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1.  Modulatory Effect of Acupuncture at Waiguan (TE5) on the Functional Connectivity of the Central Nervous System of Patients with Ischemic Stroke in the Left Basal Ganglia 
PLoS ONE  2014;9(6):e96777.
Objective
To study the influence of acupuncture at Waiguan (TE5) on the functional connectivity of the central nervous system of patients with ischemic stroke.
Methods
Twenty-four patients with ischemic stroke in the left basal ganglia were randomized based on gender to receive TE5 acupuncture (n = 12) or nonacupoint acupuncture (n = 12). Each group underwent sham acupuncture and then verum acupuncture while being scanned with functional magnetic resonance imaging. Six regions of interest (ROI) were defined, including bilateral motor, somatosensory, and bilateral basal ganglia areas. The functional connectivity between these ROIs and all voxels of the brain was analyzed in Analysis of Functional NeuroImages(AFNI) to explore the differences between verum acupuncture and sham acupuncture at TE5 and between TE5 acupuncture and nonacupoint acupuncture. The participants were blinded to the allocation.
Result
The effect of acupuncture on six seed-associated networks was explored. The result demonstrated that acupuncture at Waiguan (TE5) can regulate the sensorimotor network of the ipsilesional hemisphere, stimulate the contralesional sensorimotor network, increase cooperation of bilateral sensorimotor networks, and change the synchronization between the cerebellum and cerebrum. Furthermore, a lot of differences of effect existed between verum acupuncture and sham acupuncture at TE5, but there was little difference between TE5 acupuncture and nonacupoint acupuncture.
Conclusion
The modulation of synchronizations between different regions within different brain networks might be the mechanism of acupuncture at Waiguan (TE5). Stimulation of the contralesional sensorimotor network and increase of cooperation of bilateral hemispheres imply a compensatory effect of the intact hemisphere, whereas changes in synchronization might influence the sensorimotor function of the affected side of the body.
Trial Registration
Chinese Clinical Trial Registry ChiCTR-ONRC-08000255
doi:10.1371/journal.pone.0096777
PMCID: PMC4057077  PMID: 24927275
2.  Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study 
The lancet. Respiratory medicine  2013;1(4):309-317.
Summary
Background
Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF.
Methods
First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10−8) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes.
Findings
In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [r2=0.07], rs5743894 [r2=0.16], and rs5743890 [r2=0.01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1.72 [95% CI 1.24–2.38]; p=0.0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0.097), 40% in those with the minor allele of rs111521887 (p=3.0 × 10−4), and 50% in those with the minor allele of rs5743894 (p=2.93 × 10−5) compared with homozygous carriers of common alleles for these SNPs.
Interpretation
Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease.
Funding
National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.
doi:10.1016/S2213-2600(13)70045-6
PMCID: PMC3894577  PMID: 24429156
3.  Preclinical Detection of Porcine Circovirus Type 2 Infection Using an Ultrasensitive Nanoparticle DNA Probe-Based PCR Assay 
PLoS ONE  2014;9(5):e97869.
Porcine circovirus type 2 (PCV2) has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR) for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.
doi:10.1371/journal.pone.0097869
PMCID: PMC4026479  PMID: 24842840
4.  HIV-Infected Individuals with Low CD4/CD8 Ratio despite Effective Antiretroviral Therapy Exhibit Altered T Cell Subsets, Heightened CD8+ T Cell Activation, and Increased Risk of Non-AIDS Morbidity and Mortality 
PLoS Pathogens  2014;10(5):e1004078.
A low CD4/CD8 ratio in elderly HIV-uninfected adults is associated with increased morbidity and mortality. A subset of HIV-infected adults receiving effective antiretroviral therapy (ART) fails to normalize this ratio, even after they achieve normal CD4+ T cell counts. The immunologic and clinical characteristics of this clinical phenotype remain undefined. Using data from four distinct clinical cohorts and three clinical trials, we show that a low CD4/CD8 ratio in HIV-infected adults during otherwise effective ART (after CD4 count recovery above 500 cells/mm3) is associated with a number of immunological abnormalities, including a skewed T cell phenotype from naïve toward terminally differentiated CD8+ T cells, higher levels of CD8+ T cell activation (HLADR+CD38+) and senescence (CD28− and CD57+CD28−), and higher kynurenine/tryptophan ratio. Changes in the peripheral CD4/CD8 ratio are also reflective of changes in gut mucosa, but not in lymph nodes. In a longitudinal study, individuals who initiated ART within six months of infection had greater CD4/CD8 ratio increase compared to later initiators (>2 years). After controlling for age, gender, ART duration, nadir and CD4 count, the CD4/CD8 ratio predicted increased risk of morbidity and mortality. Hence, a persistently low CD4/CD8 ratio during otherwise effective ART is associated with increased innate and adaptive immune activation, an immunosenescent phenotype, and higher risk of morbidity/mortality. This ratio may prove useful in monitoring response to ART and could identify a unique subset of individuals needed of novel therapeutic interventions.
