ORCID Profile
0000-0002-5072-378X
Current Organisations
UNSW Sydney
,
Drop Bio Pty Ltd
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Publisher: Springer Science and Business Media LLC
Date: 10-11-2017
Publisher: Elsevier BV
Date: 04-2007
Publisher: Oxford University Press (OUP)
Date: 10-2003
Publisher: Elsevier BV
Date: 11-2004
Publisher: Springer Science and Business Media LLC
Date: 07-08-2017
Publisher: Wiley
Date: 28-07-2014
Abstract: Secretomic analysis requires removal of serum proteins from cell-culture media. We evaluate the proteins washed from cells prepared in bovine serum-supplemented medium. PBS and serum-free-medium (SFM) were the washing solutions. A Bradford assay was used for total protein concentration and a 1D gel and LC-MS/MS, to assign the protein to human or bovine origin. For both wash solutions, all bovine protein had been removed by the third wash, without compromising the number of living cells. Further washes reduced the number of living cells, especially when using PBS. Proteomic analysis of wash supernatant showed that SFM induced greater lysis of dead cells. Three washes were sufficient to minimize the effects on cell viability, while still removing serum proteins. Washing in SFM resulted in contamination of the wash supernatant with lysed dead cell proteins. Washed cells were incubated in SFM and exposed to ionizing radiation. Analysis of the supernatant showed an increase in human cytoplasmic, plasma membrane, and nuclear protein following irradiation. Secreted proteins were also detected, but in smaller quantities. The significance of these findings extend to in vitro studies of bystander phenomena, since the proteins of lysed dead cells may participate in driving bystander responses.
Publisher: Elsevier BV
Date: 12-2003
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.YMBEN.2018.08.006
Abstract: Metabolic engineering has been vital to the development of industrial microbes such as the yeast Saccharomyces cerevisiae. However, sequential rounds of modification are often needed to achieve particular industrial design targets. Systems biology approaches can aid in identifying genetic targets for modification through providing an integrated view of cellular physiology. Recently, research into the generation of commercial yeasts that can produce reduced-ethanol wines has resulted in metabolically-engineered strains of S. cerevisiae that are less efficient at producing ethanol from sugar. However, these modifications led to the concomitant production of off-flavour by-products. A combination of transcriptomics, proteomics and metabolomics was therefore used to investigate the physiological changes occurring in an engineered low-ethanol yeast strain during alcoholic fermentation. Integration of 'omics data identified several metabolic reactions, including those related to the pyruvate node and redox homeostasis, as being significantly affected by the low-ethanol engineering methodology, and highlighted acetaldehyde and 2,4,5-trimethyl-1,3-dioxolane as the main off-flavour compounds. Gene remediation strategies were then successfully applied to decrease the formation of these by-products, while maintaining the 'low-alcohol' phenotype. The data generated from this comprehensive systems-based study will inform wine yeast strain development programmes, which, in turn, could potentially play an important role in assisting winemakers in their endeavour to produce low-alcohol wines with desirable flavour profiles.
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.JPROT.2012.05.018
Abstract: Cytokines, chemokines, growth factors (CCGFs) and other low abundance proteins eptides in human body fluids or in tissues are potential biomarkers. Human body fluids such as plasma, saliva, urine, etc. are being analyzed more frequently than tissues primarily because of ease of s le collection. However, available information on concentrations of a large number of CCGFs in various body fluids of the same healthy in iduals and gender-specific CCGFs is limited. In this work concentrations of 48 CCGFs were measured using multiplex bead assays and compared between plasma, saliva and urine collected from 20 male and female healthy volunteers. Forty three CCGFs were detected at least in one s le type of which 37 were in plasma, 41 were in saliva, and 34 were in urine five CCGFs were not detected in any s le. Concentrations of detected CCGFs differed significantly between s le types but similar between gender groups. Gender-specific CCGFs were also observed. Concentrations of nine acute phase proteins were also measured from plasma, saliva and urine to determine general health conditions of the volunteers. This work will provide an idea of which CCGFs are detectable and their relative concentrations in healthy human plasma, saliva and urine and which CCGFs are gender-specific.
