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1.  Process-Based Expansion and Neural Differentiation of Human Pluripotent Stem Cells for Transplantation and Disease Modeling 
Journal of neuroscience research  2013;91(10):1247-1262.
Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.
PMCID: PMC4285152  PMID: 23893392
cGMP; cellular therapy; differentiation; cellular models of disease; iPSCs; drug discovery; neural stem cells; neurons; glia; nomenclature; methods
2.  Characteristics of Nucleocytoplasmic Transport of H1N1 Influenza A Virus Nuclear Export Protein 
Journal of Virology  2014;88(13):7455-7463.
The influenza A virus nuclear export protein (NEP) plays crucial roles in the nuclear export of the viral ribonucleoprotein complex through the chromosome region maintenance 1 (CRM1)-mediated cellular protein transport system. However, the detailed mechanism of NEP nucleocytoplasmic trafficking remains incompletely understood. Here, we investigated the subcellular localization of NEP from two strains of H1N1 influenza A virus and found that 2009 swine-origin H1N1 influenza A virus A/California/04/2009 (CA04) NEP displayed a distinct cellular distribution pattern, forming unique nuclear aggregates, compared to A/WSN/33 (H1N1) (WSN) NEP. Characterization of the nucleocytoplasmic transport pathways of these two NEPs showed that they both enter the nucleus by passive diffusion but are exported through the nuclear export receptor CRM1-mediated pathway with different efficiencies. The two identified nuclear export signals (NESs) on the two NEPs functioned similarly despite differences in their amino acid sequences. Using a two-hybrid assay, we confirmed that the CA04 NEP interacts less efficiently with CRM1 and that a threonine residue at position 48 is responsible for the nuclear aggregation. The present study revealed the dissimilarity in subcellular NEP transport processes between the 2009 pandemic (H1N1) influenza A virus CA04 and the laboratory-adapted H1N1 virus WSN and uncovered the mechanism responsible for this difference.
IMPORTANCE Because the efficiency of the nucleocytoplasmic transport of viral components is often correlated with the viral RNA polymerase activity, propagation, and host range of influenza viruses, the present study investigated the subcellular localization of NEP from two strains of H1N1 influenza virus. We found that the NEPs of both A/California/04/2009 (H1N1) (CA04) and A/WSN/33 (H1N1) (WSN) enter the nucleus by passive diffusion but are exported with different efficiencies, which were caused by weaker binding activity between the CA04 NEP and CRM1. The results of the present study revealed characteristics of the nuclear import and export pathways of NEP and the mechanism responsible for the differences in the cellular distribution of NEP between two H1N1 strains.
PMCID: PMC4054460  PMID: 24741105
3.  The Association of the MTHFR c.1625A>C Genetic Variant with the Risk of Congenital Heart Diseases in the Chinese 
The purpose of this study is to investigate the association of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with the risk of congenital heart diseases (CHD). The genotypes of the MTHFR genetic variant were determined by the polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing methods. Our data suggested that the allelic and genotypic frequencies of CHD patients were significantly different from non-CHD controls. The MTHFR c.1625A>C genetic variant was significantly associated with the increased risk of CHD (CC vs. AA: odds ratio [OR]=2.29, 95% confidence interval [CI] 1.15–4.53, p=0.016; C vs. A: OR=1.47, 95% CI 1.11–1.96, p=0.008). Results from this study indicate that the MTHFR c.1625A>C genetic variant influences the risk of CHD in the studied population.
PMCID: PMC4278078  PMID: 25494855
4.  A Highly Selective and Sensitive Turn-On Fluorescent Chemosensor Based on Rhodamine 6G for Iron(III)** 
ChemistryOpen  2014;3(6):264-268.
