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1.  Solution-phase-peptide synthesis via the Group-Assisted Purification (GAP) chemistry without using chromatography and recrystallization† 
The solution phase synthesis of N-protected amino acids and peptides has been achieved through the Group-Assisted Purification (GAP) chemistry by avoiding disadvantages of other methods in regard to the difficult scale-up, expenses of solid and soluble polymers, etc. The GAP synthesis can reduce the use of solvents, silica gels, energy and manpower. In addition, the GAP auxiliary can be conveniently recovered for re-use and is of environmentally friendly benign by substantially reducing waste production in academic labs and industry.
doi:10.1039/c3cc48509a
PMCID: PMC3929111  PMID: 24336500
2.  Glutamate Mediates the Function of Melanocortin Receptors 4 on Sim1 Neurons in Body Weight Regulation 
Cell metabolism  2013;18(6):10.1016/j.cmet.2013.11.003.
SUMMARY
The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown and as a result, little is known about the second-order neurons of the MC4R neural pathway. Single minded 1 (Sim1)-expressing brain regions, which include the paraventricular nucleus of hypothalamus (PVH), represent key brain sites that mediate melanocortin action. We conditionally restored MC4R expression in Sim1 neurons in the background of Mc4r-null mice. The restoration dramatically reduced obesity in Mc4r-null mice. The anti-obesity effect was completely reversed by selective disruption of glutamate release from those same Sim1 neurons. The reversal was caused by lower energy expenditure and hyperphagia. Corroboratively, disruption of glutamate release selectively from adult PVH neurons led to rapid obesity development via reduced energy expenditure and hyperphagia. Thus, this study establishes glutamate as the primary neurotransmitter that mediates MC4Rs on Sim1 neurons in body weight regulation.
doi:10.1016/j.cmet.2013.11.003
PMCID: PMC3880549  PMID: 24315371
glutamate; MC4R; Sim1; obesity; energy expenditure; feeding
3.  Effect of fibroblast growth factor 9 on the osteogenic differentiation of bone marrow stromal stem cells and dental pulp stem cells 
Molecular Medicine Reports  2014;11(3):1661-1668.
The role of fibroblast growth factor 9 (FGF9) in bone formation may depend on gene dosage, developmental stage, cell type or interactions with other cytokines. In the present study bone marrow stromal stem cells (BMSCs) and dental pulp stem cells (DPSCs) were cultured and osteogenically induced in vitro, treated with exogenous FGF9 at varying concentrations. Alkaline phosphatase staining, alizarin red S staining, reverse transcription quantitative polymerase chain reaction and western blot analyses were performed in order to investigate the gene expression levels of osteogenic markers. The results of the present study demonstrated that FGF9 enhanced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) during osteogenic induction in BMSCs and DPSCs, which are derived from different tissues. FGF9 also inhibited the osteogenic differentiation of BMSCs and DPSCs through the activation of ERK1/2. These findings suggested that FGF9 may be an inhibitor of osteogenesis in mesenchymal stem cells in vitro and its application in vivo requires investigation in the future.
doi:10.3892/mmr.2014.2998
PMCID: PMC4270321  PMID: 25435023
fibroblast growth factor 9; osteogenic differentiation; bone marrow stromal stem cells; dental pulp stem cells
4.  Δ9-THC-caused synaptic and memory impairments are mediated through COX-2 signaling 
Cell  2013;155(5):1154-1165.
SUMMARY
Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here we show that synaptic and cognitive impairments following repeated exposure to Δ9-tetrahydrocannabinol (Δ9-THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids, in the brain. COX-2 induction by Δ9-THC is mediated via CB1 receptor-coupled G-protein βγ subunits. Pharmacological or genetic inhibition of COX-2 blocks down-regulation and internalization of glutamate receptor subunits and alterations of the dendritic spine density of hippocampal neurons induced by repeated Δ9-THC exposures. Ablation of COX-2 also eliminates Δ9-THC-impaired hippocampal long-term synaptic plasticity, spatial, and fear memories. Importantly, the beneficial effects of decreasing β-amyloid plaques and neurodegeneration by Δ9-THC in Alzheimer’s disease animals are retained in the presence of COX-2 inhibition. These results suggest that the applicability of medical marijuana would be broadened by concurrent inhibition of COX-2.
