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1.  Recoding RNA editing of antizyme inhibitor 1 predisposes to hepatocellular carcinoma 
Nature medicine  2013;19(2):209-216.
Better understanding of human hepatocellular carcinoma (HCC) pathogenesis at the molecular level will facilitate the discovery of tumor initiating events. Herein, transcriptome sequencing revealed that adenosine (A)-to-inosine (I) RNA editing of antizyme inhibitor 1 (AZIN1) displays a high modification rate in HCC specimens. A-to-I editing of AZIN1 transcripts is specifically regulated by adenosine deaminase acting on RNA-1 (ADAR1). The serine (S) → glycine (G) substitution at residue 367, located in β-strand 15 (β15), predicted a conformational change, induced a cytoplasmic-to-nuclear translocation, and conferred “gain-of-function” phenotypes manifested by augmented tumor initiating potential and more aggressive behavior. Compared with wild-type AZIN1 protein, the edited form possesses stronger affinity to antizyme, and the resultant higher protein stability promotes cell proliferation via the neutralization of antizyme-mediated degradation of ornithine decarboxylase (ODC) and cyclin D1 (CCND1). Collectively, A-to-I RNA editing of AZIN1 may be a potential driver in the pathogenesis of human cancers, particularly HCC.
PMCID: PMC3783260  PMID: 23291631
A-to-I; RNA editing; AZIN1; ADAR1; antizyme; ODC; CCND1; HCC
3.  Intermedin in the Paraventricular Nucleus Attenuates Cardiac Sympathetic Afferent Reflex in Chronic Heart Failure Rats 
PLoS ONE  2014;9(4):e94234.
Background and Aim
Intermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide (CGRP) family together with adrenomedullin (AM) and amylin. It has a wide distribution in the central nervous system (CNS) especially in hypothalamic paraventricular nucleus (PVN). Cardiac sympathetic afferent reflex (CSAR) is enhanced in chronic heart failure (CHF) rats. The aim of this study is to determine the effect of IMD in the PVN on CSAR and its related mechanisms in CHF rats.
Methodology/Principal Findings
Rats were subjected to left descending coronary artery ligation to induce CHF or sham-operation (Sham). Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. Acute experiments were carried out 8 weeks after coronary ligation or sham surgery under anesthesia. IMD and angiotensin II (Ang II) levels in the PVN were up-regulated in CHF rats. Bilateral PVN microinjection of IMD caused greater decreases in CSAR and the baseline RSNA and MAP in CHF rats than those in Sham rats. The decrease of CSAR caused by IMD was prevented by pretreatment with AM receptor antagonist AM22-52, but not CGRP receptor antagonist CGRP8-37. Ang II in the PVN significantly enhanced CSAR and superoxide anions level, which was inhibited by PVN pretreatment with IMD or tempol (a superoxide anions scavenger) in Sham and CHF rats.
IMD in the PVN inhibits CSAR via AM receptor, and attenuates the effects of Ang II on CSAR and superoxide anions level in CHF rats. PVN superoxide anions involve in the effect of IMD on attenuating Ang II-induced CSAR response.
PMCID: PMC3978024  PMID: 24709972
4.  The Exit of Mouse Embryonic Fibroblasts from the Cell-Cycle Changes the Nature of Solvent Exposure of the 5′-Methylcytosine Epitope within Chromatin 
PLoS ONE  2014;9(4):e92523.
The methylation of CpG dinucleotides is a pervasive epigenetic signature with critical roles governing genomic stability and lineage-specific patterns of gene expression. Reprogramming the patterns of CpG methylation accompanies key developmental transitions and the onset of some pathologies, such as cancer. In this study we show that levels of immuno-detectable 5meC decreased as mouse embryonic fibroblasts withdraw from the cell-cycle (became mitotically quiescent), but increased as they aged in culture. Two pools of 5meC epitope were found to exist, one solvent exposed after acid-induced denaturation of chromatin and another that required the additional step of tryptic digestion for detection. Proliferative cells displayed a relatively greater accumulation of detectable 5meC within the trypsin-sensitive pool than did quiescent cells. A substantial proportion of the 5meC was associated with a large number of heterochromatic foci scattered throughout nuclei, yet much of this was masked in a trypsin-sensitive manner, particularly in young proliferative cells. This study showed that the growth status of cells changed the level of solvent exposure of 5meC in fibroblasts and the long-accepted conventional methods of immunolocalization underestimate the level of 5meC in cells. This resulted in an artefactual assessment of the levels and patterns of nuclear localization of the antigen. The use of an additional tryptic digestion step improved antigen retrieval and revealed a more dynamic response of 5meC levels and distribution patterns to changes in the cell's growth state. This discovery will provide a basis for investigating the role of changes in chromatin structure that underlie this dynamism.
