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1.  Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE) 
Nature biotechnology  2008;26(3):305-312.
One purpose of the biomedical literature is to report results in sufficient detail so that the methods of data collection and analysis can be independently replicated and verified. Here we present for consideration a minimum information specification for gene expression localization experiments, called the “Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)”. It is modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments. Data specifications like MIAME and MISFISHIE specify the information content without dictating a format for encoding that information. The MISFISHIE specification describes six types of information that should be provided for each experiment: Experimental Design, Biomaterials and Treatments, Reporters, Staining, Imaging Data, and Image Characterizations. This specification has benefited the consortium within which it was initially developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.
PMCID: PMC4367930  PMID: 18327244
2.  Performance of Cavopulmonary Palliation at Elevated Altitude 
Circulation  2008;118(14 Suppl):S177-S181.
Outcomes of patients undergoing cavopulmonary palliation for single ventricle physiology may be impacted by living at altitude, as the passive pulmonary circulation is dependent on the resistance of the pulmonary vascular bed. The objective of this study is to identify risk factors for failure of cavopulmonary palliation at elevated altitude.
Methods and Results
Between January 1995 and March 2007, 122 consecutive patients living at a mean altitude of 1600 m (range 305 to 2570) underwent a bidirectional Glenn (BDG). There was one in-hospital mortality and 7 late deaths. 52 have proceeded to the Fontan procedure. Survival after BDG was 92.4% at 5 years. Freedom from palliation failure, defined as death, transplant, BDG/Fontan takedown, or revision was 81% at 5 years. At a mean follow-up of 39.8 months, 90 patients (75%) were in New York Heart Association class I. Patients with failing cavopulmonary circulation had higher pre-BDG pulmonary artery pressure (PAP) (18.3±6.1 mm Hg versus 14.8±5.1 mm Hg, P=0.016) and higher pre-BDG transpulmonary gradient (TPG) (11.2±6.2 mm Hg versus 7.7±4.3 mm Hg, P=0.014). Post-BDG, patients with palliation failure had increased PAP (15.0±5.7 mm Hg versus 10.8±2.8 mm Hg, P=0.008) and indexed pulmonary vascular resistance (PVRI) (2.43±1.0 Wood U · m2 versus 1.52±0.9 Wood U · m2, P=0.007).
The majority of patients at moderate altitude have favorable outcomes after BDG or Fontan palliation. Risk factors for palliation failure at elevated altitude include PAP >15 mm Hg, TPG >8 mm Hg, and PVRI >2.5 Wood U · m2.
PMCID: PMC3129266  PMID: 18824752
Fontan procedure; single ventricle; altitude
3.  Outcomes of Reparative and Transplantation Strategies for Multilevel Left Heart Obstructions With Mitral Stenosis 
The Annals of thoracic surgery  2008;86(4):1305-1310.
Conventional management for multilevel left heart obstructions and mitral stenosis (Shone’s complex) involves multiple operations that carry additive risks. This study reviews our experience with reconstructive and transplantation approaches for Shone’s complex.
Between 1987 and 2007, 43 patients with mitral stenosis and one or more left-sided obstructions were identified: supramitral ring (n = 13), subaortic stenosis (n = 25), aortic stenosis (n = 24), hypoplastic arch (n = 20), and coarctation (n = 38). Thirty patients underwent a staged reparative approach, including 27 mitral and 51 left ventricular outflow tract operations. Thirteen patients were referred for transplantation. Patients with severe hypoplasia of the left ventricle were excluded.
There was one in-hospital death (2.5%) and six late deaths (14.2%). Actuarial 5- and 10-year survival for staged surgical and transplantation was 88% vs 61.3% and 83.1% vs 61.3% (p = 0.035). At a mean follow-up of 7.9 years, freedom from mitral reoperation was 83.3% and freedom from reoperation for subaortic stenosis was 78.0%. Wait-list mortality was 13.3% (2 of 13). Wait-list time exceeding 90 days was an incremental risk factor for death after transplantation (p = 0.005).