doi:10.1016/j.cell.2013.10.042
PMCID: PMC3918429  PMID: 24267894
5.  Spatial distributions of cone inputs to cells of the parvocellular pathway investigated with cone-isolating gratings 
Receptive fields of midget ganglion cells and parvocellular lateral geniculate nucleus (LGN) neurons show color-opponent responses because they receive antagonistic input from the middle- and long-wavelength sensitive cones. It has been controversial as to whether this opponency can derive from random connectivity; if receptive field centers of cells near the fovea are cone-specific due to midget morphology, this would confer some degree of color opponency even with random cone input to the surround. A simple test of this mixed surround hypothesis is to compare spatial frequency tuning curves for luminance gratings and gratings isolating cone input to the receptive field center. If tuning curves for luminance gratings were bandpass, then with the mixed surround hypothesis tuning curves for gratings isolating the receptive field center cone class should also be bandpass, but to a lesser extent than for luminance. Tuning curves for luminance, chromatic, and cone-isolating gratings were measured in macaque retinal ganglion cells and LGN cells. We defined and measured a bandpass index to compare luminance and center cone-isolating tuning curves. Midget retinal ganglion cells and parvocellular LGN cells had bandpass indices between 0.1 and 1 with luminance gratings, but the index was usually near 1 (meaning low-pass tuning) when the receptive field center cone class alone was modulated. This is strong evidence for a considerable degree of cone-specific input to the surround. A fraction of midget and parvocellular cells showed evidence of incomplete specificity. Fitting the data with receptive field models revealed considerable intercell variability, with indications in some cells of a more complex receptive structure than a simple difference of Gaussians model.
PMCID: PMC4237200  PMID: 22330383
6.  Diagnosis of 65 cases of ampullary renal pelvis after postnatal follow-up of 1,167 newborn infants with prenatally suspected hydronephrosis 
The aim of the present study was to assess the morbidity of ampullary renal pelvis (ARP) and document its natural history in post-natal life. A total of 1,167 newborn infants with prenatally suspected hydronephrosis were retrospectively analyzed. Of these, 65 patients were diagnosed with ARP by computed tomography urography (CTU) and/or magnetic resonance urography (MRU). All cases were followed up with ultrasonogrophy at 1, 3, 6 and 12 months after birth, and one case was followed up for 5 years. Changes in the separation of the renal pelvis collection system were recorded. Children with ARP accounted for 5.57% of the total cases (65/1,167) followed-up. No lack of connection between the renal calyces and the renal pelvis was detected. The long-term follow-up revealed that the separation of the renal pelvis collection system did not tend to increase over time. In addition to imaging examinations, long-term follow-up observation is recommended for the accurate diagnosis of pediatric ARP, particularly for differentiation from hydronephrosis.
doi:10.3892/etm.2014.2076
PMCID: PMC4247286  PMID: 25452792
hydronephrosis; ampullary renal pelvis; follow-up studies
7.  Asymmetric C-C Bond Formation between Chiral N-Phosphonyl Imines and Ni(II)-Complex of Glycine Schiff Base Provides a GAP Synthesis of α,β-syn-Diamino Acid Derivatives 
European journal of organic chemistry  2013;2013(22):4744-4747.
The GAP asymmetric synthesis of α,β-diamino acid derivatives has been achieved by reacting chiral N-phosphonyl imines with Ni(II)-complex of glycine ester-based enolate without the use of traditional purifications of chromatography and recrystallization. The successful control of synstereochemistry of vicinal diamino products complements our previous methods which afforded anti stereoisomers and enables all four individual isomers to be synthesized by simply changing enolate geometry. In contrast to our previous synthesis where required at least 5 equiv of glycine Schiff base enolate for complete conversion, the new synthesis only needs 1.1 equiv of glycine Schiff base enolate to give complete diastereoselectivity (>99% de) and yields (91% – 97%). The absolute stereochemistry has been unambiguously determined by X-ray structural analysis.
