Podstrona: Publikacje / Wizytówka pracownika PRz

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Publikacje naukowe:

2025

1. Świderski G., Kalinowska M., Zapora E., Wołkowycki M., Stocki M., Ciszkowicz E., Bocian A., Jaromin M., Tyrka M., Lecka-Szlachta K., Wołejko E., Wydro U., Pawłowska M., Golianek P., Zawadzka M., Ramshaj Q., Girometta C. E., Karadelev, M. (2025). Bioactive Properties of Selected European Phellinus Species: A Comprehensive Study. International Journal of Molecular Sciences, 26(16), 8013. https://doi.org/10.3390/ijms26168013  

This study conducted a multi-directional evaluation of the chemical potential and biological properties of selected European fungal species of the genus Phellinus. We investigated 30 samples belonging to 22 Phellinus species. Fruiting bodies were collected, among other specimens, in the Białowieża Forest (Poland); Village Kozhle (North Macedonia); Estremadura, Sesimbra, and Lagoa de Albufeira (Portugal); Zlatari close to Prishtina (Kosovo); and Spoleto and the Bosco Siro Negri State Nature Reserve (Italy). Morphological identification of the collected fungi was carried out, and genetic tests were performed to confirm the identity of the collected specimens. Methanol extracts for biological activity tests were prepared. Screening of antimicrobial activity of 30 methanolic extracts was performed on strains of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Kocuria rhizophila) and fungi (Candida albicans). Antioxidant activity tests (DPPH and ABTS) were also performed. The three most biologically active fungi species were selected (Phellinus igniarius, Fomitiporia robusta, and Porodaedalea pini) for further research. The chemical composition of the extracts was determined using GC-MS analysis. Thermal decomposition studies and spectroscopic analysis of the dry fruiting bodies were performed. The extracts were tested for their antimicrobial activity against antibiotic-resistant bacteria. Cytotoxic activity was also tested.

2. Ciszkowicz E., Miłoś A., Łyskowski A., Buczkowicz J., Nieczaj A., Lecka-Szlachta K., Hus K. K., Sikora K., Neubauer D., Bauer M., Kamysz W., Bocian, A. (2025). AMPEC4: Naja ashei Venom-Derived Peptide as a Stimulator of Fibroblast Migration with Antibacterial Activity. Molecules, 30(10), 2167. https://doi.org/10.3390/molecules30102167

The treatment of proctological conditions, including hemorrhoids, anal fissures, and perianal abscesses, is often complicated by bacterial infections, particularly those involving multidrug-resistant Escherichia coli. This study presents the synthesis, characterization, and biological evaluation of the newly designed synthetic peptide AMPEC4, inspired by cytotoxin 5 from Naja ashei snake venom. AMPEC4 demonstrated potent antimicrobial properties with MIC values of 100 and 200 µg/mL, effectively inhibiting biofilm formation (up to 84%) and eradicating the pre-formed biofilm by up to 35%. The antibacterial activity of AMPEC4 was further supported by a membrane permeabilization assay, demonstrating its capacity to disrupt bacterial membrane integrity in a dose-dependent manner. Furthermore, AMPEC4 significantly promoted fibroblast migration, a critical step in tissue regeneration, while exhibiting notable biocompatibility, as evidenced by the absence of hemolytic, cytotoxic, and genotoxic effects. By addressing both infection control and tissue regeneration, AMPEC4 represents a promising therapeutic strategy for managing chronic wounds, particularly in the challenging environment of the anorectal region. Its ability to target Escherichia coli reference and clinical strains while accelerating the wound-healing process underscores its potential for future clinical applications.

3. Świderski G., Kowalczyk N., Tyniecka G., Kalinowska M., Łyszczek R., Bocian A., Ciszkowicz E., Siergiejczyk L., Pawłowska M.,  Czerwiński J. (2025). Study of the Relationship Between the Structures and Biological Activity of Herbicides Derived from Phenoxyacetic Acid. Materials, 18(7), 1680. https://doi.org/10.3390/ma18071680

Chloroderivatives of phenoxyacetic acid are a group of compounds commonly used as plant protection products. Differences in the molecular structure of these compounds are related to varying substitution and the number of chlorine atoms in the aromatic ring. Different molecular structures may affect the activity of these compounds, their physicochemical properties, as well as their toxicity and biological effects. A group of 6 chemical compounds derived from phenoxyacetic acid was tested. The molecular structure was analysed using spectroscopic methods (FTIR, FTRaman, UV-VIS, ¹HNMR, ¹³CNMR) and quantum chemical computational methods (DFT). The reactivity of the tested compounds was determined using DFT calculations and experimentally in reaction with a hydroxyl radical. The electronic charge distribution of NBO, CHelpG and ESP was analysed and aromaticity indices were calculated for theoretically modeled structures and structures examined by X-ray diffraction (data obtained from the CSD database). Phenoxyacetic acid derivatives were tested for antimicrobial activity on soil bacterial strains. Cytotoxicity tests were performed on normal human skin fibroblasts (BJ CRL-2522) and the human prostate cancer cell line (DU-145 HTB-81). The purpose of this study was to investigate the relationship between the molecular structure of phenoxyacetic acid derivatives and their reactivity and biological activity.

4. Marczak B., Bocian A., Łyskowski A. (2025). Antimicrobial Peptide Databases as the Guiding Resource in New Antimicrobial Agent Identification via Computational Methods. Molecules 30, 1318. https://doi.org/10.3390/molecules30061318

In light of the growing interest in antimicrobial peptides (AMPs) as potential alternatives to traditional antibiotics, proteomic research has increasingly focused on this area. Addressing this significant scientific need, we undertook an initiative to review and analyze the available databases containing information on AMPs. These databases play a pivotal role as a foundation for most AMP-related studies, enabling not only the identification of new compounds, but also a deeper understanding of their properties and therapeutic potential. As part of this study, we evaluated the quality of information within selected AMP databases, considering their accessibility, content, and research potential. The initial step of the analysis involved a comparison of the per-database and cross-database peptide sequences. A diamond, high-throughput protein alignment program was used to compare the degree of sequence similarity among peptides across the individual databases. The redundancy of the data was also evaluated. Collected information was used for an in silico evaluation of the selected species’ venom proteomes in order to identify putative antimicrobial peptide candidates. An example candidate was further evaluated via a combination of structural analysis based on the computed homology based structural model, the in silico digestion of the source protein, and the antimicrobial potential.

5. Zapała L., Ciszkowicz E., Kosińska-Pezda M., Maciołek U., Kozioł A.E., Miłoś A., Woźnicka E., Bocian A., Zapała W., Rydel-Ciszek K., Perrone M.G. (2025). Novel silver(I) complexes with fenamates: Insights into synthesis, spectral characterization, and bioactivity. J Inorg Biochem 266, 112846. doi: 10.1016/j.jinorgbio.2025.112846

Six new Ag(I) ions complexes with N-phenylanthranilic, mefenamic, and niflumic acids have been synthesized. Three of them are binary complexes with the [Ag(L)] formula (where L represents N-phenylanthranilate (nfa), mefenamate (mfa), or niflumate (nif) ions), and the other three complexes are ternary with the formula [Ag(L)(phen)₂]⋅nH₂O (where phen - 1,10-phenanthroline). The complexes were characterized by elemental analysis, differential scanning calorimetry (DSC), X-ray fluorescence, powder X-ray diffraction, and single-crystal X-ray structure analysis. Additionally, techniques such as ESI-MS spectrometry, ¹H NMR, UV–Vis, and FTIR spectroscopy were employed. The X-ray crystallography showed that in the solid [Ag(nif)] complex, the cation showed an unusual structure with coordination number 5, i.e. AgO₃NC. The silver cation interacts with three niflumate anions, forming a two-dimensional coordination polymer. Complexes have potential antibacterial efficacy with varied minimum inhibitory concentration values (MIC) between 45.96 and 800 μM against multidrug-resistant Pseudomonas aeruginosa. Antibacterial combination therapy of Ag(I) complexes with chloramphenicol (CHL) and kanamycin (KAN) showed a very strong synergistic impact against P. aeruginosa with no cytotoxic effect on normal human fibroblasts. Complexes [Ag(nif)] and [Ag(nfa)] inhibit protein denaturation, bind to BSA via static quenching (k_q = 0.65–1.08 × 10¹³ M⁻¹ s⁻¹). Furthermore, the formation of these complexes enhances the penetration of the drug across human membrane monolayers, which could improve bioavailability and therapeutic potential. The [Ag(nif)] complex demonstrates significant potential for topical dermal application due to its antimicrobial and anti-inflammatory properties. Notably, among all complexes evaluated, it displays the lowest BA/AB ratio (5.41), facilitating the most efficient transdermal permeation.

