Critical Review of Selected Analytical Platforms for GC-MS Metabolomics Profiling-Case Study: HS-SPME/GC-MS Analysis of Blackberry's Aroma.

Data processing and data extraction are the first, and most often crucial, steps in metabolomics and multivariate data analysis in general. There are several software solutions for these purposes in GC-MS metabolomics. It becomes unclear which platform offers what kind of data and how that information influences the analysis's conclusions. In this study, selected analytical platforms for GC-MS metabolomics profiling, SpectConnect and XCMS as well as MestReNova software, were used to process the results of the HS-SPME/GC-MS aroma analyses of several blackberry varieties. In addition, a detailed analysis of the identification of the individual components of the blackberry aroma club varieties was performed. In total, 72 components were detected in the XCMS platform, 119 in SpectConnect, and 87 and 167 in MestReNova, with automatic integral and manual correction, respectively, as well as 219 aroma components after manual analysis of GC-MS chromatograms. The obtained datasets were fed, for multivariate data analysis, to SIMCA software, and underwent the creation of PCA, OPLS, and OPLS-DA models. The results of the validation tests and VIP-pred. scores were analyzed in detail.

Effect of Aging on Tensile and Chemical Properties of Polylactic Acid and Polylactic Acid-Like Polymer Materials for Additive Manufacturing.

Additive manufacturing, with its fast development and application of polymeric materials, led to the wide utilization of polylactic acid (PLA) materials. As a biodegradable and biocompatible aliphatic polyester, produced from renewable sources, PLA is widely used in different sectors, from industry to medicine and science. The aim of this research is to determine the differences between two forms of the PLA material, i.e., fused deposition modeling (FDM) printed filament and digital light processing (DLP) printed resin, followed by aging due to environmental and hygiene maintenance conditions for a period of two months. Specimens underwent 3D scanning, tensile testing, and Fourier transform infrared (FTIR) spectrometry to obtain insights into the material changes that occurred. Two-way Analysis of Variance (ANOVA) statistical analysis was subsequently carried out to determine the statistical significance of the determined changes. Significant impairment can be observed in the dimensional accuracies between both materials, whether they are non-aged or aged. The mechanical properties fluctuated for aged FDM specimens: 15% for ultimate tensile stress, 15% for elongation at yield, and 12% for elastic modulus. Regarding the DLP aged specimens, the UTS decreased by 61%, elongation at yield by around 61%, and elastic modulus by 62%. According to the FTIR spectral analysis, the PLA materials degraded, especially in the case of resin specimens. Aging also showed a significant influence on the elastic modulus, ultimate tensile stress, elongation at yield, elongation at break, and toughness of both materials, which was statistically shown by means of a two-way ANOVA test. The data collected in this research give a better understanding of the underlying aging mechanism of PLA materials.

Encapsulation of Fennel and Basil Essential Oils in ß-Cyclodextrin for Novel Biopesticide Formulation.

ß-cyclodextrin (ß-CD) is a good host for the encapsulation of fennel and basil essential oils (FEO and BEO, respectively) and the formation of inclusion complexes (ICs) using the co-precipitation method. According to the results of the GC/MS analysis conducted in this study, monoterpenes and monoterpenoids were the dominant chemical groups in total FEO, while in BEO, these two groups occurred along with sesquiterpenes and sesquiterpenoids. The presence of dominant compounds from both EOs was validated using the FT-IR spectra of ICs, which indicated successful complexation. Analyses conducted using SPME/GC-MS showed the continuous emission of volatiles over 24 h from both ICs. Under SEM, particles of both ICs appeared to have a rectangular or rhomboid morphology and few aggregates. The insecticidal properties of EOs and ICs with ß-CD were tested on the Colorado potato beetle (CPB) as a model pest. The inclusion complex of ß-CD with FEO altered the developmental dynamic and body mass of the CPB. The initial increase in the proteolytic activity of CPB larvae fed with potato plants sprayed with ICs was not maintained for long, and the proteolytic efficacy of treated larvae remained in line with that of the control larvae. Future investigations will focus on manipulating the volume of EOs used and the treatment duration for optimal efficacy and potential application.

Synthesis, Characterization and Application of Biobased Unsaturated Polyester Resin Reinforced with Unmodified/Modified Biosilica Nanoparticles.

