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Looking insight pathological processes, metallothioneins (MTs) are considered to be potential biomarkers for monitoring of a development of various types of diseases, such as cancer. The early identification of the MTs in biological tissues could be important tool for the estimation of appropriate clinical therapy. Therefore, here we investigated the application of matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) together with immunohistochemical analyses (IHC) using MT-1/2 antibody for MT detection in formalin-fixed paraffin-embedded (FFPE) biopsy specimens of human skin. Principal component analyses revealed differences in the peptide/protein profiles separating healthy skin from the carcinoma specimens. Statistically significant ion peaks at m/z 6038, 6300, 6676, and 7026 were more frequently detected in squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma. Using IHC, we found that MT-1/2 was significantly higher in SCC and melanoma compared to healthy skin. Surprisingly, significantly low levels of MT-1/2 were found in BCC. On one side, the results indicate important role of MTs in melanoma occurrence and progression, as on the second side, there are hidden processes associated with MTs based on differences of the occurrence of the MS peaks, which could be associated with cycling of MTs isoforms.

Metallothioneins (MTs) are small cysteine-rich intracellular proteins with four major isoforms identified in mammals, designated MT-1 through MT-4. The best known biological functions of MTs are their ability to bind and sequester metal ions as well as their active role in redox homeostasis. Despite these protective roles, numerous studies have demonstrated that changes in MT expression could be associated with the process of carcinogenesis and participation in cell differentiation, proliferation, migration, and angiogenesis. Hence, MTs have the role of double agents, i.e., working with and against cancer. In view of their rich biochemical properties, it is not surprising that MTs participate in the emergence of chemoresistance in tumor cells. Many studies have demonstrated that MT overexpression is involved in the acquisition of resistance to anticancer drugs including cisplatin, anthracyclines, tyrosine kinase inhibitors and mitomycin. The evidence is gradually increasing for a cellular switch in MT functions, showing that they indeed have two faces: protector and saboteur. Initially, MTs display anti-oncogenic and protective roles; however, once the oncogenic process was launched, MTs are utilized by cancer cells for progression, survival, and contribution to chemoresistance. The duality of MTs can serve as a potential prognostic/diagnostic biomarker and can therefore pave the way towards the development of new cancer treatment strategies. Herein, we review and discuss MTs as tumor disease markers and describe their role in chemoresistance to distinct anticancer drugs.

Background Currently, the diagnosis and treatment of neuroblastomas—the most frequent solid tumors in children—exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. Results The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg ( Conclusion This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

A capillary electrophoretic (CE) method using a short-sweep approach and laser-induced fluorescence (LIF) detection (ShortSweepCE-LIF) was developed for determination of Zn2+ and Cd2+ 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 Zn2+ and Cd2+ 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 Zn2+ and Cd2+ were 4 and 125 nM, respectively. The developed method was demonstrated in a study of interactions between metalothionein-2a isoform and metal ions Zn2+, Co2+ and Cd2+. It was found that FluoZin-3 was able to extract a single Zn2+ ion, while added Co2+ (in surplus) extracted only 2.4 Zn2+ ions, and Cd2+ extracted all 7 Zn2+ ions present in the metalothionein molecule.

Head and neck squamous cell carcinomas (HNSCC) are malignant tumors originating from the upper aerodigestive tract. The environmental pollution, high alcohol consumption and smoking are associated with this type of cancer, as well as the human papillomavirus (HPV), which is often involved in the carcinogenesis and significantly correlates with the cancer prognosis. In recent decades, the number of techniques based on diagnostic of HPV using nanotechnologies including nanoparticles and their surface modification has been developed. Herein, we describe specific biosensing system for detection of HPV. The biosensor employs gold-coated nanoparticles-coated superparamagnetic nanomaghemite (AuMNPs) covered with thiolated oligodeoxyribonucleotides complementary to DNA sequence of E6 HPV-16 oncogene. Successful binding of viral ssDNA was recognized by anti-dsDNA antibodies labeled with HWRGWVC peptide-modified-CdTe quantum dots. The recognition and detection parts were characterized and the specificity was tested using HIV, influenza and HBV-specific oligonucleotides. As a proof-of-concept, system was employed for a detection of HPV-16 E6 presence in DNA obtained by eight subjects with histologically confirmed HNSCC. The obtained results were successfully verified using polymerase chain reaction (PCR). The developed system offers a cheap alternative to conventional PCR with a comparable sensitivity and versatility.

Present study describes the preparation of a polyethylene glycol-grafted oxidized multi-walled carbon nanotubes (oMWCNTs-PEG) hybrid nanosystem as a carrier of etoposide (VP-16) and Bcl-2 phosphorothioate antisense deoxyoligonucleotides (Aso) to achieve a superior cytostastic efficacy in non-small and small cell lung cancer in vitro. We have demonstrated that the adsorption of hydrophobic VP-16 and Bcl-2 Aso results in a stable nanotransporter exhibiting good dispersion with excellent release profiles (both, in pH 7.4 and 4.8) and negligible hemolytic activity (up to 6.5%). The evaluation of cytotoxicity was carried out in in vitro using small cell (SCLC; DMS53) and non-small cell lung cancer (NSCLC; NCIH2135) cell lines. It was found that Bcl-2 interference significantly increased the anti-cancer efficiency of VP-16 in the chemoresistant NSCLC cells. This was further supported using a flow-cytometry (Annexin V/propidium iodide assay), which revealed a significant increase in apoptotic cells in both the cell lines after the co-administration of VP-16 and Bcl-2 Aso using oMWCNTs-PEG hybrid, and fluorescence microscopy, which showed an increase in reactive oxygen species identified after Bcl-2 knock-down. Overall, oMWCNTs-PEG provided an exceptional biocompatible vehicle enabling the internalization of negatively charged nucleic acids and pH-sensitive release of cargoes in a hypoxic environment of the most of solid tumors. Moreover, Aso specifically binding to the first six codons of the Bcl-2 mRNA gave a satisfactorily decrease in Bcl-2 translation and an increase in NCIH2135 chemosensitivity towards VP-16.

