Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS)

Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure turnover rates with high confidence are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared with selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data are acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focused studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways

A proteome-integrated, carbon source dependent genetic regulatory network in Saccharomyces cerevisiae

Integrated regulatory networks can be powerful tools to examine and test properties of cellular systems, such as modelling environmental effects on the molecular bioeconomy, where protein levels are altered in response to changes in growth conditions. Although extensive regulatory pathways and protein interaction data sets exist which represent such networks, few have formally considered quantitative proteomics data to validate and extend them. We generate and consider such data here using a label-free proteomics strategy to quantify alterations in protein abundance for S. cerevisiae when grown on minimal media using glucose, galactose, maltose and trehalose as sole carbon sources. Using a high quality-controlled subset of proteins observed to be differentially abundant, we constructed a proteome-informed network, comprising 1850 transcription factor interactions and 37 chaperone interactions, which defines the major changes in the cellular proteome when growing under different carbon sources. Analysis of the differentially abundant proteins involved in the regulatory network pointed to their significant roles in specific metabolic pathways and function, including glucose homeostasis, amino acid biosynthesis, and carbohydrate metabolic process. We noted strong statistical enrichment in the differentially abundant proteome of targets of known transcription factors associated with stress responses and altered carbon metabolism. This shows how such integrated analysis can lend further experimental support to annotated regulatory interactions, since the proteomic changes capture both magnitude and direction of gene expression change at the level of the affected proteins. Overall this study highlights the power of quantitative proteomics to help define regulatory systems pertinent to environmental conditions

DOSCATs: Double standards for protein quantification

The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB

Loop Quantum Gravity and the The Planck Regime of Cosmology

The very early universe provides the best arena we currently have to test quantum gravity theories. The success of the inflationary paradigm in accounting for the observed inhomogeneities in the cosmic microwave background already illustrates this point to a certain extent because the paradigm is based on quantum field theory on the curved cosmological space-times. However, this analysis excludes the Planck era because the background space-time satisfies Einstein's equations all the way back to the big bang singularity. Using techniques from loop quantum gravity, the paradigm has now been extended to a self-consistent theory from the Planck regime to the onset of inflation, covering some 11 orders of magnitude in curvature. In addition, for a narrow window of initial conditions, there are departures from the standard paradigm, with novel effects, such as a modification of the consistency relation involving the scalar and tensor power spectra and a new source for non-Gaussianities. Thus, the genesis of the large scale structure of the universe can be traced back to quantum gravity fluctuations \emph{in the Planck regime}. This report provides a bird's eye view of these developments for the general relativity community.Comment: 23 pages, 4 figures. Plenary talk at the Conference: Relativity and Gravitation: 100 Years after Einstein in Prague. To appear in the Proceedings to be published by Edition Open Access. Summarizes results that appeared in journal articles [2-13

Accuracy and completeness of patient pathways – the benefits of national data linkage in Australia

Background - The technical challenges associated with national data linkage, and the extent of cross-border population movements, are explored as part of a pioneering research project. The project involved linking state-based hospital admission records and death registrations across Australia for a national study of hospital related deaths. Methods - The project linked over 44 million morbidity and mortality records from four Australian states between 1st July 1999 and 31st December 2009 using probabilistic methods. The accuracy of the linkage was measured through a comparison with jurisdictional keys sourced from individual states. The extent of cross-border population movement between these states was also assessed. Results - Data matching identified almost twelve million individuals across the four Australian states. The percentage of individuals from one state with records found in another ranged from 3-5 %. Using jurisdictional keys to measure linkage quality, results indicate a high matching efficiency (F measure 97 to 99 %), with linkage processing taking only a matter of days. Conclusions - The results demonstrate the feasibility and accuracy of undertaking cross jurisdictional linkage for national research. The benefits are substantial, particularly in relation to capturing the full complement of records in patient pathways as a result of cross-border population movements. The project identified a sizeable ‘mobile’ population with hospital records in more than one state. Research studies that focus on a single jurisdiction will under-enumerate the extent of hospital usage by individuals in the population. It is important that researchers understand and are aware of the impact of this missing hospital activity on their studies. The project highlights the need for an efficient and accurate data linkage system to support national research across Australia