This paper presents sustainable technology for environmentally friendly composite production. Biobased unsaturated polyester resin (b-UPR), synthesized from waste polyethylene terephthalate (PET) glycosylate and renewable origin maleic anhydride (MAnh) and propylene glycol (PG), was reinforced with unmodified and vinyl-modified biosilica nanoparticles obtained from rice husk. The structural and morphological properties of the obtained particles, b-UPR, as well as composites, were characterized by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) techniques. The study of the influence of biosilica modification on the mechanical properties of composites was supported by hardness modeling. Improvement of the tensile strength of the b-UPR-based composite at 2.5 wt.% addition of biosilica modified with vinyl silane, named "b-UPR/SiO2-V" composite, has been achieved with 88% increase. The thermal aging process applied to the b-UPR/SiO2-V composite, which simulates use over the product's lifetime, leads to the deterioration of composites that were used as fillers in commercial unsaturated polyester resin (c-UPR). The grinded artificially aged b-UPR composites were used as filler in c-UPR for the production of a table top layer with outstanding mechanical properties, i.e., impact resistance and microhardness, as well as fire resistance rated in the V-0 category according to the UL-94 test. Developing sustainable composites that are chemically synthesized from renewable sources is important from the aspect of preserving the environment and existing resources as well as the extending their life cycle.

Metabolomics as a Potential Chemotaxonomical Tool: Application on the Selected Euphorbia Species Growing Wild in Serbia.

Chemotaxonomy presents various challenges that need to be overcome in order to obtain valid and reliable results. Individual genetic and environmental variations can give a false picture and lead to wrong conclusions. Applying a holistic approach, based on multivariate data analysis, these challenges can be overcome. Thus, a metabolomics approach has to be optimized depending on the subject of research. We used 1H NMR-based metabolomics as a potential chemotaxonomic tool on the selected Euphorbia species growing wild in Serbia. Principal components analysis (PCA), soft independent modeling by class analogy (SIMCA) and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) were used to analyze obtained NMR data in order to reveal chemotaxonomic biomarkers. The standard protocol for plant metabolomics was optimized aiming to extract more specific metabolites, which are characteristic for the Euphorbia genus. The obtained models were validated, which revealed that variables unique for each species were associated with certain classes of molecules according to literature data. In E. salicifolia, acacetin-7-O-glycoside (not found before in the species) was detected, and the structure of the aglycone part was solved based on 2D NMR data. In the presented paper, we have shown that metabolomics can be successfully used in Euphorbia chemotaxonomy.

Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae).

Iridoids, a class of atypical monoterpenes, exhibit exceptional diversity within the Nepeta genus (subfam. Nepetoidae, fam. Lamiaceae).The majority of these plants produce iridoids of the unique stereochemistry, with nepetalactones (NLs) predominating; however, a few Nepeta species lack these compounds. By comparatively analyzing metabolomics, transcriptomics, gene co-expression, and phylogenetic data of the iridoid-producing N. rtanjensis Diklic & Milojevic and iridoid-lacking N. nervosa Royle & Bentham, we presumed that one of the factors responsible for the absence of these compounds in N. nervosa is iridoid synthase (ISY). Two orthologues of ISY were mined from leaves transcriptome of N. rtanjensis (NrPRISE1 and NrPRISE2), while in N. nervosa only one (NnPRISE) was identified, and it was phylogenetically closer to the representatives of the Family 1 isoforms, designated as P5ßRs. Organ-specific and MeJA-elicited profiling of iridoid content and co-expression analysis of IBG candidates, highlighted NrPRISE2 and NnPRISE as promising candidates for ISY orthologues, and their function was confirmed using in vitro assays with recombinant proteins, after heterologous expression of recombinant proteins in E. coli and their His-tag affinity purification. NrPRISE2 demonstrated ISY activity both in vitro and likely in planta, which was supported by the 3D modeling and molecular docking analysis, thus reclassification of NrPRISE2 to NrISY is accordingly recommended. NnPRISE also displays in vitro ISY-like activity, while its role under in vivo conditions was not here unambiguously confirmed. Most probably under in vivo conditions the NnPRISE lacks substrates to act upon, as a result of the loss of function of some of the upstream enzymes of the iridoid pathway. Our ongoing work is conducted towards re-establishing the biosynthesis of iridoids in N. nervosa.

Metabolomics study of the desiccation and recovery process in the resurrection plants Ramonda serbica and R. nathaliae.

