Bio21 - Research Publications
Now showing items 1-12 of 232
In-cell Solid-State NMR Studies of Antimicrobial Peptides
(Frontiers Media SA, 2020)
Antimicrobial peptides (AMPs) have attracted attention as alternatives to classic antibiotics due to their expected limited pressure on bacterial resistance mechanisms. Yet, their modes of action, in particular in vivo, remain to be elucidated. In situ atomistic-scale details of complex biomolecular assemblies is a challenging requirement for deciphering the complex modes of action of AMPs. The large diversity of molecules that modulate complex interactions limits the resolution achievable using imaging methodology. Herein, the latest advances in in-cell solid-state NMR (ssNMR) are discussed, which demonstrate the power of this non-invasive technique to provide atomic details of molecular structure and dynamics. Practical requirements for investigations of intact bacteria are discussed. An overview of recent in situ NMR investigations of the architecture and metabolism of bacteria and the effect of AMPs on various bacterial structures is presented. In-cell ssNMR revealed that the studied AMPs have a disruptive action on the molecular packing of bacterial membranes and DNA. Despite the limited number of studies, in-cell ssNMR is emerging as a powerful technique to monitor in situ the interplay between bacteria and AMPs.
Glucocorticoid resistance of migration and gene expression in a daughter MDA-MB-231 breast tumour cell line selected for high metastatic potential
(NATURE PUBLISHING GROUP, 2017-03-06)
Glucocorticoids are commonly used to prevent chemotherapy-induced nausea and vomiting despite a lack of understanding of their direct effect on cancer progression. Recent studies suggest that glucocorticoids inhibit cancer cell migration. However, this action has not been investigated in estrogen receptor (ER)-negative breast tumour cells, although activation of the glucocorticoid receptor (GR) is associated with a worse prognosis in ER-negative breast cancers. In this study we have explored the effect of glucocorticoids on the migration of the ER-negative MDA-MB-231 human breast tumour cell line and the highly metastatic MDA-MB-231-HM.LNm5 cell line that was generated through in vivo cycling. We show for the first time that glucocorticoids inhibit 2- and 3-dimensional migration of MDA-MB-231 cells. Selection of cells for high metastatic potential resulted in a less migratory cell phenotype that was resistant to regulation by glucocorticoids and showed decreased GR receptor expression. The emergence of glucocorticoid resistance during metastatic selection may partly explain the apparent disparity between the clinical and in vitro evidence regarding the actions of glucocorticoids in cancer. These findings highlight the highly plastic nature of tumour cells, and underscore the need to more fully understand the direct effect of glucocorticoid treatment on different stages of metastatic progression.
Genomic characterisation of E mu-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene
(NATURE PUBLISHING GROUP, 2017-03-06)
The Eμ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eμ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eμ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.
The Core Domain of Hepatitis C Virus Glycoprotein E2 Generates Potent Cross-Neutralizing Antibodies in Guinea Pigs
A vaccine that prevents hepatitis C virus (HCV) infection is urgently needed to support an emerging global elimination program. However, vaccine development has been confounded because of HCV's high degree of antigenic variability and the preferential induction of type-specific immune responses with limited potency against heterologous viral strains and genotypes. We showed previously that deletion of the three variable regions from the E2 receptor-binding domain (Δ123) increases the ability of human broadly neutralizing antibodies (bNAbs) to inhibit E2-CD81 receptor interactions, suggesting improved bNAb epitope exposure. In this study, the immunogenicity of Δ123 was examined. We show that high-molecular-weight forms of Δ123 elicit distinct antibody specificities with potent and broad neutralizing activity against all seven HCV genotypes. Antibody competition studies revealed that immune sera raised to high-molecular-weight Δ123 was poly specific, given that it inhibited the binding of human bNAbs directed to three major neutralization epitopes on E2. By contrast, the immune sera raised to monomeric Δ123 predominantly blocked the binding of a non-neutralizing antibody to Δ123, while having reduced ability to block bNAb binding to E2, and neutralization was largely toward the homologous genotype. This increased ability of oligomeric Δ123 to generate bNAbs correlates with occlusion of the non-neutralizing face of E2 in this glycoprotein form. CONCLUSION: The results from this study reveal new information on the antigenic and immunogenic potential of E2-based immunogens and provide a pathway for the development of a simple, recombinant protein-based prophylactic vaccine for HCV with potential for universal protection. (Hepatology 2017;65:1117-1131).
