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author:("Li, taohong")
1.  MiR-200a inhibits epithelial-mesenchymal transition of pancreatic cancer stem cell 
BMC Cancer  2014;14:85.
Pancreatic cancer is one of the most aggressive cancers, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells. And miR-200 has been identified as a powerful regulator of EMT.
Cancer Stem Cells (CSCs) of human pancreatic cancer cell line PANC-1 were processed for CD24, CD44 and ESA multi-colorstaining, and sorted out on a BD FACS Aria II machine. RT-qPCR was performed using the miScript PCR Kit to assay the expression of miR-200 family. In order to find the role of miR-200a in the process of EMT, miR-200a mimic was transfected to CSCs.
Pancreatic cancer cells with EMT phenotype displayed stem-like cell features characterized by the expression of cell surface markers CD24, CD44 and epithelial-specific antigen (ESA), which was associated with decreased expression of miR-200a. Moreover, overexpression of miR-200a was resulted in down-regulation of N-cadherin, ZEB1 and vimentin, but up-regulation of E-cadherin. In addition, miR-200a overexpression inhibited cell migration and invasion in CSCs.
In our study, we found that miR-200a played an important role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in pancreatic cancer. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to mesenchymal-to-epithelial transition (MET) phenotype using novel agents would be useful for prevention and/or treatment of pancreatic cancer.
PMCID: PMC3923443  PMID: 24521357
CSC; EMT; Pancreatic cancer; miR-200a
2.  Loss of TGF-β Responsiveness in Prostate Stromal Cells Alters Chemokine Levels and Facilitates the Development of Mixed Osteoblastic/Osteolytic Bone Lesions 
Molecular cancer research : MCR  2012;10(4):494-503.
Loss of TGF-β type II receptor (TβRII, encoded by Tgfbr2) expression in the prostate stroma contributes to prostate cancer initiation, progression, and invasion. We evaluated whether TβRII loss also affected prostate cancer bone metastatic growth. Immunohistologic analysis revealed that TβRII expression was lost in cancer-associated fibroblasts in human prostate cancer bone metastatic tissues. We recapitulated the human situation with a conditional stromal Tgfbr2 knockout (Tgfbr2-KO) mouse model. Conditioned media from primary cultured Tgfbr2-KO or control Tgfbr2-flox prostatic fibroblasts (koPFCM or wtPFCM, respectively) were applied to C4-2B prostate cancer cells before grafting the cells tibially. We found that koPFCM promoted prostate cancer cell growth in the bone and development of early mixed osteoblastic/osteolytic bone lesions. Furthermore, the koPFCM promoted greater C4-2B adhesion to type-I collagen, the major component of bone matrix, compared to wtPFCM-treated C4-2B. Cytokine antibody array analysis revealed that koPFCM had more than two-fold elevation in granulocyte colony-stimulating factor and CXCL1, CXCL16, and CXCL5 expression relative to wtPFCM. Interestingly, neutralizing antibodies of CXCL16 or CXCL1 were able to reduce koPFCM-associated C4-2B type-I collagen adhesion to that comparable with wtPFCM-mediated adhesion. Collectively, our data indicate that loss of TGF-β responsiveness in prostatic fibroblasts results in upregulation of CXCL16 and CXCL1 and that these paracrine signals increase prostate cancer cell adhesion in the bone matrix. These microenvironment changes at the primary tumor site can mediate early establishment of prostate cancer cells in the bone and support subsequent tumor development at the metastatic site.
PMCID: PMC3900026  PMID: 22290877
3.  Expression and immune characterization of a novel enzyme, protein arginine methyltransferase 1, from Schistosoma japonicum 
Parasitology Research  2013;113:919-924.
Protein arginine methyltransferase 1 (PRMT1) is an arginine-specific protein methyltransferase that methylates a number of proteins involved in transcription and RNA metabolism in all parasitic helminths, including the human blood fluke, Schistosoma japonicum. To characterize the role of PRMT1 in the development of S. japonicum and to investigate its influence on parasite–host interactions, we cloned and expressed the protein from an existing cDNA library. We report that the clone encoded a polypeptide comprising 360 amino acids with a predictive Mr of 42 kDa. Bioinformatic analyses predicted that there were many potential B cell epitopes and T cell epitopes associated with SjcPRMT1, suggesting it is a potential candidate molecule for vaccine development. The purified recombinant protein of S. japonicum (Chinese strain) (rSjcPRMT1) was found to be immunogenic, eliciting a high antibody titer in mice. Moreover, Western blot analysis revealed that the protein could be recognized by the sera of infected mice. Using flow cytometry, we showed that rSjcPRMT1 slightly upregulated the expression of CD40, CD80, CD86, and MHC-II molecules of mouse bone marrow-derived dendritic cell (BMDC), indicating that rSjcPRMT1 could induce mouse BMDC to mature and, therefore, activate their immune response. Overall, our findings provide evidence that rSjcPRMT1 could serve as an effective candidate molecule for the development of a vaccine against infection with S. japonicum.
