Showing papers in "Acta Biochimica Polonica in 2015"

Adult stem cells: hopes and hypes of regenerative medicine.

Abstract: Stem cells are self-renewing cells that can differentiate into specialized cell type(s). Pluripotent stem cells, i.e. embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) differentiate into cells of all three embryonic lineages. Multipotent stem cells, like hematopoietic stem cells (HSC), can develop into multiple specialized cells in a specific tissue. Unipotent cells differentiate only into one cell type, like e.g. satellite cells of skeletal muscle. There are many examples of successful clinical applications of stem cells. Over million patients worldwide have benefited from bone marrow transplantations performed for treatment of leukemias, anemias or immunodeficiencies. Skin stem cells are used to heal severe burns, while limbal stem cells can regenerate the damaged cornea. Pluripotent stem cells, especially the patient-specific iPSC, have a tremendous therapeutic potential, but their clinical application will require overcoming numerous drawbacks. Therefore, the use of adult stem cells, which are multipotent or unipotent, can be at present a more achievable strategy. Noteworthy, some studies ascribed particular adult stem cells as pluripotent. However, despite efforts, the postulated pluripotency of such events like "spore-like cells", "very small embryonic-like stem cells" or "multipotent adult progenitor cells" have not been confirmed in stringent independent studies. Also plasticity of the bone marrow-derived cells which were suggested to differentiate e.g. into cardiomyocytes, has not been positively verified, and their therapeutic effect, if observed, results rather from the paracrine activity. Here we discuss the examples of recent studies on adult stem cells in the light of current understanding of stem cell biology.

Klebsiella pneumoniae: characteristics of carbapenem resistance and virulence factors.

Abstract: Klebsiella pneumoniae, known as a major threat to public health, is the most common factor of nosocomial and community acquired infections. In this study, 50 K. pneumoniae clinical specimens isolated from bronchial, urea, blood, catheter, rectal, bile, tracheal and wound cultures were collected. These isolates were identified and carbapenem resistance was determined via an automated system, CHROMagar Orientation and CHROMagar KPC. The carbapenemase gene regions (blaIMP, blaVIM, blaOXA, blaNDM and blaKPC) and presence of virulence factors (magA, k2A, rmpA, wabG, uge, allS, entB, ycfM, kpn, wcaG, fimH, mrkD, iutA, iroN, hly ve cnf-1) of these isolates were determined by using Multiplex-PCR. The OXA-48 carbapenemase gene regions were determined in 33 of 50 K. pneumoniae strains. In addition, NDM-1 resistance in one, OXA-48 and NDM-1 resistance in four unusual K. pneumoniae isolates were detected. Virulence gene regions that were encountered among K. pneumoniae isolates were 88% wabG, 86% uge, 80% ycfM and 72% entB, related with capsule, capsule lipoprotein and external membrane protein, responsible for enterobactin production, respectively. Even though there was no significant difference between resistant and sensitive strains due to the virulence gene regions (P≥0.05), virulence factors in carbapenem resistant isolates were found to be more diverse. This study is important for both, to prevent the spread of carbapenem resistant infections and to plan for developing effective treatments. Moreover, this study is the first detailed study of the carbapenem resistance and virulence factors in K. pneumoniae strains.

Role of pro-inflammatory cytokines of pancreatic islets and prospects of elaboration of new methods for the diabetes treatment.

Abstract: Several relations between cytokines and pathogenesis of diabetes are reviewed. In type 1 and type 2 diabetes an increased synthesis is observed and as well as the release of pro-inflammatory cytokines, which cause the damage of pancreatic islet cells and, in type 2 diabetes, the development of the insulin resistance. That process results in the disturbed balance between pro-inflammatory and protective cytokines. Pro-inflammatory cytokines such as interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), as well as recently discovered pancreatic derived factor PANDER are involved in the apoptosis of pancreatic β-cells. Inside β-cells, cytokines activate different metabolic pathways leading to the cell death. IL-1β activates the mitogen-activated protein kinases (MAPK), affects the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activates the inducible nitric oxide synthase (iNOS). TNF-α and IFN-γ in a synergic way activate calcium channels, what leads to the mitochondrial dysfunction and activation of caspases. Neutralization of pro-inflammatory cytokines, especially interleukin 1β with the IL-1 receptor antagonist (IL-1Ra) and/or IL-1β antibodies might cause the extinction of the inflammatory process of pancreatic islets, and consequently normalize concentration of glucose in blood and decrease the insulin resistance. In type 1 diabetes interleukin-6 participates in regulation of balance between Th17 and regulatory T cells. In type 2 diabetes and obesity, the long-duration increase of IL-6 concentration in blood above 5 pg/ml leads to the chronic and permanent increase in expression of SOCS3, contributing to the increase in the insulin resistance in cells of the skeletal muscles, liver and adipose tissue.

