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1.  Labeling native bacterial RNA in live cells 
Cell Research  2014;24(7):894-897.
doi:10.1038/cr.2014.47
PMCID: PMC4085757  PMID: 24732010
2.  Design and Study of Efflux Function of EGFP Fused MexAB-OprM Membrane Transporter in Pseudomonas aeruginosa Using Fluorescence Spectroscopy 
The Analyst  2014;139(12):3088-3096.
Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in ΔMexB (MexB deletion) strain of Pseudomonas aeruginosa to create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined their expression in live cells by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy. Efflux function of the new strain was studied by measuring its accumulation kinetics of ethidium bromide (EtBr, a pump substrate) using fluorescence spectroscopy, which was compared with the cells (WT, ΔMexM, ΔABM, and nalB1) with various expression levels of MexAB-OprM. The new strain shows 6-fold lower accumulation rates of EtBr (15 μM) than ΔABM, 4-fold lower than ΔMexB, but only 1.1-fold higher than WT. As EtBr concentration increases to 40 μM, the new strain has nearly the same accumulation rate of EtBr as ΔMexB, but 1.4-fold higher than WT. We observed the nearly same level of inhibitory effect of CCCP (carbonyl cyanide-m-chlorophenylhydrazone) on the efflux of EtBr by the new strain and WT. Antibiotic susceptibility study shows that the minimum inhibitory concentrations (MICs) of aztreonam (AZT) and chloramphenicol (CP) for the new strain are 6-fold or 3-fold lower than WT, respectively, and 2-fold higher than those of ΔMexB. Taken together, the results suggest that the fusion protein partially retains the efflux function of MexAB-OprM. Modeled structure of the fusion protein shows that the position and orientation of the N-terminal fused EGFP domain may either partially block the translocation pore or restrict the movement of the individual pump domains, which leads to partially restrict efflux activity.
doi:10.1039/c4an00108g
PMCID: PMC4081544  PMID: 24781334
EGPF-fused membrane transporter; efflux function; fluorescence spectroscopy; MexAB-OprM; multidrug resistance; Pseudomonas aeruginosa
3.  Increased Tim-3 expression on peripheral T lymphocyte subsets and association with higher disease activity in systemic lupus erythematosus 
Diagnostic Pathology  2015;10:71.
Background
Both the T cell immunoglobulin domain- and mucin domain-containing molecule-3 (Tim-3) and the death receptor Fas contribute to the pathogenesis of various autoimmune diseases, including systemic lupus erythematosus (SLE). The aim of the present study was to determine whether Tim-3 and Fas are co-expressed on certain peripheral T lymphocyte subsets, and whether this expression is associated with greater disease activity in SLE.
Methods
Peripheral blood mononuclear cells were isolated from 46 patients newly diagnosed with SLE and 28 age- and sex-matched healthy controls (HCs). Expression of Tim-3 and Fas on T subsets was analyzed by flow cytometry, while mRNA levels of the Tim-3 ligand galectin-9 and Fas ligand FasL were assayed using real-time RT-PCR.
Results
The proportions of CD3+CD4+ and CD3+CD4- T cells expressing Tim-3+ and Tim+Fas+ were significantly higher in patients than in HCs (p < 0.05), while the proportions of these subtypes expressing Fas were similar for the two groups. Patients with active SLE, as defined by their score on the SLE Disease Activity Index, had lower proportions of CD3+CD4+ T cells and higher proportions of CD3+CD4+Tim-3+ and CD3+CD4+Tim-3+Fas+ T cells than did patients with stable SLE. Serum levels of complement C3 and C4 proteins, considered as a marker of SLE activity, correlated negatively with proportions of CD3+CD4+ and CD3+CD4- T cells expressing Tim-3.
Conclusions
Expression of Tim-3 and co-expression of Tim-3 and Fas on certain peripheral T subsets are associated with disease activity in SLE patients. Future research should examine whether the same is true of other T subsets implicated in SLE, and should explore the potential role(s) of Tim-3 in the disease pathway.