Zinc ions are essential cofactors of a wide range of enzymes, transcription factors, and other regulatory proteins. Moreover, zinc is also involved in cellular signaling and enzymes inhibition. Zinc dysregulation, deficiency, over-supply, and imbalance in zinc ion transporters regulation are connected with various diseases including cancer. A zinc ion pool is maintained by two types of proteins: (i) zinc-binding proteins, which act as a buffer and intracellular donors of zinc and (ii) zinc transporters responsible for zinc fluxes into/from cells and organelles. The decreased serum zinc ion levels have been identified in patients suffering from various cancer diseases, including head and neck tumors and breast, prostate, liver, and lung cancer. On the contrary, increased zinc ion levels have been found in breast cancer and other malignant tissues. Zinc metalloproteomes of a majority of tumors including brain ones are still not yet fully understood. Current knowledge show that zinc ion levels and detection of certain zinc-containing proteins may be utilized for diagnostic and prognostic purposes. In addition, these proteins can also be promising therapeutic targets. The aim of the present work is an overview of the importance of zinc ions, zinc transporters, and zinc-containing proteins in brain tumors, which are, after leukemia, the second most common type of childhood cancer and the second leading cause of death in children after accidents.

Cardiotoxicity is a serious complication of anticancer therapy by anthracycline antibiotics. Except for intercalation into DNA/RNA structure, inhibition of DNA-topoisomerase and histone eviction from chromatin, the main mechanism of their action is iron-mediated formation of various forms of free radicals, which leads to irreversible damage to cancer cells. The most serious adverse effect of anthracyclines is, thus, cardiomyopathy leading to congestive heart failure, which is caused by the same mechanisms. Here, we briefly summarize the basic types of free radicals formed by anthracyclines and the main processes how to scavenge them. From these, the main attention is paid to metallothioneins. These low-molecular cysteine-rich proteins are introduced and their functions and properties are reviewed. Further, their role in detoxification of metals and drugs is discussed. Based on these beneficial roles, their use as a new therapeutic agent against oxidative stress and for cardioprotection is critically evaluated with respect to their ability to increase chemoresistance against some types of commonly used cytostatics.

Histamine is a heterocyclic amine formed by decarboxylation of the amino acid l-histidine. It is involved in the local regulation of physiological processes but also can occur exogenously in the food supply. Histamine is toxic at high intakes; therefore, determination of the histamine level in food is an important aspect of food safety. This article will review the current understanding of physiological functions of endogenous and ingested histamine with a particular focus placed on existing and emerging technologies for histamine quantification in food. Methods reported in this article are sequentially arranged and provide a brief overview of analytical methods reported, including those based on nanotechnologies.

Background Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells. Objectives The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared. Material and methods Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization. Results In chip CE analysis, changes in the range of 5-8 kDa corresponding to the MT monomer, as well as some peaks of 13-14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT. Conclusions The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of -sulfhydryl (-SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.

So far multiple differences in prostate cancer-specific amino acids metabolism have been discovered. Moreover, attempts to utilize these alterations for prostate cancer diagnosis and treatment have been made. The prostate cancer metabolism and biosynthesis of amino acids are particularly focused on anaplerosis more than on energy production. Other crucial requirements on amino acids pool come from the serine, one‑carbon cycle, glycine synthesis pathway and folate metabolism forming major sources of interproducts for synthesis of nucleobases necessary for rapidly proliferating cells. Considering the lack of some amino acids biosynthetic pathways and/or their extraordinary importance for prostate cancer cells, there is a widespread potential for targeted therapeutic applications with no effect on non-malignant cells. This review summarizes the up-to-date knowledge of the importance of amino acids for prostate cancer pathogenesis with a special emphasis on potential applications of metabolic variabilities in the new oncologic paradigm of precision medicine.

DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine- induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.

Fluorescence resonance energy transfer (FRET) in combination with quantum dots (QDs) and their superior properties has enabled designing of the new and improved sensors. In this review, the latest novelties in development and application of FRET nanosensors employing QDs are presented. QDs offer several advantages over organic dyes - broad excitation spectra, narrow defined tunable emission peak, longer fluorescence lifetime, resistance to photobleaching and 10-100 times higher molar extinction coefficient. These properties of QDs allow multicolor QDs to be excited from one source by common fluorescent dyes without emission signal overlap and results in brighter probes comparing to conventional fluorophores. Due to these benefits, QD-FRET-based nanosensors gained a wide spread popularity in a variety of scientific areas. These sensors are most frequently applied in the domain of the nucleic acid and enzyme activity detection. Other applications are detection of peptides and low-molecular compounds, environmental pollutants, viruses, microorganisms and their toxins, QD-FRET-based immunoassays, and pH sensors.

A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.

Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous versus healthy tissue of various types of malignant tumors, including head and neck cancer. Thus, we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of MT, based on affinity of its sulfhydryl groups toward gold. Biosensor was crafted from PDMS combined with technology of 3D printing and contained reservoir with volume of 50 μL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunolabeling of immobilized MT anti-MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabeling (120 min, 20°C, and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of MT in cell lines derived from spinocellular carcinoma (cell line 122P-N) and fibroblasts (122P-F) and levels of the biomarker were found to be about 90 nM in tumor cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 μL), with low acquisition costs and high portability.

The purpose of this study was to test quantum dots (QDs) separation by native and Tris–Glycine SDS-PAGE according to the protocols commonly used for protein analyses. To study the electrophoretic behaviour of quantum dots, ten samples of previously synthesized CdTe QDs stabilized with mercaptosuccinic acid (MSA) were used. Prior to electrophoresis the hydrodynamic diameters of QDs and zeta potentials were determined, as well as the fluorescence properties and stability of QDs in the running buffers. After verification of QDs stability and separation in native polyacrylamide gel, SDS-PAGE in gradient 4–20 % polyacrylamide gel was performed. Under UV irradiation a colour-dependent separation of QDs was observed, which was consistent with their hydrodynamic diameter distribution. The electrophoretic conditions were further optimized with respect to achieving the optimal colour separation, fluorescence stability and to minimize the time of analysis. Based on the results obtained, for further work 15 % polyacrylamide gels with SDS were used and the times (30–60 min) and voltage (100–150 V) used for separation were optimized. Under the optimal separation conditions (30 min, 100 V) the addition of MSA in the concentration range 0–4 mM was used to improve visualization of QDs with diameters in the range from 7 ± 2 to 4 ± 2 nm.

Human metallothionein-3 (hMT-3), also known as growth inhibitory factor, is predominantly expressed in the central nervous system. hMT-3 is presumed to participate in the processes of heavy metal detoxification, regulation of metabolism and protection against oxidative damage of free radicals in the central nervous system; thus, it could play important neuromodulatory and neuroprotective roles. However, the primary functions of hMT-3 and the mechanism underlying its multiple functions in neuroblastoma have not been elucidated so far. First, we confirmed relatively high expression of hMT-3 encoding mRNA in biopsies (n = 23) from high-risk neuroblastoma subjects. Therefore, we focused on investigation of the impact of hMT-3 up-regulation in N-Myc amplifying neuroblastoma cells. The differentially up-regulated genes involved in biological pathways related to cellular senescence and cell cycle were identified using electrochemical microarray with consequent bioinformatic processing. Further, as experimental verification of microarray data, the cytotoxicity of the cisplatin (CDDP) was examined in hMT-3 and mock cells by MTT and clonogenic assays. Overall, our data strongly suggest that up-regulation of hMT-3 positively correlates with the genes involved in oncogene-induced senescence (CDKN2B and ANAPC5) or apoptosis (CASP4). Moreover, we identified a significant increase in chemoresistance to cisplatin (CDDP) due to hMT-3 up-regulation (24IC(50): 7.5 vs. 19.8 mu g/ml), indicating its multipurpose biological significance.

Background Suitable fluorophores are the core of fluorescence imaging. Among the most exciting, yet controversial, labels are quantum dots (QDs) with their unique optical and chemical properties, but also considerable toxicity. This hinders QDs applicability in living systems. Surface chemistry has a profound impact on biological behavior of QDs. This study describes a two-step synthesis of QDs formed by CdTe core doped with Schiff base ligand for lanthanides [Ln (Yb3+, Tb3+ and Gd3+)] as novel cytocompatible fluorophores. Results Microwave-assisted synthesis resulted in water-soluble nanocrystals with high colloidal and fluorescence stability with quantum yields of 40.9–58.0%. Despite induction of endocytosis and cytoplasm accumulation of Yb- and TbQDs, surface doping resulted in significant enhancement in cytocompatibility when compared to the un-doped CdTe QDs. Furthermore, only negligible antimigratory properties without triggering formation of reactive oxygen species were found, particularly for TbQDs. Ln-doped QDs did not cause observable hemolysis, adsorbed only a low degree of plasma proteins onto their surface and did not possess significant genotoxicity. To validate the applicability of Ln-doped QDs for in vitro visualization of receptor status of living cells, we performed a site-directed conjugation of antibodies towards immuno-labeling of clinically relevant target—human norepinephrine transporter (hNET), over-expressed in neuroendocrine tumors like neuroblastoma. Immuno-performance of modified TbQDs was successfully tested in distinct types of cells varying in hNET expression and also in neuroblastoma cells with hNET expression up-regulated by vorinostat. Conclusion For the first time we show that Ln-doping of CdTe QDs can significantly alleviate their cytotoxic effects. The obtained results imply great potential of Ln-doped QDs as cytocompatible and stable fluorophores for various bio-labeling applications.

In this study, we focused on the effect of heavy metal ions in resistant strains of gram-positive bacteria Staphylococcus aureus using biochemical methods and mass spectrometry. Five nitrate solutions of heavy metals (Ag(+), Cu(2+), Cd(2+), Zn(2+) and Pb(2+)) were used to create S. aureus resistant strains. Biochemical changes of resistant strains in comparison with the non-resistant control strain of S. aureus were observed by microbiological (measuring - growth curves and inhibition zones) and spectrophotometric methods (antioxidant activity and alaninaminotransferase, aspartateaminotransferase, alkaline phosphatase, γ-glutamyltransferase activities). Mass spectrometry was employed for the qualitative analysis of the samples (changes in S. aureus protein composition) and for the identification of the strains database MALDI Biotyper was employed. Alterations, in terms of biochemical properties and protein composition, were observed in resistant strains compared to non-resistant control strain. Our results describe the possible option for the analysis of S. aureus resistant strains and may thus serve as a support for monitoring of changes in genetic information caused by the forming of resistance to heavy metals.

