Male dingo urinary scents code for age class and wild dingoes respond to this information.

Chemical information in canid urine has been implicated in territoriality and influences the spacing of individuals. We identified the key volatile organic compound (VOC) components in dingo (Canis lupus dingo) urine and investigated the potential role of scents in territorial spacing. VOC analysis, using headspace gas chromatography-mass spectrometry (GC-MS), demonstrated that the information in fresh urine from adult male dingoes was sufficient to allow statistical classification into age categories. Discriminant function analyses demonstrated that the relative amounts or combinations of key VOCs from pre-prime (3-4 years), prime (5-9 years), and post-prime (≥10 years) males varied between these age categories, and that scents exposed to the environment for 4 (but not 33) days could still be classified to age categories. Further, a field experiment showed that dingoes spent less time in the vicinity of prime male dingo scents than other scents. Collectively, these results indicate that age-related scent differences may be discriminable by dingoes. Previous authors have suggested the potential to use scent as a management tool for wild canids by creating an artificial territorial boundary/barrier. Our results suggest that identifying the specific signals in prime-age male scents could facilitate the development of scent-based tools for non-lethal management.

Analysis of sex steroids in human tears using LC-MS and GC-MS: Considerations and developments to improve method sensitivity and accuracy.

Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17ß-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.

Comparative limitations and benefits of liquid chromatography - mass spectrometry techniques for analysis of sex steroids in tears.

Sex steroids impact regulation of the ocular surface tissues and thus influence dry eye. Most tissues in the human body synthesise and metabolise active sex steroids at levels required by the tissue. This is likely to also be the case for humans in ocular surface tissues. This study investigated the presence and quantities of selected sex steroids, in addition to sex steroid precursors and metabolites, in human tears. Detection of sex steroids in tears is challenging due to trace level analyte concentrations and low volumes of available tears. Immunoassays have previously been employed to assess sex steroids in tears, however, this approach only allows a single analyte to be measured and can overestimate concentrations. This study evaluated ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) and tandem mass spectrometry (MS/MS) methods for the concurrent detection and estimation of fourteen sex steroid and metabolite compounds in human tears. Basal tears were collected and pooled from 5 healthy pre-menopausal women (total 100 µL). Following protein precipitation and centrifuging, extract volumes equivalent to 14 µL of pooled tears were analysed. A Thermo Scientific Q Exactive™ Plus MS was used to compare novel high-resolution MS atmospheric pressure chemical ionization (APCI) and electrospray ionisation (ESI) methods for detection of fourteen target sex steroids, including dehydroepiandrosterone-sulphate (DHEAS), testosterone, oestradiol and their metabolites, using standards and pooled tears. The MS was programmed to switch between positive and negative polarity ionization modes at designated times through the UPLC run, in order to detect each analyte at optimal sensitivity. Analytes were analysed using APCI in standard mixtures at concentrations of 0.1, 0.3, 1, 3, 10, 30, 100 and 1000 pg/mL, per component, to determine the limits of detection (LOD) and quantification. Both parallel reaction monitoring (PRM) MS/MS and selected ion monitoring (SIM) MS, were evaluated by plotting narrow range mass-to-charge ratio (m/z) chromatograms for each analyte. An APCI UPLC-MS method to simultaneously measure 14 sex steroids using standards was successfully developed following comparative evaluation of all available LC-MS techniques. Preliminary experiments found APCI to be more sensitive than ESI using standards. Narrow m/z range SIM MS resulted in better sensitivity, in tear samples, than PRM. One sex steroid and two androgen metabolites were detected, with the developed APCI UPLC-MS method, and their concentrations estimated in human tears that had been extracted with a protein crash. Progesterone, androsterone-glucuronide (ADT-G) and 3αDiol-G were successfully detected in the tear extract and their concentrations in the pooled tear sample were estimated (with 95% confidence intervals) to be 0.10 ±â€¯0.03 pg/µL, 30.9 ±â€¯18.3 pg/µL and 9.8 ±â€¯4.3 pg/µL respectively. The concentrations of the remaining 11 sex steroids in the tear sample were below the LODs of the method. This work shows that high mass resolution UPLC-MS can detect certain sex steroids and metabolites in tears, but that sensitivity of the technique and the low available tear volumes limit its application to a broader range of sex steroids. The investigation of sex steroids in tears and ocular surface tissue will aid understanding of the influence of sex steroids on ocular surface tissues facilitating better targeted treatment for dry eye.

