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Paulina Paprocka,1 Bonita Durnaś,1,2 Angelika Mańkowska,1 Karol Skłodowski,3 Grzegorz Król,1 Magdalena Zakrzewska,3 Michał Czarnowski,3 Patrycja Kot,1 Kamila Fortunka,1 Stanisław Góźdź,2 Paul B Savage,4 Robert Bucki1,3 1Department of Microbiology and Immunology, Institute of Medical Science, Collegium Medicum, Jan Kochanowski University in Kielce, Kielce, Poland; 2Holy Cross Oncology Center of Kielce, Kielce, Poland; 3Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Białystok, Białystok, Poland; 4Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USACorrespondence: Robert Bucki Tel +48-85-7485493Fax +48-85 748 54 16Email buckirobert@gmail.comBackground: The increasing number of infections caused by antibiotic resistant strains of Pseudomonas aeruginosa posed a very serious challenge for clinical practice. This standing is driving scientists to develop new antibiotics against these microorganisms.Methods: In this study, we measured the MIC/MBC values and estimated the ability of tested molecules to prevent bacterial biofilm formation to explore the effectiveness of β-lactam antibiotics ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, and ceragenins CSA-13, CSA-44, and CSA-131 against 150 clinical isolates of Pseudomonas aeruginosa that were divided into five groups, based on their antibiotic resistance profiles to beta-lactams. Selected strains of microorganisms from each group were also subjected to prolonged incubations (20 passages) with ceragenins to probe the development of resistance towards those molecules. Cytotoxicity of tested ceragenins was evaluated using human red blood cell (RBCs) hemolysis and microscopy observations of human lung epithelial A549 cells after ceragenin treatment. Poloxamer 407 (pluronic F-127) at concentrations ranging from 0.5% to 5% was tested as a potential drug delivery substrate to reduce ceragenin toxicity.Results: Collected data proved that ceragenins at low concentrations are highly active against clinical strains of Pseudomonas aeruginosa regardless of their resistance mechanisms to conventional antibiotics. Ceragenins also show low potential for resistance development, high antibiofilm activity, and controlled toxicity when used together with poloxamer 407.Conclusion: This data strongly supports the need for further study directed to develop this group of molecules as new antibiotics to fighting infections caused by antibiotic resistant strains of Pseudomonas aeruginosa.Keywords: ceragenin, antibacterial agents, Pseudomonas aeruginosa, antibiotic resistant bacteria, new antibiotics

Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

Anna Szlachcic,1 Katarzyna Pala,1 Malgorzata Zakrzewska,1 Piotr Jakimowicz,1 Antoni Wiedlocha,2 Jacek Otlewski11Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Poland; 2Centre for Cancer Biomedicine, Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, NorwayAbstract: Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy.Keywords: FGF1-conjugates, gold nanoparticles, FGFR-targeted cancer therapy, photothermal therapy

The aim of this study was to assess the impact of zinc smelter on heavy metals concentration in the litter fall collected from three different sites in Olkusz Forest located in southern Poland. Furthermore the study was designed to investigate the input of these metals to the forest floor. Three forest areas Podlesie I and Podlesie II located 3 and 4 km south-east from Bukowno zinc smelter and Cegielnia situated 6 km north-east from the smelter were selected. At the beginning of September 2006 five litter traps were set at each site. The quantity of litter fall, along with percentage composition of each species, were estimated for an area of 1m2. The concentration of Cd, Pb, Cu, Zn and Fe in the litter fall and leaves of four main tree species were analysed. The input of these metals to the forest floor were determined. The input of cadmium ranged from 204 μg m2 at Cegielnia to 1340 μg m2 at Podlesie I. The input of lead was higher than cadmium and varied from 1031 μg m2 at Cegielnia to 3252 μg m2 at Podlesie II.The input of zinc and iron were about 10 times than cadmium and lead. The less input of all invesigated metals was noticed in control site located at WNP. for those metals. The high input of heavy metals to the forest floor may have negative effects on investigated part of Olkusz forest ecosystem.

This article presents the results of a study on the current state and evaluation of the Polish tanning industry, focusing on its production characteristics. The research sample included 220 companies that were contacted to gather in- formation about their operations. Some of these companies have been suspended, liquidated, or have changed their business profiles. Approximately 30 % confirmed that they are still active in leather manufacturing, indicating that the Polish tanning industry is experiencing a process of deindustrialization. Surveys conducted in 20 companies revealed that Polish tanneries operate on national, European, and global scales. Most of them are micro or small enterprises with annual revenues of up to PLN 5 million. The primary factor defining their competitiveness is the high quality of the products and services they offer. They mainly process calfskin and cowhides sourced from Poland and abroad, primarily for the footwear sector. Polish tanneries are aware of global trends, including the industry's shift towards ecological practices, the adoption of modern technologies, and the introduction of innovations. Given the current challenges facing the Polish tanning industry, it is essential to take action to improve the health of this sector of the economy. [ABSTRACT FROM AUTHOR]

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Background: INI1 (hSNF5) mutations ore linked to rhobdoid tumours, but mutations in meningiomas with hot spot mutations in position 377 have also been reported. Aims: To analyse the lNl1 gene in meningiomo. Methods: Exons 1, 4, 5, and 9 of the INI 1 gene were analysed by the polymerase chain reaction and direct sequencing in 80 meningiomas. For all cases, western blotting of the INl1 protein was performed. Results: Only one of the 80 samples showed a cytosine insertion in codon 376. This mutation changed the open reading frame in almost the whole exon 9 and resulted in a longer hSNF5 protein. Complex analysis of the above described tumour sample by western blotting, DNA sequencing, and loss of heterozygosily (101-1) analysis showed that this particular meningioma consisted of heterogeneic cellular components. One of these components had a mutated INI1 gene, whereas in the other component INI1 was intact. Conclusions: INI1 mutation is a rare event in the molecular pathology of meningiomas. It is possible for the INl1 gene to be mutated in only a proportion of meningioma cells. [ABSTRACT FROM AUTHOR]

Abstract Background Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. Methods To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. Results We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. Conclusion In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

Abstract Background Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed. Methods Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed. Results In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum. Conclusion Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

Abstract Background Although extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation – allowing for an increase in percentage yield of neuronal cells. Results Uncommitted GFAP and SOX2 positive neural progenitors – so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100β-, SOX2-], or mixture of neural and non-neural cells. In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells – i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively. Conclusion We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells – such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.