Ionizing radiation affects protein composition of exosomes secreted in vitro from head and neck squamous cell carcinoma.

Abstract: Exosomes are membrane vesicles of endocytic origin that participate in inter-cellular communication. Environmental and physiological conditions affect composition of secreted exosomes, their abundance and potential influence on recipient cells. Here, we analyzed protein component of exosomes released in vitro from cells exposed to ionizing radiation (2Gy dose) and compared their content with composition of exosomes released from control not irradiated cells. Exosomes secreted from FaDu cells originating from human squamous head and neck cell carcinoma were analyzed using LC-MS/MS approach. We have found that exposure to ionizing radiation resulted in gross changes in exosomal cargo. There were 217 proteins identified in exosomes from control cells and 384 proteins identified in exosomes from irradiated cells, including 148 "common" proteins, 236 proteins detected specifically after irradiation and 69 proteins not detected after irradiation. Among proteins specifically overrepresented in exosomes from irradiated cells were those involved in transcription, translation, protein turnover, cell division and cell signaling. This indicated that exosomal cargo reflected radiation-induced changes in cellular processes like transient suppression of transcription and translation or stress-induced signaling.

The impact of polyphenols on Bifidobacterium growth.

Abstract: Polyphenols are a common group of plant based bioactive compounds, that can affect human health because of their antioxidant and antimicrobial properties as well as free-radical scavenging activity. An increasing interest is observed in the interaction between polyphenols and microbiota occurring in food and the human gut. The aim of the work presented here, was to evaluate the effect of some polyphenolic compounds on the growth of two strains of Bifidobacterium: B. adolescentis and B. bifidum. The influence of some flavonoids: naringinin, hesperidin, rutin, quercetin as well as phenolic acids: gallic, caffeic, p-coumaric, ferulic, chlorogenic, vanillic and sinapic was determined by a 96-well microtiter plate assay. In the experiments the effect of three different concentrations of polyphenols: 2, 20 and 100 µg/ml on the growth of Bifidobacterium strains was investigated. All tested compounds influenced the growth of the examined bacteria. Both stimulatory and inhibitory effects were observed in comparison to the positive control. The strongest impact on the growth of bifidobacteria was observed during the first hours of incubation. The constant inhibitory effect was observed for hesperidin and quercetin addition and was dose-dependent. B. bifidum showed a stronger dependence on phenolic acids content in the medium than B. adolescentis during the first hours of incubation.

miRNA Multiplayers in glioma. From bench to bedside.

Abstract: Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fuelled a search for a new therapeutic targets and treatment modalities. A novel small non-coding RNA molecules, microRNAs (miRNAs), appear to represent one of the most attractive target molecules contributing to the pathogenesis of various types of tumors. They play crucial roles in tumorigenesis, angiogenesis, invasion and apoptosis. Some miRNAs are also associated with clinical outcome and chemo- and radiotherapy resistance. Moreover, miRNA have the potential to affect the responses to molecular-targeted therapies and they also might be associated with cancer stem cell properties, affecting tumor maintenance and progression. The expression profiles of miRNAs are also useful for subclassification of GBM, what underscores the heterogeneity of diseases that all share the same WHO histopathological grade. Importantly, molecular subtypes of GBM appear to correlate with clinical phenotypes, tumor characteristic and treatment outcomes. miRNAs are then biological markers with possible diagnostic and prognostic potential. They could also serve as one of the promising treatment targets in human glioblastoma.

The ins and outs of maternal-fetal fatty acid metabolism.

Abstract: Fatty acids (FAs) are one the most essential substances in intrauterine human growth They are involved in a number of energetic and metabolic processes, including the growth of cell membranes, the retina and the nervous system Fatty acid deficiency and disruptions in the maternal-placental fetal metabolism of FAs lead to malnutrition of the fetus, hypotrophy and preterm birth What is more, metabolic diseases and cardiovascular conditions may appear later in life Meeting a fetus' need for FAs is dependent on maternal diet and on the efficiency of the placenta in transporting FAs to fetal circulation "Essential fatty acids" are among the most important FAs during the intrauterine growth period These are α-linolenic acid, which is a precursor of the n-3 series, linoleic acid, which is a precursor of the n-6 series and their derivatives, represented by docosahexaenoic acid and arachidonic acid The latest studies have shown that medium-chain fatty acids also play a significant role in maternal-fetal metabolism These FAs have significant effect on the transformation of the precursors into DHA, which may contribute to a relatively stable supply of DHA - even in pregnant women whose diet is low in FAs The review discusses the problem of fatty acid metabolism at the intersection between a pregnant woman and her child with reference to physiological pregnancy, giving birth to a healthy child, intrauterine growth restriction, preterm birth and giving birth to a small for gestational age child

Metal responsive transcription factor 1 (MTF-1) regulates zinc dependent cellular processes at the molecular level.