Differential injurious effects of ambient and traffic-derived particulate matter on airway epithelial cells.

BACKGROUND AND OBJECTIVE: Exposure to airborne particulate matter (PM) may promote development of childhood asthma and trigger acute exacerbations of existing asthma via injury to airway epithelial cells (AEC). METHODS: We compared the response of AEC to ambient particulates with median aerodynamic diameters of <10 µm or <2.5 µm from the Sydney metropolitan region (Sydney PM10 or PM2.5), to traffic-derived particulates from the exhaust stack of a motorway tunnel or to inert carbon black as a control. RESULTS: Sydney PM10 strongly stimulated messenger RNA expression and secretion of the pro-inflammatory cytokines interleukin 6 (IL-6) and chemokine (C-X-C motif) ligand 1 (CXCL1) by mouse tracheal AEC. In contrast, traffic-derived particulates did not. Similarly, PM10 stimulated expression of IL6, IL8 and IL1B by human AEC. Mass spectrometric analysis showed that PM10 contained much higher levels of elements associated with dusts of geological origin. In contrast, tunnel soot contained much higher levels of various organic compounds, notably including long straight-chain alkanes and diesel-derived polycyclic aromatic hydrocarbons. Sydney PM2.5, as well as PM10 collected during a period including a major dust storm, both of which contained relatively lower levels of iron but similar levels of other crustal elements, did not stimulate expression or secretion of CXCL1 by mouse AEC. CONCLUSIONS: Ambient PM10 is likely to be more important than traffic-derived PM in causing injury to AEC leading to production of pro-inflammatory cytokines. The injurious effects may be related to the presence of iron in the coarse fraction of airborne PM. These findings are likely to be relevant to the pathogenesis of asthma.

Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis.

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis, 3ß-hydroxysterol Δ24-reductase (DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its posttranslational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell-line (Chinese hamster ovary-7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling.

An easy one-pot synthesis of diverse 2,5-di(2-pyridyl)pyrroles: a versatile entry point to metal complexes of functionalised, meridial and tridentate 2,5-di(2-pyridyl)pyrrolato ligands.

A wide variety of 2,5-di(2-pyridyl)pyrroles (dppHs) substituted at the C3 and C4 positions of the pyrrole core were obtained by direct condensation of a 2-pyridylcarboxaldehyde (2 equiv), an α-methylene ketone with at least one electron-withdrawing substituent and ammonium acetate. A novel 2,5-di(1,10-phenanthrolin-2-yl)pyrrole was also characterised. The dppHs provide a direct, quick entry to dipyridylpyrrolato (dpp(-) )-metal complexes. The meridial tridentate dpp(-) ligand is a useful anionic analogue of the terpyridyl ligand. The first (dpp)Ru complexes are described; the 3,4-substitution of the central pyrrole significantly perturbs the potentials of the redox processes of these complexes. A [(dpp)Ru(bpy)(MeCN)](+) (bpy=2,2'-bipyridine) complex is an electrocatalyst for the reductive disproportionation of carbon dioxide to carbon monoxide and the carbonate ion.

Coffee brewing sonoreactor for reducing the time of cold brew from several hours to minutes while maintaining sensory attributes.

This research designed and developed an ultrasonic reactor for a fast and on demand production of cold brew coffee, remarkably reducing the brewing time from 24 h to less than 3 min. The technology was engineered by utilizing resonance to induce ultrasonic waves around the walls of the brewing basket of an espresso machine. The sound transmission system comprised a transducer, a horn and a brewing basket. This arrangement transformed the coffee basket into an effective sonoreactor that injected sound waves at multiple points through its walls, thereby generating multiple regions for acoustic cavitation within the reactor. Furthermore, acoustic streaming induced greater mixing and enhanced mass transfer during brewing. The design was accomplished by modeling the transmission of sound, and acoustic cavitation. Brew characterization and chemical composition analysis was performed, considering factors such as pH, acidity, color, and the composition of caffeine, fatty acids, and volatiles. The efficiency of the extraction increased by decreasing the basket loading percentage (BLP). For instance, sonicating at 100 W doubled the extraction yield and caffeine concentration, from 15.05 % to 33.44 % at BLP = 33 %, and from 0.91 mg/mL to 1.84 mg/mL at BLP = 67 %, respectively. The total fatty acids increased from 1.16 mg/mL to 9.20 mg/mL, representing an eightfold increase, at BLP = 33 %. Finally, a sensory analysis was conducted to evaluate appearance, aroma, texture, flavor, and aftertaste, which demonstrated that coffee brewed for 1 and 3 min in the sonoreactor exhibited almost undistinguishable properties compared to a standard 24 h brewing without ultrasound.