Using hospital discharge data for determining neonatal morbidity and mortality: a validation study

<p>Abstract</p> <p>Background</p> <p>Despite widespread use of neonatal hospital discharge data, there are few published reports on the accuracy of population health data with neonatal diagnostic or procedure codes. The aim of this study was to assess the accuracy of using routinely collected hospital discharge data in identifying neonatal morbidity during the birth admission compared with data from a statewide audit of selected neonatal intensive care (NICU) admissions.</p> <p>Methods</p> <p>Validation study of population-based linked hospital discharge/birth data against neonatal intensive care audit data from New South Wales, Australia for 2,432 babies admitted to NICUs, 1994–1996. Sensitivity, specificity and positive predictive values (PPV) with exact binomial confidence intervals were calculated for 12 diagnoses and 6 procedures.</p> <p>Results</p> <p>Sensitivities ranged from 37.0% for drainage of an air leak to 97.7% for very low birthweight, specificities all exceeded 85% and PPVs ranged from 70.9% to 100%. In-hospital mortality, low birthweight (≤1500 g), retinopathy of prematurity, respiratory distress syndrome, meconium aspiration, pneumonia, pulmonary hypertension, selected major anomalies, any mechanical ventilation (including CPAP), major surgery and surgery for patent ductus arteriosus or necrotizing enterocolitis were accurately identified with PPVs over 92%. Transient tachypnea of the newborn and drainage of an air leak had the lowest PPVs, 70.9% and 83.6% respectively.</p> <p>Conclusion</p> <p>Although under-ascertained, routinely collected hospital discharge data had high PPVs for most validated items and would be suitable for risk factor analyses of neonatal morbidity. Procedures tended to be more accurately recorded than diagnoses.</p

HIV-1 Pre-Integration Complexes Selectively Target Decondensed Chromatin in the Nuclear Periphery

Integration of the double-stranded DNA copy of the HIV-1 genome into host chromosomal DNA is a requirement for efficient viral replication. Integration preferentially occurs within active transcription units, however chromosomal site specificity does not correlate with any strong primary sequence. To investigate whether the nuclear architecture may affect viral integration we have developed an experimental system where HIV-1 viral particles can be visualized within the nuclear compartment. Fluorescently labeled HIV-1 virions were engineered by fusing integrase, the viral protein that catalyzes the integration reaction, to fluorescent proteins. Viral tests demonstrate that the infectivity of fluorescent virions, including the integration step, is not altered as compared to wild-type virus. 3-D confocal microscopy allowed a detailed analysis of the spatial and temporal distribution of the pre-integration complexes (PICs) within the nucleus at different moments following infection; the fluorescently labeled PICs preferentially distribute in decondensed areas of the chromatin with a striking positioning in the nuclear periphery, while heterochromatin regions are largely disfavored. These observations provide a first indication of how the nuclear architecture may initially orient the selection of retroviral integration sites

Study protocol for the evaluation of an Infant Simulator based program delivered in schools: a pragmatic cluster randomised controlled trial

Background: This paper presents the study protocol for a pragmatic randomised controlled trial to evaluate the impact of a school based program developed to prevent teenage pregnancy. The program includes students taking care of an Infant Simulator; despite growing popularity and an increasing global presence of such programs, there is no published evidence of their long-term impact. The aim of this trial is to evaluate the Virtual Infant Parenting (VIP) program by investigating pre-conceptual health and risk behaviours, teen pregnancy and the resultant birth outcomes, early child health and maternal health. Methods and Design: Fifty-seven schools (86% of 66 eligible secondary schools) in Perth, Australia were recruited to the clustered (by school) randomised trial, with even randomisation to the intervention and control arms. Between 2003 and 2006, the VIP program was administered to 1,267 participants in the intervention schools, while 1,567 participants in the non-intervention schools received standard curriculum. Participants were all female and aged between 13-15 years upon recruitment. Pre and post-intervention questionnaires measured short-term impact and participants are now being followed through their teenage years via data linkage to hospital medical records, abortion clinics and education records. Participants who have a live birth are interviewed by face-to-face interview. Kaplan-Meier survival analysis and proportional hazards regression will test for differences in pregnancy, birth and abortion rates during the teenage years between the study arms.Discussion: This protocol paper provides a detailed overview of the trial design as well as initial results in the form of participant flow. The authors describe the intervention and its delivery within the natural school setting and discuss the practical issues in the conduct of the trial, including recruitment. The trial is pragmatic and will directly inform those who provide Infant Simulator based programs in school settings