Abstract: Metal responsive transcription factor 1 (MTF-1) is a zinc dependent transcription factor which is involved in the regulation of intracellular signaling pathways MTF-1 regulates the expression of two streams of genes functioning in metal homeostasis and anti-oxidative response MTF-1 acts in the process of binding of toxic metal ions in the cell, due to the activation of the expression of metallothioneins (MTs) Additionally, MTF-1 regulates transcription of genes involved in the sequestration of zinc and its intracellular transport Disruption of zinc and MT homeostasis has an indispensable influence on the development of several pathological states Moreover, by increasing MT activity, MTF-1 can effectively protect cells from oxidative and hypoxic stresses The mechanism of MTF-1 action in cells includes the regulation of the proper immune response through activation/repression of anti- and pro-inflammatory cytokines MTF-1 function in immune response is related to nuclear factor-κB (NF-κB) activity Synthesis of insulin is also related to the activity of this transcription factor and zinc balance Insulin transport also depends on zinc In pancreatic β-cells, several types of the zinc transporters are found Zinc transporters coordinated action is crucial for the synthesis and secretion of insulin Disturbances in the regulation of signaling pathways connected with MTF-1 function can entail further alterations in zinc intracellular status and this growing imbalance can promote the pathophysiology of degenerative disorders

Sampling, metadata and DNA extraction - important steps in metagenomic studies.

Abstract: Metagenomic studies have become increasingly popular. They allow for the estimation of biodiversity in complex populations. This diversity presents an enormous but largely unexpected genetic and biological pool and can be exploited for the recovery of novel genes, entire metabolic pathways and their products. Generally metagenomic study is a genomic analysis of organisms by direct extraction and cloning of DNA from their natural environment. The most common problems of modern metagenomics are as follows: majority of the microorganisms present in the environment cannot be cultivated by standard techniques, DNA extraction methods are not very effective, isolated DNA is contaminated with various compounds, a choice for a screening method is not obvious.

Cell wall proteome of pathogenic fungi.

Abstract: A fast development of a wide variety of proteomic techniques supported by mass spectrometry coupled with high performance liquid chromatography has been observed in recent years. It significantly contributes to the progress in research on the cell wall, very important part of the cells of pathogenic fungi. This complicated structure composed of different polysaccharides, proteins, lipids and melanin, plays a key role in interactions with the host during infection. Changes in the set of the surface-exposed proteins under different environmental conditions provide an effective way for pathogens to respond, adapt and survive in the new niches of infection. This work summarizes the current state of knowledge on proteins, studied both qualitatively and quantitatively, and found within the cell wall of fungal pathogens for humans, including Candida albicans, Candida glabrata, Aspergillus fumigatus, Cryptococcus neoformans and other medically important fungi. The described proteomic studies involved the isolation and fractionation of particular sets of proteins of interest with various techniques, often based on differences in their linkages to the polysaccharide scaffold. Furthermore, the proteinaceous contents of extracellular vesicles ("virulence bags") of C. albicans, C. neoformans, Histoplasma capsulatum and Paracoccidioides brasiliensis are compared, because their production can partially explain the problem of non-classical protein secretion by fungi. The role assigned to surface-exposed proteins in pathogenesis of fungal infections is enormously high, thus justifying the need for further investigation of cell wall proteomes.

Antimicrobial, antiadhesive and antibiofilm potential of lipopeptides synthesised by Bacillus subtilis, on uropathogenic bacteria.