Author Summary
The CD4/CD8 ratio, a hallmark of the collection of T cell defects related to aging –“immunosenescence”- and a predictor of mortality in the general population, often fails to normalize in an important proportion of HIV-infected individuals with adequate CD4+ T cell recovery after ART initiation. However, the immunological and clinical characteristics of this clinical phenotype have not been elucidated. Herein we show that during treated HIV infection, expansion of CD8+ T cells, reflected as a low CD4/CD8 ratio, identifies a subgroup of individuals with a number of immunological abnormalities and a poor prognosis. These subjects exhibit increased innate and adaptive immune activation, an immunosenescent phenotype, CD4+ and CD8+ imbalance in the gut mucosa and higher risk of morbidity and mortality. In contrast, those who normalize the CD4/CD8 ratio have traits of a healthy immune system. We observed that early ART initiation might contribute to more rapid and robust CD4/CD8 ratio normalization compared to later initiation. Hence, the CD4/CD8 ratio might help to further discriminate the risk of disease progression of successfully treated HIV-infected individuals, and a successful response to ART may require both normalization of the peripheral CD4+ T cell count and the ratio of CD4+ to CD8+ T cell counts.
doi:10.1371/journal.ppat.1004078
PMCID: PMC4022662  PMID: 24831517
5.  Prognostic Value of Tumor-Infiltrating FoxP3+ T Cells in Gastrointestinal Cancers: A Meta Analysis 
PLoS ONE  2014;9(5):e94376.
Purpose
Tumor-infiltrating FoxP3+ T cells have been reported in various human tumors, which impaired cell-mediated immunity and promoted disease progression. However, its prognostic value for survival in patients with different gastrointestinal cancers [hepatocellular carcinoma (HCC), colorectal cancer (CRC), gastric cancer (GC)] remains controversial.
Methods
Relevant literature was searched using PubMed, Embase, Cochrane, Ovid Medline and Chinese wanfang databases. A meta-analysis was conducted to estimate pooled survival and recurrence ratios. The odds ratio (OR) and 95% confidence intervals (CI) were calculated employing fixed- or random-effects models depending on the heterogeneity of the included trials.
Results
For HCC and GC, the overall survival at 1, 3 and 5-year of high FoxP3+ T cells infiltration patients were lower than low FoxP3+ T cells infiltration patients (P<0.05). The recurrences at 1, 3 and 5-year of high FoxP3+ T cells infiltration patients were higher than low FoxP3+ T cells infiltration patients (P<0.001). But for CRC, the overall survival at 1, 3 and 5-year of high FoxP3+ T cells infiltration patients were higher than low FoxP3+ T cells infiltration patients (P<0.001). There were no differences in 1, 3 and 5-year recurrences between high and low FoxP3+ T cells infiltration patients (P>0.05).
Conclusions
Our findings suggested that tumor-infiltrating FoxP3+ T cells were a factor for a poor prognosis for HCC and GC, but a good prognosis for CRC.
