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Dr. Simone Rochfort
Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Bundoora, Victoria 3083, Australia

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0 Chromatography
0 Mass Spectrometry
0 Metabolomics
0 Proteomics
0 Quantitation

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Chromatography
Mass Spectrometry
Metabolomics
Quantitation
Proteomics
nuclear magnetic resonance

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Journal article
Published: 28 June 2021 in Molecules
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Regioisomers (or positional isomers) of triacylglycerols (TAGs) of milk are known to show differential outcome in relation to human absorption. Quantitation of TAG regioisomers remains a big challenge due to the lack of facile chromatographic separation technique. The feasibility of using fragment ion intensity ratio to determine the ratio of co-eluting AAB/ABA-type regioisomer pairs was confirmed in this study. The ability of C30 stationary phase in resolving interfering TAG isomers was demonstrated for the first time. This allowed us to reveal the complexity of using fragment ion intensity to quantify 1,2-olein-3-palmitin (OOP), 1,3-olein-2-palmitin (OPO), 1,2-olein-3-stearin (OOS), and 1,3-olein-2-stearin (OSO) regioisomers in milk samples. A novel algorithm was proposed to consider the contribution of OPO/OOP and OSO/OOS double bond (DB)-isomers and to eliminate the interference of isobaric ions from other isomers, an aspect overlooked in previous studies. This liquid chromatography-mass spectrometry method that requires no pre-fractioning and a moderate chromatographic separation time of 36 min is simple and, thus, suitable for screening a large number of samples for genetic analysis of this trait. Preliminary results using a small cohort of animals showed that OPO/OOP ratio differs significantly between Jersey and Holstein cows, and a large variation was also observed across individual Holstein cows.

ACS Style

Zhiqian Liu; Simone Rochfort. Bovine Milk Triacylglycerol Regioisomer Ratio Shows Remarkable Inter-Breed and Inter-Cow Variation. Molecules 2021, 26, 3938 .

AMA Style

Zhiqian Liu, Simone Rochfort. Bovine Milk Triacylglycerol Regioisomer Ratio Shows Remarkable Inter-Breed and Inter-Cow Variation. Molecules. 2021; 26 (13):3938.

Chicago/Turabian Style

Zhiqian Liu; Simone Rochfort. 2021. "Bovine Milk Triacylglycerol Regioisomer Ratio Shows Remarkable Inter-Breed and Inter-Cow Variation." Molecules 26, no. 13: 3938.

Author correction
Published: 25 January 2021 in Scientific Reports
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ACS Style

Roya Afshari; Christopher J. Pillidge; Elizabeth Read; Simone Rochfort; Daniel A. Dias; A. Mark Osborn; Harsharn Gill. Author Correction: New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data. Scientific Reports 2021, 11, 1 -3.

AMA Style

Roya Afshari, Christopher J. Pillidge, Elizabeth Read, Simone Rochfort, Daniel A. Dias, A. Mark Osborn, Harsharn Gill. Author Correction: New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data. Scientific Reports. 2021; 11 (1):1-3.

Chicago/Turabian Style

Roya Afshari; Christopher J. Pillidge; Elizabeth Read; Simone Rochfort; Daniel A. Dias; A. Mark Osborn; Harsharn Gill. 2021. "Author Correction: New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data." Scientific Reports 11, no. 1: 1-3.

Journal article
Published: 17 July 2020 in Metabolites
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Volatile phenols (VPs) derived from smoke-exposed grapes are known to confer a smoky flavor to wine. Current methods for determination of VPs in grape berries either involve complex sample purification/derivatization steps or employ two analytical platforms for free and bound VP fractions. We report here a simple gas chromatography-tandem mass spectrometry (GC-MS/MS) method for quantification of both free and bound VPs in grapes, based on optimized (1) GC-MS/MS parameters, (2) an analyte extraction procedure, and (3) phenol glycoside hydrolysis conditions. Requiring neither sample cleanup nor a derivatization step, this method is sensitive (LOD ≤ 1 ng/g berries) and reproducible (RSD < 12% for repeated analyses) and is expected to significantly reduce the sample turnover time for smoke taint detection in vineyards.

ACS Style

Zhiqian Liu; Vilnis Ezernieks; Priyanka Reddy; Aaron Elkins; Christian Krill; Kieran Murphy; Simone Rochfort; German Spangenberg. A Simple GC-MS/MS Method for Determination of Smoke Taint-Related Volatile Phenols in Grapes. Metabolites 2020, 10, 294 .

AMA Style

Zhiqian Liu, Vilnis Ezernieks, Priyanka Reddy, Aaron Elkins, Christian Krill, Kieran Murphy, Simone Rochfort, German Spangenberg. A Simple GC-MS/MS Method for Determination of Smoke Taint-Related Volatile Phenols in Grapes. Metabolites. 2020; 10 (7):294.