PMCID: PMC3976252  PMID: 24705067
5.  Preparation and in vitro/in vivo characterization of enteric-coated nanoparticles loaded with the antihypertensive peptide VLPVPR 
Our previous study revealed that the peptide Val-Leu-Pro-Val-Pro-Arg (VLPVPR), which was prepared using deoxyribonucleic acid recombinant technology, effectively decreased the blood pressure of spontaneous hypertensive rats; however, the effect only lasts 6 hours, likely due to its low absorption in the gastrointestinal tract. To overcome this problem, the purpose of this study was to characterize (methoxy-polyethylene glycol)-b-poly(D,L-lactide-co-glycolide)-b-poly(L-lysine) nanoparticles as in vitro and in vivo carriers for the effective delivery of VLPVPR. In our study, the VLPVPR nanoparticles were prepared using a double emulsion method, coated with Eudragit S100, and freeze-dried to produce enteric-coated nanoparticles. The optimized parameters from the double emulsion method was obtained from orthogonal experiments, including drug loading (DL) and encapsulated ratio (ER) at 6.12% and 86.94%, respectively, and the average particle size was below 100 nm. The release experiment demonstrated that the nanoparticles were sensitive to pH: almost completely released at pH 7.4 after 8 hours, but demonstrated much less release at pH 4.5 or pH 1.0 in the same amount of time. Therefore, the nanoparticles are suitable for enteric release. In vivo compared with the untreated group, the medium and high doses of orally administered VLPVPR nanoparticles reduced blood pressure for more than 30 hours, demonstrating that these nanoparticles have long-lasting and significant antihypertensive effects in spontaneously hypertensive rats.
PMCID: PMC3979782
mPEG-PLGA-PLL; in vivo studies; Val-Leu-Pro-Val-Pro-Arg peptide; enteric-coated; nanoparticle; antihypertensive peptide
6.  1,25-Dihydroxyvitamin D Promotes Negative Feedback Regulation of Toll-Like Receptor Signaling via Targeting MicroRNA-155-SOCS1 in Macrophages 
The negative feedback mechanism is essential to maintain effective immunity and tissue homeostasis. 1,25-dihydroxyvitamin D (1,25(OH)2D3) modulates innate immune response, but the mechanism remains poorly understood. Here we report that vitamin D receptor (VDR) signaling attenuates Toll-like receptor-mediated inflammation by enhancing the negative feedback inhibition. VDR inactivation leads to hyper inflammatory response in mice and macrophage cultures when challenged with lipopolysaccharide (LPS), due to miR-155 overproduction that excessively suppresses SOCS1, a key regulator that enhances the negative feedback loop. Deletion of miR-155 attenuates vitamin D suppression of LPS-induced inflammation, confirming that 1,25(OH)2D3 stimulates SOCS1 by down-regulating miR-155. 1,25(OH)2D3 down-regulates bic transcription by inhibiting NF-κB activation, which is mediated by a κB cis-DNA element located within the first intron of the bic gene. Together these data identify a novel regulatory mechanism for vitamin D to control innate immunity.
PMCID: PMC3608760  PMID: 23436936
vitamin D; inflammation; macrophage; miR-155; SOCS1; negative feedback
7.  Strategies for Rapid in vivo 1H and hyperpolarized 13C MR Spectroscopic Imaging 
Journal of magnetic resonance (San Diego, Calif. : 1997)  2013;229:10.1016/j.jmr.2013.02.003.
In vivo MRSI is an important imaging modality that has been shown in numerous research studies to give biologically relevant information for assessing the underlying mechanisms of disease and for monitoring response to therapy. The increasing availability of high field scanners and multichannel radiofrequency coils has provided the opportunity to acquire in vivo data with significant improvements in sensitivity and signal to noise ratio. These capabilities may be used to shorten acquisition time and provide increase coverage. The ability to acquire rapid, volumetric MRSI data is critical for examining heterogeneity in metabolic profiles and for relating serial changes in metabolism within the same individual during the course of the disease. In this review we discuss the implementation of strategies that use alternative k-space sampling trajectories and parallel imaging methods in order to speed up data acquisition. The impact of such methods is demonstrated using three recent examples of how these methods have been applied. These are to the acquisition of robust 3D 1H MRSI data within 5 –10 minutes at a field strength of 3T, to obtaining higher sensitivity for 1H MRSI at 7T and to using ultrafast volumetric and dynamic 13C MRSI for monitoring the changes in signals that occur following the injection of hyperpolarized 13C agents.
PMCID: PMC3808990  PMID: 23453759
spectroscopic imaging; hyperpolarized agents; spatial localization
8.  Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba 
PLoS ONE  2014;9(3):e93290.
Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels.
PMCID: PMC3970962  PMID: 24687099
9.  Retrospective analysis of adjuvant chemotherapy for curatively resected gastric cancer 
AIM: To determine the efficacy of adjuvant chemotherapy for gastric cancer in clinical practice, a retrospective analysis was conducted in a high-volume Chinese cancer center.