A capillary electrophoretic (CE) method using a short-sweep approach and laser-induced fluorescence (LIF) detection (ShortSweepCE-LIF) was developed for determination of Zn

Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.

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.

Simona Dostalova,1,2 Katerina Vasickova,1 David Hynek,1,2 Sona Krizkova,1,2 Lukas Richtera,1,2 Marketa Vaculovicova,1,2 Tomas Eckschlager,3 Marie Stiborova,4 Zbynek Heger,1,2 Vojtech Adam1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, 2Central European Institute of Technology, Brno University of Technology, Brno, 3Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, University Hospital Motol, Charles University, 4Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic Abstract: Due to many adverse effects of conventional chemotherapy, novel methods of targeting drugs to cancer cells are being investigated. Nanosize carriers are a suitable platform for this specific delivery. Herein, we evaluated the long-term stability of the naturally found protein nanocarrier apoferritin (Apo) with encapsulated doxorubicin (Dox). The encapsulation was performed using Apo’s ability to disassemble reversibly into its subunits at low pH (2.7) and reassemble in neutral pH (7.2), physically entrapping drug molecules in its cavity (creating ApoDox). In this study, ApoDox was prepared in water and phosphate-buffered saline and stored for 12 weeks in various conditions (-20°C, 4°C, 20°C, and 37°C in dark, and 4°C and 20°C under ambient light). During storage, a very low amount of prematurely released drug molecules were detected (maximum of 7.5% for ApoDox prepared in PBS and 4.4% for ApoDox prepared in water). Fourier-transform infrared spectra revealed no significant differences in any of the samples after storage. Most of the ApoDox prepared in phosphate-buffered saline and ApoDox prepared in water and stored at -20°C formed very large aggregates (up to 487% of original size). Only ApoDox prepared in water and stored at 4°C showed no significant increase in size or shape. Although this storage caused slower internalization to LNCaP prostate cancer cells, ApoDox (2.5 µM of Dox) still retained its ability to inhibit completely the growth of 1.5×104 LNCaP cells after 72 hours. ApoDox stored at 20°C and 37°C in water was not able to deliver Dox inside the nucleus, and thus did not inhibit the growth of the LNCaP cells. Overall, our study demonstrates that ApoDox has very good stability over the course of 12 weeks when stored properly (at 4°C), and is thus suitable for use as a nanocarrier in the specific delivery of anticancer drugs to patients. Keywords: anticancer therapy, doxorubicin-loaded apoferritin, encapsulation, long-term stability, protein nanocarriers

This study was focused on the application of electrochemical methods for studying of bacterial strains Escherichia coli and Escherichia coli expressing human metallothionein gene (MT-3) before and after the application of cadmium and/or lead ions in four concentrations (25, 50, 75 and 150 mikroM). Bacterial strains Escherichia coli and Escherichia coli expressing human metallothionein gene (MT-3) were used like model organisms for studying of metals influence to metallothionein expression. Metallothionein was isolated using fast protein liquid chromatography and quantified by electrochemical methods. The occurrence of metallothionein in E.coli was confirmed by gel electrophoresis by the presence of the bands at 15 (MT dimer) and 22 kDa (MT trimer). The changes in electrochemical records due to the interactions of metallothioneins (MT-3 and MT-2A) with cadmium and lead ions showed decline of Cat2 signal of MT with the increasing interaction time because of metal ions binding to cysteines. Electrochemical determination also revealed that Cd(II) remains in E. coli cells in the higher amount than Pb (II). Opposite situation was found at E. coli–MT-3 strain. The antimicrobial effect of cadmium ions was determined by IC50 and was statistically calculated as 39.2 and 95.5 mikroM for E. coli without cloned MT-3 and E. coli carrying MT-3 gene, respectively. High provided concentration IC50 in strains after lead ions application (352.5 mikroM for E. coli without cloning and 207.0 mikroM for E. coli carrying cloned MT-3 gene) indicates lower toxicity of lead ions on bacterial strains compared to the cadmium ions.

Doxorubicin (DOX) belongs to the group of anthracycline antibiotics with very effective anticancer properties. On the other hand, the cardiotoxic effects limit its application over the maximum cumulative dose. To overcome this obstacle, encapsulation of this drug into the protective nanotransporter such as apoferritin is beneficial. In this study, fluorescent behavior of DOX in various solvents was determined by fluorescence spectrometry, demonstrating the fluorescence quenching effect of water, which is often used as a solvent. It was found that by increasing the amount of the organic phase in the DOX solvent the dynamic quenching is significantly suppressed. Ethanol, acetonitrile and dimethyl sulfoxide were tested and the best linearity of the calibration curve was obtained when above 50 % of the solvent was present in the binary mixture with water. Moreover, pH influence on the DOX fluorescence was also observed within the range of 4–10. Two times higher fluorescence intensity was observed at pH 4 compared to pH 10. Further, the DOX behavior in capillary electrophoresis (CE) was investigated. Electrophoretic mobilities (CE) in various pH of the background electrolyte were determined within the range from 16.3 to −13.3 × 10 −9 m−2 V−1 s−1. Finally, CE was also used to monitor the encapsulation of DOX into the cavity of apoferritin as well as the pH-triggered release.