Virtual slides
http://www.diagnosticpathology.diagnomx.eu/vs/1855527845145188
doi:10.1186/s13000-015-0306-0
PMCID: PMC4469310  PMID: 26076826
Systemic lupus erythematosus; Tim-3; Fas; T lymphocyte subset; Disease activity
4.  Association of elevated apoA-I glycation and reduced HDL-associated paraoxonase1, 3 activity, and their interaction with angiographic severity of coronary artery disease in patients with type 2 diabetes mellitus 
Objective
To investigate whether apolipoprotein A (apoA)-I glycation and paraoxonase (PON) activities are associated with the severity of coronary artery disease (CAD) in patients with type 2 diabetes mellitus (T2DM).
Methods
Relative intensity of apoA-I glycation and activities of high-density lipoprotein (HDL)-associated PON1 and PON3 were determined in 205 consecutive T2DM patients with stable angina with (n = 144) or without (n = 61) significant CAD (luminal diameter stenosis ≥ 70 %). The severity of CAD was expressed by number of diseased coronary arteries, extent index, and cumulative coronary stenosis score (CCSS).
Results
The relative intensity of apoA-I glycation was higher but the activities of HDL-associated PON1 and PON3 were lower in diabetic patients with significant CAD than in those without. The relative intensity of apoA-I glycation increased but the activities of HDL-associated PON1 and PON3 decreased stepwise from 1 - to 3 - vessel disease patients (P for trend < 0.001). After adjusting for possible confounding variables, the relative intensity of apoA-I glycation correlated positively, while the activities of HDL-associated PON1 and PON3 negatively, with extent index and CCSS, respectively. At high level of apoA-I glycation (8.70 ~ 12.50 %), low tertile of HDL-associated PON1 (7.03 ~ 38.97U/mL) and PON3 activities (7.11 ~ 22.30U/mL) was associated with a 1.97− and 2.49− fold increase of extent index and 1.73− and 2.68− fold increase of CCSS compared with high tertile of HDL-associated PON1 (57.85 ~ 154.82U/mL) and PON3 activities (39.63 ~ 124.10U/mL), respectively (all P < 0.01).
Conclusions
Elevated apoA-I glycation and decreased activities of HDL-associated PON1 and PON3, and their interaction are associated with the presence and severity of CAD in patients with T2DM.
Electronic supplementary material
The online version of this article (doi:10.1186/s12933-015-0221-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12933-015-0221-4
PMCID: PMC4432963  PMID: 25964115
Diabetes mellitus; ApoA-I glycation; Paraoxonase; Coronary artery disease
5.  New Models of Tetrahymena Telomerase RNA from Experimentally Derived Constraints and Modeling 
Journal of the American Chemical Society  2012;134(49):20070-20080.
The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA, TER, provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment towards understanding its non-templating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base pair triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of tTERT to characterize the conformational changes of tTER that attend binding to tTERT. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footpinting allows us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major grove of the helix-capping loop.
doi:10.1021/ja305636u
PMCID: PMC4427051  PMID: 23163801
Telomerase; ribonucleoprotein complex; RNA footprinting; pseudoknot
6.  Silane-catalysed fast growth of large single-crystalline graphene on hexagonal boron nitride 
Nature Communications  2015;6:6499.
The direct growth of high-quality, large single-crystalline domains of graphene on a dielectric substrate is of vital importance for applications in electronics and optoelectronics. Traditionally, graphene domains grown on dielectrics are typically only ~1 μm with a growth rate of ~1 nm min−1 or less, the main reason is the lack of a catalyst. Here we show that silane, serving as a gaseous catalyst, is able to boost the graphene growth rate to ~1 μm min−1, thereby promoting graphene domains up to 20 μm in size to be synthesized via chemical vapour deposition (CVD) on hexagonal boron nitride (h-BN). Hall measurements show that the mobility of the sample reaches 20,000 cm2 V−1 s−1 at room temperature, which is among the best for CVD-grown graphene. Combining the advantages of both catalytic CVD and the ultra-flat dielectric substrate, gaseous catalyst-assisted CVD paves the way for synthesizing high-quality graphene for device applications while avoiding the transfer process.
The growth of high-quality graphene directly on to dielectric substrates is of key importance for future electronic and optoelectronic applications. Here, the authors use silane-catalysed chemical vapour deposition to fabricate large graphene sheets, up to 20 μm in size, on hexagonal boron nitride.
doi:10.1038/ncomms7499
PMCID: PMC4382696  PMID: 25757864
7.  Computational approaches to understanding protein aggregation in neurodegeneration 
The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggregation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute significantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both all-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.
doi:10.1093/jmcb/mju007
PMCID: PMC3995224  PMID: 24620031
protein aggregation; molecular dynamics; protein folding; neurodegeneration
8.  Early kidney injury during long-term adefovir dipivoxil therapy for chronic hepatitis B 
AIM: To evaluate urine β2-microglobulin (β2-M), retinol-binding protein (RBP) excretion, and renal impairment with adefovir dipivoxil (ADV) for chronic hepatitis B.