METHODS: Between November 1995 and June 2007, a total of 423 gastric or esophagogastric adenocarcinoma patients who did (Arm A, n = 300) or did not (Arm S, n = 123) receive radical gastrectomy followed by postoperative chemotherapy were enrolled in this retrospective analysis. In Arm A, monotherapy(fluoropyrimidines, n = 25), doublet (platinum/fluoropyrimidines, n = 164), or triplet regimens [docetaxel/cisplatin/5FU (DCF), or modified DCF, epirubicin/cisplatin/5FU (ECF) or modified ECF, etoposide/cisplatin/FU, n = 111] were administered. Disease-free survival (DFS) and overall survival (OS) were compared between the two arms. A subgroup analysis was carried out in Arm A. A multivariate analysis of prognostic factors was conducted.
RESULTS: Stage I, II and III cancers accounted for 9.7%, 35.7% and 54.6% of the cases, respectively, according to the American Joint Committee on Cancer (AJCC) staging system, 7th edition. Only 178 (42.1%) patients had more than 15 lymph nodes harvested. Hazard ratio estimates for Arm A compared with Arm S were 0.47 (P < 0.001) for OS and 0.59 (P < 0.001) for DFS. The 5-year OS rate was 52% in Arm A vs 36% in Arm S (P = 0.01); the adverse events in Arm A were mild and easily controlled. Ultimately, 73 patients (26.5%) who received doublet or triplet regimens switched to monotherapy with fluoropyrimidines. The OS and DFS did not differ between monotherapy and the combination regimens, however, both were statistically improved in the subgroup of patients who were switched to monotherapy with fluoropyrimidines after doublet or triplet regimens as well as patients who received ≥ 8 cycles of chemotherapy.
CONCLUSION: In clinical practice, platinum/fluoropyrimidines with adequate treatment duration is recommended for stage II/III gastric cancer patients accordingto the 7th edition of the AJCC staging system after curative gastrectomyeven with limited lymphadenectomy.
PMCID: PMC3964407
Adjuvant chemotherapy; Gastric cancer; Lymphadenectomy; Fluoropyrimidine; Platinum
10.  Low 2012–13 Influenza Vaccine Effectiveness Associated with Mutation in the Egg-Adapted H3N2 Vaccine Strain Not Antigenic Drift in Circulating Viruses 
PLoS ONE  2014;9(3):e92153.
Influenza vaccine effectiveness (VE) is generally interpreted in the context of vaccine match/mismatch to circulating strains with evolutionary drift in the latter invoked to explain reduced protection. During the 2012–13 season, however, detailed genotypic and phenotypic characterization shows that low VE was instead related to mutations in the egg-adapted H3N2 vaccine strain rather than antigenic drift in circulating viruses.
Component-specific VE against medically-attended, PCR-confirmed influenza was estimated in Canada by test-negative case-control design. Influenza A viruses were characterized genotypically by amino acid (AA) sequencing of established haemagglutinin (HA) antigenic sites and phenotypically through haemagglutination inhibition (HI) assay. H3N2 viruses were characterized in relation to the WHO-recommended, cell-passaged vaccine prototype (A/Victoria/361/2011) as well as the egg-adapted strain as per actually used in vaccine production. Among the total of 1501 participants, influenza virus was detected in 652 (43%). Nearly two-thirds of viruses typed/subtyped were A(H3N2) (394/626; 63%); the remainder were A(H1N1)pdm09 (79/626; 13%), B/Yamagata (98/626; 16%) or B/Victoria (54/626; 9%). Suboptimal VE of 50% (95%CI: 33–63%) overall was driven by predominant H3N2 activity for which VE was 41% (95%CI: 17–59%). All H3N2 field isolates were HI-characterized as well-matched to the WHO-recommended A/Victoria/361/2011 prototype whereas all but one were antigenically distinct from the egg-adapted strain as per actually used in vaccine production. The egg-adapted strain was itself antigenically distinct from the WHO-recommended prototype, and bore three AA mutations at antigenic sites B [H156Q, G186V] and D [S219Y]. Conversely, circulating viruses were identical to the WHO-recommended prototype at these positions with other genetic variation that did not affect antigenicity. VE was 59% (95%CI:16–80%) against A(H1N1)pdm09, 67% (95%CI: 30–85%) against B/Yamagata (vaccine-lineage) and 75% (95%CI: 29–91%) against B/Victoria (non-vaccine-lineage) viruses.
These findings underscore the need to monitor vaccine viruses as well as circulating strains to explain vaccine performance. Evolutionary drift in circulating viruses cannot be regulated, but influential mutations introduced as part of egg-based vaccine production may be amenable to improvements.
PMCID: PMC3965421  PMID: 24667168
11.  Cardiovascular Events in Patients with Atherothrombotic Disease: A Population-Based Longitudinal Study in Taiwan 
PLoS ONE  2014;9(3):e92577.
Atherothrombotic diseases including cerebrovascular disease (CVD), coronary artery disease (CAD), and peripheral arterial disease (PAD), contribute to the major causes of death in the world. Although several studies showed the association between polyvascular disease and poor cardiovascular (CV) outcomes in Asian population, there was no large-scale study to validate this relationship in this population.
