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1.  Genetics of systemic lupus erythematosus: immune responses and end organ resistance to damage 
Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disorder. Considerable progress has been made to delineate the genetic control of this complex disorder. In this review, selected aspects of human and mouse genetics related to SLE are reviewed with emphasis on genes that contribute to both innate and adaptive immunity and to genes that contribute directly to susceptibility to end organ damage. It is concluded that the interactions among these two major pathways will provide further insight into the pathogenesis of SLE. An interactive model of the two major pathways is proposed without emphasis on the importance of breaking tolerance to autoantigens.
PMCID: PMC4274270  PMID: 25458999
3.  Isolation and complete nucleotide sequence of a Batai virus strain in Inner Mongolia, China 
Virology Journal  2014;11:138.
Batai virus (BATV) is a member of the Orthobunyavirus genus of the family Bunyaviridae, and a vector-borne pathogen. Genomic variations of BATV exist in different regions of the world, due to genetic reassortment. Whole-genome sequencing of any isolate is necessary for a phylogenetic analysis. In 1998, a BATV strain was isolated from an Anopheles philippines mosquito in Yunnan Province, China. This strain has not been found to infect any other host. We investigated BATV infection in cattle in Inner Mongolia, China and performed deep sequencing of the genome of the BATV isolate.
Ninety-five blood samples were collected from cattle in Inner Mongolia, China in 2012. The BATV infection rate was 2.1%. Previously, BATV strain NM/12 was isolated from two cattle in Inner Mongolia, China, and the whole genomic sequence of the strain has been available. We determined the complete genomic nucleotide sequences of the small (S), medium (M), and large (L) genome segments using bovine blood obtained in 2012, and the nucleotide homologies of these segments with those from GenBank were 88.7%-97%, 84%-95.4%, and 72.6%-95.8%, respectively. The deduced amino acid identities were 87.2-99.7%, 64.2-96.8%, and 81.1-98.6%. Phylogenetic analyses based on full-length genomic sequences indicated that the M and L segments, and a portion of the S segment, of NM/12 are most closely related to the BATV strains isolated in Asia. The S and M segments of NM/12 were independent of phylogenetic lineages. The L segment was the most closely related to Chittoor/IG-20217 (isolated in India), and distantly related to isolated strains in Italy. Nucleotide substitution rates in the nucleotide sequences that code for the nucleocapsid, envelope glycoprotein, and polymerase protein of NM/12 strain were 2.56%, 4.69%, and 4.21%, respectively, relative to the original strain of MM2222.
A novel BATV NM/12 strain from bovine serum collected in Inner Mongolia was isolated from cattle in China for the first time. Our findings elucidate the evolutionary status of the BATV NM/12 strain among different orthobunyavirus strains and may provide some clues to prevent the emergence of BATV infection in cattle and humans.
PMCID: PMC4127039  PMID: 25100223
Batai virus; Orthobunyavirus; Bunyaviridae; Reassortment
4.  Cardiac Troponin T (TNNT2) Mutations in Chinese Dilated Cardiomyopathy Patients 
BioMed Research International  2014;2014:907360.
Background. Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. Although more than 40 genes have been reported to cause DCM, the role of genetic testing in clinical practice is not well defined. Mutations in the troponin T (TNNT2) gene represent an important subset of known disease-causing mutations associated with DCM. Therefore, the aim of the present study was to determine the genetic variations in TNNT2 and the associations of those variations with DCM in Chinese patients. Methods. An approximately 4 kb fragment of the TNNT2 gene was isolated from 103 DCM patients and 192 healthy controls and was analyzed by DNA sequence analysis for genetic variations. Results. A total of 6 TNNT2 mutations were identified in 99 patients, including a G321T missense mutation (Leu84Phe) and 5 novel intronic mutations. Alleles of two novel SNPs (c.192 + 353 C>A, OR = 0.095, 95% CI: 0.013–0.714, P = 0.022; c.192 + 463 G>A, OR = 0.090, 95% CI: 0.012–0.675, P = 0.019) and SNP rs3729843 (OR = 1.889, 95% CI: 1.252–2.852; P = 0.002) were significantly correlated with DCM. Conclusions. These results suggest that the missense mutation (Leu84Phe) and two novel SNPs (c.192 + 353 C>A, c.192 + 463 G>A) in TNNT2 gene might be associated with DCM in the Chinese population.