Recently, more and more rhodamine derivatives have been used as fluorophores to construct sensors due to their excellent spectroscopic properties. A rhodamine-based fluorescent and colorimetric Fe3+ chemosensor 3’,6’-bis(ethylamino)-2-acetoxyl-2’,7’-dimethyl-spiro[1H-isoindole-1,9’-[9H]xanthen]-3(2H)-one (RAE) was designed and synthesized. Upon the addition of Fe3+, the dramatic enhancement of both fluorescence and absorbance intensity, as well as the color change of the solution, could be observed. The detection limit of RAE for Fe3+ was around 7.98 ppb. Common coexistent metal ions showed little or no interference in the detection of Fe3+. Moreover, the addition of CN− could quench the fluorescence of the acetonitrile solution of RAE and Fe3+, indicating the regeneration of the chemosensor RAE. The robust nature of the sensor was shown by the detection of Fe3+ even after repeated rounds of quenching. As iron is a ubiquitous metal in cells and plays vital roles in many biological processes, this chemosensor could be developed to have applications in biological studies.
PMCID: PMC4280826  PMID: 25558445
chemosensors; fluorescence; iron(III); rhodamine; selective response
5.  MicroRNA-543 acts as an oncogene by targeting PAQR3 in hepatocellular carcinoma 
MicroRNAs (miRNAs) are small, non-coding RNAs that can act as oncogenes or tumor suppressor genes in human cancer. Increasing evidences indicate that deregulation of miRNAs contributes to the hepatocarcinogenesis. In this study, we demonstrated that the levels of miR-543 were dramatically increased in clinical hepatocellular carcinoma (HCC) tissues and cell lines. Moreover, forced expression of miR-543 promoted the proliferative and invasive potential of HepG2. We also identified PAQR3 as a direct target gene for miR-543 using a fluorescent reporter assay and western blot. The levels of PAQR3 were dramatically decreased in clinical hepatocellular carcinoma (HCC) tissues and cell lines. The mRNA levels of PAQR3 were inversely correlated with the miR-543 expression level.Thus, our finding provides a new insight into the mechanism of hepatocarcinogenesis, indicating a therapeutic potential of miR-543 in the treatment of HCC.
PMCID: PMC4266721  PMID: 25520877
miR-543; PAQR3; hepatocellular carcinoma; oncogene
6.  TNFα Promotes Th17 Cell Differentiation through IL-6 and IL-1β Produced by Monocytes in Rheumatoid Arthritis 
Journal of Immunology Research  2014;2014:385352.
TNFα plays an important role in autoimmune pathogenesis and is the main therapeutic target of rheumatoid arthritis. However, its underlying mechanism is not completely understood. In this study, we described that Th17 cells were accumulated in synovial fluid, which was attributable to TNFα aberrantly produced in rheumatoid synovium. Interestingly, TNFα cannot induce IL-17 production of CD4+ T cells directly, but through the monocytes high levels of IL-1β and IL-6 in a TNFRI and TNFRII dependent manner from the active RA patients are produced. TNFα was shown to enhance the phosphorylation level of STAT3 and the expression level of transcription factor RORC of CD4+ T cells when cultured with CD14+ monocytes. Treatment with an approved TNFα blocking antibody showed marked reduction in the levels of IL-6, IL-1β, and IL-17 and the expression level of STAT3 phosphorylation in relation to Th17 cell differentiation in patients with rheumatoid arthritis. The study provides new evidence supporting the critical role of TNFα in the pathogenic Th17 cell differentiation in rheumatoid arthritis.
PMCID: PMC4243768  PMID: 25436214
7.  Assessment of the Internal Genes of Influenza A (H7N9) Virus Contributing to High Pathogenicity in Mice 
Journal of Virology  2014;89(1):2-13.
The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans.
IMPORTANCE To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.
PMCID: PMC4301103  PMID: 25320305
8.  Genetic Modifiers of Cystic Fibrosis–Related Diabetes 
Diabetes  2013;62(10):3627-3635.