METHODS: We enrolled 165 patients with chronic hepatitis B infection who were treated with ADV monotherapy (n = 90) or ADV plus lamivudine combination therapy (n = 75). An additional 165 chronic hepatitis B patients treated with entecavir were recruited as controls. We detected serum creatinine, urine β2-M, and RBP levels, and estimated the glomerular filtration rate (eGFR) at the initiation of antiviral therapy and every 6 mo for a period of five years.
RESULTS: Urine β2-M abnormalities were observed in patients during the first (n = 3), second (n = 7), third (n = 11), fourth (n = 16), and fifth (n = 21) year of ADV treatment. Urinary RBP abnormalities were observed in patients during the first (n = 2), second (n = 8), third (n = 12), fourth (n = 15), and fifth (n = 22) year of ADV treatment. eGFR decreased 20%-30% from baseline in 20 patients, 30%-50% in 12 patients, and > 50% in 3 patients during the five years of treatment. Further analysis indicated that decreases in eGFR of ≥ 30% relative to the baseline level correlated significantly with urine RBP and β2-M abnormalities. In contrast, both serum creatinine and eGFR remained stable in patients treated with entecavir, and only one of these patients developed a urine β2-M abnormality, and two developed urine RBP abnormalities during the five years of treatment.
CONCLUSION: Urine RBP and β2-M are biomarkers of renal injury during long-term ADV treatment for chronic hepatitis B, and indicate when treatment should be switched to entecavir.
doi:10.3748/wjg.v21.i12.3657
PMCID: PMC4375591  PMID: 25834334
Adefovir dipivoxil; Entecavir; Retinol binding protein; Renal impairment; Urine β2-microglobulin
9.  Inhibition of IAPP aggregation by insulin depends on the insulin oligomeric state regulated by zinc ion concentration 
Scientific Reports  2015;5:8240.
While islet amyloid polypeptide (IAPP) aggregation is associated with β-cell death in type-II diabetes (T2D), environmental elements of β-cell granules — e.g. high concentrations of insulin and Zn2+ — inhibit IAPP aggregation in healthy individuals. The inhibition by insulin is experimentally known, but the role of Zn2+ is controversial as both correlations and anti-correlations at the population level are observed between T2D risk and the activity of a β-cell specific zinc ion transporter, ZnT8. Since Zn2+ concentration determines insulin oligomer equilibrium, we computationally investigated interactions of IAPP with different insulin oligomers and compared with IAPP homodimer formation. We found that IAPP binding with insulin oligomers competes with the formation of both higher-molecular-weight insulin oligomers and IAPP homodimers. Therefore, zinc deficiency due to loss-of-function ZnT8 mutations shifts insulin oligomer equilibrium toward zinc-free monomers and dimers, which bind IAPP monomers more efficiently compared to zinc-bound hexamers. The hetero-molecular complex formation prevents IAPP from self-association and subsequent aggregation, reducing T2D risk.
doi:10.1038/srep08240
PMCID: PMC4316164  PMID: 25649462
10.  Long-Term Treatment of Clonidine, Atenolol, Amlodipine and Dihydrochlorothiazide, but Not Enalapril, Impairs the Sexual Function in Male Spontaneously Hypertensive Rats 
PLoS ONE  2015;10(1):e0116155.
