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1.  A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum 
Journal of proteome research  2013;13(2):875-882.
Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase LC separations to fractionate and select target fractions for follow-on LC-SRM analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and LOQ values of ~130 pg/mL in serum and ~10 pg per 100 μg total protein mass in urine, respectively. A good correlation (R2 = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and ELISA. Based on an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between non-cancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.
doi:10.1021/pr400912c
PMCID: PMC3975687  PMID: 24251762
AGR2; PSA; prostate cancer; PRISM-SRM; human urine; human serum
2.  A Metabolomics Approach to Stratify Patients Diagnosed with Diabetes Mellitus into Excess or Deficiency Syndromes 
The prevalence of type 2 diabetes continuously increases globally. The traditional Chinese medicine (TCM) can stratify the diabetic patients based on their different TCM syndromes and, thus, allow a personalized treatment. Metabolomics is able to provide metabolite biomarkers for disease subtypes. In this study, we applied a metabolomics approach using an ultraperformance liquid chromatography (UPLC) coupled with quadruple-time-of-flight (QTOF) mass spectrometry system to characterize the metabolic alterations of different TCM syndromes including excess and deficiency in patients diagnosed with diabetes mellitus (DM). We obtained a snapshot of the distinct metabolic changes of DM patients with different TCM syndromes. DM patients with excess syndrome have higher serum 2-indolecarboxylic acid, hypotaurine, pipecolic acid, and progesterone in comparison to those patients with deficiency syndrome. The excess patients have more oxidative stress as demonstrated by unique metabolite signatures than the deficiency subjects. The results provide an improved understanding of the systemic alteration of metabolites in different syndromes of DM. The identified serum metabolites may be of clinical relevance for subtyping of diabetic patients, leading to a personalized DM treatment.
doi:10.1155/2015/350703
PMCID: PMC4312632  PMID: 25667595
3.  Complete Genome Sequence of the Fish Pathogen Yersinia ruckeri Strain SC09, Isolated from Diseased Ictalurus punctatus in China 
Genome Announcements  2015;3(1):e01327-14.
Yersinia ruckeri SC09 is a Gram-negative bacterium isolated from a moribund Ictalurus punctatus collected in Jianyang, China. Here, we report the complete genome sequence of this microorganism to facilitate the investigation of its pathogenicity and to reevaluate its taxonomic position.
doi:10.1128/genomeA.01327-14
PMCID: PMC4290980  PMID: 25573927
4.  Altered Brain Activation in Early Drug-Naive Parkinson's Disease during Heat Pain Stimuli: An fMRI Study 
Parkinson's Disease  2015;2015:273019.
Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by motor and nonmotor signs and symptoms. To date, many studies of PD have focused on its cardinal motor symptoms. To study the nonmotor signs of early PD, we investigated the reactions solicited by heat pain stimuli in early untreated PD patients without pain using fMRI. The activation patterns of contact heat stimuli (51°C) were assessed in 14 patients and 17 age- and sex-matched healthy controls. Patients with PD showed significant decreases in activation of the superior temporal gyrus (STG) and insula compared with controls. In addition, a significant relationship between activation of the insula and STG and the pain scores was observed in healthy controls but not in PD. This study provided further support that the insula and STG are important parts of the somatosensory circuitry recruited during the period of pain. The hypoactivity of the STG and insula in PD implied that functions including affective, cognitive, and sensory-discriminative processes, which are associated with the insula and STG, were disturbed. This finding supports the view that leaving early PD untreated could be tied directly to central nervous system dysfunction.
doi:10.1155/2015/273019
PMCID: PMC4299805  PMID: 25628915
5.  Proteomic approaches for site-specific O-GlcNAcylation analysis 
Bioanalysis  2014;6(19):2571-2580.
