Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes

Aims: We have developed a PCR‐based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin‐coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin‐coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin‐coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin‐coding sequences present in non‐LAB bacteriocin producers. The results suggest that several potentially new bacteriocin‐coding sequences were found. Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.     

Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor

It is the first detailed study of an inulinolytic fungus Aspergillus niger ATCC 204447 since its discovery, covering submerged cultivations both in shake flasks and a stirred tank bioreactor. Various carbon sources were applied to induce the inulinolytic activity in shake flask cultures. The highest volumetric and specific (per gram of biomass) activities (respectively 0.68 U/mL and 184 U g/X) were observed for the initial inulin and sucrose concentrations equal to 20 g/L. The fungus grew as large (>3 mm) spherical pellets. The influence of inoculum density and application of microparticle‐enhanced cultivation (MPEC) were studied in the batch bioreactor cultivations. Inoculum density moderately affected the inulinolytic activities, whose highest values were 0.7 U/mL and 165 U g/X at the lowest studied spore density of 3.33·10⁸ L^?1. Dispersed hyphae evolved in the bioreactor made the broth difficult to aerate due to high apparent viscosity (exceeding 200 Pa sⁿ at shear rate about 0.05 s^?1) and shear thinned properties (flow behavior index below 0.2). In MPEC (10 μm talc microparticles) the pellets of diameter between 1 and 2 mm were formed, which facilitated the aeration of the broth and increased the specific inulinolytic activity 3.5‐fold.     

Cytocompatibility of Medical Biomaterials Containing Nickel by Osteoblasts: a Systematic Literature Review

The present review is based on a survey of 21 studies on the cytocompatibility of medical biomaterials containing nickel, as assessed by cell culture of human and animal osteoblasts or osteoblast-like cells. Among the biomaterials evaluated were stainless steel, NiTi alloys, pure Ni, Ti, and other pure metals. The materials were either commercially available, prepared by the authors, or implanted by various techniques to generate a protective layer of oxides, nitrides, acetylides. The observation that the layers significantly reduced the initial release of metal ions and increased cytocompatibility was confirmed in cell culture experiments. Physical and chemical characterization of the materials was performed. This included, e.g., surface characterization (roughness, wettability, corrosion behavior, quantity of released ions, microhardness, and characterization of passivation layer). Cytocompatibility tests of the materials were conducted in the cultures of human or animal osteoblasts and osteoblast-like cells. The following assays were carried out: cell proliferation and viability test, adhesion test, morphology (by fluorescent microscopy or SEM). Also phenotypic and genotypic markers were investigated. In the majority of works, it was found that the most cytocompatible materials were stainless steel and NiTi alloy. Pure Ni was rendered and less cytocompatible. All the papers confirmed that the consequence of the formation of protective layers was in significant increase of cytocompatibility of the materials. This indicates the possible further modifications of the manufacturing process (formation of the passivation layer).     

Polymorphisms in base-excision repair and nucleotide-excision repair genes in relation to lung cancer risk

Polymorphisms in DNA repair genes may be associated with differences in DNA repair capacity, thereby influencing the individual susceptibility to smoking-related cancer. We investigated the association of 10 base-excision and nucleotide-excision repair gene polymorphisms (XRCC1 -77 T/C, Arg194Trp, Arg280His and Arg399Gln; APE1 Asp148Glu; OGG1 Ser326Cys; XPA -4 G/A; XPC PAT; XPD Asp312Asn and Lys751Gln) with lung cancer risk in Caucasians. Genotypes were determined by PCR-RFLP and PCR-single base extension assays in 110 lung cancer patients and 110 age- and sex-matched controls, and the results were analyzed using logistic regression adjusted for relevant covariates. A significant association between the APE1 Asp148Glu polymorphism and lung cancer risk was found, with adjusted odds ratios (OR) of 3.38 (p=0.001) for the Asp/Glu genotype and 2.39 (p=0.038) for the Glu/Glu genotype. Gene-smoking interaction analyses revealed a statistically significant interaction between cumulative cigarette smoking and the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms: these polymorphisms were significantly associated with lung cancer in nonsmokers and light smokers (<25 PY; OR=4.92, p=0.021 for XRCC1 399 Gln/Gln; OR=3.62, p=0.049 for XPD 751 Gln/Gln), but not in heavy smokers (> or =25 PY; OR=0.68, p=0.566 for XRCC1 399 Gln/Gln; OR=0.46, p=0.295 for XPD 751 Gln/Gln). Both the XRCC1 Arg194Trp and Arg280His as well as the OGG1 Ser326Cys heterozygous genotypes were associated with a significantly reduced risk for lung cancer (OR=0.32, p=0.024; OR=0.25, p=0.028; OR=0.51, p=0.033, respectively). No associations with lung cancer risk were found for the XRCC1 -77 T/C, the XPA -4 G/A and the XPC PAT polymorphisms. In conclusion, the APE1 Asp148Glu polymorphism is highly predictive for lung cancer, and cumulative cigarette smoking modifies the associations between the XRCC1 Arg399Gln and the XPD Lys751Gln polymorphisms and lung cancer risk.     