This study was designed to investigate the impact of representative antihypertensive drugs of 5 classes on the sexual function in male spontaneously hypertensive rats (SHR) at doses that achieved similar blood pressure (BP) reduction. The experiment was performed in 6 groups of male SHR. The dose are 20 μg/kg/day for clonidine, 3 mg/kg/day for enalapril, 20 mg/kg/day for atenolol, 2 mg/kg/day for amlodipine, and 10 mg/kg/day for dihydrochlorothiazide. SHR were treated for 3 months, and then the penile erection and sexual behavior were detected. After BP recording, SHR were killed to evaluate the organ-damage, weight of accessory sex organs and levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone in serum. Five drugs had the similar efficacy on BP reduction. All drugs except of enalapril, significantly prolonged the mount latency, and decreased the mount frequency (P<0.05). Clonidine also reduced the conception rate (45% vs. 80% in control group, P<0.05). Amlodipine and dihydrochlorothiazide significantly increased the testosterone level (0.79±0.30, 0.80±0.34 vs. 0.49±0.20 in control group, unit: ng/dl, P<0.05). Enalapril, atenolol and amlodipine also significantly decreased the BP variability (systolic, 8.2±2.5, 7.6±1.8, 8.9±2.0 vs. 12.2±3.8 in control group, unit: mm Hg). All these drugs significantly decreased the organ-damage (P<0.05). In conclusion, long-term treatment with 5 common antihypertensive drugs possessed obvious organ protection in SHR. Clonidine, atenolol, amlodipine and dihydrochlorothiazide, but not enalapril, impair sexual function.
doi:10.1371/journal.pone.0116155
PMCID: PMC4304790  PMID: 25615941
11.  An updated role of microRNA-124 in central nervous system disorders: a review 
MicroRNA-124 (miR-124) is the most abundant miRNA in the brain. Biogenesis of miR-124 displays specific temporal and spatial profiles in various cell and tissue types and affects a broad spectrum of biological functions in the central nervous system (CNS). Recently, the link between dysregulation of miR-124 and CNS disorders, such as neurodegeneration, CNS stress, neuroimmune disorders, stroke, and brain tumors, has become evident. Here, we provide an overview of the specific molecular function of miR-124 in the CNS and a revealing insight for the therapeutic potential of miR-124 in the treatment of human CNS diseases.
doi:10.3389/fncel.2015.00193
PMCID: PMC4438253  PMID: 26041995
microRNA-124; CNS disorders; brain development; neurodegradation; CNS stress; neuroimmunity; brain tumor; stroke
12.  Formation of Graphene Grain Boundaries on Cu(100) Surface and a Route Towards Their Elimination in Chemical Vapor Deposition Growth 
Scientific Reports  2014;4:6541.
Grain boundaries (GBs) in graphene prepared by chemical vapor deposition (CVD) greatly degrade the electrical and mechanical properties of graphene and thus hinder the applications of graphene in electronic devices. The seamless stitching of graphene flakes can avoid GBs, wherein the identical orientation of graphene domain is required. In this letter, the graphene orientation on one of the most used catalyst surface — Cu(100) surface, is explored by density functional theory (DFT) calculations. Our calculation demonstrates that a zigzag edged hexagonal graphene domain on a Cu(100) surface has two equivalent energetically preferred orientations, which are 30 degree away from each other. Therefore, the fusion of graphene domains on Cu(100) surface during CVD growth will inevitably lead to densely distributed GBs in the synthesized graphene. Aiming to solve this problem, a simple route, that applies external strain to break the symmetry of the Cu(100) surface, was proposed and proved efficient.
doi:10.1038/srep06541
PMCID: PMC4187007  PMID: 25286970
13.  Direct observation of a single nanoparticle-ubiquitin corona formation 
Nanoscale  2013;5(19):9162-9169.
The advancement of nanomedicine and the increasing applications of nanoparticles in consumer products have led to administered biological exposure and unintentional environmental accumulation of nanoparticles, causing concerns over the biocompatibility and sustainability of nanotechnology. Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Consequently, understanding the structure and dynamics of nanoparticle-protein corona is essential for predicting the fate, transport, and toxicity of nanomaterials in living systems and for enabling the vast applications of nanomedicine. Here we combined multiscale molecular dynamics simulations and complementary experiments to characterize the silver nanoparticle-ubiquitin corona formation. Notably, ubiquitins competed with citrates for the nanoparticle surface, governed by specific electrostatic interactions. Under a high protein/nanoparticle stoichiometry, ubiquitins formed a multi-layer corona on the particle surface. The binding exhibited an unusual stretched-exponential behavior, suggesting a rich binding kinetics. Furthermore, the binding destabilized the α-helices while increasing the β-sheets of the proteins. This study revealed the atomic and molecular details of the structural and dynamic characteristics of nanoparticle-protein corona formation.