PMCID: PMC3932173  PMID: 24343727
4.  Gonadotropin-Releasing Hormone for Preservation of Ovarian Function during Chemotherapy in Lymphoma Patients of Reproductive Age: A Summary Based on 434 Patients 
PLoS ONE  2013;8(11):e80444.
Gonadotropin-releasing hormone agonists (GnRHa) might play a role in preserving ovarian function in lymphoma patients by inhibiting chemotherapy-induced ovarian follicular damage. However, studies of its clinical efficacy have reported conflicting results.
We conducted a meta-analysis to determine the effect of the preservation of ovarian function by administering GnRHa in young patients with lymphoma undergoing chemotherapy. Seven studies were identified that met inclusion criteria and comprised 434 patients assigned to GnRHa combined chemotherapy or chemotherapy alone.
The incidence of women with premature ovarian failure (POF) demonstrated a statistically significant difference in favor of the use of GnRHa (OR=0.32, 95% CI 0.13-0.77). In addition, the final level of FSH in the GnRH group was significantly lower than control group. (MD= -11.73, 95% CI,-22.25- -1.20), and the final level of AMH in the GnRH group was significantly higher than control group (MD=0.80; 95% CI, 0.61–0.98). However, there was no statistically significant difference between treatment and the control groups in the incidence of a spontaneous pregnancy (OR=1.11; 95% CI, 0.55–2.26).
This meta-analysis suggests that GnRHa may be effective in protecting ovarian function during chemotherapy in lymphoma patients. More well-designed prospective studies are needed to carry out for further understanding of this topic.
PMCID: PMC3842920  PMID: 24312222
5.  Feasibility of RNA and DNA Extraction from Fresh Pipelle and Archival Endometrial Tissues for Use in Gene Expression and SNP Arrays 
Identifying molecular markers of endometrial hyperplasia (neoplasia) progression is critical to cancer prevention. To assess RNA and DNA quantity and quality from routinely collected endometrial samples and evaluate the performance of RNA- and DNA-based arrays across endometrial tissue types, we collected fresh frozen (FF) Pipelle, FF curettage, and formalin-fixed paraffin-embedded (FFPE) hysterectomy specimens (benign indications) from eight women. Additionally, neoplastic and uninvolved tissues from 24 FFPE archival hysterectomy specimens with endometrial hyperplasias and carcinomas were assessed. RNA was extracted from 15 of 16 FF and 51 of 51 FFPE samples, with yields >1.2 μg for 13/15 (87%) FF and 50/51 (98%) FFPE samples. Extracted RNA was of high quality; all samples performed successfully on the Illumina whole-genome cDNA-mediated annealing, selection, extension, and ligation (WG-DASL) array and performance did not vary by tissue type. While DNA quantity from FFPE samples was excellent, quality was not sufficient for successful performance on the Affymetrix SNP Array 6.0. In conclusion, FF Pipelle samples, which are minimally invasive, yielded excellent quantity and quality of RNA for gene expression arrays (similar to FF curettage) and should be considered for use in genomic studies. FFPE-derived DNA should be evaluated on new rapidly evolving sequencing platforms.
PMCID: PMC3825122  PMID: 24282417
6.  Prognostic impact of p16 and p21 on gastroenteropancreatic neuroendocrine tumors 
Oncology Letters  2013;6(6):1641-1645.
Aberrant expression of the cell cycle kinase inhibitors, p16 and p21, has been associated with poor prognosis in a number of human malignancies. These proteins may also be involved in the development and progression of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The present study aimed to investigate protein levels of p16 and p21 in GEP-NETs and to evaluate their clinical significance. p16 and p21 protein expression was tested immunohistochemically in the tissue samples of 68 GEP-NETs. The association between expression and clinicopathological characteristics and overall survival was assessed. Low expression of p16 (no positive nuclear staining) was found in 37 (54%) cases and high p21 expression (≥5% positive nuclear staining) was detected in 23 (34%) cases. Low p16 protein levels indicated a poorer prognosis for patients graded as G2 subgroup in the univariate analysis (relative risk, 4.4; 95% CI, 1.8–10.6). No significant correlation was found between the expression of p21 and any of the clinicopathological variables. The present study indicates a prognostic relevance for p16 immunoreactivity. Low levels of p16 protein were associated with a shorter survival in the G2 subgroup of GEP-NETs. p21 protein expression was not identified to be useful as a predictive indicator in GEP-NETs.