Chicago/Turabian Style

Zhiqian Liu; Vilnis Ezernieks; Priyanka Reddy; Aaron Elkins; Christian Krill; Kieran Murphy; Simone Rochfort; German Spangenberg. 2020. "A Simple GC-MS/MS Method for Determination of Smoke Taint-Related Volatile Phenols in Grapes." Metabolites 10, no. 7: 294.

Journal article
Published: 06 July 2020 in Metabolites
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Cannabis and its secondary metabolite content have recently seen a surge in research interest. Cannabis terpenes and terpenoids in particular are increasingly the focus of research efforts due to the possibility of their contribution to the overall therapeutic effect of medicinal cannabis. Current methodology to quantify terpenes in cannabis biomass mostly relies on large quantities of biomass, long extraction protocols, and long GC gradient times, often exceeding 60 min. They are therefore not easily applicable in the high-throughput environment of a cannabis breeding program. The method presented here, however, is based on a simple hexane extract from 40 mg of biomass, with 50 μg/mL dodecane as internal standard, and a gradient of less than 30 min. The method can detect 48 individual terpenes and terpenoids and was validated for selectivity, linearity, LOD/LOQ, precision, intermediate precision, and accuracy (recovery) for 22 terpenes and terpenoids. The validation parameters are comparable to previously published studies that employ significantly longer runtimes and/or more complex extraction protocols. It is currently being applied to medicinal cannabis precision breeding programs.

ACS Style

Christian Krill; Simone Rochfort; German Spangenberg. A High-Throughput Method for the Comprehensive Analysis of Terpenes and Terpenoids in Medicinal Cannabis Biomass. Metabolites 2020, 10, 276 .

AMA Style

Christian Krill, Simone Rochfort, German Spangenberg. A High-Throughput Method for the Comprehensive Analysis of Terpenes and Terpenoids in Medicinal Cannabis Biomass. Metabolites. 2020; 10 (7):276.

Chicago/Turabian Style

Christian Krill; Simone Rochfort; German Spangenberg. 2020. "A High-Throughput Method for the Comprehensive Analysis of Terpenes and Terpenoids in Medicinal Cannabis Biomass." Metabolites 10, no. 7: 276.

Journal article
Published: 24 June 2020 in Microorganisms
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Asexual Epichloë spp. fungal endophytes have been extensively studied for their functional secondary metabolite production. Historically, research mostly focused on understanding toxicity of endophyte-derived compounds on grazing livestock. However, endophyte-derived compounds also provide protection against invertebrate pests, disease, and other environmental stresses, which is important for ensuring yield and persistence of pastures. A preliminary screen of 30 strains using an in vitro dual culture bioassay identified 18 endophyte strains with antifungal activity. The novel strains NEA12, NEA21, and NEA23 were selected for further investigation as they are also known to produce alkaloids associated with protection against insect pests. Antifungal activity of selected endophyte strains was confirmed against three grass pathogens, Ceratobasidium sp., Dreschlera sp., and Fusarium sp., using independent isolates in an in vitro bioassay. NEA21 and NEA23 showed potent activity against Ceratobasidium sp. and NEA12 showed moderate inhibition against all three pathogens. Crude extracts from liquid cultures of NEA12 and NEA23 also inhibited growth of the phytopathogens Ceratobasidium sp. and Fusarium sp. and provided evidence that the compounds of interest are stable, constitutively expressed, and secreted. Comparative analysis of the in vitro and in planta metabolome of NEA12 and NEA23 using LCMS profile data revealed individual metabolites unique to each strain that are present in vitro and in planta. These compounds are the best candidates for the differential bioactivity observed for each strain. Novel endophyte strains show promise for endophyte-mediated control of phytopathogens impacting Lolium spp. pasture production and animal welfare.

ACS Style

Krishni Fernando; Priyanka Reddy; Inoka K. Hettiarachchige; German C. Spangenberg; Simone J. Rochfort; Kathryn M. Guthridge. Novel Antifungal Activity of Lolium-Associated Epichloë Endophytes. Microorganisms 2020, 8, 955 .

AMA Style

Krishni Fernando, Priyanka Reddy, Inoka K. Hettiarachchige, German C. Spangenberg, Simone J. Rochfort, Kathryn M. Guthridge. Novel Antifungal Activity of Lolium-Associated Epichloë Endophytes. Microorganisms. 2020; 8 (6):955.

Chicago/Turabian Style

Krishni Fernando; Priyanka Reddy; Inoka K. Hettiarachchige; German C. Spangenberg; Simone J. Rochfort; Kathryn M. Guthridge. 2020. "Novel Antifungal Activity of Lolium-Associated Epichloë Endophytes." Microorganisms 8, no. 6: 955.

