MendelNet, 2017 (vol. 24)

The aims of this study were the estimation of difference in physiochemical properties and antioxidant activity of yoghurts supplemented by different natural additives. In addition, the changes in physiochemical properties and antioxidant activity during five weeks storage were performed. The change in antioxidant activities was evaluated using the ABTS radical cations decolorizing assay. The statistical analysis showed that the yoghurt samples with the natural supplement have higher antioxidant activity than the plain yoghurt. In addition, the storage period affect the yoghurt properties by increasing its acidity and antioxidant activities by increasing the storage time. Furthermore, the plain and supplemented yoghurts are still consumable until the fifth week of storage but the favourable time to consume it with its higher antioxidant capacity is the 3^rd week of storage.

Protein and/or peptide loss is an undesired but inevitable side effect of purification procedures. We compared three different containers, three different peptide standards and a representative complex proteome digest, and show that a partial drying could improve sample recovery from a glass surface and standard polypropylene tubes. Further, we show that the partial drying minimizes differences between the low-binding and standard polypropylene tubes.

The current study demonstrates design preparation and characterization of biocompatible hybrid ruthenium core-shell nanoparticles (RuNPs) coated with polyvinylpyrrolidone (PVP) and polyoxyethylene stearate (POES). The resulting RuNPs were loaded with doxorubicin, as model anticancer drug. Resulting complex has an exceptional stability in physiological conditions. The cytotoxic effects of the complex were tested using cell lines representing breast and ovarian cancers and neuroblastoma. Although bare RuNPs had only negligible cytotoxicity, RuPDox caused an enhancement of doxorubicin cytotoxicity when compared to free doxorubicin. RuPDox promoted significantly increased stability of doxorubicin in human plasma and pronounced hemocompatibility assayed on human red blood cells.  Results demonstrate that biocompatible RuNPs could have a great potential as versatile nanoplatform to enhance efficiency of anticancer therapy.

Antioxidants are very important substances that counteract the formation of free radicals. They are divided into exogenous, such as vitamin C which the body receives with food, and endogenous. One of the most important endogenous antioxidants is glutathione (γ-glutamyl-L-cysteinyl-glycine), which plays an important role in cellular defense against oxidative damage. Free glutathione is presented within organisms in both reduced (GSH) and oxidized forms (GSSG). Oxidative stress leads to a decrease in GSH level and therefore a GSH/GSSG ratio that can be used as an indicator of oxidative stress and an indicator of various diseases. The aim of presented study was to develop a sampling method for capillary blood testing, where we have found that this amount (15 µl) is sufficient for our testing. Another aim was to supplement group of volunteers with coffee and to determine GSH levels and levels of total thiols after 0, 48 h and 96 h of supplementation in capillary blood. HPLC with electrochemical detection was used for GSH determination and Ellman’s method for determination of total thiols. We could see the GSH slight increase as well as the levels of total thiols.

Looking for strategies against the development of antibiotic resistance is a major global object of interest for the public health. This work deals with synthesis of antimicrobial composites of graphene oxide (GO) with metal nanoparticles. GO has been prepared by modified Hummers' method and characterized using scanning electron microscopy, Fourier transform infrared spectroscopy (FT-IR) and differential pulse voltammetry. Composites of GO have been syntethized with silve nanoparticles, which have been characterized. Potential antimicrobial activity of the nanocomposites was tested against Escherichia coli, Staphylococcus aureus and methicilin resistant Staphylococcus aureus (MRSA).

