The Effect of Polyene Antibiotic Amphotericin B on Erythrocyte Cytoarchitectonics and Osmotic Resistance.

We studied the effect of amphotericin B (2.5×10-5 and 5.4×10-5 M) on osmotic resistance and surface cytoarchitectonics of donor blood erythrocytes. Antibiotic at a concentration of 2.5×10-5 M induced most pronounced changes in the studied parameters, which can be related to the specifics of the spatial organization of the cholesterol-amphotericin B complexes at different stoichiometric ratios of the components and their ability to pore formation in the membranes. Cholesterol binding to the polyene antibiotic and the appearance of perforations in the plasma membrane lead to accumulation of reversibly and irreversibly deformed cells and their hemolysis. The appearance of a large number of irreversibly deformed erythrocytes indicates an impaired ability to elastic deformation in the microcirculatory stream, which can lead to disruption of their functions in vivo and intravascular hemolysis.

Contribution of packaging materials to MOSH and POSH contamination of milk powder products during storage.

Mineral oil hydrocarbons (MOH) in milk powders, particularly in infant formulas, have been and continue to be a major concern to the public worldwide. These contaminants are likely derived from environmental pollution, manufacturing process and packaging materials. In this study, 23 Chinese commercial milk powder products packaged in four types of materials, i.e. metal cans, paper containers, paperboard boxes with internal bags, and aluminium foil-plastic bags, were collected and stored for 1 year. The total and surface MOH in these samples were detected and compared before and after storage to understand the MOH migration during storage, despite no mineral oil saturated hydrocarbons (MOAH) were detected. The contents of mineral oil saturated hydrocarbons (MOSH) and polyolefin oligomeric saturated hydrocarbons (POSH) in metal cans were the least among the four packages and changed little during storage, which suggested that little MOH migration occurred in metal material. Despite all the food contact materials in the other three packagings were the aluminium foil-plastic composite, the similar low migration occurred in the aluminium foil-plastic bags and internally contained composite bag(s) in paperboard boxes. However, both total and surface MOSH and POSH easily migrated from the paper-plastic-aluminium composite of paper containers during storage. These findings are helpful for the selection of packaging materials in manufacturing milk powder products or other foods.

In-depth characterization of polyolefin plastomers/elastomers (ethylene/1-octene copolymers) through hyphenated chromatographic techniques.

The chemical composition distribution (CCD) of three single site made ethylene/1-octene copolymers was investigated through offline-hyphenation of solvent gradient interaction chromatography (SGIC) with 1H NMR. Thus, a clear, non-linear correlation between SGIC elution time and chemical composition was found under the specific measurement conditions applied here. The application of 1H NMR as detection allowed to determine the CCD with unprecedented accuracy. 2D-LC of the copolymers revealed the correlation between CCD and molar mass distribution (MMD) in a quantitative manner. Furthermore, this approach allowed a comparison between the response behavior of an evaporative light scattering detector (ELSD, semi-quantitative, commonly applied in SGIC) and that of an infrared (IR) detector (quantitative, commonly applied in SEC). As a result, it could be shown that ELSD results are close to IR results for the system investigated here, in other words, the often-criticized semi-quantitative response behavior of the ELSD is affecting results in an acceptable manner.

Solid phase microextraction as a powerful alternative for screening of secondary metabolites in actinomycetes.