Methods and Results
This retrospective cohort study included patients with a diagnosis of CVD, CAD, or PAD from the database contained in the Taiwan National Health Insurance Bureau during 2001–2004. A total of 19954 patients were enrolled in this study. The atherothrombotic disease score was defined according to the number of atherothrombotic disease. The study endpoints included acute coronary syndrome (ACS), all strokes, vascular procedures, in hospital mortality, and so on. The event rate of ischemic stroke (18.2%) was higher than that of acute myocardial infarction (5.7%) in our patients (P = 0.0006). In the multivariate Cox regression analyses, the adjusted hazard ratios (HRs) of each increment of atherothrombotic disease score in predicting ACS, all strokes, vascular procedures, and in hospital mortality were 1.41, 1.66, 1.30, and 1.14, respectively (P≦0.0169).
This large population-based longitudinal study in patients with atherothrombotic disease demonstrated the risk of subsequent ischemic stroke was higher than that of subsequent AMI. In addition, the subsequent adverse CV events including ACS, all stroke, vascular procedures, and in hospital mortality were progressively increased as the increase of atherothrombotic disease score.
PMCID: PMC3960266  PMID: 24647769
12.  The Circular F-Actin Bundles Provide a Track for Turnaround and Bidirectional Movement of Mitochondria in Arabidopsis Root Hair 
PLoS ONE  2014;9(3):e91501.
The movement of organelles in root hairs primarily occurs along the actin cytoskeleton. Circulation and “reverse fountain” cytoplasmic streaming constitute the typical forms by which most organelles (such as mitochondria and the Golgi apparatus) in plant root hair cells engage in bidirectional movement. However, there remains a lack of in-depth research regarding the relationship between the distribution of the actin cytoskeleton and turnaround organelle movement in plant root hair cells.
In this paper, Arabidopsis seedlings that had been stably transformed with a GFP-ABD2-GFP (green fluorescent protein-actin-binding domain 2-green fluorescent protein) construct were utilized to study the distribution of bundles of filamentous (F)-actin and the directed motion of mitochondria along these bundles in root hairs. Observations with a confocal laser scanning microscope revealed that there were widespread circular F-actin bundles in the epidermal cells and root hairs of Arabidopsis roots. In root hairs, these circular bundles primarily start at the sub-apical region, which is the location where the turnaround movement of organelles occurs. MitoTracker probes were used to label mitochondria, and the dynamic observation of root hair cells with a confocal laser scanning microscope indicated that turnaround mitochondrial movement occurred along circular F-actin bundles.
Relevant experimental results demonstrated that the circular F-actin bundles provide a track for the turnaround and bidirectional movement of mitochondria.
PMCID: PMC3953408  PMID: 24626218
13.  Proteomics Analysis of Normal and Senescent NG108-15 Cells: GRP78 Plays a Negative Role in Cisplatin-Induced Senescence in the NG108-15 Cell Line 
PLoS ONE  2014;9(3):e90114.
Accelerated senescence (ACS) leading to proliferative arrest is a physiological mechanism of the DNA damage response that occurs during tumor therapy. Our experiment was designed to detect unknown genes that may play important roles in cisplatin-induced senescence and to illustrate the related senescence mechanism. Using 2-dimension electrophoresis (2-DE), we identified 5 protein spots with different expression levels in the normal and senescent NG108-15 cells. According to MALDI-TOF MS analysis, the 5 proteins were determined to be peptidylprolyl isomerase A (PPIA), peroxiredoxin 1 (PRX1), glutathione S-transferase mu 1 (GSTM1), vimentin (VIM) and glucose-regulated protein 78 (GRP78). Then, we investigated how cisplatin-induced senescence was mediated by GRP78 in the NG108-15 cells. Knockdown of GRP78 significantly increased P53 expression in NG108-15 cells. Additionally, 2-deoxy-D-glucose (2DG)-induced GRP78 overexpression protected the NG108-15 cells from cisplatin-induced senescence, which was accompanied by the obvious suppression of P53 and p-CDC2 expression. Inhibition of Ca2+ release from endoplasmic reticulum (ER) stores was also found to be associated with the anti-senescence effect of 2DG-induced GRP78 overexpression. In conclusion, we found 5 proteins that were differentially expressed in normal NG108-15 cells and senescent NG108-15 cells. GRP78 plays an important role in cisplatin-induced senescence in NG108-15 cells, mainly through its regulation of P53 expression and ER calcium efflux.
PMCID: PMC3951507  PMID: 24621580
14.  Pressure-induced planar N6 rings in potassium azide 
Scientific Reports  2014;4:4358.
