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1.  Bacterial dynamics within the mucus, tissue and skeleton of the coral Porites lutea during different seasons 
Scientific Reports  2014;4:7320.
Investigation of the response of coral microbial communities to seasonal ecological environment at the microscale will advance our understanding of the relationship between coral-associated bacteria community and coral health. In this study, we examined bacteria community composition from mucus, tissue and skeleton of Porites lutea and surrounding seawater every three months for 1 year on Luhuitou fringing reef. The bacterial communities were analyzed using pyrosequencing of the V1-V2 region of the 16S rRNA gene, which demonstrated diverse bacterial consortium profiles in corals. The bacterial communities in all three coral compartments studied were significantly different from the surrounding seawater. Moreover, they had a much more dynamic seasonal response compared to the seawater communities. The bacterial communities in all three coral compartments collected in each seasonal sample tended to cluster together. Analysis of the relationship between bacterial assemblages and the environmental parameters showed that the bacterial community correlated to dissolved oxygen and rainfall significantly at our study site. This study highlights a dynamic relationship between the high complexity of coral associated bacterial community and seasonally varying ecosystem parameters.
PMCID: PMC4256709  PMID: 25475855
2.  microRNA-214 functions as a tumor suppressor in human colon cancer via the suppression of ADP-ribosylation factor-like protein 2 
Oncology Letters  2014;9(2):645-650.
microRNAs (miRNAs/miRs) are a conserved class of endogenous, short non-coding RNAs that post-transcriptionally regulate the expression of genes involved in diverse cellular processes. miR-214 has been reported to be associated with several cancers, including human colon cancer. However, the function of miR-214 in colon cancer development is poorly understood. In the current study, miR-214 was demonstrated to be downregulated in colon cancer tissues compared with healthy colon tissues. Functional studies showed that miR-214 overexpression results in the inhibition of cell viability, colony formation and proliferation, and the induction of cell apoptosis. ADP-ribosylation factor-like protein 2 (ARL2) is predicted to be a target candidate of miR-214. A luciferase reporter assay, western blot analysis and quantitative polymerase chain reaction were performed, which revealed that miR-214 negatively regulates ARL2 expression by targeting its 3′ untranslated region directly. In conclusion, the results of the present study revealed that miR-214 suppresses colon cancer cell growth via the suppression of ARL2, and indicated that miR-214 may present a significant potential therapeutic target for colon cancer.
PMCID: PMC4301474  PMID: 25621032
ADP-ribosylation factor-like protein 2; apoptosis; proliferation; miR-214; human colon cancer
3.  Antigenic and Genetic Evolution of Low-Pathogenicity Avian Influenza Viruses of Subtype H7N3 following Heterologous Vaccination 
Outbreaks of low-pathogenicity avian influenza (LPAI) viruses of the H7N3 subtype were first detected in Italy in October 2002, and the virus continued to circulate between 2002 and 2004 in a densely populated poultry area in the northeast portion of that country. This virus circulated in unvaccinated and vaccinated poultry farms, and the infection was controlled in August 2003 by culling, control of movements, improved biosecurity, and heterologous vaccination. In 2004, H7N3 reoccurred in vaccinated poultry farms in which infection had been successfully controlled by the vaccination program. To shed light on this occurrence and the temporal pattern and genetic basis of antigenic drift for avian influenza viruses (AIVs) in the absence and presence of heterologous vaccination, a collection of H7N3 viruses isolated in 2002 and 2004 were characterized genetically and antigenically. Molecular analysis showed that viruses isolated in the 2004 outbreaks after the implementation of vaccination had acquired specific amino acid signatures, most of which were located at reported antibody binding sites of the hemagglutinin (HA) protein. Antigenic characterization of these 2004 isolates showed that they were antigenically different from those isolated prior to the implementation of vaccination. This is the first report on antigenic and genetic evolution of H7 LPAI viruses following the application of heterologous vaccination in poultry. These findings may have an impact on control strategies to combat AI infections in poultry based on vaccination.