PMCID: PMC3834264  PMID: 24260058
gastroenteropancreatic neuroendocrine tumor; p16; p21; immunohistochemistry
7.  Targeted Deletion and Inversion of Tandemly Arrayed Genes in Arabidopsis thaliana Using Zinc Finger Nucleases 
G3: Genes|Genomes|Genetics  2013;3(10):1707-1715.
Tandemly arrayed genes (TAGs) or gene clusters are prevalent in higher eukaryotic genomes. For example, approximately 17% of genes are organized in tandem in the model plant Arabidopsis thaliana. The genetic redundancy created by TAGs presents a challenge for reverse genetics. As molecular scissors, engineered zinc finger nucleases (ZFNs) make DNA double-strand breaks in a sequence-specific manner. ZFNs thus provide a means to delete TAGs by creating two double-strand breaks in the gene cluster. Using engineered ZFNs, we successfully targeted seven genes from three TAGs on two Arabidopsis chromosomes, including the well-known RPP4 gene cluster, which contains eight resistance (R) genes. The resulting gene cluster deletions ranged from a few kb to 55 kb with frequencies approximating 1% in somatic cells. We also obtained large chromosomal deletions of ~9 Mb at approximately one tenth the frequency, and gene cluster inversions and duplications also were achieved. This study demonstrates the ability to use sequence-specific nucleases in plants to make targeted chromosome rearrangements and create novel chimeric genes for reverse genetics and biotechnology.
PMCID: PMC3789795  PMID: 23979943
zinc finger nuclease (ZFN); tandemly arrayed genes (TAGs); deletion; inversion; Arabidopsis
8.  Gene expression profiles in engineered cardiac tissues respond to mechanical loading and inhibition of tyrosine kinases 
Physiological Reports  2013;1(5):e00078.
Engineered cardiac tissues (ECTs) are platforms to investigate cardiomyocyte maturation and functional integration, the feasibility of generating tissues for cardiac repair, and as models for pharmacology and toxicology bioassays. ECTs rapidly mature in vitro to acquire the features of functional cardiac muscle and respond to mechanical load with increased proliferation and maturation. ECTs are now being investigated as platforms for in vitro models for human diseases and for pharmacologic screening for drug toxicities. We tested the hypothesis that global ECT gene expression patterns are complex and sensitive to mechanical loading and tyrosine kinase inhibitors similar to the maturing myocardium. We generated ECTs from day 14.5 rat embryo ventricular cells, as previously published, and then conditioned constructs after 5 days in culture for 48 h with mechanical stretch (5%, 0.5 Hz) and/or the p38 MAPK (p38 mitogen-activated protein kinase) inhibitor BIRB796. RNA was isolated from individual ECTs and assayed using a standard Agilent rat 4 × 44k V3 microarray and Pathway Analysis software for transcript expression fold changes and changes in regulatory molecules and networks. Changes in expression were confirmed by quantitative-polymerase chain reaction (q-PCR) for selected regulatory molecules. At the threshold of a 1.5-fold change in expression, stretch altered 1559 transcripts, versus 1411 for BIRB796, and 1846 for stretch plus BIRB796. As anticipated, top pathways altered in response to these stimuli include cellular development, cellular growth and proliferation; tissue development; cell death, cell signaling, and small molecule biochemistry as well as numerous other pathways. Thus, ECTs display a broad spectrum of altered gene expression in response to mechanical load and/or tyrosine kinase inhibition, reflecting a complex regulation of proliferation, differentiation, and architectural alignment of cardiomyocytes and noncardiomyocytes within ECT.
