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1.  Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus–related hepatocellular carcinoma 
Nature genetics  2012;45(1):72-75.
To identify genetic susceptibility loci for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in the Chinese population, we carried out a genome-wide association study (GWAS) in 2,514 chronic HBV carriers (1,161 HCC cases and 1,353 controls) followed by a 2-stage validation among 6 independent populations of chronic HBV carriers (4,319 cases and 4,966 controls). The joint analyses showed that HCC risk was significantly associated with two independent loci: rs7574865 at STAT4, Pmeta = 2.48 × 10−10, odds ratio (OR) = 1.21; and rs9275319 at HLA-DQ, Pmeta = 2.72 × 10−17, OR = 1.49. The risk allele G at rs7574865 was significantly associated with lower mRNA levels of STAT4 in both the HCC tissues and nontumor tissues of 155 individuals with HBV-related HCC (Ptrend = 0.0008 and 0.0002, respectively). We also found significantly lower mRNA expression of STAT4 in HCC tumor tissues compared with paired adjacent nontumor tissues (P = 2.33 × 10−14).
doi:10.1038/ng.2483
PMCID: PMC4105840  PMID: 23242368
2.  Comparative Effectiveness of Di'ao Xin Xue Kang Capsule and Compound Danshen Tablet in Patients With Symptomatic Chronic Stable Angina 
Scientific Reports  2014;4:7058.
A high proportion of patients with stable angina remains symptomatic despite multiple treatment options. Di'ao Xinxuekang (XXK) capsule and Compound Danshen (CDS) tablet have been approved for treating angina pectoris for more than 20 years in China. We compare the anti-anginal effectiveness of XXK capsule and CDS tablet in patients with symptomatic chronic stable angina. A randomized, multicenter, double-blind, parallel-group, superiority trial was conducted in 4 study sites. 733 patients with symptomatic chronic stable angina were included in the full analysis set. The primary outcomes were the proportion of patients who were angina-free and the proportion of patients with normal electrocardiogram (ECG) recordings during 20 weeks treatment. Compared with CDS, XXK significantly increased the proportion of angina-free patients, but no significant difference was noted in the proportion of patients with normal ECG recordings. Weekly angina frequency and nitroglycerin use were significantly reduced with XXK versus CDS at week 20. Moreover, XXK also improved the quality of life of angina patients as measured by the SAQ score and Xueyu Zheng (a type of TCM syndrome) score. We demonstrate that XXK capsule is more effective for attenuating anginal symptoms and improving quality of life in patients with symptomatic chronic stable angina, compared with CDS tablet.
doi:10.1038/srep07058
PMCID: PMC4231340  PMID: 25394847
3.  The impact of educational status on the clinical features of major depressive disorder among Chinese women 
Journal of Affective Disorders  2012;136(3):988-992.
Background
Years of education are inversely related to the prevalence of major depressive disorder (MDD), but the relationship between the clinical features of MDD and educational status is poorly understood. We investigated this in 1970 Chinese women with recurrent MDD identified in a clinical setting.
Methods
Clinical and demographic features were obtained from 1970 Han Chinese women with DSM-IV major depression between 30 and 60 years of age across China. Analysis of linear, logistic and multiple logistic regression models were used to determine the association between educational level and clinical features of MDD.
Results
Subjects with more years of education are more likely to have MDD, with an odds ratio of 1.14 for those with more than ten years. Low educational status is not associated with an increase in the number of episodes, nor with increased rates of co-morbidity with anxiety disorders. Education impacts differentially on the symptoms of depression: lower educational attainment is associated with more biological symptoms and increased suicidal ideation and plans to commit suicide.
Limitations
Findings may not generalize to males or to other patient populations. Since the threshold for treatment seeking differs as a function of education there may an ascertainment bias in the sample.
Conclusions
The relationship between symptoms of MDD and educational status in Chinese women is unexpectedly complex. Our findings are inconsistent with the simple hypothesis from European and US reports that low levels of educational attainment increase the risk and severity of MDD.
doi:10.1016/j.jad.2011.06.046
PMCID: PMC3314924  PMID: 21824664
Major depressive disorder; Education; Socio-economic status; Symptom
4.  Resemblance of Symptoms for Major Depression Assessed at Interview versus from Hospital Record Review 
PLoS ONE  2012;7(1):e28734.