O -GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago and its significance has been implicated in several disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically challenging to characterize comprehensively and quantitatively because of its low abundance, low stoichiometry and extremely labile nature under conventional collision-induced dissociation tandem MS conditions. Herein, we review the recent advances addressing these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, including specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification and quantitative analysis of O-GlcNAcylation.
doi:10.4155/bio.14.239
PMCID: PMC4275047  PMID: 25411699
6.  An Amino Terminal Phosphorylation Motif Regulates Intranuclear Compartmentalization of Olig2 in Neural Progenitor Cells 
The Journal of Neuroscience  2014;34(25):8507-8518.
The bHLH transcription factor Olig2 is expressed in cycling neural progenitor cells but also in terminally differentiated, myelinating oligodendrocytes. Sustained expression of Olig2 is counterintuitive because all known functions of the protein in expansion of neural progenitors and specification of oligodendrocyte progenitors are completed with the formation of mature white matter. How are the biological functions of Olig2 suppressed in terminally differentiated oligodendrocytes? In previous studies, we have shown that a triple serine motif in the amino terminus of Olig2 is phosphorylated in cycling neural progenitors but not in their differentiated progeny. We now show that phosphorylation of the triple serine motif regulates intranuclear compartmentalization of murine Olig2. Phosphorylated Olig2 is preferentially localized to a transcriptionally active “open” chromatin compartment together with coregulator proteins essential for regulation of gene expression. Unphosphorylated Olig2, as seen in mature white matter, is localized mainly within a transcriptionally inactive, chromatin fraction characterized by condensed and inaccessible DNA. Of special note is the observation that the p53 tumor suppressor protein is confined to the open chromatin fraction. Proximity ligation assays show that phosphorylation brings Olig2 within 30 nm of p53 within the open chromatin compartment. The data thus shed light on previously noted promitogenic functions of phosphorylated Olig2, which reflect, at least in part, an oppositional relationship with p53 functions.
doi:10.1523/JNEUROSCI.0309-14.2014
PMCID: PMC4061392  PMID: 24948806
bHLH; intranuclear localization; NuRD complex; Olig2; p53; phosphorylation
7.  Leukotriene C4 Activates Mouse Platelets in Plasma Exclusively Through the Type 2 Cysteinyl Leukotriene Receptor1 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(12):10.4049/jimmunol.1302187.
Leukotriene (LT)C4 and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (CysLT1R, CysLT2R, and GPR99) with overlapping ligand preferences. Here we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by WT mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2−/−) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte/platelet complexes that facilitate inflammation.
doi:10.4049/jimmunol.1302187
PMCID: PMC3869987  PMID: 24244016
8.  Proteogenomic strategies for identification of aberrant cancer peptides using large-scale Next Generation Sequencing data 
Proteomics  2014;14(0):2719-2730.
Cancer is driven by the acquisition of somatic DNA lesions. Distinguishing the early driver mutations from subsequent passenger mutations is key to molecular sub-typing of cancers, understanding cancer progression, and the discovery of novel biomarkers. The advances of genomics technologies (whole-genome exome, and transcript sequencing, collectively referred to as NGS(Next Gengeration Sequencing)) have fueled recent studies on somatic mutation discovery. However, the vision is challenged by the complexity, redundancy, and errors in genomic data, and the difficulty of investigating the proteome translated portion of aberrant genes using only genomic approaches. Combination of proteomic and genomic technologies are increasingly being employed.
Various strategies have been employed to allow the usage of large scale NGS data for conventional MS/MS searches. This paper provides a discussion of applying different strategies relating to large database search, and FDR(False Discovery Rate) based error control, and their implication to cancer proteogenomics. Moreover, it extends and develops the idea of a unified genomic variant database that can be searched by any mass spectrometry sample. A total of 879 BAM files downloaded from TCGA repository were used to create a 4.34 GB unified FASTA database which contained 2, 787, 062 novel splice junctions, 38, 464 deletions, 1, 105 insertions, and 182, 302 substitutions. Proteomic data from a single ovarian carcinoma sample (439, 858 spectra) was searched against the database. By applying the most conservative FDR measure, we have identified 524 novel peptides and 65, 578 known peptides at 1% FDR threshold. The novel peptides include interesting examples of doubly mutated peptides, frame-shifts, and non-sample-recruited mutations, which emphasize the strength of our approach.
doi:10.1002/pmic.201400206
PMCID: PMC4256132  PMID: 25263569
9.  Targeting Src and tubulin in mucinous ovarian carcinoma 
Purpose
To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma.