Recent developments in mass spectrometry have introduced clinical proteomics to the forefront of diseases diagnosis, offering reliable, robust and efficient analytical method for biomarker discovery and monitoring. MALDI-TOF is a powerful tool for surveying proteins and peptides comprising the realm for clinical analysis. MALDI-TOF has the potential to revolutionize cancer diagnostics by facilitating biomarker discovery, enabling tissue imaging and quantifying biomarker levels. Healthy (control) and cancerous tissues can be analyzed on the basis of mass spectrometry (MALDI-TOF) imaging to identify cancer-specific changes that may prove to be clinically useful. We review MALDI-TOF profiling techniques as tools for detection of cancer biomarkers in various cancers. We mainly discuss recent advances including period from 2011 to 2013.

Nanoparticles have gained increasing interest in medical and in vivo applications. Metallothionein (MT) is well known as a maintainer of metal ions balance in intracellular space. This is due to high affinity of this protein to any reactive species including metals and reactive oxygen species. The purpose of this study was to determine the metallothionein-quantum dots interactions that were investigated by spectral and electrochemical techniques. CuS, CdS, PbS, and CdTe quantum dots (QDs) were analysed. The highest intensity was shown for CdTe, than for CdS measured by fluorescence. These results were supported by statistical analysis and considered as significant. Further, these interactions were analysed using gel electrophoresis, where MT aggregates forming after interactions with QDs were detected. Using differential pulse voltammetry Brdicka reaction, QDs and MT were studied. This method allowed us to confirm spectral results and, moreover, to observe the changes in MT structure causing new voltammetric peaks called X and Y, which enhanced with the prolonged time of interaction up to 6 h. (C) 2014 Elsevier B.V. All rights reserved.

Remote-controlled robotic systems are being used for analysis of various types of analytes in hostile environment including those called extraterrestrial. The aim of our study was to develop a remote-controlled robotic platform (ORPHEUS-HOPE) for bacterial detection. For the platform ORPHEUS-HOPE a 3D printed flow chip was designed and created with a culture chamber with volume 600 μL. The flow rate was optimized to 500 μL/min. The chip was tested primarily for detection of 1-naphthol by differential pulse voltammetry with detection limit (S/N = 3) as 20 nM. Further, the way how to capture bacteria was optimized. To capture bacterial cells (Staphylococcus aureus), maghemite nanoparticles (1 mg/mL) were prepared and modified with collagen, glucose, graphene, gold, hyaluronic acid, and graphene with gold or graphene with glucose (20 mg/mL). The most up to 50% of the bacteria were captured by graphene nanoparticles modified with glucose. The detection limit of the whole assay, which included capturing of bacteria and their detection under remote control operation, was estimated as 30 bacteria per μL.

Estrogen receptors [ERs (subtypes α and β)], classified as a nuclear receptor super family, are intracellular proteins with an important biological role as the transcription factors for estrogen target genes. For ER-induced transcription, an interaction must exist between ligand and coregulators. Coregulators may stimulate (coactivators) or inhibit (corepressors) transcription, following binding with a specific region of the gene, called the estrogen response element. Misbalanced activity of coregulators or higher ligand concentrations may cause increased cell proliferation, resulting in specific types of cancer. These are exhibited as overexpression of ER proteins. Breast cancer currently ranks first in the incidence and second in the mortality of cancer in females worldwide. In addition, 70% of breast tumors are ERα positive and the importance of these proteins for diagnostic use is indisputable. Early diagnosis of the tumor and its classification has a large influence on the selection of appropriate therapy, as ER-positive tumors demonstrate a positive response to hormonal therapy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) has been hypothesized to have great potential, as it offers reliable, robust and efficient analysis methods for biomarker monitoring and identification. The present review discusses ER protein analysis by MALDI TOF MS, including the crucial step of protein separation.

Sarcosine has been identified as a potential prostate cancer marker. To provide determination of this compound, a number of methods are developing. In this study, we optimized a method for its separation by hydrophilic interaction LC with electrochemical detection (ED). Due to the fact that mobile phases commonly used for this type of separation altered the LODs measured by electrochemical detectors, we applied postcolumn dosing of buffer suitable for ED. The optimized conditions were mobile phase A acetonitrile, mobile phase B water in the ratio A/B 70:30, with postcolumn addition of mobile phase C (200 mM phosphatebufferpH9).TheoptimalmixingratiowasA +B/C1:1withaflowrateof0.80mL/min (0.40 + 0.40 mL/min) and detection potential of 1000 mV. Due to the optimization of the parameters for effective separation, which had to meet the optimal parameters of ED, we reachedagoodresolutionforseparationalsowithagoodLOD(100nM).Inaddition,wesuccessfully carried out sarcosine analysis bound on our modified paramagnetic microparticles with the ability to preconcentrate sarcosine isolated from artificial urine.

Interactions of silver phosphate nanoparticles (SPNPs) and selenium nanoparticles (SeNPs) with Staphylococcus aureus cultures have been studied at the cellular, molecular and protein level. Significant antibacterial effects of both SPNPs and SeNPs on S. aureus were observed. At a concentration of 300 μM, SPNPs caused 37.5% inhibition of bacterial growth and SeNPs totally inhibited bacterial growth. As these effects might have been performed due to the interactions of nanoparticles with DNA and proteins, the interaction of SPNPs or SeNPs with the amplified zntR gene was studied. The presence of nanoparticles decreased the melting temperatures of the nanoparticle complexes with the zntR gene by 23% for SeNPs and by 12% for SPNPs in comparison with the control value. The concentration of bacterial metallothionein was 87% lower in bacteria after application of SPNPs (6.3 μg mg(-1) protein) but was increased by 29% after addition of SeNPs (63 μg mg(-1) protein) compared with the S. aureus control (49 μg mg(-1) protein). Significant antimicrobial effects of the nanoparticles on bacterial growth and DNA integrity provide a promising approach to reducing the risk of bacterial infections that cannot be controlled by the usual antibiotic treatments.