Publisher: American Chemical Society (ACS)
Date: 06-09-2006
DOI: 10.1021/PR060305Y
Abstract: Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal s le preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.
Publisher: Springer Science and Business Media LLC
Date: 11-07-2008
Publisher: Elsevier BV
Date: 02-2015
DOI: 10.1016/J.CYTO.2014.10.030
Abstract: Within the scientific literature, analyses of data from bead based multiplex immunoassays are based on either median fluorescence intensities (MFI) or derived absolute concentration values (ACV) but no consideration of which set of data is the most appropriate for analysis has been published. Here we look at the variance of MFI versus their ACV from the expression of 14 analytes in plasma, using 6 commercially available kits, across 177 patients, recorded at two time points and the associated analyte standards. In total 60 micro titre plates were used resulting in 4965 MFI. In doing so we develop a new background subtraction procedure that reduced by 50% the number of out-of-range values observed in our data set. Using a linear mixed-effect model, which normalizes for assay-to-assay variation, MFI produced similar significant differences than that observed using absolute concentration values. We show that subtracting analyte blanks produces 15% negative MFI resulting in uncertainty of the data being analysed. We argue for analysis of protein expression values MFI are generally a better choice than absolute concentration values. It is argued that analyte standards are not required on each plate, or not at all, in multi-plate experiments, but knowledge of the concentration curve and the range of MFI values that fall within the limits of this curve for each analyte is required. The significance of using MFI over concentration values for the life scientist means higher statistical power and lower costs.
Publisher: Springer Science and Business Media LLC
Date: 06-2013
Publisher: Springer Science and Business Media LLC
Date: 31-05-2016
DOI: 10.1038/SREP26996
Abstract: Tissue s les (plasma, saliva, serum or urine) from 169 patients classified as either normal or having one of seven possible diseases are analysed across three 96-well plates for the presences of 37 analytes using cytokine inflammation multiplexed immunoassay panels. Censoring for concentration data caused problems for analysis of the low abundant analytes. Using fluorescence analysis over concentration based analysis allowed analysis of these low abundant analytes. Mixed-effects analysis on the resulting fluorescence and concentration responses reveals a combination of censoring and mapping the fluorescence responses to concentration values, through a 5PL curve, changed observed analyte concentrations. Simulation verifies this, by showing a dependence on the mean florescence response and its distribution on the observed analyte concentration levels. Differences from normality, in the fluorescence responses, can lead to differences in concentration estimates and unreliable probabilities for treatment effects. It is seen that when fluorescence responses are normally distributed, probabilities of treatment effects for fluorescence based t-tests has greater statistical power than the same probabilities from concentration based t-tests . We add evidence that the fluorescence response, unlike concentration values, doesn’t require censoring and we show with respect to differential analysis on the fluorescence responses that background correction is not required.
Publisher: Wiley
Date: 2010
Abstract: Affinity depletion of abundant proteins from human plasma has become a routine s le preparation strategy in proteomics used prior to protein identification and quantitation. To date, there have been limited published studies comparing the performance of commercially available depletion products. We conducted a thorough evaluation of six depletion columns using 2-DE combined with sophisticated image analysis software, examining the following criteria: (i) efficiency of high-abundance protein depletion, (ii) non-specific removal of other than the targeted proteins and (iii) total number of protein spots detected on the gels following depletion. From all the products investigated, the Seppro IgY system provided the best results. It displayed the greatest number of protein spots on the depleted plasma gels, minimal non-specific binding and high efficiency of abundant protein removal. Nevertheless, the increase in the number of detected spots compared with the second best performing and cheaper multiple affinity removal column (MARC) was not shown to be statistically significant. The ProteoPrep spin column, considered to be the "deepest" depletion technique available at the time of conducting the study, surprisingly displayed significantly fewer spots on the flow-through fraction gels compared with both the Seppro and the MARC. The spin column format and low plasma capacity were also found to be impractical for 2-DE. To conclude, we succeeded in providing an overview of the depletion columns performances with regard to the three examined areas. Our study will serve as a reference to other scientists when deciding on the optimal product for their particular projects.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2006