PMCID: PMC4109665  PMID: 25110706
5.  A new approach for noninvasive transdermal determination of blood uric acid levels 
The aims of this study were to investigate the most effective combination of physical forces from laser, electroporation, and reverse iontophoresis for noninvasive transdermal extraction of uric acid, and to develop a highly sensitive uric acid biosensor (UAB) for quantifying the uric acid extracted. It is believed that the combination of these physical forces has additional benefits for extraction of molecules other than uric acid from human skin. A diffusion cell with porcine skin was used to investigate the most effective combination of these physical forces. UABs coated with ZnO2 nanoparticles and constructed in an array configuration were developed in this study. The results showed that a combination of laser (0.7 W), electroporation (100 V/cm2), and reverse iontophoresis (0.5 mA/cm2) was the most effective and significantly enhanced transdermal extraction of uric acid. A custom-designed UAB coated with ZnO2 nanoparticles and constructed in a 1×3 array configuration (UAB-1×3-ZnO2) demonstrated enough sensitivity (9.4 μA/mM) for quantifying uric acid extracted by the combined physical forces of laser, electroporation, and RI. A good linear relationship (R2=0.894) was demonstrated to exist between the concentration of uric acid (0.2–0.8 mM) inside the diffusion cell and the current response of the UAB-1×3-ZnO2. In conclusion, a new approach to noninvasive transdermal extraction and quantification of uric acid has been established.
PMCID: PMC4085317  PMID: 25061289
laser; electroporation; reverse iontophoresis; noninvasive; uric acid; biosensor
6.  Trapping Cardiac Recessive Mutants via Expression-based Insertional Mutagenesis Screening 
Circulation research  2013;112(4):606-617.
Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ such as the heart, especially those exhibiting adult phenotypes upon depletion.
We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes.
Methods and Results
The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein (mRFP) reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of one embryonic lethal, one larval lethal, and one adult recessive mutant exhibiting cardiac hypertrophy at one year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, alpha a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy.
We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a Zebrafish Insertional Cardiac (ZIC) mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.
PMCID: PMC3603352  PMID: 23283723
Gene trapping; insertional mutagenesis screen; cardiac mutants; adult recessive; zebrafish; transposon
7.  SNPs in VKORC1 are Risk Factors for Systemic Lupus Erythematosus in Asians 
Arthritis and rheumatism  2013;65(1):211-215.
The increased risk of thrombosis in systemic lupus erythematosus (SLE) may be partially explained by interrelated genetic pathways for thrombosis and SLE. In a case-control analysis, we investigated whether 33 established and novel single nucleotide polymorphisms (SNP) in 20 genes involved in hemostasis pathways that have been associated with deep venous thrombosis in the general population were risk factors for SLE development among Asians.
Patients in the discovery cohort were enrolled in one of two North American SLE cohorts. Patients in the replication cohort were enrolled in one of four Asian or two North American cohorts. SLE cases met American College of Rheumatology classification criteria. We first genotyped 263 Asian SLE and 357 healthy Asian control individuals for 33 SNPs using Luminex multiplex technology in the discovery phase, and then used Taqman and Immunochip assays to examine 5 SNPs in up to an additional 1496 cases and 993 controls in the Replication phase. SLE patients were compared to healthy controls for association with minor alleles in allelic models. Principal components analysis was used to control for intra-Asian ancestry in an analysis of the replication cohort.
Two genetic variants in the gene VKORC1, rs9934438 and rs9923231, were highly significant in both the discovery and replication cohorts: OR(disc) = 2.45 (p=2×10−9), OR(rep) = 1.53 (p=5×10−6) and OR(disc) = 2.40 (p=6×10−9), OR(rep) = 1.53 (p=5×10−6), respectively. These associations were significant in the replication cohort after adjustment for intra-Asian ancestry: rs9934438 OR(adj) = 1.34 (p=0.0029) and rs9923231 OR(adj) = 1.34 (p=0.0032).
