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1.  High Throughput Screening of Disulfide-Containing Proteins in a Complex Mixture 
Proteomics  2013;13(22):3256-3260.
The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new liquid chromatography mass spectrometric (LC/MS) strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2-carboxyethyl)phosphine. LC/MS analysis of reduced and non-reduced protein mixtures quickly revealed disulfide-containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide-containing and disulfide-free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on-line LC/MS analysis) and reliable (no S-S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications.
doi:10.1002/pmic.201300242
PMCID: PMC3914208  PMID: 24030959
Disulfide; Intact Proteins; Proteomics; Liquid Chromatography; TCEP; Mass Spectrometry
2.  Docetaxel-loaded solid lipid nanoparticles suppress breast cancer cells growth with reduced myelosuppression toxicity 
Docetaxel is an adjuvant chemotherapy drug widely used to treat multiple solid tumors; however, its toxicity and side effects limit its clinical efficacy. Herein, docetaxel-loaded solid lipid nanoparticles (DSNs) were developed to reduce systemic toxicity of docetaxel while still keeping its anticancer activity. To evaluate its anticancer activity and toxicity, and to understand the molecular mechanisms of DSNs, different cellular, molecular, and whole genome transcription analysis approaches were utilized. The DSNs showed lower cytotoxicity compared with the commercial formulation of docetaxel (Taxotere®) and induced more apoptosis at 24 hours after treatment in vitro. DSNs can cause the treated cancer cells to arrest in the G2/M phase in a dose-dependent manner similar to Taxotere. They can also suppress tumor growth very effectively in a mice model with human xenograft breast cancer. Systemic analysis of gene expression profiles by microarray and subsequent verification experiments suggested that both DSNs and Taxotere regulate gene expression and gene function, including DNA replication, DNA damage response, cell proliferation, apoptosis, and cell cycle regulation. Some of these genes expressed differentially at the protein level although their messenger RNA expression level was similar under Taxotere and DSN treatment. Moreover, DSNs improved the main side effect of Taxotere by greatly lowering myelosuppression toxicity to bone marrow cells from mice. Taken together, these results expound the antitumor efficacy and the potential working mechanisms of DSNs in its anticancer activity and toxicity, which provide a theoretical foundation to develop and apply a more efficient docetaxel formulation to treat cancer patients.
doi:10.2147/IJN.S70919
PMCID: PMC4207579  PMID: 25378924
docetaxel; docetaxel-loaded solid lipid nanoparticles; breast cancer; toxicity
3.  Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season 
BMC Genomics  2014;15(1):899.
Background
Seasonal estrus is a critical limiting factor of animal fecundity, and it involves changes in both ovarian biology and hormone secretion in different seasons. Previous studies indicate that two classes of small RNAs (miRNAs and piRNAs) play important regulatory roles in ovarian biology. To understand the roles of small RNA-mediated post-transcriptional regulation in ovine seasonal estrus, the variation in expression patterns of ovarian small RNAs during anestrus and the breeding season were analyzed using Solexa sequencing technology. In addition, reproductive hormone levels were determined during ovine anestrus and the breeding season.
Results
A total of 483 miRNAs (including 97 known, 369 conserved and 17 predicated novel miRNAs), which belong to 183 different miRNA families, were identified in ovaries of Tan sheep and Small Tail Han (STH) sheep. Compared with the three stages of the breeding season, 25 shared significantly differentially expressed (including 19 up- and six down-regulated) miRNAs were identified in ovine anestrus. KEGG Pathway analysis revealed that the target genes for some of the differentially expressed miRNAs were involved in reproductive hormone related pathways (e.g. steroid biosynthesis, androgen and estrogen metabolism and GnRH signaling pathway) as well as follicular/luteal development related pathways. Moreover, the expression of the differentially expressed miRNAs and most of their target genes were negatively correlated in the above pathways. Furthermore, the levels of estrogen, progesterone and LH in ovine anestrus were significantly lower than those in the breeding season. Combining the results of pathway enrichment analysis, expression of target genes and hormone measurement, we suggest that these differentially expressed miRNAs in anestrus might participate in attenuation of ovarian activity by regulating the above pathways. Besides miRNAs, a large and unexpectedly diverse set of piRNAs were also identified.