Elemental composition of glass differs based on raw material and additives used during manufacturing process, therefore elemental analysis provides substantial information from an archaeological point of view. Major, minor and trace elements in glass samples from Moldava, Kyjov and Visegrad were determined. LA-ICP-MS was used for elemental analysis. Concentrations of Na₂O, MgO, Al₂O₃, SiO₂, K₂O, CaO, TiO₂, MnO, Fe₂O₃, CoO, CuO, SnO₂, Sb₂O₃, PbO and ⁷Li, ¹¹B, ⁴⁵Sc, ⁵¹V, ⁵²Cr, ⁶⁰Ni, ⁶⁶Zn, ⁷⁵As, ⁸⁵Rb, ⁸⁸Sr, ⁸⁹Y, ⁹⁰Zr, ⁹³Nb, ⁹⁵Mo, ¹⁰⁷Ag, ¹¹¹Cd, ¹³³Cs, ¹³⁷Ba, ¹³⁹La, ¹⁴⁰Ce, ¹⁴¹Pr, ¹⁴⁶Nd, ¹⁴⁷Sm, ¹⁵³Eu, ¹⁵⁷Gd, ¹⁵⁹Tb, ¹⁶³Dy, ¹⁶⁵Ho, ¹⁶⁶Er, ¹⁶⁹Tm, ¹⁷²Yb, ¹⁷⁵Lu, ¹⁷⁸Hf, ²³²Th, ²³⁸U in 44 glass samples were determined. Samples were discriminated according to finding sites using multivariate statistical methods. Elements used for discrimination (Sc, Zr, Ti, Zn, La) were determined by the Random Forrest algorithm. For archaeological sites discrimination, Primary Component Analysis (PCA) was used.

The aim of the experiment was to determine the content of capsaicin and dihydrocapsaicin in twelve varieties of chilli peppers (Brown Bhutlah II, Bhut Jolokia Yellow I, Pieto de Moca II, Trinidad 7 POT II, 7 POT White II, Naga Jolokia I, Habanero Orange, Naga Viper II, Bhut Jolokia II, Jalapeno, White Naga Bhut Jolokia I, Carolina Reaper II) by using high performance liquid chromatography UltiMate 3000 with UV/VIS detector. Each variety of chilli peppers was prepared without seeds and partitions (septa) and with seeds and partitions (septa) in order to compare yields of capsaicin and dihydrocapsaicin depending on sample preparation. From the contents of capsaicin and dihydrocapsaicin was calculated so-called pungency in Scoville units (SHU) in order to compare our results with previous studies.

UV spectrometry is very simple and cheap method for quantitative and qualitative analysis of compounds. Furthermore, it provides intricate information about the bonded π-electron transitions and also non-bonded n-electron transitions. The aim of this work was to identify electron transition bands in two homing peptides of the human norepinephrine transporter (hNET); namely: GASNGINAYL (978 Da) and SLWERLAYGI (1206 Da). Electron transition bands directly indicate structural conformations, particularly the ones associated with double bonds, i.e. conjugated π-bonds of aromatic and peptide bonds. The results show unusual spikes in absorbance in the far UV at low temperature for GASNGINAYL and even more at other temperatures for SLWERLAYGI peptide. The latter supports the hypothesis of a stacking between tyrosine and tryptophan resulting in helix structure. Infrared spectrometry also showed abundant helix structure in SLWERLAYGI but less abundant in GASNGINAYL peptide. Based on π-stacking, an UV spectrometry method can be developed to monitor the helicity of some peptides, such as SLWERLAYGI.

Molecular imprinting has appeared to be an effective technique for creating of selective recognition sites in synthetic polymers. This procedure comprises polymerization of monomer in a presence of target molecules (template). The subsequent template removal forms tailor-made cavities that are complementary in shape and size to the template molecules. For protein imprinting, the choice of the suitable polymers is limited and polymerization conditions need to be optimized. In our work, dopamine monomer was chosen for polymer formation due to its nontoxicity, ease of preparation and self-assembly. For the optimization of conditions, lysozyme with molecular weight 14.3 kDa was used and the functionality was evaluated by fluorimetry. Different concentration of dopamine and lysozyme for polymerization were tested. Under the optimized conditions, the limit of detection for lysozyme was found to be 7.8 µg/ml. Moreover, conditions for polymer formation for a purpose to reduce the overall time of analysis were investigated. The use of dopamine as a monomer in molecular imprinting shown to be beneficial in many aspects.

