Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4β,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC₅₀ values in the range 3.6–13.5 μM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1–3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258–propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.     

Separation of Indium from Acid Sulfate-Containing Solutions by Ion Exchange with Impregnated Resins

Indium separation using ion exchange resins from acidic polymetallic and very diluted solutions are investigated. Since the selectivity of commercial ion exchange resins have proven to be too low for an effective separation from solutions with high content of other metals, Lewatit® TP 208 was impregnated with common extractants to enhance its properties. By resin impregnation with D2EHPA and Cyanex 272, not only the selective indium recovery was reached but also the resin capacity was increased approx. two times. The best loading and elution performance were shown by Cyanex 272-impregnated Lewatit® TP 208, increasing the indium purity in the eluate from 0.75 % to 85 %.     

κ-Opioid Signaling in the Lateral Hypothalamic Area Modulates Nicotine-Induced Negative Energy Balance

Several studies have reported that nicotine, the main bioactive component of tobacco, exerts a marked negative energy balance. Apart from its anorectic action, nicotine also modulates energy expenditure, by regulating brown adipose tissue (BAT) thermogenesis and white adipose tissue (WAT) browning. These effects are mainly controlled at the central level by modulation of hypothalamic neuropeptide systems and energy sensors, such as AMP-activated protein kinase (AMPK). In this study, we aimed to investigate the kappa opioid receptor (κOR)/dynorphin signaling in the modulation of nicotine’s effects on energy balance. We found that body weight loss after nicotine treatment is associated with a down-regulation of the κOR endogenous ligand dynorphin precursor and with a marked reduction in κOR signaling and the p70 S6 kinase/ribosomal protein S6 (S6K/rpS6) pathway in the lateral hypothalamic area (LHA). The inhibition of these pathways by nicotine was completely blunted in κOR deficient mice, after central pharmacological blockade of κOR, and in rodents where κOR was genetically knocked down specifically in the LHA. Moreover, κOR-mediated nicotine effects on body weight do not depend on orexin. These data unravel a new central regulatory pathway modulating nicotine’s effects on energy balance.     

Determination of the geographical origin of processed spice using multielement and isotopic pattern on the example of Szegedi paprika

This case study presents a fast and reliable method of combining strontium isotope ratios (⁸⁷Sr/⁸⁶Sr) with a multielement pattern (Rb, Sr, Y, Zr, Mo, Cd, Ba, Pb, Th, U, Mg, Ca, Sc, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As and rare earth elements) by means of inductively coupled plasma mass spectrometry (ICP-MS) to establish a unique fingerprint of authentic Szegedi Fűszerpaprika (PDO) and classify authentic and purchased paprika from different known, declared and unknown geographical origin using multivariate statistical tools (principal component and canonical discriminant analysis). Since paprika represents a processed spice, alterations in element and Sr isotopic composition throughout the production process were investigated. The Sr source in the final product was identified to stem from bioavailable Sr sources in soil. Therefore, the ammonium nitrate extract of a soil is sufficient to establish a Sr fingerprint for agricultural products of a region. As a consequence, the spice paprika can be traced back to its geographical origin even after processing.     

Reduction of counts of Listeria monocytogenes in cheese by means of high hydrostatic pressure

Inactivation of Listeria monocytogenes (strains NCTC 11994 and Scott A) was evaluated in model cheeses submitted to 10 min HHP treatments of 300, 400 or 500 MPa at 5 or 20 degrees C. Counts were measured immediately after high hydrostatic pressure (HHP) treatment (day 1) and after 2, 15 and 30 days of storage at 8 degrees C. Both strains behaved significantly different after 400 and 500 MPa, being NCTC 11994 more sensitive. Scarce differences were found among final values at both HHP treatment temperatures. Initial reductions (log cfu/g) for 400 MPa at 20 degrees C were 2.9 +/- 0.2 for strain NCTC 11994 and 1.5 +/- 0.2 for Scott A. They reached after 30-day storage 5.3 +/- 0.2 and 4.6 +/- 0.4 log cfu/g for NCTC 11994 and Scott A, respectively. For 500 MPa treatments, day-1 reductions of both strains were around 5-log cfu/g, and counts fell below quantification limit after 30 days. Injured cells (around 0.8-log cfu/g) were mostly observed in 400 MPa treated samples on days 1 and 2. Starter cells suffered higher inactivation and injury. For 20 degrees C treatments, its final counts (log cfu/g) at 300, 400 and 500 MPa were: 8.5 +/- 0.2, 5.4 +/- 0.3 and 2.5 +/- 0.1, respectively. These figures evidence the HHP potential to improve safety of cheese products.     

Enterococcus populations in Pecorino Abruzzese cheese: biodiversity and safety aspects

The presence of enterococci in Pecorino Abruzzese cheese during ripening was evaluated. Counts were high, especially in fully ripened summer batches. Seventy strains were isolated and identified based on phenotypical and genotypical features as Enterococcus faecium (48.5%), Enterococcus faecalis (40%), and Enterococcus durans (11.5%), with the first species predominant in spring batches and the second predominant in summer batches. High biodiversity was revealed by random amplification of polymorphic DNA and a PCR assay, suggesting the presence of autochthonous strains. E. faecium isolates were the most resistant to the tested antibiotics, especially to erythromycin, chloramphenicol, and penicillin, but all strains were susceptible to vancomycin, as confirmed by the absence of vanA and vanB genes. The presence of some virulence determinants was investigated, revealing the diffusion of aggregation substance (asal) and gelatinase (gelE) genes in 37.5% of E. faecalis strains. However, none of the isolates produced gelatinase in vitro, suggesting the presence of silent genes. The virulence genes were absent in E. durans. Among E. faecium strains, only Lab 41/1 possessed gelE and asal, whose presence previously has been reported only in E. faecalis. Decarboxylating activity was revealed for phenylalanine (27% of the strains) and tyrosine (96%) but not histidine. The presence of a tyrosine decarboxylase-encoding gene was observed for all strains. A comparison of these results with those of previous studies of clinical and food isolates indicates that enterococci from Pecorino Abruzzese cheese have low pathogenic potential.     