Publisher: Springer Science and Business Media LLC
Date: 16-08-2006
Publisher: Springer Science and Business Media LLC
Date: 30-08-2017
DOI: 10.1038/S41598-017-09968-7
Abstract: Human growth hormone (GH) is a naturally occurring hormone secreted by the pituitary gland with anabolic and growth-promoting activities. Since an increased availability of recombinant GH (rGH) for the treatment of GH-deficient patients, GH has been abused in sports and it is prohibited. “GH-isoform” and “biomarkers” tests are currently available for detection of GH abuse in sports, however both methods suffer from shortcomings. Here, we report on a proteomic approach to search for novel protein biomarkers associated with rGH administration in non-elite athletes. In this study, participants received either placebo or rGH for 8 weeks, and were followed over a 6-week washout period. We used 2-D DIGE and iTRAQ LC-MS/MS analyses to expose rGH-dependent marker proteins. Eight rGH-dependent plasma proteins namely apolipoproptein-L1, alpha-HS-glycoprotein, vitamin D-binding protein, afamin, insulin-like growth factor-binding protein-3, insulin-like growth factor-binding protein-ALS, lumican and extracellular matrix proteins 1 were identified. Apolipoprotein L1 and alpha-HS-glycoprotein were validated by Western blots to confirm their identities and expression patterns in rGH- and placebo-treated subject cohorts. Independent confirmation of these putative GH-responsive biomarkers would be of value for clinical practices and may have sports anti-doping utility.
Publisher: Elsevier BV
Date: 08-2005
DOI: 10.1016/J.CCCN.2005.03.013
Abstract: We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection. The 2DE method separates HuEPO and rHuEPO isoforms by both iso-electric point and molecular mass. We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO however, they separate distinctly by the 2DE method. Alpha-2-HS-glycoprotein (HSGP) was identified by peptide mass fingerprinting as one of the urinary cross-reacting proteins, and commercially available purified HSGP was chosen to be added into urine s les as an internal standard prior to separation. Software (EpIQ) was specifically developed that applies four separate criteria to the detection of the migration of rHuEPO and HuEPO relative to the internal standard. The combination of s le preparation, two-dimensional separation, internal standard, standardized blotting procedures and image analysis software enables the 2DE test for rHuEPO in urine to be performed reproducibly and accurately.
Publisher: Elsevier BV
Date: 2011
DOI: 10.1016/J.FERTNSTERT.2010.05.016
Abstract: To investigate whether proteins secreted in urine differ between women with and without endometriosis. Laboratory study using human urine. University-based laboratory. Women with and without endometriosis undergoing laparoscopy, hysteroscopy and curettage. Urine collection from women with and without endometriosis. Proteomic techniques and mass spectrometry to identify proteins secreted in the urine of women with and without endometriosis. On average, 133 proteins were significantly different between women with and without endometriosis. Cytokeratin-19 was highly up-regulated in the urine of women with endometriosis. Cytokeratin-19 may be a valuable urinary biomarker for endometriosis.
Publisher: Elsevier BV
Date: 12-2008
DOI: 10.1016/J.MYCRES.2008.06.003
Abstract: The soil-inhabiting, nematode-trapping fungus, Monacrosporium lysipagum, captures mobile stages of nematodes using specialized morphological structures, sticky knobs, that arise from mycelia. A study was conducted to separate the proteome of M. lysipagum mycelia containing knobs on two-dimensional (2D) gels resulting in a partial map of the proteome. The fungus was grown in a liquid soy peptone medium supplemented with the amino acids phenylalanine and valine to produce mycelia with knobs. Proteins extracted from the mycelia were separated by 2D gel electrophoresis and relatively high abundant proteins were identified by peptide mass fingerprinting (PMF). Out of the 250 proteins analysed by PMF, 51 (20%) were identified by cross-species matching due to unavailability of genomic information from M. lysipagum. This is the first published report on a proteomic analysis of a nematode-trapping fungus.