Actinobacteria are one of the most promising producers of medically and industrially relevant secondary metabolites. However, screening of such compounds in actinobacteria growth demands simple, fast, and efficient extraction procedures that enable detection and precise quantification of biologically active compounds. In this regard, solid phase microextraction (SPME) emerges as an ideal extraction technique for screening of secondary metabolites in bacteria culture due to its non-exhaustive, minimally invasive, and non-destructive nature: its integrated sample preparation workflow; balanced coverage feature; metabolism quenching capabilities; and superior cleanup, as well as its versatility in configuration, which enables automation and high throughput applications. The current work provides a comparison of micro-scale and direct immersion SPME (DI-SPME) for screening of secondary metabolites, describes the optimization of the developed DI-SPME method, and introduces the developed technique for mapping of target secondary metabolites as well as its direct coupling to mass spectrometry for such applications. The optimized DI-SPME method provided higher amounts of extracted ions and intensity signals, yielding superior extraction and desorption efficiency as compared with micro-scale extraction. Studied compounds presented stability on the coating for 24 h at room temperature. The DI-SPME mapping approach revealed that lysolipin I and the lienomycin analog are distributed along the center and edges of the colony, respectively. Direct coupling of SPME to MS provided a similar ions profile as SPME-LC-MS while enabling a significant decrease in analysis time, demonstrating its suitability for such applications. DI-SPME is herein presented as an alternative to micro-scale extraction for screening of secondary metabolites in actinobacteria solid medium, as well as a feasible alternative to DESI-IMS for mapping of biologic radial distribution of secondary metabolites and cell life cycle studies. Lastly, the direct coupling of DI-SPME to MS is presented as a fast, powerful technique for high throughput analysis of secondary metabolites in this medium.

HS-SPME analysis of the volatiles profile of water celery (Apium nodiflorum), a wild vegetable with increasing culinary interest.

Water celery (Apium nodiflorum) is a wild plant traditionally harvested in some Mediterranean areas for being consumed raw. Despite its appreciated organoleptic properties, the aromatic profile of the fresh vegetable remains to be studied. In the present study, volatile compounds from five wild populations were extracted by the headspace-solid phase microextraction technique, analysed by gas cromatography-mass spectrometry, and compared to related crops. The wild species had a high number of aromatic compounds. It was rich in monoterpenes (49.2%), sesquiterpenes (39.4%) and phenylpropanoids (9.6%), with quantitative differences among populations, in absolute terms and relative abundance. On average, germacrene D was the main compound (16.6%), followed by allo-ocimene (11.9%) and limonene (11.1%). Only in one population, the levels of limonene were greater than those of germacrene D. Among phenylpropanoids, dillapiol displayed the highest levels, and co-occurred with myristicin in all populations except one. These differences may have a genetic component, which would indicate the possibility of establishing selection programmes for the development of water celery as a crop adapted to different market preferences. On the other hand, comparison with related crops revealed some similarities among individual volatiles present in the different crops, which would be responsible of the common aroma notes. However, water celery displayed a unique profile, which was in addition quantitatively richer than others. Thus, this differentiation may promote the use of water celery as a new crop.

Conclusions from a Swiss official control of the safety assessment for food contact polyolefins through the compliance documentation of the producers.

Swiss control authorities checked the safety assessment of nine major producers of polyolefin granulates for making food contact materials. It was a pilot project to gain experience on the procedure of collecting and evaluating compliance documentation, but also to obtain insight into the quality of compliance work performed by the main plastic producers. It revealed that there are fundamental problems in performing such control. These are reported with proposals for improvement. For most products, the safety assessment made available did not correspond to the requirements, as confirmed by a group of internationally recognised experts, who were asked for their opinion on whether the safety of the migrates was assessed in accordance to 'internationally accepted scientific methods on risk assessment', as required by Art. 19 of Regulation (EU) 10/2011 and specified by EFSA.

Selective determination of poly(styrene) and polyolefin microplastics in sandy beach sediments by gel permeation chromatography coupled with fluorescence detection.

Microplastics generated by plastics waste degradation are ubiquitous in marine and freshwater basins, posing serious environmental concerns. Raman and FTIR spectroscopies, along with techniques such as pyrolysis-GC/MS, are typically used for their identification. We present a procedure based on gel permeation chromatography (GPC) coupled with fluorescence detection for semi-quantitative selective determination of the most common microplastics found in marine shoreline sediments: poly(styrene) (PS) and partially degraded polyolefins (LDPEox). By operating the detector at either 260/280 or 370/420 nm excitation/emission wavelengths PS can be distinguished from LDPEox upon GPC separation. Semi-quantitative determination of microplastics contents is also possible: dichloromethane extracts of PS and LDPEox yield linear plots of fluorescence peak area vs concentration (0-5.0 mg/mL range) and were used as reference materials for quantification of the microplastics content in sand samples collected in the winter berm and dune sectors of a Tuscany beach in Italy.