Journal article
Published: 15 June 2020 in Proteomes
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Cannabis research has taken off since the relaxation of legislation, yet proteomics is still lagging. In 2019, we published three proteomics methods aimed at optimizing protein extraction, protein digestion for bottom-up and middle-down proteomics, as well as the analysis of intact proteins for top-down proteomics. The database of Cannabis sativa proteins used in these studies was retrieved from UniProt, the reference repositories for proteins, which is incomplete and therefore underrepresents the genetic diversity of this non-model species. In this fourth study, we remedy this shortcoming by searching larger databases from various sources. We also compare two search engines, the oldest, SEQUEST, and the most popular, Mascot. This shotgun proteomics experiment also utilizes the power of parallel digestions with orthogonal proteases of increasing selectivity, namely chymotrypsin, trypsin/Lys-C and Asp-N. Our results show that the larger the database the greater the list of accessions identified but the longer the duration of the search. Using orthogonal proteases and different search algorithms increases the total number of proteins identified, most of them common despite differing proteases and algorithms, but many of them unique as well.

ACS Style

Delphine Vincent; Keith Savin; Simone Rochfort; German Spangenberg. The Power of Three in Cannabis Shotgun Proteomics: Proteases, Databases and Search Engines. Proteomes 2020, 8, 13 .

AMA Style

Delphine Vincent, Keith Savin, Simone Rochfort, German Spangenberg. The Power of Three in Cannabis Shotgun Proteomics: Proteases, Databases and Search Engines. Proteomes. 2020; 8 (2):13.

Chicago/Turabian Style

Delphine Vincent; Keith Savin; Simone Rochfort; German Spangenberg. 2020. "The Power of Three in Cannabis Shotgun Proteomics: Proteases, Databases and Search Engines." Proteomes 8, no. 2: 13.

Journal article
Published: 15 June 2020 in Metabolites
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Disorders of energy metabolism, which can result from a failure to adapt to the period of negative energy balance immediately after calving, have significant negative effects on the health, welfare and profitability of dairy cows. The most common biomarkers of energy balance in dairy cows are β-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA). While elevated concentrations of these biomarkers are associated with similar negative health and production outcomes, the phenotypic and genetic correlations between them are weak. In this study, we used an untargeted 1H NMR metabolomics approach to investigate the serum metabolomic fingerprints of BHBA and NEFA. Serum samples were collected from 298 cows in early lactation (calibration dataset N = 248, validation N = 50). Metabolomic fingerprinting was done by regressing 1H NMR spectra against BHBA and NEFA concentrations (determined using colorimetric assays) using orthogonal partial least squares regression. Prediction accuracies were high for BHBA models, and moderately high for NEFA models (R2 of external validation of 0.88 and 0.75, respectively). We identified 16 metabolites that were significantly (variable importance of projection score > 1) correlated with the concentration of one or both biomarkers. These metabolites were primarily intermediates of energy, phospholipid, and/or methyl donor metabolism. Of the significant metabolites identified; (1) two (acetate and creatine) were positively correlated with BHBA but negatively correlated with NEFA, (2) nine had similar associations with both BHBA and NEFA, (3) two were correlated with only BHBA concentration, and (4) three were only correlated with NEFA concentration. Overall, our results suggest that BHBA and NEFA are indicative of similar metabolic states in clinically healthy animals, but that several significant metabolic differences exist that help to explain the weak correlations between them. We also identified several metabolites that may be useful intermediate phenotypes in genomic selection for improved metabolic health.

ACS Style

Timothy D. W. Luke; Jennie E. Pryce; William J. Wales; Simone J. Rochfort. A Tale of Two Biomarkers: Untargeted 1H NMR Metabolomic Fingerprinting of BHBA and NEFA in Early Lactation Dairy Cows. Metabolites 2020, 10, 1 .

AMA Style

Timothy D. W. Luke, Jennie E. Pryce, William J. Wales, Simone J. Rochfort. A Tale of Two Biomarkers: Untargeted 1H NMR Metabolomic Fingerprinting of BHBA and NEFA in Early Lactation Dairy Cows. Metabolites. 2020; 10 (6):1.

Chicago/Turabian Style

Timothy D. W. Luke; Jennie E. Pryce; William J. Wales; Simone J. Rochfort. 2020. "A Tale of Two Biomarkers: Untargeted 1H NMR Metabolomic Fingerprinting of BHBA and NEFA in Early Lactation Dairy Cows." Metabolites 10, no. 6: 1.