PMCID: PMC4018894  PMID: 24554694
4.  Aberrant neuronal synaptic connectivity in CA1 area of the hippocampus from pilocarpine-induced epileptic rats observed by fluorogold 
In this study, we observed synaptic connectivity among neurons in CA1 region of pilocarpine-induced chronic seizures in rats. Twenty healthy male Sprague-Dawley rats were divided randomly into an epilepsy group (n = 10) and a control group (n = 10). Approximately 60 days after status epilepticus (SE) , Fluorogold (FG) was injected into the CA1 area of the hippocampus in vivo. Somatostatin (SS) expression was observed using immunofluorescence. The distribution of FG-positive and FG/SS double-labeled neurons was observed using a confocal microscope. FG-labeled pyramidal cells could be seen remotely from the FG-injected site in the CA1 area and in the subiculum in the experimental group. FG/SS double-labeled interneurons were distributed remotely from the FG-injected site in the CA1 area in the epileptic rats. These changes suggest aberrant neuronal connectivity in CA1 region, which may lead to the formation of aberrant excitatory and inhibitory circuitry, and may play an important role in the generation or compensation for temporal lobe epilepsy.
PMCID: PMC4211776  PMID: 25356126
Temporal lobe epilepsy (TLE); fluorogold (FG); somatostatin (SS); neuronal connectivity; circuit rearrangement; interneuron; lithium chloride; pilocarpin
5.  CRISPR/Cas9 Mediates Efficient Conditional Mutagenesis in Drosophila 
G3: Genes|Genomes|Genetics  2014;4(11):2167-2173.
Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach.
PMCID: PMC4232542  PMID: 25193494
CRISPR/Cas9; conditional mutagenesis; gRNA; Gal4; Drosophila
6.  Influenza A Subtype H3 Viruses in Feral Swine, United States, 2011–2012 
Emerging Infectious Diseases  2014;20(5):843-846.
To determine whether, and to what extent, influenza A subtype H3 viruses were present in feral swine in the United States, we conducted serologic and virologic surveillance during October 2011–September 2012. These animals were periodically exposed to and infected with A(H3N2) viruses, suggesting they may threaten human and animal health.
PMCID: PMC4012812  PMID: 24751326
feral swine; H3; influenza A virus; swine influenza virus; serologic assay; seropositive; H3N2v; viruses; United States
7.  Relationship Between Changes in Brain MRI and 1H-MRS, Severity of Chronic Liver Damage, and Recovery After Liver Transplantation 
Magnetic resonance imaging (MRI) and 1H magnetic resonance spectroscopy (1H-MRS) have been used in clinics for diagnosis of chronic liver diseases. This study was designed to investigate the relationship between MRI/MRS outcomes and the severity of liver damage. Of 50 patients examined, the MRI signal intensity in the globus pallidus as determined by pallidus index (PI) increased as the disease severity (scored by Child Pugh ranking) worsened (r = 0.353, P < 0.05). The changes in PI values were also linearly associated with Mn concentrations in whole blood (MnB) (r = 0.814, P < 0.01). MRS analysis of four major brain metabolites (i.e., Cho, mI, Glx, and NAA) revealed that the ratios of Cho/Cr and mI/Cr in cirrhosis and CHE patients were significantly decreased in comparison to controls (P < 0.05), whereas the ratio of Glx/Cr was significantly increased (P < 0.05). The Child Pugh scores significantly correlated with mI/ Cr (−0.484, P < 0.01) and Glx (0.369, P < 0.05), as well as MnB (0.368, P < 0.05), but not with other brain metabolites. Three patients who received a liver transplant experienced normalization of brain metabolites within 3 months of post-transplantation; the MR imaging of Mn in the globus pallidus completely disappeared 5 months after the surgery. Taken together, this clinical study, which combined MRI/MRS analysis, autopsy exam and liver transplant, clearly demonstrates that liver injury-induced brain Mn accumulation can reversibly alter the homeostasis of brain metabolites Cho, mI and Glx. Our data further suggest that liver transplantation can restore normal brain Mn levels.
