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1.  Evidence That the Heme Regulatory Motifs in Heme Oxygenase-2 Serve as a Thiol/Disulfide Redox Switch Regulating Heme Binding*s 
The Journal of biological chemistry  2007;282(29):21056-21067.
Heme oxygenase (HO) catalyzes the O2- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568–12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys265 of HRM1 and Cys282 of HRM2, binds heme tightly. Reduction of the disulfide bond increases the Kd for ferric heme from 0.03 to 0.3 μm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the Kd for heme, they do not alter the kcat for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca2+-activated potassium channel.
doi:10.1074/jbc.M700664200
PMCID: PMC3957417  PMID: 17540772
2.  Characterization and Functional Analysis of AatB, a Novel Autotransporter Adhesin and Virulence Factor of Avian Pathogenic Escherichia coli 
Infection and Immunity  2013;81(7):2437-2447.
Autotransporter (AT) proteins constitute a large family of extracellular proteins that contribute to bacterial virulence. A novel AT adhesin gene, aatB, was identified in avian pathogenic Escherichia coli (APEC) DE205B via genomic analyses. The open reading frame of aatB was 1,017 bp, encoding a putative 36.3-kDa protein which contained structural motifs characteristic for AT proteins: a signal peptide, a passenger domain, and a translocator domain. The predicted three-dimensional structure of AatB consisted of two distinct domains, the C-terminal β-barrel translocator domain and an N-terminal passenger domain. The prevalence analyses of aatB in APEC indicated that aatB was detected in 26.4% (72/273) of APEC strains and was strongly associated with phylogenetic groups D and B2. Quantitative real-time reverse transcription-PCR analyses revealed that AatB expression was increased during infection in vitro and in vivo. Moreover, AatB could elicit antibodies in infected ducks, suggesting that AatB is involved in APEC pathogenicity. Thus, APEC DE205B strains with a mutated aatB gene and mutated strains complemented with the aatB gene were constructed. Inactivation of aatB resulted in a reduced capacity to adhere to DF-1 cells, defective virulence capacity in vivo, and decreased colonization capacity in lung during systemic infection compared with the capacities of the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatB makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, biofilm formation assays with strain AAEC189 expressing AatB indicated that AatB mediates biofilm formation.
doi:10.1128/IAI.00102-13
PMCID: PMC3697619  PMID: 23630958
3.  MicroRNA-217 Promotes Angiogenesis of Human Cytomegalovirus-Infected Endothelial Cells through Downregulation of SIRT1 and FOXO3A 
PLoS ONE  2013;8(12):e83620.
Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-1217 on angiogenesis and to disclose the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells(ECs) enhanced expression of miR-217 and reduced SIRT1 and FOXO3A protein level in 24 hours post infection(hpi). Transfection of miR-217 inhibitor not only depressed cellular migration and tube formation induced by HCMV infection, but also enhanced SIRT1 and FOXO3A protein expression. Additionally, luciferase assay confirmed that miR-217 directly targeted FOXO3A mRNA 3`UTR. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Similarly, delivery of FOXO3A overexpression lentivirus suppressed proliferative rate, migration and tube formation of HCMV-infected ECs, which reversed by transfection of FOXO3A siRNA. In summary, HCMV infection of endothelial cells induces angiogenesis by both of miR-217/SIRT1 and miR-217/FOXO3A axis.
doi:10.1371/journal.pone.0083620
PMCID: PMC3869804  PMID: 24376725
4.  Correction: The Impact of Down Syndrome Screening on Taiwanese Down Syndrome Births: A Nationwide Retrospective Study and a Screening Result from a Single Medical Centre 
PLoS ONE  2013;8(12):10.1371/annotation/f6830f30-4b8e-4d72-a8c0-53ac7e50b440.
doi:10.1371/annotation/f6830f30-4b8e-4d72-a8c0-53ac7e50b440
PMCID: PMC3894922  PMID: 24465294
5.  The Cardioprotective Effect of Hypertonic Saline Is Associated with Inhibitory Effect on Macrophage Migration Inhibitory Factor in Sepsis 
BioMed Research International  2013;2013:201614.