Journal article
Published: 04 June 2020 in Scientific Reports
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Carbon dioxide supercritical fluid extraction (CO2 SFE) is a clean and cost-effective method of extracting cannabinoids from cannabis. Using design of experiment methodologies an optimised protocol for extraction of medicinal cannabis bud material (population of mixed plants, combined THC:CBD approximately 1:1.5) was developed at a scale of one kg per extraction. Key variables investigated were CO2 flow rate, extraction time and extraction pressure. A total of 15 batches were analysed for process development using a two-level, full factorial design of experiments for three variable factors over eleven batches. The initial eleven batches demonstrated that CO2 flow rate has the most influence on the overall yield and recovery of the key cannabinoids, particularly CBD. The additional four batches were conducted as replicated runs at high flow rates to determine reproducibility. The highest extraction weight of 71 g (7.1%) was obtained under high flow rate (150 g/min), with long extraction time (600 min) at high pressure (320 bar). This method also gave the best recoveries of THC and CBD. This is the first study to report the repeated extraction of large amounts of cannabis (total 15 kg) to optimise the CO2 SFE extraction process for a pharmaceutical product.

ACS Style

Simone Rochfort; Ashley Isbel; Vilnis Ezernieks; Aaron Elkins; Delphine Vincent; Myrna A. Deseo; German C. Spangenberg. Utilisation of Design of Experiments Approach to Optimise Supercritical Fluid Extraction of Medicinal Cannabis. Scientific Reports 2020, 10, 9124 .

AMA Style

Simone Rochfort, Ashley Isbel, Vilnis Ezernieks, Aaron Elkins, Delphine Vincent, Myrna A. Deseo, German C. Spangenberg. Utilisation of Design of Experiments Approach to Optimise Supercritical Fluid Extraction of Medicinal Cannabis. Scientific Reports. 2020; 10 (1):9124.

Chicago/Turabian Style

Simone Rochfort; Ashley Isbel; Vilnis Ezernieks; Aaron Elkins; Delphine Vincent; Myrna A. Deseo; German C. Spangenberg. 2020. "Utilisation of Design of Experiments Approach to Optimise Supercritical Fluid Extraction of Medicinal Cannabis." Scientific Reports 10, no. 1: 9124.

Journal article
Published: 30 April 2020 in Metabolites
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Most livestock metabolomic studies involve relatively small, homogenous populations of animals. However, livestock farming systems are non-homogenous, and large and more diverse datasets are required to ensure that biomarkers are robust. The aims of this study were therefore to (1) investigate the feasibility of using a large and diverse dataset for untargeted proton nuclear magnetic resonance (1H NMR) serum metabolomic profiling, and (2) investigate the impact of fixed effects (farm of origin, parity and stage of lactation) on the serum metabolome of early-lactation dairy cows. First, we used multiple linear regression to correct a large spectral dataset (707 cows from 13 farms) for fixed effects prior to multivariate statistical analysis with principal component analysis (PCA). Results showed that farm of origin accounted for up to 57% of overall spectral variation, and nearly 80% of variation for some individual metabolite concentrations. Parity and week of lactation had much smaller effects on both the spectra as a whole and individual metabolites (

ACS Style

Timothy D. W. Luke; Jennie E. Pryce; Aaron C. Elkins; William J. Wales; Simone J. Rochfort. Use of Large and Diverse Datasets for 1H NMR Serum Metabolic Profiling of Early Lactation Dairy Cows. Metabolites 2020, 10, 180 .

AMA Style

Timothy D. W. Luke, Jennie E. Pryce, Aaron C. Elkins, William J. Wales, Simone J. Rochfort. Use of Large and Diverse Datasets for 1H NMR Serum Metabolic Profiling of Early Lactation Dairy Cows. Metabolites. 2020; 10 (5):180.

Chicago/Turabian Style

Timothy D. W. Luke; Jennie E. Pryce; Aaron C. Elkins; William J. Wales; Simone J. Rochfort. 2020. "Use of Large and Diverse Datasets for 1H NMR Serum Metabolic Profiling of Early Lactation Dairy Cows." Metabolites 10, no. 5: 180.

Journal article
Published: 21 February 2020 in Scientific Reports
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Cheese microbiota and metabolites and their inter-relationships that underpin specific cheese quality attributes remain poorly understood. Here we report that multi-omics and integrative data analysis (multiple co-inertia analysis, MCIA) can be used to gain deeper insights into these relationships and identify microbiota and metabolite fingerprints that could be used to monitor product quality and authenticity. Our study into different brands of artisanal and industrial cheddar cheeses showed that Streptococcus, Lactococcus and Lactobacillus were the dominant taxa with overall microbial community structures differing not only between industrial and artisanal cheeses but also among different cheese brands. Metabolome analysis also revealed qualitative and semi-quantitative differences in metabolites between different cheeses. This also included the presence of two compounds (3-hydroxy propanoic acid and O-methoxycatechol-O-sulphate) in artisanal cheese that have not been previously reported in any type of cheese. Integrative analysis of multi-omics datasets revealed that highly similar cheeses, identical in age and appearance, could be distinctively clustered according to cheese type and brand. Furthermore, the analysis detected strong relationships, some previously unknown, which existed between the cheese microbiota and metabolome, and uncovered specific taxa and metabolites that contributed to these relationships. These results highlight the potential of this approach for identifying product specific microbe/metabolite signatures that could be used to monitor and control cheese quality and product authenticity.