Experimental design
The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models.
Results
In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2/M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01-sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules.
Conclusion
Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma.
doi:10.1158/1078-0432.CCR-13-1305
PMCID: PMC3852199  PMID: 24100628
Ovarian carcinoma; Mucinous; Src kinase; Tubulin; KX-01
10.  Inhibition of itch-related responses by selectively ablated serotonergic signals at the rostral ventromedial medulla in mice 
Descending control of nociceptive processing in the rostral ventromedial medulla (RVM) has been implicated in the inhibition and facilitation of spinal nociceptive transmission. Here we investigated the contribution of serotonergic (5-HT) pathway at the RVM to pruritic behavior. Selective lesion of the descending serotonergic pathway by intra-RVM injection of focal neurotoxin 5,7-dihydroxytryptamine (2 μg/0.5 μl) attenuated pruritic behavior at the 30-min observation period following an intradermal microinjection of compound 48/80 (100 μg/100 μl) in the nape of the neck. Intradermal microinjection of compound 48/80 resulted in a dramatic increase in itch behavior between naive group and saline group. 5,7-DHT-treated mice showed profound scratching deficits after intradermal injection of compound 48/80. 5,7-DHT treatment resulted in a significant decrease in the number of 5-HT positive neurons in the RVM by using intracisternal injection of the serotonin neurotoxin 5,7-DHT. These findings demonstrate that pruritic behavior is dependent in part on descending facilitation via the RVM, and identify a modulatory role of serotonergic pathway at the RVM for pruritic behavior.
PMCID: PMC4314012
Itch; serotonergic signals; the rostral ventromedial medulla
11.  Urethral Reconstruction with Tissue-Engineered Human Amniotic Scaffold in Rabbit Urethral Injury Models 
Background
Mitigating urethral injury remains a great challenge for urologists due to lack of ideal biomaterials for urethroplasty. The application of amniotic membrane (AM) over other synthetic materials makes it a better potential source for urethral reconstruction. We separated the basement layer of AM to obtain denuded human amniotic scaffold (dHAS) and then inoculated primary rabbit urethral epithelial cells on the surface of dHAS to determine whether this strategy minimizes potential rejection and maximizes the biocompatibility of human AM.
Material/Methods
After the successful acquisition of dHAS from AM, cell-seeded dHAS were prepared and characterized. Both cell-seeded dHAS and acellular dHAS were subcutaneously implanted. Immune responses were compared by histological evaluation and CD4+ cell and CD8+ cell infiltrations. Then they were applied as urethroplastic materials in the rabbit models of urethral injury to fully explore the feasibility and efficacy of tissue-engineered dHAS xenografts in urethral substitution application.
Results
Mild inflammatory infiltration was observed in cell-seeded dHAS grafts, as revealed by fewer accumulations of CD4+ cells and CD8+ cells (or neutrophils or other immune cells). Urethral defects of rabbits in the urethroplastic group with dHAS implantation (n=6) were completely resolved in 1 month, while there were 1 infection and 1 fistula in the control group with acellular dHAS patches (n=6). Histopathological analysis revealed mild immune response in the cell-seeded dHAS group (P<0.05).
