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Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy-based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.

Neuropsychiatric disorders and type 2 diabetes (T2D) are major public health concerns proposed to be intimately connected. T2D is associated with increased risk of dementia, neuropsychiatric and mood disorders. Evidences of the involvement of insulin signaling on brain mechanisms related to depression indicate that insulin resistance, a hallmark of type 2 diabetes, could develop in the brains of depressive patients. In this article, we briefly review possible molecular mechanisms associating defective brain insulin signaling with reward system, neurogenesis, synaptic plasticity and hypothalamic-pituitary-adrenal (HPA) stress axis in depression. We further discuss the involvement of tumor necrosis factor α (TNFα) promoting defective insulin signaling and depressive-like behavior in rodent models. Finally, due to the high resistant rate of anti-depressants, novel insights into the link between insulin resistance and depression may advance the development of alternative treatments for this disease.

Proteomics is a systems physiology discipline to address the large-scale characterization of protein species within a biological system, be it a cell, a tissue, a body biofluid, an organism, or a cohort population. Building on advances from chemical analytical platforms (e.g., mass spectrometry and other technologies), proteomics approaches have contributed powerful applications in cardiovascular biomedicine, most notably in: 1) the discovery of circulating protein biomarkers of heart diseases from plasma samples; and 2) the identification of disease mechanisms and potential therapeutic targets in cardiovascular tissues, in both preclinical models and translational studies. Contemporary proteomics investigations offer powerful means to simultaneously examine tens of thousands of proteins in various samples, and understand their molecular phenotypes in health and disease. This concise review introduces study design considerations, example applications and use cases, as well as interpretation and analysis of proteomics data in cardiovascular biomedicine., (Published by Elsevier Inc.)

Proteomics plays an increasingly important role in our quest to understand cardiovascular biology. Fueled by analytical and computational advances in the past decade, proteomics applications can now go beyond merely inventorying protein species, and address sophisticated questions on cardiac physiology. The advent of massive mass spectrometry datasets has in turn led to increasing intersection between proteomics and big data science. Here we review new frontiers in technological developments and their applications to cardiovascular medicine. The impact of big data science on cardiovascular proteomics investigations and translation to medicine is highlighted.

Amidst the proteomes of human tissues lie subsets of proteins that are closely involved in conserved pathophysiological processes. Much of biomedical research concerns interrogating disease signature proteins and defining their roles in disease mechanisms. With advances in proteomics technologies, it is now feasible to develop targeted proteomics assays that can accurately quantify protein abundance as well as their post-translational modifications; however, with rapidly accumulating number of studies implicating proteins in diseases, current resources are insufficient to target every protein without judiciously prioritizing the proteins with high significance and impact for assay development. We describe here a data science method to prioritize and expedite assay development on high-impact proteins across research fields by leveraging the biomedical literature record to rank and normalize proteins that are popularly and preferentially published by biomedical researchers. We demonstrate this method by finding priority proteins across six major physiological systems (cardiovascular, cerebral, hepatic, renal, pulmonary, and intestinal). The described method is data-driven and builds upon the collective knowledge of previous publications referenced on PubMed to lend objectivity to target selection. The method and resulting popular protein lists may also be useful for exploring biological processes associated with various physiological systems and research topics, in addition to benefiting ongoing efforts to facilitate the broad translation of proteomics technologies.

Protein stability is a major regulatory principle of protein function and cellular homeostasis. Despite limited understanding on mechanisms, disruption of protein turnover is widely implicated in diverse pathologies from heart failure to neurodegenerations. Information on global protein dynamics therefore has the potential to expand the depth and scope of disease phenotyping and therapeutic strategies. Using an integrated platform of metabolic labeling, high-resolution mass spectrometry and computational analysis, we report here a comprehensive dataset of the in vivo half-life of 3,228 and the expression of 8,064 cardiac proteins, quantified under healthy and hypertrophic conditions across six mouse genetic strains commonly employed in biomedical research. We anticipate these data will aid in understanding key mitochondrial and metabolic pathways in heart diseases, and further serve as a reference for methodology development in dynamics studies in multiple organ systems.