PMCID: PMC3841024  PMID: 24303162
BIRB796; engineered cardiac tissues; gene expression; mechanical loading; p38MAPK; tyrosine kinases
9.  Effect of exercise training on long-term potentiation and NMDA receptor channels in rats with cerebral infarction 
The aim of the present study was to investigate the effects of exercise training on the characteristics of long-term potentiation (LTP) and N-methyl-D aspartate (NMDA) receptor channels in the hippocampal CA3 neurons of rats with cerebral infarction. Wistar rats were randomly allocated into the model without any training and rehabilitation with exercise training. A model of cerebral infarction was established by middle cerebral artery occlusion. Using chronically embedded electrodes combined with an electrophysiological method, the population spike (PS) amplitude and latency, as well as changes in the NMDA single channel current in the hippocampal neurons were determined prior to and following Y-maze discrimination learning 60 times in the two groups. The formation of learning-dependent LTP and synaptic efficacy in the hippocampal CA3 area after exercise training in the rehabilitation group was significantly faster compared with that in the model group without any training (P<0.05). The incubation period of the PS in the CA3 area of the rats in the rehabilitation group was significantly shorter compared with that in the model group. The PS amplitude in the rehabilitation group was significantly higher compared with that in the model group. Furthermore, the opening probability of the NMDA receptor channel in the rehabilitation group was significantly higher compared with that in the model group. In conclusion, exercise training improved the opening conductance level, time and probability of NMDA receptor channels and accelerated the formation of learning-dependent LTP in the contralateral hippocampal CA3 area.
PMCID: PMC3829711  PMID: 24250723
cerebral infarction; exercise training; long-term potentiation; N-methyl-D aspartate receptor channel
10.  Impact of obesity on endometrial blood flow in women without polycystic ovarian syndrome during intracytoplasmic sperm injection 
Obesity may exert a negative effect on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. However, the effect of obesity on the endometrium remains unknown. This study was designed to assess the effect of isolated body mass index (BMI) on endometrial blood supply in non-polycystic ovary syndrome (PCOS) women during ICSI by power Doppler Ultrasound.
An observational prospective study was carried out. A total of 206 patients without PCOS were divided into 4 groups based on Chinese BMI classification (kg/m(2): underweight (BMI < 18.5), normal weight (18.5 less than or equal to BMI < 24), overweight (24 less than or equal to BMI < 28), and obese (BMI greater than or equal to 28). Endometrial thickness, endometrial pattern, endometrial spiral arterial resistance index (RI) and pulsatility index (PI) values and systolic/diastolic ratio (S/D) were assessed on the day of human chorionic gonadotropin administration.
Obese patients required more doses of gonadotrophin and longer stimulation duration than the normal weight patients (P < 0.05). Endometrial thickness and pattern were not statistically different between the 4 BMI subgroups (P > 0.05). Subendometrial blood flow was detected in 165 (80.1%) patients and spiral arterial PI was significantly higher in the obese group than in the normal weight and underweight groups (P < 0.05). All parameters of ICSI outcome were comparable, including pregnancy and miscarriage rates.
Obesity (BMI greater than or equal to 28 kg/m(2)) appears to exert a negative effect on endometrial and subendometrial blood flow based on the Chinese standard of obesity; however, it seems to have no significant effect on ICSI outcomes in non-PCOS women.
PMCID: PMC3698014  PMID: 23803145
Endometrial and subendometrial blood flow; Obesity; Power Doppler; Intracytoplasmic sperm injection
11.  NSAIDs Modulate Clonal Evolution in Barrett's Esophagus 
PLoS Genetics  2013;9(6):e1003553.
Cancer is considered an outcome of decades-long clonal evolution fueled by acquisition of somatic genomic abnormalities (SGAs). Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce cancer risk, including risk of progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). However, the cancer chemopreventive mechanisms of NSAIDs are not fully understood. We hypothesized that NSAIDs modulate clonal evolution by reducing SGA acquisition rate. We evaluated thirteen individuals with BE. Eleven had not used NSAIDs for 6.2±3.5 (mean±standard deviation) years and then began using NSAIDs for 5.6±2.7 years, whereas two had used NSAIDs for 3.3±1.4 years and then discontinued use for 7.9±0.7 years. 161 BE biopsies, collected at 5–8 time points over 6.4–19 years, were analyzed using 1Million-SNP arrays to detect SGAs. Even in the earliest biopsies there were many SGAs (284±246 in 10/13 and 1442±560 in 3/13 individuals) and in most individuals the number of SGAs changed little over time, with both increases and decreases in SGAs detected. The estimated SGA rate was 7.8 per genome per year (95% support interval [SI], 7.1–8.6) off-NSAIDs and 0.6 (95% SI 0.3–1.5) on-NSAIDs. Twelve individuals did not progress to EA. In ten we detected 279±86 SGAs affecting 53±30 Mb of the genome per biopsy per time point and in two we detected 1,463±375 SGAs affecting 180±100 Mb. In one individual who progressed to EA we detected a clone having 2,291±78 SGAs affecting 588±18 Mb of the genome at three time points in the last three of 11.4 years of follow-up. NSAIDs were associated with reduced rate of acquisition of SGAs in eleven of thirteen individuals. Barrett's cells maintained relative equilibrium level of SGAs over time with occasional punctuations by expansion of clones having massive amount of SGAs.