doi:10.1371/journal.pone.0094376
PMCID: PMC4020764  PMID: 24827118
6.  Proteomic Identification of Nrf2-Mediated Phase II Enzymes Critical for Protection of Tao Hong Si Wu Decoction against Oxygen Glucose Deprivation Injury in PC12 Cells 
Chinese herbal medicine formula Tao Hong Si Wu decoction (THSWD) is traditionally used in China for cerebrovascular diseases. However, the molecular mechanisms of THSWD associated with the cerebral ischemia reperfusion injury are largely unknown. The current study applied the two-dimensional gel electrophoresis-based proteomics to investigate the different protein profiles in PC12 cells with and without the treatment of THSWD. Twenty-six proteins affected by THSWD were identified by MALDI-TOF mass spectrometry. Gene ontology analysis showed that those proteins participated in several important biological processes and exhibited diverse molecular functions. In particular, six of them were found to be phase II antioxidant enzymes, which were regulated by NF-E2-related factor-2 (Nrf2). Quantitative PCR further confirmed a dose-dependent induction of the six phase II enzymes by THSWD at the transcription level. Moreover, the individual ingredients of THSWD were discovered to synergistically contribute to the induction of phase II enzymes. Importantly, THSWD's protection against oxygen-glucose deprivation-reperfusion (OGD-Rep) induced cell death was significantly attenuated by antioxidant response element (ARE) decoy oligonucleotides, suggesting the protection of THSWD may be likely regulated at least in part by Nrf2-mediated phase II enzymes. Thus, our data will help to elucidate the molecular mechanisms underlying the neuroprotective effect of THSWD.
doi:10.1155/2014/945814
PMCID: PMC4037622  PMID: 24949080
7.  Tetrakis[μ3-2-(piperidin-1-yl)ethanolato]tetrakis[chloridocopper(II)] 
In the title tetra­nuclear compound, [Cu4(C7H14NO)4Cl4], each CuII cation is N,O-chelated by a piperidineethanolate anion and coordinated by a Cl− anion and two O atoms from neighboring piperidine­ethano­late anions in a distorted NO3Cl square-pyramidal geometry. The deprotonated hydroxyl groups of the piperidineethanolate anions bridge CuII cations, forming the tetra­nuclear complex. All piperidine rings display a chair conformation. In the crystal, there are no significant inter­molecular inter­actions present. The crystal studied was an inversion twin refined with a minor component of 0.18 (5).
doi:10.1107/S1600536814009052
PMCID: PMC4011270  PMID: 24860321
8.  Genetic Diversity of Salp15 in the Ixodes ricinus Complex (Acari: Ixodidae) 
PLoS ONE  2014;9(4):e94131.
Salp15, a 15-kDa tick salivary gland protein, is both essential for ticks to successfully obtain host blood and also facilitates transmission of Lyme borreliosis. To determine whether the Salp15 gene is expressed in Ixodes persulcatus and Ixodes sinensis, principle vectors of Lyme borreliosis in China, we studied transcriptions of this gene in semi-engorged larvae, nymph and adults of these two species. A total of eight Salp15 homologues, five in I. persulcatus and three in I. sinensis, were identified by reverse transcriptase–polymerase chain reaction (RT-PCR). Interestingly, the intra-species similarity of Salp15 is approximately equal to its interspecies similarity and more than one Salp15 protein is expressed in a certain tick developmental stage. Comparison of DNA and proteins with other available tick Salp15 homologues suggests that the Salp15 superfamily is genetically conserved and diverse in the Ixodes ricinus complex. These findings indicate that Salp15 proteins in the I. ricinus complex may play an essential role in interacting with the host immune system and transmission of Borrelia genospecies.
doi:10.1371/journal.pone.0094131
PMCID: PMC3979764  PMID: 24714063
9.  Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts 
Scientific Reports  2014;4:4570.
Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.
doi:10.1038/srep04570
PMCID: PMC3972505  PMID: 24691566
10.  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.
doi:10.4049/jimmunol.1203273
PMCID: PMC3608760  PMID: 23436936
vitamin D; inflammation; macrophage; miR-155; SOCS1; negative feedback
11.  Pharmacokinetics and Metabolism of 2-Aminothiazoles with Antiprion Activity in Mice 
Pharmaceutical research  2013;30(4):932-950.
Purpose
To discover drugs lowering PrPSc in prion-infected cultured neuronal cells that achieve high concentrations in brain to test in mouse models of prion disease and then treat people with these fatal diseases.
Methods
We tested 2-AMT analogs for EC50 and PK after a 40 mg/kg single dose and 40–210 mg/kg/day doses for 3 days. We calculated plasma and brain AUC, ratio of AUC/EC50 after dosing. We reasoned that compounds with high AUC/EC50 ratios should be good candidates going forward.