Genetic variants in VKORC1, involved in vitamin K reduction and associated with DVT, are associated with SLE development in Asians. These results suggest intersecting genetic pathways for the development of SLE and thrombosis.
PMCID: PMC3670944  PMID: 23124848
systemic lupus erythematosus; single nucleotide polymorphisms; genetic risk factors
8.  Differential loss of thalamostriatal and corticostriatal input to striatal projection neuron types prior to overt motor symptoms in the Q140 knock-in mouse model of Huntington's disease 
Motor slowing and forebrain white matter loss have been reported in premanifest Huntington's disease (HD) prior to substantial striatal neuron loss. These findings raise the possibility that early motor defects in HD may be related to loss of excitatory input to striatum. In a prior study, we showed that in the heterozygous Q140 knock-in mouse model of HD that loss of thalamostriatal axospinous terminals is evident by 4 months, and loss of corticostriatal axospinous terminals is evident at 12 months, before striatal projection neuron pathology. In the present study, we specifically characterized the loss of thalamostriatal and corticostriatal terminals on direct (dSPN) and indirect (iSPN) pathway striatal projection neurons, using immunolabeling to identify thalamostriatal (VGLUT2+) and corticostriatal (VGLUT1+) axospinous terminals, and D1 receptor immunolabeling to distinguish dSPN (D1+) and iSPN (D1−) synaptic targets. We found that the loss of corticostriatal terminals at 12 months of age was preferential for D1+ spines, and especially involved smaller terminals, presumptively of the intratelencephalically projecting (IT) type. By contrast, indirect pathway D1− spines showed little loss of axospinous terminals at the same age. Thalamostriatal terminal loss was comparable for D1+ and D1− spines at both 4 and 12 months. Regression analysis showed that the loss of VGLUT1+ terminals on D1+ spines was correlated with a slight decline in open field motor parameters at 12 months. Our overall results raise the possibility that differential thalamic and cortical input loss to SPNs is an early event in human HD, with cortical loss to dSPNs in particular contributing to premanifest motor slowing.
PMCID: PMC4197654  PMID: 25360089
Huntington's disease; corticostriatal; thalamostriatal; premanifest; pathology
9.  The Influence of Angiotensin Converting Enzyme and Angiotensinogen Gene Polymorphisms on Hypertrophic Cardiomyopathy 
PLoS ONE  2013;8(10):e77030.
Some studies have reported that angiotensin converting enzyme (ACE) and angiotensinogen (AGT) genes have been associated with hypertrophic cardiomyopathy (HCM). However, there have been inconsonant results among different studies. To clarify the influence of ACE and AGT on HCM, a systemic review and meta-analysis of case-control studies were performed. The following databases were searched to indentify related studies: PubMed database, the Embase database, the Cochrane Central Register of Controlled Trials database, China National Knowledge Information database, and Chinese Scientific and Technological Journal database. Search terms included “hypertrophic cardiomyopathy”, “angiotensin converting enzyme” (ACE) or “ACE” and “polymorphism or mutation”. For the association of AGT M235T polymorphism and HCM, “angiotensin converting enzyme” or “ACE” was replaced with “angiotensinogen”. A total of seventeen studies were included in our review. For the association of ACE I/D polymorphism and HCM, eleven literatures were included in the meta-analysis on association of penetrance and genotype. Similarly, six case-control studies were included in the meta-analysis for AGT M235T. For ACE I/D polymorphism, the comparison of DI/II genotype vs DD genotype was performed in the present meta-analysis. The OR was 0.73 (95% CI: 0.527, 0.998, P = 0.049, power = 94%, alpha = 0.05) after the study which deviated from Hardy-Weinberg Equilibrium was excluded, indicating that the ACE I/D gene polymorphism might be associated with HCM. The AGT M235T polymorphism did not significantly affect the risk of HCM. In addition, ACE I/D gene polymorphism did not significantly influence the interventricular septal thickness in HCM patients. In conclusion, the ACE I/D polymorphism might be associated with the risk of HCM.
PMCID: PMC3808382  PMID: 24204726
10.  Preferential Binding to Elk-1 by SLE-Associated IL10 Risk Allele Upregulates IL10 Expression 
PLoS Genetics  2013;9(10):e1003870.
Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10−8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.
Author Summary
Systemic lupus erythematosus (SLE), a debilitating autoimmune disease characterized by the production of pathogenic autoantibodies, has a strong genetic basis. Variants of the IL10 gene, which encodes cytokine interleukin-10 (IL-10) with known function of promoting B cell hyperactivity and autoantibody production, are associated with SLE and other autoimmune diseases, and serum IL-10 levels are elevated in SLE patients correlating with increased disease activity. In this study, to discover SLE-predisposing causal variant(s), we assessed variants within the genomic region containing IL10 and its gene family member IL19, IL20 and IL24 for association with SLE in case and control subjects from diverse ancestries. We identified SLE-associated SNP rs3122605 located at 9.2 kb upstream of IL10 as the most likely causal variant in subjects of European ancestry. The SLE-risk allele of rs3122605 was dose-dependently associated with elevated IL10 expression at both mRNA and protein levels in peripheral blood samples from SLE patients and controls, which could be explained, at least in part, by its preferential binding to Elk-1, a transcription factor activated in B cells during active disease of SLE patients. Elk-1-mediated IL-10 overexpression could be downregulated by inhibiting activation of mitogen-activated protein kinases, suggesting a potential therapeutic target for SLE.
PMCID: PMC3794920  PMID: 24130510
11.  CpG oligodeoxynucleotide ligand potentiates the activity of the pVAX1-Sj26GST 
Biomedical Reports  2013;1(4):609-613.
Schistosomiasis is considered one of the most important neglected tropical diseases and remains a major public health problem in endemic countries. Toll-like receptor (TLR) ligands have been investigated as potential vaccine adjuvants for tumor and virus immunotherapy. However, few TLR ligands affecting schistosoma vaccines have been characterized. In this study, we evaluated a TLR9 ligand (CpG oligodeoxynucleotide 1826, CpG) as an adjuvant for a partially protective DNA vaccine encoding a 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST). Vaccination with pVAX1-Sj26GST in combination with CpG inhibited Treg immunosuppressive function, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2 and IL-6, and decreased CD4+CD8+Foxp3+ expression in vitro, which may contribute to the escape from Treg-mediated suppression during vaccination, allowing expansion of antigen-specific T cells against pathogens. In conclusion, our data demonstrated that selective TLR ligand combination may increase protective efficacy against schistosomiasis, which may synergistically antagonize Treg-mediated suppression.
PMCID: PMC3916974  PMID: 24648995
CpG oligodeoxynucleotide ligand; pVAX1-Sj26GST; vaccine
12.  MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus 
PLoS Genetics  2013;9(2):e1003336.
We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10−10, odds ratio (OR) (95%CI) = 1.27 (1.17–1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10−11, OR = 1.24 [1.18–1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10−19, OR = 1.25 [1.20–1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.
Author Summary
Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease contributed to by excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) signaling pathways. TLR7 responds against RNA–containing nuclear antigens and activates IFN-α pathway, playing a pivotal role in the development of SLE. While a genomic duplication of Tlr7 promotes lupus-like disease in the Y-linked autoimmune accelerator (Yaa) murine model, the lack of common copy number variations at TLR7 in humans led us to identify a functional single nucleotide polymorphism (SNP), rs3853839 at 3′ UTR of the TLR7 gene, associated with SLE susceptibility in Eastern Asians. In this study, we fine-mapped the TLR7-TLR8 region and confirmed rs3853839 exhibiting the strongest association with SLE in European Americans, African Americans, and Amerindian/Hispanics. Individuals carrying the risk G allele of rs3853839 exhibited increased TLR7 expression at the both mRNA and protein level and decreased transcript degradation. MicroRNA-3148 (miR-3148) downregulated the expression of non-risk allele (C) containing transcripts preferentially, suggesting a likely mechanism for increased TLR7 levels in risk-allele carriers. This trans-ancestral mapping provides evidence for the global association with SLE risk at rs3853839, which resides in a microRNA–gene regulatory site affecting TLR7 expression.