Background
Diagnostic information for psychiatric research often depends on both clinical interviews and medical records. Although discrepancies between these two sources are well known, there have been few studies into the degree and origins of inconsistencies.
Principal findings
We compared data from structured interviews and medical records on 1,970 Han Chinese women with recurrent DSM-IV major depression (MD). Correlations were high for age at onset of MD (0.93) and number of episodes (0.70), intermediate for family history (+0.62) and duration of longest episode (+0.43) and variable but generally more modest for individual depressive symptoms (mean kappa = 0.32). Four factors were identified for twelve symptoms from medical records and the same four factors emerged from analysis of structured interviews. Factor congruencies were high but the correlation of factors between interviews and records were modest (i.e. +0.2 to +0.4).
Conclusions
Structured interviews and medical records are highly concordant for age of onset, and the number and length of episodes, but agree more modestly for individual symptoms and symptom factors. The modesty of these correlations probably arises from multiple factors including i) inconsistency in the definition of the worst episode, ii) inaccuracies in self-report and iii) difficulties in coding medical records where symptoms were recorded solely for clinical purposes.
doi:10.1371/journal.pone.0028734
PMCID: PMC3256142  PMID: 22247760
5.  Influenza H7N9 and H9N2 Viruses: Coexistence in Poultry Linked to Human H7N9 Infection and Genome Characteristics 
Journal of Virology  2014;88(6):3423-3431.
ABSTRACT
Avian influenza virus A of the novel H7N9 reassortant subtype was recently found to cause severe human respiratory infections in China. Live poultry markets were suspected locations of the human H7N9 infection sources, based on the cases' exposure histories and sequence similarities between viral isolates. To explore the role of live poultry markets in the origin of the novel H7N9 virus, we systematically examined poultry and environmental specimens from local markets and farms in Hangzhou, using real-time reverse transcription-PCR (RT-PCR) as well as high-throughput next-generation sequencing (NGS). RT-PCR identified specimens positive for the H7 and N9 genomic segments in all of the 12 poultry markets epidemiologically linked to 10 human H7N9 cases. Chickens, ducks, and environmental specimens from the markets contained heavily mixed subtypes, including H7, N9, H9, and N2 and sometimes H5 and N1. The idea of the coexistence of H7N9 and H9N2 subtypes in chickens was further supported by metagenomic sequencing. In contrast, human H7N9 infection cases (n = 31) were all negative for H9N2 virus according to real-time RT-PCR. The six internal segments were indistinguishable for the H7N9 and H9N2 viruses. The H9, N2, and internal-segment sequences were very close to the sequence of the H9N2 virus circulating in chickens in China recently. Our results provide direct evidence that H9N2 strains coexisted with the novel human-pathogenic H7N9 influenza virus in epidemiologically linked live poultry markets. Avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus and continues to do so.
IMPORTANCE Our results suggest that avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus, a novel reassortant avian influenza virus A subtype, and continues to do so. The finding helps shed light on how the H7N9 virus emerged, spread, and transmitted to humans. It is of considerable interest for assessing the risk of the possible emergence of novel reassortant viruses with enhanced transmissibility to humans.
doi:10.1128/JVI.02059-13
PMCID: PMC3957952  PMID: 24403589
6.  The Diagnostic Value of Cervical Lymph Node Metastasis in Head and Neck Squamous Carcinoma by Using Diffusion-Weighted Magnetic Resonance Imaging and Computed Tomography Perfusion 
BioMed Research International  2014;2014:260859.
Purpose. The aim of this study was to compare diffusion-weighted magnetic resonance imaging (DWI) with computed tomography perfusion (CTP) for preoperative detection of metastases to lymph nodes (LNs) in head and neck squamous cell carcinoma (SCC). Methods. Between May 2010 and April 2012, 30 patients with head and neck SCC underwent preoperative DWI and CTP. Two radiologists measured apparent diffusion coefficient (ADC) values and CTP parameters independently. Surgery and histopathologic examinations were performed on all patients. Results. On DWI, 65 LNs were detected in 30 patients. The mean ADC value of metastatic nodes was lower than benign nodes and the difference was statistically significant (P < 0.05). On CTP images, the mean value in metastatic nodes of blood flow (BF) and blood volume (BV) was higher than that in benign nodes, and mean transit time (MTT) in metastatic nodes was lower than that in benign nodes. There were significant differences in BF and MTT values between metastatic and benign LNs (P < 0.05). There were significant differences between the AUCs of DWI and CTP (Z=4.612, P < 0.001). Conclusion. DWI with ADC value measurements may be more accurate than CTP for the preoperative diagnosis of cervical LN metastases.
doi:10.1155/2014/260859
PMCID: PMC4094719  PMID: 25050333
7.  Downregulation of ROCK2 through Nanocomplex Sensitizes the Cytotoxic Effect of Temozolomide in U251 Glioma Cells 
PLoS ONE  2014;9(3):e92050.