Objective: Intervertebral disc degeneration (IDD) can lead to symptomatic conditions including sciatica and back pain. The purpose of this study is to understand the extracellular matrix (ECM) changes in disc biology through comparative proteomic analysis of degenerated and non-degenerated human intervertebral disc (IVD) tissues of different ages., Design: Seven non-degenerated (11-46 years of age) and seven degenerated (16-53 years of age) annulus fibrosus (AF) and nucleus pulposus (NP) samples were used. Proteins were extracted using guanidine hydrochloride, separated from large proteoglycans (PGs) by caesium chloride (CsCl) density gradient ultracentrifugation, and identified using liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). For quantitative comparison, proteins were labeled with iTRAQ reagents. Collagen fibrils in the NP were assessed using scanning electron microscopy (SEM)., Results: In the AF, quantitative analysis revealed increased levels of HTRA1, COMP and CILP in degeneration when compared with samples from older individuals. Fibronectin showed increment with age and degeneration. In the NP, more CILP and CILP2 were present in degenerated samples of younger individuals. Reduced protein solubility was observed in degenerated and older non-degenerated samples correlated with an accumulation of type I collagen in the insoluble fibers. Characterization of collagen fibrils in the NP revealed smaller mean fibril diameters and decreased porosity in the degenerated samples., Conclusions: Our study identified distinct matrix changes associated with aging and degeneration in the intervertebral discs (IVDs). The nature of the ECM changes, together with observed decreased in solubility and changes in fibril diameter is consistent with a fibrotic-like environment., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: Alu elements are major contributors to lineage-specific new exons in primate and human genomes. Recent studies indicate that some Alu exons have high transcript inclusion levels or tissue-specific splicing profiles, and may play important regulatory roles in modulating mRNA degradation or translational efficiency. However, the contribution of Alu exons to the human proteome remains unclear and controversial. The prevailing view is that exons derived from young repetitive elements, such as Alu elements, are restricted to regulatory functions and have not had adequate evolutionary time to be incorporated into stable, functional proteins., Results: We adopt a proteotranscriptomics approach to systematically assess the contribution of Alu exons to the human proteome. Using RNA sequencing, ribosome profiling, and proteomics data from human tissues and cell lines, we provide evidence for the translational activities of Alu exons and the presence of Alu exon derived peptides in human proteins. These Alu exon peptides represent species-specific protein differences between primates and other mammals, and in certain instances between humans and closely related primates. In the case of the RNA editing enzyme ADARB1, which contains an Alu exon peptide in its catalytic domain, RNA sequencing analyses of A-to-I editing demonstrate that both the Alu exon skipping and inclusion isoforms encode active enzymes. The Alu exon derived peptide may fine tune the overall editing activity and, in limited cases, the site selectivity of ADARB1 protein products., Conclusions: Our data indicate that Alu elements have contributed to the acquisition of novel protein sequences during primate and human evolution.

Objectives: Game playing is very popular among Hong Kong teenagers. This study aimed to investigate adolescent gaming behavior and addiction at the Internet cafe, and to explore perceived benefits and harms associated with the activity., Methods: A convenient sample of 13 male high school students aged 12-15 years (mean age = 13.6 years) were interviewed at two Internet cafes. Young's (Caught in the net, Wiley, New York, 1998) criteria of Internet addiction were modified to assess gaming addiction., Results: Internet cafes were described as a safe and ideal rendezvous for gamers. The benefits of gaming included fun and satisfaction, fostering social support and teamwork, meeting new friends and becoming sociable, boosting cognitive techniques and intellectual agility, improved responsiveness and quick thinking. Perceived harms of gaming addiction were reduced time and interest in other important activities, poor academic performance, physical harms and emotional distress, disrupted friendship with non-gaming peers, risked family relationship and financial problems. Five interviewees (38.5 %) could be categorized as pathological gamers and two were problem gamers (15.4 %). The psychological factors associated with gaming addiction include low self-esteem, a strong desire for aggressive and exciting experiences, reliance on gaming to kill time and to obtain satisfaction, coping with problems and negative emotions, and obsession with achieving higher rankings in games. The social and environmental risk factors are accessibility to the Internet cafés, aggressive promotional activities at the Internet cafes, peer pressure, family influence and early gaming experiences, perceived parental approval, lack of parental supervision, and poor family relationship., Conclusions: The study results throw light on prevention programs.