Conclusions
Tissue-engineered dHAS minimizes potential rejection and maximizes the biocompatibility of AM, which makes it a potential ideal xenograft for urethral reconstruction.
doi:10.12659/MSM.891042
PMCID: PMC4257484  PMID: 25424000
Amnion; Anastomosis, Surgical; Immunity, Active; Transplantation, Heterologous; Urethral Stricture
12.  Neural mechanisms and potential treatment of epilepsy and its complications 
The factors underlying epilepsy are multifaceted, but recent research suggests that the brain’s neural circuits, which play a key role in controlling the balance between epileptic and antiepileptic factors, may lie at the heart of epilepsy. This article provides a comprehensive review of the neural mechanisms and potential treatment of intractable epilepsy from neural inflammatory responses, melanocortin circuits in brain and pedunculopontine tegmental nucleus. Further studies should be undertaken to elucidate the nature of neural circuits so that we may more effectively apply these new preventive and symptomatic therapies to the patient suffering from medically refractory seizures and its complications.
PMCID: PMC4297332  PMID: 25628775
Intractable epilepsy; neural inflammatory responses; melanocortin circuits; pedunculopontine tegmental nucleus
13.  Comparative analysis of metazoan chromatin organization 
Ho, Joshua W. K. | Jung, Youngsook L. | Liu, Tao | Alver, Burak H. | Lee, Soohyun | Ikegami, Kohta | Sohn, Kyung-Ah | Minoda, Aki | Tolstorukov, Michael Y. | Appert, Alex | Parker, Stephen C. J. | Gu, Tingting | Kundaje, Anshul | Riddle, Nicole C. | Bishop, Eric | Egelhofer, Thea A. | Hu, Sheng’en Shawn | Alekseyenko, Artyom A. | Rechtsteiner, Andreas | Asker, Dalal | Belsky, Jason A. | Bowman, Sarah K. | Chen, Q. Brent | Chen, Ron A-J | Day, Daniel S. | Dong, Yan | Dose, Andrea C. | Duan, Xikun | Epstein, Charles B. | Ercan, Sevinc | Feingold, Elise A. | Ferrari, Francesco | Garrigues, Jacob M. | Gehlenborg, Nils | Good, Peter J. | Haseley, Psalm | He, Daniel | Herrmann, Moritz | Hoffman, Michael M. | Jeffers, Tess E. | Kharchenko, Peter V. | Kolasinska-Zwierz, Paulina | Kotwaliwale, Chitra V. | Kumar, Nischay | Langley, Sasha A. | Larschan, Erica N. | Latorre, Isabel | Libbrecht, Maxwell W. | Lin, Xueqiu | Park, Richard | Pazin, Michael J. | Pham, Hoang N. | Plachetka, Annette | Qin, Bo | Schwartz, Yuri B. | Shoresh, Noam | Stempor, Przemyslaw | Vielle, Anne | Wang, Chengyang | Whittle, Christina M. | Xue, Huiling | Kingston, Robert E. | Kim, Ju Han | Bernstein, Bradley E. | Dernburg, Abby F. | Pirrotta, Vincenzo | Kuroda, Mitzi I. | Noble, William S. | Tullius, Thomas D. | Kellis, Manolis | MacAlpine, David M. | Strome, Susan | Elgin, Sarah C. R. | Liu, Xiaole Shirley | Lieb, Jason D. | Ahringer, Julie | Karpen, Gary H. | Park, Peter J.
Nature  2014;512(7515):449-452.
doi:10.1038/nature13415
PMCID: PMC4227084  PMID: 25164756
14.  Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid 
Analytical and bioanalytical chemistry  2014;406(28):7117-7125.
Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 μL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ~6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.
doi:10.1007/s00216-014-8058-3
PMCID: PMC4208972  PMID: 25192788
Microscale depletion; IgY-14 immunoaffinity resin; Human plasma; Cerebrospinal fluid; MS
15.  Fas signaling promotes chemoresistance in gastrointestinal cancer by up-regulating P-glycoprotein 
Oncotarget  2014;5(21):10763-10777.