Results
We evaluated 27 2-AMTs in single-dose and 10 in 3-day PK studies, of which IND24 and IND81 were selected for testing in mouse models of prion disease. They had high concentrations in brain after oral dosing. Absolute bioavailability ranged from 27–40%. AUC/EC50 ratios after 3 days were >100 (total) and 48–113 (unbound). Stability in liver microsomes ranged from 30–>60 min. Ring hydroxylated metabolites were observed in microsomes. Neither was a substrate for the MDR1 transporter.
Conclusions
IND24 and IND81 are active in vitro and show high AUC/EC50 ratios (total and unbound) in plasma and brain. These will be evaluated in mouse models of prion disease.
doi:10.1007/s11095-012-0912-4
PMCID: PMC3640342  PMID: 23417511
antiprion drugs; drug discovery; IND24; IND81; prion disease
12.  A simple LC–MS/MS method for determination of kynurenine and tryptophan concentrations in human plasma from HIV-infected patients 
Bioanalysis  2013;5(11):10.4155/bio.13.74.
Background
Indoleamine 2,3-dioxygenase, catalyzing tryptophan (Trp) metabolism through the kynurenine (Kyn) metabolic pathway, plays important roles in immune suppression and the CNS. In this article, we report a simple, rapid and specific LC–MS/MS method for accurate determination of Kyn and Trp concentrations in human plasma from HIV-infected patients.
Results
The human plasma sample (100 μl) was mixed with Kyn-d4 and Trp-d5 internal standards and then precipitated with trifluoroacetic acid. The supernatant was directly analyzed by LC–MS/MS. The assay using surrogate matrix calibrators was validated for precision, accuracy, matrix effect, extraction efficiency and stability. Some assay validation issues for endogenous substance bioanalysis using an LC–MS/MS method are discussed.
Conclusion
A simple, specific and reproducible LC–MS/MS method has been developed and validated for measuring Kyn and Trp in human plasma samples.
doi:10.4155/bio.13.74
PMCID: PMC3830928  PMID: 23742309
13.  Simultaneous Laparoscopy-Assisted Resection for Rectal and Gastric Cancer 
Laparoscopy-assisted surgery for either rectal or gastric cancer has been increasingly performed. However, simultaneous laparoscopy-assisted resection for synchronous rectal and gastric cancer is rarely reported in the literature. In our study, 3 cases of patients who received simultaneous laparoscopy-assisted resection for synchronous rectal and gastric cancer were recorded. The results showed that all 3 patients recovered well, with only 253 minutes of mean operation time, 57 mL of intraoperative blood loss, 5 cm of assisted operation incision, 4 days to resume oral intake, 12 days' postoperative hospital stay, and no complication or mortality. No recurrence or metastasis was found within the follow-up period of 22 months. When performed by surgeons with plentiful experience in laparoscopic technology, simultaneous laparoscopy-assisted resection for synchronous rectal and gastric cancer is safe and feasible, with the benefits of minimal trauma, fast recovery, and better cosmetic results, compared with open surgery.
doi:10.4293/108680813X13753907291350
PMCID: PMC4035648  PMID: 24960501
Laparoscopy; Rectal cancer; Gastric cancer; Synchronous cancer
14.  Sporadic hemangioblastoma of the retroperitoneum 
Hemangioblastoma is a rare neoplasm occurred in soft tissue. Herein, we present an intriguing case of sporadic hemangioblastoma in the retroperitoneum occurring in a 59-year-old male. The tumor was characterized by an alternation of cellular and paucicellular areas surrounded by a thick fibrous capsule. The stromal cells showed a pale cytoplasm exhibiting occasional lipid droplets and hyperchromatic, irregularly nuclei. CD56, NSE, a-inhibin and S100 were positive in tumor cells. This is the third reported case of a hemangioblastoma in the retroperitoneum. Based on clinical examination, computed tomography imaging, surgical operation, and histopathologic studies, a definitive diagnosis was made. Further characteristic images and pathology of this disease are discussed.
PMCID: PMC4014262  PMID: 24817978
Hemangioblastoma; soft tissue; retroperitoneum; immunohistochemistry
15.  Gene Signature Distinguishes Patients with Chronic Ulcerative Colitis Harboring Remote Neoplastic Lesions 
Inflammatory bowel diseases  2013;19(3):10.1097/MIB.0b013e3182802bac.