Mitochondrial proteins alter in their composition and quantity drastically through time and space in correspondence to changing energy demands and cellular signaling events. The integrity and permutations of this dynamism are increasingly recognized to impact the functions of the cardiac proteome in health and disease. This article provides an overview on recent advances in defining the spatial and temporal dynamics of mitochondrial proteins in the heart. Proteomics techniques to characterize dynamics on a proteome scale are reviewed and the physiological consequences of altered mitochondrial protein dynamics are discussed. Lastly, we offer our perspectives on the unmet challenges in translating mitochondrial dynamics markers into the clinic.

Background: The use of error-prone abbreviations has led to medication errors. Many safety organisations have introduced 'Do Not Use' lists (lists of error-prone abbreviations that should be avoided by prescribers), but the effectiveness of these lists have not been studied., Objective: We assessed the effectiveness of the 'Do Not Use' list introduced to the study hospital, and sought the attitudes of healthcare professionals on other potentially dangerous abbreviations (not included in the 'Do Not Use' list) used in prescriptions., Setting: The study was conducted in a university affiliated tertiary hospital in Hong Kong., Methods: An uncontrolled observational study was conducted. In-patient prescriptions were reviewed to assess the use of error-prone abbreviations included in the 'Do Not Use' list before, after its introduction, and following the first reinforcement. An on-line survey was also conducted among prescribers, pharmacists and nurses., Main Outcome Measure: Rate of using error-prone abbreviations and other unapproved abbreviations among reviewed prescriptions., Results: 3,238 prescriptions (23,398 drug items) were reviewed. The use of abbreviations in the 'Do Not Use' list decreased from 7.8 to 3.3 % after its introduction (P < 0.001) and to 1.3 % after the first reinforcement (P < 0.001). However, unapproved abbreviations were used to denote prescribing instructions in 19.2 % of the drugs prescribed. 49 different types of unapproved abbreviations were used for drug names., Conclusions: A 'Do Not Use' list is effective in reducing error-prone abbreviations. Reinforcements of the 'Do Not Use' list further improves prescriber adherence. However, many other unapproved abbreviations (not included in current 'Do Not Use' lists) are used when prescribing. Periodic reminders on error-prone abbreviations and education of prescribers on associated risks may help to reduce the use of error-prone abbreviations in hospitals.

Purpose: High-throughput quantification of human protein turnover via in vivo administration of deuterium oxide ((2) H2 O) is a powerful new approach to examine potential disease mechanisms. Its immediate clinical translation is contingent upon characterizations of the safety and hemodynamic effects of in vivo administration of (2) H2 O to human subjects., Experimental Design: We recruited ten healthy human subjects with a broad demographic variety to evaluate the safety, feasibility, efficacy, and reproducibility of (2) H2 O intake for studying protein dynamics. We designed a protocol where each subject orally consumed weight-adjusted doses of 70% (2) H2 O daily for 14 days to enrich body water and proteins with deuterium. Plasma proteome dynamics was measured using a high-resolution MS method we recently developed., Results: This protocol was successfully applied in ten human subjects to characterize the endogenous turnover rates of 542 human plasma proteins, the largest such human dataset to-date. Throughout the study, we did not detect physiological effects or signs of discomfort from (2) H2 O consumption., Conclusions and Clinical Relevance: Our investigation supports the utility of a (2) H2 O intake protocol that is safe, accessible, and effective for clinical investigations of large-scale human protein turnover dynamics. This workflow shows promising clinical translational value for examining plasma protein dynamics in human diseases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets polyubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations., Experimental Design: Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples., Results: We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites., Conclusion and Clinical Relevance: This is the most comprehensive characterization of cardiac proteasome ubiquitination to date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein temporal dynamics play a critical role in time-dimensional pathophysiological processes, including the gradual cardiac remodeling that occurs in early-stage heart failure. Methods for quantitative assessments of protein kinetics are lacking, and despite knowledge gained from single-protein studies, integrative views of the coordinated behavior of multiple proteins in cardiac remodeling are scarce. Here, we developed a workflow that integrates deuterium oxide (2H2O) labeling, high-resolution mass spectrometry (MS), and custom computational methods to systematically interrogate in vivo protein turnover. Using this workflow, we characterized the in vivo turnover kinetics of 2,964 proteins in a mouse model of β-adrenergic-induced cardiac remodeling. The data provided a quantitative and longitudinal view of cardiac remodeling at the molecular level, revealing widespread kinetic regulations in calcium signaling, metabolism, proteostasis, and mitochondrial dynamics. We translated the workflow to human studies, creating a reference dataset of 496 plasma protein turnover rates from 4 healthy adults. The approach is applicable to short, minimal label enrichment and can be performed on as little as a single biopsy, thereby overcoming critical obstacles to clinical investigations. The protein turnover quantitation experiments and computational workflow described here should be widely applicable to large-scale biomolecular investigations of human disease mechanisms with a temporal perspective.