PMCID: PMC4005269  PMID: 19546351
hepatic encephalopathy; magnetic resonance imaging (MRI); magnetic resonance spectroscopy (MRS); Child Pugh; manganese; choline; glutamine; liver transplant
8.  Maotai Ameliorates Diethylnitrosamine-Initiated Hepatocellular Carcinoma Formation in Mice 
PLoS ONE  2014;9(4):e93599.
Consumption of alcohol is closely related to liver disease, such as hepatic fibrosis or even hepatocellular carcinoma (HCC). However, epidemiological and experimental studies indicated that consumption of Maotai, one of the famous liquors in China, exhibits no significant correlation with hepatic fibrosis or cirrhosis as other beverage sources do. This study detected the relationship of Maotai consumption and HCC development in a diethylnitrosamine (DEN)-initiated HCC animal model. DEN was given to mice at a dose of 100 mg/kg, ip, and 50 mg/kg, ip in the following week. Mice were simultaneously given Maotai or an equal amount of ethanol (53%, 5 ml/kg/day, 5days/week for up to 35weeks). At 3-week and 35- week of the experiment, serum and livers were collected for biochemical and histopathological examination of liver injury and incidence of HCC. Real-time RT-PCR, immunohistochemistry and Western blotting were used to examine the expression of metallothionein-1/2 (MT-1/2), NF-E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC) and modified subunit (GCLM). We identified tissue damage and dysfunction of liver in ethanol + DEN-treated mice, whereas the extent of injury was reduced in Maotai+ DEN –treated mice. Significant Glypican-3(GPC3) expression and precancerous injury or HCC were seen in approximately 50% of mice with ethanol+ DEN, but barely be seen in Maotai + DEN-treated mice. A higher expression of MT-1/2, Nrf2 and GCLC could be seen in Maotai + DEN-treated mice. Thus, Maotai liquor ameliorates the formation of DEN-induced HCC in mice, and the protection mechanism is possibly related with the activation of anti-oxidation factors, such as MTs, Nrf2 and GCLC.
PMCID: PMC3972115  PMID: 24690765
9.  Meta-Analysis of the rs4779584 Polymorphism and Colorectal Cancer Risk 
PLoS ONE  2014;9(2):e89736.
Several researchers have suggested that the rs4779584 (15q13.3) polymorphism is associated with an increased risk of developing colorectal cancer (CRC). However, past results remain inconclusive. We addressed this controversy by performing a meta-analysis of the relationship between rs4779584 of GREM1-SCG5 and colorectal cancer.
We selected 12 case-control studies involving 11,769 cases of CRC and 14,328 healthy controls. The association between the rs4779584 polymorphism and CRC was examined by the overall odds ratio (OR) with a 95% confidence interval (CI). We used different genetic model analyses, sensitivity analyses, and assessments of bias in our meta-analysis.
GREM1-SCG5 rs4779584 polymorphisms were associated with CRC in all of the genetic models that were examined in this meta-analysis of 12 case-control studies.
GREM1-SCG5 rs4779584 polymorphisms may increase the risk of developing colorectal cancer.
PMCID: PMC3933649  PMID: 24586997
10.  Thalamic GABA Predicts Fine Motor Performance in Manganese-Exposed Smelter Workers 
PLoS ONE  2014;9(2):e88220.