Conclusions
The miRNA profiles of ovine ovaries in anestrus were presented for the first time. The identification and characterization of miRNAs that are differentially expressed between ovine anestrus and the breeding season will help understanding of the role of miRNAs in the regulation of seasonal estrus, and provides candidates for determining miRNAs which could be potentially used to regulate ovine seasonal estrus.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-899) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-899
PMCID: PMC4287553  PMID: 25318541
Sheep; Seasonal estrus; Anestrus; Ovary; miRNA; piRNA
4.  Ultra-High Pressure Fast Size Exclusion Chromatography for Top-Down Proteomics 
Proteomics  2013;13(17):10.1002/pmic.201200594.
Top-down mass spectrometry (MS)-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the liquid chromatographic separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. Size exclusion chromatography (SEC) is a favored liquid chromatography method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein we reported the use of ultra-high pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6 – 669 kDa) was achieved in less than 7 min with high-resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size-separation of proteins with great potential for top-down proteomics.
doi:10.1002/pmic.201200594
PMCID: PMC3839113  PMID: 23794208
Protein separation; liquid chromatography; mass spectrometry; UPLC; post-translational modifications
5.  In-depth Proteomic Analysis of Human Tropomyosin by Top-down Mass Spectrometry 
Journal of muscle research and cell motility  2013;34(0):10.1007/s10974-013-9352-y.
Tropomyosins (Tms) are a family of highly conserved actin-binding proteins that play critical roles in a variety of processes, most notably, in the regulation of muscle contraction and relaxation. It is well known that different Tm isoforms have distinct functions and that altered expression of Tm isoforms could lead to changes in cardiac structure and function. To precisely define Tm isoform expression in the human heart, towards a better understanding of their functional roles, we have employed top-down mass spectrometry for in-depth proteomic characterization of Tm isoforms. Using a minimal amount of human heart tissue from rejected donor organs, we confirmed the presence of multiple Tm isoforms including α-Tm, β-Tm and κ-Tm in the human heart, with α-Tm being the predominant isoform, followed by minor isoforms of β-Tm and κ-Tm. Interestingly, our data revealed regional variations of Tm isoforms and posttranslational modifications in the human heart. Specifically, the expression level of κ-Tm was highest in the left atrium but nearly undetectable in the left ventricle. The phosphorylation level of α-Tm (pα-Tm) was significantly higher in the atria than it was in the ventricles. The sequences of all Tm isoforms were characterized and the sites of post-translational modifications were localized. Clearly, top-down mass spectrometry is an attractive method for comprehensive characterization of Tm isoforms and post-translational modifications since it can universally detect and quantify all types of protein modifications without a priori knowledge and without the need for specific antibodies.
doi:10.1007/s10974-013-9352-y
PMCID: PMC3849107  PMID: 23881156
Tropomyosin; Muscle Contraction; Isoforms; Mass Spectrometry; Post-translational modification
6.  Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development 
Molecular microbiology  2013;89(4):660-675.
Summary
Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.
doi:10.1111/mmi.12303
PMCID: PMC3757501  PMID: 23796209
7.  Nonenzymatic Polymerization of Ubiquitin: Single-Step Synthesis and Isolation of Discrete Ubiquitin Oligomers** 
We report a method based on thiol-ene chemistry that enables the synthesis and purification of ubiquitin oligomers with ≥ 4 units. This is the first time in which a free-radical polymerization is used to construct oligomers that functionally mimic natural biopolymers. This approach can be applied towards the synthesis of 6-linked ubiquitin oligomers currently inaccessible by enzymatic methods. Using these chains, one can study their roles in the ubiquitin proteasome system and the DNA damage response pathway.
doi:10.1002/anie.201207171
PMCID: PMC4083817  PMID: 23161800
ubiquitin; site-specific modification; thiol-ene polymerization
8.  Top-down Proteomics Reveals Concerted Reductions in Myofilament and Z-disc Protein Phosphorylation after Acute Myocardial Infarction* 
Molecular & Cellular Proteomics : MCP  2014;13(10):2752-2764.
Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography–mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.
doi:10.1074/mcp.M114.040675
PMCID: PMC4189000  PMID: 24969035
9.  The Impact of Antibody Selection on the Detection of Cardiac Troponin I 
Background
Cardiac troponin I (cTnI) is the current standard biomarker for diagnosing acute myocardial infarction and for risk-stratification of acute coronary syndromes in patients. However it remains unclear how the epitope specificity of antibodies in immunoassays influences the detection of various modified forms of cTnI.
Methods
Four mouse anti-human cTnI monoclonal antibodies targeting different regions of human cTnI were chosen for immunoaffinity purification of cTnI from human and swine cardiac tissue. High-resolution intact protein mass spectrometry was employed to assess the comparative performance of these four antibodies in detecting modified forms of cTnI.
Results
Our data revealed that antibody selection significantly impacts the relative protein yield of cTn from immunoaffinity purification. Remarkably, a single amino acid variation in cTnI (G->S) in the epitope region completely abolished the binding between monoclonal antibody 560 and swine cTnI in solution. Moreover, proteolytic degradation around the epitope region severely compromised the detection of proteolytic fragment forms of cTnI by monoclonal antibodies. In contrast, the phosphorylation status near the epitope region did not significantly affect the antibody recognition of cTnI.
Conclusion
Caution needs to be taken in the interpretation of the data produced by immuno-assays with monoclonal antibodies against various epitopes of cTnI.
doi:10.1016/j.cca.2012.10.034
PMCID: PMC3631584  PMID: 23107929
cardiac troponin; mass spectrometry; epitope; immunoassays; biomarker; acute myocardial infarction
10.  Lnc RNA HOTAIR functions as a competing endogenous RNA to regulate HER2 expression by sponging miR-331-3p in gastric cancer 
Molecular Cancer  2014;13:92.
Background
Accumulating evidence indicates that the long non-coding RNA HOTAIR plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of HOTAIR in gastric carcinogenesis remains largely unknown.
Methods
HOTAIR expression was measured in 78 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of HOTAIR on gastric cancer cells were studied by overexpression and RNA interference approaches in vitro and in vivo. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation (RIP). The positive HOTAIR/HER2 interaction was identified and verified by immunohistochemistry assay and bivariate correlation analysis.
Results
HOTAIR upregulation was associated with larger tumor size, advanced pathological stage and extensive metastasis, and also correlated with shorter overall survival of gastric cancer patients. Furthermore, HOTAIR overexpression promoted the proliferation, migration and invasion of gastric carcinoma cells, while HOTAIR depletion inhibited both cell invasion and cell viability, and induced growth arrest in vitro and in vivo. In particular, HOTAIR may act as a ceRNA, effectively becoming a sink for miR-331-3p, thereby modulating the derepression of HER2 and imposing an additional level of post-transcriptional regulation. Finally, the positive HOTAIR/HER2 correlation was significantly associated with advanced gastric cancers.
Conclusions
HOTAIR overexpression represents a biomarker of poor prognosis in gastric cancer, and may confer malignant phenotype to tumor cells. The ceRNA regulatory network involving HOTAIR and the positive interaction between HOTAIR and HER2 may contribute to a better understanding of gastric cancer pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics against this disease.
doi:10.1186/1476-4598-13-92
PMCID: PMC4021402  PMID: 24775712
Competing endogenous RNA; HER2; HOTAIR; Gastric cancer; Proliferation and invasion
12.  Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium* 
The Journal of Biological Chemistry  2014;289(13):8818-8827.
Background: Myocardial infarction (MI) leads to proteolytic cleavage of cMyBP-C (hC0C1f) and decreased contractility.
Results: hC0C1f can incorporate into the human cardiac sarcomere, depressing force generation and increasing tension cost.
Conclusion: Interaction between hC0C1f and both actin and α-tropomyosin causes disruption of intact cMyBP-C function.
Significance: Proteolytic cleavage of cMyBP-C is sufficient to cause contractile dysfunction following MI.
Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca2+ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca2+ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.
doi:10.1074/jbc.M113.541128
PMCID: PMC3979389  PMID: 24509847
Contractile Protein; Heart Failure; Myocardial Infarction; Protein Degradation; Protein-Protein Interactions
13.  Top-down Targeted Proteomics for Deep Sequencing of Tropomyosin Isoforms 
Journal of proteome research  2012;12(1):187-198.