Objective
Rho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells.
Methods
Glycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration.
Results
Our results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone.
Conclusions
In conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma.
doi:10.1371/journal.pone.0092050
PMCID: PMC3958422  PMID: 24642531
8.  The Use of Integrative Therapies in Patients with Amyotrophic Lateral Sclerosis in Shanghai, China 
Objective. To investigate the current use of integrative therapies (IT) in the treatment of patients with amyotrophic lateral sclerosis (ALS). Methods. A cross-sectional, multicenter clinical epidemiological survey was conducted in 12 hospitals in Shanghai. We investigated the type and frequency of IT use and determined whether the use of IT correlated with demographic, social, or disease-specific characteristics in our patient population. Results. A total of 231 (89.5%) of 258 patients with ALS were eligible for the study and 229 (99% of all) of 231 reported the use of at least one IT for the treatment of ALS. Vitamins and Chinese herb decoctions, Chinese herb compounds, massage therapy, and acupuncture were the 5 most commonly used therapies. There was a strong association between education level, income, and use of IT. A household income of more than 75,000 RMB ($49,995) correlated with multiple IT use, and married patients used IT more often than single individuals. The main reasons for using IT were to treat weakness and fatigue, muscle atrophy, the development of ALS, depression, insomnia, limb pain or numbness, and side effects associated with Riluzole. Conclusion. The use of IT is common in patients with ALS in Shanghai. Vitamins and TCM are the most used additional therapies and the widespread and largely unexamined use of IT for ALS requires more attention.
doi:10.1155/2013/613596
PMCID: PMC3865630  PMID: 24363770
9.  S100+ cells: A new neuro-immune cross-talkers in lymph organs 
Scientific Reports  2013;3:1114.
Up to now, the ‘hardwired’ neural pathway of the neuro-immune regulation is not fully understood. Here we reported a new neural pathway which links sympathetic nerves with immune cells of the lymphoid tissues. Our results demonstrated that nerve fibers derived from superior cervical ganglion directly targeted only S100+ cells in the cervical lymph nodes. Moreover, we found co-expression of neurotransmitters such as norepinephrine, vasoactive intestinal polypeptide and neuropeptide Y in the postganglionic sympathetic nerve endings that innervate S100+ cells. Our findings suggested that S100+ cells serve as a neuro-immune cross-talker in lymph organs that may play a significant role in transmitting signals of nervous cells to targeted immune cells. The new findings provide better understanding of the cross-talk mechanism between the nervous system and the immune system.
doi:10.1038/srep01114
PMCID: PMC3552286  PMID: 23346371
10.  Highlights for α-fetoprotein in determining prognosis and treatment monitoring for hepatocellular carcinoma 
AIM: To explore the prognostic value in the monitoring of treatment efficacy of serial α-fetoprotein (AFP) in hepatocellular carcinoma (HCC) patients.
METHODS: We searched MEDLINE, EMBASE and COCHRANE LIBRARY through April 21, 2012, to find qualifying articles. Our overall search strategy included terms for HCC, AFP, treatment response, and prognosis. Literature was limited to English-language, human studies. Studies reporting cumulative survival rates were summarized qualitatively. For the prognostic meta-analysis, we undertook a series of meta-analyses that summarised the Cox proportional hazard ratios (HRs) by assuming a random effects model. With regards to the correlation of AFP change with radiologic response, the categorical dichotomous variables were assessed using Poisson relative risks (RRs), which were incorporated into the random effects model meta-analysis of accuracy prediction. Between-study heterogeneity was estimated by use of the I² statistic. Publication bias was evaluated using the Begg funnel plot and Egger plot. Sensitivity analyses were conducted first by separating systemic treatment estimates from locoregional therapy estimates, evaluating different AFP response cut-off point effects, and exploring the impact of different study sizes.