Sepsis can cause myocardial dysfunction, which contributes to the high mortality of sepsis. Hypertonic saline (HS) has been reported to increase myocardial contractility in sepsis. In the present study, mechanisms of action of HS resuscitation (4 mL of 7.5% NaCl per kilogram) on cardiac function have been evaluated in septic rats. HS was administered 1 h after LPS (10 mg/kg, i.v.) challenge. The mean arterial blood pressure significantly decreased 4 h after LPS challenge, and septic shock was observed at the end of experiment (6 h). Posttreatment with HS prevented hypotension caused by LPS and significantly improved cardiac function, evidenced by increases in left ventricular developed pressure, mean +dP/dt and −dP/dt. The amplitude of electrical-stimulated intracellular Ca2+ transient in isolated single cardiomyocytes was significantly reduced after 6 h LPS insult, which was recovered by HS. In addition, LPS resulted in significant increases in neutrophil myeloperoxidase activity, macrophage migration inhibitory factor (MIF), and NF-κB phospho-p65 protein levels in myocardium at 6 h, which were significantly attenuated by HS. In conclusion, HS improved myocardial contractility and prevented circulatory failure induced by endotoxemia, which may attribute to improvement of intracellular calcium handling process and inhibitory effects on neutrophil infiltration and MIF production in hearts.
doi:10.1155/2013/201614
PMCID: PMC3858963  PMID: 24371817
6.  Overexpression of luxS Cannot Increase Autoinducer-2 Production, Only Affect the Growth and Biofilm Formation in Streptococcus suis 
The Scientific World Journal  2013;2013:924276.
LuxS/AI-2 quorum sensing (QS) system involves the production of cell signaling molecules via luxS-based autoinducer-2 (AI-2). LuxS has been reported to plays critical roles in regulating various behaviors of bacteria. AI-2 is a byproduct of the catabolism of S-adenosylhomocysteine (SAH) performed by the LuxS and Pfs enzymes. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as an inability to produce AI-2 were observed in bacteria having a luxS mutant gene. In this study, the level of AI-2 activity exhibits a growth-phase dependence with a maximum in late exponential culture in SS. An SS strain that overexpressed luxS was constructed to comprehensively understand the function of AI-2. Overexpressed luxS was not able to increase the level of pfs expression and produce additional AI-2, and the bacteria were slower growing and produced only slightly more biofilm than the wild type. Thus, AI-2 production is not correlated with luxS transcription. luxS expression is constitutive, but the transcription of pfs is perhaps correlated with AI-2 production in SS.
doi:10.1155/2013/924276
PMCID: PMC3842066  PMID: 24324385
7.  MicroRNA-124 mediates the cholinergic anti-inflammatory action through inhibiting the production of pro-inflammatory cytokines 
Cell Research  2013;23(11):1270-1283.
The vagus nerve can control inflammatory response through a 'cholinergic anti-inflammatory pathway', which is mediated by the α7-nicotinic acetylcholine receptor (α7nAChR) on macrophages. However, the intracellular mechanisms that link α7nAChR activation and pro-inflammatory cytokine production remain not well understood. In this study, we found that miR-124 is upregulated by cholinergic agonists in LPS-exposed cells and mice. Utilizing miR-124 mimic and siRNA knockdown, we demonstrated that miR-124 is a critical mediator for the cholinergic anti-inflammatory action. Furthermore, our data indicated that miR-124 modulates LPS-induced cytokine production by targeting signal transducer and activator of transcription 3 (STAT3) to decrease IL-6 production and TNF-α converting enzyme (TACE) to reduce TNF-α release. These results also indicate that miR-124 is a potential therapeutic target for the treatment of inflammatory diseases.
doi:10.1038/cr.2013.116
PMCID: PMC3817544  PMID: 23979021
micorRNA-124; cholinergic anti-inflammatory action; α7nAChR; macrophages; septic shock; STAT3; TACE
8.  The Impact of Down Syndrome Screening on Taiwanese Down Syndrome Births: A Nationwide Retrospective Study and a Screening Result from a Single Medical Centre 
PLoS ONE  2013;8(9):e75428.