ACS Style

Roya Afshari; Christopher J. Pillidge; Elizabeth Read; Simone Rochfort; Daniel A. Dias; A. Mark Osborn; Harsharn Gill. New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data. Scientific Reports 2020, 10, 1 -13.

AMA Style

Roya Afshari, Christopher J. Pillidge, Elizabeth Read, Simone Rochfort, Daniel A. Dias, A. Mark Osborn, Harsharn Gill. New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data. Scientific Reports. 2020; 10 (1):1-13.

Chicago/Turabian Style

Roya Afshari; Christopher J. Pillidge; Elizabeth Read; Simone Rochfort; Daniel A. Dias; A. Mark Osborn; Harsharn Gill. 2020. "New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data." Scientific Reports 10, no. 1: 1-13.

Journal article
Published: 16 January 2020 in Molecules
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Lolitrem B is the most potent indole-diterpene mycotoxin produced by Epichloë festucae var. lolii (termed LpTG-1), with severe intoxication cases reported in livestock. To date, there are no in vivo metabolism studies conducted for the mycotoxin. A mouse model assay established for assessing toxicity of indole-diterpenes was used to investigate metabolic products of lolitrem B. Mice were administered lolitrem B at 0.5 and 2.0 mg/kg body weight (b.wt) intraperitoneally before body and brain tissues were collected at 6 h and 24 h post-treatment. Samples were cryoground and subjected to a biphasic or monophasic extraction. The aqueous and lipophilic phases were analysed using liquid chromatography high-resolution mass spectrometry (LC–HRMS); data analysis was performed with Compound Discoverer™ software. A total of 10 novel phase I metabolic products were identified in the lipophilic phase and their distribution in the liver, kidney and various brain regions are described. The biotransformation products of lolitrem B were found to be present in low levels in the brain. Based on structure–activity postulations, six of these may contribute towards the protracted tremors exhibited by lolitrem B-exposed animals.

ACS Style

Priyanka Reddy; Aaron Elkins; Joanne Hemsworth; Kathryn Guthridge; Simone Vassiliadis; Elizabeth Read; German Spangenberg; Simone Rochfort. Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry. Molecules 2020, 25, 372 .

AMA Style

Priyanka Reddy, Aaron Elkins, Joanne Hemsworth, Kathryn Guthridge, Simone Vassiliadis, Elizabeth Read, German Spangenberg, Simone Rochfort. Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry. Molecules. 2020; 25 (2):372.

Chicago/Turabian Style

Priyanka Reddy; Aaron Elkins; Joanne Hemsworth; Kathryn Guthridge; Simone Vassiliadis; Elizabeth Read; German Spangenberg; Simone Rochfort. 2020. "Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry." Molecules 25, no. 2: 372.

Journal article
Published: 11 January 2020 in Food Chemistry
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The antioxidant activity of sugarcane molasses ethanol extract (ME) and its fraction (ME-RBF) was evaluated using ABTS, ORAC 6.0 and CAA assays and ME-RBF demonstrated 26-fold, 12-fold and 2-fold higher values, respectively than ME. Likewise, total polyphenol and flavonoid concentration in ME-RBF are more than 10-fold higher than ME, that suggested antioxidant activity is correlated with polyphenol composition. Quantitative analysis of 13 polyphenols (chlorogenic acid, caffeic acid, sinapic acid, syringic acid, vanillin, homoorientin, orientin, vitexin, swertisin, diosmin, apigenin, tricin and diosmetin) was carried out by LCMS. MS/MS analysis allowed the tentative identification of seven apigenin-C-glycosides, three methoxyluteolin-C-glycosides and three tricin-O-glycosides some of which have not been reported in sugarcane before to the best of our knowledge. The results demonstrated that sugarcane molasses can be used as potential source of polyphenols that can be beneficial to health.

ACS Style

Myrna A. Deseo; Aaron Elkins; Simone Rochfort; Barry Kitchen. Antioxidant activity and polyphenol composition of sugarcane molasses extract. Food Chemistry 2020, 314, 126180 .

AMA Style

Myrna A. Deseo, Aaron Elkins, Simone Rochfort, Barry Kitchen. Antioxidant activity and polyphenol composition of sugarcane molasses extract. Food Chemistry. 2020; 314 ():126180.

Chicago/Turabian Style

Myrna A. Deseo; Aaron Elkins; Simone Rochfort; Barry Kitchen. 2020. "Antioxidant activity and polyphenol composition of sugarcane molasses extract." Food Chemistry 314, no. : 126180.