Author Summary
Cancer is a disease that develops over decades as result of accumulation of abnormalities in the genomes of otherwise normal cells. Cells in tumors compete for space and resources. Those cells able to survive the Darwinian struggle for existence within tissues progressively evolve uncontrolled growth and in some cases this results in cancer. Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) reduce death rate from multiple types of cancer by about 20%. However, the mechanisms by which NSAIDs act to prevent cancer are not fully understood. By examining thirteen individuals with Barrett's esophagus over time, we showed that the rate of accumulation of genomic abnormalities decreased when most individuals started taking NSAIDs. We also observed that, surprisingly, the number of abnormalities in the Barrett's tissues did not increase much over decades. However, in one individual who progressed to esophageal cancer, we observed massive genomic abnormalities affecting 19% of the genome. These findings suggest that NSAIDs may prevent cancer by reducing the accumulation of genomic abnormalities over time and that detection of stable versus unstable genomes may be used in the clinic to help manage treatment options in Barrett's esophagus.
PMCID: PMC3681672  PMID: 23785299
13.  Primary osteopathy of vertebrae in a neurofibromatosis type 1 murine model 
Bone  2011;48(6):1378-1387.
Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutation of the NF1 tumor suppressor gene. Spinal deformities are common skeletal manifestations in patients with NF1. To date, the mechanism of vertebral abnormalities remains unclear because of the lack of appropriate animal models for the skeletal manifestations of NF1. In the present study, we report a novel murine NF1 model, Nf1flox/−;Col2.3Cre+ mice. These mice display short vertebral segments. In addition, a significant reduction in cortical and trabecular bone mass of the vertebrae was observed in Nf1flox/−;Col2.3Cre+ mice as measured by dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). Peak stress and peak load were also significantly reduced in Nf1flox/−;Col2.3Cre+ mice as compared to controls. Furthermore, the lumbar vertebrae showed enlargement of the inter-vertebral canal, a characteristic feature of lumbar vertebrae in NF1 patients. Finally, histologic analysis demonstrated increased numbers of osteoclasts and decreased numbers of osteoblasts in the vertebrae of Nf1flox/−;Col2.3Cre+ mice in comparison to controls. In summary, Nf1flox/−;Col2.3Cre+ mice demonstrate multiple structural and functional abnormalities in the lumbar vertebrae which recapitulate the dystrophic vertebral changes in NF1 patients. This novel murine model provides a platform to understand the cellular and molecular mechanisms underlying the pathogenesis of spinal deficits in NF1 patients.
PMCID: PMC3584682  PMID: 21439418
Neurofibromatosis type 1; Spinal deformity; Osteopathy; Osteoclast; Osteoblast
14.  Geographic and urban–rural disparities in the total prevalence of neural tube defects and their subtypes during 2006–2008 in China: a study using the hospital-based birth defects surveillance system 
BMC Public Health  2013;13:161.
Previous reports on the prevalence of neural tube defects (NTDs) in China did not include cases of NTDs that were less than 28 weeks of gestational age (GA) and hence did not accurately reflect the total prevalence of NTDs or the geographic and urban–rural disparities in their prevalence. This article includes cases of NTDs that were less than 28 weeks of GA.
Data used in this study were collected from 2006 to 2008 using a nationwide hospital-based registry, the Chinese Birth Defects Monitoring Network. The total prevalence ratio (PR) of NTDs and their subtypes, the ratios of PR (PRR), and 95% confidence intervals (CI) were used to analyse geographic disparities at both the regional (north, south) and provincial levels and to analyse disparities between rural and urban areas.
Overall, the total PR of NTDs was 14.0 per 10,000 births. The PRR of NTDs of rural women between the north and south region was 2.26 (95% CI: 2.04-2.52), which was much higher than that of urban women (PRR: 1.56, 95% CI: 1.41-1.72). The three subtypes of NTDs had different geographic distribution at the level of province. The urban–rural PRR of NTDs was 2.14 (95% CI: 1.94-2.34) in the north but only 1.47 (95% CI: 1.31-1.66) in the south.