A retrospective analysis of the Taiwanese National Birth Defect Registration and Notification System was conducted in order to determine the live birth- and stillbirth rates in infants with Down syndrome, trisomy 18, trisomy 13 and Turner syndrome between 2001 and 2010. The objective was to investigate the impact of Down syndrome screening on the Taiwanese Down syndrome live birth rate. In addition, the results of first-trimester Down syndrome screening between 2006 and 2011, and of second-trimester quadruple testing between 2008 and 2011, were obtained from the National Taiwan University Hospital. All Taiwanese infants born between 2001 and 2010 were included in the first part of the analysis, and women receiving first-trimester Down syndrome screening or second-trimester quadruple testing from the National Taiwan University Hospital were included in the second part. The live birth rate of infants with Down syndrome, per 100 000 live births, decreased from 22.28 in 2001 to 7.79 in 2010. The ratio of liveborn DS to total DS was 48.74% in 2001, and then decreased to 25.88% in 2006, when first-trimester screening was widely introduced in Taiwan. This ratio dropped to 20.64% in 2008, when the second-trimester quadruple test was implemented. The overall positive rate in first-trimester screening in the National Taiwan University Hospital was 3.1%, with a Down syndrome detection rate of 100%; the quadruple test had values of 9.0% and 75%, respectively. The use of first-trimester screening and the second-trimester quadruple test may be responsible for the marked decrease in the Taiwanese Down syndrome live birth rate observed between 2001 and 2010.
doi:10.1371/journal.pone.0075428
PMCID: PMC3798710  PMID: 24147155
9.  Abnormality in face scanning by children with autism spectrum disorder is limited to the eye region: Evidence from multi-method analyses of eye tracking data 
Journal of Vision  2013;13(10):5.
There has been considerable controversy regarding whether children with autism spectrum disorder (ASD) and typically developing children (TD) show different eye movement patterns when processing faces. We investigated ASD and age- and IQ-matched TD children's scanning of faces using a novel multi-method approach. We found that ASD children spent less time looking at the whole face generally. After controlling for this difference, ASD children's fixations of the other face parts, except for the eye region, and their scanning paths between face parts were comparable either to the age-matched or IQ-matched TD groups. In contrast, in the eye region, ASD children's scanning differed significantly from that of both TD groups: (a) ASD children fixated significantly less on the right eye (from the observer's view); (b) ASD children's fixations were more biased towards the left eye region; and (c) ASD children fixated below the left eye, whereas TD children fixated on the pupil region of the eye. Thus, ASD children do not have a general abnormality in face scanning. Rather, their abnormality is limited to the eye region, likely due to their strong tendency to avoid eye contact.
doi:10.1167/13.10.5
PMCID: PMC3739407  PMID: 23929830
autism spectrum disorder; face processing; face recognition; eye movements; eye tracking
10.  Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China 
PLoS ONE  2013;8(7):e67755.
One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae.
doi:10.1371/journal.pone.0067755
PMCID: PMC3707890  PMID: 23874442
11.  Induction of apoptosis in melanoma A375 cells by a chloroform fraction of Centratherum anthelminticum (L.) seeds involves NF-kappaB, p53 and Bcl-2-controlled mitochondrial signaling pathways 
Background
Centratherum anthelminticum (L.) Kuntze (scientific synonyms: Vernonia anthelmintica; black cumin) is one of the ingredients of an Ayurvedic preparation, called “Kayakalp”, commonly applied to treat skin disorders in India and Southeast Asia. Despite its well known anti-inflammatory property on skin diseases, the anti-cancer effect of C. anthelminticum seeds on skin cancer is less documented. The present study aims to investigate the anti-cancer effect of Centratherum anthelminticum (L.) seeds chloroform fraction (CACF) on human melanoma cells and to elucidate the molecular mechanism involved.