2024

1. Dyba B., Rudolphi-Szydło E., Kreczmer B., Barbasz A., Petrilla V., Petrillova M., Legáth J., Bocian A., Hus K.K. (2024) Exploring the effects of three-finger toxins from Naja ashei venom on neuronal and immunological cancer cell membranes. Sci Rep 14(1), 18570. doi: 10.1038/s41598-024-69459-4

Three-finger proteins are the most abundant toxins in the venom of Naja ashei, a snake species from the Elapidae family. This research aimed to describe the effects of varying charges of these proteins, isolated from Naja ashei venom using SEC and IEX chromatography. The study examined how differently charged three-finger toxin fractions interact with and affect neuroblastoma (SK-N-SH) and promyeloblast (HL-60) cells, as well as model Langmuir membranes and liposomes designed to mimic cellular lipid composition. Findings revealed that protein surface charges significantly impact cell survival (MTT assay), membrane damage (lactate dehydrogenase release, malondialdehyde formation), and the structural and electrochemical properties of model membranes (Langmuir membranes and zeta potential for liposomes and cancer cell lines). Results indicated that SK-N-SH cells, characterized by a higher negative charge on their cell membranes, interacted more effectively with positively charged toxins than HL-60 cells. However, the mechanism of these electrostatic interactions is complex. The research demonstrated that electrostatic and mechanical membrane modifications induced by venom proteins can significantly affect cell metabolism. Additionally, the total charge of the membrane, influenced by polar lipid components and phospholipid saturation, plays a decisive role in toxin interaction.

2. Hus K.K., Buczkowicz J., Pietrowska M., Petrilla V., Petrillová M., Legáth J., Litschka-Koen T., Bocian A. (2024) Venom diversity in Naja mossambica: Insights from proteomic and immunochemical analyses reveal intraspecific differences. PLoS Negl Trop Dis 18(4), e0012057. doi: 10.1371/journal.pntd.0012057

Snakebite envenoming is a pervasive global health concern, posing substantial risks, particularly in less developed regions. The intricate variations in venom composition within a single species have been well-documented, contributing significantly to the varied clinical effects experienced by envenomed patients. It is imperative to unravel these variations, as they are pivotal in the formulation of effective snakebite management strategies and the development of targeted antivenom therapies.

In this study, our focus rested on the venom of the Naja mossambica species, dwelling in diverse African regions. Our objective was to delve into the toxin composition of these venoms and understand how these toxins interact with commercially available antivenoms. This exploration was aimed to uncover which toxins, despite antivenom application, evade neutralization. This information becomes a cornerstone in the design of more potent and efficacious antivenoms, contributing to a nuanced approach in combating the complex landscape of snakebite envenoming.

2023

1. Miłek M., Mołoń M., Kielar P., Sidor E., Bocian A., Marciniak-Lukasiak K., Pasternakiewicz A., Dżugan M. (2023) The Comparison of Honey Enriched with Laboratory Fermented Pollen vs. Natural Bee Bread in Terms of Nutritional and Antioxidant Properties, Protein In Vitro Bioaccessibility, and Its Genoprotective Effect in Yeast Cells. Molecules 28, 5851. https://doi.org/10.3390/molecules28155851

The aim of the study was to compare the nutritional value and bioactivity of honey enriched with a 10% addition of natural bee bread and its substitutes obtained as a result of laboratory fermentation of bee pollen. Physicochemical parameters, antioxidant properties, as well as the bioaccessibility of proteins using an in vitro static digestion model were analyzed. The bioactivity of the obtained enriched honeys was tested using the yeast model. The research indicates the similarity of honeys with the addition of “artificial bee bread” to honey with natural ones. During in vitro digestion, good bioaccessibility of the protein from the tested products was demonstrated. The ability of the products to protect yeast cells against hydrogen superoxide-induced oxidative stress was demonstrated using a qualitative spot test, which was stronger in the case of enriched honey than in pure rapeseed control honey. Significant inhibition of the growth of both strains of yeast exposed to bee pollen-enriched honeys was also demonstrated. Furthermore, all tested samples showed significant genoprotective activity against the genotoxic effect of zeocin and the reduction of the number of DNA double-strand breaks by a minimum of 70% was observed.

2. Dżugan M., Miłek M., Sidor E., Buczkowicz J., Hęclik J., Bocian A. (2023)  The Application of SDS-PAGE Protein and HPTLC Amino Acid Profiling for Verification of Declared Variety and Geographical Origin of Honey. Food Anal Methods 16, 1157–1171  https://doi.org/10.1007/s12161-023-02489-2

Proteins and amino acids are minor components of honey that are rarely used for its quality evaluation, although these components create its biological activity as well as can serve for overheating detection. The aim of the study was to use these indicators to confirm a declared on the label variety. Fifty-eight honey samples of 8 different varieties meeting the commercial quality requirements were used, including 28 local Polish and 30 commercial (mainly imported from EU and non-EU) honeys. For honey protein profiling previously used, polyacrylamide electrophoresis in denaturing conditions (SDS-PAGE) was applied whereas the free amino acid profile was analyzed by high-performance thin-layer chromatography (HPTLC) for the first time. As auxiliary indicators, the colorimetric determination of the protein content by the Bradford method, the activity of 5 glycolytic enzymes, including diastase, β-galactosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-glucosidase, and the content of proline were used. It has been shown that based on the determined model protein SDS-PAGE profiles for selected monofloral honeys, it is possible to detect honeys of questionable variety based on the lack of specific protein bands or their diverging intensity. The HPTLC amino acid analysis can serve as a supporting control tool, capturing differences in the amino acid profile. Due to a great variation of multifloral honey, such assays are effective for monofloral honey only. The colorimetric assays, especially for total protein and β-galactosidase, can be also useful. The applied tools can be proposed for the initial verification of honey variety for cost reduction of officially recognized melissopalynological analysis.

2022

1. Dżugan M., Miłek M., Kielar P., Stępień K., Sidor E.; Bocian A. (2022) SDS-PAGE Protein and HPTLC Polyphenols Profiling as a Promising Tool for Authentication of Goldenrod Honey. Foods 11, 2390. https://doi.org/10.3390/foods11162390

The aim of the study was to use protein and polyphenolic profiles as fingerprints of goldenrod honey and to apply them for verification of the labeled variety. The markers for 10 honey samples were correlated with the standard physicochemical parameters and biological activity measured in vitro as antioxidant, antifungal and antibacterial activities. Honey proteins were examined regarding soluble protein, diastase and SDS-PAGE protein profile. The polyphenolic profile was obtained with the use of the HPTLC and the antioxidant activity was detected with standard colorimetric methods. The antimicrobial effect of representative honey samples of different chemical profiles was verified against E. coli and budding yeast. It was found that the SDS-PAGE technique allows for creating the protein fingerprint of the goldenrod honey variety which was consistent for 70% of tested samples. At the same time, the similarity of their polyphenolic profile was observed. Moreover, specific chemical composition resulted in higher bioactivity of honey against tested bacteria and yeast. The study confirmed the usefulness of both SDS-PAGE and HPTLC techniques in honey authentication, as an initial step for selection of samples which required pollen analysis.

2. Manson E.Z., Kyama M.C., Kimani J., Bocian A., Hus K.K., Petrilla V., Legáth J., Kimotho J.H. (2022) Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity. Toxins 14, 285. https://doi.org/10.3390/toxins14040285

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.

3. Manson E.Z., Mutinda K.C., Gikunju J.K., Bocian A., Hus K.K., Petrílla V., Legáth J., Kimotho J.H. (2022) Development of an Inhibition Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detecting Cytotoxic Three-Finger Toxins (3FTxs) in African Spitting Cobra Venoms. Molecules 27, https://doi.org/10.3390/molecules27030888

The administration of toxin-specific therapy in snake envenoming is predicated on improved diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay (ELISA) has been shown to offer a wider practical application. We report an inhibition ELISA for detecting three-finger toxin (3FTx) proteins in venoms of African spitting cobras. The optimized assay detected 3FTxs in N. ashei (including other Naja sp.) venoms, spiked samples, and venom-challenged mice samples. In venoms of Naja sp., the assay showed inhibition, implying the detection of 3FTxs, but showed little or no inhibition in non-Naja sp. In mice-spiked samples, one-way ANOVA results showed that the observed inhibition was not statistically significant between spiked samples and negative control (p-value = 0.164). Similarly, the observed differences in inhibition between venom-challenged and negative control samples were not statistically significant (p-value = 0.9109). At an LOD of 0.01 µg/mL, the assay was able to confirm the presence of 3FTxs in the samples. Our results show a proof of concept for the use of an inhibition ELISA model as a tool for detecting 3FTxs in the venoms of African spitting cobra snakes.