PMCID: PMC3585142  PMID: 23468661
13.  The Role of Stream Water Carbon Dynamics and Export in the Carbon Balance of a Tropical Seasonal Rainforest, Southwest China 
PLoS ONE  2013;8(2):e56646.
A two-year study (2009 ∼ 2010) was carried out to investigate the dynamics of different carbon (C) forms, and the role of stream export in the C balance of a 23.4-ha headwater catchment in a tropical seasonal rainforest at Xishuangbanna (XSBN), southwest China. The seasonal volumetric weighted mean (VWM) concentrations of total inorganic C (TIC) and dissolved inorganic C (DIC) were higher, and particulate inorganic C (PIC) and organic C (POC) were lower, in the dry season than the rainy season, while the VWM concentrations of total organic C (TOC) and dissolved organic C (DOC) were similar between seasons. With increased monthly stream discharge and stream water temperature (SWT), only TIC and DIC concentrations decreased significantly. The most important C form in stream export was DIC, accounting for 51.8% of the total C (TC) export; DOC, POC, and PIC accounted for 21.8%, 14.9%, and 11.5% of the TC export, respectively. Dynamics of C flux were closely related to stream discharge, with the greatest export during the rainy season. C export in the headwater stream was 47.1 kg C ha−1 yr−1, about 2.85% of the annual net ecosystem exchange. This finding indicates that stream export represented a minor contribution to the C balance in this tropical seasonal rainforest.
PMCID: PMC3577870  PMID: 23437195
14.  DNA based identification of medicinal materials in Chinese patent medicines 
Scientific Reports  2012;2:958.
Chinese patent medicines (CPM) are highly processed and easy to use Traditional Chinese Medicine (TCM). The market for CPM in China alone is tens of billions US dollars annually and some of the CPM are also used as dietary supplements for health augmentation in the western countries. But concerns continue to be raised about the legality, safety and efficacy of many popular CPM. Here we report a pioneer work of applying molecular biotechnology to the identification of CPM, particularly well refined oral liquids and injections. What's more, this PCR based method can also be developed to an easy to use and cost-effective visual chip by taking advantage of G-quadruplex based Hybridization Chain Reaction. This study demonstrates that DNA identification of specific Medicinal materials is an efficient and cost-effective way to audit highly processed CPM and will assist in monitoring their quality and legality.
PMCID: PMC3518818  PMID: 23233877
15.  Association of PPP2CA polymorphisms with SLE susceptibility in multiple ethnic groups 
Arthritis and rheumatism  2011;63(9):2755-2763.
T cells from patients with SLE express increased amounts of PP2Ac which contribute to decreased production of IL-2. Because IL-2 is important in the regulation of several aspects of the immune response, it has been proposed that PP2Ac contributes to the expression of SLE. This study was designed to determine whether genetic variants of PPP2AC are linked to the expression of SLE and specific clinical manifestations and account for the increased expression of PP2Ac.
We conducted a trans-ethnic study consisting of 8,695 SLE cases and 7,308 controls from four different ancestries. Eighteen single-nucleotide polymorphisms (SNPs) across the PPP2CA were genotyped using an Illumina custom array. PPP2CA expression in SLE and control T cells was analyzed by real-time PCR.
A 32-kb haplotype comprised of multiple SNPs of PPP2CA showed significant association with SLE in Hispanic Americans (HA), European Americans (EA) and Asians but not in African-Americans (AA). Conditional analyses revealed that SNP rs7704116 in intron 1 showed consistently strong association with SLE across Asian, EA and HA populations (pmeta=3.8×10−7, OR=1.3[1.14–1.31]). In EA, the largest ethnic dataset, the risk A allele of rs7704116 was associated with the presence of renal disease, anti-dsDNA and anti-RNP antibodies. PPP2CA expression was approximately 2-fold higher in SLE patients carrying the rs7704116 AG genotype than those carrying GG genotype (p = 0.008).
Our data provide the first evidence for an association between PPP2CA polymorphisms and elevated PP2Ac transcript levels in T cells, which implicates a new molecular pathway for SLE susceptibility in EA, HA and Asians.
