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Background: The emergence of antibiotic resistance in pathogenic bacteria has become a global threat, encouraging the adoption of efficient and effective alternatives to conventional antibiotics and promoting their use as replacements. Titanium dioxide nanoparticles (TiO 2 NPs) have been reported to exhibit antibacterial properties. In this study, we synthesized and characterized TiO 2 NPs in anatase and rutile forms with surface modification by geraniol (GER)., Results: The crystallinity and morphology of modified TiO 2 NPs were analyzed by UV/Vis spectrophotometry, X-ray powder diffraction (XRD), and scanning electron microscopy (SEM) with elemental mapping (EDS). The antimicrobial activity of TiO 2 NPs with geraniol was assessed against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli. The minimum inhibitory concentration (MIC) values of modified NPs ranged from 0.25 to 1.0 mg/ml against all bacterial strains, and the live dead assay and fractional inhibitory concentration (FIC) supported the antibacterial properties of TiO 2 NPs with GER. Moreover, TiO 2 NPs with GER also showed a significant decrease in the biofilm thickness of MRSA., Conclusions: Our results suggest that TiO 2 NPs with GER offer a promising alternative to antibiotics, particularly for controlling antibiotic-resistant strains. The surface modification of TiO 2 NPs by geraniol resulted in enhanced antibacterial properties against multiple bacterial strains, including antibiotic-resistant MRSA. The potential applications of modified TiO 2 NPs in the biomedical and environmental fields warrant further investigation., (© 2023. The Author(s).)

For decades, the antimicrobial applications of nanoparticles (NPs) have attracted the attention of scientists as a strategy for controlling the ever-increasing threat of multidrug-resistant microorganisms. The photo-induced antimicrobial properties of titanium dioxide (TiO 2 ) NPs by ultraviolet (UV) light are well known. This review elaborates on the modern methods and antimicrobial mechanisms of TiO 2 NPs and their modifications to better understand and utilize their potential in various biomedical applications. Additional compounds can be grafted onto TiO 2 nanomaterial, leading to hybrid metallic or non-metallic materials. To improve the antimicrobial properties, many approaches involving TiO 2 have been tested. The results of selected studies from the past few years covering the most recent trends in this field are discussed in this review. There is extensive evidence to show that TiO 2 NPs can exhibit certain antimicrobial features with disputable roles of UV light. Hence, they are effective in treating bacterial infections, although the majority of these conclusions came from in vitro studies and in the presence of some additional nanomaterials. The methods of evaluation varied depending on the nature of the research while researchers incorporated different techniques, including determining the minimum inhibitory concentration, cell count, and using disk and well diffusion methods, with a noticeable indication that cell count was the most and dominant criterion used to evaluate the antimicrobial activity. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease., (© 2022 Wiley Periodicals LLC.)

The here presented work is focused on the development of a method for detection of microbial contamination of food based on uracil-selective synthetic receptors. Because uracil may serve as an indicator of bacterial contamination, its selective and on-site detection may prevent spreading of foodborne diseases. The synthetic receptors were created by molecular imprinting. Molecularly imprinted polymers for selective uracil isolation were prepared by a non-covalent imprinting method using dopamine as a functional monomer. Detection of isolated uracil was performed by capillary electrophoresis with absorption detection (λ - 260 nm). The conditions of preparation of molecularly imprinted polymers, their binding properties, adsorption kinetics and selectivity were investigated in detail. Furthermore, the prepared polymer materials were used for selective isolation and detection of uracil from complex samples as tomato products by