RESULTS: Of 142 titles identified in our original search, 11 articles (12 clinical studies) met our criteria. Six studies investigated outcome in a total of 464 cases who underwent systemic treatment, and six studies investigated outcome in a total of 510 patients who received locoregional therapy. A random-effects model meta-analysis showed that AFP response was associated with an mortality HR of 0.55 (95%CI, 0.47-0.65) across HCC in overall survival (OS) and 0.50 (95%CI, 0.38-0.65) in progression-free survival. Restricting analysis to the six eligible analyses of systemic treatment, the pooled HRs were 0.64 (95%CI, 0.53-0.77) for OS. Limiting analysis to the six analyses of locoregional therapy, the pooled HRs for OS was 0.39 (95%CI, 0.29-0.53). We showed a larger pooled HR in the 50% definition studies (HR, 0.67, 95%CI, 0.55-0.83) compared with that from the 20% definition studies (HR, 0.41, 95%CI, 0.32-0.53). Restricting analysis to the four studies including over 100 patients individually, the pooled HR was 0.65 (95%CI, 0.54-0.79), with a pooled HR for OS of 0.35 (95%CI, 0.23-0.46) in the studies of less than 100 patients. As to radiological imaging, 43.1% (155/360) of the patients in the AFP response group presented with a radiological overall response, while the response rate decreased to 11.5% (36/313) in the patients from the AFP nonresponse group. The RR of having no overall response was significantly lower in the AFP response group than the AFP nonresponse group (RR, 0.67; 95%CI, 0.61-0.75). In terms of disease control rate, 86.9% (287/330) in the AFP response group and 51.0% (153/300) in the AFP nonresponse group showed successful disease control, respectively. The RR of disease control failure, similarly, was significantly lower in the AFP response group (RR, 0.37; 95%CI, 0.23-0.58). But these findings could be overestimates because of publication and reporting bias.
CONCLUSION: HCC patients presenting with an AFP response are at decreased risk of mortality. In addition, patients with an AFP response also present with a higher overall response rate and disease control rate.
doi:10.3748/wjg.v18.i48.7242
PMCID: PMC3544026  PMID: 23326129
Liver cancer; α-fetoprotein; Response; Prognosis; Monitoring
11.  Selection of Aptamers Specific for Adipose Tissue 
PLoS ONE  2012;7(5):e37789.
Background
Obesity has reached epidemic proportions, affecting more than one tenth of the world’s population. As such, adipose tissue is being increasingly recognized as an important therapeutic target for obesity and related metabolic disorders. While many potential targets of adipose tissue have been established and drugs developed, very few of those drugs specifically target adipose tissue without affecting other tissue. This results from a limited knowledge of both cell-surface markers and physicochemical traits specific to adipocytes that might otherwise be exploited by circulating drugs.
Methodology/Principal Findings
Here we report the use of cell-SELEX technology to select two aptamers that can specifically recognize mature adipocytes: adipo-1 and adipo-8. Adipo-8 shows high affinity for differentiated, mature 3T3-L1 adipocytes with a Kd value of 17.8±5.1 nM. The binding was sustained upon incubation at 37°C and insulin stimulation, but was lost upon trypsin treatment. The binding ability was also verified on frozen tissue slides with low background fluorescence and isolated adipocytes.
Conclusions/Significance
Aptamer adipo-8 selected from a random library appears to bind to mature differentiated adipocytes specifically. This aptamer holds great promise as a molecular recognition tool for adipocyte biomarker discovery or for targeted delivery of molecules to adipocytes.
doi:10.1371/journal.pone.0037789
PMCID: PMC3360593  PMID: 22662223
12.  The Limbic-Prefrontal Network Modulated by Electroacupuncture at CV4 and CV12 
fMRI studies showed that acupuncture could induce hemodynamic changes in brain networks. Many of these studies focused on whether specific acupoints could activate specific brain regions and were often limited to manual acupuncture at acupoints on the limbs. In this fMRI study, we investigated acupuncture's modulation effects on brain functional networks by electroacupuncture (EA) at acupoints on the midline of abdomen. Acupoints Guanyuan (CV4) and Zhongwan (CV12) were stimulated in 21 healthy volunteers. The needling sensations, brain activation, and functional connectivity were studied. We found that the limbic-prefrontal functional network was deactivated by EA at CV4 and CV12. More importantly, the local functional connectivity was significantly changed during EA stimulation, and the change persisted during the period after the stimulation. Although minor differences existed, both acupoints similarly modulated the limbic-prefrontal functional network, which is overlapped with the functional circuits associated with emotional and cognitive regulation.
doi:10.1155/2012/515893
PMCID: PMC3265182  PMID: 22291848
13.  Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine 
Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX). Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX) can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.
doi:10.3390/ijms13033341
PMCID: PMC3317715  PMID: 22489154
aptamer; SELEX; molecular medicine
14.  Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition 
PLoS Pathogens  2011;7(10):e1002249.