Diabetes is a common age-dependent complication of cystic fibrosis (CF) that is strongly influenced by modifier genes. We conducted a genome-wide association study in 3,059 individuals with CF (644 with CF-related diabetes [CFRD]) and identified single nucleotide polymorphisms (SNPs) within and 5′ to the SLC26A9 gene that associated with CFRD (hazard ratio [HR] 1.38; P = 3.6 × 10−8). Replication was demonstrated in 694 individuals (124 with CFRD) (HR, 1.47; P = 0.007), with combined analysis significant at P = 9.8 × 10−10. SLC26A9 is an epithelial chloride/bicarbonate channel that can interact with the CF transmembrane regulator (CFTR), the protein mutated in CF. We also hypothesized that common SNPs associated with type 2 diabetes also might affect risk for CFRD. A previous association of CFRD with SNPs in TCF7L2 was replicated in this study (P = 0.004; combined analysis P = 3.8 × 10−6), and type 2 diabetes SNPs at or near CDKAL1, CDKN2A/B, and IGF2BP2 were associated with CFRD (P < 0.004). These five loci accounted for 8.3% of the phenotypic variance in CFRD onset and had a combined population-attributable risk of 68%. Diabetes is a highly prevalent complication of CF, for which susceptibility is determined in part by variants at SLC26A9 (which mediates processes proximate to the CF disease-causing gene) and at four susceptibility loci for type 2 diabetes in the general population.
PMCID: PMC3781476  PMID: 23670970
9.  MiR-424-5p reversed epithelial-mesenchymal transition of anchorage-independent HCC cells by directly targeting ICAT and suppressed HCC progression 
Scientific Reports  2014;4:6248.
Resistance to anoikis and Epithelial-mesenchymal transition (EMT) are two processes critically involved in cancer metastasis. In this study, we demonstrated that after anchorage deprival, hepatocellular carcinoma (HCC) cells not only resisted anoikis, but also exhibited EMT process. Microarray expression profiling revealed that expression of miR-424-5p was significantly decreased in anoikis-resistant HCC cells. Ectopic overexpression of miR-424-5p was sufficient to reverse resistance to anoikis, block EMT process and inhibit malignant behaviors of HCC cells. Target analysis showed that a potent β-catenin inhibitor, ICAT/CTNNBIP1 was a direct target of miR-424-5p. Further study demonstrated that miR-424-5p reversed resistance to anoikis and EMT of HCCs by directly targeting ICAT and further maintaining the E-cadherin/β-catanin complex on the cellular membrance. In vivo study further demonstrated that miR-424-5p significantly inhibited the tumorigenicity of HCC cells in nude mice. Clinical investigation demonstrated that miR-424-5p was significantly downregulated in HCC tissues compared with that of the non-cancerous liver tissues, and this decreased expression of miR-424-5p was significantly correlated with higher pathological grades and more advanced TNM stages. Therefore, aberrant expression of miR-424-5p is critically involved in resistance to anoikis and EMT during the metastatic process of HCC, and its downregulation significantly contributes to liver cancer progression.
PMCID: PMC4150107  PMID: 25175916
10.  Topological Organization of Multi-chromosomal Regions by Firre 
RNA is known to be an abundant and important structural component of the nuclear matrix, including long noncoding RNAs (lncRNA). Yet the molecular identities, functional roles, and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear matrix factor hnRNPU, through a 156 bp repeating sequence and Firre localizes across a ~5 Mb domain on the X-chromosome. We further observed Firre localization across at least five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X-chromosome. Both genetic deletion of the Firre locus or knockdown of hnRNPU resulted in loss of co-localization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.
PMCID: PMC3950333  PMID: 24463464
11.  Epigenetic Regulation of SOX9 by the NF-κB Signaling Pathway in Pancreatic Cancer Stem Cells 
Stem cells (Dayton, Ohio)  2013;31(8):1454-1466.