Characteristic Aroma Components from Dried "Wakame" Undaria pinnatifida.

An essential oil from dried "wakame" (Undaria pinnatifida), prepared by a simultaneous distillation extraction method, was analyzed by GC-MS, indicating the presence of one major component of volatiles. The volatile component was identified as (6Z,9Z,12Z,15Z,18Z)-1,6,9,12,15,18-henicosahexaene by comparison with the GC-MS and NMR spectra of synthetic. The henicosahexaene showed a subtly marine aroma. (6Z,9Z,12Z,15Z)-1,6,9,12,15-Henicosapentaene was also detected as a minor polyene in the essential oils. It was suggested that these polyenes contribute to the characteristic aroma of the dried wakame.

Unsaturated Cuticular Hydrocarbon Components of the Sex Pheromone of Eggplant Fruit Borer, Leucinodes orbonalis Guenée (Lepidoptera: Crambidae).

Leucinodes orbonalis is one of the most damaging insect pests affecting eggplant in the Mekong Delta of Vietnam. While (E)-11-hexadecenyl acetate (E11-16:OAc) and its alcohol, (E)-11-hexadecenol (E11-16:OH), have been identified as major and minor sex pheromone components, respectively, few males were attracted to a blend of these compounds in Vietnamese fields. In order to utilize synthetic pheromone of L. orbonalis as a tool for sustainable pest management programs, we reexamined the pheromone of this species in order to search for other minor components. Gas chromatography-electroantennogram detection and gas chromatography/mass spectrometry analyses of abdominal tip extract revealed the presence of two electroantennogram-active compounds, E11-16:OAc and (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H) in a ratio of 100:2. An extract of the abdomen and thorax showed an additional electroantennogram-active component, (3Z,6Z,9Z)-3,6,9-docosatriene (Z3,Z6,Z9-22:H), with the three compounds (E11-16:OAc, Z3,Z6,Z9-23:H and Z3,Z6,Z9-22:H) being present in a ratio of 100:45:1, indicating that the trienes were mainly present on the cuticular surface. In the field, traps baited with E11-16:OAc and the C23 triene, in a mix of 10:1, caught more male moths than traps baited with the acetate alone. A field evaluation of other polyunsaturated hydrocarbons showed that the C22 triene found in body extract also increased catches when added to the acetate, but no other hydrocarbons did. In contrast, to other studies with this moth, the addition of E11-16:OH to E11-16:OAc plus the C22 or C23 triene, resulted in decreased trap catches.

Identification of a consistent polyene component of purple pigment in diseased sclerites of Caribbean corals across region, species, and insult agent.

Gorgonians respond to insult (damage and disease) by producing sclerites containing a purple pigment as opposed to the normal white sclerites. Raman microscopy is used to study the purple areas of three species of diseased coral, Gorgonia ventalina, Pseudoplexaura porosa, and Eunicea laciniata obtained from Puerto Rico. These spectra were compared to Gorgonia ventalina samples previously reported that were obtained from San Salvador, Bahamas. Spectra from two samples of G. ventalina that had been infected by different agents, Aspergillus sydowii and a slime mold, were also obtained. The results indicate that the purple compounds (polyenes) generated by the coral in response to infection are similar regardless of region from which the coral were harvested, of species of coral, and of the infective agent. A discussion of the Raman spectra of G. ventalina and the other coral species is presented.

Female Sex Pheromone of the Cone Moth, Dioryctria mendacella: Investigation of Synergism between Type I and Type II Pheromone Components.