Validation study
Published: 01 December 2019 in Journal of Dairy Science
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In this study, we estimated genetic parameters and genomic prediction accuracies of serum biomarkers of health in early-lactation dairy cows. A single serum sample was taken from 1,393 cows, located on 14 farms in southeastern Australia, within 30 d after calving. Sera were analyzed for biomarkers of energy balance (β-hydroxybutyrate and fatty acids), macromineral status (Ca and Mg), protein nutritional status (urea and albumin), and immune status (globulins, albumin-to-globulin ratio, and haptoglobin). After editing, 47,162 SNP marker genotypes were used to estimate genomic heritabilities and breeding values (GEBV) for these traits in ASReml. Heritabilities were low for β-hydroxybutyrate, fatty acids, Ca, Mg, and urea (0.09 ± 0.04, 0.18 ± 0.05, 0.07 ± 0.04, 0.19 ± 0.06, and 0.18 ± 0.05, respectively), and moderate for albumin, globulins, and albumin-to-globulin ratio (0.27 ± 0.06, 0.46 ± 0.06, and 0.41 ± 0.06, respectively). The heritability of haptoglobin concentration was close to 0. The magnitude of genetic correlations between traits (estimated using bivariate models) varied considerably (0.01 to 0.96), and standard errors of these correlations were high (0.02 to 0.44). Interestingly, the direction of most genetic correlations was favorable, suggesting that selecting for more optimal concentrations of one biomarker may result in more optimal concentrations of other biomarkers. Correlations between biomarker GEBV and existing breeding values for survival, somatic cell count, and daughter fertility were small to moderate (0.07 to 0.45) and favorable, whereas correlations with breeding values for milk production traits were small (≤0.15). Accuracies of GEBV were evaluated by using 5-fold cross validation, and by calculating accuracies from prediction error variances associated with the GEBV. Accuracies of GEBV predicted using 5-fold cross validation were low (0.05 to 0.27), whereas the means of individual accuracies were greater, ranging from 0.31 to 0.51. Although increasing the size of the reference population should theoretically improve accuracies, our results suggest that genomic prediction of health biomarkers may allow identification of cows that are less susceptible to diseases in early lactation.

ACS Style

T.D.W. Luke; T.T.T. Nguyen; S. Rochfort; W.J. Wales; C.M. Richardson; M. Abdelsayed; J.E. Pryce. Genomic prediction of serum biomarkers of health in early lactation. Journal of Dairy Science 2019, 102, 11142 -11152.

AMA Style

T.D.W. Luke, T.T.T. Nguyen, S. Rochfort, W.J. Wales, C.M. Richardson, M. Abdelsayed, J.E. Pryce. Genomic prediction of serum biomarkers of health in early lactation. Journal of Dairy Science. 2019; 102 (12):11142-11152.

Chicago/Turabian Style

T.D.W. Luke; T.T.T. Nguyen; S. Rochfort; W.J. Wales; C.M. Richardson; M. Abdelsayed; J.E. Pryce. 2019. "Genomic prediction of serum biomarkers of health in early lactation." Journal of Dairy Science 102, no. 12: 11142-11152.

Journal article
Published: 13 November 2019 in Microorganisms
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Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I–IV in Epichloë endophytes is proposed.

ACS Style

Emma J. Ludlow; Simone Vassiliadis; Piyumi N. Ekanayake; Inoka K. Hettiarachchige; Priyanka Reddy; Tim I. Sawbridge; Simone J. Rochfort; German C. Spangenberg; Kathryn M. Guthridge. Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes. Microorganisms 2019, 7, 560 .

AMA Style

Emma J. Ludlow, Simone Vassiliadis, Piyumi N. Ekanayake, Inoka K. Hettiarachchige, Priyanka Reddy, Tim I. Sawbridge, Simone J. Rochfort, German C. Spangenberg, Kathryn M. Guthridge. Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes. Microorganisms. 2019; 7 (11):560.

Chicago/Turabian Style

Emma J. Ludlow; Simone Vassiliadis; Piyumi N. Ekanayake; Inoka K. Hettiarachchige; Priyanka Reddy; Tim I. Sawbridge; Simone J. Rochfort; German C. Spangenberg; Kathryn M. Guthridge. 2019. "Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes." Microorganisms 7, no. 11: 560.