The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.
Author Summary
The influenza neuraminidase (NA) enzyme is the most successful drug target against the seasonal and pandemic flu. The 2009 H1N1 flu pandemic led to record sales of the NA inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza). Recently, a new drug, laninamivir (Inavir), has been approved for use in Japan can also be administered effectively using a single dose via its octanoate prodrug (CS-8958), however its effectiveness against oseltamivir-resistant influenza infection has not been demonstrated in clinical studies. In this study we comprehensively evaluate the effectiveness of laninamivir and its prodrug using NA from different groups with different active site features. We expressed and purified a group 2 NA from the 1957 pandemic H2N2, an atypical group 1 NA from the 2009 H1N1 pandemic and a group 1 NA from avian H12N5. NA inhibition was assayed and NAs were further crystallized with each inhibitor to determine the structural basis of their action. We found that laninamivir inhibition is highly potent for each NA, however binding and inhibition of laninamivir and its prodrug showed group specific preferences. Our results provide the structural and functional basis of NA inhibition using classical and novel inhibitors, with NAs from multiple serotypes with different properties.
doi:10.1371/journal.ppat.1002249
PMCID: PMC3197600  PMID: 22028647
15.  ARGONAUTE10 and ARGONAUTE1 Regulate the Termination of Floral Stem Cells through Two MicroRNAs in Arabidopsis 
PLoS Genetics  2011;7(3):e1001358.
Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, we reveal a role of the type III HD-Zip genes, previously known to specify lateral organ polarity, in stem cell termination. Both reduction in HD-Zip expression by over-expression of miR165/166 and mis-expression of HD-Zip genes by rendering them resistant to miR165/166 lead to prolonged floral stem cell activity, indicating that the expression of HD-Zip genes needs to be precisely controlled to achieve floral stem cell termination. We also show that both the ubiquitously expressed ARGONAUTE1 (AGO1) gene and its homolog AGO10, which exhibits highly restricted spatial expression patterns, are required to maintain the correct temporal program of floral stem cells. We provide evidence that AGO10, like AGO1, associates with miR172 and miR165/166 in vivo and exhibits “slicer” activity in vitro. Despite the common biological functions and similar biochemical activities, AGO1 and AGO10 exert different effects on miR165/166 in vivo. This work establishes a network of microRNAs and transcription factors governing the temporal program of floral stem cells and sheds light on the relationships among different AGO genes, which tend to exist in gene families in multicellular organisms.
Author Summary
Stem cells have the capacity to self renew while producing daughter cells that undergo differentiation. While some stem cells remain as stem cells throughout the life of an organism, others are programmed to terminate within developmental contexts. It is presumed that stem cell termination is simply the differentiation of stem cells into a specific cell type(s). Using floral stem cells as a model, we show that the temporally regulated termination of floral stem cells is genetically separable from stem cell differentiation, and thus we reveal the presence of a temporal program of stem cell regulation. We show that two microRNAs, miR172 and miR165/166, and two argonaute family proteins, ARGONAUTE1 (AGO1) and AGO10, regulate the termination of floral stem cells. We establish the homeodomain-leucine zipper (DH-Zip) genes, targets of miR165/166, as crucial factors in floral stem cell termination. While AGO1 is the major miRNA effector, the molecular function of AGO10 has been elusive. Here we demonstrate that AGO10 is also a miRNA effector in that AGO10 is associated with miRNAs in vivo and exhibits “slicer” activity in vitro. Despite the similar biochemical activities, AGO1 and AGO10 promote floral stem cell termination by exerting opposite effects on miR165/166.
doi:10.1371/journal.pgen.1001358
PMCID: PMC3069122  PMID: 21483759
16.  An Oestrogen Receptor α-bound Human Chromatin Interactome 
Nature  2009;462(7269):58-64.
