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1.  Mechanical Behaviour of Umbrella-Shaped, Ni-Ti Memory Alloy Femoral Head Support Device during Implant Operation: A Finite Element Analysis Study 
PLoS ONE  2014;9(6):e100765.
A new instrument used for treating femoral head osteonecrosis was recently proposed: the umbrella-shaped, Ni-Ti memory femoral head support device. The device has an efficacy rate of 82.35%. Traditional radiographic study provides limited information about the mechanical behaviour of the support device during an implant operation. Thus, this study proposes a finite element analysis method, which includes a 3-step formal head model construction scheme and a unique material assignment strategy for evaluating mechanical behaviour during an implant operation. Four different scenarios with different constraints, initial positions and bone qualities are analyzed using the simulation method. The max radium of the implanted device was consistent with observation data, which confirms the accuracy of the proposed method. To ensure that the device does not unexpectedly open and puncture the femoral head, the constraint on the impact device should be strong. The initial position of sleeve should be in the middle to reduce the damage to the decompression channel. The operation may fail because of poor bone quality caused by severe osteoporosis. The proposed finite element analysis method has proven to be an accurate tool for studying the mechanical behaviour of umbrella-shaped, Ni-Ti memory alloy femoral head support device during an implant operation. The 3-step construct scheme can be implemented with any kind of bone structure meshed with multiple element types.
PMCID: PMC4069114  PMID: 24960038
2.  Progranulin Deficiency Exaggerates, whereas Progranulin-derived Atsttrin Attenuates, Severity of Dermatitis in Mice 
FEBS letters  2013;587(12):1805-1810.
PGRN and its derived engineered protein, Atsttrin, were reported to antagonize TNFα and protect against inflammatory arthritis (Tang, W., et al, Science, 2011). Here we found that PGRN level was also significantly elevated in skin inflammation. PGRN−/− mice exhibited more severe inflammation following induction of oxazolone. In contrast, recombinant Atsttrin protein effectively attenuated inflammation in mice dermatitis model. In addition, the protective role of PGRN and Atsttrin in dermatitis was probably due to their inhibition on NF-κB signaling. Collectively, PGRN, especially its derived engineered protein, Atsttrin, may represent a potential molecular target for prevention and treatment of inflammatory skin diseases.
PMCID: PMC3683372  PMID: 23669357
Progranulin; Atsttrin; dermatitis; NF-κB signaling
3.  Replication of 6 Obesity Genes in a Meta-Analysis of Genome-Wide Association Studies from Diverse Ancestries 
PLoS ONE  2014;9(5):e96149.
Obesity is a major public health problem with a significant genetic component. Multiple DNA polymorphisms/genes have been shown to be strongly associated with obesity, typically in populations of European descent. The aim of this study was to verify the extent to which 6 confirmed obesity genes (FTO, CTNNBL1, ADRB2, LEPR, PPARG and UCP2 genes) could be replicated in 8 different samples (n = 11,161) and to explore whether the same genes contribute to obesity-susceptibility in populations of different ancestries (five Caucasian, one Chinese, one African-American and one Hispanic population). GWAS-based data sets with 1000 G imputed variants were tested for association with obesity phenotypes individually in each population, and subsequently combined in a meta-analysis. Multiple variants at the FTO locus showed significant associations with BMI, fat mass (FM) and percentage of body fat (PBF) in meta-analysis. The strongest association was detected at rs7185735 (P-value = 1.01×10−7 for BMI, 1.80×10−6 for FM, and 5.29×10−4 for PBF). Variants at the CTNNBL1, LEPR and PPARG loci demonstrated nominal association with obesity phenotypes (meta-analysis P-values ranging from 1.15×10−3 to 4.94×10−2). There was no evidence of association with variants at ADRB2 and UCP2 genes. When stratified by sex and ethnicity, FTO variants showed sex-specific and ethnic-specific effects on obesity traits. Thus, it is likely that FTO has an important role in the sex- and ethnic-specific risk of obesity. Our data confirmed the role of FTO, CTNNBL1, LEPR and PPARG in obesity predisposition. These findings enhanced our knowledge of genetic associations between these genes and obesity-related phenotypes, and provided further justification for pursuing functional studies of these genes in the pathophysiology of obesity. Sex and ethnic differences in genetic susceptibility across populations of diverse ancestries may contribute to a more targeted prevention and customized treatment of obesity.
PMCID: PMC4039436  PMID: 24879436
4.  Characterization of the DNA methylome and its interindividual variation in human peripheral blood monocytes 
Epigenomics  2013;5(3):10.2217/epi.13.18.
Peripheral blood monocytes (PBMs) play multiple and critical roles in the immune response, and abnormalities in PBMs have been linked to a variety of human disorders. However, the DNA methylation landscape in PBMs is largely unknown. In this study, we characterized epigenome-wide DNA methylation profiles in purified PBMs.
Materials & methods
PBMs were isolated from freshly collected peripheral blood from 18 unrelated healthy postmenopausal Caucasian females. Epigenome-wide DNA methylation profiles (the methylome) were characterized by using methylated DNA immunoprecipitation combined with high-throughput sequencing.