There is a high total prevalence of NTDs, which remains one of the major public health concerns in China. Eliminating the geographic and urban–rural disparities in the disease burden is a priority for future intervention.
PMCID: PMC3599801  PMID: 23433029
Neural tube defects; Prevalence ratio; Geographic disparity; Urban–rural disparity
15.  Chloromethylated Activated Carbon: A Useful New Synthon for Making a Novel Class of Sorbents for Heavy Metal Separations 
Separation science and technology  2010;45(2):228-235.
The chloromethylation of activated carbon is described. Chloromethylation was found to produce a carbon derivative with a surface area of 1310 m2/g and no significant change in the pore structure. The product was found to contain ~1.5 mmole of –CH2Cl groups per g of material, similar to the functional density reported in the original Merrifield resin synthesis. Displacement of the benzylic chloride was achieved by treating this material with an excess of sodium thiosulfate in refluxing aqueous methanol. The resulting Bunte salt was then hydrolyzed by treatment with warm 3 M HCl to afford the corresponding thiol (“AC-CH2-SH”) cleanly and in high yield. AC-CH2-SH was found to be an effective heavy metal sorbent, efficiently capturing Hg, Pb, Ag, and Cu. Sorption kinetics were rapid, with equilibrium achieved in less than 30 minutes.
PMCID: PMC3563250  PMID: 23390326
activated carbon; chemically modified; chloromethylation; thiol; nanoporous; sorbent; mercury; heavy metal
16.  Association between paraoxonase gene and stroke in the Han Chinese population 
BMC Medical Genetics  2013;14:16.
The human paraoxonase (PON) gene family has three isoforms: PON1, PON2 and PON3. These genes are implicated as potential risk factors of cerebrovascular disease and can prevent oxidative modification of low-density lipoproteins and atherosclerosis. This study evaluated the association between the genetic variants of all three PON genes and the risks of total stroke, ischemic stroke and hemorrhagic stroke in the Han Chinese population.
A total of 1016 subjects were recruited, including 508 healthy controls and 498 patients (328 with ischemic stroke and 170 with hemorrhagic stroke). A total of 11 single nucleotide polymorphisms (SNPs) covering the PON genes were genotyped for statistical analysis. Two of the 11 SNPs (rs662 and rs854560) were contextualized in a meta-analysis of ischemic stroke.
The presence of rs705381 (−162) in the promoter region of PON1 was significantly associated with total stroke (Padjusted = 0.0007, OR = 0.57 [95% CI = 0.41-0.79]) and ischemic stroke (Padjusted = 0.0017, OR = 0.54 [95% CI = 0.37-0.79]) when analyzed using a dominant model, but was not associated with hemorrhagic stroke. There was also a nominal association between rs854571 (−824) and total stroke. Meta-analysis demonstrated a significant nominal association between rs662 and ischemic stroke, but there was no evidence of an association between rs662 and ischemic stroke risk in a single site association study.
These findings indicate that polymorphisms of PON1 gene may be a risk factor of stroke.
PMCID: PMC3562169  PMID: 23356507
Polymorphisms; Paraoxanase gene; Hemorrhagic stroke; Ischemic stroke; Association
17.  ENU-induced phenovariance in mice: inferences from 587 mutations 
BMC Research Notes  2012;5:577.
We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype.
In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied.
Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.
PMCID: PMC3532239  PMID: 23095377
N-ethyl-N-nitrosourea; Mouse; C57BL/6J; Mutagenesis; Genetic screen; PolyPhen-2; Strand asymmetry; Phenotype
18.  Receptor protein tyrosine phosphatases are novel components of a polycystin complex 
Biochimica et biophysica acta  2010;1812(10):1225-1238.
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, but the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling.
PMCID: PMC3156852  PMID: 21126580
polycystins; tyrosine kinase; tyrosine phosphatase; adherens junctions; primary cilium; G-protein coupled signaling
19.  Kinase Suppressor of Ras (KSR1) modulates multiple Kit-ligand-dependent mast cell functions 
Experimental hematology  2011;39(10):969-976.