Fas signaling promotes metastasis of gastrointestinal (GI) cancer cells by inducing epithelial-mesenchymal transition (EMT), and EMT acquisition has been found to cause cancer chemoresistance. Here, we demonstrated that the response to chemotherapy of GI cancer patients with higher expression of FasL was significantly worse than patients with lower expression. Fas-induced activation of the ERK1/2-MAPK pathway decreased the sensitivity of GI cancer cells to chemotherapeutic agents and promoted the expression of P-glycoprotein (P-gp). FasL promoted chemoresistance of GI cancer cell via upregulation of P-gp by increasing β-catenin and decreasing miR-145. β-catenin promoted P-gp gene transcription by binding with P-gp promoter while miR-145 suppressed P-gp expression by interacting with the mRNA 3′UTR of P-gp. Immunostaining and qRT-PCR analysis of human GI cancer samples revealed a positive association among FasL, β-catenin, and P-gp, but a negative correlation between miR-145 and FasL or P-gp. Altogether, our results showed Fas signaling could promote chemoresistance in GI cancer through modulation of P-gp expression by β-catenin and miR-145. Our findings suggest that Fas signaling-based cancer therapies should be administered cautiously, as activation of this pathway may not only lead to apoptosis but also induce chemoresistance.
PMCID: PMC4279408  PMID: 25333257
Fas signaling; epithelial-mesenchymal transition; chemoresistance; gastrointestinal cancer
16.  The spatial distribution of health vulnerability to heat waves in Guangdong Province, China 
Global Health Action  2014;7:10.3402/gha.v7.25051.
Background
International literature has illustrated that the health impacts of heat waves vary according to differences in the spatial variability of high temperatures and the social and economic characteristics of populations and communities. However, to date there have been few studies that quantitatively assess the health vulnerability to heat waves in China.
Objectives
To assess the spatial distribution of health vulnerability to heat waves in Guangdong Province, China.
Methods
A vulnerability framework including dimensions of exposure, sensitivity, and adaptive capacity was employed. The last two dimensions were called social vulnerability. An indicator pool was proposed with reference to relevant literatures, local context provided by relevant local stakeholder experts, and data availability. An analytic hierarchy process (AHP) and a principal component analysis were used to determine the weight of indicators. A multiplicative vulnerability index (VI) was constructed for each district/county of Guangdong province, China.
Results
A total of 13 items (two for exposure, six for sensitivity, and five for adaptive capacity) were proposed to assess vulnerability. The results of an AHP revealed that the average VI in Guangdong Province was 0.26 with the highest in the Lianzhou and Liannan counties of Qingyuan (VI=0.50) and the lowest in the Yantian district of Shenzhen (VI=0.08). Vulnerability was gradiently distributed with higher levels in northern inland regions and lower levels in southern coastal regions. In the principal component analysis, three components were isolated from the 11 social vulnerability indicators. The estimated vulnerability had a similar distribution pattern with that estimated by AHP (Intraclass correlation coefficient (ICC)=0.98, p<0.01).
Conclusions
Health vulnerability to heat waves in Guangdong Province had a distinct spatial distribution, with higher levels in northern inland regions than that in the southern coastal regions.
doi:10.3402/gha.v7.25051
PMCID: PMC4212080  PMID: 25361724
vulnerability assessment; heat waves; climate change; analytic hierarchy process; principal component analysis
17.  MicroRNA-21 Promotes Cell Growth and Migration by Targeting Programmed Cell Death 4 Gene in Kazakh's Esophageal Squamous Cell Carcinoma 
Disease Markers  2014;2014:232837.
Esophageal cancer (EC) is the eighth most common cancer worldwide and the sixth most common cause of cancer death. There are two main types of EC—squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). Although some advances in the exploration of its possible etiological mechanism were made recently including behaviors and environmental risk factors as well as gene alterations, the molecular mechanism underlying ESCC carcinogenesis and progression remains poorly understood. It has been reported that miR-21 was upregulated in most malignant cancers, the proposed mechanism of which was through suppressing expression of programmed cell death 4 (PDCD4). In present study, it is firstly reported that miR-21 was upregulated in Kazakh's ESCC and that miR-21 played a negative role in regulating PDCD4 using in situ hybridization (ISH) and luciferase reporter approach. Morever, in model of ESCC xenografted nude mice, miR-21 maybe used as an effective target in the treatment. The present results demonstrated that miR-21 may be a potential therapeutic target in management of ESCC.
doi:10.1155/2014/232837
PMCID: PMC4221975  PMID: 25400316
18.  Production of salidroside in metabolically engineered Escherichia coli 
Scientific Reports  2014;4:6640.