Background
Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer. The standard method of surveillance for neoplasia in UC by colonoscopy is invasive and can miss flat lesions. We sought to identify a gene expression signature in non-dysplastic mucosa without active inflammation that could serve as a marker for remote neoplastic lesions.
Methods
Gene expression was analyzed by cDNA microarray in 5 normal controls, 4 UC patients without dysplasia, and 11 UC patients harboring remote neoplasia. Common gene ontology pathways of significantly differentially expressed genes were identified. Expression of genes which were progressively and significantly up-regulated from controls, to UC without neoplasia, to UC with remote neoplasia were evaluated by real time PCR. Several gene products were also examined by immunohistochemistry.
Results
468 genes were significantly up-regulated and 541 genes were significantly down-regulated in UC patints with neoplasia compared to UC patients without neoplasia. Nine genes (ACSL1, BIRC3, CLC, CREM, ELTD1, FGG, S100A9, THBD, and TPD52L1) were progressively and significantly up-regulated from controls to non-dysplastic UC to UC with neoplasia. Immunostaining of proteins revealed increased expression of S100A9 and REG1α in UC-associated cancer and in non-dysplastic tissue from UC patients harboring remote neoplasia, compared to UC patients without neoplasia and controls.
Conclusions
Gene expression changes occurring as a field effect in the distal colon of patients with chronic UC identify patients harboring remote neoplastic lesions. These markers may lead to a more accurate and less invasive method of detection of neoplasia in patients with inflammatory bowel disease.
doi:10.1097/MIB.0b013e3182802bac
PMCID: PMC3836269  PMID: 23388545
Inflammatory bowel disease; ulcerative colitis; dysplasia; colorectal cancer; gene expression
16.  Construction of a Chimeric Secretory IgA and Its Neutralization Activity against Avian Influenza Virus H5N1 
Journal of Immunology Research  2014;2014:394127.
Secretory immunoglobulin A (SIgA) acts as the first line of defense against respiratory pathogens. In this assay, the variable regions of heavy chain (VH) and Light chain (VL) genes from a mouse monoclonal antibody against H5N1 were cloned and fused with human IgA constant regions. The full-length chimeric light and heavy chains were inserted into a eukaryotic expressing vector and then transfected into CHO/dhfr-cells. The chimeric monomeric IgA antibody expression was confirmed by using ELISA, SDS-PAGE, and Western blot. In order to obtain a dimeric secretory IgA, another two expressing plasmids, namely, pcDNA4/His A-IgJ and pcDNA4/His A-SC, were cotransfected into the CHO/dhfr-cells. The expression of dimeric SIgA was confirmed by using ELISA assay and native gel electrophoresis. In microneutralization assay on 96-well immunoplate, the chimeric SIgA showed neutralization activity against H5N1 virus on MDCK cells and the titer was determined to be 1 : 64. On preadministrating intranasally, the chimeric SIgA could prevent mice from lethal attack by using A/Vietnam/1194/04 H5N1 with a survival rate of 80%. So we concluded that the constructed recombinant chimeric SIgA has a neutralization capability targeting avian influenza virus H5N1 infection in vitro and in vivo.
doi:10.1155/2014/394127
PMCID: PMC3987799  PMID: 24741594
17.  Glucose Sensor O-GlcNAcylation Coordinates with Phosphorylation to Regulate Circadian Clock 
Cell metabolism  2013;17(2):291-302.
SUMMARY
Post-translational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β, hence OGT and GSK3β exhibit reciprocal regulation. Modulating OGlcNAcylation levels alter circadian period length in both mice and Drosophila, and conversely protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662–S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine tune circadian clock.
doi:10.1016/j.cmet.2012.12.017
PMCID: PMC3597447  PMID: 23395175
18.  Identification of Cinnabarinic Acid as a Novel Endogenous Aryl Hydrocarbon Receptor Ligand That Drives IL-22 Production 
PLoS ONE  2014;9(2):e87877.