Abstract: The aim of this study was to investigate the antimicrobial effect of lipopeptide biosurfactants from surfactin, iturin and fengycin families, synthesised by the Bacillus subtilis I'1a strain, on uropathogenic bacteria, including the effects on planktonic growth, processes of biofilm formation and dislodging. Antimicrobial activity was tested against 32 uropathogenic strains belonging to 12 different species of Gram-negative and Gram-positive bacteria. The sensitivity of 25 tested bacterial strains to the B. subtilis I'1a filtrate was confirmed by an agar diffusion assay. None of the strains seemed to be sensitive to pure surfactin at concentrations ranging from 0.1 mg × ml(-1) to 0.4 mg ml(-1). After the treatment of uropathogens with B. subtilis lipopeptides, the metabolic activity of planktonic cells was inhibited by 88.05±3.96% in the case of 21 studied uropathogens, the process of biofilm formation was reduced by 88.15±4.77% in the case of 24 uropathogens and mature biofilms of 18 strains were dislodged by about 81.20±4.72%. Ten strains of uropathogenic bacteria were selected to study the antimicrobial activity of surfactin (concentrations 0.1, 0.2 and 0.4 mg × ml(-1)). Surfactin had no influence on the metabolic activity of planktonic forms of uropathogens, however, biofilms of 5 tested strains were reduced by 64.77±9.05% in the presence of this biosurfactant at the concentration 0.1 mg × ml(-1). The negative effect of the compound on the biofilm formation process was observed at all concentrations used. The above-described results were fully confirmed by CLSM. It could suggest that synergistic application of biosurfactants could be efficient in uropathogen eradication.

Antibacterial activity of caffeine against plant pathogenic bacteria.

Abstract: The objective of the present study was to evaluate the antibacterial properties of a plant secondary metabolite - caffeine. Caffeine is present in over 100 plant species. Antibacterial activity of caffeine was examined against the following plant-pathogenic bacteria: Ralstonia solanacearum (Rsol), Clavibacter michiganesis subsp. sepedonicus (Cms), Dickeya solani (Dsol), Pectobacterium atrosepticum (Pba), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pseudomonas syringae pv. tomato (Pst), and Xanthomonas campestris subsp. campestris (Xcc). MIC and MBC values ranged from 5 to 20 mM and from 43 to 100 mM, respectively. Caffeine increased the bacterial generation time of all tested species and caused changes in cell morphology. The influence of caffeine on the synthesis of DNA, RNA and proteins was investigated in cultures of plant pathogenic bacteria with labelled precursors: [(3)H]thymidine, [(3)H]uridine or (14)C leucine, respectively. RNA biosynthesis was more affected than DNA or protein biosynthesis in bacterial cells treated with caffeine. Treatment of Pba with caffeine for 336 h did not induce resistance to this compound. Caffeine application reduced disease symptoms caused by Dsol on chicory leaves, potato slices, and whole potato tubers. The data presented indicate caffeine as a potential tool for the control of diseases caused by plant-pathogenic bacteria, especially under storage conditions.

Multidirectional effects of triterpene saponins on cancer cells - mini-review of in vitro studies.

Abstract: Triterpene saponins (saponosides) are found in a variety of higher plants and display a wide range of pharmacological activities, including expectorant, anti-inflamatory, vasoprotective, gastroprotective and antimicrobial properties. Recently, a potential anticancer activity of saponins has been suggested by their cytotoxic, cytostatic, pro-apoptotic and anti-invasive effects. At high concentrations (more than 100 µM) saponins exert cytotoxic and haemolytic effects via permeabilization of the cell membranes. Noteworthy, the inhibition of cancer cell proliferation, the induction of apoptosis and attenuation of cell invasiveness is observed in the presence of low saponin concentrations. Saponins might affect the expression of genes associated with malignancy. These alterations are directly related to the invasive phenotype of cancer cells and depend on "cellular context". It illustrates the relationships between the action of saponins, and the momentary genomic/proteomic status of cancer cells. Here, we discuss the hallmarks of anti-cancer activity of saponins with the particular emphasis on anti-invasive effect of diverse groups of saponins that have been investigated in relation to tumor therapy.

Activity and kinetic properties of phosphotransacetylase from intestinal sulfate-reducing bacteria.

Abstract: Phosphotransacetylase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. has never been well-characterized and has not been studied yet. In this paper, the specific activity of phosphotransacetylase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains were presented at the first time. The microbiological, biochemical, biophysical and statistical methods in this work were used. The optimal temperature and pH for enzyme reaction was determined. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period, τ) and maximum velocity of the phosphotransacetylase reaction (Vmax) were defined. Michaelis constants (Km) of the enzyme reaction (3.36 ± 0.35 mM for D. piger Vib-7, 5.97 ± 0.62 mM for Desulfomicrobium sp. Rod-9) were calculated. The studies of the phosphotransacetylase in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in intestinal sulfate-reducing bacteria, their production of acetate in detail can be perspective for clarification of their etiological role in the development of the humans and animals bowel diseases. These studies might help in predicting the development of diseases of the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.

Selected mucolytic, anti-inflammatory and cardiovascular drugs change the ability of neutrophils to form extracellular traps (NETs).