4. Tomczyk M., Bocian A., Sidor E., Miłek M., Zaguła G., Dżugan M. (2022) The Use of HPTLC and SDS-PAGE Methods for Coniferous Honeydew Honey Fingerprinting Compiled with Mineral Content and Antioxidant Activity. Molecules 27, 720. https://doi.org/10.3390/molecules27030720

Fir honeydew honey is a uniquely beneficial product which is often subjected to adulteration; however, pollen analysis is not useful to verify this honey type. Fourteen samples of EU protected designation of origin fir honeydew honey gathered directly from apiaries were studied. Standards of legal requirements and additional parameters, i.e., specific optical rotation, mineral content, and antioxidant activity, were tested. Five nectar honeys of different varieties were used as a comparative material. HPTLC and SDS-PAGE methods were used to fingerprint the honey types. All honeys tested fulfilled the quality requirements in terms of water content, pH, total acidity, conductivity, HMF, and diastase number. They were defined as dark amber on the Pfund scale and exhibited positive specific rotation (+2.5 to 25). Honeydew honey surpassed the tested nectar honeys in terms of mineral content and antioxidant activity as well as total polyphenolic content, except for buckwheat honey. The sugar and polyphenolic profile obtained by HPTLC allowed to distinguish honeydew from nectar honeys. The same was achieved by SDS-PAGE protein profiling. Both techniques seem to be cheap and quick tools for precisely distinguishing honeydew honey.

2021

1. Dyba B., Rudolphi-Szydło E., Barbasz A., Czyżowska A., Hus K.K., Petrilla V., Petrillová M., Legáth J., Bocian A.(2021) Effects of 3FTx Protein Fraction from Naja ashei Venom on the Model and Native Membranes: Recognition and Implications for the Mechanisms of Toxicity. Molecules, 26, 2164. https://doi.org/10.3390/molecules26082164

Three-finger toxins are naturally occurring proteins in Elapidae snake venoms. Nowadays, they are gaining popularity because of their therapeutic potential. On the other hand, these proteins may cause undesirable reactions inside the body's cells. A full assessment of the safety of Naja ashei venom components for human cell application is still unknown. The aim of the study was to determine the effect of the exogenous application of three-finger toxins on the cells of monocytes (U-937) and promyelocytes (HL-60), with particular emphasis on the modification of their membranes under the influence of various doses of 3FTx protein fraction (0-120 ng/mL). The fraction exhibiting the highest proportion of 3FTx proteins after size exclusion chromatography (SEC) separation was used in the experiments. The structural response of cell membranes was described on the basis of single-component and multi-component Langmuir monolayers that mimicked the native membranes. The results show that the mechanism of protein-lipid interactions depends on both the presence of lipid polar parts (especially zwitterionic type of lipids) and the degree of membrane saturation (the greatest-for unsaturated lipids). The biochemical indicators reflecting the tested cells (MDA, LDH, cell survival, induction of inflammation, LD50) proved the results that were obtained for the model.

2. Miłek M., Bocian A., Kleczyńska E., Sowa P., Dżugan M. (2021) The Comparison of Physicochemical Parameters, Antioxidant Activity and Proteins for the Raw Local Polish Honeys and Imported Honey Blends. Molecules 26, 2423. https://doi.org/10.3390/molecules26092423

Many imported honeys distributed on the Polish market compete with local products mainly by lower price, which can correspond to lower quality and widespread adulteration. The aim of the study was to compare honey samples (11 imported honey blends and 5 local honeys) based on their antioxidant activity (measured by DPPH, FRAP, and total phenolic content), protein profile obtained by native PAGE, soluble protein content, diastase, and acid phosphatase activities identified by zymography. These indicators were correlated with standard quality parameters (water, HMF, pH, free acidity, and electrical conductivity). It was found that raw local Polish honeys show higher antioxidant and enzymatic activity, as well as being more abundant in soluble protein. With the use of principal component analysis (PCA) and stepwise linear discriminant analysis (LDA) protein content and diastase number were found to be significant (p < 0.05) among all tested parameters to differentiate imported honey from raw local honeys.

2020

1. Hus K.K., Marczak Ł., Petrílla , Petrillova M., Legáth J. Bocian A. (2020). Different research approaches in unraveling the venom proteome of Naja ashei. Biomolecules 10(9), 1282; https://doi.org/10.3390/biom10091282

The dynamic development of venomics in recent years has resulted in a significant increase in publicly available proteomic data. The information contained therein is often used for comparisons between different datasets and to draw biological conclusions therefrom. In this article, we aimed to show the possible differences that can arise, in the final results of the proteomic experiment, while using different research workflows. We applied two software solutions (PeptideShaker and MaxQuant) to process data from shotgun LC-MS/MS analysis of Naja ashei venom and collate it with the previous report concerning this species. We were able to provide new information regarding the protein composition of this venom but also present the qualitative and quantitative limitations of currently used proteomic methods. Moreover, we reported a rapid and straightforward technique for the separation of the fraction of proteins from the three-finger toxin family. Our results underline the necessary caution in the interpretation of data based on a comparative analysis of data derived from different studies.

2. Kuna E., Bocian A., Hus, K.K., Petrilla V., Petrillova M., Legath J., Lewinska A., Wnuk M. (2020) Evaluation of Antifungal Activity of Naja pallida and Naja mossambica Venoms against Three Candida Species. Toxins 12, 500. doi: 10.3390/toxins12080500

In contrast to comprehensively investigated antibacterial activity of snake venoms, namely crude venoms and their selected components, little is known about antifungal properties of elapid snake venoms. In the present study, the proteome of two venoms of red spitting cobra Naja pallida (NPV) and Mozambique spitting cobra Naja mossambica (NMV) was characterized using LC-MS/MS approach, and the antifungal activity of crude venoms against three Candida species was established. A complex response to venom treatment was revealed. NPV and NMV, when used at relatively high concentrations, decreased cell viability of C. albicans and C. tropicalis, affected cell cycle of C. albicans, inhibited C. tropicalis-based biofilm formation and promoted oxidative stress in C. albicans, C. glabrata and C. tropicalis cells. NPV and NMV also modulated ammonia pulses during colony development and aging in three Candida species. All these observations provide evidence that NPV and NMV may diminish selected pathogenic features of Candida species. However, NPV and NMV also promoted the secretion of extracellular phospholipases that may facilitate Candida pathogenicity and limit their usefulness as anti-candidal agents. In conclusion, antifungal activity of snake venoms should be studied with great caution and a plethora of pathogenic biomarkers should be considered in the future experiments.

3. Bocian A., Sławek S., Jaromin M., Hus K. K., Buczkowicz J., Łysiak D., Petrílla V., Petrillova M., Legáth L., Legáth J. (2020) Comparison of Methods for Measuring Protein Concentration in Venom Samples. Animals 10(3), 448. doi: 10.3390/ani10030448

Snake venom is mostly composed of proteins and peptides, which are of interest to many researchers due to their potential pharmacological properties. Due to their biochemical character, these components are analyzed using proteomic techniques such as electrophoresis, chromatography and mass spectrometry. A very important stage of such studies is the measurement of protein concentration in the sample, which is most often performed by colorimetric methods. In the presented article, we used five such techniques on venoms of two snake species, namely Agkistrodon contortrix and Naja ashei. In the case of A. contortrix venom, four methods provide similar concentration values, whereas, in the case of N. ashei, the differences between results are very significant. The source of these differences should probably be seen in the differences in amino acid composition of proteins of these two venoms. With this report, we would like to draw attention to the need to select an appropriate method for measuring the concentration of protein in the venom, especially in the case of Elapid species.