Distinct patterns were revealed at different genomic features. For instance, promoters were commonly (~58%) found to be unmethylated; whereas protein coding regions were largely (~84%) methylated. Although CpG-rich and -poor promoters showed distinct methylation patterns, interestingly, a negative correlation between promoter methylation levels and gene transcription levels was consistently observed across promoters with high to low CpG densities. Importantly, we observed substantial interindividual variation in DNA methylation across the individual PBM methylomes and the pattern of this interindividual variation varied between different genomic features, with highly variable regions enriched for repetitive DNA elements. Furthermore, we observed a modest but significant excess (p < 2.2 × 10−16) of genes showing a negative correlation between interindividual promoter methylation and transcription levels. These significant genes were enriched in biological processes that are closely related to PBM functions, suggesting that alteration in DNA methylation is likely to be an important mechanism contributing to the interindividual variation in PBM function, and PBM-related phenotypic and disease-susceptibility variation in humans.
This study represents a comprehensive analysis of the human PBM methylome and its interindividual variation. Our data provide a valuable resource for future epigenomic and multiomic studies, exploring biological and disease-related regulatory mechanisms in PBMs.
PMCID: PMC3874233  PMID: 23750642
DNA methylation; interindividual variation; peripheral blood monocyte
5.  Genome-Wide Association Study Identified Copy Number Variants Important for Appendicular Lean Mass 
PLoS ONE  2014;9(3):e89776.
Skeletal muscle is a major component of the human body. Age-related loss of muscle mass and function contributes to some public health problems such as sarcopenia and osteoporosis. Skeletal muscle, mainly composed of appendicular lean mass (ALM), is a heritable trait. Copy number variation (CNV) is a common type of human genome variant which may play an important role in the etiology of many human diseases. In this study, we performed genome-wide association analyses of CNV for ALM in 2,286 Caucasian subjects. We then replicated the major findings in 1,627 Chinese subjects. Two CNVs, CNV1191 and CNV2580, were detected to be associated with ALM (p = 2.26×10−2 and 3.34×10−3, respectively). In the Chinese replication sample, the two CNVs achieved p-values of 3.26×10−2 and 0.107, respectively. CNV1191 covers a gene, GTPase of the immunity-associated protein family (GIMAP1), which is important for skeletal muscle cell survival/death in humans. CNV2580 is located in the Serine hydrolase-like protein (SERHL) gene, which plays an important role in normal peroxisome function and skeletal muscle growth in response to mechanical stimuli. In summary, our study suggested two novel CNVs and the related genes that may contribute to variation in ALM.
PMCID: PMC3953533  PMID: 24626161
6.  Genome-Wide Association Study Identified UQCC Locus for Spine Bone Size in Humans 
Bone  2012;53(1):129-133.
Bone size (BS) contributes significantly to the risk of osteoporotic fracture. Osteoporotic spine fracture is one of the most disabling outcomes of osteoporosis. This study aims to identify genomic loci underlying spine BS variation in humans.
We performed a genome-wide association scan in 2,286 unrelated Caucasians using Affymetrix 6.0 SNP arrays. Areal BS (cm2) at lumbar spine was measured using dual energy X-ray absorptiometry scanners. SNPs of interest were subjected to replication analyses and meta-analyses with additional two independent Caucasian populations (N = 1,000 and 2,503) and one Chinese population (N = 1,627).
In the initial GWAS, 91 SNPs were associated with spine BS (P<1.0E-4). Eight contiguous SNPs were found clustering in a haplotype block within UQCC gene (ubiquinol-cytochrome creductase complex chaperone). Association of the above eight SNPs with spine BS were replicated in one Caucasian and one Chinese populations. Meta-analyses (N = 7,416) generated much stronger association signals for these SNPs (e.g., P = 1.86E-07 for SNP rs6060373), supporting association of UQCC with spine BS across ethnicities.
This study identified a novel locus, i.e., the UQCC gene, for spine BS variation in humans. Future functional studies will contribute to elucidating the mechanisms by which UQCC regulates bone growth and development.
PMCID: PMC3682469  PMID: 23207799
Spine bone size; GWAS; UQCC
7.  Suggestion of GLYAT gene underlying variation of bone size and body lean mass as revealed by a bivariate genome-wide association study 
Human genetics  2012;132(2):189-199.
Bone and muscle, two major tissue types of musculoskeletal system, have strong genetic determination. Abnormality in bone and/or muscle may cause musculoskeletal diseases such as osteoporosis and sarcopenia. Bone size phenotypes (BSPs), such as hip bone size (HBS), appendicular bone size (ABS), are genetically correlated with body lean mass (mainly muscle mass). However, the specific genes shared by these phenotypes are largely unknown. In this study, we aimed to identify the specific genes with pleiotropic effects on BSPs and appendicular lean mass (ALM).
We performed a bivariate genome-wide association study (GWAS) by analyzing ~690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) followed by a replication study in 2,286 unrelated US Caucasians (558 males and 1728 females).