PMCID: PMC4192728  PMID: 25309122
8.  Methy-Pipe: An Integrated Bioinformatics Pipeline for Whole Genome Bisulfite Sequencing Data Analysis 
PLoS ONE  2014;9(6):e100360.
DNA methylation, one of the most important epigenetic modifications, plays a crucial role in various biological processes. The level of DNA methylation can be measured using whole-genome bisulfite sequencing at single base resolution. However, until now, there is a paucity of publicly available software for carrying out integrated methylation data analysis. In this study, we implemented Methy-Pipe, which not only fulfills the core data analysis requirements (e.g. sequence alignment, differential methylation analysis, etc.) but also provides useful tools for methylation data annotation and visualization. Specifically, it uses Burrow-Wheeler Transform (BWT) algorithm to directly align bisulfite sequencing reads to a reference genome and implements a novel sliding window based approach with statistical methods for the identification of differentially methylated regions (DMRs). The capability of processing data parallelly allows it to outperform a number of other bisulfite alignment software packages. To demonstrate its utility and performance, we applied it to both real and simulated bisulfite sequencing datasets. The results indicate that Methy-Pipe can accurately estimate methylation densities, identify DMRs and provide a variety of utility programs for downstream methylation data analysis. In summary, Methy-Pipe is a useful pipeline that can process whole genome bisulfite sequencing data in an efficient, accurate, and user-friendly manner. Software and test dataset are available at http://sunlab.lihs.cuhk.edu.hk/methy-pipe/.
doi:10.1371/journal.pone.0100360
PMCID: PMC4063866  PMID: 24945300
9.  SHANK3 overexpression causes manic-like behavior with unique pharmacogenetic properties 
Nature  2013;503(7474):72-77.
Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, suggesting that proper SHANK3 dosage is critical for normal brain function. SHANK3 overexpression per se has not been established as a cause of human disorders, however, because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modeling a human SHANK3 duplication exhibit manic-like behavior and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings suggest SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behavior of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.
doi:10.1038/nature12630
PMCID: PMC3923348  PMID: 24153177
10.  Asymmetric synthesis of novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluation of their anti-inflammatory activity 
Life sciences  2012;92(8-9):506-511.
Aims
To design and synthesize novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluate their analgesic and anti-inflammatory potential.
Main methods
The murine macrophage cell line RAW 264.7 has been widely used as a model for inflammatory responses in vitro. Our model consists of cultured monolayers of RAW 264.7 cells in which media concentrations of 15-deoxy-Δ13, 14-PGJ2 (PGJ) are measured by ELISA following LPS (10 ng/ml) stimulation and treatment with 0.1, 0.3, 1.0, 3.0 and 10 μM concentrations of the compounds.
Key findings
Our data indicate that several of our compounds have the capacity to increase production of PGJ and may also increase the occurrence of programmed cell death (apoptosis).
Significance
Thus these agents are potential candidates for the therapy of conditions characterized by ongoing (chronic) inflammation and its associated pain.
doi:10.1016/j.lfs.2012.06.040
PMCID: PMC3516634  PMID: 22820546
Cannabinoid receptor; Anandamide; Arachidonyl amide; 15-deoxy-Δ13; 14-PGJ2 (PGJ) Stimulation; Apoptosis
11.  Modulation of pyramidal cell output in the medial prefrontal cortex by mGluR5 interacting with CB1 
Neuropharmacology  2012;66:170-178.
The medial prefrontal cortex (mPFC) serves executive cognitive functions such as decision-making that are impaired in neuropsychiatric disorders and pain. We showed previously that amygdala-driven abnormal inhibition and decreased output of mPFC pyramidal cells contribute to pain-related impaired decision-making (Ji et al., 2010). Therefore, modulating pyramidal output is desirable therapeutic goal. Targeting metabotropic glutamate receptor subtype mGluR5 has emerged as a cognitive-enhancing strategy in neuropsychiatric disorders, but synaptic and cellular actions of mGluR5 in the mPFC remain to be determined. The present study determined synaptic and cellular actions of mGluR5 to test the hypothesis that increasing mGluR5 function can enhance pyramidal cell output.
