selected by centrifugation. The aqueous suspension of the nanoparticles was functionalized with human immunoglobulins or FITC-labeled human immunoglobulins and was then added to the MC38 murine colon carcinoma and to the RAW 264.7 cell line of mouse macrophages. Flow cytometry analysis was used to determine interactions between the functionalized boron carbide nanoparticles and respective cells. It was shown that B4C-IgG nanoconjugates may bind to phagocytic cells to be internalized by them, at least partially, whereas such nanoconjugates can only slightly interact with molecules on the cancer cells' surface.

2.
Molecules ; 25(19)2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987777

RESUMO

For the first time, we are introducing TTPBgp12 and TFPgp17 as new members of the tail tubular proteins B (TTPB) and tail fiber proteins (TFP) family, respectively. These proteins originate from Yersinia enterocolitica phage φYeO3-12. It was originally thought that these were structural proteins. However, our results show that they also inhibit bacterial growth and biofilm formation. According to the bioinformatic analysis, TTPBgp12 is functionally and structurally similar to the TTP of Enterobacteria phage T7 and adopts a ß-structure. TFPgp17 contains an intramolecular chaperone domain at its C-terminal end. The N-terminus of TFPgp17 is similar to other representatives of the TFP family. Interestingly, the predicted 3D structure of TFPgp17 is similar to other bacterial S-layer proteins. Based on the thermal unfolding experiment, TTPBgp12 seems to be a two-domain protein that aggregates in the presence of sugars such as maltose and N-acetylglucosamine (GlcNAc). These sugars cause two unfolding events to transition into one global event. TFPgp17 is a one-domain protein. Maltose and GlcNAc decrease the aggregation temperature of TFPgp17, while the presence of N-acetylgalactosamine (GalNAc) increases the temperature of its aggregation. The thermal unfolding analysis of the concentration gradient of TTPBgp12 and TFPgp17 indicates that with decreasing concentrations, both proteins increase in stability. However, a decrease in the protein concentration also causes an increase in its aggregation, for both TTPBgp12 and TFPgp17.


Assuntos
Caudovirales , Proteínas Estruturais Virais , Yersinia enterocolitica/virologia , Caudovirales/química , Caudovirales/genética , Caudovirales/metabolismo , Domínios Proteicos , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
3.
Front Microbiol ; 11: 1356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636826

RESUMO

We report here the complete genome sequence and characterization of Yersinia bacteriophage vB_YenP_ϕ80-18. ϕ80-18 was isolated in 1991 using a Y. enterocolitica serotype O:8 strain 8081 as a host from a sewage sample in Turku, Finland, and based on its morphological and genomic features is classified as a podovirus. The genome is 42 kb in size and has 325 bp direct terminal repeats characteristic for podoviruses. The genome contains 57 predicted genes, all encoded in the forward strand, of which 29 showed no similarity to any known genes. Phage particle proteome analysis identified altogether 24 phage particle-associated proteins (PPAPs) including those identified as structural proteins such as major capsid, scaffolding and tail component proteins. In addition, also the DNA helicase, DNA ligase, DNA polymerase, 5'-exonuclease, and the lytic glycosylase proteins were identified as PPAPs, suggesting that they might be injected together with the phage genome into the host cell to facilitate the take-over of the host metabolism. The phage-encoded RNA-polymerase and DNA-primase were not among the PPAPs. Promoter search predicted the presence of four phage and eleven host RNA polymerase -specific promoters in the genome, suggesting that early transcription of the phage is host RNA-polymerase dependent and that the phage RNA polymerase takes over later. The phage tolerates pH values between 2 and 12, and is stable at 50°C but is inactivated at 60°C. It grows slowly with a 50 min latent period and has apparently a low burst size. Electron microscopy revealed that the phage has a head diameter of about 60 nm, and a short tail of 20 nm. Whole-genome phylogenetic analysis confirmed that ϕ80-18 belongs to the Autographivirinae subfamily of the Podoviridae family, that it is 93.2% identical to Yersinia phage fHe-Yen3-01. Host range analysis showed that ϕ80-18 can infect in addition to Y. enterocolitica serotype O:8 strains also strains of serotypes O:4, O:4,32, O:20 and O:21, the latter ones representing similar to Y. enterocolitica serotype O:8, the American pathogenic biotype 1B strains. In conclusion, the phage ϕ80-18 is a promising candidate for the biocontrol of the American biotype 1B Y. enterocolitica.

