A caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical 'death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [ 18 F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The 'death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Deathswitch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of 'death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [ 18 F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [ 18 F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers. © 2013 Macmillan Publishers Limited. All rights reserved

The Application of a Hypothesis-driven Strategy to the Sensitive Detection and Location of Acetylated Lysine Residues

The application of a hypothesis-driven method for the sensitive determination of lysine acetylation sites on enzymatically digested proteins is described. Comparative sensitivity tests were carried out using serial dilution of an acetylated bovine serum albumin (AcBSA) digest to assess the performance of a multiple reaction monitoring (MRM)–based approach as compared to a more conventional precursor scanning (PS) method. Both methods were capable of selectively detecting an acetylated peptide at the low femtomole level when spiked into a background of 500 fmol six-protein tryptic digest. The MRM approach was roughly tenfold more sensitive than precursor scanning with one acetylated peptide detected and sequenced at the level of 2 fmol on-column. The technique was subsequently applied to a gel-derived sample of cytokeratin-8 (CK8) shown to contain acetylated lysine residues by Western blot analysis. The strategy applied herein, termed MRM-initiated detection and sequencing (MIDAS), resulted in the facile identification of novel sites of acetylation on this protein

An introduction to Trends in Extreme Weather and Climate Events: Observations, Socioeconomic Impacts, Terrestrial Ecological Impacts, and Model Projections

Weather and climatic extremes can have serious and damaging effects on human society and infrastructure as well as on ecosystems and wildlife. Thus, they are usually the main focus of attention of the news media in reports on climate. There are some indications from observations concerning how climatic extremes may have changed in the past. Climate models show how they could change in the future either due to natural climate fluctuations or under conditions of greenhouse gas-induced warming. These observed and modeled changes relate directly to the understanding of socioeconomic and ecological impacts related to extremes.Integrative Biolog

Chd1 protects genome integrity at promoters to sustain hypertranscription in embryonic stem cells

Stem and progenitor cells undergo a global elevation of nascent transcription, or hyper- transcription, during key developmental transitions involving rapid cell proliferation. The chromatin remodeler Chd1 mediates hypertranscription in pluripotent cells but its mechanism of action remains poorly understood. Here we report a novel role for Chd1 in protecting genome integrity at promoter regions by preventing DNA double-stranded break (DSB) accumulation in ES cells. Chd1 interacts with several DNA repair factors including Atm, Parp1, Kap1 and Topoisomerase 2βand its absence leads to an accumulation of DSBs at Chd1-bound Pol II-transcribed genes and rDNA. Genes prone to DNA breaks in Chd1 KO ES cells are longer genes with GC-rich promoters, a more labile nucleosomal structure and roles in chromatin regulation, transcription and signaling. These results reveal a vulnerability of hypertranscribing stem cells to accumulation of endogenous DNA breaks, with important implications for developmental and cancer biology

Candidate plasma biomarkers for predicting ascending aortic aneurysm in bicuspid aortic valve disease.

BACKGROUND: Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality affecting 1-2% of the population and is associated with a significantly increased risk of ascending aortic aneurysm. However, predicting which patients will develop aneurysms remains a challenge. This pilot study aimed to identify candidate plasma biomarkers for monitoring ascending aortic diameter and predicting risk of future aneurysm in BAV patients. METHODS: Plasma samples were collected pre-operatively from BAV patients undergoing aortic valve surgery. Maximum ascending aortic diameter was measured on pre-operative transoesophageal echocardiography. Maximum diameter ≥ 45 mm was classified as aneurysmal. Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH-MS), an advanced mass spectrometry technique, was used to identify and quantify all proteins within the samples. Protein abundance and aortic diameter were correlated using logistic regression. Levene's test was used to identify proteins demonstrating low abundance variability in the aneurysmal patients (consistent expression in disease), and high variability in the non-aneurysmal patients (differential expression between 'at risk' and not 'at risk' patients). RESULTS: Fifteen plasma samples were collected (seven non-aneurysmal and 8 aneurysmal BAV patients). The mean age of the patients was 55.5 years and the majority were female (10/15, 67%). Four proteins (haemoglobin subunits alpha, beta and delta and mannan-binding lectin serine protease) correlated significantly with maximal ascending aortic diameter (p < 0.05, r = 0.5-0.6). Five plasma proteins demonstrated significantly lower variability in the aneurysmal group and may indicate increased risk of aneurysm in non-aneurysmal patients (DNA-dependent protein kinase catalytic subunit, lumican, tetranectin, gelsolin and cartilage acidic protein 1). A further 7 proteins were identified only in the aneurysmal group (matrin-3, glucose-6-phosphate isomerase, coactosin-like protein, peptidyl-prolyl cis-trans isomerase A, golgin subfamily B member 1, myeloperoxidase and 2'-deoxynucleoside 5'-phosphate N-hydrolase 1). CONCLUSIONS: This study is the first to identify candidate plasma biomarkers for predicting aortic diameter and risk of future aneurysm in BAV patients. It provides valuable pilot data and proof of principle that could be used to design a large-scale prospective investigation. Ultimately, a more affordable 'off-the-shelf' follow-on blood assay could then be developed in place of SWATH-MS, for use in the healthcare setting

Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia

Relationship between the Plasma Proteome and Changes in Inflammatory Markers after Bariatric Surgery

Severe obesity is a disease associated with multiple adverse effects on health. Metabolic bariatric surgery (MBS) can have significant effects on multiple body systems and was shown to improve inflammatory markers in previous short-term follow-up studies. We evaluated associations between changes in inflammatory markers (CRP, IL6 and TNFα) and circulating proteins after MBS. Methods: Sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics was performed on plasma samples taken at baseline (pre-surgery) and 6 and 12 months after MBS, and concurrent analyses of inflammatory/metabolic parameters were carried out. The change in absolute abundances of those proteins, showing significant change at both 6 and 12 months, was tested for correlation with the absolute and percentage (%) change in inflammatory markers. Results: We found the following results: at 6 months, there was a correlation between %change in IL-6 and fold change in HSPA4 (rho = −0.659; p = 0.038) and in SERPINF1 (rho = 0.714, p = 0.020); at 12 months, there was a positive correlation between %change in IL-6 and fold change in the following proteins—LGALS3BP (rho = 0.700, p = 0.036), HSP90B1 (rho = 0.667; p = 0.05) and ACE (rho = 0.667, p = 0.05). We found significant inverse correlations at 12 months between %change in TNFα and the following proteins: EPHX2 and ACE (for both rho = −0.783, p = 0.013). We also found significant inverse correlations between %change in CRP at 12 months and SHBG (rho = −0.759, p = 0.029), L1CAM (rho = −0.904, p = 0.002) and AMBP (rho = −0.684, p = 0.042). Conclusion: Using SWATH-MS, we identified several proteins that are involved in the inflammatory response whose levels change in patients who achieve remission of T2DM after bariatric surgery in tandem with changes in IL6, TNFα and/or CRP. Future studies are needed to clarify the underlying mechanisms in how MBS decreases low-grade inflammation

Climate Variability and Ross River Virus Transmission in Townsville Region, Australia 1985 to 1996

Background How climate variability affects the transmission of infectious diseases at a regional level remains unclear. In this paper, we assessed the impact of climate variation on the Ross River virus (RRv) transmission in the Townsville region, Queensland, north-east Australia. Methods Population-based information was obtained on monthly variations in RRv cases, climatic factors, sea level, and population growth between 1985 and 1996. Cross-correlations were computed for a series of associations between climate variables (rainfall, maximum temperature, minimum temperature, relative humidity and high tide) and the monthly incidence of RRv disease over a range of time lags. The impact of climate variability on RRv transmission was assessed using the seasonal auto-regressive integrated moving average (SARIMA) model. Results There were significant correlations of the monthly incidence of RRv to rainfall, maximum temperature, minimum temperature and relative humidity, all at a lag of 2 months, and high tide in the current month. The results of SARIMA models show that monthly average rainfall (β=0.0012, p=0.04) and high tide (β=0.0262, p=0.01) were significantly associated with RRv transmission, although temperature and relative humidity did not seem to have played an important role in the Townsville region. Conclusions Rainfall, and high tide were likely to be key determinants of RRv transmission in the Townsville region