Chemotherapy often results in various side effects, which can negatively affect health. Neuroblastoma, one of the most common types of childhood cancer, is but one of the examples, where side effects of chemotherapeutic treatment lower the quality of patient’s life. Modern way how to fight that is to enclose cytotoxic drug into some nanocarrier and its targeting to receptors overexpressed in membranes of cancer cells. Apoferritin (Apo), a natural protein cage, is very suitable as a nanocarrier, as it has no toxicity, immune system does not react to it, and drug can easily be loaded into its cavity. We enclosed ellipticine, clinical tested anti-cancer drug, into Apo cavity (creating ApoElli). The percentage of encapsulation was 61 % and size and transmission electron microscopy analysis showed the preserved Apo ~12 nm icosahedral structure after this encapsulation. Then we modified Apo outer surface with in silico-modelled peptides with hNET affinity and tested its toxicity and hemolytic activity. ApoElli modified with anti-hNET peptides was able to internalize into neuroblastoma cells and to deliver the drug. However, it proved to be safe for human RBC, unlike pure ellipticine, which caused observable hemolysis at the same concentration.

A hyphenated method consisting of transient electrokinetic dosing on-line coupled to isotachophoretic analysis was developed for the pre-concentration, pre-separation and analytical determination of a model substance – anionic herbicide glyphosate (gly) – in aqueous samples containing very low concentration of the analyte of interest. Various parameters were investigated in the framework of an optimisation study; the aim was to reach minimal concentration limit of detection decreasing in minimum time. The developed method consists of 2 phases. In the first one, a sample with addition of convenient buffer (dosing electrolyte) is electrokinetically dosed to the isotachophoretic column with proper leading electrolyte. During the dosing time, a moving-boundary electrophoresis zone of accumulated sample is created in the column and it is slowly moving through column. The accumulation of zone is proportional to the time and driving current and to the composition of dosing electrolyte. After some time of accumulation of the zone, dosing electrolyte is replaced with terminating one. Now, the regular isotachophoretic separation and analysis starts. The electrolyte composition and the dosing time were thoroughly optimized and 14 fold of accumulation was reached in 25 minutes in comparison to classical isotachophoretic analysis. The method is simple and applicable to all commercial isotachophoretic analysers.

The aim of this study is to obtain data based on an experimental procedure and check the effectiveness of some antibacterial agents against pathogenic bacteria. In the present experiment, two different nanoparticles (Cadmium Telluride Quantum dots with Lanthanides: Gadolinium and Terbium) were used to check their antibacterial properties on Pseudomonas aeruginosa. The Cadmium Telluride Quantum dots without the Lanthanides, Gadolinium nitrate and Terbium nitrate were also tested on those bacteria as control. In the present experiment, the following methods were employed: disc-diffusion test, the determination of the growth properties of the bacteria and comparison of absorbance after treatment with antimicrobial agent. From the results it has been found that the tested Cadmium Telluride Quantum dots with Lanthanides (Gadolinium and Terbium) have good antimicrobial effects. Additionally, GdQDs show stronger antibacterial effect than Tb QD and other tested compounds.

This work is aimed at the possibilities of targeted drug delivery into the tumour tissue. This approach can greatly reduce the otherwise serious side effects of conventional treatment – systemic toxicity. For this purpose, ubiquitous protein cage apoferritin was employed as a carrier of cytotoxic drugs. Its molecule size of 10–12 nm allows it to employ the effect of increased permeability and retention as well as to avoid renal clearance. The cellular uptake of this carrier is known to be mediated via the transferrin receptor 1 (TfR1), which is overexpressed on metabolically highly active cells, such as cancer cells. Therefore, apoferritin’s ability to deliver drug molecules to site-of-action was tested using cell lines with high, medium and low expression of TfR1. The optimal conditions for studying the expression of TfR1 using western blotting were as follows: lysate of 50,000 cells applied in non-reducing non-denaturing buffer and the concentration of the primary antibody of 1.0 µg/mL. The properties of encapsulated doxorubicin were not affected by apoferritin, thus preserving its toxicity for cells with high level of TfR1expression (30% growth inhibition of these cells after 24 h of treatment). The suitable usage of apoferritin as a nanocarrier for chemotherapeutic delivery was confirmed in this work.