We identified 14 interesting single nucleotide polymorphisms (SNPs) that may contribute to variation of both BSPs and ALM, with p values <10−6 in discovery stage. Among them, the association of three SNPs (rs2507838, rs7116722, and rs11826261) in/near GLYAT (glycine-N-acyltransferase) gene was replicated in US Caucasians, with p values ranging from 1.89×10−3 to 3.71×10−4 for ALM-ABS, from 5.14×10−3 to 1.11×10−2 for ALM-HBS, respectively. Meta-analyses yielded stronger association signals for rs2507838, rs7116722, and rs11826261, with pooled p values of 1.68×10−8, 7.94×10−8, 6.80×10−8 for ALB-ABS and 1.22×10−4, 9.85×10−5, 3.96×10−4 for ALM-HBS, respectively. Haplotype allele ATA based on these three SNPs were also associated with ALM-HBS and ALM-ABS in both discovery and replication samples. Interestingly, GLYAT was previously found to be essential to glucose metabolism and energy metabolism, suggesting the gene’s dual role in both bone development and muscle growth.
Our findings, together with the prior biological evidence, suggest the importance of GLYAT gene in co-regulation of bone phenotypes and body lean mass.
PMCID: PMC3682481  PMID: 23108985
Bivariate GWAS; Bone size; Lean mass; GLYAT
8.  Total parenteral nutrition caused Wernicke’s encephalopathy accompanied by wet beriberi 
Patient: Female, 17
Final Diagnosis: Wernicke’s encephalopathy
Symptoms: Blurred vision • dizziness • nystygmus • tachycardia
Medication: —
Clinical Procedure: —
Specialty: Neurology
Mistake in diagnosis
Wernicke’s encephalopathy (WE) is an acute and life-threatening illness which is not only seen in alcoholics, but also in persons with poor nutrition lacking thiamine (vitamin B1).
Case Report:
Here, we presented a case of WE in a patient who received parenteral nutrition without complement of thiamine. Besides neuropsychiatric problems, she also manifested prominent cardiovascular abnormalities, which were consistent with wet beriberi.
This case emphasizes the need for thiamine supplementation in prolonged total parenteral nutrition, and also highlights the awareness of WE in persons with parenteral nutrition lacking thiamine. More importantly, we call for attention to wet beriberi in such persons.
PMCID: PMC3917879  PMID: 24516692
Parenteral Nutrition; Total – adverse effects; Thiamine Deficiency; Beriberi; Wernicke Encephalopathy
9.  eRF3b, a Biomarker for Hepatocellular Carcinoma, Influences Cell Cycle and Phosphoralation Status of 4E-BP1 
PLoS ONE  2014;9(1):e86371.
Hepatitis B virus (HBV) infection and its sequelae are now recognized as serious problems globally. Our aime is to screen hepatocellular carcinoma (HCC) from chronic hepatitis B (CHB) and identify the characteristics of proteins involved.
Methodology/Principal Findings
We affinity-purified sample serum with weak cation-exchange (WCX) magnetic beads and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis to search for potential markers. The 4210 Da protein, which differed substantially between HCC and CHB isolates, was later identified to be eukaryotic peptide chain release factor GTP-binding subunit eRF3b. Further research showed that eRF3b/GSPT2 was positively expressed in liver tissues. GSPT2 mRNA was, however differentially expressed in blood. Compared with normal controls, the relative expression of GSPT2/18s rRNA was higher in CHB patients than in patients with either LC or HCC (P = 0.035 for CHB vs. LC; P = 0.020 for CHB vs. HCC). The data of further research showed that eRF3b/GSPT2 promoted the entrance of the HepG2 cells into the S-phase and that one of the substrates of the mTOR kinase, 4E-BP1, was hyperphosphorylated in eRF3b-overexpressing HepG2 cells.
Overall, the differentially expressed protein eRF3b, which was discovered as a biomarker for HCC, could change the cell cycle and influence the phosphorylation status of 4E-BP1 on Ser65 in HepG2.
PMCID: PMC3900531  PMID: 24466059
10.  EPAC Inhibition of SUR1 Receptor Increases Glutamate Release and Seizure Vulnerability 
The Journal of Neuroscience  2013;33(20):8861-8865.
EPAC (Exchange Proteins Activated by cAMP) regulates glutamate transmitter release in the central neurons, but a role underlying this regulation has yet to be identified. Here we show that EPAC binds directly to the intracellular loop of an ATP-sensitive potassium (KATP) channel type-1 sulfonylurea receptor (SUR1) receptor consisting of amino acids 859–881 (SUR1859–881). Ablation of EPAC or expression of SUR1859–881, which intercepts EPAC-SUR1 binding, increases the open probability of KATP channels consisting of the Kir6.1 subunit and SUR1. Opening of KATP channels inhibits glutamate release and reduces seizure vulnerability in adult mice. Therefore, EPAC interaction with SUR1 controls seizure susceptibility and possibly acts via regulation of glutamate release.