Stiffness alterations of single cells induced by UV in the presence of nanoTiO2

Nanocrystalline titanium dioxide (nanoTiO2) has been reported to generate reactive oxygen species (ROS) under UV illumination. In our studies, changes in mechanical properties of human skin fibroblasts, exposed to the oxidative stress induced in the presence of nanoTiO2 and UV light, were studied using atomic force microscopy (AFM). The exposure of cells to the action of ROS was performed at low TiO2 concentration (4 microg/mL) and under illumination with low-intensity UVA (8 and 20 mW/cm2) or UVC (0.1 mW/ cm2). AFM measurements of the cell stiffness were carried out immediately after exposure of cells to the oxidative stress. The data suggest that under illumination with low-intensity UVA nanoTiO2 generates ROS, which, in turn, damage cellular and subcellular structures. This process was detected by AFM as a marked drop in the cellular stiffness of ca. 30-75%, which occurred rapidly, in the time frame of 1 min. The photo-oxidative stress inducing the decrease of cell stiffness was cancelled in the presence of a well-established antioxidant, beta-carotene. The results highlight the sensitivity of AFM to detect early changes in mechanical properties of cells exposed to oxidative stress.     

Phage-resistant mutants of Lactobacillus delbrueckii may have functional properties that differ from those of parent strains

Three commercial phage sensitive Lactobacillus delbrueckii strains (identified as Ab(1), YSD V and Ib(3)), and four spontaneous phage-resistant mutants isolated from them were tested for their capacity to activate the gut mucosal immune response in mice, as indicated by the numbers of IgA-producing cells. Random Amplified Polymorphic DNA (RAPD) analysis revealed a strong genetic homology between the sensitive strains and their respective derivatives. The phage-resistant mutants exhibited high levels of phage resistance, elevated stability of this phenotype and technological properties comparable to those of their respective parent strains. The tolerance to acidic conditions, bile salts and lysozyme was strain dependent and total cell viability losses as a result of exposure to all three stresses ranged from 2.0 to 3.7 log units. All the strains were highly resistant to a simulated gastric solution of pH 3, while significant additional losses in cell viability were observed when acid treated cells were exposed to bile salts and lysozyme. BALB/c mice received pure cultures of Lb. delbrueckii sensitive and phage-resistant strains for 2, 5 or 7 consecutive days. The ability of the parent strains to activate the small intestine immune response was preserved or enhanced in phage-resistant mutants. The maximal proliferation of IgA(+) cells was observed at day 5 or 7, depending on the strain. Mutants isolated in this study using natural selection strategies had improved phage resistance, adequate technological properties and satisfactory gut mucosal immunostimulation ability, and so would be good candidates for industrial applications in functional foods.     

Biofilms of Listeria monocytogenes produced at 12 °C either in pure culture or in co-culture with Pseudomonas aeruginosa showed reduced susceptibility to sanitizers

The biofilm-forming ability of 21 Listeria monocytogenes isolates, previously pulsotyped and corresponding to 16 strains, from different origins was evaluated using the Calgary Biofilm Device, at 37 °C. Biofilms of 4 selected strains were also produced either on pure cultures or on co-cultures with Pseudomonas aeruginosa (PAO1), at 12 °C and at 37 °C. For these biofilms, the minimum biofilm eradication concentrations (MBECs) of 4 commercial dairy sanitizers (1 alkyl amine acetate based--T99, 2 chlorine based--T66 and DD, and 1 phosphoric acid based--BP) were determined. Listeria monocytogenes biofilms grown, either at 37 °C or 12 °C, were able to achieve similar cell densities by using different incubation periods (24 h and 7 d, respectively). In co-culture biofilms, P. aeruginosa was the dominant species, either at 37 °C or at 12 °C, representing 99% of a total biofilm population of 6 to 7 log CFU/peg. Co-culture biofilms were generally less susceptible than L. monocytogenes pure cultures. More interestingly, the biofilms produced at 12 °C were usually less susceptible to the sanitizers than when produced at 37 °C. Single or co-culture biofilms of L. monocytogenes and PAO1, particularly produced at 12 °C, retrieved MBEC values for agents T99 and BP that were, at times, above the maximum in-use recommended concentrations for these agents. The results presented here reinforce the importance of the temperature used for biofilm formation, when susceptibility to sanitizers is being assessed. PRACTICAL APPLICATION: Since most food plants have cold wet growth niches in production and storage areas, susceptibility testing should be performed on biofilms produced at refrigeration temperatures. Moreover, the efficiency of the sanitizers used in food industries should be performed on mixed culture biofilms, since in field conditions these will predominate. The results presented here highlight the importance of the temperature used for biofilm formation, when susceptibility to disinfectants is being assessed, as biofilms produced at lower temperature were less susceptible to sanitizers.     

Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.