Liquid chromatography (LC) is essential for sample fractionation in shotgun proteomics applications. With suitable design, common LC separation chemistries, including reversed-phase (RP) and strong cation exchange (SCX) mode, can be combined in online multidimensional LC to greatly enhance the overall separation power and, thus, proteome coverage. This protocol describes the design and assembly of a flexible online multidimensional RP-SCX-RP LC system that is compatible with deep proteome profiling on common shotgun proteomics platforms.

Study Question: Does CD147 regulate trophoblast functions in vitro?, Summary Answer: CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast., What Is Known Already: CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear., Study Design, Size, Duration: A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts., Participants/materials, Setting Methods: CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs., Main Results and the Role of Chance: CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin β1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by siRNA significantly (P < 0.05) reduced trophoblast-endometrial cell interaction, cell invasion, syncytialization, differentiation and ERK activation of BeWo cells. Consistently, anti-CD147 functional blocking antibody suppressed the invasiveness of primary human cytotrophoblasts. The reduced invasiveness was probably due to the restrained (P < 0.05) enzyme activities of MMP-2, MMP-9 and uPA., Limitations, Reasons for Caution: Most of the above findings are based on BeWo cell lines. These results need to be confirmed with human first trimester primary cytotrophoblast., Wider Implications of the Findings: This is the first study on the role of CD147 in trophoblast function. Further investigation on the function of CD147 and its associated protein complexes will enhance our understanding on human placentation., Study Funding/competing Interest(s): This work was supported in part by the University of Hong Kong Grant 201011159200. The authors have no competing interests to declare.

Rationale: Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts., Objective: The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine., Methods and Results: We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest., Conclusions: COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.

Allopurinol is a drug that has been used for decades to lower serum urate levels in patients with gout or chronic renal failure and in cancer patients undergoing chemotherapy at risk of tumor lysis syndrome. Patients may develop cutaneous hypersensitivity reactions, ranging from mild rashes to potentially fatal severe cutaneous adverse reactions (SCARs) namely drug hypersensitivity syndrome, Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). Recent studies have demonstrated the association between human leukocyte antigen (HLA) B*58:01 allele and allopurinol-induced SCARs, which might explain ethnic differences in their incidences. Genotyping is now required before starting abacavir and carbamazepine so as to identify individuals susceptible to SJS. However, no genetic screening is advocated before commencement of allopurinol. The lack of availability of a rapid and inexpensive screening test for the HLA-B*58:01 allele is one of the obstacles to such screening. Development of a test that is quick, accurate, and cost-effective is warranted., (© The Author(s) 2013.)