4. Bocian A., Ciszkowicz E., Hus K. K., Buczkowicz J., Lecka-Szlachta K., Pietrowska M., Petrílla V., Petrillova M., Legáth L., Legáth J. (2020) Antimicrobial activity of protein fraction from Naja ashei venom against Staphylococcus epidermidis. Molecules, 25(2), 293. doi: 10.3390/molecules25020293

One of the key problems of modern infectious disease medicine is the growing number of drug-resistant and multi-drug-resistant bacterial strains. For this reason, many studies are devoted to the search for highly active antimicrobial substances that could be used in therapy against bacterial infections. As it turns out, snake venoms are a rich source of proteins that exert a strong antibacterial effect, and therefore they have become an interesting research material. We analyzed Naja ashei venom for such antibacterial properties, and we found that a specific composition of proteins can act to eliminate individual bacterial cells, as well as the entire biofilm of Staphylococcus epidermidis. In general, we used ion exchange chromatography (IEX) to obtain 10 protein fractions with different levels of complexity, which were then tested against certified and clinical strains of S. epidermidis. One of the fractions (F2) showed exceptional antimicrobial effects both alone and in combination with antibiotics. The protein composition of the obtained fractions was determined using mass spectrometry techniques, indicating a high proportion of phospholipases A₂, three-finger toxins, and L-amino acids oxidases in F2 fraction, which are most likely responsible for the unique properties of this fraction. Moreover, we were able to identify a new group of low abundant proteins containing the Ig-like domain that have not been previously described in snake venoms.

5. Bocian A., Hus K.K. (2020) Antibacterial properties of snake venom components. Chemical Papers 74(2), 407-419. https://doi.org/10.1007/s11696-019-00939-y

An increasing problem in the field of health protection is the emergence of drug-resistant and multi-drug-resistant bacterial strains. They cause a number of infections, including hospital infections, which currently available antibiotics are unable to fight. Therefore, many studies are devoted to the search for new therapeutic agents with bactericidal and bacteriostatic properties. One of the latest concepts is to search for this type of substances among toxins produced by venomous animals. In this approach, however, special attention is paid to snake venom because it contains molecules with antibacterial properties. Thorough investigations have shown that the phospholipases A₂ (PLA₂) and l-amino acids oxidases (LAAO), as well as fragments of these enzymes, are mainly responsible for the bactericidal properties of snake venoms. Some preliminary research studies also suggest that fragments of three-finger toxins (3FTx) are bactericidal. It has also been proven that some snakes produce antibacterial peptides (AMP) homologous to human defensins and cathelicidins. The presence of these proteins and peptides means that snake venoms continue to be an interesting material for researchers and can be perceived as a promising source of antibacterial agents.

2019

1. Bocian A., Buczkowicz J., Jaromin M., Hus K.K., Legáth, J. (2019) An effective method of isolating honey proteins. Molecules 24 (13), 2399. doi: 10.3390/molecules24132399

Honey is a natural sweetener composed mostly of sugars, but it contains also pollen grains, proteins, free amino acids, and minerals. The amounts and proportions of these components depend on the honey type and bee species. Despite the low content of honey protein, they are becoming a popular study object, and have recently been used as markers of the authenticity and quality of honey. Currently, the most popular methods of protein isolation from honey are dialysis against distilled water, lyophilization of dialysate, or various precipitation protocols. In this work, we propose a new method based on saturated phenol. We tested it on three popular polish honey types and we proved its compatibility with both 1D and 2D polyacrylamide gel electrophoresis (PAGE) and MS (mass spectrometry) techniques. The elaborated technique is also potentially less expensive and less time-consuming than other previously described methods, while being equally effective.

2. Lewinska A., Bocian A., Petrilla V., Adamczyk-Grochala J., Szymura K., Hendzel W., Kaleniuk E., Hus K.K., Petrillova M., Wnuk M. (2019) Snake venoms promote stress-induced senescence in human fibroblasts. J Cell Physiol 234(5),6147-6160. doi: 10.1002/jcp.27382

Snake venoms are widely studied in terms of their systemic toxicity and proteolytic, hemotoxic, neurotoxic, and cytotoxic activities. However, little is known about snake-venom-mediated effects when used at low, noncytotoxic concentrations. In the current study, two human fibroblast cell lines of different origin, namely WI-38 fetal lung fibroblasts and BJ foreskin fibroblasts were used to investigate snake-venom-induced adaptive response at a relatively noncytotoxic concentration (0.01 µg/ml). The venoms of Indochinese spitting cobra ( Naja siamensis), western green mamba ( Dendroaspis viridis), forest cobra ( Naja melanoleuca), and southern copperhead ( Agkistrodon contortrix) were considered. Snake venoms promoted FOXO3a-mediated oxidative stress response and to a lesser extent DNA damage response, which lead to changes in cell cycle regulators both at messenger RNA and protein levels, limited cell proliferation and migration, and induced cellular senescence. Taken together, we have shown for the first time that selected snake venoms may also exert adverse effects when used at relatively noncytotoxic concentrations.

3. Hus K.K., Bocian A. (2019) Potencjał farmakologiczny składników jadu węży. Kosmos, 68 (1), 57-64

Spośród wszystkich zwierząt jadowitych i produkujących toksyny węże należą do grupy zwierząt dysponujących najbardziej złożonym jadem. W jego skład wchodzi ponad 100 różnych komponentów, do których należą zarówno toksyczne, jak i nietoksyczne białka i peptydy, a także małocząsteczkowe węglowodany, lipidy, aminy i kationy metali. Ze względu na specyficzność działania i rodzaj atakowanych tkanek wyróżnia się jady działające cytotoksycznie, hemotoksycznie, neurotoksycznie i miotoksycznie. Skład jadu różni się nie tylko pomiędzy grupami systematycznymi, ale także zmienia się pod wpływem licznych czynników m.in. wieku, płci czy rodzaju spożywanego pokarmu. Przedstawiony powyżej podział został dokonany na podstawie klinicznych objawów ukąszenia, jednak w rzeczywistości większość jadów zawiera składniki klasyfikujące się do wszystkich wymienionych kategorii. Nierzadko też jedno białko może wykazywać więcej niż jedną funkcję, w zależności od tego czy działa samodzielnie, czy z innymi białkami. Niektóre białka jadu są enzymami, inne nie mają aktywności katalitycznej, ale potrafią np. bardzo specyficznie wiązać się z określonymi receptorami, jeszcze inne działają hamująco na wzrost bakterii. Szeroki wachlarz właściwości oraz równie szerokie spektrum działania białek i peptydów pochodzących z jadu sprawia, że jad jest niezwykle interesującym materiałem badawczym o wysokim potencjale farmakologicznym. Obecnie dostępne są już na rynku produkty lecznicze opracowane na bazie składników jadu, a koronnym przykładem takiego leku jest captopril.

4. Hus K.K., Bocian A. (2019) Dlaczego jady węży wywołują krwotoki? Krótka historia metaloproteinaz z jadów węży. Kosmos, 68 (3), 409-420

Każdego roku w wyniku ukąszeń węży na całym świecie umiera około 100.000 ludzi, a co najmniej cztery razy tyle zostaje poważnie okaleczonych. Do najczęstszych objawów obserwowanych po ugryzieniach przez węże z rodziny żmijowatych zalicza się m.in. krwotoki lokalne i ogólnosystemowe. Bezpośrednią przyczyną ich powstawania są metaloproteinazy z jadów węży (SVMPs) i niniejsza praca podsumowuje dotychczas zebraną wiedzę o tych białkach. Ponadto, prezentuje najnowsze hipotezy dotyczące przyczyn powstawania całego procesu, z uwzględnieniem mechanizmów zachodzących na poziomie molekularnym i makroskopowym. Artykuł opowiada również historię odkryć i postępów, które na przestrzeni lat stopniowo wzbogacały wiedzę, ostatecznie prowadząc do zrozumienia przyczyn i skutków aktywności krwotocznej metaloproteinaz z jadów węży.

2018

1. Hus K.K., Buczkowicz J., Petrilla V., Petrillová M., Łyskowski A., Legáth J., Bocian A.(2018) First look at the venom of Naja ashei. Molecules 23 (3), 609. doi: 10.3390/molecules23030609

Naja ashei is an African spitting cobra species closely related to N. mossambica and N. nigricollis. It is known that the venom of N. ashei, like that of other African spitting cobras, mainly has cytotoxic effects, however data about its specific protein composition are not yet available. Thus, an attempt was made to determine the venom proteome of N. ashei with the use of 2-D electrophoresis and MALDI ToF/ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry techniques. Our investigation revealed that the main components of analysed venom are 3FTxs (Three-Finger Toxins) and PLA₂s (Phospholipases A₂). Additionally the presence of cysteine-rich venom proteins, 5'-nucleotidase and metalloproteinases has also been confirmed. The most interesting fact derived from this study is that the venom of N. ashei includes proteins not described previously in other African spitting cobras-cobra venom factor and venom nerve growth factor. To our knowledge, there are currently no other reports concerning this venom composition and we believe that our results will significantly increase interest in research of this species.