Pancreatic cancer is the fourth leading cause of cancer-related mortality in the world. Pancreatic cancer can be localized, locally advanced or metastatic. The median 1- and 5-year survival rates are 25% and 6%, respectively. Epigenetic modifications such as DNA methylation play a significant role during both normal human development and cancer progression. To investigate epigenetic regulation of genes in the tumor-initiating population of pancreatic cancer cells, which are also termed cancer stem cells (CSCs), we conducted epigenetic arrays in PANC1 and HPAC pancreatic cancer cell lines and compared the global DNA methylation status of CpG promoters in invasive cells, demonstrated to be CSCs, to their non-invasive counterparts, or non-CSCs. Our results suggested that the NF-κB pathway is one of the most activated pathways in pancreatic CSCs. In agreement with this, we determined that upon treatment with NF-κB pathway inhibitors, the stem cell-like properties of cells are significantly disrupted. Moreover, SOX9, demethylated in CSCs, is shown to play a crucial role in the invasion process. Additionally, we found a potential NF-κB binding site located in the SOX9 promoter, and determined that the NF-κB subunit p65 positively regulates SOX9 expression by binding to its promoter directly. This interaction can be efficiently blocked by NF-κB inhibitors. Thus, our work establishes a link between the classical NF-κB signaling transduction pathway and the invasiveness of pancreatic CSCs, which may result in the identification of novel signals and molecules that function at an epigenetic level, and could potentially be targeted for pharmaceutical investigations and clinical trials.
PMCID: PMC3775871  PMID: 23592398
Pancreatic cancer; Cancer stem cells; Invasion; Epigenetic regulation; NF-κB signaling; SOX9
12.  A Digital Multigate Doppler Method for High Frequency Ultrasound 
Sensors (Basel, Switzerland)  2014;14(8):13348-13360.
Noninvasive visualization of blood flow with high frequency Doppler ultrasound has been extensively used to assess the morphology and hemodynamics of the microcirculation. A completely digital implementation of multigate pulsed-wave (PW) Doppler method was proposed in this paper for high frequency ultrasound applications. Analog mixer was eliminated by a digital demodulator and the same data acquisition path was shared with traditional B-mode imaging which made the design compact and flexible. Hilbert transform based quadrature demodulation scheme was employed to achieve the multigate Doppler acquisition. A programmable high frequency ultrasound platform was also proposed to facilitate the multigate flow visualization. Experimental results showed good performance of the proposed method. Parabolic velocity gradient inside the vessel and velocity profile with different time slots were acquired to demonstrate the functionality of the multigate Doppler. Slow wall motion was also recorded by the proposed method.
PMCID: PMC4178981  PMID: 25061836
flow visualization; multigate PW Doppler; digital quadrature demodulation; high frequency ultrasound
13.  High Performance Relaxor-Based Ferroelectric Single Crystals for Ultrasonic Transducer Applications 
Sensors (Basel, Switzerland)  2014;14(8):13730-13758.
Relaxor-based ferroelectric single crystals Pb(Mg1/3Nb2/3)O3-PbTiO3 (PMN-PT) have drawn much attention in the ferroelectric field because of their excellent piezoelectric properties and high electromechanical coupling coefficients (d33∼2000 pC/N, kt∼60%) near the morphotropic phase boundary (MPB). Ternary Pb(In1/2Nb1/2)O3-Pb(Mg1/3Nb2/3)O3-PbTiO3 (PIN-PMN-PT) single crystals also possess outstanding performance comparable with PMN-PT single crystals, but have higher phase transition temperatures (rhombohedral to tetragonal Trt, and tetragonal to cubic Tc) and larger coercive field Ec. Therefore, these relaxor-based single crystals have been extensively employed for ultrasonic transducer applications. In this paper, an overview of our work and perspectives on using PMN-PT and PIN-PMN-PT single crystals for ultrasonic transducer applications is presented. Various types of single-element ultrasonic transducers, including endoscopic transducers, intravascular transducers, high-frequency and high-temperature transducers fabricated using the PMN-PT and PIN-PMN-PT crystals and their 2-2 and 1-3 composites are reported. Besides, the fabrication and characterization of the array transducers, such as phased array, cylindrical shaped linear array, high-temperature linear array, radial endoscopic array, and annular array, are also addressed.
PMCID: PMC4178991  PMID: 25076222
PMN-PT; PIN-PMN-PT; single crystals; composites; ultrasonic transducers
14.  Occurrence of mycotoxins in feed ingredients and complete feeds obtained from the Beijing region of China 
The current study was carried out to provide a reference for the control of mycotoxin contamination in feed ingredients and complete feeds for swine.