Despite the challenges of a reparative strategy for Shone’s complex, favorable survival and durability outcomes can be expected. Heart transplantation, although avoiding the pitfalls of staged repair, confers increased risks from ongoing physiologic derangements due to uncorrected left heart inflow and outflow obstructions during the wait for donor heart availability.
PMCID: PMC3128450  PMID: 18805182
4.  Differential Plasma Glycoproteome of p19ARF Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform 
Clinical proteomics  2008;4(3-4):105.
A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.
PMCID: PMC2821048  PMID: 20157627
Skin cancer; LC-MS; Label-free protein quantification; Biomarker discovery; Systems biology; Targeted peptide sequencing; Glycoproteomics; Plasma
7.  Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex ▿ ‡  
Molecular and Cellular Biology  2008;29(5):1202-1211.
Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2′-O-ribose methylation of the first four nucleotides and pseudouridine (ψ) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2′-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL ψ28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3′-end maturation, and ψ28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.
PMCID: PMC2643836  PMID: 19103757
8.  Detailed Characterization and Profiles of Crankcase and Diesel Particular Matter Exhaust Emissions Using Speciated Organics 
Environmental science & technology  2008;42(15):5661-5666.
A monitoring campaign was conducted in August-September 2005 to compare different experimental approaches quantifying school bus self-pollution. As part of this monitoring campaign, a detailed characterization of PM2.5 diesel engine emissions from the tailpipe and crankcase emissions from the road draft tubes was performed. To distinguish between tailpipe and crankcase vent emissions, a deuterated alkane, n-hexatriacontane-d74 (n-C36D74) was added to the engine oil to serve as intentional quantitative tracers for lubricating oil PM emissions. This paper focuses on the detailed chemical speciation of crankcase and tailpipe PM emissions from two school buses used in this study. We found that organic carbon emission rates were generally higher from the crankcase than from the tailpipe for these two school buses, while elemental carbon contributed significantly only in the tailpipe emissions. The n-C36D74 that was added to the engine oil was emitted at higher rates from the crankcase than the tailpipe. Tracers of engine oil (hopanes, and steranes) were present in much higher proportion in crankcase emissions. Particle-associated PAH emission rates were generally very low (< 1 μg/km), but more PAH species were present in crankcase than in tailpipe emissions. The speciation of samples collected in the bus cabins was consistent with most of the bus self-pollution originating from crankcase emissions.
PMCID: PMC2614383  PMID: 18754490
School bus self-pollution; tailpipe and crankcase emissions; organic speciation; tracers
9.  Corra: Computational framework and tools for LC-MS discovery and targeted mass spectrometry-based proteomics 
BMC Bioinformatics  2008;9:542.
Quantitative proteomics holds great promise for identifying proteins that are differentially abundant between populations representing different physiological or disease states. A range of computational tools is now available for both isotopically labeled and label-free liquid chromatography mass spectrometry (LC-MS) based quantitative proteomics. However, they are generally not comparable to each other in terms of functionality, user interfaces, information input/output, and do not readily facilitate appropriate statistical data analysis. These limitations, along with the array of choices, present a daunting prospect for biologists, and other researchers not trained in bioinformatics, who wish to use LC-MS-based quantitative proteomics.
We have developed Corra, a computational framework and tools for discovery-based LC-MS proteomics. Corra extends and adapts existing algorithms used for LC-MS-based proteomics, and statistical algorithms, originally developed for microarray data analyses, appropriate for LC-MS data analysis. Corra also adapts software engineering technologies (e.g. Google Web Toolkit, distributed processing) so that computationally intense data processing and statistical analyses can run on a remote server, while the user controls and manages the process from their own computer via a simple web interface. Corra also allows the user to output significantly differentially abundant LC-MS-detected peptide features in a form compatible with subsequent sequence identification via tandem mass spectrometry (MS/MS). We present two case studies to illustrate the application of Corra to commonly performed LC-MS-based biological workflows: a pilot biomarker discovery study of glycoproteins isolated from human plasma samples relevant to type 2 diabetes, and a study in yeast to identify in vivo targets of the protein kinase Ark1 via phosphopeptide profiling.