Genomes are organized into high-level 3-dimensional structures, and DNA elements separated by long genomic distances could functionally interact. Many transcription factors bind to regulatory DNA elements distant from gene promoters. While distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Therefore, we developed Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) for de novo detection of global chromatin interactions, and comprehensively mapped the chromatin interaction network bound by oestrogen receptor α (ERα) in the human genome. We found that most high-confidence remote ERα binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ERα functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.
doi:10.1038/nature08497
PMCID: PMC2774924  PMID: 19890323
17.  Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT 
PLoS ONE  2015;10(1):e0114423.
Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.
doi:10.1371/journal.pone.0114423
PMCID: PMC4300223  PMID: 25602959
18.  Multimodal Magnetic Resonance Imaging: The Coordinated Use of Multiple, Mutually Informative Probes to Understand Brain Structure and Function 
Human brain mapping  2011;34(2):253-271.
Differing imaging modalities provide unique channels of information to probe differing aspects of the brain’s structural or functional organization. In combination, differing modalities provide complementary and mutually informative data about tissue organization that is more than their sum. We acquired and spatially coregistered data in four MRI modalities – anatomical MRI, functional MRI, diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS) – from 20 healthy adults to understand how inter-individual variability in measures from one modality account for variability in measures from other modalities at each voxel of the brain. We detected significant correlations of local volumes with the magnitude of functional activation, suggesting that underlying variation in local volumes contributes to individual variability in functional activation. We also detected significant inverse correlations of NAA (a putative measure of neuronal density and viability) with volumes of white matter in the frontal cortex, with DTI-based measures of tissue organization within the superior longitudinal fasciculus, and with the magnitude of functional activation and default-mode activity during simple visual and motor tasks, indicating that substantial variance in local volumes, white matter organization, and functional activation derives from an underlying variability in the number or density of neurons in those regions. Many of these imaging measures correlated with measures of intellectual ability within differing brain tissues and differing neural systems, demonstrating that the neural determinants of intellectual capacity involve numerous and disparate features of brain tissue organization, a conclusion that could be made with confidence only when imaging the same individuals with multiple MRI modalities.
doi:10.1002/hbm.21440
PMCID: PMC4284056  PMID: 22076792
multimodal MRI; anatomical MRI; functional MRI; diffusion tensor imaging; magnetic resonance spectroscopy; correlation; brain structure; brain function
19.  Phosphine-Catalyzed [3+2] and [4+3]Annulation Reactions of C,N-Cyclic Azomethine Imines with Allenoates 
Advanced synthesis & catalysis  2012;354(6):1023-1034.
Phosphine-catalyzed [3+2] and [4+3]annulation reactions of C,N-cyclic azomethine imines with allenoates have been developed to give a variety of pharmaceutically attractive tetrahydroisoquinoline derivatives in moderate to excellent yields. The two distinct reaction pathways, [3+2] and [4+3]cyclization, depend on the nature of the nucleophilic phosphine and the allenoate. Generally, for α-alkylallenoates, the reactions always proceed with [3 +2]cyclization as the major pathway no matter what phosphine was used; for α-ArCH2-substituted allenoates, the reaction pathway was controlled by the phosphine catalyst used.
doi:10.1002/adsc.201100831
PMCID: PMC4266944  PMID: 25525424
allenoates; annulation; azomethine imines; catalysis; phosphines
20.  The Pseudomonas syringae Type III Effector HopF2 Suppresses Arabidopsis Stomatal Immunity 
PLoS ONE  2014;9(12):e114921.
Pseudomonas syringae subverts plant immune signalling through injection of type III secreted effectors (T3SE) into host cells. The T3SE HopF2 can disable Arabidopsis immunity through Its ADP-ribosyltransferase activity. Proteomic analysis of HopF2 interacting proteins identified a protein complex containing ATPases required for regulating stomatal aperture, suggesting HopF2 may manipulate stomatal immunity. Here we report HopF2 can inhibit stomatal immunity independent of its ADP-ribosyltransferase activity. Transgenic expression of HopF2 in Arabidopsis inhibits stomatal closing in response to P. syringae and increases the virulence of surface inoculated P. syringae. Further, transgenic expression of HopF2 inhibits flg22 induced reactive oxygen species production. Intriguingly, ADP-ribosyltransferase activity is dispensable for inhibiting stomatal immunity and flg22 induced reactive oxygen species. Together, this implies HopF2 may be a bifunctional T3SE with ADP-ribosyltransferase activity required for inhibiting apoplastic immunity and an independent function required to inhibit stomatal immunity.
doi:10.1371/journal.pone.0114921
PMCID: PMC4263708  PMID: 25503437
21.  Utilisation of adsorption and desorption for simultaneously improving protein crystallisation success rate and crystal quality 
Scientific Reports  2014;4:7308.