Salidroside (1) is the most important bioactive component of Rhodiola (also called as “Tibetan Ginseng”), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.
doi:10.1038/srep06640
PMCID: PMC4200411  PMID: 25323006
19.  Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer 
Molecular oncology  2014;8(7):1169-1180.
Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing “signature” peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.
doi:10.1016/j.molonc.2014.02.004
PMCID: PMC4183720  PMID: 25266362
TMPRSS2-ERG gene fusion; ERG protein isoform; PRISM-SRM; Targeted quantification; Prostate cancer
20.  Phylogeny of C4-Photosynthesis Enzymes Based on Algal Transcriptomic and Genomic Data Supports an Archaeal/Proteobacterial Origin and Multiple Duplication for Most C4-Related Genes 
PLoS ONE  2014;9(10):e110154.
Both Calvin-Benson-Bassham (C3) and Hatch-Slack (C4) cycles are most important autotrophic CO2 fixation pathways on today’s Earth. C3 cycle is believed to be originated from cyanobacterial endosymbiosis. However, studies on evolution of different biochemical variants of C4 photosynthesis are limited to tracheophytes and origins of C4-cycle genes are not clear till now. Our comprehensive analyses on bioinformatics and phylogenetics of novel transcriptomic sequencing data of 21 rhodophytes and 19 Phaeophyceae marine species and public genomic data of more algae, tracheophytes, cyanobacteria, proteobacteria and archaea revealed the origin and evolution of C4 cycle-related genes. Almost all of C4-related genes were annotated in extensive algal lineages with proteobacterial or archaeal origins, except for phosphoenolpyruvate carboxykinase (PCK) and aspartate aminotransferase (AST) with both cyanobacterial and archaeal/proteobacterial origin. Notably, cyanobacteria may not possess complete C4 pathway because of the flawed annotation of pyruvate orthophosphate dikinase (PPDK) genes in public data. Most C4 cycle-related genes endured duplication and gave rise to functional differentiation and adaptation in different algal lineages. C4-related genes of NAD-ME (NAD-malic enzyme) and PCK subtypes exist in most algae and may be primitive ones, while NADP-ME (NADP-malic enzyme) subtype genes might evolve from NAD-ME subtype by gene duplication in chlorophytes and tracheophytes.
doi:10.1371/journal.pone.0110154
PMCID: PMC4196954  PMID: 25313828
21.  Expediting SRM assay development for large-scale targeted proteomics experiments 
Journal of proteome research  2014;13(10):4479-4487.
Due to their high sensitivity and specificity, selected reaction monitoring (SRM) based targeted proteomics has become increasingly popular for biological and translational applications. Selection of optimal transitions and optimization of collision energy (CE) are important assay development steps for achieving sensitive detection and accurate quantification; however, these steps can be labor-intensive, especially, for large-scale applications. Herein, we explored several options for accelerating SRM assay development evaluated in the context of a relatively large set of 215 synthetic peptide targets. We first showed that HCD fragmentation is very similar to CID in triple quadrupole (QQQ) instrumentation, and by selection of the top six y fragment ions from HCD spectra, >86% of the top transitions optimized from direct infusion on QQQ instrument are covered. We also demonstrated that the CE calculated by existing prediction tools was less accurate for 3+ precursors, and a significant increase in intensity for transitions could be obtained using a new CE prediction equation constructed from the present experimental data. Overall, our study illustrated the feasibility of expediting the development of larger numbers of high-sensitivity SRM assays through automation of transition selection and accurate prediction of optimal CE to improve both SRM throughput and measurement quality.
