Analysis of CYP1B1 Polymorphisms in Lung Cancer Patients Using Novel, Quick and Easy Methods Based on CAPS and ACRS-PCR Techniques.

Within the sequence of the CYP1B1 gene, more than 50 polymorphisms, resulting from single-nucleotide polymorphisms (SNPs), have been described. Some of them play an important role as specific genetic markers in the process of carcinogenesis and for therapeutic purposes. In this publication, we present methods we have developed that enable the specific and unambiguous identification of four polymorphisms that result in amino acid changes: c. 142C > G, c. 355G > T, c. 1294C > G, and c. 1358A > G. Our studies are based on cleaved amplified polymorphic sequences (CAPSs) and artificially created restriction site (ACRS) PCR techniques; therefore, they require only basic laboratory equipment and low financial outlays. Utilizing the described methods allows for the reduction of research time and cost, and the minimization of errors. Their effectiveness and efficiency depend on the careful design of appropriate primers and the precise selection of suitable restriction enzymes. As a result, further confirmation by sequencing is not necessary. Using the developed method, we examined 63 patients diagnosed with lung cancer and observed a 1.5 to 2.1 times higher frequency of the analyzed single-nucleotide polymorphisms compared to the frequency in the European population.

Phenolic and Nonpolar Fractions of Elaeagnus rhamnoides (L.) A. Nelson Extracts as Virulence Modulators-In Vitro Study on Bacteria, Fungi, and Epithelial Cells.

Butanol extracts from leaves, twigs, and fruits of Elaeagnus rhamnoides (L.) A. Nelson (sea buckthorn, SBT) were fractionated into phenolic and nonpolar lipid components, the chemical composition of which was analyzed. Assuming that an effect on natural microbiota and host epithelial cells needs to be assessed, regardless of the purpose of using SBT formulations in vivo, the minimal inhibitory/biocidal/fungicidal concentrations (MICs/MBCs/MFCs) of the fractions and reference phytocompounds were screened, involving 17 species of Gram-positive and Gram-negative bacteria and Candida species. The MICs of SBT extracts were in the range of 0.25⁻2.0 mg∙mL−1. Since direct antimicrobial activity of the extracts was quite low and variable, the impact of subMIC on the important in vivo persistence properties of model microorganisms S. aureus and C. albicans was evaluated. Tests for adhesion and biofilm formation on an abiotic surface and on surfaces conditioned with fibrinogen, collagen, plasma, or artificial saliva showed the inhibitory activity of the fractions. The effects on fluorescein isothiocyanate (FITC)-labeled staphylococci adhesion to fibroblasts (HFF-1) and epithelial cells (Caco-2), and on fungal morphogenesis, indicated that SBT extracts have high antivirulence potential. Cytotoxicity tests (MTT reduction) on the standard fibroblast cell line showed variable biological safety of the fractions depending on their composition and concentration. The new information afforded by this study, additional to that already known, is of potential practical value in the application of SBT-derived preparations as antivirulence agents.

Novel properties of Hippophae rhamnoides L. twig and leaf extracts - anti-virulence action and synergy with antifungals studied in vitro on Candida spp. model.

Original, chemically characterized Sea buckthorn (SBT) twig and leaf extracts were in vitro studied in terms of anti-Candida activity. Minimum inhibitory concentrations (MICs) of the extracts against C. albicans ATCC 10231 ranged: 250 µg/ml (twig), 31.5 µg/ml (leaf), and against C. glabrata G1 (clinical isolate) - 15.6 µg/ml (twig), 3.9 µg/ml (leaf). Next the extracts have been used at their subMIC. Both extracts significantly enhanced activity of fluconazole (FLC) and caspofungin (CAS) against C. albicans and increased their efficacy against C. glabrata, measured by an agar dilution assay combined with the E-test. The extracts inhibited C. albicans morphogenesis such as germ tube and hyphae formation as well as invasion to the "Spider" Agar. Antiadhesive and anti-biofilm activities of the extracts were evaluated by Alamar Blue reduction assay. It showed not significant reduction in the degree of cell adhesion (by 10-15%) but noticeable decrease of biofilm formation (by 80% in the case of SBT-twig extract). In conclusion, this study provided the evidence that SBT extracts, used at non-cytotoxic concentrations for the fibroblasts (IC50 from 664.8 µg/ml to 1060.4 µg/ml), targeted some of Candida spp. virulence factors essential for the establishment of the infection. SBT twigs, previously regarded as waste material, were shown to be also a valuable source of the substances with promising antimicrobial activity.