High-quality protein crystals of suitable size are an important prerequisite for applying X-ray crystallography to determine the 3-dimensional structure of proteins. However, it is often difficult to obtain protein crystals of appropriate size and quality because nucleation and growth processes can be unsuccessful. Here, we show that by adsorbing proteins onto porous polystyrene-divinylbenzene microspheres (SDB) floating on the surface of the crystallisation solution, a localised high supersaturation region at the surface of the microspheres and a low supersaturation region below the microspheres can coexist in a single solution. The crystals will easily nucleate in the region of high supersaturation, but when they grow to a certain size, they will sediment to the region of low supersaturation and continue to grow. In this way, the probability of crystallisation and crystal quality can be simultaneously increased in a single solution without changing other crystallisation parameters.
doi:10.1038/srep07308
PMCID: PMC4255177  PMID: 25471817
22.  Lyn mitigates mouse airway remodeling by down-regulating the TGF-β3 isoform in house dust mite models 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(11):10.4049/jimmunol.1301596.
Chronic airway remodeling is a serious consequence of asthma, which is caused by complex but largely unknown mechanisms. Despite versatile functions, the role of Lyn in chronic airway remodeling remains undefined. Using Lyn−/− mice, we show that continual exposure (for 8 weeks) of house dust mite (HDM) extracts induced a severe phenotype of chronic airway remodeling including exacerbated mucus production, collagen deposition, dysregulated cytokine secretion, and elevated inflammation. Strikingly, a significant increase in TGF-β3 rather than TGF-β1 was observed in Lyn−/− mouse lungs compared to wild-type mice. Furthermore, TGF-β3 neutralizing antibodies not only inhibited the expression of STAT6 and Smad2/3, but also decreased phosphorylation of Smad2 and NFκB in Lyn−/− mouse lungs. In addition, both recombinant and adenoviral TGF-β3 significantly promoted epithelial to mesenchymal transition (EMT) and intensified collagen I production and MUC5AC expression. Further examining chronic asthma patients showed that a decreased Lyn correlated with the severity of airway inflammation and mucus hypersecretion. Finally, Lyn may critically regulate airway remodeling by directly interacting with TGF-β3. Collectively, these findings revealed that Lyn regulates TGF-β3 isoform and modulates the development of airway remodeling, which may have therapeutic indications for severe chronic asthma.
doi:10.4049/jimmunol.1301596
PMCID: PMC3863691  PMID: 24127553
knockout mice; Th2 cytokines; HDM extracts; oxidative stress; airway remodeling; EMT
23.  The Effects of Changing Water Content, Relaxation Times, and Tissue Contrast on Tissue Segmentation and Measures of Cortical Anatomy in MR Images 
Magnetic resonance imaging  2013;31(10):1709-1730.
Water content is the dominant chemical compound in the brain and it is the primary determinant of tissue contrast in magnetic resonance (MR) images. Water content varies greatly between individuals, and it changes dramatically over time from birth through senescence of the human life span. We hypothesize that the effects that individual- and age-related variations in water content have on contrast of the brain in MR images also has important, systematic effects on in vivo, MRI-based measures of regional brain volumes. We also hypothesize that changes in water content and tissue contrast across time may account for age-related changes in regional volumes, and that differences in water content or tissue contrast across differing neuropsychiatric diagnoses may account for differences in regional volumes across diagnostic groups.
We demonstrate in several complementary ways that subtle variations in water content across age and tissue compartments alter tissue contrast, and that changing tissue contrast in turn alters measures of the thickness and volume of the cortical mantle: (1) We derive analytic relations describing how age-related changes in tissue relaxation times produce age-related changes in tissue gray-scale intensity values and tissue contrast; (2) We vary tissue contrast in computer-generated images to assess its effects on tissue segmentation and volumes of gray matter and white matter; and (3) We use real-world imaging data from adults with either Schizophrenia or Bipolar Disorder and age- and sex-matched healthy adults to assess the ways in which variations in tissue contrast across diagnoses affects group differences in tissue segmentation and associated volumes.