A total of 55 feed ingredients, including 14 corn, 13 wheat bran, 11 soybean meal and 17 dried distillers grains with solubles (DDGS) as well as 76 complete swine feeds including 7 creep feeds, 14 starter feeds, 14 grower feeds, 18 grower-finisher feeds, 10 gestating sow feeds, and 13 lactating sow feeds were randomly collected from 15 swine farms located in the Beijing region of China from July to August 2011. Immunoaffinity clean-up, using High-Performance Liquid Chromatography (HPLC) in combination with UV or Fluorescence Detection, was used for quantitative analysis of aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEA) and ochratoxin A (OTA) in the ingredients and complete feeds.
DON and ZEA were the most prevalent mycotoxins found. DON was detected at percentages of 93, 92, 54, 100 and 97% with a mean level of 1.01, 0.44, 0.05, 1.36 and 0.65 ppm in the samples of corn, wheat bran, soybean meal, DDGS and complete feeds, respectively. The detected percentages of ZEA were 100, 100, 54, 100 and 100 with mean levels of 109.1, 14.9, 9.2, 882.7 and 58.9 ppb in the same samples. In the wheat bran and soybean meal samples, the content of all four mycotoxins were below the maximum limits set by Chinese regulations while the percentage of samples that exceeded regulatory limits were 7, 57 and 7% for corn, and 7, 14 and 3% for the complete feeds for AFB1, DON and OTA respectively. DDGS showed the most serious mycotoxin contamination and the percentage of samples that exceeded regulatory limits were 6, 88 and 41%, for AFB1, DON and ZEA, respectively.
This paper is the first to present data on the natural occurrence of AFB1, DON, ZEA and OTA in ingredients and complete feeds obtained from swine farms in China’s Beijing region. The data shows that feed ingredients and complete swine feeds obtained from these farms are most often contaminated with DON followed by contamination with AFB1 and ZEA.
PMCID: PMC4123309  PMID: 25101169
AflatoxinB1; Beijing region; Deoxynivalenol; Ochratoxin A; Swine feeds; Zearalenone
15.  Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux 
Nature cell biology  2013;15(2):143-156.
Many unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell C.elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1 overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity. AQP-8 is transiently recruited to the lumen by ERM-1, co-localizing in peri-lumenal cuffs interspaced along expanding canals. An ERM-1[++]-mediated increase in the number of lumen-associated canaliculi is reversed by AQP-8 depletion. We propose that the ERM-1-AQP-8 interaction propels lumen extension by translumenal flux, suggesting a direct morphogenetic effect of water-channel-regulated fluid pressure.
PMCID: PMC4091717  PMID: 23334498
16.  A Study of the Adult Zebrafish Ventricular Function by Retrospective Doppler-Gated Ultrahigh-Frame-Rate Echocardiography 
The zebrafish (Danio rerio) has become a preferred animal model for studying various human diseases, particularly those related to cardiovascular regeneration; therefore, a noninvasive imaging modality is needed for observing the cardiac function of zebrafish. Because of its high resolution, high-frequency ultrasound B-mode imaging has recently been used successfully to observe the heart of adult zebrafish. However, ultrahigh-frame-rate echocardiography combining B-mode imaging and color flow imaging is still needed to observe the detailed transient motions of the zebrafish ventricle. This study develops an 80-MHz ultrahigh-frame-rate echocardiography system for this purpose, based on retrospective Doppler- gated technology. B-mode and color flow images of the cardiovascular system of the zebrafish were reconstructed by two-dimensional autocorrelation at maximum frame rates of up to 40 000 and 400 fps, respectively. The timings of end diastole (ED) and end systole (ES) of ventricle can be determined by using this high-resolution image system. Two ventricular function parameters—fractional shortening (FS) and fractional area change (FAC)—were measured for evaluating the ventricular function by using ED and ES with their corresponding ventricular dimensions. The experimental results indicated that the measured FS values were 42 ± 4% (mean ± standard deviation) and 60 ± 13% for the long axis and short axis of the ventricle, respectively, and that FAC was 77 ± 9%. This is the first report of these ventricular function parameters for a normal adult zebrafish. The results showed that retrospective high-frequency echocardiography is a useful tool for studying the cardiac function of normal adult zebrafish.