Candida albicans/Staphylococcus aureus Dual-Species Biofilm as a Target for the Combination of Essential Oils and Fluconazole or Mupirocin.

The effectiveness of essential oils (EOs), fluconazole (FLU) and mupirocin (MUP) used alone or in combination against mono-species and mixed Candida albicans/Staphylococcus aureus biofilms was examined. An experimentally established dual-species biofilm model, verified by fluorescence microscopy and viable cell counting, was used. Selected commercial EOs were tested: geranium, citronella and clove oils, which have been chemically characterized and found to differ in the content of the main components (qualitative and quantitative). As expected, C. albicans and S. aureus biofilms were less susceptible to fluconazole and mupirocin action, respectively, compared to the planktonic counterparts. However, the drug effectiveness in combination with the EOs was significantly improved, giving enhancement of biofilm eradication than caused by the antibiotics alone. Moreover, dual-species biofilm formation was limited by sub-MIC of EOs, and preformed mixed biofilm was eliminated more efficiently by combined action of drugs and EOs.

New biological potential of abietane diterpenoids isolated from Salvia austriaca against microbial virulence factors.

The increasing importance of multi-resistant strains and microbial biofilms in the development of chronic infections has driven the search for more effective alternative therapy including plant-origin preparations. The present study evaluates the broadly-defined antimicrobial activity of two abietane diterpenoids isolated from Salvia austriaca transformed roots: taxodone and 15-deoxy-fuerstione. The direct biostatic/biocidal effect of these phytocompounds and their influence on Staphylococcus aureus and Candida albicans virulence factors/mechanisms (adhesion, biofilm formation, agglutination in human plasma, survival in the blood, germ tube and mycelium formation) were tested using in vitro assays. Both phytocompounds significantly inhibited microbial adhesion and biofilm formation when used at ½ and » MIC. Additionally, taxodone was able to limit staphylococcal survival in human blood, as well as C. albicans germ tube formation and hyphal growth. The tested diterpenoids express significant anti-biofilm activity against both staphylococci and yeast, and adversely affect their virulence factors/mechanisms, which are relevant in the course of the infection in vivo. Therefore, they demonstrate considerable biomedical potential as complements for classic therapy with antibiotics.

In vitro studies of epithelium-associated crystallization caused by uropathogens during urinary calculi development.

Infectious urinary stones account for about 10% of all urinary stones. In 50% of cases urolithiasis is a recurrent illness, which can lead to the loss of a kidney if not properly treated. One of the reasons for recurrence of the disease may be the ability of bacteria to invade urothelial cells, persist in the host cells and serve as potential reservoirs for infection. Various uropathogens are associated with the formation of bacteria-induced urinary stones but Proteus mirabilis is the most commonly isolated (70%). An in vitro model was used in this study to analyze intracellular growth and crystallization in the presence of P. mirabilis, Klebsiella pneumoniae and Escherichia coli. Human ureter (Hu 609) and bladder (HCV 29) epithelial cell lines were infected with bacteria and incubated (3-72 h) in the presence of synthetic urine and amikacin to prevent extracellular bacterial growth. During the incubation the number of bacteria (CFU/ml) inside epithelial cells and the intensity of crystallization were established. Crystallization was determined as an amount of a calcium radioisotope. The chosen strains of uropathogens were able to invade both types of epithelial cells but the Hu 609 cells were invaded to a higher extent. However, crystallization occurred only in the presence of P. mirabilis strains which were invasive and urease-positive. The highest intensity of cell-associated crystallization was observed when the number of bacteria within the urothelium remained stable during the time of incubation. These results show that P. mirabilis has an ability to form crystals inside the host cells. Under these conditions bacteria are protected from antibiotic killing, which leads to persistent and recurrent infections. We also suspect that this phenomenon may be an important stage of kidney stones formation.