The first-principles method and the evolutionary algorithm are used to identify stable high pressure phases of potassium azide (KN3). It has been verified that the stable phase with space group I4/mcm below 22 GPa, which is consistent with the experimental result, will transform into the C2/m phase with pressure increasing. These two phases are insulator with anions. A metallic phase with P6/mmm symmetry is preferred above 40 GPa, and the N atoms in this structure form six-membered rings which are important for understanding the pressure effect on anions and phase transitions of KN3. Above the studied pressure (100 GPa), a polymerization of N6 rings may be obtained as the result of the increasing compactness.
PMCID: PMC3950634  PMID: 24619232
15.  Quantum dot-based immunofluorescent imaging of Ki67 and identification of prognostic value in HER2-positive (non-luminal) breast cancer 
The immunohistochemical assessment of Ki67 antigen (Ki67) is the most widely practiced measurement of breast cancer cell proliferation; however, it has some disadvantages and thus the prognostic value of Ki67 in breast cancer remains controversial. Our previous studies confirmed the advantages of quantum dots-based nanotechnology for quantitative analysis of biomarkers compared with conventional immunohistochemistry (IHC). This study was designed to assess Ki67 by quantum dot-immunohistochemistry (QD-IHC) and investigate the prognostic value of the Ki67 score in human epidermal growth factor receptor 2 (HER2)-positive (non-luminal) breast cancer.
Ki67 expression in 108 HER2-positive (non-luminal) breast cancer specimens was detected by IHC and QD-IHC. Two observers assessed the Ki67 score independently and comparisons between the two methods were made. The prognostic value of the Ki67 score for five-year disease-free survival was estimated.
The same antigen localization, high correlation of staining rates (r=0.993), and high agreement of measurements (κ=0.874) of Ki67 expression (cutoff: 30%) in breast cancer were found by QD-IHC and conventional IHC. The QD-IHC had a better interobserver agreement for the Ki67 score than conventional IHC (t=−7.280, P<0.01). High Ki67 expression (cutoff: 30%) was associated with shorter disease-free survival (log-rank test; IHC, P=0.026; QD-IHC, P=0.001), especially in the lymph node-negative subgroups (log-rank test; IHC, P=0.017; QD-IHC, P=0.002).
QD-IHC imaging of Ki67 was an easier and more accurate method for detecting and assessing Ki67. The Ki67 score was an independent prognosticator in the HER2-positive (non-luminal) breast cancer patients.
PMCID: PMC3956684  PMID: 24648732
quantum dots; breast cancer; Ki67; disease-free survival; prognosis
16.  Use of confocal laser endomicroscopy to predict relapse of ulcerative colitis 
BMC Gastroenterology  2014;14:45.
Assessment of inflammatory activity in patients with ulcerative colitis (UC) is crucial to the prediction of relapse. Confocal laser endomicroscopy (CLE) is an accurate tool for assessing inflammatory activity in UC patients. This study aimed to evaluate whether CLE could be used to predict UC relapse reliably.
In total, forty-three patients with documented UC were analyzed in this study. Patients identified as having obvious active inflammation by conventional colonoscopy were excluded. The mucosa of each patient’s sigmoid colon and rectum was assessed by CLE before targeted biopsies were taken. The patients were then followed up for at least 12 months to evaluate relapse according to the Simple Clinical Colitis Activity Index. The correlation between CLE classification and UC relapse was evaluated.
Seventeen of 20 patients with histologically confirmed normal or chronic inflammation were diagnosed as having non-active inflammation by real-time CLE and 22 of 23 patients with histologically confirmed acute inflammation were diagnosed as having active inflammation by CLE. The sensitivity, specificity, and accuracy of CLE in real-time diagnosis of active inflammation were 95.7%, 85%, and 90.7%, respectively. The agreement between CLE and conventional histology was excellent (kappa value = 0.812). Two of 18 (11.1%) patients who were classified as having non-active inflammation by CLE relapsed, while 16 of 25 (64%) patients classified as having as active inflammation relapsed. The relapse rate of patients with active inflammation was significantly higher than of those with non-active inflammation (P < 0.001).
CLE is comparable to conventional histology in predicting relapse in patients with UC.
PMCID: PMC3975275  PMID: 24618122
Ulcerative colitis; Relapse; Confocal laser endomicroscopy; UC; CLE
17.  CaMKII Mediates Recruitment and Activation of the Deubiquitinase CYLD at the Postsynaptic Density 
PLoS ONE  2014;9(3):e91312.
NMDA treatment of cultured hippocampal neurons causes recruitment of CYLD, as well as CaMKII, to the postsynaptic density (PSD), as shown by immunoelectron microscopy. Recruitment of CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is blocked by pre-treatment with tatCN21, a CaMKII inhibitor, at a concentration that inhibits the translocation of CaMKII to the PSD. Furthermore, CaMKII co-immunoprecipitates with CYLD from solubilized PSD fractions, indicating an association between the proteins. Purified CaMKII phosphorylates CYLD on at least three residues (S-362, S-418, and S-772 on the human CYLD protein Q9NQC7-1) and promotes its deubiquitinase activity. Activation of CaMKII in isolated PSDs promotes phosphorylation of CYLD on the same residues and also enhances endogenous deubiquitinase activity specific for K63-linked polyubiquitins. Since K63-linked polyubiquitin conjugation to proteins inhibits their interaction with proteasomes, CaMKII-mediated recruitment and upregulation of CYLD is expected to remove K63-linked polyubiquitins and facilitate proteasomal degradation at the PSD.