2017

1. Ciura J., Bocian A., Kononiuk A., Szeliga M., Jaromin M., Tyrka M. (2017) Proteomic signature of fenugreek treated by methyl jasmonate and cholesterol. Acta Physiologiae Plantarum 39 (5), 112. doi:10.1007/s11738-017-2416-7

In this study, the fenugreek plants were treated with methyl jasmonate (as an elicitor) and cholesterol (as a precursor of steroids and steroidal saponins) to check reaction at the level of the proteome to stress and to investigate steroidal saponin (diosgenin) biosynthesis. Proteins were separated by two-dimensional electrophoresis (2-DE) and identified by MALDI-ToF/ToF followed by database searches using Mascot search engine. Totally, 63 and 41 protein spots were differentially expressed after methyl jasmonate and cholesterol treatment, respectively. These proteins were classified into seven groups: photosynthesis, energy, metabolism, protein metabolism, secondary metabolism, stress and defense, and other. We found that 9 proteins were responsive to all treatments, and 18 proteins expressed in treatment-specific manner. Higher level of photosynthetic proteins sensitive to both biotic and abiotic stimuli was detected. In addition, proteins related to the stress (especially oxidative) and defense, protein, and secondary metabolism were overexpressed. The results indicate that methyl jasmonate and cholesterol elicited a defense reaction at the proteome level as a response to stress. The usefulness of 2-DE method for identification of proteins related with species-specific metabolic pathways is restricted. Integration of transcriptome data with proteomic analysis improved annotation process.

2. Bocian A., Hus K., Jaromin M., Tyrka M., Łyskowski A. (2017). Identification of proteins differentially accumulated in Enterococcus faecalis under acrylamide exposure. Turk J Biol 41, 166-177. doi: 10.3906/biy-1606-23

Analysis of bacterial proteomes can be used to obtain large amounts of information about adaptive microbial mechanisms to changing extracellular conditions. In the past, many bacterial species with the ability to degrade acrylamide were isolated. In this study differences in the Enterococcus faecalis proteome upon acrylamide exposure were investigated. We revealed substantial changes in the proteome of bacteria cultured in different environmental conditions. Microorganisms exposed to acrylamide showed higher accumulation of proteins associated with energy metabolism and its regulation. Moreover, several proteins involved in protection of cells from stress conditions were also identified. These biomacromolecules are involved in proper folding of newly synthesized polypeptides like chaperones or participate in mechanisms of DNA repair. In contradiction with previous reports, the presence of amidase was not detected. However, identification of aminopeptidase with activity to hydrolyze amino acid amides can indicate that it can degrade acrylamide to acrylic acid and ammonia instead of amidase. According to identified proteomic profiles, a new mechanism of acrylamide degradation by Enterococcus faecalis is proposed.

3. Hus K. K., Ossoliński K., Jaromin M., Ossoliński T., Ossolińska A., Dobrowolski Z., Groszek G., Bocian A., Łyskowski A. (2017). Comparison of protein isolation methods from clear cell Renal Cell Carcinoma tissue. MicroMed 5(1),  12. doi: http://dx.doi.org/10.5281/zenodo.572500

This study presents the comparison of three protein extraction methods in the investigations of clear cell Renal Cell Carcinoma tissue. For protein isolation, we applied: phenol extraction according to Hurkman and Tanaka (1986) protocol (method 1), whole tissue lysis in urea-containing buffer (method 2) and commercially available protein isolation kit (2-D Clean-up Kit) (method 3). Statistical analysis indicated that the additional preparation steps including extraction and purification of proteins by 2-D Clean-up Kit significantly increased the quality of obtained data. We believe that gathered information could be a valuable lead for researchers involved in proteomic studies of renal tissue.

4. Hus K., Bocian A. (2017) Mechanizmy adaptacyjne umożliwiające życie bakterii w wysokich temperaturach. Kosmos 66, 2

Z antropocentrycznego punktu widzenia, środowiska cechujące się wysokimi temperaturami opisywane są jako ekstremalne. Pierwotnie uważano, że są one zbyt niekorzystne dla rozwoju życia, jednakże wiele badań naukowych dowiodło, iż istnieje spora grupa mikroorganizmów, które mogą przetrwać w tak trudnych warunkach. Jednakże aby było to możliwe, organizmy te wykształciły wiele mechanizmów i strategii ochrony komórki przed niekorzystnymi warunkami środowiska. Zaliczyć tu można: produkcję białek szoku cieplnego, stabilizację struktury DNA, błyskawiczną resyntezę ATP, aminokwasów i innych termolabilnych składników komórki, syntezę trehalozy i innych cząsteczek stabilizujących struktury komórkowe, zwiększoną syntezę specyficznych proteaz, zastąpienie nukleotydów nikotynamidowych przez stabilniejszą ferredoksynę czy zmianę ekspresji genów w komórce. Enzymy produkowane przez mikroorganizmy termofilne są obecnie źródłem intensywnych badań, głównie ze względu na swoje wyjątkowe właściwości i szerokie zastosowanie w przemyśle.

2016

1. Bocian A., Urbanik M., Hus K., Łyskowski A., Petrilla V., Andrejcakova Z., Petrillova M., Legath J. (2016) Proteomic analyses of Agkistrodon contortrix contortrix venom using 2D electrophoresis and MS techniques. Toxins 8, 372. doi: 10.3390/toxins8120372

Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A₂ and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5′-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor.

2. Bocian A., Urbanik M., Hus K., Łyskowski A., Petrilla V., Andrejcakova Z., Petrillova M., Legath J. (2016) Proteome and peptidome of Vipera berus berus venom. Molecules 21, 1398. doi: 10.3390/molecules21101398

Snake venom is a rich source of peptides and proteins with a wide range of actions. Many of the venom components are currently being tested for their usefulness in the treatment of many diseases ranging from neurological and cardiovascular to cancer. It is also important to constantly search for new proteins and peptides with properties not yet described. The venom of Vipera berus berus has hemolytic, proteolytic and cytotoxic properties, but its exact composition and the factors responsible for these properties are not known. Therefore, an attempt was made to identify proteins and peptides derived from this species venom by using high resolution two-dimensional electrophoresis and MALDI ToF/ToF mass spectrometry. A total of 11 protein classes have been identified mainly proteases but also l-amino acid oxidases, C-type lectin like proteins, cysteine-rich venom proteins and phospholipases A₂ and 4 peptides of molecular weight less than 1500 Da. Most of the identified proteins are responsible for the highly hemotoxic properties of the venom. Presence of venom phospholipases A₂ and l-amino acid oxidases cause moderate neuro-, myo- and cytotoxicity. All successfully identified peptides belong to the bradykinin-potentiating peptides family. The mass spectrometry data are available via ProteomeXchange with identifier PXD004958.

3. Kononiuk A., Bocian A., Karwowska M., Drzazga T. (2016) Białkowe markery gatunkowe jako potencjalne narzędzie molekularne do kontroli autentyczności produktów orkiszowych. ŻYWNOŚĆ Nauka. Technologia. Jakość 5 (108), 95

Coraz większe zainteresowanie żywnością ekologiczną powoduje wzrost popularności produktów pochodzących z ziarna pszenicy orkisz, które charakteryzują się dobrymi właściwościami żywieniowymi. Ze względu na duże podobieństwo pszenicy zwyczajnej i pszenicy orkisz występują trudności z ich odróżnieniem w produktach mącznych, celowe jest więc poszukiwanie markerów gatunkowych umożliwiających ich zróżnicowanie. W pracy podjęto próbę rozróżnienia pszenicy zwyczajnej (Triticum aestivum) i pszenicy orkisz (Triticum spelta) na podstawie analiz elektroforetycznych białek zapasowych występujących w ziarniakach. Materiał badawczy stanowiły ziarniaki 10 linii hodowlanych pszenicy zwyczajnej oraz 8 linii hodowlanych pszenicy orkisz. Do izolacji białek zastosowano metody frakcjonowania białek zapasowych. Następnie przeprowadzono rozdziały elektroforetyczne kolejnych frakcji białek: albumin i globulin, gliadyn oraz glutenin. Po zakończonej elektroforezie żele wybarwiono i dokonano porównania profili białkowych uzyskanych z T. aestivum i T. spelta w celu identyfikacji potencjalnych markerów białkowych. Uzyskane wyniki nie pozwalają na jednoznaczne określenie występowania białkowego markera odróżniającego pszenicę zwyczajną od pszenicy orkisz. Niemniej jednak mogą stanowić podstawę do rozważań dotyczących wykorzystania bardziej zaawansowanych technik elektroforetycznych (A-PAGE i elektroforezy 2D) do opracowania markerów. Przedstawione wyniki mogą pomóc w opracowaniu metody identyfikacji podjednostek białkowych specyficznych dla pszenicy orkisz, ułatwiającej wykrywanie zafałszowań żywności.