doi:10.1039/c3nr02147e
PMCID: PMC4037870  PMID: 23921560
14.  Predicting binding affinity of CSAR ligands using both structure-based and ligand-based approaches 
We report on the prediction accuracy of ligand-based (2D QSAR) and structure-based (MedusaDock) methods used both independently and in consensus for ranking the congeneric series of ligands binding to three protein targets (UK, ERK2, and CHK1) from the CSAR 2011 benchmark exercise. An ensemble of predictive QSAR models was developed using known binders of these three targets extracted from the publicly-available ChEMBL database. Selected models were used to predict the binding affinity of CSAR compounds towards the corresponding targets and rank them accordingly; the overall ranking accuracy evaluated by Spearman correlation was as high as 0.78 for UK, 0.60 for ERK2, and 0.56 for CHK1, placing our predictions in top-10% among all the participants. In parallel, MedusaDock designed to predict reliable docking poses was also used for ranking the CSAR ligands according to their docking scores; the resulting accuracy (Spearman correlation) for UK, ERK2, and CHK1 were 0.76, 0.31, and 0.26, respectively. In addition, performance of several consensus approaches combining MedusaDock and QSAR predicted ranks altogether has been explored; the best approach yielded Spearman correlation coefficients for UK, ERK2, and CHK1 of 0.82, 0.50, and 0.45, respectively. This study shows that (i) externally validated 2D QSAR models were capable of ranking CSAR ligands at least as accurately as more computationally intensive structure-based approaches used both by us and by other groups and (ii) ligand-based QSAR models can complement structure-based approaches by boosting the prediction performances when used in consensus.
doi:10.1021/ci400216q
PMCID: PMC3779696  PMID: 23809015
15.  Genetics of rheumatoid arthritis contributes to biology and drug discovery 
Okada, Yukinori | Wu, Di | Trynka, Gosia | Raj, Towfique | Terao, Chikashi | Ikari, Katsunori | Kochi, Yuta | Ohmura, Koichiro | Suzuki, Akari | Yoshida, Shinji | Graham, Robert R. | Manoharan, Arun | Ortmann, Ward | Bhangale, Tushar | Denny, Joshua C. | Carroll, Robert J. | Eyler, Anne E. | Greenberg, Jeffrey D. | Kremer, Joel M. | Pappas, Dimitrios A. | Jiang, Lei | Yin, Jian | Ye, Lingying | Su, Ding-Feng | Yang, Jian | Xie, Gang | Keystone, Ed | Westra, Harm-Jan | Esko, Tõnu | Metspalu, Andres | Zhou, Xuezhong | Gupta, Namrata | Mirel, Daniel | Stahl, Eli A. | Diogo, Dorothée | Cui, Jing | Liao, Katherine | Guo, Michael H. | Myouzen, Keiko | Kawaguchi, Takahisa | Coenen, Marieke J.H. | van Riel, Piet L.C.M. | van de Laar, Mart A.F.J. | Guchelaar, Henk-Jan | Huizinga, Tom W.J. | Dieudé, Philippe | Mariette, Xavier | Bridges, S. Louis | Zhernakova, Alexandra | Toes, Rene E.M. | Tak, Paul P. | Miceli-Richard, Corinne | Bang, So-Young | Lee, Hye-Soon | Martin, Javier | Gonzalez-Gay, Miguel A. | Rodriguez-Rodriguez, Luis | Rantapää-Dahlqvist, Solbritt | Ärlestig, Lisbeth | Choi, Hyon K. | Kamatani, Yoichiro | Galan, Pilar | Lathrop, Mark | Eyre, Steve | Bowes, John | Barton, Anne | de Vries, Niek | Moreland, Larry W. | Criswell, Lindsey A. | Karlson, Elizabeth W. | Taniguchi, Atsuo | Yamada, Ryo | Kubo, Michiaki | Liu, Jun S. | Bae, Sang-Cheol | Worthington, Jane | Padyukov, Leonid | Klareskog, Lars | Gregersen, Peter K. | Raychaudhuri, Soumya | Stranger, Barbara E. | De Jager, Philip L. | Franke, Lude | Visscher, Peter M. | Brown, Matthew A. | Yamanaka, Hisashi | Mimori, Tsuneyo | Takahashi, Atsushi | Xu, Huji | Behrens, Timothy W. | Siminovitch, Katherine A. | Momohara, Shigeki | Matsuda, Fumihiko | Yamamoto, Kazuhiko | Plenge, Robert M.