Folate analysis in foods by UPLC-MS/MS: development and validation of a novel, high throughput quantitative assay; folate levels determined in Australian fortified breads.

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed, optimised and validated for the quantification of synthetic folic acid (FA), also called pteroyl-L: -glutamic acid or vitamin B9 and naturally occurring 5-methyltetrahydrofolate (5-MTHF) found in folate-fortified breads. Optimised sample preparation prior to analysis involved addition of (13)C(5) labelled internal standards, treatments with α-amylase and rat serum, solid-phase extraction using aromatic-selective cartridges and ultra-filtration. Analytes were separated on a Waters ACQUITY HSS T3 column during a 6-min run and analysed by positive ion electrospray selected reaction monitoring MS/MS. Standard calibration curves for the two analytes were linear over the range of 0.018-14 µg FA/g of fresh bread (r(2) = 0.997) and 9.3-900 ng 5-MTHF/g of fresh bread (r(2) = 0.999). The absolute recoveries were 90% and 76% for FA and 5-MTHF, respectively. Intra-day coefficients of variation were 3% for FA and 18% for 5-MTHF. The limit of detection was 9.0 ng/g for FA and 4.3 ng/g for 5-MTHF, determined using pre-extracted tapioca starch as the blank matrix. The assay is rugged, fast, accurate and sensitive, applicable to a variety of food matrices and is capable of the detection and quantification of the naturally occurring low levels of 5-MTHF in wheat breads. The findings of this study revealed that the FA range in Australian fortified breads was 79-110 µg/100 g of fresh bread and suggest that the flour may not have the mandated FA fortification level (200-300 µg/100 g of flour), though this cannot be determined conclusively from experimental bread data alone, as variable baking losses have been documented by other authors.

Brewing coffee? - Ultra-sonication has clear beneficial effects on the extraction of key volatile aroma components and triglycerides.

Ultrasound has been investigated as a new technique for brewing coffee. A two-level factorial experimental design was conducted to identify the effects of ultra-sonication on the extraction of coffee components during ultrasonically-assisted coffee brewing. Different brews were produced by aqueous extraction from roasted ground coffee beans with sonication, and without it as a control, by varying coffee concentration (5% and 10% w/w), temperature (25 and 50 °C) and sonication time (1 and 5 min). These brews were tested for antioxidant capacity (using the ABTS assay), caffeine and triglycerides (using quantitative NMR spectroscopy) and specific aroma/flavour volatiles (using headspace SPME-GC-MS). Additional observations of colour, foaming, body and flavour were also reported. Ultrasound was found to significantly increase the extraction of caffeine, triglycerides and several of the key volatile compounds from coffee, although it did appear to decrease the concentration of antioxidants over the controls, especially with longer time and higher temperature. Furthermore, all the sonicated samples exhibited a lighter caramel colour and lower foam formation which were attributed to their higher triglyceride content. The increased concentration of triglycerides and volatiles were by far the most outstanding responses.

Gas chromatography-mass spectrometry analyses of encapsulated stable perovskite solar cells.

Although perovskite solar cells have produced remarkable energy conversion efficiencies, they cannot become commercially viable without improvements in durability. We used gas chromatography-mass spectrometry (GC-MS) to reveal signature volatile products of the decomposition of organic hybrid perovskites under thermal stress. In addition, we were able to use GC-MS to confirm that a low-cost polymer/glass stack encapsulation is effective in suppressing such outgassing. Using such an encapsulation scheme, we produced multi-cation, multi-halide perovskite solar cells containing methylammonium that exceed the requirements of the International Electrotechnical Commission 61215:2016 standard by surviving more than 1800 hours of the Damp Heat test and 75 cycles of the Humidity Freeze test.