Saponins of Trifolium spp. aerial parts as modulators of Candida albicans virulence attributes.

The aim was to provide the insight into the biology of C. albicans influenced by undescribed yet properties of saponin-rich (80%-98%) fractions (SAPFs), isolated from extracts of Trifolium alexandrinum, T. incarnatum, T. resupinatum var. resupinatum aerial parts. Their concentrations below 0.5 mg/mL were arbitrarily considered as subMICs for C. albicans ATCC 10231 and were further used. SAPFs affected yeast enzymatic activity, lowered tolerance to the oxidative stress, to the osmotic stress and to the action of the cell wall disrupting agent. In their presence, germ tubes formation was significantly and irreversibly inhibited, as well as Candida invasive capacity. The evaluation of SAPFs interactions with anti-mycotics showed synergistic activity, mainly with azoles. Fluconazole MIC was lowered-susceptible C. albicans ATCC 10231 was more susceptible, and resistant C. glabrata (clinical strain) become more susceptible (eightfold). Moreover, the tested samples showed no hemolytic activity and at the concentrations up to 0.5 mg/mL did not reduce viability of fibroblasts L929. This study provided the original evidence that SAPFs of Trifolium spp. aerial part exhibit significant antimicrobial activity, by reduce the expression/quantity of important Candida virulence factors and have good potential for the development of novel antifungal products supporting classic drugs.

[In vitro efficacy analysis of absorbent dressing modified with essential oils, against Staphylococcus aureus and Candida albicans]. / Analiza in vitro skutecznosci modyfikowanych olejkami eterycznymi opatrunków absorpcyjnych wobec Staphylococcus aureus i Candida albicans.

INTRODUCTION: The widespread use of antiseptics for wound dressings, unfortunately, not always effective, prompted us to search for alternative solutions, tailored to individual patient's needs. The aim of the study was checking the validity of the idea to apply some selected essential oils in order to modify active dressings which are routinely used in the care of chronically infected wounds. Our choice is commercially available an absorptive wound dressing which does not contain antiseptics (Sorbact). METHODS: The proposed is modification of dressing by its immersion in essential oil solution and then estimation of the biocide availability and stability during storage. Evaluation of inhibition of microbial surface growth (zone inhibition) and survival of absorbed microorganisms (retentivity by CFU counting) was performed directly after modification and repeated after 7 days of their storage at 4 degrees C. RESULTS: This study indicated that the dressings containing essential oils can keep absorbed bacteria/fungi inside and efficiently limit their growth. Depending on the properties (composition of volatile fraction) of the tested essential oil, saturated dressings were more active when stored at 4 degrees C for 7 days after their modification. The differences of antimicrobial strength, duration of the effect and retentivity between essential oils used for dressing modification have been shown. CONCLUSIONS: Modification of absorbent dressings with essential oils is a good option to achieve better therapeutic effect. Using a mixture of these four essential in several different quantitative ratios can be considered and is worthy of further research.

[The immunomodulatory role of plant polyphenols]. / Immunomodulacyjna rola polifenoli roslinnych.