The intricately regulated Ras pathway coordinates multiple kit-ligand induced mast cell functions, including chemotaxis, proliferation and degranulation. However, the intracellular proteins that modulate the intensity and duration of SCF-induced signals and the consequent cellular response are incompletely understood. Scaffolding proteins coordinate the spatial organization of MAPK proteins that may potentiate and/or inhibit cell functions. The Kinase Suppressor of Ras (KSR1) protein is known to function as a molecular scaffold and coordinates the organization of Raf/Mek/Erk in response to receptor tyrosine kinases. However, the impact of KSR1 in myeloid mast cell functions and in response to stem cell factor remains unknown. In the present study, we investigated the role of KSR1 in regulating cellular functions of bone marrow derived mast cells (BMMCs) of KSR1 deficient (−/−) mice. Genetic disruption of KSR1 resulted in both striking reductions in kit-ligand mediated proliferation and degranulation that are commonly attributed to MAPK signals. Surprisingly, disruption of the KSR1 scaffold also resulted in a decline in migration that is generally not linked to Raf-Erk signals. We found that loss of KSR1 does impact the biochemical activation of Pak kinase, a kinase that is known to modulate Raf-Erk signals and also F-actin polymerization key to mast cell migration. Collectively, these studies demonstrate that the scaffolding protein KSR1 has an important role in multiple kit-ligand mediated mast cell functions. This study elucidates varied mast cell physiological functions for KSR1, including those related to cytoskeletal organization, and it suggests a novel molecular target for attenuating mast cell-mediated inflammation.
PMCID: PMC3182112  PMID: 21726514
20.  Alteration of astrocytes and Wnt/β-catenin signaling in the frontal cortex of autistic subjects 
Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. To date the etiology of this disorder is poorly understood. Studies suggest that astrocytes play critical roles in neural plasticity by detecting neuronal activity and modulating neuronal networks. Recently, a number of studies suggested that an abnormal function of glia/astrocytes may be involved in the development of autism. However, there is yet no direct evidence showing how astrocytes develop in the brain of autistic individuals.
Study subjects include brain tissue from autistic subjects, BTBR T + tfJ (BTBR) and Neuroligin (NL)-3 knock-down mice. Western blot analysis, Immunohistochemistry and confocal microscopy studies have be used to examine the density and morphology of astrocytes, as well as Wnt and β-catenin protein expression.
In this study, we demonstrate that the astrocytes in autisitcsubjects exhibit significantly reduced branching processes, total branching length and cell body sizes. We also detected an astrocytosis in the frontal cortex of autistic subjects. In addition, we found that the astrocytes in the brain of an NL3 knockdown mouse exhibited similar alterations to what we found in the autistic brain. Furthermore, we detected that both Wnt and β-catenin proteins are decreased in the frontal cortex of autistic subjects. Wnt/β-catenin pathway has been suggested to be involved in the regulation of astrocyte development.
Our findings imply that defects in astrocytes could impair neuronal plasticity and partially contribute to the development of autistic-like behaviors in both humans and mice. The alteration of Wnt/β-catenin pathway in the brain of autistic subjects may contribute to the changes of astrocytes.
PMCID: PMC3544729  PMID: 22999633
Autism; Astrocytes; Morphology; Wnt/β-catenin pathway; Neural plasticity
21.  Improved deposition and deprotection of silane tethered 3,4 hydroxypyridinone (HOPO) ligands on functionalized nanoporous silica 
An improved synthesis of a 3,4 hydroxypyridinone (HOPO) functionalized mesoporous silica is described. Higher 3,4-HOPO monolayer ligand loadings have been achieved, resulting in better performance. Performance improvements were demonstrated with the capture of U(VI) from human blood, plasma and filtered river water.
PMCID: PMC3430465  PMID: 22942668
Nanoporous sorbent; Uranium removal; HOPO; HOPO; Blood
22.  Estimating the Numbers of Malaria Infections in Blood Samples Using High-Resolution Genotyping Data 
PLoS ONE  2012;7(8):e42496.
People living in endemic areas often habour several malaria infections at once. High-resolution genotyping can distinguish between infections by detecting the presence of different alleles at a polymorphic locus. However the number of infections may not be accurately counted since parasites from multiple infections may carry the same allele. We use simulation to determine the circumstances under which the number of observed genotypes are likely to be substantially less than the number of infections present and investigate the performance of two methods for estimating the numbers of infections from high-resolution genotyping data.
The simulations suggest that the problem is not substantial in most datasets: the disparity between the mean numbers of infections and of observed genotypes was small when there was 20 or more alleles, 20 or more blood samples, a mean number of infections of 6 or less and where the frequency of the most common allele was no greater than 20%. The issue of multiple infections carrying the same allele is unlikely to be a major component of the errors in PCR-based genotyping.