PMCID: PMC4091976  PMID: 24658716
17.  Preserving Sialic Acid-dependent Pattern Recognition by CD24-Siglec G Interaction for Therapy of Polybacterial Sepsis 
Nature biotechnology  2011;29(5):428-435.
Control of inflammation is critical for therapy of infectious diseases. Pathogen-associated and/or danger-associated molecular patterns (PAMPs and DAMPs, respectively) are the two major inducers of inflammation. Because the CD24-Siglec G/10 interactions selectively repress inflammatory response to DAMPs, microbial disruption of the negative regulation would provide a general mechanism to exacerbate inflammation. Here we show that the sialic acid-based pattern recognitions of CD24 by Siglec G/10 are targeted by sialidases in polybacterial sepsis. Sialidase inhibitors protect mice against sepsis by a CD24-Siglecg-dependent mechanism, whereas a targeted mutation of either CD24 or Siglecg exacerbates sepsis. Bacterial sialidase and host CD24 and Siglecg genes interact to determine pathogen virulence. Our data demonstrate a critical role for disrupting sialic acid-based pattern recognitions in microbial virulence and suggest a therapeutic approach to dampen harmful inflammatory response during infection.
PMCID: PMC4090080  PMID: 21478876
18.  The differentiation of pancreatic tumor-initiating cells by vitronectin can be blocked by Cilengitide 
Pancreas  2013;42(5):861-870.
Pancreatic cancer is a leading cancer type and its molecular pathology is poorly understood. The only potentially curative therapeutic option available is complete surgical resection; however, this is inadequate as a majority of patients are diagnosed at an advanced or metastatic stage. Tumor-initiating cells constitute a subpopulation of cells within a solid tumor that sustain tumor growth, metastasis and chemo-/radio-resistance. Within pancreatic cancer, tumor-initiating cells have been identified based on the expression of specific cell surface markers.
We utilize a sphere formation assay to enrich for putative TICs and use human serum as a driver of differentiation. We demonstrate using specific blocking reagents that we can inhibit the differentiation process and maintain tumor-initiating cell associated markers and genes.
We can induce differentiation of pancreatospheres with the addition of human serum and identified vitronectin as an inducer of differentiation. We inhibit differentiation by human serum using an arginine-glycine-aspartate specific peptide, Cilengitide; hence, demonstrating this differentiation is mediated via specific integrin receptors.
Overall, our studies further the definition of pancreatic tumor-initiating cells and provide further insight into both the maintenance and differentiation of this lethal population.
PMCID: PMC3676482  PMID: 23462327
Pancreatic cancer; TICs; vitronectin; differentiation; Cilengitide
19.  Using a Bayesian latent variable approach to detect pleiotropy in the Genetic Analysis Workshop 18 data 
BMC Proceedings  2014;8(Suppl 1):S77.
Pleiotropy, which occurs when a single genetic factor influences multiple phenotypes, is present in many genetic studies of complex human traits. Longitudinal family data, such as the Genetic Analysis Workshop 18 data, combine the features of longitudinal studies in individuals and cross-sectional studies in families, thus providing richer information about the genetic and environmental factors associated with the trait of interest. We recently proposed a Bayesian latent variable methodology for the study of pleiotropy, in the presence of longitudinal and family correlation. The purpose of this work is to evaluate the Bayesian latent variable method in a real data setting using the Genetic Analysis Workshop 18 blood pressure phenotypes and sequenced genotype data. To detect single-nucleotide polymorphisms with pleiotropic effect on both diastolic and systolic blood pressure, we focused on a set of 6 single-nucleotide polymorphisms from chromosome 3 that was reported in the literature to be significantly associated with either diastolic blood pressure or the binary hypertension trait. Our analysis suggests that both diastolic blood pressure and systolic blood pressure are associated with the latent hypertension severity variable, but the analysis did not find any of the 6 single-nucleotide polymorphisms to have statistically significant pleiotropic effect on both diastolic blood pressure and systolic blood pressure.