Treating seeds with activators of plant defence generates long-lasting priming of resistance to pests and pathogens

? Priming of defence is a strategy employed by plants exposed to stress to enhance resistance against future stress episodes with minimal associated costs on growth. Here, we test the hypothesis that application of priming agents to seeds can result in plants with primed defences. ? We measured resistance to arthropod herbivores and disease in tomato (Solanum lycopersicum) plants grown from seed treated with jasmonic acid (JA) and/or β-aminobutryric acid (BABA). ? Plants grown from JA-treated seed showed increased resistance against herbivory by spider mites, caterpillars and aphids, and against the necrotrophic fungal pathogen, Botrytis cinerea. BABA seed treatment provided primed defence against powdery mildew disease caused by the biotrophic fungal pathogen, Oidium neolycopersici. Priming responses were long-lasting, with significant increases in resistance sustained in plants grown from treated seed for at least 8 wk, and were associated with enhanced defence gene expression during pathogen attack. There was no significant antagonism between different forms of defence in plants grown from seeds treated with a combination of JA and BABA. ? Long-term defence priming by seed treatments was not accompanied by reductions in growth, and may therefore be suitable for commercial exploitation.     

Carbon uptake in Eurasian boreal forests dominates the high-latitude net ecosystem carbon budget

Arctic-boreal landscapes are experiencing profound warming, along with changes in ecosystem moisture status and disturbance from fire. This region is of global importance in terms of carbon feedbacks to climate, yet the sign (sink or source) and magnitude of the Arctic-boreal carbon budget within recent years remains highly uncertain. Here, we provide new estimates of recent (2003–2015) vegetation gross primary productivity (GPP), ecosystem respiration (R_eco), net ecosystem CO₂ exchange (NEE; R_eco − GPP), and terrestrial methane (CH₄) emissions for the Arctic-boreal zone using a satellite data-driven process-model for northern ecosystems (TCFM-Arctic), calibrated and evaluated using measurements from >60 tower eddy covariance (EC) sites. We used TCFM-Arctic to obtain daily 1-km² flux estimates and annual carbon budgets for the pan-Arctic-boreal region. Across the domain, the model indicated an overall average NEE sink of −850 Tg CO₂-C year⁻¹. Eurasian boreal zones, especially those in Siberia, contributed to a majority of the net sink. In contrast, the tundra biome was relatively carbon neutral (ranging from small sink to source). Regional CH₄ emissions from tundra and boreal wetlands (not accounting for aquatic CH₄) were estimated at 35 Tg CH₄-C year⁻¹. Accounting for additional emissions from open water aquatic bodies and from fire, using available estimates from the literature, reduced the total regional NEE sink by 21% and shifted many far northern tundra landscapes, and some boreal forests, to a net carbon source. This assessment, based on in situ observations and models, improves our understanding of the high-latitude carbon status and also indicates a continued need for integrated site-to-regional assessments to monitor the vulnerability of these ecosystems to climate change.     

Partial Purification and Characterization of Indol-3-Ylacetylglucosemyo-Inositol Indol-3-Ylacetyltransferase (Indoleacetic Acid-Inositol Synthase)

A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-β-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-β-d-glucose is, K_m = 30 micromolar, and for myo-inositol is, K_m = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.     

Two‐dimensional fluorescence as soft sensor in the monitoring of biotransformation performed by yeast

Soft sensors are powerful tools for bioprocess monitoring due to their ability to perform online, noninvasive measurement, and possibility of detection of multiple components in cultivation media, which in turn can provide tools for the quantification of more than one metabolite/substrate/product in real time. In this work, soft sensor based on excitation‐emission fluorescence is for the first time applied for the monitoring of biotransformation production of 2‐phenylethanol (2‐PE) by yeast strains. Main process parameters—such as optical density, glucose, and 2‐PE concentrations—were determined with high accuracy and precision by fluorescence fingerprinting coupled with partial least squares regression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:299–307, 2017     

Anti-HmuY Antibodies Specifically Recognize Porphyromonas gingivalis HmuY Protein but Not Homologous Proteins in Other Periodontopathogens

Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.     

Refined model of optical activity of cis-dihydrodiol metabolites: role of 1,3-diene conformation in the electronic circular dichroism spectra

The effect of the secondary structural feature, that is, nonplanarity of carbon-carbon double bonds, on the rotatory strength of the long-wavelength π-π* electronic transition has been investigated for the series of monocyclic cis-dihydrodiol arene metabolites and model compounds. The contribution from nonplanar C=C bonds to the overall rotatory strength of the π-π* electronic transition is more significant than the contribution from twisted 1,3-diene chromophore. The effect of the substitution pattern in the cyclohexadiene skeleton may be decisive for the sign of the long-wavelength π-π* Cotton effect.