The aryl hydrocarbon receptor (AHR) binds to environmental toxicants including synthetic halogenated aromatic hydrocarbons and is involved in a diverse array of biological processes. Recently, the AHR was shown to control host immunity by affecting the balance between inflammatory T cells that produce IL-17 (Th17) and IL-22 versus regulatory T cells (Treg) involved in tolerance. While environmental AHR ligands can mediate this effect, endogenous ligands are likely to be more relevant in host immune responses. We investigated downstream metabolites of tryptophan as potential AHR ligands because (1) tryptophan metabolites have been implicated in regulating the balance between Th17 and Treg cells and (2) many of the AHR ligands identified thus far are derivatives of tryptophan. We characterized the ability of tryptophan metabolites to bind and activate the AHR and to increase IL-22 production in human T cells. We report that the tryptophan metabolite, cinnabarinic acid (CA), is an AHR ligand that stimulates the differentiation of human and mouse T cells producing IL-22. We compare the IL-22-stimulating activity of CA to that of other tryptophan metabolites and define stimulation conditions that lead to CA production from immune cells. Our findings link tryptophan metabolism to AHR activation and define a novel endogenous AHR agonist with potentially broad biological functions.
doi:10.1371/journal.pone.0087877
PMCID: PMC3912126  PMID: 24498387
19.  Preclinical Evaluation of HIV Eradication Strategies in the Simian Immunodeficiency Virus-Infected Rhesus Macaque: A Pilot Study Testing Inhibition of Indoleamine 2,3-Dioxygenase 
Abstract
Even in the setting of maximally suppressive antiretroviral therapy (ART), HIV persists indefinitely. Several mechanisms might contribute to this persistence, including chronic inflammation and immune dysfunction. In this study, we have explored a preclinical model for the evaluation of potential interventions that might serve to eradicate or to minimize the level of persistent virus. Given data that metabolic products of the inducible enzyme indoleamine 2,3-dioxygeanse (IDO) might foster inflammation and viral persistence, chronically simian immunodeficiency virus (SIV)-infected, ART-treated rhesus macaques were treated with the IDO inhibitor 1-methyl tryptophan (1mT). Orally administered 1mT achieved targeted plasma levels, but did not impact tryptophan metabolism or decrease viral RNA or DNA in plasma or in intestinal tissues beyond levels achieved by ART alone. Animals treated with 1mT showed no difference in the levels of T cell activation or differentiation, or in the kinetics or magnitude of viral rebound following cessation of ART. Notwithstanding these negative results, our observations suggest that the chronically SIV-infected rhesus macaque on suppressive ART can serve as a tractable model in which to test and to prioritize the selection of other potential interventions designed to eradicate HIV in vivo. In addition, this model might be used to optimize the route and dose by which such interventions are administered and the methods by which their effects are monitored.
doi:10.1089/aid.2012.0162
PMCID: PMC3552181  PMID: 22924680
20.  Characteristics of Heavy Metals and Pb Isotopic Composition in Sediments Collected from the Tributaries in Three Gorges Reservoir, China 
The Scientific World Journal  2014;2014:685834.
The concentrations, distribution, accumulation, and potential ecological risk of heavy metals (Cr, Cu, Zn, Ni, As, Pb, Cd, and Hg) in sediments from the Three Gorges Reservoir (TGR) tributaries were determined and studied. Pb isotopic compositions in sediments were also measured to effectively identify the potential Pb sources. The results showed that the average concentrations of heavy metals in sediment of TGR tributaries were higher than the local background values of soils and sediments in China. The assessment by Geoaccumulation Index indicated that Cu, Ni, and Hg were at the “slightly polluted” level and Cd was ranked as the “moderately polluted” level in tributary sediments of TGR. The assessment by Potential Ecological Risk Index showed that Hg and Cd were the predominant elements in tributary sediments in TGR. The Pb isotopic ratios in sediments varied from 1.171 to 1.202 for 206Pb/207Pb and from 2.459 to 2.482 for 208Pb/207Pb in TGR. All Pb isotopic ratios in sediments were similar to those from coal combustion, lead ores (the mining activities and smelting process), and cement material, indicating that these anthropogenic inputs may be the main sources for Pb pollution in sediments of TGR tributaries.
doi:10.1155/2014/685834
PMCID: PMC3927837  PMID: 24624045
21.  Morphological changes of cortical pyramidal neurons in hepatic encephalopathy 
BMC Neuroscience  2014;15:15.
Background
Hepatic encephalopathy (HE) is a reversible neuropsychiatric syndrome associated with acute and chronic liver diseases. It includes a number of neuropsychiatric disturbances including impaired motor activity and coordination, intellectual and cognitive function.