A next generation, pilot-scale continuous sterilization system for fermentation media

A new continuous sterilization system was designed, constructed, started up, and qualified for media sterilization for secondary metabolite cultivations, bioconversions, and enzyme production. An existing Honeywell Total Distributed Control 3000-based control system was extended using redundant High performance Process Manager controllers for 98 I/O (input/output) points. This new equipment was retrofitted into an industrial research fermentation pilot plant, designed and constructed in the early 1980s. Design strategies of this new continuous sterilizer system and the expanded control system are described and compared with the literature (including dairy and bio-waste inactivation applications) and the weaknesses of the prior installation for expected effectiveness. In addition, the reasoning behind selection of some of these improved features has been incorporated. Examples of enhancements adopted include sanitary heat exchanger (HEX) design, incorporation of a “flash” cooling HEX, on-line calculation of F(o) and R(o), and use of field I/O modules located near the vessel to permit low-cost addition of new instrumentation. Sterilizer performance also was characterized over the expected range of operating conditions. Differences between design and observed temperature, pressure, and other profiles were quantified and investigated

Voting with their feet - predictors of discharge against medical advice in Aboriginal and non-Aboriginal ischaemic heart disease inpatients in Western Australia: an analytic study using data linkage

Background: Discharge Against Medical Advice (DAMA) from hospital is associated with adverse outcomes and is considered an indicator of the responsiveness of hospitals to the needs of Aboriginal and Torres Strait Islander Australians, the indigenous people of Australia. We investigated demographic and clinical factors that predict DAMA in patients experiencing their first-ever inpatient admission for ischaemic heart disease (IHD). The study focuse sparticularly on the differences in the risk of DAMA in Aboriginal and non-Aboriginal patients while also investigating other factors in their own right. Methods: A cross-sectional analytical study was undertaken using linked hospital and mortality data with complete coverage of Western Australia. Participants included all first-ever IHD inpatients (aged 25–79 years) admitted between 2005 and 2009, selected after a 15-year clearance period and who were discharged alive. The main outcome measure was DAMA as reflected in the hospital record. Multiple logistic regression was used to determine disparities in DAMA between Aboriginal and non-Aboriginal patients, adjusting for a range of demographic and clinical factors, including comorbidity based on 5-year hospitalization history. A series of additional models were run on subgroups of the cohort to refine the analysis. Ethics approval was granted by the WA Human Research and the WA Aboriginal Health Ethics Committees.Results: Aboriginal patients comprised 4.3% of the cohort of 37,304 IHD patients and 23% of the 224 DAMAs. Emergency admission (OR=5.9, 95% CI 2.9-12.2), alcohol admission history (alcohol-related OR=2.9, 95% CI 2.0-4.2) and Aboriginality (OR 2.3, 95% CI 1.5-3.5) were the strongest predictors of DAMA in the multivariate model. Patients living in rural areas while attending non-metropolitan hospitals had a 50% higher risk of DAMA than those living and hospitalised in metropolitan areas. There was consistency in the ORs for Aboriginality in the different multivariate models using restricted sub-cohorts and different Aboriginal identifiers. Sex, IHD diagnosis type and co-morbidity scores imparted different risks in Aboriginal versus non-Aboriginal patients. Conclusions: Understanding the risks and reasons for DAMA is important for health system policy and proactive management of those at risk of DAMA. Improving care to prevent DAMA should target unplanned admissions, rural hospitals and young men, Aboriginal people and those with alcohol and mental health comorbidities