4.
Front Immunol ; 10: 211, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30814999

RESUMO

Recent developments demonstrate that tumor-derived extracellular vesicles (EVs) could become a highly effective tool for delivery of antitumor factors. The main objective of the study was to determine whether EVs secreted by MC38 colon carcinoma cells genetically engineered for overproduction of interleukin (IL-)12 and/or shRNA targeting TGF-ß1 are effectively loaded with these molecules and whether the obtained EVs could be an efficient tool for antitumor therapy. Fractions of EVs released by genetically modified MC38 cells [both modified tumor-derived exosomes (mTEx) and modified microvesicles (mTMv)] and those released by unmodified, wild-type MC38 cells were characterized in terms of loading efficacy, using real-time PCR and ELISA, as well as their antitumor potential. In order to examine the therapeutic potential of mTEx, they were applied in the form of sole treatment as well as in combination with dendritic cell (DC)-based vaccines stimulated with mTMv in the therapy of mice with subcutaneously growing MC38 tumors. The results demonstrated that genetic modification of wild-type MC38 tumor cells is an effective method of loading the molecules of interest into extracellular vesicles secreted by the cells (both TEx and TMv). The results also showed that mTEx secreted by cells engineered for overproduction of IL-12 and/or shRNA for TGF-ß1 are able to induce tumor growth inhibition as opposed to TEx from unmodified MC38 cells. Additionally, antitumor therapy composed of mTEx (especially those deprived of TGF-ß1) and DC-based vaccines allowed for regeneration of antitumor immunity and induction of the systemic Th1 response responsible for the sustained effect of the therapy. In conclusion, tumor-derived exosomes loaded with IL-12 and/or deprived of TGF-ß1 could become an efficient adjuvant supporting induction of a specific antitumor response in both immuno- and chemotherapeutic schemes of treatment.


Assuntos
Vesículas Extracelulares/metabolismo , Expressão Gênica , Interleucina-12/genética , Neoplasias/genética , Neoplasias/metabolismo , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta1/genética , Animais , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Vesículas Extracelulares/ultraestrutura , Feminino , Inativação Gênica , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Interferência de RNA , Fator de Crescimento Transformador beta1/metabolismo
5.
Methods Mol Biol ; 1600: 107-112, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28478561

RESUMO

Lipopolysaccharide (LPS, endotoxin, pyrogen) which is a component of the outer membrane of most Gram-negative bacteria is a troubling contaminant of crude bacteriophage suspension. Therefore, its removal is important for bacteriophage applications especially in preparations dedicated for use in therapy with bacterial infections treatment. The method presented here is used for extractive removal of endotoxins from bacteriophage preparations with a water immiscible solvent such as 1-octanol. During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin in the aqueous bacteriophage rich fraction are determine by Limulus Amebocyte Lysate or EndoLISA assay and are extremely low.


Assuntos
Escherichia coli/metabolismo , Lipopolissacarídeos/química , Bacteriófagos/metabolismo , Lipopolissacarídeos/isolamento & purificação
6.
J Nanobiotechnology ; 15(1): 32, 2017 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-28438164