Results
In the present study, we used a chronic rat HE model by ligation of the bile duct (BDL) for 4 weeks. These rats showed increased plasma ammonia level, bile duct hyperplasia and impaired spatial learning memory and motor coordination when tested with Rota-rod and Morris water maze tests, respectively. By immunohistochemistry, the cerebral cortex showed swelling of astrocytes and microglia activation. To gain a better understanding of the effect of HE on the brain, the dendritic arbors of layer V cortical pyramidal neurons and hippocampal CA1 pyramidal neurons were revealed by an intracellular dye injection combined with a 3-dimensional reconstruction. Although the dendritic arbors remained unaltered, the dendritic spine density on these neurons was significantly reduced. It was suggested that the reduction of dendritic spines may be the underlying cause for increased motor evoked potential threshold and prolonged central motor conduction time in clinical finding in cirrhosis.
Conclusions
We found that HE perturbs CNS functions by altering the dendritic morphology of cortical and hippocampal pyramidal neurons, which may be the underlying cause for the motor and intellectual impairments associated with HE patients.
doi:10.1186/1471-2202-15-15
PMCID: PMC3898242  PMID: 24433342
Primary sensorimotor cortex; Hippocampus; Pyramidal neuron; Dendritic spine; Liver failure; Bile duct ligation
22.  Assessing Quantitative Resistance against Leptosphaeria maculans (Phoma Stem Canker) in Brassica napus (Oilseed Rape) in Young Plants 
PLoS ONE  2014;9(1):e84924.
Quantitative resistance against Leptosphaeria maculans in Brassica napus is difficult to assess in young plants due to the long period of symptomless growth of the pathogen from the appearance of leaf lesions to the appearance of canker symptoms on the stem. By using doubled haploid (DH) lines A30 (susceptible) and C119 (with quantitative resistance), quantitative resistance against L. maculans was assessed in young plants in controlled environments at two stages: stage 1, growth of the pathogen along leaf veins/petioles towards the stem by leaf lamina inoculation; stage 2, growth in stem tissues to produce stem canker symptoms by leaf petiole inoculation. Two types of inoculum (ascospores; conidia) and three assessment methods (extent of visible necrosis; symptomless pathogen growth visualised using the GFP reporter gene; amount of pathogen DNA quantified by PCR) were used. In stage 1 assessments, significant differences were observed between lines A30 and C119 in area of leaf lesions, distance grown along veins/petioles assessed by visible necrosis or by viewing GFP and amount of L. maculans DNA in leaf petioles. In stage 2 assessments, significant differences were observed between lines A30 and C119 in severity of stem canker and amount of L. maculans DNA in stem tissues. GFP-labelled L. maculans spread more quickly from the stem cortex to the stem pith in A30 than in C119. Stem canker symptoms were produced more rapidly by using ascospore inoculum than by using conidial inoculum. These results suggest that quantitative resistance against L. maculans in B. napus can be assessed in young plants in controlled conditions. Development of methods to phenotype quantitative resistance against plant pathogens in young plants in controlled environments will help identification of stable quantitative resistance for control of crop diseases.
doi:10.1371/journal.pone.0084924
PMCID: PMC3893142  PMID: 24454767
23.  Strong Relationship between Oral Dose and Tenofovir Hair Levels in a Randomized Trial: Hair as a Potential Adherence Measure for Pre-Exposure Prophylaxis (PrEP) 
PLoS ONE  2014;9(1):e83736.
Background
Pre-exposure prophylaxis (PrEP) trials using tenofovir-based regimens have demonstrated that high levels of adherence are required to evaluate efficacy; the incorporation of objective biomarkers of adherence in trial design has been essential to interpretation, given the inaccuracy of self-report. Antiretroviral measurements in scalp hair have been useful as a marker of long-term exposure in the HIV treatment setting, and hair samples are relatively easy and inexpensive to collect, transport, and store for analysis. To evaluate the relationship between dose and tenofovir concentrations in hair, we examined the dose proportionality of tenofovir in hair in healthy, HIV-uninfected adults.