Polyunsaturated hydrocarbons (Type II pheromone components) have been reported to be synergists for unsaturated acetates, alcohols or aldehydes (Type I components) in the sex pheromones of several species of Lepidoptera. However, there is some debate over whether the active components are the hydrocarbons themselves or more volatile degradation products. Extracts of pheromone glands of adult females of the cone moth, Dioryctria mendacella (Lepidoptera: Pyralidae), contain (Z,E)-9,11-tetradecadienyl acetate (ZE9,11-14:Ac) and at least ten times as much (Z,Z,Z,Z,Z)-3,6,9,12,15-pentacosapentaene (ZZZZZ3,6,9,12,15-25:H). The former elicits a strong electroantennogram response from males while no response could be recorded to the latter. In field trapping tests, both compounds were individually unattractive to male D. mendacella moths, but blends of the two compounds containing at least a 10:1 ratio of ZZZZZ3,6,9,12,15-25:H : ZE9,11-14:Ac were highly attractive. The relatively involatile hydrocarbon was shown to be released from the dispensers used and no significant degradation could be detected. Furthermore, blends of ZE9,11-14:Ac and analogs of ZZZZZ3,6,9,12,15-25:H with fewer carbons and/or double bonds that might be expected to produce similar degradation products to ZZZZZ3,6,9,12,15-25:H were unattractive. This indicated a specific response to the hydrocarbon itself, further substantiated by the observation that related hydrocarbons did not interfere with the activity of ZZZZZ3,6,9,12,15-25:H. Thus a three-step conversion of fish oil was used to produce a blend of unsaturated hydrocarbons containing ZZZZZ3,6,9,12,15-25:H as the major component, albeit only 30% of the total, and a blend of this material with ZE9,11-14:Ac was as attractive to male D. mendacella moths as blends with an equivalent amount of the purified material. This mixture of unsaturated hydrocarbons is much cheaper to produce than the pure pentaene, and may be useful in lures for other species using these compounds. Dioryctria mendacella is a major constraint to production of edible pine kernels throughout the Mediterranean region. Pheromone traps will provide a means to improve monitoring of seasonal flight patterns and changes in population abundance of this pest.

In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line.

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.

Argentation high performance liquid chromatography on-line coupled to gas chromatography for the analysis of monounsaturated polyolefin oligomers in packaging materials and foods.

Multidimensional chromatography based on two-dimensional high performance liquid chromatography on-line coupled to gas chromatography (on-line HPLC-HPLC-GC) enables the separate analysis of saturated, monounsaturated and aromatic hydrocarbons in packaging materials like polyolefins or paperboard and their migrates into foods. Since normal-phase HPLC on silica gel did not preseparate saturated from monounsaturated hydrocarbons, a separation step on a normal-phase HPLC column treated in the laboratory with an optimized amount of silver nitrate was added. The preparation of this HPLC column and the instrumental set-up are described, followed by examples showing the potential of the method. In a preliminary investigation of 11 polyolefin granulates for food contact up to 40% monounsaturated hydrocarbons among the oligomers C16-35 were determined.

Characterization and quantitation of polyolefin microplastics in personal-care products using high-temperature gel-permeation chromatography.

In recent years, the development of reliable methods for the quantitation of microplastics in different samples, including evaluating the particles' adverse effects in the marine environment, has become a great concern. Because polyolefins are the most prevalent type of polymer in personal-care products containing microplastics, this study presents a novel approach for their quantitation. The method is suitable for aqueous and hydrocarbon-based products, and includes a rapid sample clean-up involving twofold density separation and a subsequent quantitation with high-temperature gel-permeation chromatography. In contrast with previous procedures, both errors caused by weighing after insufficient separation of plastics and matrix and time-consuming visual sorting are avoided. In addition to reliable quantitative results, in this investigation a comprehensive characterization of the polymer particles isolated from the product matrix, covering size, shape, molecular weight distribution and stabilization, is provided. Results for seven different personal-care products are presented. Recoveries of this method were in the range of 92-96 %.

Raman spectroscopy as a tool in differentiating conjugated polyenes from synthetic and natural sources.