SUMMARY
Tropomyosins (Tm) constitute a family of ubiquitous and highly conserved actin-binding proteins, playing essential roles in a variety of biological processes. Tm isoforms produced by multiple Tm encoding genes and alternatively expressed exons along with post-translational modifications (PTMs) regulate Tm function. Therefore, to gain a better understanding of the functional role of Tm, it is essential to fully characterize Tm isoforms. Herein, we developed a top-down high-resolution mass spectrometry (MS) based targeted proteomics method for comprehensive characterization of Tm isoforms. α–Tm was identified to be the predominant isoform in swine cardiac muscle. We further characterized its sequence and localized the PTMs such as acetylation and phosphorylation as well as amino acid polymorphisms. Interestingly, we discovered a “novel” Tm isoform that does not match with any of the currently available swine Tm sequences. A deep sequencing of this isoform by top-down MS revealed an exact match with mouse β–Tm sequence, suggesting that this “novel” isoform is swine β–Tm which is 100% conserved between swine and mouse. Taken together, we demonstrated that top-down targeted proteomics provides a powerful tool for deep sequencing of Tm isoforms from genetic variations together with complete mapping of the PTM sites.
doi:10.1021/pr301054n
PMCID: PMC3596867  PMID: 23256820
Tropomyosin; Muscle Contraction; Actin Filament; Mass Spectrometry; Electron Capture Dissociation
14.  Value of multidetector computed tomography in the diagnosis of mucosa-associated lymphoid tissue-lymphomas in the parotid gland: A case report and review of the literature 
Oncology Letters  2013;7(3):781-786.
The present study aimed to review the multidetector computed tomography (MDCT) imaging features of eight mucosa-associated lymphoid tissue (MALT)-lymphoma cases of the parotid gland and to explore the diagnostic value of MDCT. A total of eight patients with pathologically confirmed MALT-lymphomas of the parotid gland underwent pre-operative MDCT plain and dual-phase scans. The changes in the CT values and enhancement patterns of the tumors were assessed. Quantitative analysis was performed to determine the CT value changes of the tumors in the various enhanced phases compared with the plain scan. The MALT-lymphomas of the parotid gland exhibited even density isodense or hyperdense nodules, with occasional calcification and necrosis. The dual-phase scan of the MALT-lymphomas revealed a pattern of lower or moderate enhancement, circumambient enhancement or delayed enhancement. The MALT-lymphomas were closely associated with Sjögre’s syndrome and demonstrated malignant features and isodense or hyperdense nodules and lower or moderate enhancement on the CT scans.
doi:10.3892/ol.2013.1769
PMCID: PMC3919924  PMID: 24520295
parotid gland; mucosa-associated lymphoid tissue lymphomas; computer tomography; enhancement
15.  HNO, Redox Switches, Cardiac Myofilaments, and Heart Failure: A Prequel to Novel Therapeutics? 
Circulation research  2012;111(8):954-956.
doi:10.1161/CIRCRESAHA.112.278416
PMCID: PMC3494458  PMID: 23023505
heart failure; contractility; nitroxyl; disulfide; mass spectrometry
16.  The role of industrial nitrogen in the global nitrogen biogeochemical cycle 
Scientific Reports  2013;3:2579.
Haber-Bosch nitrogen (N) has been increasingly used in industrial products, e.g., nylon, besides fertilizer. Massive numbers of species of industrial reactive N (Nr) have emerged and produced definite consequences but receive little notice. Based on a comprehensive inventory, we show that (1) the industrial N flux has increased globally from 2.5 to 25.4 Tg N yr−1 from 1960 through 2008, comparable to the NOx emissions from fossil fuel combustion; (2) more than 25% of industrial products (primarily structural forms, e.g., nylon) tend to accumulate in human settlements due to their long service lives; (3) emerging Nr species define new N-assimilation and decomposition pathways and change the way that Nr is released to the environment; and (4) the loss of these Nr species to the environment has significant negative human and ecosystem impacts. Incorporating industrial Nr into urban environmental and biogeochemical models could help to advance urban ecology and environmental sciences.
doi:10.1038/srep02579
PMCID: PMC3759834  PMID: 23999540
17.  CistromeMap: a knowledgebase and web server for ChIP-Seq and DNase-Seq studies in mouse and human 
Bioinformatics  2012;28(10):1411-1412.