Publisher: Hindawi Limited
Date: 29-04-2021
DOI: 10.1155/2021/5535562
Abstract: Purpose. Sepsis originates from the host inflammatory response, especially to bacterial infections, and is considered one of the main causes of death in intensive care units. Various agents have been developed to inhibit mediators of the inflammatory response one prospective agent is β-sitosterol (βS), a phytosterol with a structure similar to cholesterol. This study is aimed at evaluating the effects of βS on the biomarkers of inflammation and liver function in cecal ligation and puncture- (CLP-) induced septic rats. Methods. Thirty male Wistar rats were ided equally into six groups as follows: sham, CLP, CLP+dexamethasone (DX, 0.2 mg/kg), CLP+βS (1 mg/kg), CLP+imipenem (IMI, 20 mg/kg), and CLP+IMI (20 mg/kg)+βS (1 mg/kg). Serum levels of IL-1β, IL-6, IL-10, AST, ALT, and liver glutathione (GSH) were assessed by ELISA. Liver expression levels of TNF-α and NF-κBi mRNAs were evaluated by RT-qPCR. Results. Serum concentrations of IL-1β, IL-6, IL-10, ALT, and AST and mRNA levels of TNF-α and NF-κBi were all significantly higher in septic rats than in normal rats ( p 0.05 ). Liver GSH content was markedly lower in the CLP group than that in the sham group. βS-treated rats had remarkably lower levels of IL-1β, IL-6, IL-10, TNF-α, NF-κBi, AST, and ALT (51.79%, 62.63%, 41.46%, 54.35%, 94.37%, 95.30%, 34.87%, and 46.53% lower, respectively) and greater liver GSH content (35.71% greater) compared to the CLP group ( p 0.05 ). Conclusion. βS may play a protective role in the septic process by mitigating inflammation. This effect is at least partly mediated by inhibition of the NF-κB signaling pathway. Thus, βS can be considered as a supplementary treatment in septic patients.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2008
DOI: 10.1007/S00134-008-1156-Y
Abstract: Critical illness is associated with tissue damage, inflammation and the development of immune dysfunction. Leukocyte reprogramming occurs leading to insufficient production of pro-inflammatory cytokines upon subsequent stimulation. Cellular nucleotides released during tissue damage act via purinergic receptors to modulate immune function. We hypothesized that extracellular nucleotides in concentrations similar to those found near injured and ischemic tissues will modulate cytokine secretion. Bench study in 28 healthy human volunteers using standardized lipopolysaccharide (LPS) stimulated ex vivo whole blood cultures (ILCS). The Nepean Hospital Laboratories, University of Sydney. Nucleotides ATP, ADP and other P2 purinergic receptor agonists ATPgammaS, BzATP, UTP and P1 agonist CV1808 were injected into the ILCS, and cultured for 6, 12 and 24 h as indicated. ATP (100 muM) reduced the LPS stimulated secretion of TNFalpha at 6 and 12 h, as well as IL-12(p70) and IFNgamma at 24 h of incubation. ADP, ATPgammaS, BzATP, and CV1808, but not UTP displayed IL-12(p70) and IFNgamma reducing effect similar to ATP. Higher ATP concentration (500 muM) had even more pronounced immunosuppressive effect. Nucleotides had variable effect on stimulated IL-10 secretion. ATP and ADP at high-micromolar concentrations reduce secretion of the main Th1 cytokines TNFalpha, IL-12(p70) and IFNgamma in LPS stimulated human blood. As immune dysfunction associated with critical illness is characterized by decreased TNFalpha, IL-12 and IFNgamma production by leukocytes, extracellular nucleotides might contribute to its pathogenesis [corrected]
Publisher: Elsevier BV
Date: 12-2009
DOI: 10.1016/J.MIMET.2009.10.009
Abstract: Secreted fungal proteins with mannanase activity were identified by mass spectrometry of bands excised from a Congo Red stained zymogram containing locust bean gum as substrate. This technique circumvents the need to locate corresponding bands on a parallel gel without substrate and provides good accuracy in targeting proteins for identification.