Methods
A chloroform fraction was extracted from C. anthelminticum (CACF). Bioactive compounds of the CACF were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human melanoma cell line A375 was treated with CACF in vitro. Effects of CACF on growth inhibition, morphology, stress and survival of the cell were examined with MTT, high content screening (HSC) array scan and flow cytometry analyses. Involvement of intrinsic or extrinsic pathways in the CACF-induced A375 cell death mechanism was examined using a caspase luminescence assay. The results were further verified with different caspase inhibitors. In addition, Western blot analysis was performed to elucidate the changes in apoptosis-associated molecules. Finally, the effect of CACF on the NF-κB nuclear translocation ability was assayed.
Results
The MTT assay showed that CACF dose-dependently inhibited cell growth of A375, while exerted less cytotoxic effect on normal primary epithelial melanocytes. We demonstrated that CACF induced cell growth inhibition through apoptosis, as evidenced by cell shrinkage, increased annexin V staining and formation of membrane blebs. CACF treatment also resulted in higher reactive oxygen species (ROS) production and lower Bcl-2 expression, leading to decrease mitochondrial membrane potential (MMP). Disruption of the MMP facilitated the release of mitochondrial cytochrome c, which activates caspase-9 and downstream caspase-3/7, resulting in DNA fragmentation and up-regulation of p53 in melanoma cells. Moreover, CACF prevented TNF-α-induced NF-κB nuclear translocation, which further committed A375 cells toward apoptosis.
Conclusions
Together, our findings suggest CACF as a potential therapeutic agent against human melanoma malignancy.
doi:10.1186/1472-6882-13-166
PMCID: PMC3718627  PMID: 23837445
Centratherum anthelminticum; Melanoma; Caspase cascade; Apoptosis; Bcl-2; p53; NF-κB
12.  Downregulation of KLF8 expression by shRNA induces inhibition of cell proliferation in CAL27 human oral cancer cells 
Objectives: KLF8 is a member of KLF transcription factors which play an important tolr in oncogenesis. It is barely expressed in normal human epithelial cells but highly overexpressed in several types of human cancer cell lines. In the present study, we investigate the role of KLF8 in oral cancer and the effects of KLF8 knockdown via lentivirus mediated siRNA infection in human adenosquamos carcinoma CAL 27 cells. Study Design: We developed a vector-based siRNA expression system that can induce RNAi in CAL 27 oral cancer cells. Downregulation of KLF8 was confirmed by evaluating GFP expressions, RT-PCR and western blot analysis. Finally, the effects of KLF8 downregulation were analyzed by MTT assay and colony formation assays. Results: The expression levels of KLF8 mRNA and proteins are reduced in CAL 27 cells that transfected with 21-nt siRNA against KLF8. Lentivirus-mediated silencing of KLF8 reduces cell proliferation and colonies number, thereby indicating the role of KLF8 in cell proliferation and tumorigenesis. Conclusions: These results strongly suggest that KLF8 is essential for growth of CAL 27 cancer cells. A better understanding of KLF8 function and processing may provide novel insights into the clinical therapy of oral cancer.
Key words:KLF8, lentivirus, CAL 27, oral cancer, cell proliferation.
doi:10.4317/medoral.18736
PMCID: PMC3731086  PMID: 23722127
13.  Differential effects of selective and non-selective nitric oxide synthase inhibitors on the blood perfusion of ischemia-reperfused myocardium in dogs 
Background
Nitric oxide (NO) is protective for the cardiovascular system, and excessive NO exerts negative effects on the circulatory system. This study aimed to compare the effects of selective or non-selective NO synthase (NOS) inhibitors on blood flow perfusion of ischemia-reperfused myocardium.
Materials/Methods
Male mongrel dogs were randomly assigned to 4 groups: only ischemia-reperfusion (control), ischemia-reperfusion plus Nω-nitro-L-arginine methyl ester (NAME) treatment, ischemia-reperfusion plus aminoguanidine (AMD) treatment, and sham operation group. Myocardial contrast echocardiography (MCE) was performed. Blood samples were taken for measurement of NO. Background-subtracted peak videointensity (PVI) and PVI ratio in myocardium were measured.