Whole-cell voltage- and current-clamp recordings were made from visually identified pyramidal neurons in layer V of the mPFC in rat brain slices. Both the prototypical mGluR5 agonist CHPG and a positive allosteric modulator (PAM) for mGluR5 (VU0360172) increased synaptically evoked spiking (E–S coupling) in mPFC pyramidal cells. The facilitatory effects of CHPG and VU0360172 were inhibited by an mGluR5 antagonist (MTEP). CHPG, but not VU0360172, increased neuronal excitability (frequency– current [F–I] function). VU0360172, but not CHPG, increased evoked excitatory synaptic currents (EPSCs) and amplitude, but not frequency, of miniature EPSCs, indicating a postsynaptic action. VU0360172, but not CHPG, decreased evoked inhibitory synaptic currents (IPSCs) through an action that involved cannabinoid receptor CB1, because a CB1 receptor antagonist (AM281) blocked the inhibitory effect of VU0360172 on synaptic inhibition. VU0360172 also increased and prolonged CB1-mediated depolarization-induced suppression of synaptic inhibition (DSI). Activation of CB1 with ACEA decreased inhibitory transmission through a presynaptic mechanism.
The results show that increasing mGluR5 function enhances mPFC output. This effect can be accomplished by increasing excitability with an orthosteric agonist (CHPG) or by increasing excitatory synaptic drive and CB1-mediated presynaptic suppression of synaptic inhibition (“dis-inhibition”) with a PAM (VU0360172). Therefore, mGluR5 may be a useful target in conditions of impaired mPFC output.
doi:10.1016/j.neuropharm.2012.03.024
PMCID: PMC3568505  PMID: 22521499
mGluR5; Positive allosteric modulator; PAM; CB1; Prefrontal cortex; Synaptic transmission
12.  Efficacy and Safety of Therapies for Acute Ischemic Stroke in China: A Network Meta-Analysis of 13289 Patients from 145 Randomized Controlled Trials 
PLoS ONE  2014;9(2):e88440.
Background
Many of these therapies have been compared against placebos, but have not been directly compared against each other. To evaluate the efficacy and safety of several commonly used drugs for AIS directly or indirectly.
Methods
A systematic literature review was performed to identify randomized controlled trials (RCTs) published prior to April 2013 for AIS therapies. The primary outcome measures were the National Institutes of Health Stroke Scale (NIHSS) scores and the clinical effective rate. A fixed-effects meta-analysis and meta-regression are performed; lastly, performed a mixed treatment comparison was performed through the Bayesian methods.
Results
Outcome of Efficacy of therapies for acute ischemic stroke are as followed: All of the therapies mentioned above yielded results a more effective result than placebo, Sodium ozagrel (RR 3.86, 95%CI 3.18–4.61); Sodium ozagrel + edaravone (RR 9.60, 95%CI 7.04–13.06); Edaravone (RR 4.07, 95%CI 3.30–5.01); Edaravone + Kininogenase (RR 15.33, 95%CI 10.03–23.05). The significant difference in efficacy between edaravone monotherapy and Sodium ozagrel + edaravone was evident (RR 0.43, 95%CI 0.08–0.61) and was also significant between efficacy of edaravone + Kininogenase and Sodium ozagrel (RR 4.00, 95%CI 2.47–6.24). The differences between the risk and benefit were not significant when comparing Sodium ozagrel and edaravone or edaravone + Kininogenase and Sodium ozagrel + Edaravone for AIS. Outcome of the defect of neurological function: Placebo served a significant difference in treating the defects of neurological function compared with Sodium ozagrel (WMD = −3.11, 95%CI −4.43 to −1.79), Sodium ozagrel + edaravone (WMD = −6.25, 95%CI −7.96 to −4.54) and Edaravone + Kininogenase (WMD =  −3.47, 95%CI −5.73 to −1.21).