A Review of Analytical Techniques and Their Application in Disease Diagnosis in Breathomics and Salivaomics Research
(MDPI AG, 2017-01-01)
The application of metabolomics to biological samples has been a key focus in systems biology research, which is aimed at the development of rapid diagnostic methods and the creation of personalized medicine. More recently, there has been a strong focus towards this approach applied to non-invasively acquired samples, such as saliva and exhaled breath. The analysis of these biological samples, in conjunction with other sample types and traditional diagnostic tests, has resulted in faster and more reliable characterization of a range of health disorders and diseases. As the sampling process involved in collecting exhaled breath and saliva is non-intrusive as well as comparatively low-cost and uses a series of widely accepted methods, it provides researchers with easy access to the metabolites secreted by the human body. Owing to its accuracy and rapid nature, metabolomic analysis of saliva and breath (known as salivaomics and breathomics, respectively) is a rapidly growing field and has shown potential to be effective in detecting and diagnosing the early stages of numerous diseases and infections in preclinical studies. This review discusses the various collection and analyses methods currently applied in two of the least used non-invasive sample types in metabolomics, specifically their application in salivaomics and breathomics research. Some of the salient research completed in this field to date is also assessed and discussed in order to provide a basis to advocate their use and possible future scientific directions.
Association between dairy intake, lipids and vascular structure and function in diabetes
(BAISHIDENG PUBLISHING GROUP INC, 2017-05-15)
AIM: To determine lipid species that change in response to a change in dairy consumption. In addition, to investigate whether dairy associated lipid species are correlated with changes in measures of vascular structure and function. METHODS: A 12-mo randomised controlled trial was conducted to determine the effect of increased consumption of fruit, vegetables and dairy, compared to usual diet, on measures of vascular structure and function in adults with type 1 and type 2 diabetes (n = 108). This paper comprises post-hoc analyses investigating the relationship between dairy intake, serum lipid species and vascular health. Central and peripheral blood pressure, carotid femoral pulse wave velocity, augmentation index, serum lipid species and dietary intake were measured at baseline and 3-mo. Common carotid artery intima media thickness was measured at baseline and 12-mo. RESULTS: Serum lipid species [lysophosphatidylcholine (LPC) 14:0, LPC 15:0, LPC 16:1, phosphatidylcholine (PC) 29:0 PC 30:0, PC 31:0 and cholesterol ester (CE) 14:0] were associated with the change in full fat dairy consumption (rho 0.19-0.25; P < 0.05). The 3-mo change in some lipids was positively associated with the 3-mo change in central systolic [LPC 14:0 (rho 0.30; P = 0.007), PC 30:0 (rho 0.28; P = 0.010)] and diastolic blood pressure [LPC 14:0 (rho 0.32; P = 0.004), LPC 15:0 (rho 0.23; P = 0.04), LPC 16:1 (rho 0.23; P = 0.035), PC 29:0 (rho 0.28; P = 0.01), PC 30:0 (rho 0.36; P = 0.001), PC 31:0 (rho 0.30; P = 0.007)] and 12-mo change in common carotid artery intimal medial thickness [CE 14:0 (rho 0.22; P = 0.02)]. Pulse wave velocity and augmentation index were unrelated to dairy and lipid species. CONCLUSION: An increase in dairy associated lipids appears to be associated with an increase in blood pressure and common carotid intimal medial thickness.
A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS
A simple method for tracing carbon fixation and lipid synthesis in microalgae was developed using a combination of solid-phase extraction (SPE) and negative ion chemical ionisation gas chromatography mass spectrometry (NCI-GC-MS). NCI-GC-MS is an extremely sensitive technique that can produce an unfragmented molecular ion making this technique particularly useful for stable isotope enrichment studies. Derivatisation of fatty acids using pentafluorobenzyl bromide (PFBBr) allows the coupling of the high separation efficiency of GC and the measurement of unfragmented molecular ions for each of the fatty acids by single quadrupole MS. The key is that isotope spectra can be measured without interference from co-eluting fatty acids or other molecules. Pre-fractionation of lipid extracts by SPE allows the measurement of 13C isotope incorporation into the three main lipid classes (phospholipids, glycolipids, neutral lipids) in microalgae thus allowing the study of complex lipid biochemistry using relatively straightforward analytical technology. The high selectivity of GC is necessary as it allows the collection of mass spectra for individual fatty acids, including cis/trans isomers, of the PFB-derivatised fatty acids. The combination of solid-phase extraction and GC-MS enables the accurate determination of 13C incorporation into each lipid pool. Three solvent extraction protocols that are commonly used in lipidomics were also evaluated and are described here with regard to extraction efficiencies for lipid analysis in microalgae.