2015

1. Bocian A., Zwierzykowski Z., Rapacz M., Koczyk G., Ciesiołka D., Kosmala A. (2015). Metabolite profiling during cold acclimation of Lolium perenne genotypes distinct in the level of frost tolerance. J Appl Genet 56(4), 439-449. doi: 10.1007/s13353-015-0293-6

Abiotic stresses, including low temperature, can significantly reduce plant yielding. The knowledge on the molecular basis of stress tolerance could help to improve its level in species of relatively high importance to agriculture. Unfortunately, the complex research performed so far mainly on model species and also, to some extent, on cereals does not fully cover the demands of other agricultural plants of temperate climate, including forage grasses. Two Lolium perenne (perennial ryegrass) genotypes with contrasting levels of frost tolerance, the high frost tolerant (HFT) and the low frost tolerant (LFT) genotypes, were selected for comparative metabolomic research. The work focused on the analysis of leaf metabolite accumulation before and after seven separate time points of cold acclimation. Gas chromatography-mass spectrometry (GC/MS) was used to identify amino acids (alanine, proline, glycine, glutamic and aspartic acid, serine, lysine and asparagine), carbohydrates (fructose, glucose, sucrose, raffinose and trehalose) and their derivatives (mannitol, sorbitol and inositol) accumulated in leaves in low temperature. The observed differences in the level of frost tolerance between the analysed genotypes could be partially due to the time point of cold acclimation at which the accumulation level of crucial metabolite started to increase. In the HFT genotype, earlier accumulation was observed for proline and asparagine. The increased amounts of alanine, glutamic and aspartic acids, and asparagine during cold acclimation could be involved in the regulation of photosynthesis intensity in L. perenne. Among the analysed carbohydrates, only raffinose revealed a significant association with the acclimation process in this species.

2013

1. Kosmala A., Bocian A., Rapacz M., Jurczyk B., Marczak Ł., Zwierzykowski Z. (2013) Similarities and Differences in Leaf Proteome Response to Cold Acclimation Between Festuca pratensis and Lolium perenne. Breeding strategies for sustainable forage and turf grass improvement, 01/2013; ISBN: 978-94-007-4554-4.

Lolium perenne is used extensively as a forage and turf grass due to its high nutritive values, rapid establishment and persistence. Festuca pratensis, a species closely related to L. perenne, has lower nutritive values but is one of the most winter-hardy species within the Lolium-Festuca complex. Frost tolerance is the main component of winter-hardiness and the plant species growing in temperate regions can acquire it through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation. Herein, we review our recent results of two proteomic projects, focused on F. pratensis and L. perenne. Each project involved the comparison of leaf protein accumulation profiles during cold acclimation between plants with different levels of frost tolerance by the use of two-dimensional electrophoresis and identification of differentially accumulated proteins by mass spectrometry. In the present paper similarities and differences in leaf proteome response to cold acclimation between F. pratensis and L. perenne are summarized.

2011

1. Bocian A., Kosmala A., Rapacz M., Jurczyk B., Marczak Ł., Zwierzykowski Z. (2011). Differences in leaf proteome response to cold acclimation between Lolium perenne plants with distinct levels of frost tolerance. J Plant Physiol 168, 1271-1279. doi: 10.1016/j.jplph.2011.01.029

Perennial ryegrass (Lolium perenne) is a high quality forage and turf grass mainly due to its excellent nutritive values and rapid establishment rate. However, this species has limited ability to perform in harsh winter climates. Though winter hardiness is a complex trait, it is commonly agreed that frost tolerance (FT) is its main component. Species growing in temperate regions can acquire FT through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation (CA). The research on molecular basis of FT has been performed on the model plants, but they are not well adapted to extreme winter climates. Thus, the mechanisms of cell response to low temperature in winter crops and agronomically important perennial grasses have yet to be revealed. Here, two L. perenne plants with contrasting levels of FT, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on analyses of leaf protein accumulation before and after 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA, using a high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry (MS). Analyses of 580 protein profiles revealed a total of 42 (7.2%) spots that showed at a minimum of 1.5-fold differences in protein abundance, at a minimum of at one time point of CA between HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analyzed plants appeared most often on the 5th (18 proteins) and the 7th (19 proteins) day of CA. The proteins derived from 35 (83.3%) spots were successfully identified by the use of MS and chloroplast proteins were shown to be the major group selected as differentially accumulated during CA. The functions of the identified proteins and their probable influence on the level of FT in L. perenne are discussed.

2009

1. Kosmala A., Bocian A., Rapacz M., Jurczyk B., Zwierzykowski Z. (2009). Identification of leaf proteins differentially accumulated during cold acclimation between Festuca pratensis genotypes plants with distinct levels of frost tolerance. J Exp Bot 60, 3595-3609. doi: 10.1093/jxb/erp205

Festuca pratensis (meadow fescue) as the most frost-tolerant species within the Lolium–Festuca complex was used as a model for research aimed at identifying the cellular components involved in the cold acclimation (CA) of forage grasses. The work presented here also comprises the first comprehensive proteomic research on CA in a group of monocotyledonous species which are able to withstand winter conditions. Individual F. pratensis plants with contrasting levels of frost tolerance, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on the analysis of leaf protein accumulation before and after 2, 8, and 26 h, and 3, 5, 7, 14, and 21 d of CA, using high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry. The analyses of approximately 800 protein profiles revealed a total of 41 (5.1%) proteins that showed a minimum of a 1.5-fold difference in abundance, at a minimum of one time point of CA for HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analysed plants appeared relatively early during cold acclimation, most often after 26 h (on the 2nd day) of CA and one-half of the differentially accumulated proteins were all parts of the photosynthetic apparatus. Several proteins identified here have been reported to be differentially accumulated during cold conditions for the first time in this paper. The functions of the selected proteins in plant cells and their probable influence on the level of frost tolerance in F. pratensis, are discussed.

2. Kosmala A., Bocian A., Zwierzykowski Z. (2009). Activities of proteolytic enzymes during cold acclimation of Festuca pratensis. W: P. Krajewski, P. Kachlicki, B. Naganowska (red.), Genetyka i Genomika w Doskonaleniu Roślin Uprawnych - od Rośliny Modelowej do Nowej Odmiany. Instytut Genetyki Roślin Polskiej Akademii Nauk. Seria: Rozprawy i monografie Nr 18: 131-137

2008

1. Kosmala A., Bocian A., Rapacz M., Wolanin B., Kachlicki P., Zwierzykowski Z. (2008). Analysis of Festuca pratensis proteom during cold acclimation. In: J. Prohens and M.L. Badens (eds), Modern Variety Breeding for Present and Future Needs, pp. 408-412. Editorial Universidad Politécnica de Valencia, Valencia, Spain. [ISBN: 978-84-8363-302-1]

2007

1. Kalinowski A., Bocian A., Kosmala A., Winiarczyk K. (2007). Two-dimensional patterns of soluble proteins including three hydrolytic enzymes of mature pollen of tristylous Lythrum salicaria. Sex Plant Reprod 20, 51- 62. doi: https://doi.org/10.1007/s00497-006-0042-4

Lythrum salicaria, now a widespread invasive species, exhibits tristyly, a form of heteromorphic selfincompatibility. In tristyly, each plant exhibits one (and only one) of three morphologically different floral forms. Moreover, each flower produces two types of stamens, and these two exhibit different incompatibility reactions. Differences between stamens of a single flower must be the result of epigenetic phenomena and for that reason, we performed two-dimensional gel electrophoresis (2-DE) to analyze fractions of soluble proteins derived from the pollen coat and protoplast including three hydrolytic enzymes from the six different stamen types (two from each of three floral forms). There were significant differences in the 2-D protein profiles both between pollen from the same flower and between the same type of pollen from two different flowers, in the pollen coat as well as in the protoplast extracts. In five of the six samples of pollen fractions, characteristic peptides were found. Quantitative differences between pollen from the same flower were observed in case of esterases. Furthermore, analysis of proteases and acid phosphatases revealed also qualitative differences between these enzymes in pollen from the same flower.