Conclusions
It provides that the efficacy of edaravone monotherapy in treatment was not more effective than Sodium ozagrel + edaravone.The efficacy of edaravone + Kininogenase monotherapy in treatment was more effective than Sodium ozagrel. Edaravone + Kininogenase and Sodium ozagrel + Edaravone appeared the most effective treatments. And Sodium ozagrel, Sodium ozagrel + edaravone, Edaravone + Kininogenase can improve the nerve dysfunction.
doi:10.1371/journal.pone.0088440
PMCID: PMC3923787  PMID: 24551100
13.  Cyanophycin Mediates the Accumulation and Storage of Fixed Carbon in Non-Heterocystous Filamentous Cyanobacteria from Coniform Mats 
PLoS ONE  2014;9(2):e88142.
Thin, filamentous, non-heterocystous, benthic cyanobacteria (Subsection III) from some marine, lacustrine and thermal environments aggregate into macroscopic cones and conical stromatolites. We investigate the uptake and storage of inorganic carbon by cone-forming cyanobacteria from Yellowstone National Park using high-resolution stable isotope mapping of labeled carbon (H13CO3−) and immunoassays. Observations and incubation experiments in actively photosynthesizing enrichment cultures and field samples reveal the presence of abundant cyanophycin granules in the active growth layer of cones. These ultrastructurally heterogeneous granules rapidly accumulate newly fixed carbon and store 18% of the total particulate labeled carbon after 120 mins of incubation. The intracellular distribution of labeled carbon during the incubation experiment demonstrates an unexpectedly large contribution of PEP carboxylase to carbon fixation, and a large flow of carbon and nitrogen toward cyanophycin in thin filamentous, non-heterocystous cyanobacteria. This pattern does not occur in obvious response to a changing N or C status. Instead, it may suggest an unusual interplay between the regulation of carbon concentration mechanisms and accumulation of photorespiratory products that facilitates uptake of inorganic C and reduces photorespiration in the dense, surface-attached communities of cyanobacteria from Subsection III.
doi:10.1371/journal.pone.0088142
PMCID: PMC3917874  PMID: 24516596
14.  Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors 
Nucleic Acids Research  2014;42(7):4375-4390.
The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.
doi:10.1093/nar/gku109
PMCID: PMC3985678  PMID: 24500196
15.  An Approach to Vicinal t-Boc-Amino Dibromides via Catalytic Aminobromination of Nitrostyrenes without using Chromatography and Recrystallization 
The Journal of organic chemistry  2013;78(3):1171-1175.
1.0 % Mol of K3PO4·3H2O was found to catalyze aminohalogenation reaction of nitrostyrenes with N,N-dibromo-tert-butylcarbamate (t-Boc-NBr2) in dichloroethane system. Good to excellent yields and complete regioselectivity have been achieved by taking advantage of the GAP work-up without using traditional purification techniques such as column chromatography and recrystallization. New mechanism was proposed involving radical and ionic catalytic cycles and an intramolecular migration.
doi:10.1021/jo302727v
PMCID: PMC3568972  PMID: 23311641
Aminohalogenation; bromoamine; Group-Assistant-Purification (GAP) chemistry; N,N-dibromo-tert-butylcarbamate; nitrostyrenes
16.  Sebnif: An Integrated Bioinformatics Pipeline for the Identification of Novel Large Intergenic Noncoding RNAs (lincRNAs) - Application in Human Skeletal Muscle Cells 
PLoS ONE  2014;9(1):e84500.