miniaturized electrophoretic system suggesting the potential of in situ analysis of real samples., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Enhanced antibacterial properties of nanomaterials such as TiO 2 nanotubes (TNTs) and silver nanoparticles (AgNPs) have attracted much attention in biomedicine and industry. The antibacterial properties of nanoparticles depend, among others, on the functionalization layer of the nanoparticles. However, the more complex information about the influence of different functionalization layers on antibacterial properties of nanoparticle decorated surfaces is still missing. Here we show the array of ∼50 nm diameter TNTs decorated with ∼50 nm AgNPs having different functionalization layers such as polyvinylpyrrolidone, branched polyethyleneimine, citrate, lipoic acid, and polyethylene glycol. To assess the antibacterial properties, the viability of Gram-positive ( Staphylococcus aureus ) and Gram-negative bacteria ( Escherichia coli and Pseudomonas aeruginosa ) has been assessed. Our results showed that the functional layer of nanoparticles plays an important role in antibacterial properties and the synergistic effect such nanoparticles and TiO 2 nanotubes have had different effects on adhesion and viability of G - and G + bacteria. These findings could help researchers to optimally design any surfaces to be used as an antibacterial including the implantable titanium biomaterials., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In the present study, the thermal decomposition of citric acid in the presence of biogenic amine was used to synthesize four different functionalized carbon quantum dots (CQDs), namely, histamine-(HCQDs), putrescine-(PCQDs), cadaverine-(CCQDs) and spermine-(SCQDs). The thermal decomposition of the precursors resulted in a decrease in stability and the formation of surface amides via a cross-linking process between the carboxyl and amine groups. The deposition of biogenic amines was confirmed by a structural characterization of the synthesized CQDs. The resulting CQDs, with a net zero charge, exhibited excellent stability in environments with different pH values. Through a set of different cytotoxicity tests, the absence of gene mutations, apoptosis, necrosis or disruption in cell membranes revealed the high biocompatibility of the CQDs. The antimicrobial activity of the synthesized CQDs was investigated against different bacterial species (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia). We determined the growth kinetics, production of reactive oxygen species (ROS), cell viability and changes in membrane integrity by scanning electron microscopy (SEM). The minimal inhibitory concentrations (MICs) for S. aureus ranged from 3.4 to 6.9 µg/mL. Regarding E.coli and K. pneumonia, all CQD formulations reduced growth, and the MICs were determined for CCQDs and HCQDs (6.9-19.4 µg/mL). The antibacterial activity mechanism was attributed to the oxidative stress generated after CQD treatment, which resulted in the destabilization of the bacterial membrane. The bacterial permeability to propidium iodide indicated a change in membrane integrity, and the effect of CQDs on the morphology of the bacterial cells was evidenced by SEM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

With a growing health threat of bacterial resistance to antibiotics, the nanomaterials have been extensively studied as an alternative. It is assumed that antimicrobial nanomaterials can affect bacteria by several mechanisms simultaneously and thereby overcome antibiotic resistance. Another promising potential use is employing nanomaterials as nanocarriers for antibiotics in order to overcome bacterial defense mechanisms. The passive targeting of nanomaterials is the often used strategy for bacterial treatment, including intracellular infections of macrophages. Furthermore, the specific targeting enhances the efficacy of antimicrobials and reduces side effects. This review aims to discuss advantages, disadvantages, and challenges of nanomaterials in the context of the targeting strategies for antimicrobials as advanced tools for treatments of bacterial infections. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease., (© 2020 Wiley Periodicals, Inc.)