Results
In the NAME-treated group, the PVI at 5 min reperfusion did not significantly differ from pre-LAD-occlusion, but declined to and retained at a level obviously lower than the pre-LAD-occlusion. In the AMD-treated group, the PVI at 5 min reperfusion was significantly higher than at pre-LAD-occlusion, and then restored to and remained at the pre-LAD-occlusion level. The changes of PVI ratios in the 3 groups were similar to PVI values. In the AMD-treated group, the curve width increased in the early reperfusion, but returned to the pre-LAD-occlusion level at 90 min reperfusion. The plasma NO concentration in the NAME-treated group greatly decreased and remained low during the whole period of reperfusion. In the AMD-treated group, there were only slight increases in NO concentrations during reperfusion.
Conclusions
NAME totally inhibited NO production and attenuated myocardial blood flow perfusion. Aminoguanidine significantly relieved the increase in NO production and alleviated the congestion of reperfused myocardium. Selective inhibitors of iNOS might be useful in the management of certain diseases associated with ischemia-reperfusion.
doi:10.12659/MSMBR.883964
PMCID: PMC3699539  PMID: 23807023
myocardial ischemia; NAME; myocardial reperfusion; nitric oxide; nitric oxide synthase inhibitor
14.  Genome Sequence of Mink Enteritis Virus Strain SD 12/01, Isolated from a Mink with Severe Diarrhea in China 
Genome Announcements  2013;1(3):e00306-13.
The mink enteritis virus (MEV) SD12/01 strain was isolated from a mink showing clinical and pathological signs of enteritis in Shandong, China, in 2012. The genome of MEV SD12/01 was sequenced and analyzed, which will promote a better understanding of the molecular epidemiology and genetic diversity of MEV field isolates in northern China.
doi:10.1128/genomeA.00306-13
PMCID: PMC3707587  PMID: 23788538
15.  Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246 
BMC Genomics  2013;14:377.
Background
Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus.
Results
Genome of S. zooepidemicus ATCC35246 (Sz35246) comprises 2,167,264-bp of a single circular chromosome, with a GC content of 41.65%. Comparative genome analysis of Sz35246, S. zooepidemicus MGCS10565 (Sz10565), Streptococcus equi. ssp. equi. 4047 (Se4047) and S. zooepidemicus H70 (Sz70) identified 320 Sz35246-specific genes, clustered into three toxin-antitoxin (TA) systems PAIs and one restriction modification system (RM system) PAI. These four acquired PAIs encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. Analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed PAI genes and non-PAI genes, suggesting that Sz35246 possess mechanisms for infecting animals and adapting to a wide range of host environments. Analysis of the genome identified potential Sz35246 virulence genes. Genes of the Fim III operon were presumed to be involved in breaking the host-restriction of Sz35246.
Conclusion
Genome wide comparisons of Sz35246 with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. Four specific PAIs, which were judged to have been transferred into Sz35246 genome through HGT, were identified for the first time. Further analysis of the TA and RM systems in the PAIs will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines.
doi:10.1186/1471-2164-14-377
PMCID: PMC3750634  PMID: 23742619
16.  Complete Nucleotide Sequence of Canine Distemper Virus CDV-PS, Isolated from Dogs in China 
Genome Announcements  2013;1(3):e00232-13.
