Stability study in selected conditions and biofilm-reducing activity of phages active against drug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is currently a serious threat to human health, especially to people with immunodeficiency as well as patients with prolonged hospital stays and those undergoing invasive medical procedures. The ever-increasing percentage of strains characterized by multidrug resistance to widely used antibiotics and their ability to form biofilms make it difficult to fight infections with traditional antibiotic therapy. In view of the above, phage therapy seems to be extremely attractive. Therefore, phages with good storage stability are recommended for therapeutic purposes. In this work, we present the results of studies on the stability of 12 phages specific for A. baumannii under different conditions (including temperature, different pH values, commercially available disinfectants, essential oils, and surfactants) and in the urine of patients with urinary tract infections (UTIs). Based on our long-term stability studies, the most optimal storage method for the A. baumannii phage turned out to be - 70 °C. In contrast, 60 °C caused a significant decrease in phage activity after 1 h of incubation. The tested phages were the most stable at a pH from 7.0 to 9.0, with the most inactivating pH being strongly acidic. Interestingly, ethanol-based disinfectants caused a significant decrease in phage titers even after 30 s of incubation. Moreover, copper and silver nanoparticle solutions also caused a decrease in phage titers (which was statistically significant, except for the Acba_3 phage incubated in silver solution), but to a much lesser extent than disinfectants. However, bacteriophages incubated for 24 h in essential oils (cinnamon and eucalyptus) can be considered stable.

Novel Pyrimidine Derivatives as Potential Anticancer Agents: Synthesis, Biological Evaluation and Molecular Docking Study.

In the present paper, new pyrimidine derivatives were designed, synthesized and analyzed in terms of their anticancer properties. The tested compounds were evaluated in vitro for their antitumor activity. The cytotoxic effect on normal human dermal fibroblasts (NHDF) was also determined. According to the results, all the tested compounds exhibited inhibitory activity on the proliferation of all lines of cancer cells (colon adenocarcinoma (LoVo), resistant colon adenocarcinoma (LoVo/DX), breast cancer (MCF-7), lung cancer (A549), cervical cancer (HeLa), human leukemic lymphoblasts (CCRF-CEM) and human monocytic (THP-1)). In particular, their feature stronger influence on the activity of P-glycoprotein of cell cultures resistant to doxorubicin than doxorubicin. Tested compounds have more lipophilic character than doxorubicin, which determines their affinity for the molecular target and passive transport through biological membranes. Moreover, the inhibitory potential against topoisomerase II and DNA intercalating properties of synthesized compounds were analyzed via molecular docking.

Qualitative and Quantitative Assessment of Buckwheat Husks as a Material for Use in Therapeutic Mattresses.

Buckwheat husks are used in many therapeutic products such as pillows, mattresses, seats, etc. This material is proposed by producers for example for discopathy, back pain and head vasomotor disorders. Our studies evaluated the impact of using cotton mattresses with buckwheat husk fillings on people's health condition. The main research was carried out on the group of 60 people divided into 3 groups (1-people with skeletal system problems, 2-people spending a lot of time lying with the probability of pressure ulcer formation and 3-healthy people). In addition, different tests have been carried out on the possibility of colonization of mattresses by fungi, bacteria and arthropod pests, and rheological, chemical and flammability tests. The research material in the form of buckwheat husks was tested in a diverse way. All tests indicate high usefulness of husks for therapeutic activity. This material was contaminated with fungi, bacteria and pests at a very low level, related to the natural colonization of buckwheat nuts during harvest and storage. The quality of the husks was also confirmed in rheological, chemical and flammability studies. Finally, this has also been confirmed in surveys conducted on people with health problems. The analyses show that the buckwheat husk is an excellent material that can be used to fill prophylactic mattresses. This has been confirmed by the results of laboratory tests and opinions of respondents using mattresses filled with buckwheat husk.

Antimicrobial chloro-hydroxylactones derived from the biotransformation of bicyclic halolactones by cultures of Pleurotus ostreatus.

The aim of this research was to test the ability of cultures of edible fungi to biotransform three bicyclic halolactones. The substrates (2-chloro-, 2-bromo- and 2-iodo-4,4,6,7-tetramethyl-9-oxabicyclo[4.3.0]nonan-8-one) received by means of synthesis were transformed by oyster mushroom Pleurotus ostreatus and edible mushrooms of the genus Armillaria mellea, Marasmius scorodonius and Laetiporus sulfureus. The substrates were converted to hydroxyl derivatives only by the cultures of oyster mushroom. Out of seven strains of Pleurotus ostreatus - three were capable of hydroxylation of all substrates with the most effective conversion of chlorolactone. Bromo- and iodolactone were transformed to a small extent. Four new chloro-hydroxylactones were obtained as biotransformation products. The structures of substrates and products were established on the basis of spectroscopic data. Studies of antimicrobial activity performed on reference strains of pathogenic microorganisms showed that halolactones caused complete inhibition of growth of A. alternata and F. linii strains. On the other hand, chloro-hydroxylactones were able to completely inhibit the growth of A. alternata and F. linii strains and also C. albicans strain.