PMCID: PMC3865516  PMID: 23678128
11.  The let-7 microRNA Enhances Heme Oxygenase-1 by Suppressing Bach1 and Attenuates Oxidant Injury in Human Hepatocytes 
Biochimica et biophysica acta  2012;1819(11-12):1113-1122.
The let-7 microRNA (miRNA) plays important roles in human liver development and disease such as hepatocellular carcinoma, liver fibrosis and hepatitis wherein oxidative stress accelerates the progression of these diseases. To date, the role of the let-7 miRNA family in modulation of heme oxygenase 1 (HMOX1), a key cytoprotective enzyme, remains unknown. Our aims were to determine whether let-7 miRNA directly regulates Bach1, a transcriptional repressor of the HMOX1 gene, and whether indirect up-regulation of HMOX1 by let-7 miRNA attenuates oxidant injury in human hepatocytes. The effects of let-7 miRNA on Bach1 and HMOX1 gene expression in Huh-7 and HepG2 cells were determined by real-time qRT-PCR, Western blot, and luciferase reporter assays. Dual luciferase reporter assays revealed that let-7b, let-7c, or miR-98 significantly decreased Bach1 3’-untranslated region (3’-UTR)-dependent luciferase activity but not mutant Bach1 3’-UTR-dependent luciferase activity, whereas mutant let-7 miRNA containing base complementarity with mutant Bach1 3’-UTR restored its effect on mutant reporter activity. let-7b, let-7c, or miR-98 down-regulated Bach1 protein levels by 50–70%, and subsequently up-regulated HMOX1 gene expression by 3–4 fold, compared with non-specific controls. Furthermore, Huh-7 cells transfected with let-7b, let-7c or miR-98 mimic showed increased resistance against oxidant injury induced by tert-butyl-hydroperoxide (tBuOOH), whereas the protection was abrogated by over-expression of Bach1. In conclusion, let-7 miRNA directly acts on the 3’-UTR of Bach1 and negatively regulates expression of this protein, and thereby up-regulates HMOX1 gene expression. Over-expression of the let-7 miRNA family members may represent a novel approach to protecting human hepatocytes from oxidant injury.
PMCID: PMC3480558  PMID: 22698995
let-7; HMOX1; Bach1; microRNA; oxidative stress; Huh-7 cell
12.  Expression of Tau40 Induces Activation of Cultured Rat Microglial Cells 
PLoS ONE  2013;8(10):e76057.
Accumulation of microtubule-associated protein tau has been observed in the brain of aging and tauopathies. Tau was observed in microglia, but its role is not illustrated. By immunofluorescence staining and the fractal dimension value assay in the present study, we observed that microglia were activated in the brains of rats and mice during aging, simultaneously, the immunoreactivities of total tau and the phosphorylated tau were significantly enhanced in the activated microglia. Furtherly by transient transfection of tau40 (human 2N/4R tau) into the cultured rat microglia, we demonstrated that expression of tau40 increased the level of Iba1, indicating activation of microglia. Moreover, expression of tau40 significantly enhanced the membranous localization of the phosphorylated tau at Ser396 in microglia possibly by a mechanism involving protein phosphatase 2A, extracellular signal-regulated kinase and glycogen synthase kinase-3β. It was also found that expression of tau40 promoted microglial migration and phagocytosis, but not proliferation. And we observed increased secretion of several cytokines, including interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor-α and nitric oxide after the expression of tau40. These data suggest a novel role of human 2N/4R tau in microglial activation.
PMCID: PMC3795725  PMID: 24146816
13.  Melatonin in Alzheimer’s Disease 
Alzheimer’s disease (AD), an age-related neurodegenerative disorder with progressive cognition deficit, is characterized by extracellular senile plaques (SP) of aggregated β-amyloid (Aβ) and intracellular neurofibrillary tangles, mainly containing the hyperphosphorylated microtubule-associated protein tau. Multiple factors contribute to the etiology of AD in terms of initiation and progression. Melatonin is an endogenously produced hormone in the brain and decreases during aging and in patients with AD. Data from clinical trials indicate that melatonin supplementation improves sleep, ameliorates sundowning and slows down the progression of cognitive impairment in AD patients. Melatonin efficiently protects neuronal cells from Aβ-mediated toxicity via antioxidant and anti-amyloid properties. It not only inhibits Aβ generation, but also arrests the formation of amyloid fibrils by a structure-dependent interaction with Aβ. Our studies have demonstrated that melatonin efficiently attenuates Alzheimer-like tau hyperphosphorylation. Although the exact mechanism is still not fully understood, a direct regulatory influence of melatonin on the activities of protein kinases and protein phosphatases is proposed. Additionally, melatonin also plays a role in protecting the cholinergic system and in anti-inflammation. The aim of this review is to stimulate interest in melatonin as a potentially useful agent in the prevention and treatment of AD.