We conclude that in vivo MRI-based morphological measures of the brain, including regional volumes and measures of cortical thickness, are a product of, or at least are confounded by, differences in tissue contrast across individuals, ages, and diagnostic groups, and that differences in tissue contrast in turn likely derive from corresponding differences in water content of the brain across individuals, ages, and diagnostic groups.
doi:10.1016/j.mri.2013.07.017
PMCID: PMC4241465  PMID: 24055410
Anatomical MRI; Tissue Contrast; Segmentation; Markov Random Field; Expectation Maximization
24.  Feasibility and safety of sorafenib treatment in hepatocellular carcinoma patients with spontaneous rupture 
World Journal of Gastroenterology : WJG  2014;20(43):16275-16281.
AIM: To report the outcome of patients with ruptured hepatocellular carcinoma (HCC) treated at a single center during a 5-year period.
METHODS: We retrospectively analyzed 32 patients who presented with ruptured HCC at Shandong Provincial Hospital Affiliated to Shandong University between 2008 and 2013.
RESULTS: The mean age of the patients was 53 years (range 39-71 years). Of these patients, 22 received surgical management, 10 underwent transarterial embolization (TAE) or transarterial chemoembolization (TACE), and 12 received sorafenib after surgery, TAE or TACE. Cumulative survival rates at 4, 8 and 12 mo were 72.9%, 50.0% and 33.3%, respectively, in the surgery only group and were 90.0%, 80.6% and 64.1%, respectively, in the surgery plus sorafenib group. Cumulative survival rates at 4, 8 and 12 mo were 68.4%, 43.6% and 19.4%, respectively, in the surgery only or TAE/TACE only groups, and were 91.7%, 75.0% and 60.2%, respectively, in the sorafenib combination groups (P = 0.04). No unexpected side effects due to sorafenib were observed. The most common side effect was hand-foot skin reaction. To date, 5 patients have died. Median follow-up from the start of sorafenib therapy for the remaining 7 patients is 12.7 mo (range 5.8-32.2 mo).
CONCLUSION: Sorafenib can be used in patients with ruptured HCC as it has interesting activity and is well tolerated; dose adjustment is generally not required. However, a larger prospective study is necessary to determine the efficacy of sorafenib in this group of patients.
doi:10.3748/wjg.v20.i43.16275
PMCID: PMC4239517  PMID: 25473183
Sorafenib; Hepatocellular carcinoma; Spontaneous rupture; Efficacy; Safety
25.  CarSPred: A Computational Tool for Predicting Carbonylation Sites of Human Proteins 
PLoS ONE  2014;9(10):e111478.
Protein carbonylation is one of the most pervasive oxidative stress-induced post-translational modifications (PTMs), which plays a significant role in the etiology and progression of several human diseases. It has been regarded as a biomarker of oxidative stress due to its relatively early formation and stability compared with other oxidative PTMs. Only a subset of proteins is prone to carbonylation and most carbonyl groups are formed from lysine (K), arginine (R), threonine (T) and proline (P) residues. Recent advancements in analysis of the PTM by mass spectrometry provided new insights into the mechanisms of protein carbonylation, such as protein susceptibility and exact modification sites. However, the experimental approaches to identifying carbonylation sites are costly, time-consuming and capable of processing a limited number of proteins, and there is no bioinformatics method or tool devoted to predicting carbonylation sites of human proteins so far. In the paper, a computational method is proposed to identify carbonylation sites of human proteins. The method extracted four kinds of features and combined the minimum Redundancy Maximum Relevance (mRMR) feature selection criterion with weighted support vector machine (WSVM) to achieve total accuracies of 85.72%, 85.95%, 83.92% and 85.72% for K, R, T and P carbonylation site predictions respectively using 10-fold cross-validation. The final optimal feature sets were analysed, the position-specific composition and hydrophobicity environment of flanking residues of modification sites were discussed. In addition, a software tool named CarSPred has been developed to facilitate the application of the method. Datasets and the software involved in the paper are available at https://sourceforge.net/projects/hqlstudio/files/CarSPred-1.0/.
doi:10.1371/journal.pone.0111478
PMCID: PMC4210226  PMID: 25347395

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