RESUMO

BACKGROUND: Bacteriophage survives in at least two extremes of ionic environments: bacterial host (high ionic-cytosol) and that of soil (low ionic-environmental water). The impact of ionic composition in the micro- and macro-environments has not so far been addressed in phage biology. RESULTS: Here, we discovered a novel mechanism of aggregation/disaggregation transitions by phage virions. When normal sodium levels in phage media (150 mM) were lowered to 10 mM, advanced imaging by scanning electron microscopy, atomic force microscopy and dynamic light scattering all revealed formation of viral packages, each containing 20-100 virions. When ionic strength was returned from low to high, the aggregated state of phage reversed to a dispersed state, and the change in ionic strength did not substantially affect infectivity of the phage. By providing the direct evidence, that lowering of the sodium ion below the threshold of 20 mM causes rapid aggregation of phage while returning Na+ concentration to the values above this threshold causes dispersion of phage, we identified a biophysical mechanism of phage aggregation. CONCLUSIONS: Our results implicate operation of group behavior in phage and suggest a new kind of quorum sensing among its virions that is mediated by ions. Loss of ionic strength may act as a trigger in an evolutionary mechanism to improve the survival of bacteriophage by stimulating aggregation of phage when outside a bacterial host. Reversal of phage aggregation is also a promising breakthrough in biotechnological applications, since we demonstrated here the ability to retain viable virion aggregates on standard micro-filters.


Assuntos
Bacteriófago T4/fisiologia , Sódio/metabolismo , Bacteriófago T4/ultraestrutura , Cátions Monovalentes/metabolismo , Concentração Osmolar , Percepção de Quorum
7.
J Gen Appl Microbiol ; 61(3): 75-81, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26227910

RESUMO

The aim of this study was to develop a minimal medium for the cultivation of Escherichia coli B, which could be especially suitable for the industrial propagation of bacteriophage T4. The new defined, minimal SM-1 culture medium, contains free amino acids as the only nitrogen source and enables the bacteria generation time to be prolonged and satisfactory phage titers to be achieved. The presence of organic ingredients, such as meat extracts, yeast hydrolysates, enzymatic protein hydrolysates, in a culture medium may cause problems in the case of bacteria or phage cultures for therapeutic purposes. In the present study, we introduce a new medium, together with some procedures and applications for its usage. We also present new kinetics of E. coli B growth. Some traits such as the lack of high molecular proteins, a bacterial growth comparable to that in a rich medium, and the cost effectiveness of the medium, makes it highly competitive with currently used microbiological media. The surprisingly high titers of bacteriophage T4 obtained in our experiments suggest that SM-1 medium has the potential to find a broad application in medicine, especially in infectious disease therapy, pharmacy and biotechnology.


Assuntos
Bacteriófago T4/crescimento & desenvolvimento , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Aminoácidos/análise , Aminoácidos/metabolismo , Bacteriófago T4/isolamento & purificação , Reatores Biológicos , Meios de Cultura/química , Meios de Cultura/economia , Escherichia coli/metabolismo , Carga Viral
8.
PLoS One ; 10(3): e0122672, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25811193

RESUMO

Lipopolysaccharide (LPS, endotoxin, pyrogen) constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol). During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU) in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 10(3) and 10(5) EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 10(3)-10(5) EU/10(9) PFU (plaque forming units) down to an average of 2.8 EU/10(9) PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli) and F8 (P. aeruginosa).


Assuntos
Bacteriófagos/isolamento & purificação , Endotoxinas , Extração Líquido-Líquido , Compostos Orgânicos/química , Solventes/química , Bacteriófagos/crescimento & desenvolvimento , Escherichia coli/metabolismo , Escherichia coli/virologia , Ensaio de Placa Viral
9.
Postepy Hig Med Dosw (Online) ; 68: 1392-6, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25531702

RESUMO

Determination of the number of cultured bacteria is essential for scientific and industrial practice. A spread plate technique is the most common and accurate method for counting of microorganisms. However, time consuming incubation does not allow for a quick estimation of the number of bacteria in a growing culture. In the present study, the results of photometric measurements: direct optical density method (OD at 585 nm), UV absorbance at 260 and/or 280 nm of separated and lysed bacteria by sodium hydroxide and surfactant with the spread plate technique were compared. The linear regression model for bacterial strains Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was used to compare these three methods. The UV measurement method enabled determination of the number of bacteria with similar precision. The procedure for solubilized bacteria UV measurement is robust, and is not influenced by dispersions in the original culture medium.


Assuntos
Técnicas Bacteriológicas , Contagem de Colônia Microbiana/métodos , Escherichia coli/crescimento & desenvolvimento , Fotometria/métodos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Raios Ultravioleta
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