Summary: Transcription and chromatin regulators, and histone modifications play essential roles in gene expression regulation. We have created CistromeMap as a web server to provide a comprehensive knowledgebase of all of the publicly available ChIP-Seq and DNase-Seq data in mouse and human. We have also manually curated metadata to ensure annotation consistency, and developed a user-friendly display matrix for quick navigation and retrieval of data for specific factors, cells and papers. Finally, we provide users with summary statistics of ChIP-Seq and DNase-Seq studies.
Availability: Freely available on the web at http://cistrome.dfci.harvard.edu/pc/
Contact: yzhang@tongji.edu.cn; xsliu@jimmy.harvard.edu
doi:10.1093/bioinformatics/bts157
PMCID: PMC3348563  PMID: 22495751
18.  The vasodilatory effect of testosterone on renal afferent arterioles 
Gender Medicine  2012;9(2):103-111.
Background
Gender differences exist in a variety of cardiovascular and renal diseases, and testosterone may contribute to the discrepancy. Afferent arterioles (Af-Art) are the major resistance vessels in the kidney, and play an important role in the development of renal injury and hypertension.
Objective
The present study aimed to determine the acute effect and underlying mechanism(s) of testosterone on Af-Art.
Methods
The mRNA expression of androgen receptors (AR) in microdissected Af-Art was measured by RT-PCR. An in vitro microperfusion model was used to measure the diameter of Ar-Art in mice. Nitric oxide (NO) was evaluated by an NO-sensitive fluorescent dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate.
Results
Testosterone had no effect on microperfused Af-Art when added into the bath. Therefore we pre-constricted the Af-Art to about 30% with norepinephrine (NE, 10−6mol/L); administration of testosterone (10−9 to 10−7mol/L) subsequently dilated the Af-Art in a dose-dependent manner (p<0.001; n=7). AR mRNA was expressed in microdissected Af-Art measured by RT-PCR. An AR antagonist, flutamide (10−5mol/L), totally blocked testosterone (10−8mol/L)-induced vasodilator effect. NO production of Af-Art wall was increased when testosterone was added into the bath solution after NE treatment, from 278.4 ± 12.1 units/min to 351.2 ± 33.1 units/min (p<0.05; n=3). In the presence of NO inhibition with NG-nitro-L-arginine methyl ester (L-NAME, 3×10−4mol/L), the testosterone-induced dilatation was blunted compared with NE (p<0.05).
Conclusions
We conclude that testosterone dilates pre-constricted mouse Af-Art in a dose-dependent manner by activation of AR and partially mediated by NO.
doi:10.1016/j.genm.2012.02.003
PMCID: PMC3322301  PMID: 22445695
kidney; testosterone; androgen receptors; nitric oxide
19.  The Role of Community Mixing Styles in Shaping Epidemic Behaviors in Weighted Networks 
PLoS ONE  2013;8(2):e57100.
The dynamics of infectious diseases that are spread through direct contact have been proven to depend on the strength of community structure or modularity within the underlying network. It has been recently shown that weighted networks with similar modularity values may exhibit different mixing styles regarding the number of connections among communities and their respective weights. However, the effect of mixing style on epidemic behavior was still unclear. In this paper, we simulate the spread of disease within networks with different mixing styles: a dense-weak style (i.e., many edges among the communities with small weights) and a sparse-strong style (i.e., a few edges among the communities with large weights). Simulation results show that, with the same modularity: 1) the mixing style significantly influences the epidemic size, speed, pattern and immunization strategy; 2) the increase of the number of communities amplifies the effect of the mixing style; 3) when the mixing style changes from sparse-strong to dense-weak, there is a ‘saturation point’, after which the epidemic size and pattern become stable. We also provide a mean-field solution of the epidemic threshold and size on weighted community networks with arbitrary external and internal degree distribution. The solution explains the effect of the second moment of the degree distribution, and a symmetric effect of internal and external connections (incl. degree distribution and weight). Our study has both potential significance for designing more accurate metrics for the community structure and exploring diffusion dynamics on metapopulation networks.