PMCID: PMC3163110  PMID: 21590681
16.  3-Eth­oxy­carbonyl-2-hy­droxy-6-meth­oxy-4-methyl­benzoic acid 
The title compound, C12H14O6, a substituted isophthalic acid monoester which was isolated from the lichen Thamnolia vermicularis var. subuliformis, displays intra­molecular carbox­yl–meth­oxy O—H⋯O and hy­droxy–carboxyl O—H⋯O hydrogen-bonding inter­actions. The terminal methyl group of the ethyl ester is disordered over two sets of sites with occupancies of 0.599 (19) and 0.401 (19).
PMCID: PMC3344543  PMID: 22590305
17.  Gene Clusters Located on Two Large Plasmids Determine Spore Crystal Association (SCA) in Bacillus thuringiensis Subsp. finitimus Strain YBT-020 
PLoS ONE  2011;6(11):e27164.
Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named ‘spore crystal association’ (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.
PMCID: PMC3208593  PMID: 22076131
18.  Complete Genome Sequence of Bacillus thuringiensis Serovar finitimus Strain YBT-020▿ 
Journal of Bacteriology  2011;193(9):2379-2380.
Bacillus thuringiensis is a Gram-positive, spore-forming bacterium that forms parasporal crystals at the onset of the sporulation phase of its growth. Here, we report the complete genome sequence of B. thuringiensis serovar finitimus strain YBT-020, whose parasporal crystals consist of Cry26Aa and Cry28Aa crystal proteins and are located between the exosporium and the spore coat and remain adhering to the spore after sporulation.
PMCID: PMC3133068  PMID: 21398543
19.  Complete Genome Sequence of Bacillus subtilis BSn5, an Endophytic Bacterium of Amorphophallus konjac with Antimicrobial Activity for the Plant Pathogen Erwinia carotovora subsp. carotovora ▿  
Journal of Bacteriology  2011;193(8):2070-2071.
Here, we present the complete genome sequence of Bacillus subtilis strain BSn5, isolated from Amorphophallus konjac calli tissue and showing strong inhibitory activity to Erwinia carotovora subsp. carotovora, which causes Amorphophallus soft rot disease and affects the industry development of this organism.
PMCID: PMC3133036  PMID: 21317323
20.  Immunohistochemical Localization of AMPA Type Glutamate Receptor Subunits in the Striatum of Rhesus Monkey 
Brain research  2010;1344:104-123.
Corticostriatal and thalamostriatal projections utilize glutamate as their neurotransmitter. Their influence on striatum is mediated, in part, by ionotropic AMPA-type glutamate receptors, which are heteromers composed of GluR1-4 subunits. While the cellular localization of AMPA-type subunits in the basal ganglia has been well characterized in rodents, the cellular localization of AMPA subunits in primate basal ganglia is not. We thus carried out immunohistochemical studies of GluR1-4 distribution in rhesus monkey basal ganglia in conjunction with characterization of each major neuron type. In striatum, about 65% of striatal neurons immunolabeled for GluR1, 75%-79% immunolabeled for GluR2 or GluR2/3, and only 2.5% possessed GluR4. All neurons the large size of cholinergic interneurons (mean diameter 26.1μm) were moderately labeled for GluR1, while all neurons in the size range of parvalbuminergic interneurons (mean diameter 13.8μm) were intensely rich in GluR1. Additionally, somewhat more than half of neurons in the size range of projection neurons (mean diameter 11.6μm) immunolabeled for GluR1, and about one third of these were very rich in GluR1. About half of neurons the size of cholinergic interneurons were immunolabeled for GluR2, and the remainder of the neurons that were immunolabeled for GluR2 coincided with projection neurons in size and shape (GluR2 diameter=10.7μm), indicating that the vast majority of striatal projection neurons possess immunodectible GluR2. Similar results were observed with GluR2/3 immunolabeling. Half of the neurons the size of cholinergic interneurons immunolabeled for GluR4 and seemingly all neurons in the size range of parvalbuminergic interneurons possessed GluR4. These results indicate that AMPA receptor subunit combinations for striatal projection neurons in rhesus monkey are similar to those for the corresponding neuron types in rodents, and thus their AMPA responses to glutamate likely to be similar to those demonstrated in rodents.