Effects on intestinal cellular bioaccessibility of carotenoids and cellular biological activity as a consequence of co-ingestion of anthocyanin- and carotenoid-rich vegetables.

The effects of co-digestion of a carotenoid-rich vegetable such as carrot, cherry tomato or baby spinach with an anthocyanin-rich vegetable such as red cabbage with and without salad dressing on the intestinal cellular bioaccessibility (cBAC) of carotenoids and the resultant cellular antioxidant and anti-inflammatory activities were investigated. The % cBAC of lutein from the tested vegetables was 0.23-1.42%, lycopene 0.07-0.39%, α-carotene 0.01-0.12% and ß-carotene 0.03-0.61% respectively. The % cBAC of each of these carotenoids from the co-digested vegetables was significantly higher (p < 0.05) than from carrot, cherry tomato or baby spinach digested alone. % cBAC of total carotenoids was significantly increased by 46-191% (p < 0.05) as a result of the co-digestion. The vegetable co-digestion did not result in any impairment on the resultant cellular anti-oxidation and anti-inflammation (NO, IL-8 secretion). Among the tested vegetables, baby spinach co-digested with red cabbage showed synergistic bioactivities in all tested assays.

Interferences of anthocyanins with the uptake of lycopene in Caco-2 cells, and their interactive effects on anti-oxidation and anti-inflammation in vitro and ex vivo.

Lycopene was combined with the glucosides of each of the six common anthocyanidins at 3 different ratios to investigate their interactions on antioxidant and anti-inflammatory activities, and cellular uptake. The bioactivity interaction between lycopene and anthocyanins was studied in both chemical and cellular models. Anti-oxidative synergy was not seen in any of the tested lycopene-anthocyanin mixtures, nor in the models studied. When lycopene was paired with the methoxylated anthocyanins, the anti-inflammatory effect on the inhibition of the cytokine IL-8, which is a pro-inflammatory biomarker, was increased by 15-69% of the expected additive activity, indicating synergistic interaction between the compounds. The cellular uptake of lycopene was significantly impaired by the presence of the anthocyanins: reduced by 50-80% at the lycopene: anthocyanin combinatory ratios of 2.5:7.5 µM (1:3) or 5:5 µM (1:1). The reduced intracellular lycopene content might be partly responsible for the antagonistic cellular antioxidant property seen in some of the tested mixtures.

Co-ingestion of red cabbage with cherry tomato enhances digestive bioaccessibility of anthocyanins but decreases carotenoid bioaccessibility after simulated in vitro gastro-intestinal digestion.

The effects of co-digestion of red cabbage with carrot, baby spinach and/or cherry tomato on the bioaccessibility of anthocyanins and carotenoids such as α-carotene, ß-carotene, lutein and lycopene were examined using a simulated in vitro gastro-intestinal digestion model. The individual vegetables and their mixtures were digested with and without added a standardised salad dressing. Bioaccessibility of total anthocyanins was enhanced by 10-15% (p < 0.05) when red cabbage was co-digested with the carotenoid-rich vegetables, except with carrot. In contrast, the co-digestion of red cabbage with carrot decreased bioaccessibility of total carotenoids by 21-33% (p < 0.05), and with cherry tomato by 42-56% (p < 0.05). The bioaccessibility of a given carotenoid varied depending on the vegetable matrix. Among the tested vegetable mixtures, red cabbage and baby spinach when co-digested demonstrated that anthocyanins and carotenoids were equally bioaccessible (total anthocyanin bioaccessibility of 62-66% and total carotenoid bioaccessibility of 66%).

Profile changes in banana flavour volatiles during low temperature drying.

Headspace gas chromatography mass spectrometry (HS-GC-MS) was used to measure changes in selected volatile flavour compounds in fresh banana during low temperature heat pump drying. Ten compounds from a range of chemical classes were measured during drying at three different drying conditions. Ester compounds were found to be the most affected, with losses varying from 25 to 87% during drying. Three patterns of depletion were observed in this study. Ester and aldehyde levels reduced quickly during the early stages of drying, but levels stabilised at non-zero values towards the end of drying; alcohol levels initially increased, then decreased and stabilised; whilst high molecular weight compounds, such as elemicine and eugenol, were not affected significantly. Selective diffusion and volatility affected the degree of flavour retention.