Polyphenols, plant secondary metabolites, are present in human diet and have been widely used for medical and cosmetic purposes. They possess beneficial features such as antioxidant, immunomodulatory, anti-cancer and antibacterial activity. There is some evidence that these phytochemicals can improve wound healing. However, more and more data suggest that, under certain conditions, they can act in a different, often unpredictable way. Some investigations indicate that polyphenols, generally known as antioxidants, can exhibit pro-oxidant, and therefore cytotoxic, activity. Hence, the ability of phytochemicals to induce apoptosis of cancer cells and bacterial cell damage may be, at least partly, due to their prooxidant properties. Phytocompounds enter the body through the digestive system where they undergo metabolic processes that often change their chemical features. The gastrointestinal microbiome interacts with phytochemicals and influences their bioavailability and absorption in the gut. Except for biochemical changes of plant polyphenols in the host, the achievement of therapeutic concentration in vivo may be the main problem in the determination of their real efficacy. Ambiguous results of some studies demonstrate the need for the development of more accurate and standardized methods for the evaluation of polyphenols' properties. Better understanding of human body-polyphenol interactions is crucial for more effective use of these phytochemicals in disease prevention and therapy. 

Synthetic 3-arylideneflavanones as inhibitors of the initial stages of biofilm formation by Staphylococcus aureus and Enterococcus faecalis.

The antimicrobial activity of twenty two synthetic flavonoids is reported. Among them three 3-arylideneflavanones, 2b, 2c, and 2i, were shown to be highly active against Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis reference strains, with MIC (minimal inhibitory concentration) values ranging from 4.68 microg/ml (14.3 microM) to 37.5 microg/ml (119.7 microM). The synergy of oxacillin and vancomycin with 2c, evaluated as fractional inhibitory concentration index (FICI) was shown (against planktonic culture of S. aureus A3 and E. faecium 138/09 clinical strains). The presence of 2c in the culture medium diminished the initial adhesion of bacteria to an abiotic surface. Such an effect resulted in a decrease in biofilm formation during prolonged culture. Unfortunately, 2e failed to eradicate the S. aureus mature biofilm which was already preformed, however, decreased the number of live biofilm cells. The biofilm of E. faecalis was more susceptible to the action of 3-arylideneflavanone 2c than the S. aureus biofilm. The finding that 3-arylideneflavanones are lipophilic, cause bacterial aggregation, and influence the integrity of membranes making them permeable to SYTO 9/propidium iodide dyes may implicate the cytoplasmic membrane as a target site for these compounds activity.

Antibiofilm activity of selected plant essential oils and their major components.

The aim of the study was to examine the antibiofilm activity of selected essential oils (EO): Lavandula angustifblia (LEO), Melaleuca alternifolia (TTO), Melissa officinalis (MEO) and some of their major constituents: linalool, linalyl acetate, alpha-terpineol, terpinen-4-ol. Biofilms were formed by Staphylococcus aureus ATCC 29213 and Escherichia coli NCTC 8196 on the surface of medical biomaterials (urinary catheter, infusion tube and surgical mesh). TTC reduction assay was used for the evaluation of mature biofilm eradication from these surfaces. Moreover, time-dependent eradication ofbiofilms preformed in polystyrene 96-well culture microplates was examined and expressed as minimal biofilm eradication concentration (evaluated by MTT reduction assay). TTO, alpha-terpineol and terpinen-4-ol as well as MEO, showed stronger anti-biofilm activity than LEO and linalool or linalyl acetate. Among the biomaterials tested, surgical mesh was the surface most prone to persistent colonization since biofilms formed on it, both by S. aureus and E. coli, were difficult to destroy. The killing rate studies of S. aureus biofilm treated with TTO, LEO, MEO and some of their constituents revealed that partial (50%) destruction of 24-h-old biofilms (MBEC50) was achieved by the concentration 4-8 x MIC after 1 h, whereas 2-4 x MIC was enough to obtain 90% reduction in biomass metabolic activity (MBEC90) after just 4 h of treatment. A similar dose-dependent effect was observed for E. coli biofilm which, however, was more susceptible to the action of phytochemicals than the biofilms of S. aureus. It is noteworthy that an evident decrease in biofilm cells metabolic activity does not always lead to their total destruction and eradication.