PMCID: PMC2905043  PMID: 20460117
Basal Ganglia; glutamate receptors; glutamate; monkey
21.  Genetic susceptibility to systemic lupus erythematosus in the genomic era 
Nature reviews. Rheumatology  2010;6(12):683-692.
Our understanding of the genetic basis of systemic lupus erythematosus (SLE) has been rapidly advanced using large-scale, case–control, candidate gene studies as well as genome-wide association studies during the past 3 years. These techniques have identified more than 30 robust genetic associations with SLE including genetic variants of HLA and Fcγ receptor genes, IRF5, STAT4, PTPN22, TNFAIP3, BLK, BANK1, TNFSF4 and ITGAM. Most SLE-associated gene products participate in key pathogenic pathways, including Toll-like receptor and type I interferon signaling pathways, immune regulation pathways and those that control the clearance of immune complexes. Disease-associated loci that have not yet been demonstrated to have important functions in the immune system might provide new clues to the underlying molecular mechanisms that contribute to the pathogenesis or progression of SLE. Of note, genetic risk factors that are shared between SLE and other immune-related diseases highlight common pathways in the pathophysiology of these diseases, and might provide innovative molecular targets for therapeutic interventions.
PMCID: PMC3135416  PMID: 21060334
22.  Biotransformation of aesculin by human gut bacteria and identification of its metabolites in rat urine 
AIM: To observe the biotransformation process of a Chinese compound, aesculin, by human gut bacteria, and to identify its metabolites in rat urine.
METHODS: Representative human gut bacteria were collected from 20 healthy volunteers, and then utilized in vitro to biotransform aesculin under anaerobic conditions. At 0, 2, 4, 8, 12, 16, 24, 48 and 72 h post-incubation, 10 mL of culture medium was collected. Metabolites of aesculin were extracted 3 × from rat urine with methanol and analyzed by HPLC. For in vivo metabolite analysis, aesculetin (100 mg/kg) was administered to rats via stomach gavage, rat urine was collected from 6 to 48 h post-administration, and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes.
RESULTS: Human gut bacteria could completely convert aesculin into aesculetin in vitro. The biotransformation process occurred from 8 to 24 h post-incubation, with its highest activity was seen from 8 to 12 h. The in vitro process was much slower than the in vivo process. In contrast to the in vitro model, six aesculetin metabolites were identified in rat urine, including 6-hydroxy-7-gluco-coumarin (M1), 6-hydroxy-7-sulf-coumarin (M2), 6, 7-di-gluco-coumarin (M3), 6-glc-7-gluco-coumarin (M4), 6-O-methyl-7-gluco-coumarin (M5) and 6-O-methyl-7-sulf-coumarin (M6). Of which, M2 and M6 were novel metabolites.
CONCLUSION: Aesculin can be transferred into aesculetin by human gut bacteria and is further modified by the host in vivo. The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.
PMCID: PMC2665149  PMID: 19322928
Aesculin; Biotransformation; Human gut bacteria; Rat urine; Sulfated derivatives; LC/ESI-MS; Aseculetin
23.  A Web Based Resource Characterizing the Zebrafish Developmental Profile of over 16,000 transcripts 
Gene expression patterns : GEP  2007;8(3):171-180.