In this study, the effect of selected phenolic and flavonoid compounds of black pepper and caraway seeds on prostate cells (PNT1A, 22RV1 and PC3) was observed. Synthetic standards of 3,4-dihydroxybenzaldehyde and naringenin chalcone, identified previously by HPLC-MS in black pepper seeds extracts, and neochlorogenic acid and apigenin, identified in caraway seeds extracts, were applied. For the evaluation of the potential inhibitory effect of selected compounds on PNT1A, 22RV1 and PC3 cells, the clonogenic assay and the microscopic observation of cells were done. The results of clonogenic assay showed that phenolic compounds had the strongest inhibitory effect on 22RV1 and PC3 cells, while the flavonoid compounds had the strongest inhibitory effect on PNT1A cells.

The first aim of the experiment was an optimization of the method for determination of hydroxyproline in a pig skin using high-performance liquid chromatography with fluorescence detector (HPLC-FLD) and ion-exchange liquid chromatography with VIS detector (IEC-VIS). On the basis of the experiments performed, it was found that HPLC-FLD method is three times more sensitive than IEC-VIS method. For IEC analysis of hydroxyproline, the limit of detection (LOD) was 4.10 μg/ml whereas the limit of quantification (LOQ) was 13.50 μg/ml.  For HPLC analysis of hydroxyproline, the limit of detection (LOD) was 1 ng/ml whereas the limit of quantification (LOQ) was 3 ng/ml. The second aim was the testing of influence of different volume of 6M HCl on extraction of sample (50 mg) for analysis using HPLC-FLD. Here it was found that the best volume was 250 µl 6M HCl.

Protein identification and quantitation is routinely based on oligopeptides longer than six amino acids. Here, we examined theoretical tryptic digests of five contrasting model proteomes and evaluated potential benefits of shorter peptide sequences for proteome analyses. Our results indicate that pentapeptides should not be excluded and shorter sequences may present valid targets in a dedicated targeted analysis.

Zinc is an important biogenic element which is able to connect with indicators such as a spiropyran. The spiropyran is known for its fluorescent character and selectivity towards zinc ions (and some other metal ions, e.g. cadmium). This complex, especially with zinc, gives a high fluorescence intensity which was analyzed by fluorescence spectra and by capillary electrophoresis with laser induced fluorescence detection. In addition, the influence of visible light irradiation on zinc release was tested. The spiropyran specificity to zinc was also investigated by coupling with copper and cadmium.

This study pays attention to the possibilities of using column tests in order to determine parameters of contaminant migration through soils. The column tests have been done in the Trautwein apparatus by constant head method. In this research solution of chlorides prepared on the basis of distilled water has been used as a conservative marker. Based on breakthrough curves, velocity of flow, dispersion coefficient, longitudinal dispersivity and Peclet number were calculated for each sample tested using CXTFIT-STANMOD package. Obtained Peclet numbers (Pe = 0.82, Pe = 0.57, Pe = 19.01, respectively) have indicated that advection is dominant mechanism of chloride transport in sand whereas diffusion dominates during the chlorides transport through silt loam. Velocity of chlorides flow in silt loam samples were almost equal to 5 × 10^-7 m/s. Velocity of chlorides flow in sand was equal to 3.1 × 10^-4 m/s. It was concluded that the column experiment can be used as an efficient method to provide input data for numerical modeling of non-reactive tracers transport and fate in the soil-water system for the purpose of surface and groundwater protection.

It has been shown that sarcosine suplementation stimulates the proliferation of prostate cells and also their invassiveness. In present study we show that enzymes conected with sarcosine conversion to glycine (sarcosine dehydrogenase, pipecolic acid oxidase) are stimulated due to sarcosine treatment.  Further, sarcosine treatment increases S-adenosylmethioneine-to-S-adenosylhomocysteine ratio, which indicates a release and utilization of free methyl groups from sarcosine degradation pathway. We identified the highest induction of global methylation in non-malignant PNT1A cells, but global methylation profiles were altered also in malignant (22Rv1) and metastatic (LNCaP) cells. The influence on methylation changes was further verified using hypomethylating agent 5-azacytidine (5-aza). Co-treatment of prostate cells with 5-aza and sarcosine resulted in decrease in cells invassiveness when compared to treatment with sarcosine alone. This correlates with sarcosine-related hypermethylation of genes involved in cells growth and cell cycle.

