Petr Michalek

Effect of Ampicillin, Streptomycin, Penicillin and Tetracycline on Metal Resistant and Non-Resistant Staphylococcus aureus

There is an arising and concerning issue in the field of bacterial resistance, which is confirmed by the number of deaths associated with drug-resistant bacterial infections. The aim of this study was to compare the effects of antibiotics on Staphylococcus aureus non-resistant strain and strains resistant to cadmium or lead ions. Metal resistant strains were created by the gradual addition of 2 mM solution of metal ions (cadmium or lead) to the S. aureus culture. An increasing antimicrobial effect of ampicillin, streptomycin, penicillin and tetracycline (0, 10, 25, 50, 75, 150, 225 and 300 µM) on the resistant strains was observed using a method of growth curves. A significant growth inhibition (compared to control) of cadmium resistant cells was observed in the presence of all the four different antibiotics. On the other hand, the addition of streptomycin and ampicillin did not inhibit the growth of lead resistant strain. Other antibiotics were still toxic to the bacterial cells. Significant differences in the morphology of cell walls were indicated by changes in the cell shape. Our data show that the presence of metal ions in the urban environment may contribute to the development of bacterial strain resistance to other substances including antibiotics, which would have an impact on public health.

Synthesis of carbon quantum dots for DNA labeling and its electrochemical, fluorescent and electrophoretic characterization

Nanoparticles as a progressively developing branch offer a tool for studying the interaction of carbon quantum dots (CQDs) with DNA. In this study, fluorescent CQDs were synthesized using citric acid covered with polyethylene glycol (PEG) as the source of carbon precursors. Furthermore, interactions between CQDs and DNA (double-stranded DNA and single-stranded DNA) were investigated by spectral methods, gel electrophoresis, and electrochemical analysis. Primarily, the fluorescent behavior of CQDs in the presence of DNA was monitored and major differences in the interaction of CQDs with tested single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) were observed at different amounts of CQDs (µg mL−1: 25, 50, 100, 250, 500). It was found that the interaction of ssDNA with CQDs had no significant influence on the CQDs fluorescence intensity measured at the excitation wavelengths of 280 nm, 350 nm, and 400 nm. However, in the presence of dsDNA, the fluorescence intensity of CQDs was significantly increased. Our results provide basic understanding of the interaction between CQDs and DNA. Such fabricated CQDs-DNA might be of great benefit for the emerging nanomaterials based biosensing methods.

Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to β-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)—ampicillin, oxacillin and penicillin—and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99% ± 7% for S. aureus and up to 94% ± 4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79% ± 5% and MRSA up to 16% ± 2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs.

Sarcosine Up-Regulates Expression of Genes Involved in Cell Cycle Progression of Metastatic Models of Prostate Cancer

The effects of sarcosine on the processes driving prostate cancer (PCa) development remain still unclear. Herein, we show that a supplementation of metastatic PCa cells (androgen independent PC-3 and androgen dependent LNCaP) with sarcosine stimulates cells proliferation in vitro. Similar stimulatory effects were observed also in PCa murine xenografts, in which sarcosine treatment induced a tumor growth and significantly reduced weight of treated mice (p < 0.05). Determination of sarcosine metabolism-related amino acids and enzymes within tumor mass revealed significantly increased glycine, serine and sarcosine concentrations after treatment accompanied with the increased amount of sarcosine dehydrogenase. In both tumor types, dimethylglycine and glycine-N-methyltransferase were affected slightly, only. To identify the effects of sarcosine treatment on the expression of genes involved in any aspect of cancer development, we further investigated expression profiles of excised tumors using cDNA electrochemical microarray followed by validation using the semi-quantitative PCR. We found 25 differentially expressed genes in PC-3, 32 in LNCaP tumors and 18 overlapping genes. Bioinformatical processing revealed strong sarcosine-related induction of genes involved particularly in a cell cycle progression. Our exploratory study demonstrates that sarcosine stimulates PCa metastatic cells irrespectively of androgen dependence. Overall, the obtained data provides valuable information towards understanding the role of sarcosine in PCa progression and adds another piece of puzzle into a picture of sarcosine oncometabolic potential.

Nanotechnology-based analytical approaches for detection of viruses

The risks posed by viral diseases have been recognised as critical threats to mankind. On the other hand, using nanotechnology approaches, viruses are used as tools and building blocks for electronics, chemistry, and biomedical science. Virus-based nanotechnology is benefiting from the natural circulatory and targeting properties of viruses in order to design drugs and vaccines that specifically target tissues of interest in vivo. This review details the exploitation of nanomaterials for the detection of viruses in general, with a focus on four of the most common and damaging viruses, namely influenza, hepatitis, HIV, and dengue. As the principles behind the techniques used in the area of detection and analysis are interconnected with the principles used in some other areas, the review also outlines the use of viral nanoparticles as nanocarriers for targeted delivery as well as their utilization in nanomaterial engineering.

Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection.

The majority of carcinomas that were developed due to the infection with human papillomavirus (HPV) are caused by high-risk HPV types, HPV16 and HPV18. These HPV types contain the E6 and E7 oncogenes, so the fast detection of these oncogenes is an important point to avoid the development of cancer. Many different HPV tests are available to detect the presence of HPV in biological samples. The aim of this study was to design a fast and low cost method for HPV identification employing magnetic isolation, polymerase chain reaction (PCR) and electrochemical detection. These assays were developed to detect the interactions between E6-HPV16 oncogene and magnetizable particles (MPs) using commercial Dynabeads M-280 Streptavidin particles and laboratory-synthesized “homemade” particles called MANs (MAN-37, MAN-127 and MAN-164). The yields of PCR amplification of E6-HPV16 oncogene bound on the particles and after the elution from the particles were compared. A highest yield of E6-HPV16 DNA isolation was obtained with both MPs particles commercial M-280 Streptavidin and MAN-37 due to reducing of the interferents compared with the standard PCR method. A biosensor employing the isolation of E6-HPV16 oncogene with MPs particles followed by its electrochemical detection can be a very effective technique for HPV identification, providing simple, sensitive and cost-effective analysis.

Exposure to 17β-Oestradiol Induces Oxidative Stress in the Non-Oestrogen Receptor Invertebrate Species Eisenia fetida

Background The environmental impacts of various substances on all levels of organisms are under investigation. Among these substances, endocrine-disrupting compounds (EDCs) present a threat, although the environmental significance of these compounds remains largely unknown. To shed some light on this field, we assessed the effects of 17β-oestradiol on the growth, reproduction and formation of free radicals in Eisenia fetida. Methodology/Principal Findings Although the observed effects on growth and survival were relatively weak, a strong impact on reproduction was observed (50.70% inhibition in 100 μg/kg of E2). We further demonstrated that the exposure of the earthworm Eisenia fetida to a contaminant of emerging concern, 17β-oestradiol (E2), significantly affected the molecules involved in antioxidant defence. Exposure to E2 results in the production of reactive oxygen species (ROS) and the stimulation of antioxidant systems (metallothionein and reduced oxidized glutathione ratio) but not phytochelatins at both the mRNA and translated protein levels. Matrix-assisted laser desorption/ionization (MALDI)-imaging revealed the subcuticular bioaccumulation of oestradiol-3,4-quinone, altering the levels of local antioxidants in a time-dependent manner. Conclusions/Significance The present study illustrates that although most invertebrates do not possess oestrogen receptors, these organisms can be affected by oestrogen hormones, likely reflecting free diffusion into the cellular microenvironment with subsequent degradation to molecules that undergo redox cycling, producing ROS, thereby increasing environmental contamination that also perilously affects keystone animals, forming lower trophic levels.

Study of Linkage between Glutathione Pathway and the Antibiotic Resistance of Escherichia coli from Patients' Swabs

In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase—GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (Km 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of “more resistant” E. coli exhibited differences in Km between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group.

Bioconjugation of peptides using advanced nanomaterials to examine their interactions in 3D printed flow‐through device

Peptide–peptide interactions are crucial in the living cell as they lead to the formation of the numerous types of complexes. In this study, synthetic peptides containing 11 of cysteines (α‐domain of metallothionein (MT)) and sialic acid binding region (130‐loop of hemagglutinin (HA)) were employed. The aim of the experiment was studying the interactions between MT and HA‐derived peptides. For this purpose, fragments were tagged with cysteines at C‐terminal part to serve as ligand sites for PbS and CuS quantum dots (QDs), and therefore these conjugates can be traced and quantified during wide spectrum of methods. As a platform for interaction, γ‐Fe2O3 paramagnetic particles modified with tetraethyl orthosilicate and (3‐aminopropyl)triethoxysilane (hydrodynamic diameter 30–40 nm) were utilized and MT/HA interactions were examined using multi‐instrumental approach including electrochemistry, electrophoretic methods, and MALDI‐TOF/TOF mass spectrometry. It was found that peptides enter mutual creation of complexes, which are based on some of nonbonded interactions. The higher willingness to interact was observed in MT‐derived peptides toward immobilized HA. Finally, we designed and manufactured flow‐through electrochemical 3D printed device (reservoir volume 150 μL) and utilized it for automated analysis of the HA/MT metal labels. Under the optimal conditions, (deposition time and flow rate 80 s and 1.6 mL/min for CuS and 120 s and 1.6 mL/min PbS, respectively), the results of peptide‐conjugated QDs were comparable with atomic absorption spectrometry.

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes

Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non‐malignant and non‐metastatic cells.