INTRODUCTION: Ramonda serbica and R. nathaliae are resurrection plants that have the remarkable ability to survive the complete desiccation of their vegetative organs (i.e. leaves, stem, roots) during periods of drought and rapidly revive when rewatered and rehydrated. OBJECTIVE: To investigate metabolic changes in R. serbica and R. nathaliae during their desiccation and recovery process METHODS: Proton nuclear magnetic resonance (1 H-NMR) and gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach coupled with multivariate data analysis was utilised to identify the metabolomes of the plants from 90 biological replicates. RESULTS: Sucrose and the polyphenolic glycoside myconoside were predominant in almost equal amounts in all samples studied, regardless of their water content at sampling. During the dehydration process, a decrease in the relative content of fructose, galactose, and galactinol was observed while the contents of those metabolites were preserved in the partially rehydrated plants. Raffinose and myo-inositol were accumulated in dry samples. CONCLUSION: Using 1 H-NMR and GC-MS as two complementary analytical platforms provided a more complete picture of the metabolite composition for investigation of the desiccation and recovery process in resurrection plants.

"Scent of a fruit fly": Cuticular chemoprofiles after mating in differently fed Drosophila melanogaster (Diptera: Drosophilidae) strains.

In the world of complex smells in natural environment, feeding and mating represent two important olfactory-guided behaviors in Drosophila melanogaster (Diptera: Drosophilidae). Diet affects the chemoprofile composition of the individuals, which, indirectly, may significantly affect their mating success. In this study, chemoprofiles of recently mated flies belonging to four D. melanogaster strains, which were fed for many generations on different substrates (standard cornmeal-S strain; banana-B strain; carrot-C strain; tomato-T strain) were identified and quantified. In total, 67 chemical compounds were identified: 48 compounds were extracted from males maintained on banana and carrot, and 47 compounds from males maintained on cornmeal and tomato substrates, while total of 60 compounds were identified in females from all strains. The strains and the sexes significantly differed in qualitative nature of their chemoprofiles after mating. Significant differences in the relative amount of three major male pheromones (cis-vaccenyl acetate-cVA, (Z)-7-pentacosene, and (Z)-7-tricosene) and in female pheromone (Z,Z)-7,11-nonacosadiene among strains were also recorded. Furthermore, multivariate analysis of variance (MANOVA) pointed to significant differences between virgin and mated individuals of all strains and within both sexes. Differences in some of the well known sex pheromones were also identified when comparing their relative amount before and after mating. The presence of typical male pheromones in females, and vice versa may indicate their bidirectional transfer during copulation. Our results confirm significant effect of mating status on cuticular hydrocarbon (CHC) phenotypes in differently fed D. melanogaster flies.

Antimicrobial and Immunomodulating Activities of Two Endemic Nepeta Species and Their Major Iridoids Isolated from Natural Sources.

Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources-cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

Cytotoxic triterpenoids and triterpene sugar esters from the medicinal mushroom Fomitopsis betulina.

Thirteen undescribed 24-methylene lanostane triterpenoids, named polyporenic acids E-M and fomitosides L-O, as well as seventeen known analogues, were isolated from the fruiting bodies of the mushroom Fomitopsis betulina. Their structures were determined using 1D, 2D NMR, IR, and HRESIMS. Fomitoside L and fomitoside N exhibited cytotoxicity against HL60 leukemia cells (IC50 = 15.8 and 23.7 µM, respectively). Among the known compounds, notable cytotoxicities against HL60 leukemia cells and selectivity with respect to MRC-5 healthy cells were noticed for dehydropachymic acid (IC50 = 10.9 µM, SI 8.6), pachymic acid (IC50 = 11.0 µM, SI 9.8), 3-epi-dehydrotumulosic acid (IC50 = 19.9 µM, SI 5.8) and 12α-hydroxy-3α-(3'-hydroxy-4'-methoxycarbonyl-3'-methylbutyryloxy)-24-methyllanosta-8,24 (31)-dien-26-oic acid (IC50 = 19.2 µM, SI 2.2).

Fungal transformation and reduction of phytotoxicity of grape pomace waste.

Grape pomace (GP) from Vitis labrusca, the main byproduct from "American table wine" production, is recalcitrant to degradation, and its accumulation is a serious problem with negative environmental impacts. In this work, transformation of grape pomace using a steam pretreatment followed by incubation of GP during a 90-day period with six different fungi were evaluated. Several fungi tested reduced the phytotoxicity of water-soluble fraction (WSFd) from steam-pretreated GP after 90 days' incubation to lettuce and tomato seeds. U. botrytis caused the largest effective phytotoxicity reduction of WSFd (used in the concentration range of 10-1.25% p/v) and was the only fungus causing the removal of monoaromatic compounds. Therefore, this procedure with U. botrytis effectively reduces the availability of phytotoxic monoaromatic compounds in GP, which opens a way for the development of guidelines for the management of these wastes and their potential use as organic amendments in agricultural soil.