PMCID: PMC3948843  PMID: 24614225
18.  Dopamine transporter deficiency syndrome: phenotypic spectrum from infancy to adulthood 
Brain  2014;137(4):1107-1119.
Dopamine transporter deficiency syndrome is an SLC6A3-related progressive infantile-onset parkinsonism-dystonia that mimics cerebral palsy. Ng et al. describe clinical features and molecular findings in a new cohort of patients. They report infants with classical disease, as well as young adults manifesting as atypical juvenile-onset parkinsonism-dystonia, thereby expanding the disease spectrum.
Dopamine transporter deficiency syndrome due to SLC6A3 mutations is the first inherited dopamine ‘transportopathy’ to be described, with a classical presentation of early infantile-onset progressive parkinsonism dystonia. In this study we have identified a new cohort of patients with dopamine transporter deficiency syndrome, including, most significantly, atypical presentation later in childhood with a milder disease course. We report the detailed clinical features, molecular genetic findings and in vitro functional investigations undertaken for adult and paediatric cases. Patients presenting with parkinsonism dystonia or a neurotransmitter profile characteristic of dopamine transporter deficiency syndrome were recruited for study. SLC6A3 mutational analysis was undertaken in all patients. The functional consequences of missense variants on the dopamine transporter were evaluated by determining the effect of mutant dopamine transporter on dopamine uptake, protein expression and amphetamine-mediated dopamine efflux using an in vitro cellular heterologous expression system. We identified eight new patients from five unrelated families with dopamine transporter deficiency syndrome. The median age at diagnosis was 13 years (range 1.5–34 years). Most significantly, the case series included three adolescent males with atypical dopamine transporter deficiency syndrome of juvenile onset (outside infancy) and progressive parkinsonism dystonia. The other five patients in the cohort presented with classical infantile-onset parkinsonism dystonia, with one surviving into adulthood (currently aged 34 years) and labelled as having ‘juvenile parkinsonism’. All eight patients harboured homozygous or compound heterozygous mutations in SLC6A3, of which the majority are previously unreported variants. In vitro studies of mutant dopamine transporter demonstrated multifaceted loss of dopamine transporter function. Impaired dopamine uptake was universally present, and more severely impacted in dopamine transporter mutants causing infantile-onset rather than juvenile-onset disease. Dopamine transporter mutants also showed diminished dopamine binding affinity, reduced cell surface transporter, loss of post-translational dopamine transporter glycosylation and failure of amphetamine-mediated dopamine efflux. Our data series expands the clinical phenotypic continuum of dopamine transporter deficiency syndrome and indicates that there is a phenotypic spectrum from infancy (early onset, rapidly progressive disease) to childhood/adolescence and adulthood (later onset, slower disease progression). Genotype–phenotype analysis in this cohort suggests that higher residual dopamine transporter activity is likely to contribute to postponing disease presentation in these later-onset adult cases. Dopamine transporter deficiency syndrome remains under-recognized and our data highlights that dopamine transporter deficiency syndrome should be considered as a differential diagnosis for both infantile- and juvenile-onset movement disorders, including cerebral palsy and juvenile parkinsonism.
PMCID: PMC3959557  PMID: 24613933
dopamine; dopamine transporter (DAT); juvenile; parkinsonism; dystonia; SLC6A3
19.  Serum level of DKK-1 and its prognostic potential in non–small cell lung cancer 
Diagnostic Pathology  2014;9:52.
The aim of the present study was to measure the serum level of dickkopf-1(DKK-1) in patients with non-small cell lung cancer (NSCLC), and to determine the prognostic potential of serum DKK-1 in NSCLC.
Material and methods
The present study included a total of 150 patients with NSCLC and 150 healthy controls. Serum level of DKK-1 was measured by enzyme-linked immunosorbent assay (ELISA). Numerical variables were recorded as means ± standard deviation (SD) and analyzed by independent t-tests. Categorical variables were presented as rates and analyzed by using the chi-square test or Fisher’s exact test. The overall survival was analyzed by log-rank test, and survival curves were plotted according to Kaplan–Meier.
We found that serum DKK-1 level was significantly higher in patients with NSCLC than healthy controls. Mean serum DKK-1 level was 31.42 ± 6.32 ng/ml in the NSCLC group and 14.12 ± 3.29 ng/ml in the healthy control group (p <0.01). Serum DKK-1 level expression level was significantly positively correlated with TNM stage (p = 0.009), lymph node involvement(p = 0.001), and distant metastases(p < 0.001).
