Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H₂O₂), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H₂O₂, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H₂O₂ production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.     

Curated MicroRNAs in Urine and Blood Fail to Validate as Predictive Biomarkers for High-Risk Prostate Cancer

Purpose

The purpose of this study was to determine if microRNA profiling of urine and plasma at radical prostatectomy can distinguish potentially lethal from indolent prostate cancer.

Materials and Methods

A panel of microRNAs was profiled in the plasma of 70 patients and the urine of 33 patients collected prior to radical prostatectomy. Expression of microRNAs was correlated to the clinical endpoints at a follow-up time of 3.9 years to identify microRNAs that may predict clinical response after radical prostatectomy. A machine learning approach was applied to test the predictive ability of all microRNAs profiled in urine, plasma, and a combination of both, and global performance assessed using the area under the receiver operator characteristic curve (AUC). Validation of urinary expression of miRNAs was performed on a further independent cohort of 36 patients.

Results

The best predictor in plasma using eight miRs yielded only moderate predictive performance (AUC = 0.62). The best predictor of high-risk disease was achieved using miR-16, miR-21 and miR-222 measured in urine (AUC = 0.75). This combination of three microRNAs in urine was a better predictor of high-risk disease than any individual microRNA. Using a different methodology we found that this set of miRNAs was unable to predict high-volume, high-grade disease.

Conclusions

Our initial findings suggested that plasma and urinary profiling of microRNAs at radical prostatectomy may allow prognostication of prostate cancer behaviour. However we found that the microRNA expression signature failed to validate in an independent cohort of patients using a different platform for PCR. This highlights the need for independent validation patient cohorts and suggests that urinary microRNA signatures at radical prostatectomy may not be a robust way to predict the course of clinical disease after definitive treatment for prostate cancer.     

Protein signatures of centenarians and their offspring suggest centenarians age slower than other humans

Using samples from the New England Centenarian Study (NECS), we sought to characterize the serum proteome of 77 centenarians, 82 centenarians'' offspring, and 65 age‐matched controls of the offspring (mean ages: 105, 80, and 79 years). We identified 1312 proteins that significantly differ between centenarians and their offspring and controls (FDR < 1%), and two different protein signatures that predict longer survival in centenarians and in younger people. By comparing the centenarian signature with 2 independent proteomic studies of aging, we replicated the association of 484 proteins of aging and we identified two serum protein signatures that are specific of extreme old age. The data suggest that centenarians acquire similar aging signatures as seen in younger cohorts that have short survival periods, suggesting that they do not escape normal aging markers, but rather acquire them much later than usual. For example, centenarian signatures are significantly enriched for senescence‐associated secretory phenotypes, consistent with those seen with younger aged individuals, and from this finding, we provide a new list of serum proteins that can be used to measure cellular senescence. Protein co‐expression network analysis suggests that a small number of biological drivers may regulate aging and extreme longevity, and that changes in gene regulation may be important to reach extreme old age. This centenarian study thus provides additional signatures that can be used to measure aging and provides specific circulating biomarkers of healthy aging and longevity, suggesting potential mechanisms that could help prolong health and support longevity.     

Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung

Background

Chronic alveolar hypoxia, due to residence at high altitude or chronic obstructive lung diseases, leads to pulmonary hypertension, which may be further complicated by right heart failure, increasing morbidity and mortality. In the non-diseased lung, angiogenesis occurs in chronic hypoxia and may act in a protective, adaptive manner. To date, little is known about the behaviour of individual vascular endothelial growth factor (VEGF) family ligands in hypoxia-induced pulmonary angiogenesis. The aim of this study was to examine the expression of placenta growth factor (PlGF) and VEGFB during the development of hypoxic pulmonary angiogenesis and their functional effects on the pulmonary endothelium.

Methods

Male Sprague Dawley rats were exposed to conditions of normoxia (21% O₂) or hypoxia (10% O₂) for 1-21 days. Stereological analysis of vascular structure, real-time PCR analysis of vascular endothelial growth factor A (VEGFA), VEGFB, placenta growth factor (PlGF), VEGF receptor 1 (VEGFR1) and VEGFR2, immunohistochemistry and western blots were completed. The effects of VEGF ligands on human pulmonary microvascular endothelial cells were determined using a wound-healing assay.

Results

Typical vascular remodelling and angiogenesis were observed in the hypoxic lung. PlGF and VEGFB mRNA expression were significantly increased in the hypoxic lung. Immunohistochemical analysis showed reduced expression of VEGFB protein in hypoxia although PlGF protein was unchanged. The expression of VEGFA mRNA and protein was unchanged. In vitro PlGF at high concentration mimicked the wound-healing actions of VEGFA on pulmonary microvascular endothelial monolayers. Low concentrations of PlGF potentiated the wound-healing actions of VEGFA while higher concentrations of PlGF were without this effect. VEGFB inhibited the wound-healing actions of VEGFA while VEGFB and PlGF together were mutually antagonistic.

Conclusions

VEGFB and PlGF can either inhibit or potentiate the actions of VEGFA, depending on their relative concentrations, which change in the hypoxic lung. Thus their actions in vivo depend on their specific concentrations within the microenvironment of the alveolar wall during the course of adaptation to pulmonary hypoxia.     

Comparative efficacy of a secretory phospholipase A<Subscript>2</Subscript> inhibitor with conventional anti-inflammatory agents in a rat model of antigen-induced arthritis

Introduction  

Previously, secretory phospholipase A₂ (sPLA₂) inhibition has been used as an adjunct to conventional rheumatoid arthritis therapy in human clinical trials without significant improvement of arthritic pathology. In this study, we compared the efficacy of a potent and orally active group IIa secretory phospholipase A₂ inhibitor (sPLA₂I) to conventional anti-arthritic agents; infliximab, leflunomide and prednisolone, in a rat model of antigen-induced arthritis.     

The human proteome project: current state and future direction

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.     

Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis

Disease-associated HLA-DR molecules, which may present autoantigens, constitute the greatest genetic risk factor for rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). The peptides presented by HLA-DR molecules in synovia have not previously been defined. Using tandem mass spectrometry, rigorous database searches, and manual spectral interpretation, we identified 1,427 HLA-DR-presented peptides (220-464 per patient) from the synovia of four patients, two diagnosed with RA and two diagnosed with LA. The peptides were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. A few epitopes were found only in RA or LA patients. However, two patients with different diseases who had the same HLA allele had the largest number of epitopes in common. In one RA patient, peptides were identified as originating from source proteins that have been reported to undergo citrullination under other circumstances, yet neither this post-translational modification nor anti-cyclic citrullinated peptide antibodies were detected. Instead, peptides with the post-translational modification of S-cysteinylation were identified. We conclude that a wide range of proteins enter the HLA-DR pathway of antigen-presenting cells in the patients' synovial tissue, and their HLA-DR genotype, not the disease type, appears to be the primary determinant of their HLA-DR-peptide repertoire. New insights into the naturally presented HLA-DR epitope repertoire in target tissues may allow the identification of pathogenic T cell epitopes, and this could lead to innovative therapeutic interventions.     

Mass spectrometry of glycans

Powerful new strategies based on mass spectrometry are revolutionizing the structural analysis and profiling of glycans and glycoconjugates. We survey here the major biosynthetic pathways that underlie the biological diversity in glycobiology, with emphasis on glycoproteins, and the approaches that can be used to address the resulting heterogeneity. Included among these are derivatizations, on- and off-line chromatography, electrospray and matrix-assisted laser desorption/ionization, and a variety of dissociation methods, the recently introduced electron-based techniques being of particular interest.     

Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.