Ab initio assembly of transcriptome sequencing data has been widely used to identify large intergenic non-coding RNAs (lincRNAs), a novel class of gene regulators involved in many biological processes. To differentiate real lincRNA transcripts from thousands of assembly artifacts, a series of filtering steps such as filters of transcript length, expression level and coding potential, need to be applied. However, an easy-to-use and publicly available bioinformatics pipeline that integrates these filters is not yet available. Hence, we implemented sebnif, an integrative bioinformatics pipeline to facilitate the discovery of bona fide novel lincRNAs that are suitable for further functional characterization. Specifically, sebnif is the only pipeline that implements an algorithm for identifying high-quality single-exonic lincRNAs that were often omitted in many studies. To demonstrate the usage of sebnif, we applied it on a real biological RNA-seq dataset from Human Skeletal Muscle Cells (HSkMC) and built a novel lincRNA catalog containing 917 highly reliable lincRNAs. Sebnif is available at http://sunlab.lihs.cuhk.edu.hk/sebnif/.
doi:10.1371/journal.pone.0084500
PMCID: PMC3882232  PMID: 24400097
17.  Antioxidant activities of chick embryo egg hydrolysates 
Food Science & Nutrition  2013;2(1):58-64.
Chick embryo egg hydrolysates (CEEH) were obtained by enzymatic hydrolysis of chick embryo egg in vitro-simulated gastrointestinal digestion. The antioxidant activities of CEEH were investigated by employing three in vitro assays, including the 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate)/1,1-diphenyl-2-picrylhydrazyl (ABTS/DPPH)/hydroxyl radical-scavenging assays. The radical-scavenging effect of CEEH (1.0 mg/mL) was in a dose-dependent manner, with the highest trolox equivalent antioxidant capacity for ABTS, DPPH, and that of hydroxyl radicals found to be 569, 2097, and 259.6 μmol/L, respectively; whereas the trolox equivalent antioxidant capacity of unhatched egg for ABTS, DPPH, and that of hydroxyl radicals were found to be 199, 993, and 226.5 μmol/L, respectively. CEEH showed stronger scavenging activity than the hydrolysates of unhatched egg against free radicals such as ABTS, DPPH, and hydroxyl radicals. The antioxidant amino acid analysis indicated that the 14-day CEEH possess more antioxidant amino acids than that of the unhatched egg. In addition, essential amino acids analysis showed that the 14-day CEEH have the highest nutritional value. Combined with the results of the amino acid profiles, CEEH were believed to have higher nutritive value in addition to antioxidant activities than the unhatched egg.
doi:10.1002/fsn3.77
PMCID: PMC3951551  PMID: 24804065
Antioxidant activities; chick embryo egg hydrolysates; nutritive value
18.  128-Slice Acceletated-Pitch Dual Energy CT Angiography of the Head and Neck: Comparison of Different Low Contrast Medium Volumes 
PLoS ONE  2013;8(11):e80939.
Background
Our study aims to evaluate the image quality and feasibility of 128-slice dual-energy CTA (DE-CTA) for supra-aortic arteries using reduced amounts of contrast medium (CM).
Methods
A prospective study was performed in 54 patients receiving CTA of the head and neck with a 128-slice dual-source CT system. Patients were randomized into two groups with a volume of either 40 mL of CM (Group I) or 50 mL of CM (Group II). Arterial and venous enhancements were recorded for quantitative assessment. Qualitative assessments for images without bone removal (BR) were based on a) the visualization of the circle of Willis and b) streak artifacts due to residual CM in the subclavian or internal jugular veins ipsilateral to injection of CM. Qualitative assessment of dual-energy images using BR was based on the presence of bone remnants and vessel integrity. Quantitative data was compared using the Student t test. The χ2 test was used for the qualitative measurements of streak artifacts in veins while the Mann-Whitney U test was used for the qualitative measurements of images with BR.
Results
Arterial and venous attenuation was significantly higher in Group II (P=0.000). Image quality regarding the circle of Willis was excellent in both groups (3.90±0.30 for Group I and 4.00±0 for Group II) . Imaging of the internal jugular veins was scored higher in Group I (1.87±0.72) compared with Group II (1.48±0.51) (P=0.021). Within Group I using BR, mean scores for bone remnants did not differ significantly (P>0.05) but mean scores of vessel integrity (P<0.05) did.