The Corra computational framework leverages computational innovation to enable biologists or other researchers to process, analyze and visualize LC-MS data with what would otherwise be a complex and not user-friendly suite of tools. Corra enables appropriate statistical analyses, with controlled false-discovery rates, ultimately to inform subsequent targeted identification of differentially abundant peptides by MS/MS. For the user not trained in bioinformatics, Corra represents a complete, customizable, free and open source computational platform enabling LC-MS-based proteomic workflows, and as such, addresses an unmet need in the LC-MS proteomics field.
PMCID: PMC2651178  PMID: 19087345
10.  Displacement affinity chromatography of protein phosphatase one (PP1) complexes 
BMC Biochemistry  2008;9:28.
Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif.
We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex.
This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.
PMCID: PMC2587467  PMID: 19000314
11.  Pre-surgical radiologic identification of peri-prosthetic osteolytic lesions around TKRs: a pre-clinical investigation of diagnostic accuracy 
Emerging longitudinal data appear to demonstrate an alarming trend towards an increasing prevalence of osteolysis-induced mechanical failure, following total knee replacement (TKR). Even with high-quality multi-plane X-rays, accurate pre-surgical evaluation of osteolytic lesions is often difficult. This is likely to have an impact on surgical management and provides reasonable indication for the development of a model allowing more reliable lesion assessment. The aim of this study, using a simulated cadaver model, was to explore the accuracy of rapid spiral computed tomography (CT) examination in the non-invasive evaluation of peri-prosthetic osteolytic lesions, secondary to TKR, and to compare this to conventional X-ray standards.
A series of nine volume-occupying defects, simulating osteolytic lesions, were introduced into three human cadaveric knees, adjacent to the TKR implant components. With implants in situ, each knee was imaged using a two-stage conventional plain X-ray series and rapid-acquisition spiral CT. A beam-hardening artefact removal algorithm was employed to improve CT image quality.
After random image sorting, 12 radiologists were independently shown the series of plain X-ray images and asked to note the presence, anatomic location and 'size' of osteolytic lesions observed. The same process was repeated separately for review of the CT images. The corresponding X-ray and CT responses were directly compared to elicit any difference in the ability to demonstrate the presence and size of osteolytic lesions.
Access to CT images significantly improved the accuracy of recognition of peri-prosthetic osteolytic lesions when compared to AP and lateral projections alone (P = 0.008) and with the addition of bi-planar oblique X-rays (P = 0.03). No advantage was obtained in accuracy of identification of such lesions through the introduction of the oblique images when compared with the AP and lateral projections alone (P = 0.13)
The findings of this study suggest that peri-prosthetic osteolytic lesions can be reliably described non-invasively using a simple, rapid-acquisition CT-based imaging approach. The low sensitivity of conventional X-ray, even with provision of supplementary bi-planar 45° oblique views, suggests a limited role for use in situ for TKR implant screening where peri-prosthetic osteolytic lesions are clinically suspected. In contrast, the accuracy of CT evaluation, linked to its procedural ease and widespread availability, may provide a more accurate way of evaluating osteolysis around TKRs, at routine orthopaedic follow up. These findings have direct clinical relevance, as accurate early recognition and classification of such lesions influences the timing and aggressiveness of surgical and non-operative management strategies, and also the nature and appropriateness of planned implant revision or joint-salvaging osteotomy procedures.
PMCID: PMC2570664  PMID: 18834525
12.  Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate 
BMC Genomics  2008;9:246.
Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes.
Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance.
Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.
PMCID: PMC2413246  PMID: 18501003

Results 1-12 (12)