doi:10.1021/pr500500d
PMCID: PMC4184450  PMID: 25145539
SRM; MRM; HCD; QQQ; transition selection; optimization; CE prediction; targeted quantification
22.  Role of Focal Adhesion Kinase in Regulating YB–1–Mediated Paclitaxel Resistance in Ovarian Cancer 
Background
We previously found focal adhesion kinase (FAK) inhibition sensitizes ovarian cancer to taxanes; however, the mechanisms are not well understood.
Methods
We characterized the biologic response of taxane-resistant and taxane-sensitive ovarian cancer models to a novel FAK inhibitor (VS-6063). We used reverse-phase protein arrays (RPPA) to identify novel downstream targets in taxane-resistant cell lines. Furthermore, we correlated clinical and pathological data with nuclear and cytoplasmic expression of FAK and YB-1 in 105 ovarian cancer samples. Statistical tests were two-sided, and P values were calculated with Student t test or Fisher exact test.
Results
We found that VS-6063 inhibited FAK phosphorylation at the Tyr397 site in a time- and dose-dependent manner. The combination of VS-6063 and paclitaxel markedly decreased proliferation and increased apoptosis, which resulted in 92.7% to 97.9% reductions in tumor weight. RPPA data showed that VS-6063 reduced levels of AKT and YB-1 in taxane-resistant cell lines. FAK inhibition enhanced chemosensitivity in taxane-resistant cells by decreasing YB-1 phosphorylation and subsequently CD44 in an AKT-dependent manner. In human ovarian cancer samples, nuclear FAK expression was associated with increased nuclear YB-1 expression (χ 2 = 37.7; P < .001). Coexpression of nuclear FAK and YB-1 was associated with statistically significantly worse median overall survival (24.9 vs 67.3 months; hazard ratio = 2.64; 95% confidence interval = 1.38 to 5.05; P = .006).
Conclusions
We have identified a novel pathway whereby FAK inhibition with VS-6063 overcomes YB-1–mediated paclitaxel resistance by an AKT-dependent pathway. These findings have implications for clinical trials aimed at targeting FAK.
doi:10.1093/jnci/djt210
PMCID: PMC3787907  PMID: 24062525
23.  Long-gradient separations coupled with selected reaction monitoring for highly sensitive, large scale targeted protein quantification in a single analysis 
Analytical chemistry  2013;85(19):10.1021/ac402105s.
Long-gradient separations coupled to tandem MS were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional LC-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in LOQ for target proteins in human female serum. Based on at least one surrogate peptide per protein, an LOQ of 10 ng/mL was achieved for the two spiked proteins in non-depleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or sub-ng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and ELISA measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM potentially offers much higher multiplexing capacity than conventional LC-SRM due to an increase in average peak widths (~3-fold) for a 300-min gradient compared to a 45-min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.
doi:10.1021/ac402105s
PMCID: PMC3839867  PMID: 24004026
long-gradient; targeted quantification; low-abundance protein; human serum; sensitivity; reproducibility
24.  The prenatal origins of cancer 
Nature reviews. Cancer  2014;14(4):277-289.
The concept that some childhood malignancies arise from postnatally persistent embryonal remnant or rest cells has a long history. Recent research has strengthened the links between driver mutations, and, embryonal and early postnatal development. This evidence, coupled with much greater detail on the cell of origin and the initial steps in embryonal cancer initiation, has identified important therapeutic targets and provided renewed interest in strategies for the early detection and prevention of childhood cancer.
doi:10.1038/nrc3679
PMCID: PMC4041218  PMID: 24599217
25.  TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells 
Oncotarget  2014;5(20):10127-10139.
High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma.
PMCID: PMC4259410  PMID: 25333256
Melanoma; TRIM16; BRAF inhibitor; cell migration; IFNβ1

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