Journal article
Published: 11 November 2019 in International Journal of Molecular Sciences
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Earlier this year we published a method article aimed at optimising protein extraction from mature buds of medicinal cannabis for trypsin-based shotgun proteomics (Vincent, D., et al. Molecules 2019, 24, 659). We then developed a top-down proteomics (TDP) method (Vincent, D., et al. Proteomes 2019, 7, 33). This follow-up study aims at optimising the digestion of medicinal cannabis proteins for identification purposes by bottom-up and middle-down proteomics (BUP and MDP). Four proteases, namely a mixture of trypsin/LysC, GluC, and chymotrypsin, which target different amino acids (AAs) and therefore are orthogonal and cleave proteins more or less frequently, were tested both on their own as well as sequentially or pooled, followed by nLC-MS/MS analyses of the peptide digests. Bovine serum albumin (BSA, 66 kDa) was used as a control of digestion efficiency. With this multiple protease strategy, BSA was reproducibly 97% sequenced, with peptides ranging from 0.7 to 6.4 kD containing 5 to 54 AA residues with 0 to 6 miscleavages. The proteome of mature apical buds from medicinal cannabis was explored more in depth with the identification of 27,123 peptides matching 494 unique accessions corresponding to 229 unique proteins from Cannabis sativa and close relatives, including 130 (57%) additional annotations when the list is compared to that of our previous BUP study (Vincent, D., et al. Molecules 2019, 24, 659). Almost half of the medicinal cannabis proteins were identified with 100% sequence coverage, with peptides composed of 7 to 91 AA residues with up to 9 miscleavages and ranging from 0.6 to 10 kDa, thus falling into the MDP domain. Many post-translational modifications (PTMs) were identified, such as oxidation, phosphorylations, and N-terminus acetylations. This method will pave the way for deeper proteome exploration of the reproductive organs of medicinal cannabis, and therefore for molecular phenotyping within breeding programs.

ACS Style

Delphine Vincent; Vilnis Ezernieks; Simone Rochfort; German Spangenberg. A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds. International Journal of Molecular Sciences 2019, 20, 5630 .

AMA Style

Delphine Vincent, Vilnis Ezernieks, Simone Rochfort, German Spangenberg. A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds. International Journal of Molecular Sciences. 2019; 20 (22):5630.

Chicago/Turabian Style

Delphine Vincent; Vilnis Ezernieks; Simone Rochfort; German Spangenberg. 2019. "A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds." International Journal of Molecular Sciences 20, no. 22: 5630.

Journal article
Published: 07 November 2019 in Toxins
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The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography–mass spectrometry (LC–MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC–MS (for peramine). This work provides the first highly sensitive quantitative LC–MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

ACS Style

Simone Vassiliadis; Aaron C. Elkins; Priyanka Reddy; Kathryn M. Guthridge; German C. Spangenberg; Simone J. Rochfort. A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass. Toxins 2019, 11, 649 .

AMA Style

Simone Vassiliadis, Aaron C. Elkins, Priyanka Reddy, Kathryn M. Guthridge, German C. Spangenberg, Simone J. Rochfort. A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass. Toxins. 2019; 11 (11):649.

Chicago/Turabian Style

Simone Vassiliadis; Aaron C. Elkins; Priyanka Reddy; Kathryn M. Guthridge; German C. Spangenberg; Simone J. Rochfort. 2019. "A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass." Toxins 11, no. 11: 649.

Journal article
Published: 24 September 2019 in Proteomes
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The revised legislation on medicinal cannabis has triggered a surge of research studies in this space. Yet, cannabis proteomics is lagging. In a previous study, we optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we developed a top-down mass spectrometry (MS) proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different source-induced dissociation (SID), collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) parameters on infused known protein standards, we devised three LC-MS/MS methods for top-down sequencing of cannabis proteins. Different MS/MS modes produced distinct spectra, albeit greatly overlapping between SID, CID, and HCD. The number of fragments increased with the energy applied; however, this did not necessarily translate into greater sequence coverage. Some precursors were more amenable to fragmentation than others. Sequence coverage decreased as the mass of the protein increased. Combining all MS/MS data maximised amino acid (AA) sequence coverage, achieving 73% for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. A total of 46 cannabis proteins were identified with 136 proteoforms bearing different post-translational modifications (PTMs), including the excision of N-terminal M, the N-terminal acetylation, methylation, and acetylation of K resides, and phosphorylation. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase.

ACS Style

Delphine Vincent; Steve Binos; Simone Rochfort; German Spangenberg. Top-Down Proteomics of Medicinal Cannabis. Proteomes 2019, 7, 33 .

AMA Style

Delphine Vincent, Steve Binos, Simone Rochfort, German Spangenberg. Top-Down Proteomics of Medicinal Cannabis. Proteomes. 2019; 7 (4):33.

Chicago/Turabian Style

Delphine Vincent; Steve Binos; Simone Rochfort; German Spangenberg. 2019. "Top-Down Proteomics of Medicinal Cannabis." Proteomes 7, no. 4: 33.