Abstract: Neutrophils form the first line of host defense against infections that combat pathogens using two major mechanisms, the phagocytosis or the release of neutrophil extracellular traps (NETs). The netosis (NET formation) exerts additional, unfavorable effects on the fitness of host cells and is also involved at the sites of lung infection, increasing the mucus viscosity and in the circulatory system where it can influence the intravascular clot formation. Although molecular mechanisms underlying the netosis are still incompletely understood, a role of NADPH oxidase that activates the production of reactive oxygen species (ROS) during the initiation of NETs has been well documented. Since several commonly used drugs can affects the netosis, our current study was aimed to determine the effects of selected mucolytic, anti-inflammatory and cardiovascular drugs on NET formation, with a special emphasis on ROS production and NADPH oxidase activity. The treatment of neutrophils with N-acetylcysteine, ketoprofen and ethamsylate reduced the production of ROS by these cells in a dose-dependent manner. NET formation was also modulated by selected drugs. N-acetylcysteine inhibited the netosis but in the presence of H2O2 this neutrophil ability was restored, indicating that N-acetylcysteine may influence the NET formation by modulating ROS productivity. The administration of ethamsylate led to a significant reduction in NET formation and this effect was not restored by H2O2 or S. aureus, suggesting the unexpected additional side effects of this drug. Ketoprofen seemed to promote ROS-independent NET release, simultaneously inhibiting ROS production. The results, obtained in this study strongly suggest that the therapeutic strategies applied in many neutrophil-mediated diseases should take into account the NET-associated effects.

Influence of silver nanoparticles on metabolism and toxicity of moulds.

Abstract: The unique antimicrobial features of silver nanoparticles (AgNPs) are commonly applied in innumerable products. The lack of published studies on the mechanisms of AgNPs action on fungi resulted in identification of the aim of this study, which was: the determination of the influence of AgNPs on the mould cytotoxicity for swine kidney cells (MTT test) and the production of selected mycotoxins, organic acids, extracellular enzymes by moulds. The conducted study had shown that silver nanoparticles can change the metabolism and toxicity of moulds. AgNPs decrease the mycotoxin production of Aspergillus sp. (81-96%) and reduce mould cytotoxicity (50-75%). AgNPs influence the organic acid production of A. niger and P. chrysogenum by decreasing their concentration (especially of the oxalic and citric acid). Also, a change in the extracellular enzyme profile of A. niger and P. chrysogenum was observed, however, the total enzymatic activity was increased.

Proteomics in studies of Staphylococcus aureus virulence.

Abstract: Staphylococcus aureus is a widespread, opportunistic pathogen that causes community and hospital acquired infections. Its high pathogenicity is driven by multifactorial and complex mechanisms determined by the ability of the bacterium to express a wide variety of virulence factors. The proteome secreted into extracellular milieu is a rich reservoir of such factors which include mainly nonenzymatic toxins and enzymes. Simultaneously, membrane proteins, membrane-cell wall interface proteins and cell wall-associated proteins also strongly influence staphylococcal virulence. Proteomics shows a great potential in exploring the role of the extracellular proteome in cell physiology, including the pathogenic potential of particular strains of staphylococci. In turn, understanding the bacterial physiology including the interconnections of particular factors within the extracellular proteomes is a key to the development of the ever needed, novel antibacterial strategies. Here, we briefly overview the latest applications of gel-based and gel-free proteomic techniques in the identification of the virulence factors within S. aureus secretome and surfacome. Such studies are of utmost importance in understanding the host-pathogen interactions, analysis of the role of staphylococcal regulatory systems and also the detection of posttranslational modifications emerging as important modifiers of the infection process.

Chlamydial plasmids and bacteriophages.

Abstract: Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

Application of the BIOLOG system for characterization of Serratia marcescens ss marcescens isolated from onsite wastewater technology (OSWT)

Abstract: The scope of this study was to apply the Biolog system to identify and characterize a Serratia strain isolated from the surface of black plastic pieces which constitute the fluidized bed filter (onsite wastewater technology, OSWT). The preliminary isolation of the strain was done in the medium with tetracycline at a 16 mg/l concentration. To characterize the isolated strain, the following Biolog methods were applied: (1) EcoPlates microplates for evaluation of physiological profiling, (2) GEN III OmniLog® ID System for identification of the isolate, and (3) phenotypic microarrays (PM) technology for evaluation of sensitivity to antibiotics (PM11 and PM12). Results were recorded using the original OmniLog® software. The Serratia strain was identified as Serratia marcescens ss marcescens with similarity index 0.569. The same identification was obtained by the 16S rDNA analysis. PM analysis showed an enhancement of phenotype (resistance or growth) of this strain to 35 antibiotics. The loss of phenotype (sensitivity or non-growth) was observed only for 5 antibiotics: lomefloxacin (0.4 µg/ml), enoxacin (0.9 µg/ml), nalidixic acid (18.0 µg/ml), paromomycin (25.0 µg/ml) and novobiocin (1100 µg/ml). This study acknowledges that the methods proposed by the Biolog system allow correct and complete identification and characterization of the microbes isolated from different environments. Phenotypic microarrays could be successfully used as a new tool for identification of the multi-antibiotic resistance of bacteria and for determination of the minimal inhibition concentrations (MIC).