Comparative analysis of airborne bacteria and fungi in two salt mines in Poland.

The aim of this work was to determine the genera or species composition and the number of colony forming units of airborne bacteria and fungi, respectively, in two salt mines in Poland "Wieliczka" (Lesser Poland) and "Polkowice-Sieroszowice" (Lower Silesia). Both of them are working environments characterized by extreme conditions, and additionally "Wieliczka," officially placed on the UNESCO World Heritage Sites' list, plays a role of tourist attraction. There are also some curative chambers located in this mine. Air samples were taken once in December 2015, between 6:00 a.m. and 9:00 a.m. There were nine measurement points located about 200 m underground in "Wieliczka" and six measurement points located in the working shafts about 400 m underground in "Polkowice-Sieroszowice." The total volume of each air sample was 150 L. Air samples, collected in individual measurement points of both salt mines, were inoculated on two microbiological media: potato dextrose agar and tryptic soy agar using the impact method. We identified 10 and 3 fungal genera in the "Wieliczka" Salt Mine and in "Polkowice-Sieroszowice," respectively. The most common were fungi of the Penicillium genus. In both mines, the Gram-positive bacteria of genus Micrococcus were detected most frequently. Among identified microorganisms, there were neither pathogenic fungi nor bacteria. The most prevalent microorganisms detected in indoor air were Gram-positive cocci, which constituted up to 80% of airborne microflora. Our results showed that microorganisms recorded in the air samples are not a threat to workers, tourists or patients. Neither pathogens nor potentially pathogenic microorganisms, listed as BSL-2, BSL-3 or BSL-4, were detected. The microbes identified during our analysis commonly occur in such environments as the soil, water and air. Some of the detected bacteria are component of natural microflora of human skin and mucous membranes, and they can cause only opportunistic infections in individuals depending on their health condition.

Salt mine microorganisms used for the biotransformation of chlorolactones.

The aim of the project was to find new catalysts capable of chlorolactone biotransformation. Three bicyclic chlorolactones with structures possessing one or two methyl groups in their cyclohexane ring were subjected to screening biotransformation using seven bacterial strains and one fungal strain from a salt mine. Three strains of bacteria (Micrococcus luteus Pb10, Micrococcus luteus WSP45, Gordonia alkanivorans Pd25) and one fungal strain (Aspergillus sydowii KGJ10) were able to catalyse hydrolytic dehalogenation of one substrate. The classification of the strains that were effective biocatalysts was confirmed by 16S rDNA analysis. The best result (76%) was obtained using Aspergillus sydowii KGJ10. All strains catalysed hydrolytic dehalogenation without changing the conformation. The equatorial position of the chlorine atom in the substrate turned out to be warrant of the positive result of the biotransformation process.

Dehalogenation Activity of Selected Fungi Toward δ-Iodo-γ-Lactone Derived from trans,trans-Farnesol.

Time-course of biotransformation of racemic trans-4-((E)-4',8'-dimethylnona-3',7'-dien-1-yl)-5-iodomethyl-4-methyldihydrofuran-2-one (1) in fungal and yeast cultures was investigated. In these conditions, the substrate 1 was enantioselectively dehalogenated yielding 4-((E)-4',8'-dimethylnona-3',7'-dien-1-yl)-4-methyl-5-methylenedihydrofuran-2-one (2) and its structure was established based on the spectroscopic data. The most effective biocatalyst used was Didymosphaeria igniaria, which catalyzed the process with highest rate and enantioselectivity (ee of product = 76%). The antiproliferative activity of δ-iodo-γ-lactone 1, product of its biotransformation 2, and starting substrate (farnesol) were evaluated toward two cancer cell lines: A549 (human lung adenocarcinoma) and HL-60 (human promyelocytic leukemia).

In vitro and in vivo matrix metalloproteinase expression after photodynamic therapy with a liposomal formulation of aminolevulinic acid and its methyl ester.

Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including nonmelanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.

Effect of melatonin on melanoma cells subjected to UVA and UVB radiation in In vitro studies.