This work presents the Raman spectroscopic characterization of synthetic analogs of natural conjugated polyenals found in octocorals, focusing the unequivocal identification of the chemical species present in these systems. The synthetic material was produced by the autocondensation reaction of crotonaldehyde, generating a demethylated conjugated polyene containing 11 carbon-carbon double bonds, with just a methyl group on the end of the carbon chain. The resonance Raman spectra of such pigment has shown the existence of enhanced modes assigned to ν1(CC) and ν2(CC) modes of the main chain. For the resonance Raman spectra of natural pigments from octocorals collected in the Brazilian coast, besides the previously cited bands, it could be also observed the presence of the ν4(CCH3), related to the vibrational mode who describes the vibration of the methyl group of the central carbon chain of carotenoids. Other interesting point is the observation of overtones and combination bands, which for carotenoids involves the presence of the ν4 mode, whereas for the synthetic polyene this band, besides be seen at a slightly different wavenumber position, does not appear as an enhanced mode and also as a combination, such as for the natural carotenoids. Theoretical molecular orbital analysis of polyenal-11 and lycopene has shown the structural differences which are also responsible for the resonance Raman data, based on the appearance of the (CH3) vibrational mode in the resonant transition only for lycopene. At last, the Raman band at ca. 1010 cm(-1), assigned to the (CH3) vibrational mode, can be used for attributing the presence of each one of the conjugated polyenes: the resonance Raman spectrum containing the band at ca. 1010 cm(-1) refers to the carotenoid (in this case lycopene), and the absence of such band in resonance conditions refers to the polyenal (in this case the polyenal-11).

[The condition of the system "peroxide oxidation of lipids-antioxidant defense" in mixed saliva of patients with chronic generalized periodontitis].

The comparative analysis was made concerning conditions of the system "Peroxide oxidation of lipids-antioxidant defenses in mixed saliva of patients with chronic disseminated periodontitis of light (n = 45), mean (n = 36) and severe (n = 18) degrees. The control group consisted of 25 clinically healthy persons with intact periodontium. The study revealed that in comparison with control group, in patients with chronic disseminated periodontitis occurred an increasing of content of heptane-soluble diene conjugates, isopropanol-soluble ketodienes and conjugated trienes, ceruloplasmin and a decreasing of level of alpha-tocopherol, especially under severe degree of disease. The study also established a non-linear U-dependence between indicators of the system "Peroxide oxidation of lipids-antioxidant defenses" in mixed saliva and severity of affection of tissues of periodontium in patients with chronic disseminated dermatosis.

Reexamination of the female sex pheromone of the sweet potato vine borer moth: identification and field evaluation of a tricosatriene.

The sweet potato vine borer moth, Omphisa anastomosalis (Pyraloidea: Crambidae), is a serious pest in tropical and subtropical Asia-Pacific regions. In previous work using a population from Okinawa, Japan, (10E,14E)-10,14-hexadecadienal (E10,E14-16:Ald) was identified as the major pheromone component, with hexadecanal, (E)-10-hexadecenal, and (E)-14-hexadecenal as minor components. However, traps baited with the synthetic compounds were less effective at attracting males in the field than those baited with virgin females. While Pyraloidea females usually produce only Type I pheromone components (unsaturated fatty alcohols and their derivatives), the pheromones of some Pyraloidea species have been shown to involve a combination of both Type I and Type II components (unsaturated hydrocarbons and their epoxides). We examined an extract of the pheromone glands of female O. anastomosalis from Vietnam by gas chromatography coupled to mass spectrometry and detected (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H) in addition to the compounds identified previously. All four isomers of 10,14-16:Ald were synthesized. A mixture of synthetic E10,E14-16:Ald and Z3,Z6,Z9-23:H in a ratio of 1:0.2-1:2 was attractive to male moths in Vietnam, indicating the strong synergistic effect of the Type II compound. Addition of the other minor pheromone components to the binary blend did not increase the number of male moths captured. Combinations of Z3,Z6,Z9-23:H with the other three geometrical isomers of E10,E14-16:Ald attracted no males, further substantiating the 10E,14E configuration of the natural diene component. E10,E14-16:Ald mixed with other polyunsaturated hydrocarbons showed that mixtures that included a C21 triene, a C22 triene, or a C23 pentaene attracted as many males as did the mixture with Z3,Z6,Z9-23:H. The identification of a highly attractive sex pheromone will help in developing efficient strategies for monitoring and control of O. anastomosalis populations in sweet potato fields.