We report the development of a multiple-reaction monitoring (MRM) strategy specifically tailored to the detection and quantification of mitochondrial protein phosphorylation. We recently derived 68 MRM transitions specific to protein modifications in the respiratory chain, voltage-dependent anion channel, and adenine nucleotide translocase. Here, we have now expanded the total number of MRM transitions to 176 to cover proteins from the tricarboxylic acid cycle, pyruvate dehydrogenase complex, and branched-chain alpha-keto acid dehydrogenase complex. We utilized the transition set to analyze endogenous protein phosphorylation in human heart, mouse heart, and mouse liver. The data demonstrate the potential utility of the MRM workflow for studying the functional details of mitochondrial phosphorylation signaling. This article is part of a Special Issue entitled: From protein structures to clinical applications., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is an important process during placentation. Dysregulation of the process is associated with a wide range of pregnancy complications. Adrenomedullin (ADM) is a polypeptide expressed most abundantly in first-trimester placentas. We hypothesized that ADM modulated the invasion of human EVCT. Our results showed that ADM enhanced invasion and migration but not proliferation in two EVCT cell lines, JEG-3 and TEV-1. Similar observation can also be obtained in primary EVCTs. JEG-3 and TEV-1 cells expressed ADM receptor components as demonstrated by immunostaining, Western blotting, and RT-PCR. The ADM antagonist ADM(22-52) (ADM C-terminal 22-52 amino acid fragment) suppressed ADM-induced invasion and migration, confirming that ADM exerted its biological effects through its classical receptors. The stimulatory effect of ADM on EVCT invasiveness was associated with induction (P < 0.05) of urokinase plasminogen activator (uPA) and nitric oxide synthase (NOS) expression and activity. Silencing of uPA by siRNA transfection abolished the stimulatory effect of ADM, suggesting that uPA is the key mediator for ADM-induced invasion. The involvement of NO in enhancing the invasion and biosynthesis of uPA in EVCT cell lines was confirmed by using pharmacological inhibitors of NOS and NO donors. ADM-mediated NO production also increased protein S-nitrosylation of JEG-3 cells. S-nitrosylation activated uPA in vitro and induced a higher proteinase activity. These findings provide indications that ADM and its downstream NO signaling may play an important role in modulating human EVCT functions.

Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water ((2)H(2)O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with (2)H(2)O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level (2)H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d(-1) and 0.163 d(-1), respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (≈ 60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) - using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) - with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC-reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC-reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 10(6) copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 10(4) . This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationale: Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart., Objective: To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms., Methods and Results: Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities., Conclusions: The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

A newly reorganized HUPO Cardiovascular Initiative was announced at the HUPO 2011 Cardiovascular Initiative Workshop at Geneva. The new initiative is now part of the biology- and disease-driven component of the HUPO Human Proteome Project (B/D-HPP). Here we report the recent achievements and future directions of the initiative, and offer a perspective on the present challenges of cardiovascular proteomics and its integration with the cardiovascular biology community at large., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Warfarin is a commonly used oral anticoagulant with a narrow therapeutic range and large interindividual variability in daily dose. Compared with Caucasians, Chinese are known to require lower doses of warfarin. Differences between Caucasians and Chinese in the allelic frequencies of two genes, CYP2C9 and VKORC1, largely explain the difference in dose requirement. There are other genetic polymorphisms that may further explain the response to warfarin. The VKORC1 genotype is an important determinant of response to warfarin in Chinese, but some genetic variants found in other ethnic groups that have a large effect on warfarin response and dosing are not commonly found in Chinese. Therefore, it is important to recognize and beware of ethnic differences in the pharmacogenetics of the response to warfarin, especially in the design of algorithms to aid dosing in clinical practice., (© 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.)

Although numerous initiatives and interventions have been developed to promote medication safety, medication incidents still remain an important cause of hospitalization. To avoid this, it is important for physicians to prescribe safely. To date, the Beers criteria have been the most widely used explicit criteria for assessing the appropriateness of medications in the elderly, but they do have limitations. The more recent STOPP/START criteria were developed in the hope of addressing the deficiencies observed in the Beers criteria. This article gives an overview of STOPP/START criteria and its applications, and reviews the studies that assessed medication appropriateness using STOPP/START and/or the Beers criteria.