Nature  2013;506(7488):376-381.
A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological datasets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA)1. Here, we performed a genome-wide association study (GWAS) meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ~10 million single nucleotide polymorphisms (SNPs). We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 1012–4. We devised an in-silico pipeline using established bioinformatics methods based on functional annotation5, cis-acting expression quantitative trait loci (cis-eQTL)6, and pathway analyses7–9 – as well as novel methods based on genetic overlap with human primary immunodeficiency (PID), hematological cancer somatic mutations and knock-out mouse phenotypes – to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery.
doi:10.1038/nature12873
PMCID: PMC3944098  PMID: 24390342
16.  Rhesus monkey is a new model of secondary lymphedema in the upper limb 
Objective: This study is to establish the rhesus monkey model of lymphedema in the upper limbs, and assess the suitability of this model. Methods: An animal model of lymphedema was established by the combined irradiation and surgical techniques in the upper limbs of these rhesus monkeys. Physical examination, high-resolution MR lymphangiography, bioelectrical impedance analysis (BIA), and immunohistochemical staining were performed to determine the severity of the edema in the upper limbs of the animal model. Results: Our results from physical examination indicated that the rhesus monkey model present with typical appearance and features of lymphedema. MR lymphangiography further demonstrated pathologically modified lymphatic vessels in our rhesus monkey model. BIA revealed increased water content in the upper limb in these rhesus monkeys, which was in line with the pathology of lymphedema. Immunohistochemical staining showed the curvature of the lymphatic vessels in the rhesus monkey model, typical pathological changes in lymphedema. Conclusion: Rhesus monkey lymphedema model provides a more consistent background to elucidate the pathophysiology of the disease. This new model would help to increase our understanding of acquired upper limb lymphedema, and promote the development of new treatments for this intractable disorder.
PMCID: PMC4203178  PMID: 25337207
Secondary lymphedema; rhesus monkeys; upper extremity; breast cancer; animal model
17.  Protein Kinase D3 Is Essential for Prostratin-Activated Transcription of Integrated HIV-1 Provirus Promoter via NF-κB Signaling Pathway 
BioMed Research International  2014;2014:968027.
Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKCε/PKD3/NF-κB signaling pathway.
doi:10.1155/2014/968027
PMCID: PMC4127265  PMID: 25136641
18.  Adolescent Mouse Takes on An Active Transcriptomic Expression During Postnatal Cerebral Development 
Postnatal cerebral development is a complicated biological process precisely controlled by multiple genes. To understand the molecular mechanism of cerebral development, we compared dynamics of mouse cerebrum transcriptome through three developmental stages using high-throughput RNA-seq technique. Three libraries were generated from the mouse cerebrum at infancy, adolescence and adulthood, respectively. Consequently, 44,557,729 (infancy), 59,257,530 (adolescence) and 72,729,636 (adulthood) reads were produced, which were assembled into 15,344, 16,048 and 15,775 genes, respectively. We found that the overall gene expression level increased from infancy to adolescence and decreased later on upon reaching adulthood. The adolescence cerebrum has the most active gene expression, with expression of a large number of regulatory genes up-regulated and some crucial pathways activated. Transcription factor (TF) analysis suggested the similar dynamics as expression profiling, especially those TFs functioning in neurogenesis differentiation, oligodendrocyte lineage determination and circadian rhythm regulation. Moreover, our data revealed a drastic increase in myelin basic protein (MBP)-coding gene expression in adolescence and adulthood, suggesting that the brain myelin may be generated since mouse adolescence. In addition, differential gene expression analysis indicated the activation of rhythmic pathway, suggesting the function of rhythmic movement since adolescence; Furthermore, during infancy and adolescence periods, gene expression related to axon repulsion and attraction showed the opposite trends, indicating that axon repulsion was activated after birth, while axon attraction might be activated at the embryonic stage and declined during the postnatal development. Our results from the present study may shed light on the molecular mechanism underlying the postnatal development of the mammalian cerebrum.
doi:10.1016/j.gpb.2014.04.004
PMCID: PMC4411375  PMID: 24953867
Cerebrum; Postnatal development; Gene expression; Transcriptome; High-throughput sequencing
19.  Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis 
BMC Genomics  2014;15(1):431.
Background
Alternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.
Results
To analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.