Immune response gene polymorphisms in tuberculosis

Abstract: Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M.tb), remains a leading public health problem in most parts of the world. Despite the discovery of the bacilli over 100 years ago, there are still many unanswered questions about the host resistance to TB. Although one third of the world's population is infected with virulent M.tb, no more than 5-10% develop active disease within their lifetime. A lot of studies suggest that host genetic factors determine the outcome of M.tb-host interactions, however, specific genes and polymorphisms that govern the development of TB are not completely understood. Strong evidence exists for genes encoding pattern recognition receptors (TLR, CD14), C-type lectins, cytokines/chemokines and their receptors (IFN-γ, TNF-α, IL-12, IL-10, MCP-1, MMP-1), major histocompatibility complex (MHC) molecules, vitamin D receptor (VDR), and proton-coupled divalent metal ion transporters (SLC11A1). Polymorphisms in these genes have a diverse influence on the susceptibility to or protection against TB among particular families, ethnicities and races. In this paper, we review recent discoveries in genetic studies and correlate these findings with their influence on TB susceptibility.

PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

Abstract: Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens.

Optimization of culture conditions for flexirubin production by Chryseobacterium artocarpi CECT 8497 using response surface methodology

Abstract: Flexirubins are the unique type of bacterial pigments produced by the bacteria from the genus Chryseobacterium, which are used in the treatment of chronic skin disease, eczema etc. and may serve as a chemotaxonomic marker. Chryseobacterium artocarpi CECT 8497, an yellowish-orange pigment producing strain was investigated for maximum production of pigment by optimizing medium composition employing response surface methodology (RSM). Culture conditions affecting pigment production were optimized statistically in shake flask experiments. Lactose, l-tryptophan and KH2PO4 were the most significant variables affecting pigment production. Box Behnken design (BBD) and RSM analysis were adopted to investigate the interactions between variables and determine the optimal values for maximum pigment production. Evaluation of the experimental results signified that the optimum conditions for maximum production of pigment (521.64 mg/L) in 50 L bioreactor were lactose 11.25 g/L, l-tryptophan 6 g/L and KH2PO4 650 ppm. Production under optimized conditions increased to 7.23 fold comparing to its production prior to optimization. Results of this study showed that statistical optimization of medium composition and their interaction effects enable short listing of the significant factors influencing maximum pigment production from Chryseobacterium artocarpi CECT 8497. In addition, this is the first report optimizing the process parameters for flexirubin type pigment production from Chryseobacterium artocarpi CECT 8497.

Surfaceome of pathogenic yeasts, Candida parapsilosis and Candida tropicalis, revealed with the use of cell surface shaving method and shotgun proteomic approach.

Abstract: In the course of infections caused by pathogenic yeasts from the genus Candida, the fungal cell surface is the first line of contact with the human host. As the surface-exposed proteins are the key players in these interactions, their identification can significantly contribute to discovering the mechanisms of pathogenesis of two emerging pathogens from this genus, C. parapsilosis and C. tropicalis. Therefore, the aim of the present study was to identify the cell wall-attached proteins of these two species with the use of cell surface shaving and a shotgun proteomic approach. Different morphological forms of C. parapsilosis and C. tropicalis cells obtained after growth under various conditions were subjected to this treatment. This allowed to indicate the most abundant cell surface proteins on the basis of the normalized spectral abundance factors. In case of yeast-like forms these were, among others, proteins similar to a chitinase, glyceraldehyde-3-phosphate dehydrogenase and an inducible acid phosphatase for C. parapsilosis, and a constitutive acid phosphatase, pyruvate decarboxylase and glyceraldehyde-3-phosphate dehydrogenase for C. tropicalis. In case of pseudohyphal forms, proteins similar to a cell surface mannoprotein Mp65, chitinase and glycosylphosphatidylinositol-anchored transglycosylase Crh11 were identified at the cell surface of C. parapsilosis. The Rbt1 cell wall protein, a hyphally regulated cell wall protein and proteins from agglutinin-like sequence protein family were found as the most abundant on C. tropicalis pseudohyphae. Apart from the abovementioned proteins, several additional covalently bound and atypical cell wall proteins were also identified. These results extend the current knowledge regarding the molecular basis of virulence of these two non-albicans Candida species.