In this work, a novel method was developed, for isolation of S. aureus from complex (food) samples using molecular imprinting.  Dopamine was used as a functional monomer and fluorescence microscopy was used for detection. Conditions for preparation of molecularly imprinted polymers (MIPs), adsorption performance, adsorption kinetic, and selectivity of the polymeric layers were investigated. The various procedures were combined in a single extraction process, with the imprinted layer on the surface of the magnetic particles (magnetic MIPs). Subsequently, MIPs were used for extraction of S. aureus from milk and rice. Moreover, raw milk from cows with mastitis was tested successfully. Using this novel MIP-based method, it was possible to detect bacteria in milk at 1 × 10 3 CFU·ml -1 , which corresponds to the limit set in European Union legislation for microbial control of food., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The high doses of zinc oxide (ZnO) administered orally to piglets for the prevention of diarrhea and increase of growth rate can contaminate pig farms and the surrounding environment. Therefore, there is a need to find a replacement of high doses of dietary ZnO with an equally effective alternative. In the present study, the effect of two formulations of zinc phosphate-based nanoparticles (ZnA and ZnC NPs) on growth performance, intestinal microbiota, antioxidant status, and intestinal and liver morphology was evaluated. A total of 100 weaned piglets were randomly divided into 10 equal groups with the base diet (control) or the base diet supplemented with ZnA, ZnC, or ZnO at concentrations 500, 1000, and 2000 mg Zn per kilogram of diet. Supplements were given to animals for 10 days. Fecal samples were collected on day 0, 5, 10 and 20. At the end of the treatment (day 10), three piglets from each group were sacrificed and analyzed., Results: Comparing to that of control, the significantly higher piglet weight gain was observed in all piglet groups fed with ZnA ( P  < 0.05). Differences in the total aerobic bacteria and coliform counts in piglet feces after NPs supplementation compared to that of control and ZnO groups were also found ( P  < 0.05). The majority of aerobic culturable bacteria from the feces represented Escherichia (28.57-47.62%), Enterococcus (3.85-35.71%), and Streptococcus (3.70-42.31%) spp. A total of 542 Escherichia coli isolates were screened for the virulence genes STa , STb , Stx2 , F4 , and F18 . The substantial occurrence of E. coli virulence factors was found on day 5, mainly in fimbrillary antigen and thermostable toxins, except for piglets fed by ZnC. Zn treatment decreased Zn blood levels in piglets fed with ZnO and ZnA (500 mg/kg) and increased in ZnC (2000 mg/kg) compared to that of control ( P  < 0.05). The antioxidant status of piglets was affected only by ZnA. While some changes in the liver and the intestinal morphology of piglets with NPs were observed, none were serious as reflected by the normal health status and increased weigh gain performance., Conclusions: Our results indicate that ZnA NPs have a positive effect on the piglet growth performance even at the lowest concentration. The prevalence of E. coli virulence factors was lowest in pigs supplemented with ZnC. Zinc phosphate-based nanoparticles may be an effective alternative to ZnO., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Short non-coding RNAs, specifically microRNAs (miRNAs), are of a great interest due to their presumed function in genome regulation. Moreover, miRNAs are currently perceived as potential biomarkers for numerous diseases; a variety of detection methods and sensing systems have therefore been studied. We present a magnetic-bead-based assay for specific miRNA isolation coupled with sensitive electrophoretic analysis with fluorescence detection. The magnetic separation step involves creating a duplex with targeted miR-141, which is subsequently cleaved from the magnetic bead surface with a specific endonuclease. The duplex is then determined using capillary electrophoresis with laser-induced fluorescence detection in the presence of the fluorescent dye PicoGreen for quantitating double-stranded DNA. The benefits of using microcolumn separation technique coupled with sensitive detection over traditionally used determination by fluorescence spectrometry include the fact that there is no need for a specific pre-labeled fluorescent probe. This significantly simplifies the method and reduces the costs. Cross-reactivity with mismatched oligonucleotides (3 and 5 mismatched bases) and different miRNAs (miR-124 and miR-150) was tested, demonstrating the specificity of the developed method for miRNA-141. This magnetic extraction method was demonstrated for the direct isolation and determination of miR-141 at different concentration levels from urine samples and the achieved nanomolar detection limit.

Nanocomposite films that were based on furcellaran (FUR) and nanofillers (carbon quantum dots (CQDs), maghemite nanoparticles (MAN), and graphene oxide (GO)) were obtained by the casting method. The microstructure, as well as the structural, physical, mechanical, antimicrobial, and antioxidant properties of the films was investigated. The incorporation of MAN and GO remarkably increased the tensile strength of furcellaran films. However, the water content, solubility, and elongation at break were significantly reduced by the addition of the nanofillers. Moreover, furcellaran films containing the nanofillers exhibited potent free radical scavenging ability. FUR films with CQDs showed an inhibitory effect on the growth of Staphylococcus aureus and Escherichia coli . The nanocomposite films were used to cover transparent glass containers to study the potential UV-blocking properties in an oil oxidation test and compare with tinted glass. The samples were irradiated for 30 min. with UV-B and then analyzed for oxidation markers (peroxide value, free fatty acids, malondialdehyde content, and degradation of carotenoids). The test showed that covering the transparent glass with MAN films was as effective in inhibiting the oxidation as the use of tinted glass, while the GO and CQDs films did not inhibit oxidation. It can be concluded that the active nanocomposite films can be used as a desirable material for food packaging., Competing Interests: The authors declare no conflict of interest.

Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.

The ever increasing scenario of bacterial resistance against commonly available antibiotics is becoming a global threat of major concern, which necessitates the development of new strategies to overcome this hurdle. Conjugation of nanoparticles (NPs) with antimicrobial moieties, such as antibiotics, peptides or different biomolecules, has been one of the successful techniques in targeting antibiotic resistance. This review mainly focusses on the possible nanoparticle-drug conjugates with their activity against pathogenic bacterial infections. Nanoparticles play an array of roles, e.g. as a carrier, synergistically acting agent and as theranostic agent, henceforth facilitates the efficacy of therapy. Moreover, this review elaborates the studies with reported nanoparticles-drug conjugates that include their possible synthesis methodologies and applications. In most of the cases, the nanoparticles were found to increase the permeability of bacterial cell membrane, which enables higher uptake of antibiotics inside the bacterial cells which in return showed better effects. Even the conjugates were found to efficiently kill the antibiotic-resistant strains. Since several limitations are exerted by the biological systems, there is an urge for the advancement of nanoparticle-drug conjugates for better proficiency., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Environmental pollutants, including antibiotics (ATBs), have become an increasingly common health hazard in the last several decades. Overdose and abuse of ATBs led to the emergence of antibiotic-resistant genes (ARGs), which represent a serious health threat. Moreover, water bodies and reservoirs are places where a wide range of bacterial species with ARGs originate, owing to the strong selective pressure from presence of ATB residues. In this regard, graphene oxide (GO) has been utilised in several fields including remediation of the environment. In this review, we present a brief overview of resistant genes of frequently used ATBs, their occurrence in the environment and their behaviour. Further, we discussed the factors influencing the binding of nucleic acids and the response of ARGs to GO, including the presence of salts in the water environment or water pH, because of intrinsic properties of GO of not only binding to nucleic acids but also catalysing their decomposition. This would be helpful in designing new types of water treatment facilities.

Essential oils (EOs) are now a hot topic in finding modern substitutes for antibiotics. Many studies have shown positive results and confirmed their high antibacterial activity both in vitro and in vivo. Deservedly, there is an attempt to use EOs as a substitute for antibiotics, which are currently limited by legislation in animal breeding. Given the potential of EOs, studies on their fate in the body need to be summarized. The content of EO ' s active substances varies depending on growing conditions and consequently on processing and storage. Their content also changes dynamically during the passage through the gastrointestinal tract and their effective concentration can be noticeably diluted at their place of action (small intestine and colon). Based on the solubility of the individual EO's active substances, they are eliminated from the body at different rates. Despite a strong antimicrobial effect, some oils can be toxic to the body and cause damage to the liver, kidneys, or gastrointestinal tissues. Reproductive toxicity has been reported for Origanum vulgare and Mentha arvensis . Several publications also address the effect on the genome. It has been observed that EOs can show both genoprotective effects ( Syzygium aromaticum ) and genotoxicity, as is the case of Cinnamomum camphor . This review shows that although oils are mainly studied as promising antimicrobials, it is also important to assess animal safety., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Background: Development of new nanomaterials that inhibit or kill bacteria is an important and timely research topic. For example, financial losses due to infectious diseases, such as diarrhea, are a major concern in livestock productions around the world. Antimicrobial nanoparticles (NPs) represent a promising alternative to antibiotics and may lower antibiotic use and consequently