[Assessment of essential oils activity used in liquid or volatile phase against biofilm cultures of Staphylococcus aureus and Candida albicans. The methodological study]. / Ocena bójczego dzialania fazy plynnej i frakcji lotnej olejków eterycznych na hodowle biofilmowe Staphylococcus aureus oraz Candida albicans. Wlasne rozwiazania metodyczne.

Biofilm formation is a significant factor in chronic infections with fungal and bacterial pathogens. Due to the high drug resistance of biofilm populations and the frequent failures of chemotherapy i such infections, it seems necessary to take recourse to unconventional treatment methods involving e.g. the use of some phytocompounds such as essential oils or their components. In order to evaluate the effect of their action on the microbial biomass a variety of techniques are used. However, there is still a need to develop new tests or modifications of these known, for the biofilm viability assessment. They should be adapted to the physico-chemical nature of the tested compounds and should decrease the risk ofbiofilm damage during staining procedure. We described a test assessing the effect of essential oils on bacterial and fungal biofilm formed on the membrane of cell culture inserts. The proposed model provides a minimal violation of the biofilm integrity during the test. It allows easily explore the activity of essential oil volatile fraction and is useful in determination of the kinetics of their action. Using this test it is also easy to examine the relationship between antimicrobial activity and the cytotoxic effect, known as the biocompatibility index (BI, biocompatibility index). Moreover, it allows qualitative and quantitative analysis of metabolic products, released into the growth medium from biofilm's cells. In successively repeated experiments high reproducibility of results has been obtained, thus the developed methodology seems to be useful in our future studies in this field.

[The synergism of antifungals and essential oils against Candida spp. evaluated by a modified gradient-diffusion method]. / Synergizm leków przeciwgrzybiczych i olejków eterycznych wobec Candida sp., oceniany zmodyfikowana metoda gradientowo-dyfuzyjna.

The usefulness of modified method of MIC Test Strip, for determining the synergistic effect of essential oils in the liquid or volatile phase with fluconazole and voriconazole, was evaluated. Geranium oil used against C. albicans in agar dilution test, at a concentration of 1/2 MIC caused a drop in the value of fluconazole MIC from 12.0 mg/l to 0.064 mg/l and voriconazole from 0.125 mg/l to 0.006 mg/l. A similar effect of drug combinations with essential oils was obtained in the case of C. glabrata study. Volatile Clove oil and cytronelal, applied in subMIC concentrations, also caused a reduction offluconazole and voriconazole MICs. Thus, utility of this simple methods developed by us for testing the effectiveness of combinations of known drugs and new compounds with antifungal activity, has been confirmed.

[The optimization of methods utilized for testing the antibacterial activity of essential oils]. / Weryfikacja metodycznych parametrów oznaczania przeciwbakteryjnej aktywnosci olejków eterycznych.

The antibacterial activity of essential oils: Lavandulae aetherleum, Limonis aetherleum and Melaleucae aetherleum was determined against Staphylococcus aureus Gram (+) and Escherichia coli Gram (-) bacteria, using four methods: agar diffusion (paper disk and agar wells) and broth dilution (turbidimetric and rezazurin reduction assay). This study revealed that the sensitivity of the agar diffusion techniques was much lower than the broth dilution methods. This was mainly due to partitioning of the oil components in the agar according to their affinity with water. This limitation could be lessened by the selection of suitable oil emulsifier/solvent, which itself not influence the bacterial growth. By initial oil solubilization with 96% ethanol (1:1), the increase of agar dilution methods sensitivity could be achieved. The broth microdilution methods, using 0.5% Tween 20 or 0.15% agar to emulsify the oils, were optimized and shown to be most accurate for testing their antimicrobial activity. By introducing resazurin, as an MIC endpoint indicator, the sensitivity of these methods was further enhanced.

Biogenic nanosilver synthesized in Metarhizium robertsii waste mycelium extract - As a modulator of Candida albicans morphogenesis, membrane lipidome and biofilm.