A new strain of canine distemper virus, CDV-PS, has been isolated from dogs in China, and its complete genome has been sequenced and analyzed. The phylogenetic analysis suggests that CDV-PS belongs to the Asia-1 cluster and has low identity to the vaccine strain.
doi:10.1128/genomeA.00232-13
PMCID: PMC3656203  PMID: 23682141
17.  Dietary Intake and Risk for Reflux Esophagitis: A Case-Control Study 
Background. Specific dietary components have been associated with gastroesophageal reflux disease (GERD) in Europe and the United States. However, the relationship between dietary components and GERD in Chinese still remains unclear. Methods. A total of 268 patients who were newly diagnosed as reflux esophagitis (RE) in Outpatient Endoscopy Center of Tongji Hospital were recruited. In addition, 269 sex- and age-matched subjects were also recruited as controls. The body measurements were determined, and the dietary intake during the previous year was evaluated using food frequency questionnaire (FFQ). Stepwise multiple logistic regression analysis was performed to examine the association between nutrients and RE. Results. After adjustment for WC, WHR, total energy intake, and demographics, there were a positive dose-response relationship between RE and calcium, meat, oils, and salt and a negative dose-response relationship between RE and protein, carbohydrate, calories from protein (%), vitamin C, grains and potatoes, fruits, and eggs. Conclusion. High intake of meat, oils, salt, and calcium is associated with an increased risk for RE while high intake of protein, carbohydrate, calories from protein (%), vitamin C, grains and potatoes, fruits, and eggs correlates with a reduced risk for RE.
doi:10.1155/2013/691026
PMCID: PMC3652144  PMID: 23690762
18.  Nobiletin Ameliorates the Deficits in Hippocampal BDNF, TrkB, and Synapsin I Induced by Chronic Unpredictable Mild Stress 
Background. Our previous study has demonstrated that nobiletin could reverse the behavioral alterations in stressed mice. However, the relation of its antidepressant-like action with neurotrophic molecular expression remains unknown. This study aimed to explore the antidepressant-like mechanism of nobiletin related to the neurotrophic system in rats exposed to chronic unpredictable mild stress (CUMS). Methods. Depressive-like anhedonia (assessed by sucrose preference) and serum corticosterone secretion were evaluated in the CUMS, followed by brain-derived neurotrophic factor (BDNF), its tropomyosin-related kinase receptor B (TrkB), and the downstream target synapsin I expressions in the hippocampus. Results. Anhedonia, which occurred within week 2, was rapidly ameliorated by nobiletin. While fluoxetine needed additional 2 weeks to improve the anhedonia. In addition, nobiletin administration for 5 weeks significantly ameliorated CUMS-induced increase in serum corticosterone levels. Furthermore, we also found that CUMS-induced deficits of hippocampal BDNF, TrkB, and synapsin I were ameliorated by nobiletin. Conclusions. Taken together, these findings suggest that nobiletin produces rapidly acting antidepressant-like responses in the CUMS and imply that BDNF-TrkB pathway may play an important role in the antidepressant-like effect of nobiletin.
doi:10.1155/2013/359682
PMCID: PMC3613093  PMID: 23573124
19.  Hashimoto’s Thyroiditis, microcalcification and raised thyrotropin levels within normal range are associated with thyroid cancer 
Background
To confirm whether clinical and biochemical parameters or Hashimoto’s thyroiditis (HT) could predict the risks of malignancy among subjects who underwent thyroidectomy, as well as to determine the influence of HT on the biological behavior of papillary thyroid cancer (PTC).
Methods
A total of 2,052 patients who underwent initial thyroidectomy were enrolled between June 2006 and August 2008. Serum free T4, free T3, thyrotropin (TSH), thyroglobulin, thyroglobulin antibody, antimicrosomal antibody, tumor-associated status, and thyroid disorders were documented.
Results
Binary logistic regression analysis was performed to define the risk predictors for thyroid cancer. Finally, calcification, HT, TSH, and age, were entered into the multivariate model. Multivariate logistic regression analysis revealed the risk of thyroid cancer increases in parallel with TSH concentration within normal range, and the risk for malignancy significantly increased with serum TSH 1.97–4.94 mIU/L, compared with TSH less than 0.35 mIU/L (OR = 1.951, 95% CI = 1.201–3.171, P = 0.007). Increased risks of thyroid cancer were also detected among the patients with HT (OR = 3.732, 95% CI = 2.563–5.435), and microcalcification (OR = 14.486, 95% CI = 11.374–18.449). The effects of HT on the aggressiveness of PTC were not observed in extrathyroidal invasion (P = 0.347), capsular infiltration (P = 0.345), angioinvasion (P = 0.512), and lymph node metastases (P = 0.634).