Publisher: Oxford University Press (OUP)
Date: 25-04-2017
Abstract: Yeast AP-1 transcription factor (Yap1p) and the enigmatic oxidoreductases Oye2p and Oye3p are involved in counteracting lipid oxidants and their unsaturated breakdown products. In order to uncover the response to linoleic acid hydroperoxide (LoaOOH) and the roles of Oye2p, Oye3p and Yap1p, we carried out proteomic analysis of the homozygous deletion mutants oye3Δ, oye2Δ and yap1Δ alongside the diploid parent strain BY4743. The findings demonstrate that deletion of YAP1 narrowed the response to LoaOOH, as the number of proteins differentially expressed in yap1Δ was 70% of that observed in BY4743. The role of Yap1p in regulating the major yeast peroxiredoxin Tsa1p was demonstrated by the decreased expression of Tsa1p in yap1Δ. The levels of Ahp1p and Hsp31p, previously shown to be regulated by Yap1p, were increased in LoaOOH-treated yap1Δ, indicating their expression is also regulated by another transcription factor(s). Relative to BY4743, protein expression differed in oye3Δ and oye2Δ under LoaOOH, underscored by superoxide dismutase (Sod1p), multiple heat shock proteins (Hsp60p, Ssa1p, and Sse1p), the flavodoxin-like protein Pst2p and the actin stabiliser tropomyosin (Tpm1p). Proteins associated with glycolysis were increased in all strains following treatment with LoaOOH. Together, the dataset reveals, for the first time, the yeast proteomic response to LoaOOH, highlighting the significance of carbohydrate metabolism, as well as distinction between the roles of Oye3p, Oye2p and Yap1p.
Publisher: Elsevier BV
Date: 06-2014
Publisher: Brill
Date: 1999
Abstract: The in vitro interaction of Paecilomyces lilacinus strain 251 with eggs, 3rd and 4th stage juveniles and adult females of Meloidogyne javanica was studied. Eggs of all stages, including those containing unhatched juveniles, were infected by P. lilacinus. Infection of eggs occurred following flattening of hyphae to the egg surface and formation of appressoria. Sometimes these occurred within extensive networks of hyphae of the egg surface. Hyphae later grew out of the egg to continue growing or form conidiophores. Third and 4th stage juveniles and adult females were readily infected, with hyphae and conidiophores penetrating the body wall. Die Infektion von Meloidogyne javanica durch Paecilomyces lilacinus - Es wurden die in vitro auftretenden Wechselwirkungen zwischen Paecilomyces lilacinus Stamm 251 und den Eiern, J3, J4 und adulten Weibchen von Meloidogyne javanica untersucht. Eier wurden in allen Stadien von P. lilacinus infiziert einschliesslich der ungeschlupfte J2 enthaltenden Eier. Die Infektion erfolgte anschliessend an eine Abflachung von Hyphen auf der Eioberflache und eine Appressorienbildung. Manchmal erschienen diese innerhalb eines ausgedehnten Netzwerkes von Hyphen auf der Eioberflache. Spater wuchsen Hyphen aus dem Ei heraus, wuchsen weiter oder bildeten Konidiophoren. Juvenile des dritten oder vierten Stadiums und adulte Weibchen wurden ohne weiteres befallen, wobei Hyphen und Konidiophoren durch die Korperwand drangen.
Publisher: Elsevier BV
Date: 11-2006
Publisher: Elsevier BV
Date: 03-2015
DOI: 10.1016/J.FOODCHEM.2014.07.044
Abstract: Wheat cv. H45 was grown under ambient CO2 concentration and Free Air CO2 Enrichment (FACE e[CO2], ∼550 μmol CO2 mol(-1)). The effect of FACE on wheat grain proteome and associated changes in the flour rheological properties was investigated. A comparative proteomic analysis was performed using 2-D-DIGE followed by MALDI/TOF-MS. Total grain protein concentration was decreased by 9% at e[CO2]. Relative abundance of three high molecular weight glutenin sub units (HMW-GS) were decreased at e[CO2]. In contrast, relative abundance of serpins Z1C and 1-Cys peroxiredoxin was increased at e[CO2]. Elevated [CO2] also decreased the bread volume (by 11%) and dough strength (by 7%) while increased mixing time. However, dough extensibility and dough stability were unchanged at elevated [CO2]. These findings suggest that e[CO2] has a major impact on gluten protein concentration which is associated lower bread quality at e[CO2].
Location: Australia
Start Date: 2011
End Date: 2011
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2009
End Date: 2010
Funder: Macquarie University
View Funded Activity