Identification of cytotoxic metabolites from Mahonia aquifolium using 1H NMR-based metabolomics approach.

Herein, we propose a 1H NMR-based metabolomics method to reveal cytotoxic metabolites from Mahonia aquifolium stem-bark. Primary and secondary metabolites in the Mahonia aquifolium extracts were identified by thorough analysis of 1H and 2D NMR spectra, without prior isolation. An OPLS multivariate analysis method was used to correlate the chemical composition of the plant extracts with the results of cytotoxic activity against Human cervical adenocarcinoma cell line. Protoberberine alkaloids berberinе and palmatine, along with bisbenzylisoquinoline alkaloid berbamine were identified as the most influential in the OPLS model, with the highest cytotoxic activity.

NMR chemical shift and J coupling parameterization and quantum mechanical reference spectrum simulation for selected nerve agent degradation products in aqueous conditions.

The spectral parameters of selected nerve agent degradation products relevant to the Chemical Weapons Convention, namely, ethyl methylphosphonate, isopropyl methylphosphonate, pinacolyl methylphosphonate and methylphosphonic acid, were studied in wide range of pH conditions and selected temperatures. The pH and temperature dependence of chemical shifts and J couplings was parameterized using Henderson-Hasselbalch-based functions. The obtained parameters allowed calculation of precise chemical shifts and J coupling constants in arbitrary pH conditions and typical measurement temperatures, thus facilitating quantum mechanical simulation of reference spectra in the chosen magnetic field strength for chemical verification. Copyright © 2017 John Wiley & Sons, Ltd.

Chemical Ecology of Cave-Dwelling Millipedes: Defensive Secretions of the Typhloiulini (Diplopoda, Julida, Julidae).

Cave animals live under highly constant ecological conditions and in permanent darkness, and many evolutionary adaptations of cave-dwellers have been triggered by their specific environment. A similar "cave effect" leading to pronounced chemical interactions under such conditions may be assumed, but the chemoecology of troglobionts is mostly unknown. We investigated the defensive chemistry of a largely cave-dwelling julid group, the controversial tribe "Typhloiulini", and we included some cave-dwelling and some endogean representatives. While chemical defense in juliform diplopods is known to be highly uniform, and mainly based on methyl- and methoxy-substituted benzoquinones, the defensive secretions of typhloiulines contained ethyl-benzoquinones and related compounds. Interestingly, ethyl-benzoquinones were found in some, but not all cave-dwelling typhloiulines, and some non-cave dwellers also contained these compounds. On the other hand, ethyl-benzoquinones were not detected in troglobiont nor in endogean typhloiuline outgroups. In order to explain the taxonomic pattern of ethyl-benzoquinone occurrence, and to unravel whether a cave-effect triggered ethyl-benzoquinone evolution, we classed the "Typhloiulini" investigated here within a phylogenetic framework of julid taxa, and traced the evolutionary history of ethyl-benzoquinones in typhloiulines in relation to cave-dwelling. The results indicated a cave-independent evolution of ethyl-substituted benzoquinones, indicating the absence of a "cave effect" on the secretions of troglobiont Typhloiulini. Ethyl-benzoquinones probably evolved early in an epi- or endogean ancestor of a clade including several, but not all Typhloiulus (basically comprising a taxonomic entity known as "Typhloiulus sensu stricto") and Serboiulus. Ethyl-benzoquinones are proposed as novel and valuable chemical characters for julid systematics.

Metabolomics study of Populus type propolis.

Herein, we propose rapid and simple spectroscopic methods to determine the chemical composition of propolis derived from various Populus species using a metabolomics approach. In order to correlate variability in Populus type propolis composition with the altitude of its collection, NMR, IR, and UV spectroscopy followed by OPLS was conducted. The botanical origin of propolis was established by comparing propolis spectral data to those of buds of various Populus species. An O2PLS method was utilized to integrate two blocks of data. According to OPLS and O2PLS, the major compounds in propolis samples, collected from temperate continental climate above 500m, were phenolic glycerides originating from P. tremula buds. Flavonoids were predominant in propolis samples collected below 400m, originating from P. nigra and P. x euramericana buds. Samples collected at 400-500m were of mixed origin, with variable amounts of all detected metabolites.

Metabolic changes in Euphorbia palusrtis latex after fungal infection.