Overexposure to manganese (Mn) may lead to parkinsonian symptoms including motor deficits. The main inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is known to play a pivotal role in the regulation and performance of movement. Therefore this study was aimed at testing the hypothesis that an alteration of GABA following Mn exposure may be associated with fine motor performance in occupationally exposed workers and may underlie the mechanism of Mn-induced motor deficits. A cohort of nine Mn-exposed male smelter workers from an Mn-iron alloy factory and 23 gender- and age-matched controls were recruited and underwent neurological exams, magnetic resonance spectroscopy (MRS) measurements, and Purdue pegboard motor testing. Short-echo-time MRS was used to measure N-Acetyl-aspartate (NAA) and myo-inositol (mI). GABA was detected with a MEGA-PRESS J-editing MRS sequence. The mean thalamic GABA level was significantly increased in smelter workers compared to controls (p = 0.009). Multiple linear regression analysis reveals (1) a significant association between the increase in GABA level and the duration of exposure (R2 = 0.660, p = 0.039), and (2) significant inverse associations between GABA levels and all Purdue pegboard test scores (for summation of all scores R2 = 0.902, p = 0.001) in the smelter workers. In addition, levels of mI were reduced significantly in the thalamus and PCC of smelter workers compared to controls (p = 0.030 and p = 0.009, respectively). In conclusion, our results show clear associations between thalamic GABA levels and fine motor performance. Thus in Mn-exposed subjects, increased thalamic GABA levels may serve as a biomarker for subtle deficits in motor control and may become valuable for early diagnosis of Mn poisoning.
PMCID: PMC3913772  PMID: 24505436
11.  Antigenic Characterization of H3N2 Influenza A Viruses from Ohio Agricultural Fairs 
Journal of Virology  2013;87(13):7655-7667.
The demonstrated link between the emergence of H3N2 variant (H3N2v) influenza A viruses (IAVs) and swine exposure at agricultural fairs has raised concerns about the human health risk posed by IAV-infected swine. Understanding the antigenic profiles of IAVs circulating in pigs at agricultural fairs is critical to developing effective prevention and control strategies. Here, 68 H3N2 IAV isolates recovered from pigs at Ohio fairs (2009 to 2011) were antigenically characterized. These isolates were compared with other H3 IAVs recovered from commercial swine, wild birds, and canines, along with human seasonal and variant H3N2 IAVs. Antigenic cartography demonstrated that H3N2 IAV isolates from Ohio fairs could be divided into two antigenic groups: (i) the 2009 fair isolates and (ii) the 2010 and 2011 fair isolates. These same two antigenic clusters have also been observed in commercial swine populations in recent years. Human H3N2v isolates from 2010 and 2011 are antigenically clustered with swine-origin IAVs from the same time period. The isolates recovered from pigs at fairs did not cross-react with ferret antisera produced against the human seasonal H3N2 IAVs circulating during the past decade, raising the question of the degree of immunity that the human population has to swine-origin H3N2 IAVs. Our results demonstrate that H3N2 IAVs infecting pigs at fairs and H3N2v isolates were antigenically similar to the IAVs circulating in commercial swine, demonstrating that exhibition swine can function as a bridge between commercial swine and the human population.
PMCID: PMC3700273  PMID: 23637412
12.  Research of the Methylation Status of miR-124a Gene Promoter among Rheumatoid Arthritis Patients 
Objective. To analyze the methylation status of miR-124a loci in synovial tissues of rheumatoid arthritis (RA) patients using methylation-specific polymerase chain reaction (MSP). Materials and Methods. DNA obtained from the frozen tissue of 7 RA samples, 6 osteoarthritis (OA) samples, and 3 healthy controls were undergoing bisulfite conversion and then analyzed for miR-124a promoter methylation using MSP assay. Results. miR-124-a1 and miR-124-a2 promoter methylation were both seen in 71.4% of RA samples compared to 16.7% of OA samples. miR-124-a3 promoter methylation was seen in 57.1% of RA samples and 0% of OA samples. All the three loci were unmethylated in 3 healthy controls. Conclusion. The methylation status of miR-124a seen in this study concurs with that reported in tumor cells, indicating epigenetic dysregulation constituents, a mechanism in the development of rheumatoid arthritis.