Transformed MDCK cells secrete elevated MMP1 that generates LAMA5 fragments promoting endothelial cell angiogenesis
(NATURE PUBLISHING GROUP, 2016-06-21)
Epithelial-mesenchymal transition (EMT) enhances the migration and invasion of cancer cells, and is regulated by various molecular mechanisms including extracellular matrix metalloproteinase (MMP) activity. Previously, we reported transformation of epithelial Madin-Darby canine kidney (MDCK) cells with oncogenic H-Ras (21D1 cells) induces EMT, and significantly elevates MMP1 expression. To explore the biological significance, in this study we characterized 21D1 cells with knocked-down MMP1 expression (21D1(-MMP1)). MMP1 silencing diminished 21D1 cell migration, invasion and anchorage-independent growth in vitro. Additionally, 21D1(-MMP1) cells displayed reduced tumour volume when grown as in vivo subcutaneous xenografts in mice. Depletion of MMP1 lowered the ability of the cellular secretome (extracellular culture medium) to influence recipient cell behaviour. For example, supplementation with 21D1 secretome elevated cell migration of recipient fibroblasts, and enhanced endothelial cell angiogenesis (vessel length and branching). By contrast, 21D1(-MMP1) secretome was less potent in both functional assays. We reveal laminin subunit alpha-5 (LAMA5) as a novel biological substrate of MMP1, that generates internal and C-terminal proteolytic fragments in 21D1 secretome. Furthermore, antibody-based inhibition of integrin αvβ3 on endothelial cells nullified the angiogenic capability of 21D1 secretome. Therefore, we report this as a new VEGF-independent mechanism that oncogenic cells may employ to promote tumour angiogenesis.
Establishing multiple omics baselines for three Southeast Asian populations in the Singapore Integrative Omics Study
(NATURE PUBLISHING GROUP, 2017-09-21)
The Singapore Integrative Omics Study provides valuable insights on establishing population reference measurement in 364 Chinese, Malay, and Indian individuals. These measurements include > 2.5 millions genetic variants, 21,649 transcripts expression, 282 lipid species quantification, and 284 clinical, lifestyle, and dietary variables. This concept paper introduces the depth of the data resource, and investigates the extent of ethnic variation at these omics and non-omics biomarkers. It is evident that there are specific biomarkers in each of these platforms to differentiate between the ethnicities, and intra-population analyses suggest that Chinese and Indians are the most biologically homogeneous and heterogeneous, respectively, of the three groups. Consistent patterns of correlations between lipid species also suggest the possibility of lipid tagging to simplify future lipidomics assays. The Singapore Integrative Omics Study is expected to allow the characterization of intra-omic and inter-omic correlations within and across all three ethnic groups through a systems biology approach.The Singapore Genome Variation projects characterized the genetics of Singapore's Chinese, Malay, and Indian populations. The Singapore Integrative Omics Study introduced here goes further in providing multi-omic measurements in individuals from these populations, including genetic, transcriptome, lipidome, and lifestyle data, and will facilitate the study of common diseases in Asian communities.