Conclusions
Contrast-enhanced head and neck CTA is feasible using a scan protocol with low amounts of contrast medium (40 mL) on a 128-slice dual-energy CTA. The 40-mL protocol provides satisfactory image quality before and after dual-energy bone-removal post-processing.
doi:10.1371/journal.pone.0080939
PMCID: PMC3834309  PMID: 24260516
19.  NF-κB–YY1–miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma 
Cancer cell  2008;14(5):10.1016/j.ccr.2008.10.006.
SUMMARY
Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and Rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts miR-29 is repressed by NF-κB acting through YY1 and the Polycomb. During myogenesis, NF-κB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-κB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its pro-myogenic function. Together, results identify a NF-κB–YY1–miR-29 regulatory circuit whose disruption may contribute to RMS.
SIGNIFICANCE
MicroRNAs regulate skeletal myogenesis, but their impact in muscle diseases is not well understood. Here we describe miR-29 as an enhancer of myogenic differentiation and a suppressor of RMS. We find that miR-29 exists in a regulatory circuit involving NF-κB and YY1. In myoblasts NF-B acts through YY1 to epigenetically suppress miR-29, while during differentiation miR-29 is induced to facilitate myogenesis by a negative feedback on YY1. Significantly, RMS tumors lose miR-29 due to an elevation in NF-B and YY1, and readjustment of miR-29 levels in RMS stimulates differentiation. Thus, myogenesis is dependent on NF-κB–YY1–miR-29 circuitry whose dysfunction may contribute to RMS pathogenesis. Such findings offer potential avenues for the diagnosis and treatment of muscle relevant cancers.
doi:10.1016/j.ccr.2008.10.006
PMCID: PMC3829205  PMID: 18977326
microRNA; differentiation; myogenesis; YY1; NF-kappaB; Rhabdomyosarcoma
20.  Insight into the Intermolecular Recognition Mechanism between Keap1 and IKKβ Combining Homology Modelling, Protein-Protein Docking, Molecular Dynamics Simulations and Virtual Alanine Mutation 
PLoS ONE  2013;8(9):e75076.
Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1), a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2) and IκB kinase β (IKKβ), which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI), the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.
doi:10.1371/journal.pone.0075076
PMCID: PMC3774807  PMID: 24066166
21.  mGluR5 Ablation in Cortical Glutamatergic Neurons Increases Novelty-Induced Locomotion 
PLoS ONE  2013;8(8):e70415.
The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the pathology of various neurological disorders including schizophrenia, ADHD, and autism. mGluR5-dependent synaptic plasticity has been described at a variety of neural connections and its signaling has been implicated in several behaviors. These behaviors include locomotor reactivity to novel environment, sensorimotor gating, anxiety, and cognition. mGluR5 is expressed in glutamatergic neurons, inhibitory neurons, and glia in various brain regions. In this study, we show that deleting mGluR5 expression only in principal cortical neurons leads to defective cannabinoid receptor 1 (CB1R) dependent synaptic plasticity in the prefrontal cortex. These cortical glutamatergic mGluR5 knockout mice exhibit increased novelty-induced locomotion, and their locomotion can be further enhanced by treatment with the psychostimulant methylphenidate. Despite a modest reduction in repetitive behaviors, cortical glutamatergic mGluR5 knockout mice are normal in sensorimotor gating, anxiety, motor balance/learning and fear conditioning behaviors. These results show that mGluR5 signaling in cortical glutamatergic neurons is required for precisely modulating locomotor reactivity to a novel environment but not for sensorimotor gating, anxiety, motor coordination, several forms of learning or social interactions.
doi:10.1371/journal.pone.0070415
PMCID: PMC3734292  PMID: 23940572
22.  Human Umbilical Cord Blood-Derived Stromal Cells Are Superior to Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Inducing Myeloid Lineage Differentiation In Vitro 
Stem Cells and Development  2011;21(9):1429-1440.
Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34+ hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.
doi:10.1089/scd.2011.0348
PMCID: PMC3359623  PMID: 22023173
23.  Autosomal STRs Provide Genetic Evidence for the Hypothesis That Tai People Originate from Southern China 
PLoS ONE  2013;8(4):e60822.
Tai people are widely distributed in Thailand, Laos and southwestern China and are a large population of Southeast Asia. Although most anthropologists and historians agree that modern Tai people are from southwestern China and northern Thailand, the place from which they historically migrated remains controversial. Three popular hypotheses have been proposed: northern origin hypothesis, southern origin hypothesis or an indigenous origin. We compared the genetic relationships between the Tai in China and their “siblings” to test different hypotheses by analyzing 10 autosomal microsatellites. The genetic data of 916 samples from 19 populations were analyzed in this survey. The autosomal STR data from 15 of the 19 populations came from our previous study (Lin et al., 2010). 194 samples from four additional populations were genotyped in this study: Han (Yunnan), Dai (Dehong), Dai (Yuxi) and Mongolian. The results of genetic distance comparisons, genetic structure analyses and admixture analyses all indicate that populations from northern origin hypothesis have large genetic distances and are clearly differentiated from the Tai. The simulation-based ABC analysis also indicates this. The posterior probability of the northern origin hypothesis is just 0.04 [95%CI: (0.01–0.06)]. Conversely, genetic relationships were very close between the Tai and populations from southern origin or an indigenous origin hypothesis. Simulation-based ABC analyses were also used to distinguish the southern origin hypothesis from the indigenous origin hypothesis. The results indicate that the posterior probability of the southern origin hypothesis [0.640, 95%CI: (0.524–0.757)] is greater than that of the indigenous origin hypothesis [0.324, 95%CI: (0.211–0.438)]. Therefore, we propose that the genetic evidence does not support the hypothesis of northern origin. Our genetic data indicate that the southern origin hypothesis has higher probability than the other two hypotheses statistically, suggesting that the Tai people most likely originated from southern China.
doi:10.1371/journal.pone.0060822
PMCID: PMC3620166  PMID: 23593317
24.  GPR55, a G-Protein Coupled Receptor for Lysophosphatidylinositol, Plays a Role in Motor Coordination 
PLoS ONE  2013;8(4):e60314.
The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.
doi:10.1371/journal.pone.0060314
PMCID: PMC3614963  PMID: 23565223
25.  Luminance and chromatic contributions to a hyperacuity task: isolation by contrast polarity and target separation 
Vision Research  2012;56:28-37.
Vernier thresholds are known to be elevated when a target pair has opposite contrast polarity. Polarity reversal is used to assess the role of luminance and chromatic pathways in hyperacuity performance. Psychophysical hyperacuity thresholds were measured for pairs of gratings of various combinations of luminance (Lum) and chromatic (Chr) contrast polarities, at different ratios of luminance to chromatic contrast. With two red-green gratings of matched luminance and chromatic polarity (+Lum+Chr), there was an elevation of threshold at isoluminance. When both luminance and chromatic polarity were mismatched (−Lum−Chr), thresholds were substantially elevated under all conditions. With the same luminance contrast polarity and opposite chromatic polarity (+Lum−Chr) thresholds were only elevated close to isoluminance; in the reverse condition (−Lum+Chr), thresholds were elevated as in the −Lum−Chr condition except close to equiluminance. Similar data were obtained for gratings isolating the short-wavelength cone mechanism. Further psychophysical measurements assessed the role of target separation with matched or mismatched contrast polarity; similar results were found for luminance and chromatic gratings. Comparison physiological data were collected from parafoveal ganglion cells of the macaque retina. Positional precision of ganglion cell signals was assessed under conditions related to the psychophysical measurements. On the basis of these combined observations, it is argued that both magnocellular, parvocellular, and koniocellular pathways have access to cortical positional mechanisms associated with vernier acuity.
doi:10.1016/j.visres.2012.01.007
PMCID: PMC3294055  PMID: 22306680
hyperacuity; vernier; contrast; polarity; magnocellular; parvocellular; luminance; chromatic

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