PMCID: PMC3810484  PMID: 24223605
13.  Upregulated MicroRNA-155 Expression in Peripheral Blood Mononuclear Cells and Fibroblast-Like Synoviocytes in Rheumatoid Arthritis 
Objective. This study was to screen for the miRNAs differently expressed in peripheral blood mononuclear cells (PBMC) of RA, to further identify the expression of miR-155 in RA PBMC and fibroblast-like synoviocytes (FLS), and to evaluate the function of miR-155 in RA-FLS. Methods. Microarray was used to screen for differentially expressed miRNAs in RA PBMC. miR-155 expression in PBMC and FLS of RA were identified by real-time PCR. Enforced overexpression and downexpression of miR-155 were used to investigate the function of miR-155 in RA-FLS. Expression of IKBKE which was previously identified as the actual target of miR-155 was examined by Western blot and real-time PCR in RA-FLS. Results. miR-155 levels were increased in both PBMC and FLS of RA and could be induced by TNF-α. Upregulation of miR-155 decreased MMP-3 levels and suppressed proliferation and invasion of RA-FLS. Inverse relationship between the expressions of miR-155 and the MMPs production-related protein IKBKE was found. Conclusion. An inflammatory milieu may alter miRNA expression profiles in rheumatoid arthritis. miR-155 is upregulated in RA-FLS, and it may be a protective factor against the inflammatory effect in part by attenuating expression of IKBKE.
PMCID: PMC3789322  PMID: 24151514
14.  Identifying antigenicity associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning 
Journal of molecular biology  2012;422(1):145-155.
Since the isolation of A/goose/Guangdong/1/1996 (H5N1) in farmed geese in southern China, highly pathogenic H5N1 avian influenza viruses have posed a continuous threat to both public and animal health. The non-synonymous mutation of the H5 hemagglutinin gene has resulted in antigenic drift, leading to difficulties in both clinical diagnosis and vaccine strain selection. Characterizing H5N1’s antigenic profiles would help resolve these problems. In this study, a novel sparse learning method was developed to identify antigenicity associated sites in influenza A viruses on the basis of immunologic datasets (i.e., from hemagglutination inhibition and microneutralization assays) and HA protein sequences. Twenty-one potential antigenicity associated sites were identified. A total of seventeen H5N1 mutants were used to validate the effects of eleven of these predicted sites on H5N1’s antigenicity, including seven newly identified sites not located in reported antibody binding sites. The experimental data confirmed that mutations of these tested sites lead to changes in viral antigenicity, validating our method.
PMCID: PMC3412944  PMID: 22609437
15.  Highly Heterogeneous Bacterial Communities Associated with the South China Sea Reef Corals Porites lutea, Galaxea fascicularis and Acropora millepora 
PLoS ONE  2013;8(8):e71301.
Coral harbor diverse and specific bacteria play significant roles in coral holobiont function. Bacteria associated with three of the common and phylogenetically divergent reef-building corals in the South China Sea, Porites lutea, Galaxea fascicularis and Acropora millepora, were investigated using 454 barcoded-pyrosequencing. Three colonies of each species were sampled, and 16S rRNA gene libraries were constructed individually. Analysis of pyrosequencing libraries showed that bacterial communities associated with the three coral species were more diverse than previous estimates based on corals from the Caribbean Sea, Indo-Pacific reefs and the Red Sea. Three candidate phyla, including BRC1, OD1 and SR1, were found for the first time in corals. Bacterial communities were separated into three groups: P. lutea and G. fascicular, A. millepora and seawater. P. lutea and G. fascicular displayed more similar bacterial communities, and bacterial communities associated with A. millepora differed from the other two coral species. The three coral species shared only 22 OTUs, which were distributed in Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Chloroflexi, Actinobacteria, Acidobacteria and an unclassified bacterial group. The composition of bacterial communities within each colony of each coral species also showed variation. The relatively small common and large specific bacterial communities in these corals implies that bacterial associations may be structured by multiple factors at different scales and that corals may associate with microbes in terms of similar function, rather than identical species.