doi:10.1371/journal.pone.0057100
PMCID: PMC3577779  PMID: 23437321
20.  Comprehensive Analysis of Protein Modifications by Top-down Mass Spectrometry 
Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research.
doi:10.1161/CIRCGENETICS.110.957829
PMCID: PMC3320739  PMID: 22187450
Cardiovascular diseases; Proteomics; Electron Capture dissociation; Post-translational modification; Top-Down Mass Spectrometry
21.  A comparative study of 22-channel water-perfusion system and solid-state system with 36-sensors in esophageal manometery 
BMC Gastroenterology  2012;12:157.
Background
To compare the characteristics between 22-channel water-perfusion manometry (WPM) and solid-state manometry (SSM) with 36 sensors of the pressure measurements, as well as patients’ discomfort indices in nose and pharynx, the preparation and operation time of the manometry.
Methods
12 volunteers were included in the study. Each of the volunteers underwent esophageal manometry by both 22-channel water-perfusion catheter (WPC) and solid-state catheter (SSC) with 36 sensors in random order, and separated by 30 min. The subjects gave a VAS score soon after each test. Non-parametric tests were used to analyze the differences and Bland-Altman plots were used to assess the consistency of the two systems.
Results
During the wet swallows, there were significant differences between the two systems in three measurements of location of lower esophageal sphincter (LES) upper margin (Z = -2.11, P = 0.035), LES relax ratio (Z = -2.20, P = 0.028) and IRP4s (Z = -2.05, P = 0.041). During the jelly pocket swallows, LES relax ratio measurements of the two systems showed significant differences (Z = -2.805, P = 0.005). Further Bland–Altman plots analysis presented good agreement between the two systems measurements of location of LES upper margin, LES relax ratio and IRP4s. The discomfort indices of subjects’ nasal sensation were higher when inserting the solid-state catheter [5(3.75-5)] than water-perfusion one (2.5(2-4)) (Z = -2.471, P = 0.013), as well as the discomfort indices of pharyngeal sensation (7.5(4.75-9) vs. 4.5(3.75-6.5)), (Z = -2.354, P = 0.019). The preparation time for WPC was 40(39-41) minutes, which was much longer than that for SSC 32.5(31.75-33) minutes, (Z = -3.087, P = 0.002). And the nurses reported it’s much easier to insert WPC (Z = -3.126, P = 0.002).
Conclusions
In conclusion, most pressure measurements were consistent between WPM and SSM. Patients tolerated better with WPC, while for operators, the SSC presented more convenient.
doi:10.1186/1471-230X-12-157
PMCID: PMC3503791  PMID: 23134719
22-channel water-perfusion manometry; Solid-state manometry (SSM) with 36 sensors; Pressure measurements; Patients’ tolerance; Operators’ convenience; Comparative study
22.  Esophageal intraluminal baseline impedance is associated with severity of acid reflux and epithelial structural abnormalities in patients with gastroesophageal reflux disease 
Journal of Gastroenterology  2012;48(5):601-610.
Background
The esophageal intraluminal baseline impedance may be used to evaluate the status of mucosa integrity. Esophageal acid exposure decreases the baseline impedance. We aimed to compare baseline impedance in patients with various reflux events and with different acid-related parameters, and investigate the relationships between epithelial histopathologic abnormalities and baseline impedance.
Methods
A total of 229 GERD patients and 34 controls underwent 24-h multichannel intraluminal impedance and pH monitoring (MII–pH monitoring), gastroendoscopy, and completed a GERD questionnaire (GerdQ). We quantified epithelial intercellular spaces (ICSs) and expression of tight junction (TJ) proteins by histologic techniques.