In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationale: The endogenous opioid and corticotropin-releasing hormone (CRH) systems, present in the central amygdala (CeA), are implicated in alcohol consumption., Objectives: The purpose of this study is to investigate the hypothesis that, in CeA, alcohol stimulates CRH release, which then stimulates β-endorphin release., Materials and Methods: Rats were unilaterally implanted with a guide cannula to aim microdialysis probes in CeA. Experiment 1: rats received an intraperitoneal (IP) injection of various ethanol doses (0.0, 2.0, 2.4, or 2.8 g ethanol/kg body weight) and microdialysates were sampled at 30-min intervals to determine the effects over time of acute alcohol on the extracellular CRH concentrations in CeA. Experiment 2: phosphate-buffered saline, CRH, or CRH receptor (CRHR) antagonists (antalarmin or anti-sauvagine-30) was microinjected into CeA followed by a saline or 2.8 g/kg ethanol IP injection to determine the effects of CRHR activation or blockade in CeA on the basal and alcohol-stimulated release of β-endorphin. CRH and β-endorphin dialysate contents were determined using specific radioimmunoassays., Results: Acute alcohol induced a delayed increase in the extracellular CRH levels in CeA. Behavioural data showed no difference in locomotion between alcohol- and saline-treated rats. However, a transient increase in grooming was observed which did not correspond with alcohol-induced changes in CRH. Local CRH microinjections increased the extracellular β-endorphin concentrations in CeA. CRHR1 and CRHR2 blockade with microinjections of antalarmin and anti-sauvagine-30, respectively, attenuated the alcohol-induced increase of extracellular β-endorphin in CeA., Conclusions: Acute alcohol exerts indirect actions on CRH release and induced interactions of the CRH and β-endorphin systems in CeA.

Neurons in the central amygdala (CeA) co-express dynorphin and corticotropin-releasing hormone (CRH). Moreover, the activity of both the CRH and dynorphin systems in CeA is altered by alcohol treatments, effects suggesting interactions between the CRH and dynorphin systems. Thus, the objectives of the present study were to investigate the effects of (1) activating CRH receptors (CRHRs) by microinjection of CRH in CeA and (2) blocking CRHRs by local microinjections of CRHR antagonists in the CeA on the alcohol-induced changes in the extracellular concentrations of dynorphin A1-8 with in vivo microdialysis experiments. Microdialysis probes with a microinjection port were implanted in the CeA of alcohol-naïve Sprague-Dawley rats. Microinjections of CRH or antalarmin, a CRH receptor type 1 (CRHR1) antagonist, or anti-sauvagine-30, a CRH receptor type 2 (CRHR2) antagonist, at the level of CeA were followed by an intraperitoneal injection of either saline or 2.8 g ethanol/kg body weight. The content of dynorphin A1-8 was determined in dialyzate samples obtained prior to and following the various treatments using a specific radioimmunoassay. Activation of CRHRs in CeA induced an increase in the extracellular concentrations of dynorphin A1-8. Moreover, acute alcohol administration increased the extracellular concentrations of dynorphin A1-8 in CeA, an effect that was attenuated by blocking CRHR2 with anti-sauvagine-30 microinjection but not blocking CRHR1 with antalarmin microinjection. Therefore, the findings suggest an interaction between the CRH and dynorphin A1-8 systems at the level of CeA in response to acute alcohol exposure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC--with only a single sample injection event--as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.

Objective: To investigate the effect of antibiotic lock solutions for preventing catheter-related bacteraemia in patients receiving haemodialysis., Design: Retrospective study., Setting: University teaching hospital, Hong Kong., Patients: Consecutive patients from March 2006 to April 2007 who had central venous catheter insertion for haemodialysis in our centre were included in this historically controlled study. In all, 75 patients had catheters with heparin solution alone and 74 had catheters with a gentamicin antibiotic lock. The majority of catheters were non-tunnelled (95%). Cumulative catheter survival free of catheter-related bacteraemia in the two groups was compared., Results: Baseline characteristics in the two groups were similar apart from a slightly lower serum albumin level in those with gentamicin locks. There were 18 and five catheter-related bacteraemia episodes before and after recourse to gentamicin antibiotic locks, respectively. Staphylococcus aureus contributed to over half (65%) of the total bacteraemia episodes. Use of gentamicin antibiotic locks significantly reduced catheter-related bacteraemia episodes per 1000 catheter days from 4.6 to 1.5 (P=0.002). Kaplan-Meier survival analysis using the log rank test showed significantly better bloodstream infection-free survival associated with using gentamicin antibiotic locks (P=0.032). A similar survival advantage was associated with gentamicin antibiotic locks when the analysis was restricted to non-tunnelled catheters. There was no significant association of catheter-related bacteraemia with patient age, obesity, gender, baseline serum albumin level, or diabetes mellitus. No serious adverse events were attributable to the use of gentamicin antibiotic locks., Conclusion: Use of gentamicin lock solutions effectively reduced catheter-related bacteraemia in haemodialysis patients, including those with non-tunnelled catheters.