The variations of metabolic profile of the latex of wild-growing Euphorbia palustris was carried out using multivariate analysis of 1H NMR spectral data. One population was infected with fungi Fusarium sporotrichioides, Fusarium proliferatum and Alternaria alternata, while the other consisted of healthy plant species. The non-polar metabolites of latex extracts such as benzoyl ingenol-laurate, amyrin decadienoate esters, cis-1,4-polyisoprene, and 24-methylenecycloartanol were identified using 1H and 2D NMR spectra. Principal component analysis of 1H NMR data provided a clear discrimination between the latex of infected and healthy plants. Minimum inhibitory concentration and minimum fungicidal concentration values of the latex extracts of healthy and infected plants were determined. The latex of infected plants was found to contain higher levels of benzoyl ingenol-laurate and 24-methylenecycloartanol, of which concentrations were strongly correlated with the antifungal activities of the latex.

'Does my Diet Affect my Perfume?' Identification and Quantification of Cuticular Compounds in Five Drosophila melanogaster Strains Maintained over 300 Generations on Different Diets.

Cuticular hydrocarbons (CHCs) in Drosophila melanogaster represent the basis of chemical communication being involved in many important biological functions. The aim of this study was to characterize chemical composition and variation of cuticular profiles in five D. melanogaster strains. These strains were reared for approximately 300 generations on five diets: standard cornmeal medium and substrates prepared with apple, banana, tomato, and carrot. Differences in quantity and/or quality in CHCs were assumed as a result of activation of different metabolic pathways involved in food digestion and adaptations to the particular diet type. In total, independently of sex and strain, 66 chemical compounds were identified. In females of all strains, 60 compounds were identified, while, in males, 47 compounds were extracted. Certain new chemical compounds for D. melanogaster were found. MANOVA confirmed that CHC amounts significantly depend on sex and substrates, as well as on their interactions. Discriminant analysis revealed that flies belonging to 'apple' and 'carrot' strains exhibited the most noticeable differences in CHC repertoires. A non-hydrocarbon pheromone, cis-vaccenyl acetate (cVA) also contributed to the variation in the pheromone bouquet among the strains. Variability detected in CHCs and cVA may be used in the explanation of differences in mating behaviour previously determined in analyzed fly strains.

Phenolic Compounds from Atriplex littoralis and Their Radiation-Mitigating Activity.

From the aerial parts of Atriplex littoralis, three new flavonoid glycosides named atriplexins I-III (1-3), a known flavonoid glycoside, spinacetin 3-O-ß-d-glucopyranoside (4), arbutin (5), and 4-hydroxybenzyl-ß-d-glucopyranoside (6) were isolated. Their structures were elucidated on the basis of detailed spectroscopic analysis, including 1D and 2D NMR (COSY, NOESY, TOCSY, HSQC, HMBC) and HRESITOF MS data. The compounds were tested for in vitro protective effects on chromosome aberrations in peripheral human lymphocytes using a cytochalasin-B-blocked micronucleus (MN) assay in a concentration range of 0.8-7.4 µM of final culture solution. Chromosomal damage was induced by 2 Gy of γ-radiation on binucleated human lymphocytes, and the effects of the compounds were tested 2 to 19 h after irradiation. The frequency of micronuclei (MNi) was scored in binucleated cells, and the nuclear proliferation index was calculated. The highest prevention of in vitro biochemical and cytogenetic damage of human lymphocytes induced by γ-radiation was exhibited by 3 (reduction of MN frequency by 31%), followed by 4 and 6.

Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage.

Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 °C to 60 °C with ΔG°321 of 13.9±0.3 kJ/mol and Tm value of 84±1 °C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular ß-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry.

Chemical composition, cytotoxic and antioxidative activities of ethanolic extracts of propolis on HCT-116 cell line.

BACKGROUND: Propolis is a complex resinous sticky substance that honeybees collect from buds and exudates of various plants. Owing to its versatile biological and pharmacological activities, propolis is widely used in medicines, cosmetics and foods. The aim of this study was to evaluate the cytotoxic and antioxidative effects of various ethanolic extracts of propolis (EEPs) on human colon cancer cell line HCT-116 and compare them with their composition determined by HPLC-DAD. RESULTS: The most abundant flavonoids in all samples were chrysin, pinocembrin and galangin (12.697-40.811 µg mg⁻¹), while the main phenolic acids were caffeic acid, ferulic acid and isoferulic acid. Dose- and time-dependent inhibition of growth of HCT-116 cells was observed for all propolis samples, with IC50 values ranging from 26.33 to 143.09 µg mL⁻¹. Differences in cytotoxic activity of propolis samples were associated with differences in their composition. All EEP samples reduced both superoxide anion radical and nitrite levels and also had strong DPPH-scavenging activity. CONCLUSION: All tested propolis samples had pronounced cytotoxic and antioxidative activities.