In this study we describe a beads-based assay for rapid, sensitive and specific isolation and detection of influenza vaccine hemagglutinin (HA). Amplification of the hemagglutinin signal resulted from binding of an electrochemical label as quantum dots (QDs). For detection of the metal and protein part of the resulting HA-CdTe complex, two differential pulse voltammetric methods were used. The procedure includes automated robotic isolation and electrochemical analysis of the isolated product. The isolation procedure was based on the binding of paramagnetic particles (MPs) with glycan (Gly), where glycan was used as the specific receptor for linkage of the QD-labeled hemagglutinin. V této studii jsme popsali test korálky založené na rychlou, citlivou a specifickou izolaci a detekci vakcíny proti chřipce hemaglutininu (HA). Amplifikace hemaglutinin signálu důsledkem vazby elektrochemického štítku podle kvantových teček (QDS). Pro detekci kovů a proteinové části výsledného HA-CdTe komplexu, byly použity dvě metody diferenciální pulsní voltametrické.Postup zahrnuje automatizované robotizované izolaci a elektrochemické analýze izolovaného produktu. Postup izolace byl založen na vázání paramagnetických částic (MPS) s glykan (Gly), kde byl použit glykanů jako specifický receptor pro propojení na QD-značeného hemaglutininu.

The bioconjugation of quantum dots (QDs) is a key process in their application for bioanalysis as well as imaging. The coupling of QDs with biologically active molecules such as peptides, nucleic acids, and/or antibodies enables their fluorescent labeling, and therefore, selective and sensitive tracking during the bioanalytical process, however, the efficiency of the labeling and preservation of the biological activity of the bioconjugate have to be considered. In this study, a new approach of the bioconjugation of CdTe-QDs and human immunoglobulin employing a small peptide is described. The heptapeptide (HWRGWVC) was synthesized and characterized by mass spectrometry, liquid chromatography, and capillary electrophoresis. Moreover, the peptide was used as a capping agent for QDs synthesis. The CdTe-QDs were synthesized by microwave synthesis (600 W, 20 min) using 3.2 mM CdCl2 and 0.8 mM Na2TeO3. The bioconjugation of QDs capped by this peptide with immunoglobulin was investigated by capillary electrophoresis and magnetic immunoextraction coupled with electrochemical detection by differential pulse voltammetry. Furthermore, the applicability of prepared bioconjugates for fluorescent immunodetection was verified using immobilized goat antihuman IgG antibody.

Lactoferrin (LF) is approximately 80 kDa iron-binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of LF from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimised steps. Protein G was bound on the paramagnetic particles, on which goat antibody (10 μg) was linked. LF was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30 min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase reducing 3,3′,5,5′-tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng/mL. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1 μg/mL. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-LF-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R2 higher than 0.95, indicating a very good agreement of the results obtained.

In this study, we optimized method for the isolation and detection of lactoferrin from human saliva using 3 mm short monolithic disc. We optimized the conditions for separation as flow rate 4 mL min−1 and ionic strength of effluent as 2 M·NaCl. We estimated limit of detection of whole method, which was hyphenated to the Bradford’s assay, down to 100 ng mL−1. The purity of the isolated fractions was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and recovery of isolation was found to be 51 % using minimally processed sample of saliva. Further, we tested the optimized method on group of healthy volunteers (n = 7). We were able to distinguish between the healthy subjects and subject suffering from celiac disease, which reported at least 2.5× higher level of lactoferrin in comparison to healthy ones. The results were correlated with standard enzyme-linked immunosorbent assay (ELISA) kit with obtained correlation coefficient R2 = 0.8446. Analysis of lactoferrin in saliva by monolithic disc and subsequent offline photometric detection is faster and cheaper method compared to ELISA commercial kit. The total analysis of one sample takes

Quantum dots (QDs) belong to a new class of fluorescent agent for biochemical, medicinal or other purposes. However, QDs based on cadmium or other metals can be risky for an organism. As one of the mechanism how to detoxify cadmium-based QDs expression of metallothioneins (MT) can be considered. Due to high affinity of metallothionein to cadmium(II) ions, we attempted to develop an approach for studying of possible interaction with QDs. We prepared QDs with CdTe core and studied the interaction with MT, which we isolated from livers of Cd-administered rabbits. To study the interaction, we used the mixture of both components MT (3.6 μM): CdTe QDs (0, 0.34, 0.68, 1.02, 1.36, 1.7, 2.04 and 2.47 μM). The mixtures were studied by spectrophotometry within the range from 200 to 750 nm with detected maxima at 260 and 505 nm. Same mixtures were also analysed by differential pulse voltammetry Brdicka reaction, which supported data from spectrophotometry. Subsequently, we used fast protein liquid chromatography for purification of protein–quantum dot conjugates. We obtained the different chromatograms for (1) Apo MT, (2) CdTe QDs and (3) MT–QD complex. We also collected the fractions and subsequently analysed them on the content of Cd and MT, which confirmed the formation of CdTe QDs–MT complex.