Monomeric and gemini surfactants as antimicrobial agents - influence on environmental and reference strains.

Abstract: Quaternary ammonium salts (QAS) belong to surfactant commonly used both, in the household and in different branches of industry, primarily in the process of cleaning and disinfection. They have several positive features inter alia effectively limiting the development of microorganisms on many surfaces. In the present work, two compounds were used as biocides: hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) that belongs to the gemini surfactant (GS), and its single analogue - dodecyl(trimethyl)ammonium bromide (DTAB). Two fold dilution method was used to determine the minimum concentration of compounds (MIC) which inhibit the growth of bacteria: Staphylococcus aureus (ATCC 6538 and an environmental strain), Pseudomonas aeruginosa (ATCC 85327 and an environmental strain), and yeast Candida albicans (ATCC 11509 and an environmental strain). The viability of cells in liquid cultures with addition of these substances at ¼ MIC, ½ MIC and MIC concentrations were also determined. The obtained results show that DTAB inhibits the growth of bacteria at the concentration of 0.126-1.010 µM/ml, and gemini surfactant is active at 0.036-0.029 µM/ml. Therefore, GS is active at more than 17-70-fold lower concentrations than its monomeric analogue. Strains isolated from natural environment are less sensitive upon testing biocides than the references strains. Both compounds at the MIC value reduced the number of cells of all strains. The use of too low concentration of biocides can limit the growth of microorganisms, but often only for a short period of time in case of special environmental strains. Later on, they can adapt to adverse environmental conditions and begin to evolve defence mechanisms.

Examination of cyp51A and cyp51B expression level of the first Polish azole resistant clinical Aspergillus fumigatus isolate.

Abstract: Aspergillus fumigatus is one of the most prevalent airborne fungal pathogens causing infections worldwide. Most A. fumigatus strains are susceptible to azoles, which are administered as the first line therapeutics. However, during last decade the acquired resistance to triazoles by these species has been described. There is a number of publications concerning the examination of clinical A. fumigatus strains from different countries, however there has been no report from Poland. Here, we describe for the first time, an examination of cyp51A and cyp51B expression level of 11 clinical A. fumigatus strains isolated during 2007-2014 period from the collection of Medical University in Wroclaw. Their susceptibility to itraconazole, voriconazole and posaconazole has been examined. The MIC values of triazoles for one of the examined isolates were respectively: > 8 mg/L for itraconazole, 2 mg/L for voriconazole and 0.5 mg/L for posaconazole. The cyp51A gene with its promoter region of all isolates was sequenced. It was found that the resistant isolate harbors the TR34/L98H mutation in the cyp51A gene and when cultured on media supplemented with voriconazole exhibits overexpression of both, cyp51A and cyp51B genes. The level of cyp51A gene expression was about 50 times higher than cyp51B.

Inhibition of cell proliferation and induction of apoptosis in K562 human leukemia cells by the derivative (3-NpC) from dihydro-pyranochromenes family.

Abstract: Leukemia is a particular type of cancer characterized by the failure of cell death or disability in differentiation of hematopoietic cells. Chronic myelogenus leukemia (CML) is the most studied kind of this cancer. In this study, anti-cancer effect of dihydro-pyranochromenes derivatives were investigated in the human leukemia K562 cells. These compounds were found to be active cell proliferation inhibitors using MTT assay. Among these compounds, 3-NpC was determined as stronger compound with IC50 value of 100 ± 3.1 µM and was chosen for further studies. Induction of apoptosis was analyzed by AO/EtBr staining, DNA fragmentation assay, Annexin V/PI double staining and cell cycle analysis. Furthermore, Western Blot analysis showed that treatment of the cells with 3-NpC led to up-regulation and activation of caspase-3. The results of this investigation clearly indicated that dihydro-pyranochromenes derivatives induce apoptosis in the K562 cell line. This information signalizes also that these compounds may prepare a new therapeutic approach for the treatment of leukemia.

Analysis of uropathogenic Escherichia coli biofilm formation under different growth conditions.