Simulations also showed that, with heterogeneity in allele frequencies, the observed frequencies are not a good approximation of the true allele frequencies. The first method that we proposed to estimate the numbers of infections assumes that they are a good approximation and hence did poorly in the presence of heterogeneity. In contrast, the second method by Li et al estimates both the numbers of infections and the true allele frequencies simultaneously and produced accurate estimates of the mean number of infections.
PMCID: PMC3430681  PMID: 22952595
23.  ZBTB1 is a determinant of lymphoid development 
ZBTB1, a BTB-ZF family member, is essential for T cell development and for complete B and NK cell differentiation.
In this study, we describe a chemically induced mouse mutation that caused a complete and cell-intrinsic T cell deficiency. Development of other lymphoid lineages was also partially impaired and was severely compromised under competitive conditions. Positional cloning, retroviral transduction, and a somatic reversion event revealed that the causative mutation lay within Zbtb1 (zinc finger and BTB domain containing 1), a gene conserved throughout vertebrate evolution. Our data establish ZBTB1 as a critical determinant of T cell development and lymphopoiesis in general, most likely by acting as a transcriptional regulator.
PMCID: PMC3260866  PMID: 22201126
24.  Conversion of Isoprenoid Oil by Catalytic Cracking and Hydrocracking over Nanoporous Hybrid Catalysts 
In order to produce petroleum alternatives from biomass, a significant amount of research has been focused on oils from microalgae due to their origin, which would not affect food availability. Nanoporous hybrid catalysts composed of ns Al2O3 and zeolites have been proven to be very useful compared to traditional catalysts in hydrotreating (HT), hydrocracking (HC), and catalytic cracking (CC) of large molecules. To evaluate the reaction scheme and products from model isoprenoid compounds of microalgae oil, nanoporous hybrid catalyst technologies (CC: ns Al2O3/H-USY and ns Al2O3/H-GaAlMFI; HC: [Ni-Mo/γ-Al2O3]/ns Al2O3/H-beta) were studied. The major product from CC on ns Al2O3/H-USY was highly aromatic gasoline, while the product from HC was half-isoparaffinic/olefinic kerosene. Although more than 50 wt% of the products from HT/CC on the USY catalyst was liquefied petroleum gas due to overcracking, the product from HT/CC on the MFI catalyst was high-octane-number gasoline. Delightfully, the product from HT/HC was kerosene and its average number was 11, with more than 80 wt% being isoparaffinic. As a result, it was demonstrated that hydrotreating may convert isoprenoid oil from microalgae over nanoporous hybrid catalysts into a variety of products.
PMCID: PMC3391972  PMID: 22791962
25.  The Changes in Maternal Mortality in 1000 Counties in Mid-Western China by a Government-Initiated Intervention 
PLoS ONE  2012;7(5):e37458.
Since 2000, the Chinese government has implemented an intervention program to reduce maternal mortality and eliminate neonatal tetanus in accordance with the Millennium Development Goals 5. To assess the effectiveness of this intervention program, we analyzed the level, trend and reasons defining the maternal mortality ratio (MMR) in the 1,000 priority counties before and after implementation of the intervention between 1999 and 2007.
Methodology/Principal Findings
The data was obtained from the National Maternal and Child Health Routine Reporting System. The intervention included providing basic and emergency obstetric equipment and supplies to local medical hospitals, and also included providing professional training to local obstetric doctors, development of obstetric emergency centers and “green channel” express referral networks, reducing or waiving the cost of hospital delivery, and conducting community health education. Based on the initiation time of the intervention and the level of poverty, 1,000 counties, containing a total population of 300 million, were categorized into three groups. MMR significantly decreased by about 50%, with an average reduction rate of 9.24%, 16.06%, and 18.61% per year in the three county groups, respectively. The hospital delivery rate significantly increased. Obstetric hemorrhage was the leading cause of maternal deaths and significantly declined, with an average decrease in the MMR of 11.25%, 18.03%, and 24.90% per year, respectively. The magnitude of the MMR, the average reduction rate of the MMR, and the occurrence of the leading causes of death were closely associated with the percentage of poverty.
The intervention program implemented by the Chinese government has significantly reduced the MMR in mid-western China, suggesting that well-targeted interventions could be an efficient strategy to reducing MMR in resource-poor areas. Reduction of the MMR not only depends on conducting proven interventions, but also relies on economic development in rural areas with a high burden of maternal death.
PMCID: PMC3357422  PMID: 22629398

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