The nanotechnological concept is based on size-dependent properties of particles in the 1–100 nm range. Nevertheless, the connection between their size and effect is still not clear. Thus, we focused on reductive colloidal synthesis, characterization and biological testing of Pt nanoparticles (PtNPs) capped with biocompatible polymer polyvinylpyrrolidone (PVP). Synthesized PtNPs were of 3 different primary sizes (approx. ∼10; ∼14 and > 20 nm) and demonstrated exceptional haemocompatibility. In vitro treatment of three different types of malignant cells (prostate – LNCaP, breast – MDA-MB-231 and neuroblastoma – GI-ME-N) revealed that even marginal differences in PtNPs diameter resulted in changes in their cytotoxicity. The highest cytotoxicity was observed using the smallest PtNPs-10, where 24IC 50 was lower (3.1–6.2 μg/mL) than for cisplatin (8.1–19.8 μg/mL). In contrast to MDA-MB-231 and LNCaP cells, in GI-ME-N cells PtNPs caused noticeable changes in their cellular structure without influencing their viability. Post-exposure analyses revealed that PtNPs-29 and PtNPs-40 were capable of forming considerably higher amount of reactive oxygen species with consequent stimulation of expression of metallothionein (MT1/2 and MT3), at both mRNA and protein level. Overall, our pilot study demonstrates that in the nanoscaled world even the smallest differences can have crucial biological effect.

Herein we describe a novel alternative synthesis route of polyvinylpyrrolidone nanoparticles using salting-out method at a temperature close to polyvinylpyrrolidone decomposition. At elevated temperatures, the stability of polyvinylpyrrolidone decreases and the opening of pyrrolidone ring fractions occurs. This leads to cross-linking process, where separate units of polyvinylpyrrolidone interact among themselves and rearrange to form nanoparticles. The formation/stability of these nanoparticles was confirmed by transmission electron microscopy, X-ray photoelectron spectroscopy, mass spectrometry, infrared spectroscopy, and spectrophotometry. The obtained nanoparticles possess exceptional biocompatibility. No toxicity and genotoxicity was found in normal human prostate epithelium cells (PNT1A) together with their high hemocompatibility. The antimicrobial effects of polyvinylpyrrolidone nanoparticles were tested on bacterial strains isolated from the wounds of patients suffering from hard-to-heal infections. Molecular analysis (qPCR) confirmed that the treatment can induce the regulation of stress-related survival genes. Our results strongly suggest that the polyvinylpyrrolidone nanoparticles have great potential to be developed into a novel antibacterial compound.

Great boom of nanotechnologies impacts almost all areas of science and therefore detail understanding of the properties of nanomaterials as well as their interaction abilities is required. Surface modification and functionalization of nanoparticles is of a great interest due to the wide range of applications in the area of nanomedicine, nanobiology, and/or biochemistry. In this study, CdTe QDs were synthesized using microwave reactor and their surface was modified by streptavidin to ensure further suitability for bioconjugation with biotin-labelled oligonucleotides. For characterization of the synthesized QDs and for monitoring of the interaction with the oligonucleotide, capillary and gel electrophoresis was used. Moreover, complementary advantages of absorption (CE–UV) and laser-induced fluorescence detection (CE–LIF) were exploited. Comparison the electrophoretic mobilities obtained for streptavidin-modified QDs by CE–LIF (−9.87 × 10−9 m2/V/s) and by CE–UV (−10.02 × 10−9 m2/V/s) was in a good agreement enabling us to identify the peak of streptavidin-modified QDs in the CE–UV electropherogram containing also the peak of unreacted streptavidin. Subsequent conjugation of streptavidin-modified QDs with two model biotinylated oligonucleotides (BCL-2 and HBV) led to formation of the complex represented in the electropherograms as a very sharp peak. This peak height increased with time for 15.5 and 27 mAU using BCL-2 oligonucleotide and HBV oligonucleotide, respectively during 30 min interaction.

Pathogenic bacteria have become a serious socio-economic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn-proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn-proteins by superparamagnetic beads modified with chicken anti-Zn(II) antibody. S. aureus culture was treated with ZnCl(2). Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle-surface interactions were tested and associated protein profiles were identified using SDS-PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS-PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.

Metallothioneins (MTs) are involved in protection against oxidative stress (OS) and toxic metals and they participate in zinc metabolism and its homeostasis. Disturbing of zinc homeostasis can lead to formation of reactive oxygen species, which can result in OS causing alterations in immunity, aging, and civilization diseases, but also in cancer development. It is not surprising that altered zinc metabolism and expression of MTs are of great interest in the case of studying of oncogenesis and cancer prognosis. The role of MTs and zinc in cancer development is tightly connected, and the structure and function of MTs are strongly dependent on Zn²⁺ redox state and its binding to proteins. Antiapoptic effects of MTs and their interactions with proteins nuclear factor kappa B, protein kinase C, esophageal cancer-related gene, and p53 as well as the role of MTs in their proliferation, immunomodulation, enzyme activation, and interaction with nitric oxide are reviewed. Utilization of MTs in cancer diagnosis and therapy is summarized and their importance for chemoresistance is also mentioned.