Zinc oxide nanoparticles are one of the most versatile materials, due to their diverse properties, functionalities, and applications. Their potencial in agriculture is also not negligible. The zinc in form of nanoparticles is more available for plants due to its skill to penetrate roots better. In this work we focused on the evaluation and comparison of effect of common zinc source (ZnSO₄.7H₂O), and zinc in form of nanoparticles (nanoZnO). Our pilot results show that it is very important to deal with the toxicity of zinc in the form of nanoparticles, as this form is most likely to be more toxic to plants than its equivalents in the form of simple inorganic salts.

Many researchers are developing nanocarriers in order to minimalize side effects of cytotoxic drugs during cancer treatment via chemotherapy. Nanocarriers can serve as a suitable platform for targeted drug delivery. To overcome their failure in in vivo use, the effects of surface modifications (PEGylation and PASylation) of natural nanocarriers based on apoferritin were tested in this work. Various properties of these modified apoferitin nanoparticles were studied, such as their size or degree of hemolysis. TEM characterization was also performed. The formation of hard coronas on these particles in plasma environment was evaluated using SDS-PAGE electrophoresis. The best biocompatibility results were obtained using apoferritin nanoparticles with PEG surface modification.

The classical hand pipetting approach can be partially substituted in several cases by sophisticated devices that are taking over the necessity of sample manipulation. The developed pipetting method can facilitate routine laboratory work, but the created method has to meet several criteria including – minimizing the possibility of contamination. In this work is briefly summarized four years experiences with the liquid handling system BioNex Nanodrop II. This device is able to pipette (using eight pipetting tips) small volumes in the range of 0.100 µl up to 500 µl into the three common microplate formats – MTP 96, MTP 384 and MTP 1536. The possibility of sample (bacteria, bacterial plasmids and DNA templates for PCR) cross-contamination was estimated in three experiments. The cross-contamination was not presented or it was detected at very low level. The main advantages – speed and accuracy – of the automatic pipetting are described in the last experiment where the enzyme activity was measured.

The aim of this study is to investigate the applicability of fluorescence imaging for monitoring of the abilities of various water-retention additives to supply water to plants protecting them against drought. Rhodamine B solution was used as a fluorophore and its transport through the plants was monitored. Mainly the fluorophore optimal concentration and image acquisition parameters were investigated.

Upconversion nanoparticles (UCNPs) are a novel class of luminescent tags for applications in life and material sciences. Unlike traditional fluorophores, UCNPs exhibit emission of shorter wavelength under near-infrared excitation (typically 980 nm). In this work, we have examined these unique photophysical properties by fluorescence spectrometry and capillary electrophoresis. UCNPs co-doped with Yb(III) and Er(III) were characterized using laboratory-made fluorescence spectrometer. We have exploited and evaluated two excitation sources and the dependence of the fluorescence of UCNPs on the relative excitation power. Moreover, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was for the first time used for characterization of the nanoparticles. It was proved that CE-LIF is a valuable method to be used for investigation of upconversion luminiscence and monitoring of the interactions of UCNPs with other molecules of interest.

Platinum-based cytostatics are the metal-containing anticancer cytostatic drugs that have found application in clinical practice. Antitumor activity of platinum-based drugs is caused by the crosslinking of DNA and formation of DNA adducts with subsequent triggering the apoptosis leading to cell death. Disadvantage of this type of cytostatics is that some kind of cancer is resistant against them. This resistance can be potentially caused by metalloproteins such as metallothioneins (MTs) that bind platinum to their structure and make the interaction with DNA of cell impossible. MTs are low molecular mass, intracellular cysteine-rich, metal-binding proteins and ensure a number of functions in body for example detoxification of heavy metals or maintenance cellular zinc homeostasis. In this work, the interaction between two isoforms of MTs (MT3 and MT2) and several types of platinum cytostatics (oxaliplatin, carboplatin and cisplatin) as well as platinum nanoparticles (size of 10 and 40 nm) were examined by fluorimetric analysis using a fluorescence zinc indicator (Fluozin-3). Fluorescence spectrometry with laser-induced fluorescence detection (ex-488 nm, em-520 nm) was used in the study.