In the multivariate Cox proportional hazards analysis, high DKK-1 expression was independently associated with poor survival (P < 0.001; HR = 3.98; 95% CI =2.19-4.83).
In conclusion, our results showed that DKK-1 was overexpressed in NSCLC, and DKK-1 in serum was a good predictor of poor prognosis in patients with NSCLC. More researches are needed in the future to clarify the detailed mechanism of DKK-1 in the carcinogenesis and metastasis of NSCLC.
Virtual slides
The virtual slides for this article can be found here:
PMCID: PMC3975329  PMID: 24612589
Dickkopf-1; Non-small cell lung cancer; Prognosis
20.  Intestinal obstruction due to migration of a thermometer from bladder to abdominal cavity: A case report 
Intraperitoneal foreign bodies such as retained surgical instruments can cause intestinal obstruction. However, intestinal obstruction due to transmural migration of foreign bodies has rarely been reported. Here, we report a case of intestinal obstruction due to a clinical thermometer which migrated from the bladder into the abdominal cavity. A 45-year-old man was admitted to our hospital with a one-year history of recurrent lower abdominal cramps. Two days before admission, the abdominal cramps aggravated. Intestinal obstruction was confirmed with upright abdominal radiography and computerized tomography scan which showed dilation of the small intestines and a thermometer in the abdominal cavity. Then laparotomy was performed. A scar was observed at the fundus of the bladder and a thermometer was adhering to the small bowels and mesentery which resulted in intestinal obstruction. Abdominal cramps were eliminated and defecation and flatus recovered soon after removal of the thermometer.
PMCID: PMC3942848  PMID: 24605042
Intestinal obstruction; Foreign body; Thermometer; Transmural migration; Bladder
21.  CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses 
Nature neuroscience  2013;17(1):56-64.
Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca2+/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.
PMCID: PMC3943352  PMID: 24336150
22.  Variations in the MHC Region Confer Risk to Esophageal Squamous Cell Carcinoma on the Subjects from High-Incidence Area in Northern China 
PLoS ONE  2014;9(3):e90438.
The human major histocompatibility complex (MHC) is the most important region in vertebrate genome, and is crucial in innate immunity. Recent studies have demonstrated the possible role of polymorphisms in the MHC region to high risk for esophageal squamous cell carcinoma (ESCC). Our previous genome-wide association study (GWAS) has indicated that the MHC region may confer important risk loci for ESCC, but without further fine mapping. The aim of this study is to further identify the risk loci in the MHC region for ESCC in Chinese population.
Conditional logistic regression analysis (CLRA) was performed on 24 single nucleotide polymorphisms (SNPs) within the MHC region, which were obtained from the genetically matched 937 cases and 692 controls of Chinese Han population. The identified promising SNPs were further correlated with clinical and clinicopathology characteristics. Immunohistochemistry was performed to explore the protein expression pattern of the related genes in ESCC and neighboring normal tissues.
Of the 24 promising SNPs analyzed, we identified three independent SNPs in the MHC region associated with ESCC: rs35399661 (P = 6.07E-06, OR = 1.71, 95%CI = 1.36–2.17), rs3763338 (P = 1.62E-05, OR = 0.63, 95%CI = 0.50–0.78) and rs2844695 (P = 7.60E-05, OR = 0.74, 95%CI = 0.64–0.86). These three SNPs were located at the genes of HLA-DQA1, TRIM27, and DPCR1, respectively. Further analyses showed that rs2844695 was preferentially associated with younger ESCC cases (P = 0.009). The positive immunostaining rates both for HLA-DQA1 and TRIM27 were much higher in ESCC tissues than in neighboring normal tissues (69.4% vs. 26.8% for HLA-DQA1 and 77.6% vs. 47.8% for TRIM27, P<0.001). Furthermore, the overexpression of HLA-DQA1 is correlated significantly with age (P = 0.001) and family history (P<0.001).
This study for the first time provides evidence that multiple genetic factors within the MHC region confer risk to ESCC on the subjects from high-risk area in northern China.
PMCID: PMC3942432  PMID: 24595008
23.  Development of HuMiChip for Functional Profiling of Human Microbiomes 
PLoS ONE  2014;9(3):e90546.
Understanding the diversity, composition, structure, function, and dynamics of human microbiomes in individual human hosts is crucial to reveal human-microbial interactions, especially for patients with microbially mediated disorders, but challenging due to the high diversity of the human microbiome. Here we have developed a functional gene-based microarray for profiling human microbiomes (HuMiChip) with 36,802 probes targeting 50,007 protein coding sequences for 139 key functional gene families. Computational evaluation suggested all probes included are highly specific to their target sequences. HuMiChip was used to analyze human oral and gut microbiomes, showing significantly different functional gene profiles between oral and gut microbiome. Obvious shifts of microbial functional structure and composition were observed for both patients with dental caries and periodontitis from moderate to advanced stages, suggesting a progressive change of microbial communities in response to the diseases. Consistent gene family profiles were observed by both HuMiChip and next generation sequencing technologies. Additionally, HuMiChip was able to detect gene families at as low as 0.001% relative abundance. The results indicate that the developed HuMiChip is a useful and effective tool for functional profiling of human microbiomes.