Due to low efficacy of classic antimicrobial drugs, finding new active preparations attracts much attention. In this study an innovative, cost-effective and environmentally friendly method was applied to produce silver nanoparticles (AgNPs) using filamentous fungi Metarhizium robertsii biomass waste. It was shown that these NPs possess prominent antifungal effects against C. albicans, C. glabrata and C. parapsilosis reference strains. Further detailed studies were performed on C. albicans ATCC 90028. AgNPs kill curve (CFU method and esterase-mediated reduction of fluorescein diacetate); fractionally inhibitory concentration index (FICI) with fluconazole (FLC); effect on fungal cell membrane permeability (propidium iodide (PI) staining), membrane lipids profile (HPLC-MS), yeast morphotypes and intracellular reactive oxygen species level (H2DCFDA probe) were investigated. Anti-adhesive and anti-biofilm properties of AgNPs (alone and in combination with FLC) were also tested. Biosafety of AgNPs use was assessed in vitro in cytotoxicity tests against L929 fibroblasts, pulmonary epithelial A549 cell line, and red blood cells. Significant reduction in the viability of yeast cells treated with AgNPs was shown within 6 h. The proportion of C. albicans PI-positive cells increased in a dose and time-dependent manner. Changes in the qualitative and quantitative profile of cell membrane lipids, including significant decline in the quantity of most phospholipid species containing C18:2 and an increase in the amount of phospholipids containing C18:1 acyl species were observed after yeast exposure to AgNPs. CLSM images showed an enhancement in ROS intracellular accumulation in C. albicans treated with biogenic nanosilver. C. albicans transformation from yeast to hyphal forms was also reduced. AgNPs decreased adhesion of yeast to abiotic surfaces, as well as acted synergistically with FLC against sessile population. At fungicidal and fungistatic concentrations, they were non-toxic to mammalian cells. Obtained results confirm suitability of our "green synthesis" method to produce AgNPs with therapeutic potential against fungal infections.

Enzymatic profile, adhesive and invasive properties of Candida albicans under the influence of selected plant essential oils.

The influence of essential oils (EOs) used at sublethal level, on the presence and intensity of Candida albicans virulence factors was evaluated. Minimal inhibitory concentrations (MICs) of Lemon balm, Citronella, Geranium and Clove oils were established as 0.097% (v/v). Using the agar plates with substrates for proteases, phospholipases and hemolysins it was shown that C. albicans ATCC 10231 and C. albicans ATCC 90028 strains differed in the type and amount of enzymes produced. No significant difference in their total amount could be detected after pretreatment for 24 h with EOs at ½ MIC. However, the short-term (1 h) acting oils at MIC caused a statistically significant reduction in this activity. In the API ZYM test it was demonstrated that both strains exhibited activity of the same 9 out of 19 enzyme types and that EOs caused a significant decrease in the release of some of them. In the presence of subMIC of EOs, or when the fungus had previously been exposed to the MIC of oil, germ tubes formation was significantly and irreversibly reduced. Such C. albicans spotted on the Spider agar containing EOs at subMICs were unable to penetrate the agar. A significant decrease in the C. albicans adhesion to the fibroblast monolayer with respect to controls was also demonstrated when yeasts had been exposed to EOs at MIC (1 h) in liquid medium. Thus, it has been shown that tested oils, used even at subMIC, exhibit significant activity reducing the presence/quantity of important C. albicans virulence factors.

New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans.

The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa (aerial parts and roots) and Saponaria officinalis (used as a well-known source of plant saponins) against Candida albicans reference and clinical strains, their yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated. Direct fungicidal/fungistatic properties of the tested phytochemicals used alone, as well as their synergy with azoles (probably resulting from yeast cell wall instability) were demonstrated. Here, to the best of our knowledge, we report for the first time the ability of saponin-rich extracts of M. sativa and S. officinalis to inhibit C. albicans germ tube formation, limit hyphal growth, reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the range of concentrations which were active modulators of Candida virulence factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These properties seem to be very promising in the context of using plant-derived SFs as potential novel antifungal therapeutics supporting classic drugs or as ingredients of disinfectants.