Methods
A phase I, crossover pharmacokinetic study was performed in 24 HIV-negative adults receiving directly-observed oral tenofovir tablets administered 2, 4, and 7 doses/week for 6 weeks, with a ≥3-week break between periods. Small samples of hair were collected after each six-week period and analyzed for tenofovir concentrations. Geometric-mean-ratios compared levels between each pair of dosing conditions. Intensive plasma pharmacokinetic studies were performed during the daily-dosing period to calculate areas-under-the-time-concentration curves (AUCs).
Results
Over 90% of doses were observed per protocol. Median tenofovir concentrations in hair increased monotonically with dose. A log-linear relationship was seen between dose and hair levels, with an estimated 76% (95% CI 60–93%) increase in hair level per 2-fold dose increase. Tenofovir plasma AUCs modestly predicted drug concentrations in hair.
Conclusions
This study found a strong linear relationship between frequency of dosing and tenofovir levels in scalp hair. The analysis of quantitative drug levels in hair has the potential to improve adherence measurement in the PrEP field and may be helpful in determining exposure thresholds for protection and explaining failures in PrEP trials. Hair measures for adherence monitoring may also facilitate adherence measurement in real-world settings and merit further investigation in upcoming PrEP implementation studies and programs.
Trial Registration
ClinicalTrials.gov +NCT00903084.
doi:10.1371/journal.pone.0083736
PMCID: PMC3885443  PMID: 24421901
24.  Expression profile of microRNAs in c-Myc induced mouse mammary tumors 
Breast cancer research and treatment  2008;118(1):10.1007/s10549-008-0171-6.
c-Myc is a transcription factor overexpression of which induces mammary cancer in transgenic mice. To explore whether certain microRNAs (mirRNA) mediate c-Myc induced mammary carcinogenesis, we studied mir-RNA expression profile in mammary tumors developed from MMTV-c-myc transgenic mice, and found 50 and 59 mirRNAs showing increased and decreased expression, respectively, compared with lactating mammary glands of wild type mice. Twenty-four of these mirRNAs could be grouped into eight clusters because they had the same chromosomal localizations and might be processed from the same primary RNA transcripts. The increased expression of mir-20a, mir-20b, and mir-9 as well as decreased expression of mir-222 were verified by RT-PCR, real-time RT-PCR, and cDNA sequencing. Moreover, we fortuitously identified a novel non-coding RNA, the level of which was decreased in proliferating mammary glands of MMTV-c-myc mice was further decreased to undetectable level in the mammary tumors. Sequencing of this novel RNA revealed that it was transcribed from a region of mouse chromosome 19 that harbored the metastasis associated lung adenocarcinoma transcript-1 (Malat-1), a non-protein-coding gene. These results suggest that certain mirRNAs and the chromosome 19 derived non-coding RNAs may mediate c-myc induced mammary carcinogenesis.
doi:10.1007/s10549-008-0171-6
PMCID: PMC3882315  PMID: 18777135
c-myc; MicroRNA; Breast cancer; Microarray
25.  Sapphire ball lensed fiber probe for common-path optical coherence tomography in ocular imaging and sensing 
Proceedings of SPIE  2013;8567:10.1117/12.2005099.
We describe a novel common-path optical coherence tomography (CP-OCT) fiber probe design using a sapphire ball lens for cross-sectional imaging and sensing in retina vitrectomy surgery. Single mode Gaussian beam (TEM00) simulation was used to optimize lateral resolution and working distance (WD) of the common-path probe. A theoretical sensitivity model for CP-OCT was prosed to assess its optimal performance based an unbalanced photodetector configuration. Two probe designs with working distances (WD) 415μm and 1221μm and lateral resolution 11μm and 18μm, respectively were implemented with sensitivity up to 88dB. The designs are also fully compatible with conventional Michelson interferometer based OCT configurations. The reference plane of the probe, located at the distal beam exit interface of the single mode fiber (SMF), was encased within a 25-gauge hypodermic needle by the sapphire ball lens facilitates its applications in bloody and harsh environments. The performances of the fiber probe with 11μm of lateral resolution and 19μm of axial resolution were demonstrated by cross-sectional imaging of a cow cornea and retina in vitro with a 1310nm swept source OCT system. This probe was also attached to a piezoelectric motor for active compensation of physiological tremor for handheld retinal surgical tools.
doi:10.1117/12.2005099
PMCID: PMC3877324  PMID: 24392202
optical coherence tomography; retina imaging; fiber probe

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