PMCID: PMC3737133  PMID: 23940737
16.  Using Sequence Data To Infer the Antigenicity of Influenza Virus 
mBio  2013;4(4):e00230-13.
The efficacy of current influenza vaccines requires a close antigenic match between circulating and vaccine strains. As such, timely identification of emerging influenza virus antigenic variants is central to the success of influenza vaccination programs. Empirical methods to determine influenza virus antigenic properties are time-consuming and mid-throughput and require live viruses. Here, we present a novel, experimentally validated, computational method for determining influenza virus antigenicity on the basis of hemagglutinin (HA) sequence. This method integrates a bootstrapped ridge regression with antigenic mapping to quantify antigenic distances by using influenza HA1 sequences. Our method was applied to H3N2 seasonal influenza viruses and identified the 13 previously recognized H3N2 antigenic clusters and the antigenic drift event of 2009 that led to a change of the H3N2 vaccine strain.
This report supplies a novel method for quantifying antigenic distance and identifying antigenic variants using sequences alone. This method will be useful in influenza vaccine strain selection by significantly reducing the human labor efforts for serological characterization and will increase the likelihood of correct influenza vaccine candidate selection.
PMCID: PMC3705446  PMID: 23820391
17.  A Perspective on Multiple Waves of Influenza Pandemics 
PLoS ONE  2013;8(4):e60343.
A striking characteristic of the past four influenza pandemic outbreaks in the United States has been the multiple waves of infections. However, the mechanisms responsible for the multiple waves of influenza or other acute infectious diseases are uncertain. Understanding these mechanisms could provide knowledge for health authorities to develop and implement prevention and control strategies.
Materials and Methods
We exhibit five distinct mechanisms, each of which can generate two waves of infections for an acute infectious disease. The first two mechanisms capture changes in virus transmissibility and behavioral changes. The third mechanism involves population heterogeneity (e.g., demography, geography), where each wave spreads through one sub-population. The fourth mechanism is virus mutation which causes delayed susceptibility of individuals. The fifth mechanism is waning immunity. Each mechanism is incorporated into separate mathematical models, and outbreaks are then simulated. We use the models to examine the effects of the initial number of infected individuals (e.g., border control at the beginning of the outbreak) and the timing of and amount of available vaccinations.
Four models, individually or in any combination, reproduce the two waves of the 2009 H1N1 pandemic in the United States, both qualitatively and quantitatively. One model reproduces the two waves only qualitatively. All models indicate that significantly reducing or delaying the initial numbers of infected individuals would have little impact on the attack rate. Instead, this reduction or delay results in a single wave as opposed to two waves. Furthermore, four of these models also indicate that a vaccination program started earlier than October 2009 (when the H1N1 vaccine was initially distributed) could have eliminated the second wave of infection, while more vaccine available starting in October would not have eliminated the second wave.
PMCID: PMC3634039  PMID: 23637746
18.  Bt Crops Producing Cry1Ac, Cry2Ab and Cry1F Do Not Harm the Green Lacewing, Chrysoperla rufilabris 
PLoS ONE  2013;8(3):e60125.
The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.
PMCID: PMC3609736  PMID: 23544126
19.  MicroRNA expression profile of the hippocampus in a rat model of temporal lobe epilepsy and miR-34a-targeted neuroprotection against hippocampal neurone cell apoptosis post-status epilepticus 
BMC Neuroscience  2012;13:115.
The expression pattern and function of miRNAs in the rat model of temporal lobe epilepsy have not been well defined. Profiling miRNA expression in the rat model of temporal lobe epilepsy and investigating the function of specific miRNAs in epilepsy offers the prospect of a deeper understanding of the mechanisms of epilepsy.