PMCID: PMC3742260  PMID: 23857055
Alzheimer’s disease; melatonin; tau hyperphosphorylation; beta amyloid; antioxidation; cholinergic; neuroinflammation
14.  Comprehensive Characterization of Human Genome Variation by High Coverage Whole-Genome Sequencing of Forty Four Caucasians 
PLoS ONE  2013;8(4):e59494.
Whole genome sequencing studies are essential to obtain a comprehensive understanding of the vast pattern of human genomic variations. Here we report the results of a high-coverage whole genome sequencing study for 44 unrelated healthy Caucasian adults, each sequenced to over 50-fold coverage (averaging 65.8×). We identified approximately 11 million single nucleotide polymorphisms (SNPs), 2.8 million short insertions and deletions, and over 500,000 block substitutions. We showed that, although previous studies, including the 1000 Genomes Project Phase 1 study, have catalogued the vast majority of common SNPs, many of the low-frequency and rare variants remain undiscovered. For instance, approximately 1.4 million SNPs and 1.3 million short indels that we found were novel to both the dbSNP and the 1000 Genomes Project Phase 1 data sets, and the majority of which (∼96%) have a minor allele frequency less than 5%. On average, each individual genome carried ∼3.3 million SNPs and ∼492,000 indels/block substitutions, including approximately 179 variants that were predicted to cause loss of function of the gene products. Moreover, each individual genome carried an average of 44 such loss-of-function variants in a homozygous state, which would completely “knock out” the corresponding genes. Across all the 44 genomes, a total of 182 genes were “knocked-out” in at least one individual genome, among which 46 genes were “knocked out” in over 30% of our samples, suggesting that a number of genes are commonly “knocked-out” in general populations. Gene ontology analysis suggested that these commonly “knocked-out” genes are enriched in biological process related to antigen processing and immune response. Our results contribute towards a comprehensive characterization of human genomic variation, especially for less-common and rare variants, and provide an invaluable resource for future genetic studies of human variation and diseases.
PMCID: PMC3618277  PMID: 23577066
15.  Bivariate Genome-Wide Association Analyses Identified Genes with Pleiotropic Effects for Femoral Neck Bone Geometry and Age at Menarche 
PLoS ONE  2013;8(4):e60362.
Femoral neck geometric parameters (FNGPs), which include cortical thickness (CT), periosteal diameter (W), buckling ratio (BR), cross-sectional area (CSA), and section modulus (Z), contribute to bone strength and may predict hip fracture risk. Age at menarche (AAM) is an important risk factor for osteoporosis and bone fractures in women. Some FNGPs are genetically correlated with AAM. In this study, we performed a bivariate genome-wide association study (GWAS) to identify new candidate genes responsible for both FNGPs and AAM. In the discovery stage, we tested 760,794 SNPs in 1,728 unrelated Caucasian subject, followed by replication analyses in independent samples of US Caucasians (with 501 subjects) and Chinese (with 826 subjects). We found six SNPs that were associated with FNGPs and AAM. These SNPs are located in three genes (i.e. NRCAM, IDS and LOC148145), suggesting these three genes may co-regulate FNGPs and AAM. Our findings may help improve the understanding of genetic architecture and pathophysiological mechanisms underlying both osteoporosis and AAM.
PMCID: PMC3617200  PMID: 23593202
16.  Heme status affects human hepatic messenger RNA and microRNA expression 
AIM: To assess effects of heme on messenger RNA (mRNA) and microRNA (miRNA) profiles of liver cells derived from humans.
METHODS: We exposed human hepatoma cell line Huh-7 cells to excess iron protoporphyrin (heme) (10 μmol/L) or induced heme deficiency by addition of 4, 6-dioxoheptanoic acid (500 μmol/L), a potent inhibitor of aminolevulinic acid dehydratase, for 6 h or 24 h. We harvested total RNA from the cells and performed both mRNA and miRNA array analyses, with use of Affymetrix chips, reagents, and instruments (human genome U133 plus 2.0 and miRNA 2.0 arrays). We assessed changes and their significance and interrelationships with Target Scan, Pathway Studios, and Ingenuity software.
RESULTS: Changes in mRNA levels were most numerous and striking at 6 h after heme treatment but were similar and still numerous at 24 h. After 6 h of heme exposure, the increase in heme oxygenase 1 gene expression was 60-fold by mRNA and 88-fold by quantitative reverse transcription-polymerase chain reaction. We found striking changes, especially up-regulation by heme of nuclear erythroid-2 related factor-mediated oxidative stress responses, protein ubiquitination, glucocorticoid signaling, P53 signaling, and changes in RNAs that regulate intermediary metabolism. Fewer mRNAs were down-regulated by heme, and the fold decreases were less exuberant than were the increases. Notable decreases after 24 h of heme exposure were patatin-like phospholipase domain-containing protein 3 (-6.5-fold), neuronal PAS domain protein 2 (-1.93-fold), and protoporphyrinogen oxidase (-1.7-fold).
CONCLUSION: Heme excess exhibits several toxic effects on liver and kidney, which deserve study in humans and in animal models of the human porphyrias or other disorders.