Rationale: The selectively bred lines of alcohol-preferring alko alcohol (AA) and alcohol-avoiding alko nonalcohol (ANA) rats have been used to demonstrate differences in relevant neurotransmitters which could account for their difference in alcohol consumption. Studies have demonstrated differences in distinct components of the endogenous opioid system in various brain regions associated with the process of reinforcement between the AA and ANA lines of rats., Objectives: The goal of this current study was to investigate the hypotheses that the AA and ANA rats will show differences in the release of beta-endorphin at the level of nucleus accumbens (NAC) and in locomotor activity in response to acute systemic administration of ethanol., Materials and Methods: AA and ANA rats were unilaterally implanted with a guide cannula to aim microdialysis probes at the level of NAC. Intraperitoneal injections of 0.0, 1.5, 2.0, and 2.5 g ethanol/kg body weight were administered. Dialysate samples were collected at 30-min intervals prior to and following the injection. Radioimmunoassay specific for beta-endorphin was used to determine the dialysate beta-endorphin content., Results: The 2.5-g/kg ethanol dose induced a transient increase in extracellular beta-endorphin at the level of NAC of AA but not of ANA rats. The 2.5-g/kg ethanol dose also attenuated locomotor activity in the AA but not in the ANA rats., Conclusions: The lack of an increase in the beta-endorphin concentration in the NAC of ANA rats in response to ethanol may partially account for their lower alcohol consumption and lower alcohol-induced attenuation of locomotor activity compared to AA rats.

Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.

Rationale: There is experimental evidence that indicates that the endogenous opioid system of the central nucleus of the amygdala (CeA) may mediate some of the reinforcing effects of ethanol. However, the precise interactions of ethanol with the endogenous opioid system at the level of the CeA have not been investigated., Objectives: The aim of the current study was to investigate the hypothesis that acute systemic ethanol administration will increase the release of endogenous opioid peptides at the level of the CeA in a time- and dose-dependent manner., Materials and Methods: Rats were implanted with a unilateral guide cannula to aim microdialysis probes at the CeA. Intraperitoneal injections of saline and various doses of ethanol (0.8, 1.6, 2.0, 2.4, and 2.8 g ethanol/kg body weight) were administered to the rats. Dialysate samples were collected at 30-min intervals at distinct time points prior to and following treatment. Radioimmunoassays specific for beta-endorphin, met-enkephalin, and dynorphin A1-8 were used to determine the effect of ethanol on the content of the opioid peptides in the dialysate., Results: We report that the 2.8-g/kg dose of ethanol induced a long-lasting increase in beta-endorphin release from 60 min onwards following administration and, later, an ongoing increase in dynorphin A1-8 release. None of the ethanol doses tested elicited significant changes in dialysate met-enkephalin content compared to the saline treatment., Conclusions: Acute systemic ethanol administration induced a dose- and time-dependent increase in beta-endorphin and dynorphin A1-8 release at the level of the CeA, which may be involved in ethanol consumption.

In 1995 Chan and Hui examined the responses of a sample of Chinese teachers on the Maslach Burnout Inventory and recommended a possible 2-factor rather than the original 3-factor model for the assessment of burnout among Chinese teachers. In the present study, the factor structure of responses to the Chinese version of the Maslach Burnout Inventory in a sample of 1,398 Chinese secondary school guidance teachers was examined using the EQS approach to confirmatory factor analysis. The results showed that a 3-factor model (Emotional Exhaustion, Depersonalization, and Personal Accomplishment) provided the best fit, with the first two factors highly correlated (r = .80). Internal consistencies for the subscales ranged from .80 to .88.