Results
Mean baseline values in reflux esophagitis (RE) (1752 ± 1018 Ω) and non-erosive reflux disease (NERD) (2640 ± 1143 Ω) were significantly lower than in controls (3360 ± 1258 Ω; p < 0.001 and p = 0.001, respectively). Among NERD subgroups, mean baselines in the acid reflux group (2510 ± 1239 Ω) and mixed acid/weakly acidic reflux group (2393 ± 1009 Ω) were much lower than in controls (3360 ± 1258 Ω; p = 0.020 and p < 0.001, respectively). The mean baseline in severe RE patients was significantly lower than in mild RE patients (LA-C/D vs. LA-A/B: 970 ± 505 Ω vs. 1921 ± 1024 Ω, p < 0.001). There was a significant negative correlation between baseline value and acid exposure time (AET) (r = −0.41, p < 0.001), and a weak but significant correlation (r = −0.20, p = 0.007) between baseline value and weakly AET. Negative correlations were observed between ICS and the baseline impedance (r = −0.637, p < 0.001) and claudin-1 and the baseline impedance (r = −0.648, p < 0.001).
Conclusions
Patients with dominant acid reflux events and with longer AET have low baseline impedance. Baseline values are correlated with esophageal mucosal histopathologic changes such as dilated ICS and TJ alteration.
Electronic supplementary material
The online version of this article (doi:10.1007/s00535-012-0689-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00535-012-0689-6
PMCID: PMC3654188  PMID: 23076541
Baseline impedance; Acid reflux; Intercellular spaces; Tight junction
23.  Top-Down Quantitative Proteomics Identified Phosphorylation of Cardiac Troponin I as a Candidate Biomarker for Chronic Heart Failure 
Journal of proteome research  2011;10(9):4054-4065.
The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We have systematically analyzed thirty-six clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%Ptotal) were 56.4±3.5%, 36.9±1.6%, 6.1±2.4%, and 1.0±0.6% for postmortem hearts with normal cardiac function (n=7), early-stage of mild hypertrophy (n=5), severe hypertrophy/dilation (n=4), and end-stage CHF (n=6), respectively. In fresh transplant samples, the %Ptotal of cTnI from non-failing donor (n=4), and end-stage failing hearts (n=10) were 49.5±5.9% and 18.8±2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTM as disease biomarkers.
doi:10.1021/pr200258m
PMCID: PMC3170873  PMID: 21751783
Heart failure; Phosphorylation; Quantitative Proteomics; Top-Down Mass Spectrometry; Post-translational Modification; Cardiac troponin I
24.  In Vivo Phosphorylation Site Mapping in Mouse Cardiac Troponin I by High Resolution Top-Down Electron Capture Dissociation Mass Spectrometry: Ser22/23 Are the Only Sites Basally Phosphorylated† 
Biochemistry  2009;48(34):8161-8170.
Cardiac troponin I (cTnI) is the inhibitory subunit of cardiac troponin, a key myofilament regulatory protein complex located on the thin filaments of the contractile apparatus. cTnI is uniquely specific for the heart and is widely used in clinics as a serum biomarker for cardiac injury. Phosphorylation of cTnI plays a critical role in modulating cardiac function. cTnI is known to be regulated by protein kinase A and protein kinase C at five sites, Ser22/Ser23, Ser42/44, and Thr143, primarily based on results from in vitro phosphorylation assays by the specific kinase(s). However, a comprehensive characterization of phosphorylation of mouse cTnI occurring in vivo has been lacking. Herein, we have employed top-down mass spectrometry (MS) methodology with electron capture dissociation for precise mapping of in vivo phosphorylation sites of cTnI affinity purified from wild-type and transgenic mouse hearts. As demonstrated, top-down MS (analysis of intact proteins) is an extremely valuable technology for global characterization of labile phosphorylation occurring in vivo without a priori knowledge. Our top-down MS data unambiguously identified Ser22/23 as the only two sites basally phosphorylated in wild-type mouse cTnI with full sequence coverage, which was confirmed by the lack of phosphorylation in cTnI-Ala2 transgenic mice where Ser22/23 in cTnI have been rendered nonphosphorylatable by mutation to alanine.
doi:10.1021/bi900739f
PMCID: PMC3341416  PMID: 19637843
25.  The Protein Partners of GTP Cyclohydrolase I in Rat Organs 
PLoS ONE  2012;7(3):e33991.
Objective
GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat.
Methods and Results
A pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria.
Conclusion
GCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis.
doi:10.1371/journal.pone.0033991
PMCID: PMC3313957  PMID: 22479495

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