Conclusions
The risk of malignancy increases in patients with higher level TSH within normal range, as well as the presence of HT and microcalcification. No evidence suggests that coexistent HT alleviates the aggressiveness of PTC.
doi:10.1186/1477-7819-11-56
PMCID: PMC3717052  PMID: 23496874
Thyroid cancer; Thyroiditis; Papillary thyroid cancer; Thyrotropin
20.  Redox, haem and CO in enzymatic catalysis and regulation 
Biochemical Society transactions  2012;40(3):501-507.
The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood–Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 Å (1 Å = 0.1 nm) channel guides CO, generated at the active site of CODH, to a CO ‘cage’ near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol–disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.
doi:10.1042/BST20120083
PMCID: PMC3553215  PMID: 22616859
acetyl-CoA synthase (ACS); carbon monoxide; carbon monoxide dehydrogenase (CODH); catalysis; haem; redox regulation
21.  Extensive Ethnogenomic Diversity of Endothelial Nitric Oxide Synthase (eNOS) Polymorphisms 
Nitric oxide (NO) is highly reactive, produced in endothelial cells by endothelial NO synthase (eNOS) and has been implicated in sickle cell pathophysiology. We evaluated the distribution of functionally significant eNOS variants (the T786C variant in the promoter region, the Glu298Asp variant in exon 7, and the variable number of tandem repeats (VNTR) in intron 4) in Africans, African Americans and Caucasians. The C-786 variant was more common in Caucasians than in Africans and African Americans. Consistent with other findings, the Asp-298 variant had the highest frequency in Caucasians followed by African Americans, but was completely absent in Africans. The very rare intron 4 allele, eNOS 4c, was found in some Africans and African Americans, but not in Caucasians. eNOS 4d allele was present in 2 Africans. These findings suggest a consistent and widespread genomic diversity in the distribution of eNOS variants in Africans, comparative to African Americans and Caucasians.
doi:10.4137/GRSB.S10857
PMCID: PMC3562081  PMID: 23400313
polymorphisms; ethnicity; endothelial nitric oxide synthase; haplotypes
22.  Screening of the antidepressant-like effect of the traditional Chinese medicinal formula Si-Ni-San and their possible mechanism of action in mice 
Pharmacognosy Research  2013;5(1):36-42.
Background:
The traditional Chinese medicine formula Si-Ni-San has well therapeutic applications in improvement of mental diseases including depression. However, the neuropharmacological and neuroendocrine mechanisms of the formula on antidepressant-like action have not been reported.
Objective:
Herein, we explored the antidepressant-like effect and its mechanism of Si-Ni-San.
Materials and Methods:
Acute effect of Si-Ni-San on the immobility time was assessed in the mouse forced swim test (FST) and tail suspension test (TST). Moreover, we investigated the neurochemical, neuroendocrine, and neurotrophin systems involved in the antidepressant-like effect of this formula.
Results:
Si-Ni-San significantly decreased the immobility time after acute treatment in the mouse TST (1300 mg/kg) but not in the FST compared with the control group. In addition, pretreatment of mice with PCPA or AMPT prevented the anti-immobility effect of Si-Ni-San (1300 mg/kg) in the TST. Moreover, acute Si-Ni-San (1300 mg/kg) decreased serum corticosterone levels, elevated serotonin (5-HT), norepinephrine (NE), and dopamine (DA) levels without affecting brain-derived neurotrophic factor (BDNF) levels in the whole brain exposed to TST.