Better Indigenous Risk stratification for Cardiac Health study (BIRCH) protocol: rationale and design of a cross-sectional and prospective cohort study to identify novel cardiovascular risk indicators in Aboriginal Australian and Torres Strait Islander adults
BACKGROUND: Of the estimated 10-11 year life expectancy gap between Indigenous (Aboriginal and Torres Strait Islander people) and non-Indigenous Australians, approximately one quarter is attributable to cardiovascular disease (CVD). Risk prediction of CVD is imperfect, but particularly limited for Indigenous Australians. The BIRCH (Better Indigenous Risk stratification for Cardiac Health) project aims to identify and assess existing and novel markers of early disease and risk in Indigenous Australians to optimise health outcomes in this disadvantaged population. It further aims to determine whether these markers are relevant in non-Indigenous Australians. METHODS/DESIGN: BIRCH is a cross-sectional and prospective cohort study of Indigenous and non-Indigenous Australian adults (≥ 18 years) living in remote, regional and urban locations. Participants will be assessed for CVD risk factors, left ventricular mass and strain via echocardiography, sleep disordered breathing and quality via home-based polysomnography or actigraphy respectively, and plasma lipidomic profiles via mass spectrometry. Outcome data will comprise CVD events and death over a period of five years. DISCUSSION: Results of BIRCH may increase understanding regarding the factors underlying the increased burden of CVD in Indigenous Australians in this setting. Further, it may identify novel markers of early disease and risk to inform the development of more accurate prediction equations. Better identification of at-risk individuals will promote more effective primary and secondary preventive initiatives to reduce Indigenous Australian health disadvantage.
Changes in Lipids and Inflammatory Markers after Consuming Diets High in Red Meat or Dairy for Four Weeks
(MDPI AG, 2017-08-01)
There is a body of evidence linking inflammation, altered lipid metabolism, and insulin resistance. Our previous research found that insulin sensitivity decreased after a four-week diet high in dairy compared to a control diet and to one high in red meat. Our aim was to determine whether a relationship exists between changes in insulin sensitivity and inflammatory biomarkers, or with lipid species. Fasting Tumor Necrosis Factor alpha (TNF-α), Tumor Necrosis Factor Receptor II (sTNF-RII), C-reactive protein (CRP), and lipids were measured at the end of each diet. TNF-α and the ratio TNF-α/sTNF-RII were not different between diets and TNF-α, sTNF-RII, or the ratio TNF-α/sTNF-RII showed no association with homeostasis model assessment-estimated insulin resistance (HOMA-IR). A number of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) species differed between dairy and red meat and dairy and control diets, as did many phosphatidylcholine (PC) species and cholesteryl ester (CE) 14:0, CE15:0, lysophosphatidylcholine (LPC) 14:0, and LPC15:0. None had a significant relationship (p = 0.001 or better) with log homeostasis model assessment-estimated insulin resistance (HOMA-IR), although LPC14:0 had the strongest relationship (p = 0.004) and may be the main mediator of the effect of dairy on insulin sensitivity. LPC14:0 and the whole LPC class were correlated with CRP. The correlations between dietary change and the minor plasma phospholipids PI32:1 and PE32:1 are novel and may reflect significant changes in membrane composition. Inflammatory markers were not altered by changes in protein source while the correlation of LPC with CRP confirms a relationship between changes in lipid profile and inflammation.
Statin action favors normalization of the plasma lipidome in the atherogenic mixed dyslipidemia of MetS: potential relevance to statin-associated dysglycemia
(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2015-12-01)
The impact of statin treatment on the abnormal plasma lipidome of mixed dyslipidemic patients with metabolic syndrome (MetS), a group at increased risk of developing diabetes, was evaluated. Insulin-resistant hypertriglyceridemic hypertensive obese males (n = 12) displaying MetS were treated with pitavastatin (4 mg/day) for 180 days; healthy normolipidemic age-matched nonobese males (n = 12) acted as controls. Statin treatment substantially normalized triglyceride (-41%), remnant cholesterol (-55%), and LDL-cholesterol (-39%), with minor effect on HDL-cholesterol (+4%). Lipidomic analysis, normalized to nonHDL-cholesterol in order to probe statin-induced differences in molecular composition independently of reduction in plasma cholesterol, revealed increment in 132 of 138 lipid species that were subnormal at baseline and significantly shifted toward the control group on statin treatment. Increment in alkyl- and alkenylphospholipids (plasmalogens) was prominent, and consistent with significant statin-induced increase in plasma polyunsaturated fatty acid levels. Comparison of the statin-mediated lipidomic changes in MetS with the abnormal plasma lipidomic profile characteristic of prediabetes and T2D in the Australian Diabetes, Obesity, and Lifestyle Study and San Antonio Family Heart Study cohorts by hypergeometric analysis revealed a significant shift toward the lipid profile of controls, indicative of a marked trend toward a normolipidemic phenotype. Pitavastatin attenuated the abnormal plasma lipidome of MetS patients typical of prediabetes and T2D.