2006

1. Kalinowski A., Radłowski M., Bocian A. (2006) Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants of Lolium multiflorum Lam. J Appl Genet 47 (4), 319- 329. doi: 10.1007/BF03194641

Two-dimensional electrophoresis (2-DE) of soluble proteins and enzymes was performed and specific activities of 5 enzymes (esterase, pectinesterase, acid phosphatase, protease and diaphorase) were determined in stigmas ofLolium multiflorum (Italian ryegrass) treated with self or foreign pollen coat eluates (pc). Also, a low-molecular-weight fraction of the treated self-compatible (SC) and self-incompatible (SI) stigmas was analyzed by high-pressure liquid chromatography (HPLC). The treatment of stigmas with foreign pollen induced the loss of 42% of the control sample proteins in SC plants but only of 5.5% in SI plants. In contrast, the treatment of stigmas with foreign pollen induced the loss of 15% proteins in SC plants and of 29% in SI plants. Specific activities of esterase, pectinesterase and diaphorase were higher in SC than in SI stigmas. The 2-DE enzyme patterns indicated qualitative relationships between the presence of some isoforms of acid phosphatase or protease and the treatment with self or foreign pc in SC and SI stigmas. No changes were observed in HPLC profiles of the low-molecular-weight fraction from SC and SI stigmas treated or not with pc. The presented results revealed different reactions of SC and SI stigmas to the treatment with self or foreign pc. Further investigations may explain if any of the observed reactions represent specific reorientations in the style, facilitating cross- or self-pollination.

2004

1. Kalinowski A., Bocian A., Borzyszkowska E. (2004). Wpływ eluatu płaszcza pyłku na białka znamion u samozgodnych i samoniezgodnych roślin Festuca pratensis Huds. W: P. Krajewski, Z. Zwierzykowski, P. Kachlicki (red.), Genetyka w ulepszaniu roślin użytkowych. Instytut Genetyki Roślin Polskiej Akademii Nauk. Seria: Rozprawy i Monografie Nr 11: 171-178.

Wystąpienia konferencyjne (wykłady):

1. Wykorzystanie technik proteomicznych w badaniach żywności, w ramach cyklu wykładów otwartych „Jakość żywności – świadome odżywianie” realizowanego przez Podkarpacki Oddział PTTŻ w Instytucie Technologii Żywności i Żywienia Uniwersytetu Rzeszowskiego, Rzeszów, 12 grudnia 2019.
2. Analysis of the antibacterial properties of Naja ashei venom components", 13^th Central and Eastern European Conference, Ustroń, 23-25 września 2019.
3. Biotechnologiczny potencjał jadu węży, KonTeCh Konferencja Technologii Chemicznej i Biotechnologii, Wrocław, 9-10 czerwca 2018.
4. Biologiczne i molekularne aspekty toksyczności jadu węży, seminarium naukowe w Instytucie Genetyki Roślin PAN w Poznaniu, 13 października 2017.
5. Proteomiczne badania jadu węży tropikalnych, w ramach ogólnopolskiego seminarium "Zastosowanie proteomiki i metod analizy danych w badaniach biologicznych", Gliwice, 4-5 czerwca 2014.
6. Identyfikacja genów związanych z aklimatyzacją do chłodu i odpornością na mróz u życicy trwałej (Lolium perenne). III Sesja Sprawozdawcza z realizacji projektu badawczego zamawianego „Nowe metody genetyki molekularnej i genomiki służące doskonaleniu odmian roślin uprawnych”, Falenty, 17-20 stycznia 2011.
7. Analiza proteomu Lolium perenne w trakcie hartowania na mróz. II Sesja Sprawozdawcza z realizacji projektu badawczego zamawianego „Nowe metody genetyki molekularnej i genomiki służące doskonaleniu odmian roślin uprawnych”, Zakopane, 8-13 lutego 2010.
8. The proteins involved in cold acclimation of perennial ryegrass (Lolium perenne L.). 28^th Meeting of the Fodder Crops and Amenity Grasses Section of EUCARPIA, La Rochelle, France, 11-14 May 2009.

Wystąpienia konferencyjne (Postery):

1. Bocian A.,  Hus K.K., Buczkowicz J., Petrílla V., Legáth J. (2024) hat we have discovered about Naja ashei venom so far… 2nd European Venom Network Congress, Neapol, 23-25.09. 2024
   

2. Bocian A.,  Hus K.K., Buczkowicz J., Petrílla V., Legáth J. (2022) 5 years of delving into the Naja ashei venom proteome, 16th Central and Eastern European Proteomic Conference, Praga, 29.09.2022 - 1.10.2022
   

3. Bocian A., Buczkowicz J., Hus K.K., Petrílla V., Legáth J. (2022) Interaction studies of Naja ashei venom proteins with commercially available antivenom. 9th International Toxicology Meeting Venoms and Toxins 2022, Oxford, 23rd – 25th August 2022 
   

4. Hus K.K., Marczak Ł., Petrílla V., Petrillová M., Legáth J., Bocian A. (2021). How your research strategy can influence your data. 1^st International Congress European Venom Network, on-line, 14-16 September 2021

5. Bocian A., Hus K.K., Buczkowicz J., Petrílla V., Petrillová M., Legáth J. (2021). Enzymatic activity of phospholipases A2 from Naja ashei venom. 1^st International Congress European Venom Network, on-line, 14-16 September 2021

6. Ciszkowicz E., Bocian A., Miłoś A., Hus K.K., Buczkowicz J., Petrílla V., Petrillová M., Legáth L., Legáth J. (2021) Cytotoxicity of Phospholipases A2 and 3FTxs from Naja ashei venom. 1^st International Congress European Venom Network, on-line, 14-16 September 2021