Conclusions
Our study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-431) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-431
PMCID: PMC4079960  PMID: 24897929
Alternative splicing; Pre-mRNA; SR proteins; Salt stress; Arabidopsis thaliana
20.  Involvement of arterial baroreflex in the protective effect of dietary restriction against stroke 
Dietary restriction (DR) protects against neuronal dysfunction and degeneration, and reduces the risk of ischemic stroke. This study examined the role of silent information regulator T1 (SIRT1) and arterial baroreflex in the beneficial effects of DR against stroke, using two distinct stroke models: stroke-prone spontaneously hypertensive rats (SP-SHRs) and Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Sirt1 knockout (KO) mice were used to examine the involvement of sirt1. Sinoaortic denervation was used to inactivate arterial baroreflex. Dietary restriction was defined as 40% reduction of dietary intake. Briefly, DR prolonged the life span of SP-SHRs and reduced the infarct size induced by MCAO. Dietary restriction also improved the function arterial baroreflex, decreased the release of proinflammatory cytokines, and reduced end-organ damage. The beneficial effect of DR on stroke was markedly attenuated by blunting arterial baroreflex. Lastly, the infarct area in sirt1 KO mice was significantly larger than in the wild-type mice. However, the beneficial effect of DR against ischemic injury was still apparent in sirt1 KO mice. Accordingly, arterial baroreflex, but not sirt1, is important in the protective effect of DR against stroke.
doi:10.1038/jcbfm.2013.28
PMCID: PMC3677110  PMID: 23443169
arterial baroreflex; dietary restriction; sirt1; stroke
21.  Arterial Baroreflex Dysfunction Impairs Ischemia‐Induced Angiogenesis 
Background
Endothelium‐derived acetylcholine (eACh) plays an important role in the regulation of vascular actions in response to hypoxia, whereas arterial baroreflex (ABR) dysfunction impairs the eACh system. We investigated the effects of ABR dysfunction on ischemia‐induced angiogenesis in animal models of hindlimb ischemia with a special focus on eACh/nicotinic ACh receptor (nAChR) signaling activation.
Methods and Results
Male Sprague‐Dawley rats were randomly assigned to 1 of 3 groups that received (1) sham operation (control group), (2) sinoaortic denervation (SAD)‐induced ABR dysfunction (SAD group), or (3) SAD rats on diet with an acetylcholinesterase inhibitor pyridostigmine (30 mg/kg per day, SAD+Pyr group). After 4 weeks of the SAD intervention, unilateral limb ischemia was surgically induced in all animals. At postoperative day 14, SAD rats exhibited impaired angiogenic action (skin temperature and capillary density) and decreased angiogenic factor expressions (vascular endothelial growth factor [VEGF] and hypoxic inducible factor [HIF]‐1α) in ischemic muscles. These changes were restored by acetylcholinesterase inhibition. Rats with ABR dysfunction had lower eACh levels than did control rats, and this effect was recovered in SAD+Pyr rats. In α7‐nAChR knockout mice, pyridostigmine improved ischemia‐induced angiogenic responses and increased the levels of VEGF and HIF‐1α. Moreover, nicotinic receptor blocker inhibited VEGF expression and VEGF receptor 2 phosphorylation (p‐VEGFR2) induced by ACh analog.
Conclusions
Thus, ABR dysfunction appears to impair ischemia‐induced angiogenesis through the reduction of eACh/α7‐nAChR‐dependent and ‐independent HIF‐1α/VEGF‐VEGFR2 signaling activation.
doi:10.1161/JAHA.114.000804
PMCID: PMC4309071  PMID: 24820655
acetylcholinesterase inhibitor; angiogenesis; arterial baroreflex; non‐neural cholinergic system; peripheral vascular disease
22.  De Novo Characterization of the Spleen Transcriptome of the Large Yellow Croaker (Pseudosciaena crocea) and Analysis of the Immune Relevant Genes and Pathways Involved in the Antiviral Response 
PLoS ONE  2014;9(5):e97471.