spread of antibiotic resistance traits among bacteria, including pathogens., Results: Four formulations of zinc nanoparticles (ZnA, ZnB, ZnC, and ZnD) based on phosphates with spherical (ZnA, ZnB) or irregular (ZnC, ZnD) morphology were prepared. The highest in vitro inhibitory effect of our NPs was observed against Staphylococcus aureus (inhibitory concentration values, IC 50 , ranged from 0.5 to 1.6 mmol/L) , followed by Escherichia coli (IC 50 0.8-1.5 mmol/L). In contrast, methicillin resistant S. aureus (IC 50 1.2-4.7 mmol/L) was least affected and this was similar to inhibitory patterns of commercial ZnO-based NPs and ZnO. After the successful in vitro testing, the in vivo study with rats based on dietary supplementation with zinc NPs was conducted. Four groups of rats were treated by 2,000 mg Zn/kg diet of ZnA, ZnB, ZnC, and ZnD, for comparison two groups were supplemented by 2,000 mg Zn/kg diet of ZnO-N and ZnO, and one group (control) was fed only by basal diet. The significantly higher ( P  < 0.05) Zn level in liver and kidney of all treated groups was found, nevertheless Zn NPs did not greatly influence antioxidant status of rats. However, the total aerobic and coliform bacterial population in rat feces significantly decreased ( P  < 0.05) in all zinc groups after 30 d of the treatment. Furthermore, when compared to the ZnO group, ZnA and ZnC nanoparticles reduced coliforms significantly more ( P  < 0.05)., Conclusions: Our results demonstrate that phosphate-based zinc nanoparticles have the potential to act as antibiotic agents., Competing Interests: All animal management and experiments were performed with the approval of the Ethics Commission at the Faculty of AgriSciences, Mendel University in Brno, Czech Republic in accordance with Act No. 246/1992 Coll. on the protection of animals against cruelty.Not applicable.The authors declare that they have no competing interests.

In the present study, two binuclear copper(II) coordination compounds bridged by hydroxy- and thiodipropionic acid have been synthesized. The structure of compounds was determined by X-ray crystallography. The central copper atoms exist in square pyramidal surroundings. Basal plane is formed by nitrogen atoms of amines and oxygen atoms of bridges, whereas apical positions are occupied by oxygen atoms of coordinated water molecules. Temperature dependence study of magnetic susceptibility proved strong antiferromagnetic exchange between copper atoms in hydroxy-bridged complex. These coordination compounds were also tested for their biological activities in vitro. Both coordination compounds exhibit pronounced cytocompatibility in mammalian epithelial cells with no induction of oxidative stress and DNA fragmentation. Moreover, synthesized compounds are hemocompatible and do not alter expression of a marker of multiple cellular stress, p53. On the other hand, both compounds had stimulatory effect on expression of metallothioneins (MT-1/2 and MT-3). Antimicrobial testing on Escherichia coli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus revealed that both copper compounds exhibit antibacterial activity regardless the cell wall composition. Overall, current work presents a synthesis of Cu(II) coordination compounds with interesting biological behavior and with a promising potential to be further tested in pre-clinical models., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The presented study deals with the observation of properties of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in the toxic arsenic environment and influence of arsenic on antioxidant capacity. Two forms of arsenic (As(III), As(V)) with different concentrations were used for induction of the oxidative stress in tested strains. Microbiological methods showed that the growth inhibition of MSSA was higher than that of MRSA in presence of both arsenic ions. As(III) showed 24% and 33% higher anti-microbial effects than As(V) against MSSA and MRSA respectively. A similar result was found also in the experiment of reduction of biofilm-formation. By using spectrophotometry, it was revealed that As(III) induced higher antioxidant production in both bacterial cultures. Methicillin-susceptible S. aureus produced an app. 50 mg equivalent of gallic acid (GAE/1 mg of protein) and MRSA produced an app. 15 mg of GAE/1 mg of protein. The productions of