PMCID: PMC3602476  PMID: 23538684
Delta-aminolevulinic acid synthase; Heme; Heat shock proteins; Hepatotoxicity; Messenger RNA; MicroRNA
17.  EPAC Null Mutation Impairs Learning and Social Interactions via Aberrant Regulation of miR-124 and Zif268 Translation 
Neuron  2012;73(4):774-788.
EPAC proteins are the guanine nucleotide exchange factors that act as the intracellular receptors for cyclic AMP. Two variants of EPAC genes including EPAC1 and EPAC2 are cloned and are widely expressed throughout the brain. But, their functions in the brain remain unknown. Here, we genetically delete EPAC1 (EPAC1-/-), or EPAC2 (EPAC2-/-) or both EPAC1 and EPAC2 genes (EPAC-/-) in the forebrain of mice. We show that EPAC null mutation impairs long-term potentiation (LTP) and that this impairment is paralleled with the severe deficits in spatial learning and social interactions and is mediated in a direct manner by miR-124 transcription and Zif268 translation. Knockdown of miR-124 restores Zif268 and hence reverses all aspects of the EPAC-/- phenotypes, whereas expression of miR-124 or knockdown of Zif268 reproduces the effects of EPAC null mutation. Thus, EPAC proteins control miR-124 transcription in the brain for processing spatial learning and social interactions.
PMCID: PMC3307595  PMID: 22365550
18.  Astragalus polysaccharides can regulate cytokine and P-glycoprotein expression in H22 tumor-bearing mice 
AIM: To investigate the adjunct anticancer effect of Astragalus polysaccharides in H22 tumor-bearing mice.
METHODS: To establish a solid tumor model, 5.0 × 106/mL H22 hepatoma cells were inoculated subcutaneously into the right armpit region of Kunming mice (6-12 wk old, 18-22 g). When the tumors reached a size of 100 mm3, the animals were treated as indicated, and the mice were randomly assigned to seven groups (n = 10 each). After ten days of treatment, blood samples were collected from mouse eyes, and serum was harvested by centrifugation. Mice were sacrificed, and the whole body, tumor, spleen and thymus were weighed immediately. The rate of tumor inhibition and organ indexes were calculated. The expression levels of serum cytokines, P-glycoprotein (P-GP) and multidrug resistance (MDR) 1 mRNA in tumor tissues were detected using enzyme-linked immunosorbent assay, Western blotting, and quantitative myeloid-derived suppressor cells reverse transcription-polymerase chain reaction, respectively.
RESULTS: The tumor inhibition rates in the treatment groups of Adriamycin (ADM) + Astragalus polysaccharides (APS) (50 mg/kg), ADM + APS (100 mg/kg), and ADM + APS (200 mg/kg) were significantly higher than in the ADM group (72.88% vs 60.36%, P = 0.013; 73.40% vs 60.36%, P = 0.010; 77.57% vs 60.36%, P = 0.001). The spleen indexes of the above groups were also significantly higher than in the ADM group (0.65 ± 0.22 vs 0.39 ± 0.17, P = 0.023; 0.62 ± 0.34 vs 0.39 ± 0.17, P = 0.022; 0.67 ± 0.20 vs 0.39 ± 0.17, P = 0.012), and the thymus indexes of the ADM + APS (100 mg/kg) and ADM + APS (200 mg/kg) groups were significantly higher than in the ADM group (0.20 ± 0.06 vs 0.13 ± 0.04, P = 0.029; 0.47 ± 0.12 vs 0.13 ± 0.04, P = 0.000). APS was found to exert a synergistic anti-tumor effect with ADM and to alleviate the decrease in the sizes of the spleen and thymus induced by AMD. The expression of interleukin-1α (IL-1α), IL-2, IL-6, and tumor necrosis factor-α (TNF-α) was significantly higher in the ADM + APS (50 mg/kg), ADM + APS (100 mg/kg) and ADM + APS (200 mg/kg) groups than in the ADM group; and IL-10 was significantly lower in the above groups than in the ADM group. APS could increase IL-1α, IL-2, IL-6, and TNF-α expression and decrease IL-10 levels. Compared with the ADM group, APS treatment at a dose of 50-200 mg/kg could down-regulate MDR1 mRNA expression in a dose-dependent manner (0.48 ± 0.13 vs 4.26 ± 1.51, P = 0.000; 0.36 ± 0.03 vs 4.26 ± 1.51, P = 0.000; 0.21 ± 0.04 vs 4.26 ± 1.51, P = 0.000). The expression level of P-GP was significantly lower in the ADM + APS (200 mg/kg) group than in the ADM group (137.35 ± 9.20 mg/kg vs 282.19 ± 20.54 mg/kg, P = 0.023).
CONCLUSION: APS exerts a synergistic anti-tumor effect with ADM in H22 tumor-bearing mice. This may be related to its ability to enhance the expression of IL-1α, IL-2, IL-6, and TNF-α, decrease IL-10, and down-regulate MDR1 mRNA and P-GP expression levels.