Using a spotted 65-mer oligonucleotide microarray, we have characterized the developmental expression profile from mid-gastrulation (75% epiboly) to 5 days post-fertilization (dpf) for >16,000 unique transcripts in the zebrafish genome. Microarray profiling data sets are often immense, and one challenge is validating the results and prioritizing genes for further study. The purpose of the current study was to address such issues, as well as to generate a publicly available resource for investigators to examine the developmental expression profile of any of the over 16,000 zebrafish genes on the array. On the chips, there are 16,459 printed spots corresponding to 16,288 unique transcripts and 172 β-actin (AF025305) spots spatially distributed throughout the chip as a positive control. We have collected 55 microarray gene expression profiling results from various zebrafish laboratories and created a Perl/CGI-based software tool ( for researchers to look for the expression patterns of their gene of interest. Users can search for their genes of interest by entering the accession numbers or the nucleotide sequences and the expression profiling will be reported in the form of expression intensities versus time-course graphical displays. In order to validate this web tool, we compared seventy-four genes’ expression results between our web tool and the in situ hybridization results from Thisse et al. (2004) as well as those reported by Mathavan et al. (2005). The comparison indicates the expression patterns are 80% and 75% in agreement between our web resource with the in situ database (Thisse et al. 2004) and with those reported by Mathavan et al. (2005), respectively. Those genes that conflict between our web tool and the in situ database either have high sequence similarity with other genes or the in situ probes are not reliable. Among those genes that disagree between our web tool and those reported by Mathavan et al. (2005), 93% of the genes are in agreement between our web tool and the in situ database, indicating our web tool results are quite reliable. Thus, this resource provides a user-friendly web based platform for researchers to determine the developmental profile of their gene of interest and to prioritize genes identified in microarray analyses by their developmental expression profile.
PMCID: PMC2253684  PMID: 18068546
24.  Phylogeny and Biogeography of the Genus Ainsliaea (Asteraceae) in the Sino-Japanese Region based on Nuclear rDNA and Plastid DNA Sequence Data 
Annals of Botany  2007;101(1):111-124.
Background and Aims
The flora of the Sino-Japanese plant region of eastern Asia is distinctively rich compared with other floristic regions in the world. However, knowledge of its floristic evolution is fairly limited. The genus Ainsliaea is endemic to and distributed throughout the Sino-Japanese region. Its interspecific phylogenetic relationships have not been resolved. The aim is to provide insight into floristic evolution in eastern Asia on the basis of a molecular phylogenetic analysis of Ainsliaea species.
Cladistic analyses of the sequences of two nuclear (ITS, ETS) and one plastid (ndhF) regions were carried out individually and using the combined data from the three markers.
Key Results
Phylogenetic analyses of three DNA regions confirmed that Ainsliaea is composed of three major clades that correspond to species distributions. Evolution of the three lineages was estimated to have occurred around 1·1 MYA during the early Pleistocene.
The results suggest that Ainsliaea species evolved allopatrically and that the descendants were isolated in the eastern (between SE China and Japan, through Taiwan and the Ryukyu Islands) and western (Yunnan Province and its surrounding areas, including the Himalayas, the temperate region of Southeast Asia, and Sichuan Province) sides of the Sino-Japanese region. The results suggest that two distinct lineages of Ainsliaea have independently evolved in environmentally heterogeneous regions within the Sino-Japanese region. These regions have maintained rich and original floras due to their diverse climates and topographies.
PMCID: PMC2701833  PMID: 17981878
Ainsliaea; ETS; ITS; ndhF; phylogeography; rheophytes; Sino-Japanese region
25.  In situ Hybridization Histochemical and Immunohistochemical Evidence that Striatal Projection Neurons Co-containing Substance P and Enkephalin are Overrepresented in the Striosomal Compartment of Striatum in Rats 
Neuroscience letters  2007;425(3):195-199.
In a prior study, we showed that the few striatal projection neurons that contain both substance P (SP) and enkephalin (ENK) in rats may preferentially project to the substantia nigra pars compacta. Since striatal neurons that project to the pars compacta are thought to preferentially reside in the striosomal compartment, we investigated if striatal neurons that contain both SP and ENK are preferentially localized to the patch compartment. We used in situ hybridization histochemistry to double-label sections for SP and ENK to identify SP/ENK co-containing neurons, and immunolabeling of adjacent sections for the mu opiate receptor (MOR) to define the striosomal compartment. We found that 32.3% of neurons containing both SP and ENK were localized to the striosomal compartment, which itself only made up 12.8% of the striatum. Our results further showed that the density of neurons co-containing SP and ENK was three-fold higher in striosomes than in the matrix compartment. These results are consistent with the notion that SP/ENK colocalizing neurons preferentially project to pars compacta, and these and our prior results additionally raise the possibility that neurons of this type in the striatal matrix may also project to the pars compacta.
PMCID: PMC2034403  PMID: 17868995
Striatum; Substance P; Enkephalin; Projection Neuron; Colocalization

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