PMCID: PMC3942451  PMID: 24595026
24.  Glutathione S-transferase L1 multiplex serology as a measure of cumulative infection with human papillomavirus 
BMC Infectious Diseases  2014;14:120.
Several assays are used to measure type-specific serological responses to human papillomavirus (HPV), including the bead-based glutathione S-transferase (GST)-L1 multiplex serology assay and virus-like particle (VLP)-based ELISA. We evaluated the high-throughput GST-L1, which is increasingly used in epidemiologic research, as a measure of cumulative HPV infection and future immune protection among HPV-unvaccinated women.
We tested enrollment sera from participants in the control arm of the Costa Rica Vaccine Trial (n = 488) for HPV16 and HPV18 using GST-L1, VLP-ELISA, and two assays that measure neutralizing antibodies (cLIA and SEAP-NA). With statistical adjustment for sampling, we compared GST-L1 serostatus to established HPV seropositivity correlates and incident cervical HPV infection using odds ratios. We further compared GST-L1 to VLP-ELISA using pair-wise agreement statistics and by defining alternate assay cutoffs.
Odds of HPV16 GST-L1 seropositivity increased with enrollment age (OR = 1.20 per year, 95%CI 1.03-1.40) and lifetime number of sexual partners (OR = 2.06 per partner, 95%CI 1.49-2.83), with similar results for HPV18. GST-L1 seropositivity did not indicate protection from incident infection over 4 years of follow-up (HPV16 adjusted OR = 1.72, 95%CI 0.95-3.13; HPV18 adjusted OR = 0.38, 95%CI 0.12-1.23). Seroprevalence by GST-L1 (HPV16 and HPV18, respectively) was 5.0% and 5.2%, compared to 19.4% and 23.8% by VLP-ELISA, giving positive agreement of 39.2% and 20.8%. Lowering GST-L1 seropositivity cutoffs improved GST-L1/VLP-ELISA positive agreement to 68.6% (HPV16) and 61.5% (HPV18).
Our data support GST-L1 as a marker of cumulative HPV infection, but not immune protection. At lower seropositivity cutoffs, GST-L1 better approximates VLP-ELISA.
PMCID: PMC3973893  PMID: 24588945
25.  Synthesis, identification and in vivo studies of tumor-targeting agent peptide doxorubicin (PDOX) to treat peritoneal carcinomatosis of gastric cancer with similar efficacy but reduced toxicity 
Molecular Cancer  2014;13:44.
This work aimed to synthesize a cathepsin B (CTSB)-cleavable tumor-targeting prodrug peptide doxorubicin (PDOX) and study the in vivo efficacy and toxicities on an animal model of gastric peritoneal carcinomatosis (PC).
PDOX was synthesized using doxorubicin (DOX) attaching to a CTSB-cleavable dipeptide Ac-Phe-Lys and a para-amino-benzyloxycarbonyl (PABC) spacer. PC model was established by injecting VX2 tumor cells into the gastric sub-mucosa of 40 rabbits, which then were randomized into 4 groups: the Control (n = 10) without treatment, the HIPEC (n = 10) receiving cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC), the PDOX (n = 10) and the DOX (n = 10) receiving systemic chemotherapy with PDOX 50.0 mg/kg or DOX 5.0 mg/kg, respectively, after CRS + HIPEC.
The median overall survivals (OS) were 23.0 d (95% CI: 19.9 d - 26.1 d) in the Control, 41.0 d (36.9 d - 45.1 d) in the HIPEC, 65.0 d (44.1 d - 71.9 d) in the PDOX, and 58.0 d (39.6 d - 54.4 d) in the DOX. Compared with the Control, the OS was extended by 70% in the HIPEC (p < 0.001) and further extended by 40% in the DOX (p = 0.029) and by 58% in the PDOX (p = 0.021), and the PC severity was decreased in the HIPEC and further decreased in the PDOX and DOX. Animals receiving DOX treatment showed hematological toxicities with marked reduction of white blood cells and platelets, as well as cardiac toxicities with significant increases in creatine kinase mb isoenzyme, evident myocardium coagulation necrosis, significant nuclear degeneration, peri-nucleus mitochondria deletion, mitochondria-pyknosis, and abnormal intercalated discs. But these toxicities were not evident in the PDOX.
PDOX is a newly synthesized tumor-targeting prodrug of DOX. Compared with DOX, PDOX has similar efficacy but reduced hematological and cardiac toxicities in treating rabbit model of gastric PC.
PMCID: PMC3984748  PMID: 24588871
Peritoneal carcinomatosis; Gastric cancer; Peptide doxorubicin; Cytoreductive surgery; Hyperthermic intraperitoneal chemotherapy

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