The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. To understand the molecular basis for antiviral defense in this species, we used Illumia paired-end sequencing to characterize the spleen transcriptome of polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced large yellow croakers. The library produced 56,355,728 reads and assembled into 108,237 contigs. As a result, 15,192 unigenes were found from this transcriptome. Gene ontology analysis showed that 4,759 genes were involved in three major functional categories: biological process, cellular component, and molecular function. We further ascertained that numerous consensus sequences were homologous to known immune-relevant genes. Kyoto Encyclopedia of Genes and Genomes orthology mapping annotated 5,389 unigenes and identified numerous immune-relevant pathways. These immune-relevant genes and pathways revealed major antiviral immunity effectors, including but not limited to: pattern recognition receptors, adaptors and signal transducers, the interferons and interferon-stimulated genes, inflammatory cytokines and receptors, complement components, and B-cell and T-cell antigen activation molecules. Moreover, the partial genes of Toll-like receptor signaling pathway, RIG-I-like receptors signaling pathway, Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway, and T-cell receptor (TCR) signaling pathway were found to be changed after poly(I:C) induction by real-time polymerase chain reaction (PCR) analysis, suggesting that these signaling pathways may be regulated by poly(I:C), a viral mimic. Overall, the antivirus-related genes and signaling pathways that were identified in response to poly(I:C) challenge provide valuable leads for further investigation of the antiviral defense mechanism in the large yellow croaker.
doi:10.1371/journal.pone.0097471
PMCID: PMC4018400  PMID: 24820969
23.  hSulf-1 inhibits cell proliferation and migration and promotes apoptosis by suppressing stat3 signaling in hepatocellular carcinoma 
Oncology Letters  2014;7(4):963-969.
Human sulfatase-1 (hSulf-1) has been shown to desulfate cellular heparin sulfate proteoglycans and modulate several growth factors and cytokines. However, hSulf-1 has not been previously shown to mediate the signal transducer and activator of transcription 3 (stat3) signaling pathway, which is known to regulate cell proliferation, motility and apoptosis. The present study investigated the role of hSulf-1 in stat3 signaling in hepatocellular cancer. hSulf-1 expression vector and stat3 small interfering RNA (siRNA) were constructed to control the expression of hSulf-1 and stat3 in HepG2 cells. hSulf-1 was found to inhibit the phosphorylation of stat3 and downregulate its targeted protein. MTT and Transwell chamber assays, as well as Annexin V/propidium iodide double-staining methods, were used to examine the effects of hSulf-1 on stat3-mediated motility, proliferation and apoptosis in HepG2 cells. Transfection with hSulf-1 cDNA and/or stat3 siRNA inhibited cell proliferation and motility, concurrent with G0/G1 and G2/M phase cell cycle arrest and apoptosis. Overall, the results of the current study suggested that hSulf-1 functions as a negative regulator of proliferation and migration and as a positive regulator of apoptosis in hepatocellular carcinoma, at least partly via the downregulation of stat3 signaling.
doi:10.3892/ol.2014.1848
PMCID: PMC3961425  PMID: 24944651
human sulfatase-1; hepatocellular carcinoma; stat3 signaling
24.  Statistical analysis of SHAPE-directed RNA secondary structure modeling 
Biochemistry  2013;52(4):596-599.
The ability to predict RNA secondary structure is fundamental for understanding and manipulating RNA function. The structural information obtained from selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments greatly improves the accuracy of RNA secondary structure prediction. Recently, Das and colleagues [Kladwang et al., Biochemistry 50:8049 (2011)] proposed a “bootstrapping” approach to estimate the variance and helix-by-helix confidence levels of predicted secondary structures based on resampling (randomizing and summing) the measured SHAPE data. We show that the specific resampling approach described by Kladwang et al. introduces systematic errors and underestimates confidence in secondary structure prediction using SHAPE data. Instead, a leave-data-out jackknife approach better estimates the influence of a given experimental dataset on SHAPE-directed secondary structure modeling. Even when 35% of the data were left out in the jackknife approach, the confidence levels of SHAPE-directed secondary structure prediction were significantly higher than those calculated by Das and colleagues using bootstrapping. Helix confidence levels were thus significantly underestimated in the recent study, and resampling approach implemented by Kladwang et al. is not an appropriate metric for assigning confidences in SHAPE-directed secondary structure modeling.
doi:10.1021/bi300756s
PMCID: PMC3558531  PMID: 23286327
25.  Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption 
Background
Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.
Results
KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g · L-1 · h-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.
Conclusions
Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.
doi:10.1186/1754-6834-7-5
PMCID: PMC3891980  PMID: 24401161
Biofilm reactor; Clostridium acetobutylicum; Simultaneous product recovery; Acetoin; Adsorption; Redox modulation

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