metallothionein in MSSA and MRSA were decreased up to 62.41% and 55.84% respectively in presence of As ions. Reduction of As(III) and As(V) concentrations leads to a decrease in antioxidant production and increased the formation of metallothionein. All of these changes in the results were found to be significant statistically. Taken together, these experiments proved that in comparison with MSSA, MRSA is less susceptible not only to the antimicrobial effects of antibiotics but also against effects caused by metalloids, as arsenic. Thus, it can be stated that MRSA abounds with complex defensive mechanisms, which may in the future constitute significant problem in the efficiency of antibiotics alternatives as metal ions or nanoparticles., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Upconversion nanoparticles (UCNPs) are an emerging class of optical materials with high potential in bioimaging due to practically no background signal and high penetration depth. Their excellent optical properties and easy surface functionalization make them perfect for conjugation with targeting ligands. In this work, capillary electrophoretic (CE) method with laser-induced fluorescence detection was used to investigate the behavior of carboxyl-silica-coated UCNPs. Folic acid, targeting folate receptor overexpressed by wide variety of cancer cells, was used for illustrative purposes and assessed by CE under optimized conditions. Peptide-mediated bioconjugation of antibodies to UCNPs was also investigated. Despite the numerous advantages of CE, this is the first time that CE was employed for characterization of UCNPs and their bioconjugates. The separation conditions were optimized including the background electrolyte concentration and pH. The optimized electrolyte was 20 mM borate buffer with pH 8., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A capillary electrophoretic (CE) method using a short-sweep approach and laser-induced fluorescence (LIF) detection (ShortSweepCE-LIF) was developed for determination of Zn 2+ and Cd 2+ as complexes with highly selective and sensitive fluorescent probe FluoZin-3. The ShortSweepCE-LIF method, established in this work, can be used for examining competitive Zn 2+ and Cd 2+ binding properties of metalloproteins or peptides. The parameters including background electrolyte composition, injection pressure and time as well as separation voltage were investigated. Under the optimized conditions, 80 mM HEPES, pH 7.4, with 1.5 μM FluoZin-3 was used as an electrolyte, hydrodynamic injection was performed at 50 mbar for 5 s, and separation voltage of 25 kV. Limits of detection for Zn 2+ and Cd 2+ were 4 and 125 nM, respectively. The developed method was demonstrated in a study of interactions between metalothionein-2a isoform and metal ions Zn 2+ , Co 2+ and Cd 2+ . It was found that FluoZin-3 was able to extract a single Zn 2+ ion, while added Co 2+ (in surplus) extracted only 2.4 Zn 2+ ions, and Cd 2+ extracted all 7 Zn 2+ ions present in the metalothionein molecule., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The main mechanism of action of platinum-based cytostatic drugs - cisplatin, oxaliplatin and carboplatin - is the formation of DNA cross-links, which restricts the transcription due to the disability of DNA to enter the active site of the polymerase. The polymerase chain reaction (PCR) was employed as a simplified model of the amplification process in the cell nucleus. PCR with fluorescently labelled dideoxynucleotides commonly employed for DNA sequencing was used to monitor the effect of platinum-based cytostatics on DNA in terms of decrease in labeling efficiency dependent on a presence of the DNA-drug cross-link. It was found that significantly different amounts of the drugs - cisplatin (0.21 μg/mL), oxaliplatin (5.23 μg/mL), and carboplatin (71.11 μg/mL) - were required to cause the same quenching effect (50%) on the fluorescent labelling of 50 μg/mL of DNA. Moreover, it was found that even though the amounts of the drugs was applied to the reaction mixture differing by several orders of magnitude, the amount of incorporated platinum, quantified by inductively coupled plasma mass spectrometry, was in all cases at the level of tenths of μg per 5 μg of DNA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8-11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent.