Studies performed in vivo and in vitro have shown that exogenous melatonin (Mel) exerts oncostatic effects on melanoma cells. Although the protective effect of Mel on skin and cells exposed mainly to UVB has been documented, effects of Mel have not yet been examined on melanoma cells exposed to UVA. Our investigations aimed at examination of the effect of Mel alone (0, 10(-3), 10(-6) and 10(-9) M), and after its addition to the culture medium for 30 minutes before exposure of melanoma cells to UVA (15 J/cm(2)) or UVB (30 mJ/cm(2), 60 mJ/cm(2)). Viability of the cells was examined using the colorimetric sulphorhodamine B test. Mel added to the medium at concentrations of 10(-3)-10(-8) M was found to increase the number of melanoma cells as compared to the control (cells with no Mel) after 24 hours. Compared to exposed cells without Mel, at 10(-3) M Mel, melanoma cells exposed to 30 mJ/cm(2) UVB increased in number and at 10(-9) M increased the survival of those exposed to 60 mJ/cm(2) UVB. The cells exposed to UVA (15 J/cm(2)) were protected by Mel at 10(-6) and 10(-9) M. Physiological concentrations of Mel exerted no oncostatic effects on the BM cell line used here. In addition, the pharmacological Mel concentrations stimulated proliferation of the cells. Beyond doubt, we have confirmed that Mel represents a substance which protects cells from UVA and UVB action in in vitro experiments.

Effect of melatonin on human keratinocytes and fibroblasts subjected to UVA and UVB radiation In vitro.

Skin represents one of the extrapineal sites of melatonin (Mel) synthesis. In the skin Mel plays, for example, the role of an antioxidant which scavenges and inactivates free radicals arising due to UV irradiation. Although the protective effect of Mel on skin and cells irradiated mainly with UVB has been documented, to date no comparison has been made for the effects of Mel on cells exposed to UVA. Our study aimed at evaluating the effect of Mel (0, 10(-3), 10(-6) or 10(-9) M) added to culture medium 30 minutes before exposure of keratinocytes and fibroblasts to irradiation with UVA (15 J/cm(2)) and UVB (30 mJ/cm(2), 60 mJ/cm(2)). Viability of the cells was evaluated using sulphorhodamine (SRB) colorimetric test. Mel at 10(-3) M increased the number of surviving keratinocytes and at 10(-6) M increased the number of surviving fibroblasts exposed to UVB (30 mJ/cm(2), 60 mJ/cm(2)) as compared to cells exposed only to radiation. In addition, 10(-6) M protected keratinocytes exposed to the dose of 30 mJ/cm(2). Mel at 10(-3) M exerted a protective effect on both types of cells irradiated with UVA (15 J/cm(2)). As documented by our studies, Mel protects skin cells from the action of UVA and UVB. The protective effect of different Mel concentrations might result from variable expression of melatonin receptors.

Effect of melatonin on cytotoxicity of doxorubicin toward selected cell lines (human keratinocytes, lung cancer cell line A-549, laryngeal cancer cell line Hep-2).

The pineal hormone melatonin (MLT) has been recognised as a substance capable of alleviating in vivo nephro-, cardio- and myelotoxicity of doxorubicin (DOX) and of other anthracyclines in animal models. However, few data are available on the effects of MLT on cytotoxicity of antineoplastic drugs toward tumor cells in vitro. The present study aimed at the evaluation of effects of MLT and of DOX on selected cell lines. The experiments were conducted on human keratinocytes (primary culture), non-small cell lung cancer (A-549) and laryngeal cancer cell lines (HEp-2). In keratinocytes and in A-549 cells, MLT used at pharmacological concentrations (0.1 and 1.0 mM) was observed to intensify apoptotic lesions. MLT exerted no clear-cut effects on the HEp-2 cell line. In contrast, DOX at concentrations of 0.1 and 1.0 microg/ml intensified apoptosis and augmented the frequency of necrotic lesions in cell nuclei in all the examined cell lines. MLT intensified cytotoxicity of DOX in all cell lines, significantly decreasing cell numbers and promoting apoptosis. The effect was MLT concentration-dependent. MLT decreased the proportion of cells with necrotic lesions.

ABCC2 (MRP2, cMOAT) can be localized in the nuclear membrane of ovarian carcinomas and correlates with resistance to cisplatin and clinical outcome.