PMCID: PMC3531697  PMID: 23323011
Astragalus polysaccharides; Tumor inhibition rate; Cytokines; P-glycoprotein; Adjunct anticancer
19.  Genome-Wide Association Study Identified CNP12587 Region Underlying Height Variation in Chinese Females 
PLoS ONE  2012;7(9):e44292.
Human height is a highly heritable trait considered as an important factor for health. There has been limited success in identifying the genetic factors underlying height variation. We aim to identify sequence variants associated with adult height by a genome-wide association study of copy number variants (CNVs) in Chinese.
Genome-wide CNV association analyses were conducted in 1,625 unrelated Chinese adults and sex specific subgroup for height variation, respectively. Height was measured with a stadiometer. Affymetrix SNP6.0 genotyping platform was used to identify copy number polymorphisms (CNPs). We constructed a genomic map containing 1,009 CNPs in Chinese individuals and performed a genome-wide association study of CNPs with height.
We detected 10 significant association signals for height (p<0.05) in the whole population, 9 and 11 association signals for Chinese female and male population, respectively. A copy number polymorphism (CNP12587, chr18:54081842-54086942, p = 2.41×10−4) was found to be significantly associated with height variation in Chinese females even after strict Bonferroni correction (p = 0.048). Confirmatory real time PCR experiments lent further support for CNV validation. Compared to female subjects with two copies of the CNP, carriers of three copies had an average of 8.1% decrease in height. An important candidate gene, ubiquitin-protein ligase NEDD4-like (NEDD4L), was detected at this region, which plays important roles in bone metabolism by binding to bone formation regulators.
Our findings suggest the important genetic variants underlying height variation in Chinese.
PMCID: PMC3434125  PMID: 22957059
20.  Mitochondria-wide Association Study of Common Variants in Osteoporosis 
Annals of human genetics  2011;75(5):569-574.
Many lines of evidence suggest that mitochondrial DNA (mtDNA) variants are involved in the pathogenesis of human complex diseases, especially for age-related disorders. Osteoporosis is a typical age-related complex disease. However, the role of mtDNA variants in the susceptibility of osteoporosis is largely unknown. In this study, we performed a mitochondria-wide association study for osteoporosis in Caucasians. A total of 445 mitochondrial single nucleotide polymorphisms (mtSNPs) were genotyped in a large sample of 2,286 unrelated Caucasian subjects by using the Affymetrix Genome-Wide SNP Array 6.0, and 72 mtSNPs survived the quality control. We first tested for association between single-mtSNP and bone mineral density (BMD), and identified that, a mtSNP within the NADH dehydrogenase 2 gene (ND2), mt4823 C/A polymorphism, was strongly associated with hip BMD (P = 2.05 × 10−4), even after conservative Bonferroni correction‥ The C allele of mt4823 was associated with reduced hip BMD and the effect size (β) was estimated to be ~0.044. Another SNP mt15885 within the Cytochrome b gene (Cytb) was found to be associated both with spine (P = 1.66×10−3) and hip BMD (P = 0.023). The T allele of mt15885 had a protective effect on spine (β = 0.064) and hip BMD (β = 0.038). Next, we classified subjects into the nine common European haplogroups and conducted association analyses. Subjects classified as haplogroup X had significantly lower mean hip BMD values than others (P = 0.040). Our results highlighted the importance of mtDNA variants in influencing BMD variation and risk to osteoporosis.
PMCID: PMC3155639  PMID: 21762117
mtSNP; haplogroup; osteoporosis; BMD; association
21.  Effects of Astragalus polysaccharides on P-glycoprotein efflux pump function and protein expression in H22 hepatoma cells in vitro 
Astragalus polysaccharides (APS) are active constituents of Astragalus membranaceus. They have been widely studied, especially with respect to their immunopotentiating properties, their ability to counteract the side effects of chemotherapeutic drugs, and their anticancer properties. However, the mechanism by which APS inhibit cancer and the issue of whether that mechanism involves the reversal of multidrug resistance (MDR) is not completely clear. The present paper describes an investigation of the effects of APS on P-glycoprotein function and expression in H22 hepatoma cell lines resistant to Adriamycin (H22/ADM).
H22/ADM cell lines were treated with different concentrations of APS and/or the most common chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine. Chemotherapeutic drug sensitivity, P-glycoprotein function and expression, and MDR1 mRNA expression were detected using MTT assay, flow cytometry, Western blotting, and quantitative RT-PCR.
When used alone, APS had no anti-tumor activity in H22/ADM cells in vitro. However, it can increase the cytotoxicity of certain chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine, in H22/ADM cells. It acts in a dose-dependent manner. Compared to a blank control group, APS increased intracellular Rhodamine-123 retention and decreased P-glycoprotein efflux function in a dose-dependent manner. These factors were assessed 24 h, 48 h, and 72 h after administration. APS down regulated P-glycoprotein and MDR1 mRNA expression in a concentration-dependent manner within a final range of 0.8–500 mg/L and in a time-dependent manner from 24–72 h.