Background: Infections, mostly those associated with colonization of wound by different pathogenic microorganisms, are one of the most serious health complications during a medical treatment. Therefore, this study is focused on the isolation, characterization, and identification of microorganisms prevalent in superficial wounds of patients (n=50) presenting with bacterial infection., Methods: After successful cultivation, bacteria were processed and analyzed. Initially the identification of the strains was performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on comparison of protein profiles (2-30kDa) with database. Subsequently, bacterial strains from infected wounds were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequencing of 16S rRNA gene 108., Results: The most prevalent species was Staphylococcus aureus (70%), and out of those 11% turned out to be methicillin-resistant (mecA positive). Identified strains were compared with patients' diagnoses using the method of artificial neuronal network to assess the association between severity of infection and wound microbiome species composition. Artificial neuronal network was subsequently used to predict patients' prognosis (n=9) with 85% success., Conclusions: In all of 50 patients tested bacterial infections were identified. Based on the proposed artificial neuronal network we were able to predict the severity of the infection and length of the treatment., (Copyright © 2015 Elsevier Editora Ltda. All rights reserved.)

Doxorubicin (DOX) is an effective antitumor drug employed for treatment of a wide range of cancers types such as neuroblastoma, osteosarcoma, breast and esophageal carcinomas. On the other hand, the cumulative dose is restricted (300-550 mg/m(2)) and its amount administered to a patient has to be closely controlled due to its cardiotoxicity. To understand the mechanisms of the DOX side effects as well as to reveal the ways how to reduce its adverse impact on cardiomyocytes, the interactions with particular components of the blood and tissues have to be studied in greater detail. In this work, microdialysis technique was optimized to extract DOX from samples and subsequently monitor its interaction with BSA. Finally, the microdialysis probe was connected on-line to the LIF detector to ensure the real-time detection. The best flow rate was 1 μL/min and after 120 min of microdialysis 28% of the DOX was dialyzed out from the sample. The results from investigation of the DOX-BSA interaction indicate that the interaction occurs in less than 30 min, causing marked decrease in the amount of DOX extracted by microdialysis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

MicroRNAs (miRNAs) are becoming a very important group of molecules especially since their connection to numerous diseases has been revealed. The potential in gene therapy as well as in diagnostics is being widely investigated leading to the demand of sensitive, selective and simple methods of isolation and detection. The combined advantages of magnetic particle-based separation with sensitive electrochemical detection may offer a very valuable tool for these purposes. In this study, the miR‑124 was targeted as an example analyte for development and optimization of the isolation procedure coupled to the electrochemical detection. The sensitivity of the method was demonstrated by the limit of detection at the level of nanomolar concentration (4 nM). To verify the applicability of the procedure to the real samples, miR‑124 was isolated from the human embryonic kidney cells naturally expressing this miRNA molecule and the results were compared to the amount of miR‑124 isolated from the cells transfected by the pENTR-miR‑124 plasmid leading to the overexpression of miR‑124.

Cisplatin belongs to the most widely used cytostatic drugs. The determination of the presence of the DNA-cisplatin adducts may not only signal the guanine-rich regions but also monitor the interaction reaction between DNA and the drug in terms of speed of interaction. In this work, the combined advantages of magnetic particles-based isolation/purification with fluorescent properties of quantum dots (QDs) and antibodies targeted on specific recognition of DNA-cisplatin adducts are demonstrated. The formation of a complex between magnetic particles with surface modified by anti-dsDNA antibody, cisplatin-modified DNA and QDs labelled anti-cisplatin-modified DNA antibody was suggested and optimized., Competing Interests: The authors declare no conflicts of interest.