Evaluating folate extraction from infant milk formulae and adult nutritionals: Enzymatic digestion versus enzyme-free heat treatment.

This study compares enzymatic treatments to release folic acid (FA) and endogenous 5-methyltetrahydrofolate (5-MTHF) from infant milk formulae with enzyme-free heat extraction. The limits of detection and quantitation of FA were 1.4ng/mL and 3.1ng/mL, respectively; 7.5ng/mL and 16.2ng/mL for 5-MTHF. Absolute mean recoveries were 85% (FA) and 95% (5-MTHF). The RSD of the within-run variability was 6% and the inter-day variability was 8%. Averaged measurements of FA and 5-MTHF in SRM-1849a were within the certified value range. Analysed folate levels in three brands were greater than label values, because of inherently high 5-MTHF occurring in samples. The results indicate that enzyme-free heat treatment prior to UPLC-MS/MS analysis gives better sensitivity and reduces chromatographic interferences for the determination of FA and 5-MTHF in milk formulae than enzymatic treatments. Enzyme-free heat treatment is more compatible with UPLC-MS/MS than folate extraction techniques involving the addition of enzymes to milk.

Strategic design of a ruthenium catalyst for both CO2 reduction and H2O oxidation: the electronic influence of the co-ligands.

A new ruthenium(ii) complex capable of catalysing both CO2 reduction and water oxidation was designed and synthesised. The electro-catalytic efficiency and robustness of the complex together with the electronic effect of its co-ligands were investigated to develop next generation dual activity electrocatalysts.

Novel predators emit novel cues: a mechanism for prey naivety towards alien predators.

Detecting enemies is crucial for survival and a trait that develops over an evolutionary timeframe. Introduced species disrupt coevolved systems of communication and detection in their new ranges, often leading to devastating impacts. The classic example is prey naivety towards alien predators, whereby prey fail to recognise a new predator. Yet exactly why native prey fail to recognise alien predators remains puzzling. Naivety theory predicts that it is because novel predators emit novel cues. Distantly related animals have distinct evolutionary histories, physiologies and ecologies, predicting they will emit different cues. Yet it also possible that all predators emit similar cues because they are carnivorous. We investigate whether odour cues differ between placental and marsupial carnivores in Australia, where native prey experienced only marsupial mammal predation until ~4000 years ago. We compared volatile chemical profiles of urine, scats and bedding from four placental and three marsupial predators. Chemical profiles showed little overlap between placental and marsupial carnivores across all odour types, suggesting that cue novelty is a plausible mechanism for prey naivety towards alien predators. Our results also suggest a role for olfactory cues to complement visual appearance and vocalisations as biologically meaningful ways to differentiate species.

On the use of test gases of various radii to investigate molecular sieving in leak channels.

Evidence of the effect of molecule size (molecular sieving) was discovered in leak channels similar to those found in hermetically sealed implantable bionics. A range of test gases of different molecular sizes was used to investigate the relative leak rates of several different samples. A contemporary model of molecular sieving is shown to be in partial agreement with our data.

Induction of settlement of larvae of the sea urchin Holopneustes purpurascens by histamine from a host alga.

Larvae of the Australian sea urchin Holopneustes purpurascens are induced to settle and metamorphose (termed settlement herein) by a water-soluble compound produced by the red alga Delisea pulchra, the main host plant of new recruits. The settlement cue for H. purpurascens had previously been identified as a floridoside-isethionic acid complex, and this paper presents new evidence correcting that finding. The actual settlement cue produced by D. pulchra was isolated from the polar extract by cation-exchange chromatography and identified as histamine, using one- and two-dimensional nuclear magnetic resonance spectrometry. The chemical identity of the cue was confirmed by gas chromatography-mass spectrometry (GC-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Synthetic histamine and histamine at 4.5 microM isolated from D. pulchra both induced rapid settlement in 80%-100% of the larvae of H. purpurascens. Lower concentrations of histamine (0.9-2.3 micro M) induced larval settlement, but this response varied from 0%-90%. The histamine content of two host plants of H. purpurascens--D. pulchra and Ecklonia radiata--and of four other common species was quantified using GC-MS. D. pulchra had the highest histamine content, which is consistent with H. purpurascens recruiting to this species. Histamine was also detected in the seawater surrounding these host algae. This is the first time that a settlement cue has been quantified in the habitat of a marine organism.