Journal article
Published: 13 August 2019 in Metabolites
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Lentils are a high-protein plant food and a valuable source of human nutrition, particularly in the Indian subcontinent. However, beyond sustenance, there is evidence that the consumption of lentils (and legumes in general) is associated with decreased risk of diseases, such as diabetes and cardiovascular disease. Lentils contain health-promoting phytochemicals, such as trigonelline and various polyphenolics. Fourteen lentil genotypes were grown at three locations to explore the variation in phytochemical composition in hulls and cotyledons. Significant differences were measured between genotypes and environments, with some genotypes more affected by environment than others. However, there was a strong genetic effect which indicated that future breeding programs could breed for lentils that product more of these health-promoting phytochemicals.

ACS Style

Simone Rochfort; Simone Vassiliadis; Pankaj Maharjan; Jason Brand; Joe Panozzo. NMR Based Metabolomic Analysis of Health Promoting Phytochemicals in Lentils. Metabolites 2019, 9, 168 .

AMA Style

Simone Rochfort, Simone Vassiliadis, Pankaj Maharjan, Jason Brand, Joe Panozzo. NMR Based Metabolomic Analysis of Health Promoting Phytochemicals in Lentils. Metabolites. 2019; 9 (8):168.

Chicago/Turabian Style

Simone Rochfort; Simone Vassiliadis; Pankaj Maharjan; Jason Brand; Joe Panozzo. 2019. "NMR Based Metabolomic Analysis of Health Promoting Phytochemicals in Lentils." Metabolites 9, no. 8: 168.

Journal article
Published: 27 June 2019 in Scientific Reports
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ACS Style

Priyanka Reddy; Simone Rochfort; Elizabeth Read; Myrna Deseo; Emily Jaehne; Maarten Van Den Buuse; Kathryn Guthridge; Martin Combs; German Spangenberg; Jane Quinn. Tremorgenic effects and functional metabolomics analysis of lolitrem B and its biosynthetic intermediates. Scientific Reports 2019, 9, 1 .

AMA Style

Priyanka Reddy, Simone Rochfort, Elizabeth Read, Myrna Deseo, Emily Jaehne, Maarten Van Den Buuse, Kathryn Guthridge, Martin Combs, German Spangenberg, Jane Quinn. Tremorgenic effects and functional metabolomics analysis of lolitrem B and its biosynthetic intermediates. Scientific Reports. 2019; 9 (1):1.

Chicago/Turabian Style

Priyanka Reddy; Simone Rochfort; Elizabeth Read; Myrna Deseo; Emily Jaehne; Maarten Van Den Buuse; Kathryn Guthridge; Martin Combs; German Spangenberg; Jane Quinn. 2019. "Tremorgenic effects and functional metabolomics analysis of lolitrem B and its biosynthetic intermediates." Scientific Reports 9, no. 1: 1.

Review
Published: 27 May 2019 in Toxins
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Indole-diterpenes are an important class of chemical compounds which can be unique to different fungal species. The highly complex lolitrem compounds are confined to Epichloë species, whilst penitrem production is confined to Penicillium spp. and Aspergillus spp. These fungal species are often present in association with pasture grasses, and the indole-diterpenes produced may cause toxicity in grazing animals. In this review, we highlight the unique structural variations of indole-diterpenes that are characterised into subgroups, including paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines. A detailed description of the unique biological activities has been documented where even structurally related compounds have displayed unique biological activities. Indole-diterpene production has been reported in two classes of ascomycete fungi, namely Eurotiomycetes (e.g., Aspergillus and Penicillium) and Sordariomycetes (e.g., Claviceps and Epichloë). These compounds all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from tryptophan. Structure diversity is generated from the enzymatic conversion of different sites on the basic indole-diterpene structure. This review highlights the wide-ranging biological versatility presented by the indole-diterpene group of compounds and their role in an agricultural and pharmaceutical setting.

ACS Style

Priyanka Reddy; Kathryn Guthridge; Simone Vassiliadis; Joanne Hemsworth; Inoka Hettiarachchige; German Spangenberg; Simone Rochfort. Tremorgenic Mycotoxins: Structure Diversity and Biological Activity. Toxins 2019, 11, 302 .

AMA Style

Priyanka Reddy, Kathryn Guthridge, Simone Vassiliadis, Joanne Hemsworth, Inoka Hettiarachchige, German Spangenberg, Simone Rochfort. Tremorgenic Mycotoxins: Structure Diversity and Biological Activity. Toxins. 2019; 11 (5):302.

Chicago/Turabian Style

Priyanka Reddy; Kathryn Guthridge; Simone Vassiliadis; Joanne Hemsworth; Inoka Hettiarachchige; German Spangenberg; Simone Rochfort. 2019. "Tremorgenic Mycotoxins: Structure Diversity and Biological Activity." Toxins 11, no. 5: 302.