We focused on interactions of Zn(II) with DNA in this study. These interactions were monitored using UV/vis spectrophotometry and gel electrophoresis. Firstly, we isolated and amplified 498 bp fragment of DNA. Samples were obtained by incubation of DNA fragment with Zn(II) for 60 min at 25 °C. After incubation, the samples were dialyzed and analyzed immediately. In this way, DNA was converted into a metal bound DNA (Zn-DNA). Interaction of Zn(II) with DNA caused change in the absorption spectrum (190-350 nm) and decrease in the melting temperature (Tm) of Zn-DNA. Spectrophotometric (UV/vis) analysis showed that increasing concentrations of zinc(II) ions led to the increase in the absorbance at 200 nm and decrease in absorbance at 251 nm. Application of zinc(II) ions at 5.5 μM concentration caused decrease in Tm for app. 7.5 °C in average in comparison with control (75.5 ± 3 °C). The lowest melting temperature (60.5 ± 2.5 °C) was observed after application of zinc(II) ions at 33 μM concentration. Gel electrophoresis proved significance of Zn(II) in the renaturation of DNA. Samples of Zn-DNA (15 μM DNA+5.5-55 μM Zn(II)) caused significant changes in the renaturation of DNA in comparison with the control, untreated DNA (15 μM DNA)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin-modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20 , serum vs. oligo(dT)20 , serum and SMPs). RNA content was measured, and the expression of metallothionein-2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT-PCR detection. Isolation is possible on serum volume range (10-200 μL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 μL, with optimal results within 10-30 μL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1-0.4 μL. This optimized protocol was also modified to fit needs of automated one-step single-tube analysis with identical efficiency compared to conventional setup. One-step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nucleic acids belong to the most important molecules and therefore the understanding of their properties, function and behavior is crucial. Even though a range of analytical and biochemical methods have been developed for this purpose, one common step is essential for all of them - isolation of the nucleic acid from the from complex sample matrix. The use of magnetic particles for the separation of nucleic acids has many advantages over other isolation methods. In this study, an isolation procedure for extraction of DNA was optimized. Each step of the isolation process including washing, immobilization and elution was optimized and therefore the efficiency was increased from 1.7% to 28.7% and the total time was shortened from 75 to 30min comparing to the previously described method. Quantification of the particular parameter influence was performed by square-wave voltammetry using hanging drop mercury electrode. Further, we compared the optimized method with standard chloroform extraction and applied on isolation of DNA from Staphylococcus aureus and Escherichia coli., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Rapid and sensitive detection is a key step in the effective and early response to the global hazard of various viral diseases. In this study, an integrated isolation of hepatitis B virus (HBV)-specific DNA fragment by magnetic nanoparticles (MNPs) and its immediate analysis by microchip CGE was performed. Microfluidic CE chip was used to accommodate the complete process of viral DNA isolation by MNPs including hybridization and thermal denaturation followed by CE separation. Beforehand, calibration curves of HBV fragments were constructed. For isolation by MNPs, specific streptavidin-biotin interaction was used to bind complementary HBV fragment to magnetic particles. After analysis of isolated HBV by regular MNPs method, innovative approach was performed. The commercial CE chip (Bio-rad) was successfully used to execute HBV fragment isolation. Detection using LIF with detection limit of 1 ng/mL was accomplished., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cisplatin, carboplatin, and oxaliplatin represent three generations of platinum based drugs applied successfully for cancer treatment. As a consequence of the employment of platinum based cytostatics in the cancer treatment, it became necessary to study the mechanism of their action. Current accepted opinion is the formation of Pt-DNA adducts, but the mechanism of their formation is still unclear. Nanomaterials, as a progressively developing branch, can offer a tool for studying the interactions of these drugs with DNA. In this study, fluorescent CdTe quantum dots (QDs, λem = 525 nm) were employed to investigate the interactions of platinum cytostatics (cisplatin, carboplatin, and oxaliplatin) with DNA fragment (500 bp, c = 25 μg/mL). Primarily, the fluorescent behavior of QDs in the presence of platinum cytostatics was monitored and major differences in the interaction of QDs with tested drugs were observed. It was found that the presence of carboplatin (c = 0.25 mg/mL) had no significant influence on QDs fluorescence; however cisplatin and oxaliplatin quenched the fluorescence significantly (average decrease of 20%) at the same concentration. Subsequently, the amount of platinum incorporated in DNA was determined by QDs fluorescence quenching. Best results were reached using oxaliplatin (9.4% quenching). Linear trend (R(2) = 0.9811) was observed for DNA platinated by three different concentrations of oxaliplatin (0.250, 0.125, and 0.063 mg/mL). Correlation with differential pulse voltammetric measurements provided linear trend (R(2) = 0.9511). As a conclusion, especially in the case of oxaliplatin-DNA adducts, the quenching was the most significant compared to cisplatin and nonquenching carboplatin., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)