Currently, the influenza virus infects millions of individuals every year. Since the influenza virus represents one of the greatest threats, it is necessary to develop a diagnostic technique that can quickly, inexpensively, and accurately detect the virus to effectively treat and control seasonal and pandemic strains. This study presents an alternative to current detection methods. The flow-injection analysis-based biosensor, which can rapidly and economically analyze a wide panel of influenza virus strains by using paramagnetic particles modified with glycan, can selectively bind to specific viral A/H5N1/Vietnam/1203/2004 protein-labeled quantum dots. Optimized detection of cadmium sulfide quantum dots (CdS QDs)-protein complexes connected to paramagnetic microbeads was performed using differential pulse voltammetry on the surface of a hanging mercury drop electrode (HMDE) and/or glassy carbon electrode (GCE). Detection limit (3 S/N) estimations based on cadmium(II) ions quantification were 0.1 μg/mL or 10 μg/mL viral protein at HMDE or GCE, respectively. Viral protein detection was directly determined using differential pulse voltammetry Brdicka reaction. The limit detection (3 S/N) of viral protein was estimated as 0.1 μg/mL. Streptavidin-modified paramagnetic particles were mixed with biotinylated selective glycan to modify their surfaces. Under optimized conditions (250 μg/mL of glycan, 30-min long interaction with viral protein, 25°C and 400 rpm), the viral protein labeled with quantum dots was selectively isolated and its cadmium(II) content was determined. Cadmium was present in detectable amounts of 10 ng per mg of protein. Using this method, submicrogram concentrations of viral proteins can be identified.

The present paper is focused on zinc(ii) treatment effects on prostatic cell lines PC-3 (tumour) and PNT1A (non-tumour). Oxidative status of cells was monitored by evaluation of expression of metallothionein (MT) isoforms 1A and 2A at the mRNA and protein level, glutathione (oxidised and reduced), and intracellular zinc(ii) after exposition to zinc(ii) treatment at concentrations of 0-150 μM using electrochemical methods, western blotting and fluorescent microscopy. A novel real-time impedance-based growth monitoring system was compared with widely used end-point MTT assay. Impedance-based IC(50) for zinc(ii) is 55.5 and 150.8 μM for PC-3 and PNT1A, respectively. MTT-determined IC(50) are1.3-fold higher. Impedance-based viability correlates with viable count (r0.92; p0.03), not with MTT. Two-fold lower intracellular zinc(ii) in the tumour PC-3 cell line was found. After zinc(ii) treatment2.6-fold increase of intracellular zinc(ii) was observed in non-tumour PNT1A and in tumour PC-3 cells. In PC-3 cells, free and bound zinc(ii) levels were enhanced more markedly as compared to PNT1A. PNT1A produced 4.2-fold less MT compared to PC3. PNT1A cells showed a 4.8-fold increase trend (r = 0.94; p = 0.005); PC-3 did show a significant trend at MT1 and MT2 protein levels (r = 0.93; p = 0.02) with nearly ten-fold increase after 100 μM zinc(ii) treatment. In terms of redox state, PNT1A had a predominance of reduced GSH forms (GSH : GSSG ratio1), when exposed to zinc(ii) compared to PC3, where predominance of oxidised forms remains at all concentrations. IC(50) differs significantly when determined by MTT and real-time impedance-based assays due to dependence of impedance on cell morphology and adhesion. When real-time growth monitoring, precise electrochemical methods and fluorescent microscopy are performed together, accurate information for metal fluxes, their buffering by thiol compounds and monitoring of the redox state become a powerful tool for understanding the role of oxidative stress in carcinogenesis.

Matrix metalloproteinases (MMPs) are involved in many physiological and pathological processes. Due to their ability to cleave and to remodel components of surrounding tissues, MMPs may affect cell migration, differentiation, growth, inflammatory processes, neovascularization, wound healing, apoptosis, the uterine cycle and many other actions within the body, including those needed for tumorigenesis and other diseases. MMPs can therefore be used as potential markers for detecting various cancers, neurodegenerative, and immune and cardiovascular diseases. Numerous MMP assays were developed for clinical and research purposes, but far more attention has been devoted to understanding their biological functions. Due to differences in methodology, results obtained in various laboratory settings are difficult to compare because of the lack of standards and analytical methods of validation. Limits of detection of particular methods used for identifying MMPs are also disputable. Enzymatic, immunochemical and fluorimetric methods are particularly suitable for clinical use. In-vivo imaging methods offer many potential advantages in cancer research and diagnostics. Other methods are subject to investigation [e.g., phage display, multiple-enzyme/multiple-reagent assay system (MEMRAS) and activity-based profiling].

Guanosine derivatives are important for diagnosis of oxidative DNA damage including 8-hydroxy-2'-deoxyguanosine (8-OHdG) as one of the most abundant products of DNA oxidation. This compound is commonly determined in urine, which makes 8-OHdG a good non-invasive marker of oxidation stress. In this study, we optimized and tested the isolation of 8-OHdG from biological matrix by using paramagnetic particles with an antibody-modified surface. 8-OHdG was determined using 1-naphthol generated by alkaline phosphatase conjugated with the secondary antibody. 1-Naphthol was determined by stopped flow injection analysis (SFIA) with electrochemical detector using a glassy carbon working electrode and by stationary electrochemical detection using linear sweep voltammetry. A special modular electrochemical SFIA system which needs only 10 μL of sample including working buffer for one analysis was completely designed and successfully verified. The recoveries in different matrices and analyte concentration were estimated. Detection limit (3 S/N) was estimated as 5 pg/mL of 8-OHdG. This method promises to be very easily modified to microfluidic systems as "lab on valve". The optimized method had sufficient selectivity and thus could be used for determination of 8-OHDG in human urine and therefore for estimation of oxidative DNA damage as a result of oxidation stress in prostate cancer patients.

In this study, two methods for metallothioneins (MT) determination in a biological sample were compared. Particularly, twenty five human and nine pig blood serum samples and liver and kidney samples from thirty five carps (Cyprinus carpio) were analyzed by enzyme-linked immunosorbent assay and differential pulse voltammetry