7. Bocian A., Hus K. K., Petrilla V., Legáth J. (2017). Proteomic research of African elapid snakes venom. 11^th Central and Eastern European Proteomic Conference, Koszyce, 27-29 September 2017.
8. Hus K. K., Bocian A., Petrilla V., Legath J., Łyskowski (2017). Comparative analysis of the proteomic composition of the Vipera berus berus venom obtained by different analytical sampling. 11^th Central and Eastern European Proteomic Conference, Koszyce, 27-29 September 2017.
9. Hus K.K., Bocian A., Petrilla V., Buczkowicz J., Semik M., Legath J. (2017). Discovering the venom protein profiles of Naja species using proteomic tools. 11^th Central and Eastern European Proteomic Conference, Koszyce, 27-29 September 2017.
10. Hus K.K., Buczkowicz J., Łyskowski A., Petrilla.V., Petrillova M., Legath J., Bocian A. (2017). Preliminary analysis of Naja ashei venom proteome in terms of its pharmacological potential. EUROBIOTECH 6^th Central European Congress of Life Science, Kraków, 11-14 September 2017.
11. Sowa P., Bocian A., Hus K.K., Dżugan M. (2017). The comparison of protein profiles of selected monofloral honeys using SDS-PAGE. Scientific Researches in Food Production, Nitra, 6 November 2017.
12. Bocian A., Urbanik M., Hus K., Łyskowski A., Legath J. (2016). Comparative analysis of protein venom composition between members of the Viperidae family: Vipera berus berus and Agkistrodon contortrix 10^th Central and Eastern European Proteomic Conference, Budapest, 11-14 October 2016.
13. Łyskowski A., Bocian A., Hus K., Legath J. (2016). In-silico characterization of selected protein components from venom of two members of viperidae family. 10^th Central and Eastern European Proteomic Conference, Budapest, 11-14 October 2016.
14. Hus K., Jaromin M., Bocian A., Łyskowski A., Ossoliński K., Ossoliński T., Ossolińska A., Groszek G. (2016). Comparison of protein isolation methods in studies of rcc biomarkers discovery. 10^th Central and Eastern European Proteomic Conference, Budapest, 11-14 October 2016.
15. Hus K., Bocian A., Jaromin M., Tyrka M., Łyskowski A. (2016). Tracking changes in protein accumulation of Enterococcus faecalis after exposure to acrylamide. 11^th International Conference of Young Naturalists – from Biotechnology to Environmental Protection. Interdisciplinary Meeting of Young Naturalists, Zielona Góra, 23-26 November 2016.
16. Bocian A., Sowa P. (2016). Optimization of protein denaturation conditions for identification on MALDI ToF mass spectrometer. 6^th International Young Scientists Conference Human – Nutrition – Environment, Rzeszów, 21-22 April 2016.
17. Kononiuk A., Bocian A., Karwowska M. (2016). Białkowe markery gatunkowe pszenicy orkisz jako potencjalne narzędzie do kontroli autentyczności produktów orkiszowych. I Sympozjum Naukowe Bezpieczeństwo Żywnościowe i Żywności, Kiry, 17-19 April 2016.
18. Bocian A., Ciura J., Kononiuk A., Szeliga M., Jaromin M., Tyrka M. (2015). Changes in the proteome of fenugreek under the influence of selected effectors. 9^th Central and Eastern European Proteomic Conference, Poznań, 15-18 June 2015.
19. Bocian A., Kosmala A., Rapacz M., Zwierzykowski Z. (2010). Accumulation of the selected amino acids and sugars during cold acclimation of perennial ryegrass distinct in the level of frost tolerance. Abstracts of the 1^st Festulolium Working Group Workshop, Poznań, Poland, 7-8 October 2010, p. 22.
20. Kosmala A., Bocian A., Rapacz M., Jurczyk B., Zwierzykowski Z. (2010). Proteome analysis during cold acclimation of Lolium-Festuca Abstracts of the 1^st Festulolium Working Group Workshop, Poznań, Poland, 7-8 October 2010, p. 4.
21. Bocian A., Kosmala A., Rapacz M., Zwierzykowski Z. (2010). Analiza akumulacji wybranych cukrów w stresie chłodu u życicy trwałej (Lolium perenne ). III Polski Kongres Genetyki, Lublin, 12-15 września 2010. Streszczenia, s. 207.
22. Kosmala A., Bocian A., Pawłowicz I., Kachlicki P., Zwierzykowski Z. (2010). Identyfikacja białek zaangażowanych w ekspresję odporności na mróz u traw kompleksu Lolium-Festuca. Materiały IV Sympozjum Naukowego Jakość Środowiska, Surowców i Żywności, Kraków, 22-23 kwietnia 2010, s. 198-200.
23. Bocian A., Kosmala A., Zwierzykowski Z. (2010). Accumulation of the selected primary metabolites during cold acclimation in perennial ryegrass (Lolium perenne). 2^nd Conference of Polish Mass Spectrometry Society, Poznań, 24-26 March 2010. Acta Biochimica Polonica vol. 57 (suppl. 1/2010), p. 35.
24. Bocian A., Kosmala A., Rapacz M., Zwierzykowski Z. (2010). Analiza profili akumulacji wybranych białek w trakcie hartowania na mróz u Lolium perenne i Festuca pratensis. II Sesja Sprawozdawcza – „Nowe metody genetyki molekularnej i genomiki służące doskonaleniu odmian roślin uprawnych”, Zakopane, 8-13 lutego 2010. Streszczenia, s. 23.
25. Bocian A., Kosmala A., Zwierzykowski Z. (2009). Protease activities during cold acclimation of perennial ryegrass (Lolium perenne ). Proc. of the 4^th Conference of Polish Society of Experimental Biology, September 21-25, 2009, Kraków, p. 50-51.
26. Bocian A., Kosmala A., Rapacz M., Zwierzykowski Z. (2009). The proteins involved in cold acclimation of perennial ryegrass (Lolium perenne ). Proc. of the XXVIII Meeting of the Fodder Crops and Amenity Grasses Section of Eucarpia, La Rochelle France, 11-14 May 2009, p. 70.
27. Kosmala A., Bocian A., Rapacz M., Zwierzykowski Z. (2009). Differences in leaf proteome response to cold acclimation in two Festuca pratensis plants distinct in the level of frost tolerance. Proc. of the XXVIII Meeting of the Fodder Crops and Amenity Section of Eucarpia, La Rochelle, France, 11-14 May 2009, p. 111.
28. Kosmala A., Bocian A., Rapacz M., Jurczyk B., Zwierzykowski Z. (2009). Identification of leaf proteins differentially accumulated during cold acclimation between Festuca pratensis plants with distinct levels of frost tolerance. The Conference on The Challenges of Contemporary Cell Biology, Łódź, April 20-21, 2009, p. 71.
29. Kosmala A., Bocian A., Kachlicki P., Zwierzykowski Z. (2009). Identyfikacja niektórych białek zaangażowanych w ekspresję odporności na mróz u traw kompleksu Lolium-Festuca. Materiały III Sympozjum naukowego Jakość Środowiska, Surowców i Żywności, Lublin, 30-31 marca 2009, s. 199-201.
30. Kosmala A., Bocian A., Rapacz M., Zwierzykowski Z. (2009). Identyfikacja genów związanych z aklimatyzacją do chłodu i odpornością na mróz
    u życicy trwałej (Lolium perenne). I sesja sprawozdawcza zespołów realizujących projekt badawczy zamawiany PBZ-MNiSW-2/3/2006: „Nowe metody genetyki molekularnej i genomiki służące doskonaleniu odmian roślin uprawnych”, Szczecin, 25-27 lutego 2009.
31. Kosmala A., Bocian A., Rapacz M., Zwierzykowski Z. (2009). Proteome response to cold acclimation in Lolium-Festuca Proc. of the 5^th International Conference on Plant and Microbe Adaptation to the Cold, 4-8 December 2008, Ås, Norway, p 56.
32. Bocian A., Kosmala A., Rapacz M., Wolanin B., Zwierzykowski Z. (2008). Frost tolerance of Festuca pratensis and Lolium perenne. Proc. of the 2^nd National Conference Genetics and genomics in improving of cultivated plants – from a model plant to a new cultivar, Poznań, 24-26 November 2008, p. 90.
33. Kosmala A., Bocian A. (2008). Activities of proteolytic enzymes during cold acclimation of Festuca pratensis. Proc. of the 2^nd National Conference Genetics and Genomics in Improving of Cultivated Plants – from a model plant to a new cultivar, Poznań, 24-26 November 2008, p. 91.
34. Bocian A., Kosmala , Kachlicki P., Marczak Ł., Zwierzykowski Z. (2008) Identification of proteins involved in cold acclimation of Festuca pratensis using MALDI-ToF and LC/MS/MS mass spectrometers. Abstracts of the 1^st Conference of Polish Mass Spectrometry Society, Acta Biochim. Polon. 55 (supplement 2), p. 23.
35. Kosmala A., Bocian A., Kachlicki P., Zwierzykowski Z. (2008). Identyfikacja niektórych białek zaangażowanych w ekspresję odporności na mróz u traw kompleksu Lolium-Festuca. Materiały II Sympozjum naukowego Jakość środowiska, surowców i żywności, Turwia, 1-3 kwietnia 2008, s. 229-231.
36. Bocian A., Kalinowski A., Kosmala A. (2007). Analysis of three groups of pollen hydrolytic enzymes of tristylous Lythrum salicaria using two-dimensional electrophoresis. Abstracts of the 42^nd Meeting of the Polish Biochemical Society, Szczecin, Poland, 18-21 September 2007; Acta Biochim. Polon. 54 (suppl. 4): p. 74.
37. Bocian A., Kalinowski A., Kosmala A. (2007). Analiza białek protoplastu i płaszcza pyłkowego Lythrum salicaria przy zastosowaniu metody dwukierunkowej elektroforezy. Streszczenia 54 Zjazdu Polskiego Towarzystwa Botanicznego „Botanika w Polsce - sukcesy, problemy, perspektywy”, Szczecin, 3-8 września 2007, s. 46.
38. Kosmala A., Bocian , Rapacz M., Wolanin B., Zwierzykowski Z. (2007). Qualitative and quantitative changes in 2-D protein profiles during cold acclimation of frost tolerant Festuca pratensis genotype. Abstracts of the 3^rd Conference of Polish Society of Experimental Plant Biology; Warsaw, Poland, 26-30 August 2007, p. 69.
39. Kosmala A., Bocian A., Rapacz M., Wolanin B., Zwierzykowski Z. (2007). The comparisons of 2-D protein profiles during cold acclimation of Festuca pratensis genotypes distinct in the level of freezing tolerance. Proc. of the 5^th International Symposium on the Molecular Breeding of Forage and Turf (MBFT), Sapporo, Japan, 1-6 July 2007, p. 60.