Abstract: The ability to form different types of biofilm enables bacteria to survive in a harsh or toxic environment. Different structures of biofilms are related to different surfaces and environment of bacterial growth. The aim of this study was analysis of the biofilm formation of 115 clinical uropathogenic Escherichia coli strains under different growth conditions: surface for biofilm formation, medium composition and time of incubation. The biofilm formation after 24 h, 48 h, 72 h and 96 h was determined spectrophotometrically (A531) after crystal violet staining and it was correlated with bacterial growth (A600). The live and dead cells in biofilm structures was also observed on the glass surface by an epi-fluorescence microscope. Additionally, the presence of rpoS, sdiA and rscA genes was analyzed. The statistical significance was estimated by paired T-test. The observed biofilms were different for each particular strain. The biofilm formation was the highest in the rich medium (LB) after 24 h and its level hasn't changed in time. When biofilm level was compared to bacterial growth (relative biofilm) - it was higher in a minimal medium in comparison to enriched medium. These results suggest that most of the bacterial cells prefer to live in a biofilm community under the difficult environmental conditions. Moreover, biofilm formation on polyurethane surface did not correlate with biofilm formation on glass. It suggests that mechanisms of biofilm formation can be correlated with other bacterial properties. This phenomenon may explain different types of biofilm formation among one species and even one pathotype - uropathogenic Escherichia coli.

Protective effects of quercetin on cadmium fluoride induced oxidative stress at different intervals of time in mouse liver.

Abstract: Quercetin, a member of the flavonoid family is a major antioxidant acquired in humans by food consumption, while Cadmium fluoride (CdF 2) is one of the naturally occurring chemicals having adverse effects. The protec tive effect of quercetin on time dependent oxidative damage induced in mice liver by CdF2 was studied in the following groups of mice consisting of six mice each: (i) control group; (ii) mice treated with single i.p injection of 2 mg/kg bw CdF2 for 24 h; (iii) mice treated with single i.p injection of 2 mg/kg bw CdF2 for 48 h; (iv) mice treated with single i.p injection of quercetin (100 mg/ kg bw); (v) mice treated with i.p injection of 100 mg/kg bw of quercetin followed by i.p injection of CdF2 (2 mg/ kg bw) for 24 h; and (vi) mice treated with i.p injection of 100mg/kg bw of quercetin followed by CdF2 (2 mg/ kg bw) for 48 h. Administration of quercetin two hours before CdF2 significantly reduced the biochemical altera tions in reduced glutathione, ascorbic acid, lipid peroxidation, super oxide dismutase, catalase and total protein (p<0.05). Histopathology also showed the protective effect of quercetin. The livers treated with CdF 2 were atrophic, markedly nodular, inflamed and necrotic. How ever, this effect was reduced to a minimum in the mice pre-treated for two hours with quercetin.

Biotechnological conversion of glycerol from biofuels to 1,3-propanediol using Escherichia coli.

Abstract: In the face of shortage of fossil fuel supplies and climate warming triggered by excessive carbon dioxide emission, alternative resources for chemical industry have gained considerable attention. Renewable resources and their derivatives are of particular interest. Glycerol, which constitutes one of the by-products during biodiesel production, is such a substrate. Thus, generated excess glycerol may become an environmental problem, since it cannot be disposed of in the environment. The most promising products obtained from glycerol are polyols, including 1,3-propanediol, an important substrate in the production of synthetic materials, e.g. polyurethanes, unsaturated polyesters, and epoxy resins. Glycerol can be used as a carbon and energy source for microbial growth in industrial microbiology to produce 1,3-propanediol. This paper is a review of metabolic pathways of native producers and E. coli with the acquired ability to produce the diol via genetic manipulations. Culture conditions during 1,3-PDO production and genetic modifications of E. coli used in order to increase efficiency of glycerol bioconversion are also described in this paper.

Synthesis of kaempferide Mannich base derivatives and their antiproliferative activity on three human cancer cell lines

Abstract: Kaempferide (3,5,7-trihydroxy-4'-methoxyflavone, 1), a naturally occurring flavonoid with potent anticancer activity in a number of human tumour cell lines, was first semisynthesized from naringin. Based on Mannich reaction of kaempferide with various secondary amines and formaldehyde, nine novel kaempferide Mannich base derivatives 2-10 were synthesized. The aminomethylation occurred preferentially in the position at C-6 and C-8 of the A-ring of kaempferide. All the synthetic compounds were tested for antiproliferative activity against three human cancer cell lines (Hela, HCC1954, SK-OV-3) by the standard MTT method. The results showed that compounds 1, 2 and 5-10 were more potent against Hela cells with IC50 values of 12.47-28.24 μM than the positive control cis-platin (IC50 41.25 μM), compounds 5, 6, 8 and 10 were more potent against HCC1954 cells with IC50 values of 8.82-14.97 μM than the positive control cis-platin (IC50 29.68 μM), and compounds 2, 3, 5, 6 and 10 were more potent against SK-OV-3 cells with IC50 values of 7.67-18.50 μM than the positive control cis-platin (IC50 21.27 μM).