The lithium-pilocarpine-induced status epilepticus model and the temporal lobe epilepsy model were established in Sprague–Dawley rats. Samples were analysed to detect deregulated miRNAs in the hippocampal temporal lobe, and several of these deregulated miRNAs were confirmed by qPCR. The expression of the pro-apoptotic miR-34a was detected at 1 day, 7 days and 2 weeks post-status epilepticus and at 2 months after temporal lobe epilepsy. The antagomir of miR-34a was then utilised. The expression of miR-34a after targeting and the expression change of activated caspase-3 protein were examined. The effects of altering the expression of miR-34a and activated caspase-3 protein on neuronal survival and neuronal death or apoptosis post-status epilepticus were assessed.
The miRNA microarray detected 9 up-regulated miRNAs (miR-146a, -211, -203, -210, -152, -31, -23a, -34a, -27a) and 15 down-regulated miRNAs (miR-138*, -301a, -136, -153, -19a, -135b, -325-5p, -380, -190, -542-3p, -33, -144, -542-5p, -543, -296*). Some of the deregulated miRNAs (miR-146a, miR-210, miR-27a, miR-135b and miR-33) were confirmed using qPCR. Furthermore, an increase in expression of the pro-apoptotic miR-34a was demonstrated in the post-status epilepticus rat hippocampus. miR-34a was significantly up-regulated at 1 day, 7 days and 2 weeks post-status epilepticus and at 2 months after temporal lobe epilepsy. Experiments with the miR-34a antagomir revealed that targeting miR-34a led to an inhibition of activated caspase-3 protein expression, which may contribute to increased neuronal survival and reduced neuronal death or apoptosis.
Our study showed the expression profile of miRNAs in the hippocampus in a rat model of temporal lobe epilepsy and an increase in the expression of the pro-apoptotic miR-34a in post-status epilepticus rats. The results show that miR-34a is up-regulated during seizure-induced neuronal death or apoptosis, and targeting miR-34a is neuroprotective and is associated with an inhibition of an increase in activated caspase-3 protein.
PMCID: PMC3471047  PMID: 22998082
MiRNA; Epilepsy; Hippocampus; Apoptosis; Status epilepticus
20.  Identification of an H6N6 swine influenza virus in southern China 
This is the first report of avian-like H6N6 swine influenza virus from swine in southern China. Phylogenetic analysis indicated that this virus might originate from domestic ducks. Serological surveillance suggested there had been sporadic H6 swine influenza infections in this area. Continuing study is required to determine if this virus could be established in the swine population and pose potential threats to public health.
PMCID: PMC3104128  PMID: 21382518
H6N6; swine influenza virus; influenza A virus; influenza pandemic
21.  Indications that Live Poultry Markets Are a Major Source of Human H5N1 Influenza Virus Infection in China ▿ †  
Journal of Virology  2011;85(24):13432-13438.
Human infections of H5N1 highly pathogenic avian influenza virus have continued to occur in China without corresponding outbreaks in poultry, and there is little conclusive evidence of the source of these infections. Seeking to identify the source of the human infections, we sequenced 31 H5N1 viruses isolated from humans in China (2005 to 2010). We found a number of viral genotypes, not all of which have similar known avian virus counterparts. Guided by patient questionnaire data, we also obtained environmental samples from live poultry markets and dwellings frequented by six individuals prior to disease onset (2008 and 2009). H5N1 viruses were isolated from 4 of the 6 live poultry markets sampled. In each case, the genetic sequences of the environmental and corresponding human isolates were highly similar, demonstrating a link between human infection and live poultry markets. Therefore, infection control measures in live poultry markets are likely to reduce human H5N1 infection in China.
PMCID: PMC3233185  PMID: 21976646
22.  Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition 
Acta Pharmacologica Sinica  2012;33(5):594-605.
To identify the mechanisms underlying the elevation of intracellular Ca2+ level ([Ca2+]i) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons.
Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca2+]i was measured using fura-2 AM. Ca2+ current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay.
Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca2+]i in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca2+]i in ARC neurons depended on extracellular Ca2+, and was blocked by P/Q-type Ca2+channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca2+ channel blocker nifedipine (10 μmol/L) or N-type Ca2+channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca2+ current in ARC neurons. The low-glucose induced elevation of [Ca2+]i in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L).
Lowering glucose level enhances the activity of P/Q type Ca2+channels and elevates [Ca2+]i level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β.
PMCID: PMC4010362  PMID: 22504905
hypothalamic arcuate nucleus; NPY neurons; glucose; calcium imaging; P/Q type Ca2+ channel; GSK3β; AMPK; whole-cell patch clamp
23.  Avian-origin H3N2 canine influenza A viruses in Southern China 
This study reports four sporadic cases of H3N2 canine influenza in southern China, which were identified from sick dogs from May 2006 to October 2007. The evolutionary analysis showed that all eight segments of these four viruses are avian-origin and phylogenetically close to the H3N2 canine influenza viruses reported earlier in South Korea. Systematic surveillance is required to monitor the disease and evolutionary behavior of this virus in canine populations in China.
PMCID: PMC2950248  PMID: 20732458
H3N2; Canine influenza virus; phylogenetics; influenza A virus; pathogenesis; avian-origin
24.  2-Methyl-6-nitro-2H-indazole 
In the title compound, C8H7N3O2, the mol­ecular skeleton is almost planar with a maximum deviation of 0.0484 (9) Å for the methyl C atom. In the crystal, weak inter­molecular C—H⋯N and C—H⋯O hydrogen bonds help to establish the packing.
PMCID: PMC3120619  PMID: 21754905
25.  In Vivo Measurement of Brain GABA Concentrations by Magnetic Resonance Spectroscopy in Smelters Occupationally Exposed to Manganese 
Environmental Health Perspectives  2010;119(2):219-224.
Exposure to excessive levels of manganese (Mn) is known to induce psychiatric and motor disorders, including parkinsonian symptoms. Therefore, finding a reliable means for early detection of Mn neurotoxicity is desirable.
Our goal was to determine whether in vivo brain levels of γ-aminobutyric acid (GABA), N-acetylaspartate (NAA), and other brain metabolites in male smelters were altered as a consequence of Mn exposure.
We used T1-weighted magnetic resonance imaging (MRI) to visualize Mn deposition in the brain. Magnetic resonance spectroscopy (MRS) was used to quantify concentrations of NAA, glutamate, and other brain metabolites in globus pallidus, putamen, thalamus, and frontal cortex from a well-established cohort of 10 male Mn-exposed smelters and 10 male age-matched control subjects. We used the MEGA-PRESS MRS sequence to determine GABA levels in a region encompassing the thalamus and adjacent parts of the basal ganglia [GABA-VOI (volume of interest)].
Seven of 10 exposed subjects showed clear T1-hyperintense signals in the globus pallidus indicating Mn accumulation. We found a significant increase (82%; p = 0.014) in the ratio of GABA to total creatine (GABA/tCr) in the GABA-VOI of Mn-exposed subjects, as well as a distinct decrease (9%; p = 0.04) of NAA/tCr in frontal cortex that strongly correlated with cumulative Mn exposure (R = −0.93; p < 0.001).
We demonstrated elevated GABA levels in the thalamus and adjacent basal ganglia and decreased NAA levels in the frontal cortex, indicating neuronal dysfunction in a brain area not primarily targeted by Mn. Therefore, the noninvasive in vivo MRS measurement of GABA and NAA may prove to be a powerful tool for detecting presymptomatic effects of Mn neurotoxicity.
PMCID: PMC3040609  PMID: 20876035
GABA; imaging; manganese; metabolism; MRI; MRS; NAA; occupational health; parkinsonism; smelters

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