Conclusion:
The acute antidepressant-like action of Si-Ni-San is mediated by the monoaminergic and neuroendocrine systems although underlying mechanism still remains to be further elucidated, and this formula should be further investigated as an alternative therapeutic approach for the treatment of depression.
doi:10.4103/0974-8490.105647
PMCID: PMC3579018  PMID: 23598923
BDNF; corticosterone; depression; monoamine neurotransmitter; Si-Ni-San
23.  Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode 
Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A∗0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies.
doi:10.3389/fimmu.2013.00168
PMCID: PMC3693486  PMID: 23805144
human T leukocyte virus type 1; crystal structure; peptide-major histocompatibility complex; surface plasmon resonance; T cell; T cell receptor; A6 TCR; high affinity TCR
24.  The Nuclear Chaperone Nucleophosmin Escorts an Epstein-Barr Virus Nuclear Antigen to Establish Transcriptional Cascades for Latent Infection in Human B Cells 
PLoS Pathogens  2012;8(12):e1003084.
Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.
Author Summary
Epstein-Barr Virus (EBV) infects human B cells to establish a permanent infection in hosts, which can cause diseases ranging from infectious mononucleosis to a broad spectrum of human malignancies. The conversion of human primary B cells into indefinitely proliferating lymphoblastoid cell lines (LCLs) by in vitro EBV infection provides a suitable model for virus-mediated cellular transformation. Epstein-Barr nuclear antigen (EBNA) 2-mediated transcription is essential for the establishment and maintenance of EBV latent infection. In this report, we have extensively explored the mechanism by which EBNA2 activates the latency-specific LMP1 promoter to establish a permanent infection in B cells. We have identified and characterized the protein-protein interaction of EBNA2 with the nuclear shuttle protein nucleophosmin (NPM1) in vivo and in vitro. In particular, we have determined that the expression of NPM1 is promptly induced upon EBV infection and that EBNA2 has a role in activating NPM1 gene expression. Furthermore, we have shown that oligomerized NPM1 is charged by ATP and binds to EBNA2, which is crucial for its ability to stabilize its interaction with the DNA binding protein RBP-Jκ, which is in turn essential for supporting the transcriptional cascades of EBV latent infection. Our findings provide striking evidence to illustrate a new model for understanding EBV pathology.
doi:10.1371/journal.ppat.1003084
PMCID: PMC3521654  PMID: 23271972
25.  Different distribution of breast ductal carcinoma in situ, ductal carcinoma in situ with microinvasion, and invasion breast cancer 
Background
Breast ductal cancer in situ (DCIS) can recur or progress to invasive ductal cancer (IDC), and the interim stage include DCIS with microinvasion (DCIS-Mi). In this article, we attempt to study the study the differences of clinicopathological features, imaging data, and immunohistochemical-based subtypes among DCIS, DCIS-Mi, and IDC.
Methods
In this retrospective study, we attempt to compare the clinicopathological features, immunohistochemical results and imaging data of 866 patients (included 73 DCIS, 72 DCIS-Mi, and 721 IDC).
Results
Patients with DCIS and DCIS-Mi were younger than those with IDC (P = 0.007). DCIS and DCIS-Mi often happened in premenopausal women while IDC was opposite (P <0.001). The incidence of IDC with node-positive was significantly higher than it in DCIS and DCIS-Mi (P <0.001). We also observed that the Her2-positive was more often found in patients with pure DCIS compared to those with DCIS-Mi and DCIS-I (P <0.001). There was a significant difference between the four subgroups (Luminal-A, Luminal-B, ERBB2+, Basal-like) from DCIS, DCIS-Mi, and IDC (P <0.001). Basal-like patients were fewer than other subgroups in DCIS, DCIS-Mi, and IDC. The incidence of the first performance of ultrasound (catheter winded and nodular mass) and mammography (nodular mass) had significantly difference among patients with DCIS, DCIS-Mi, and IDC (P <0.001).
Conclusions
Different clinicopathological, immunohistochemical, and imaging features among DCIS, DCIS-Mi, and IDC indicate that they are distinct entities. A larger sample size is needed for further study.
doi:10.1186/1477-7819-10-262
PMCID: PMC3543195  PMID: 23216911
Breast neoplasms; Ductal carcinoma in situ; Ductal carcinoma in situ with microinvasion; Invasion breast cancer

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