PURPOSE: Cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma. ABCC2 is commonly localized in apical cell membranes and could confer cisplatin resistance. Here, we show that ABCC2 can be localized in the cytoplasmic membrane as well as in the nuclear membrane of various human tissues including ovarian carcinoma cells. EXPERIMENTAL DESIGN: For the subcellular detection of ABCC2, immunohistochemistry was done using 41 Federation Internationale des Gynaecologistes et Obstetristes stage III ovarian carcinoma specimens prepared before treatment with cisplatin-based schemes and 35 specimens from the same group after chemotherapy. Furthermore, 11 ovarian carcinoma cell lines as well as tissue microarrays consisting of various human tissues were analyzed. RESULTS: Nuclear membranous localization of ABCC2 was associated with response to first-line chemotherapy at primary (P = 0.0013) and secondary surgery (P = 0.0060). Cases with relapse showed higher nuclear membrane expression at primary (P = 0.0003) and secondary surgery (P = 0.0024). Kaplan-Meier analyses showed that weak nuclear membrane ABCC2 expression before treatment was associated with significantly longer overall (P = 0.04) and progression-free survival (P = 0.001); following chemotherapy, it correlated with significantly longer progression-free survival (P = 0.038). Tissue microarrays confirmed nuclear membranous localization of ABCC2, in particular, in poorly differentiated cells. In ovarian carcinoma cells, it correlated with resistance against cisplatin, whereas localization in the cytoplasmic membrane did not. CONCLUSIONS: ABCC2 confers resistance to cisplatin of ovarian carcinoma in cell culture systems and in clinics when expressed in the nuclear membrane. Thus, ABCC2 localization can predict platinum therapy outcome. Furthermore, expression of ABCC2 in nuclear membranes in human tissues is specific for poorly differentiated cells including stem cells.

The effect of extracellular substance components on the proliferation and expression of hormones in cultured cells of medullary thyroid carcinomas.

The effects were examined of selected extracellular medium (ECM) components on the proliferation of medullary thyroid carcinoma cells and on the production of calcitonin and CGRP. Human TT cells and rat rMTC cells were cultured for 24, 48 and 72 hours on glass coated with type I collagen, fibronectin or poly-D-lysine. More pronounced proliferation was demonstrated by TT cells grown on poly-Dlysine or collagen in comparison with the control and less pronounced proliferation was typical of cells grown on fibronectin. On the other hand, rMTC cells were more markedly manifest on any ECM substrates than that on glass. Alterations in the proliferation were paralleled by changes in the expression of CT and CGRP.

The effects of quercetin vs cisplatin on proliferation and the apoptotic process in A549 and SW1271 cell lines in in vitro conditions.

Experience over several years has indicated that chemotherapy, even if widely used, does not always remain effective in the therapy of lung tumours and, in addition, is linked to serious side effects. In parallel, some plant polyphenols are known to exert a proapoptotic action on tumour cells while, in contrast, representing anti-cancerogenic anti-oxidants in living organisms. Our studies were aimed at comparing the effects of a polyphenol, quercetin, and cisplatin on cells of various types of lung cancer in in vitro conditions. In these studies we also attempted to define the relationship between the dose and the duration of the activity of the compounds. Cisplatin alone was found to induce only a small reaction in the cells, while in combination with quercetin its anti-proliferative and pro-apoptotic effects were amplified, depending upon the type of tumour, the dose and the duration of the drug's action.

Induction of apoptosis by EGCG in selected tumour cell lines in vitro.

One of the best recognised polyphenols of plant origin, epigallocatechin-3-gallate (EGCG) is contained mainly in green tea and in grapes. Studies performed in vivo and in vitro have demonstrated high probability of anti-neoplastic potential of the compound, due to its capacity to induce programmed cell death. The present studies were aimed at evaluation of apoptosis induction in cells of three selected tumour cell lines, subjected to action of various concentrations of EGCG. The experiment was performed on cultures of HEp-2 laryngeal carcinoma cells, LoVo colon carcinoma cells, HeLa cervical carcinoma cells and on normal myoepithelial cell line, HS. EGCG was found to induce apoptosis in cells of the examined neoplastic lines in a dose-related manner. Moreover, effect of EGCG on normal cells of HS line was found to be much less pronounced as compared to effects exerted on sensitive neoplastic cells.

The effect of calcitriol and its analogues on proliferation and hormone expression in cultured cells of thyroid medullary carcinomas.

The study aimed at evaluating the effects of calcitriol and of its analogues on the proliferation of TT and rMTC cells (human and rat line tumour cells originating from thyroid medullary carcinoma) and at examining the effects of the substances on the secretion of the principal hormones of the cells, calcitonin (CT) and calcitonin gene-related peptide (CGRP). Cells of thyroid medullary carcinoma (human TT cells and rat rMTC cells) were cultured for 5 days in the absence or in the presence of calcitriol and of its two analogues (PRI-1906 and PRI-2191) in concentrations of 10(-9) to 10(-6) M. Calcitriol and the applied analogues weakly inhibited proliferation of thyroid medullary carcinoma in in vitro conditions. The evident effect of analogues on hormone secretion points to their effect on the process of CT gene expression.