Roots of the invasive species Carduus nutans L. and C. acanthoides L. produce large amounts of aplotaxene, a possible allelochemical.

The invasive thistle Carduus nutans has been reported to be allelopathic, yet no allelochemicals have been identified from the species. In a search for allelochemicals from C. nutans and the closely related invasive species C. acanthoides, bioassay-guided fractionation of roots and leaves of each species were conducted. Only dichloromethane extracts of the roots of both species contained a phytotoxin (aplotaxene, (Z,Z,Z)-heptadeca-1,8,11,14-tetraene) with sufficient total activity to potentially act as an allelochemical. Aplotaxene made up 0.44 % of the weight of greenhouse-grown C. acanthoides roots (ca. 20 mM in the plant) and was not found in leaves of either species. It inhibited growth of lettuce 50 % (I 50) in soil at a concentration of ca. 0.5 mg g(-1) of dry soil (ca. 6.5 mM in soil moisture). These values gave a total activity in soil value (molar concentration in the plant divided by the molarity required for 50 % growth inhibition in soil = 3.08) similar to those of some established allelochemicals. The aplotaxene I 50 for duckweed (Lemna paucicostata) in nutrient solution was less than 0.333 mM, and the compound caused cellular leakage of cucumber cotyledon discs in darkness and light at similar concentrations. Soil in which C. acanthoides had grown contained aplotaxene at a lower concentration than necessary for biological activity in our short-term soil bioassays, but these levels might have activity over longer periods of time and might be an underestimate of concentrations in undisturbed and/or rhizosphere soil.

[Application of wavenumber-linear scaling to the calculated Raman frequencies of polyenes and carotenoids].

Raman spectra of two typical carotenoids (beta-carotene and lutein) and some short (n = 2-5) polyenes were calculated using density functional theory. The wavenumber-linear scaling (WLS) and other frequency scaling methods were used to calibrate the calculated frequencies. It was found that the most commonly used uniform scaling (UFS) method can only calibrate several individual frequencies perfectly, and the systematic result of this method is not very good. The fitting parameters obtained by the WLS method are upsilon(obs)/upsilon(calc)) = 0.999 9-0.000 027 4upsilon(calc) and upsilon(obs)/upsilon(calc)= 0.993 8-0.000 024 8upsilon(calc) for short polyenes and carotenoids, respectively. The calibration results of the WLS method are much better than the UFS method. This result suggests that the WLS method can be used for the frequency scaling of the molecules as large as carotenoids. The similar fitting parameters for short polyenes and carotenoids indicate that the fitting parameters obtained by WLS for short polyenes can be used for calibrating the calculated vibrational frequencies of carotenoids. This presents a new frequency scaling method for vibrational spectroscopic analysis of carotenoids.

Raman spectrometric discrimination of flexirubin pigments from two genera of Bacteroidetes.

Flexirubins are specific polyene pigments produced by several genera of Bacteroidetes. Colonies and cell extracts of Flavobacterium johnsoniae and Flexibacter elegans have been investigated by Raman spectroscopy to show that this fast and non-destructive technique can be used to differentiate these pigments from carotenoids and to compare the flexirubin content of the two microorganisms. The presence or absence of certain distinguishing features in the CH combination band region at 2500-2750 cm(-1) can assist in the discrimination between the two flexirubins investigated. Raman spectroscopy is thus a suitable tool not only to detect flexirubin pigments in bacterial cells, but also to further characterize the pigments present in members of the Bacteroidetes genera that are rich in flexirubins.