APS can enhance the chemosensitivity of H22/ADM cells. This may involve the downregulation of MDR1 mRNA expression, inhibition of P-GP efflux pump function, or both, which would decrease the expression of the MDR1 protein.
PMCID: PMC3493361  PMID: 22784390
Astragalus polysaccharides; Multidrug resistance; P-glycoprotein
23.  Genome-Wide Association Study of Copy Number Variants Suggests LTBP1 and FGD4 Are Important for Alcohol Drinking 
PLoS ONE  2012;7(1):e30860.
Alcohol dependence (AD) is a complex disorder characterized by psychiatric and physiological dependence on alcohol. AD is reflected by regular alcohol drinking, which is highly inheritable. In this study, to identify susceptibility genes associated with alcohol drinking, we performed a genome-wide association study of copy number variants (CNVs) in 2,286 Caucasian subjects with Affymetrix SNP6.0 genotyping array. We replicated our findings in 1,627 Chinese subjects with the same genotyping array. We identified two CNVs, CNV207 (combined p-value 1.91E-03) and CNV1836 (combined p-value 3.05E-03) that were associated with alcohol drinking. CNV207 and CNV1836 are located at the downstream of genes LTBP1 (870 kb) and FGD4 (400 kb), respectively. LTBP1, by interacting TGFB1, may down-regulate enzymes directly participating in alcohol metabolism. FGD4 plays a role in clustering and trafficking GABAA receptor and subsequently influence alcohol drinking through activating CDC42. Our results provide suggestive evidence that the newly identified CNV regions and relevant genes may contribute to the genetic mechanism of alcohol dependence.
PMCID: PMC3266269  PMID: 22295116
24.  Decrease in retinal neuronal cells in streptozotocin-induced diabetic mice 
Molecular Vision  2012;18:1411-1420.
Little is known about retinal neuronal loss in the retinas of diabetic mice. The purpose of this study was the quantitative assessment of retinal neural cell number in diabetic mice.
Five-week-old C57BL/6 mice were used as a diabetic model with streptozotocin. Mice were studied over the course of 6 and 12 weeks after the onset of diabetes. Intraocular pressure (IOP) was measured with a noninvasive TonoLab tonometer. The retinal ganglion cells (RGCs) were counted at two different time points after the induction of diabetes and examined using the immunofluorescence technique and quantitative analysis.
The diabetic mice had significantly elevated IOP levels at 6 and 12 weeks after the onset of diabetes compared with the age-matched control mice (p<0.01 and p<0.001, respectively). The temporal course of Brn3a+ RGC and Neuronal Nuclei+RGC (NeuN+ RGC) loss induced by intraperitoneal injection of streptozotocin followed a similar trend. At 6 and 12 weeks after the onset of diabetes, the number of Brn3a+ RGCs (p<0.05 at 6 weeks; p<0.001 at 12 weeks) and NeuN+ RGCs (p<0.05 at 6 weeks; p<0.001 at 12 weeks) was significantly lower in diabetic mice than age-matched control mice. In the retinal flatmounts, the number of Brn3a+ RGCs (p<0.05 at 6 weeks, p<0.01 at 12 weeks) was also significantly lower in diabetic mice than control mice. The IOP in diabetic mice was negatively related with RGCs in cross sections. The cut-off value of IOP was 14.2 mmHg for diabetes.
This is a specific quantitative study of neural cell loss in the retina during diabetes. These data suggest that retinal neural cell reduction occurs in diabetic mice. It indicates that RGC loss may be an important component of diabetic retinopathy.
PMCID: PMC3369894  PMID: 22690119
25.  The Fat Mass and Obesity Associated Gene, FTO, Is Also Associated with Osteoporosis Phenotypes 
PLoS ONE  2011;6(11):e27312.
Obesity and osteoporosis are closely correlated genetically. FTO gene has been consistently identified to be associated with obesity phenotypes. A recent study reported that the mice lacking Fto could result in lower bone mineral density (BMD). Thus, we hypothesize that the FTO gene might be also important for osteoporosis phenotypes. To test for such a hypothesis, we performed an association analyses to investigate the relationship between SNPs in FTO and BMD at both hip and spine. A total of 141 SNPs were tested in two independent Chinese populations (818 and 809 unrelated Han subjects, respectively) and a Caucasian population (2,286 unrelated subjects). Combining the two Chinese samples, we identified 6 SNPs in FTO to be significantly associated with hip BMD after multiple testing adjustments, with the combined P values ranged from 4.99×10−4–1.47×10−4. These 6 SNPs are all located at the intron 8 of FTO and in high linkage disequilibrium. Each copy of the minor allele of each SNP was associated with increased hip BMD values with the effect size (beta) of ∼0.025 and ∼0.015 in the Chinese sample 1 and 2, respectively. However, none of these 6 SNPs showed significant association signal in the Caucasian sample, by presenting some extent of ethnic difference. Our findings, together with the prior biological evidence, suggest that the FTO gene might be a new candidate for BMD variation and osteoporosis in Chinese populations.
PMCID: PMC3220685  PMID: 22125610

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