PMCID: PMC4143687  PMID: 25519405
20.  Evaluation of gene-based association tests for analyzing rare variants using Genetic Analysis Workshop 18 data 
BMC Proceedings  2014;8(Suppl 1):S9.
The focus of our work is to evaluate several recently developed pooled association tests for rare variants and assess the impact of different gene annotation methods and binning strategies on the analyses of rare variants under Genetic Analysis Workshop 18 real and simulated data settings. We considered the sample of 103 unrelated individuals with sequence data, genotypes of rare variants from chromosome 3, real phenotype of hypertension status and simulated phenotypes of systolic blood pressure (SBP) and diastolic blood pressure (DBP), and covariates of age, sex, and the interaction between age and sex. In the analysis of real phenotype data, we did not obtain significant results for any binning strategy; however, we observed a slight deviation of the p-values from the uniform distribution based on the protein-damaging variant grouping strategy. Evaluation of methods using simulated data showed lack of power even at the conservative level of 0.05 for most of the causal genes on chromosome 3. Nevertheless, analysis of MAP4 produced good power for all tests at various levels of the tests for both DBP and SBP. Our results also confirmed that Fisher's method is not only robust but can also improve power over individual pooled linear and quadratic tests and is often better than other robust tests such as SKAT-O.
PMCID: PMC4143759  PMID: 25519417
21.  PREST-plus identifies pedigree errors and cryptic relatedness in the GAW18 sample using genome-wide SNP data 
BMC Proceedings  2014;8(Suppl 1):S23.
Pedigree errors and cryptic relatedness often appear in families or population samples collected for genetic studies. If not identified, these issues can lead to either increased false negatives or false positives in both linkage and association analyses. To identify pedigree errors and cryptic relatedness among individuals from the 20 San Antonio Family Studies (SAFS) families and cryptic relatedness among the 157 putatively unrelated individuals, we apply PREST-plus to the genome-wide single-nucleotide polymorphism (SNP) data and analyze estimated identity-by-descent (IBD) distributions for all pairs of genotyped individuals. Based on the given pedigrees alone, PREST-plus identifies the following putative pairs: 1091 full-sib, 162 half-sib, 360 grandparent-grandchild, 2269 avuncular, 2717 first cousin, 402 half-avuncular, 559 half-first cousin, 2 half-sib+first cousin, 957 parent-offspring and 440,546 unrelated. Using the genotype data, PREST-plus detects 7 mis-specified relative pairs, with their IBD estimates clearly deviating from the null expectations, and it identifies 4 cryptic related pairs involving 7 individuals from 6 families.
PMCID: PMC4143714  PMID: 25519375
22.  Genetic Analysis Workshop 18: Methods and strategies for analyzing human sequence and phenotype data in members of extended pedigrees 
BMC Proceedings  2014;8(Suppl 1):S1.
Genetic Analysis Workshop 18 provided a platform for developing and evaluating statistical methods to analyze whole-genome sequence data from a pedigree-based sample. In this article we present an overview of the data sets and the contributions that analyzed these data. The family data, donated by the Type 2 Diabetes Genetic Exploration by Next-Generation Sequencing in Ethnic Samples Consortium, included sequence-level genotypes based on sequencing and imputation, genome-wide association genotypes from prior genotyping arrays, and phenotypes from longitudinal assessments. The contributions from individual research groups were extensively discussed before, during, and after the workshop in theme-based discussion groups before being submitted for publication.
PMCID: PMC4143625  PMID: 25519310
23.  Neural stem cell transplantation in a double-layer collagen membrane with unequal pore sizes for spinal cord injury repair 
Neural Regeneration Research  2014;9(10):1014-1019.
A novel double-layer collagen membrane with unequal pore sizes in each layer was designed and tested in this study. The inner, loose layer has about 100-μm-diameter pores, while the outer, compact layer has about 10-μm-diameter pores. In a rat model of incomplete spinal cord injury, a large number of neural stem cells were seeded into the loose layer, which was then adhered to the injured side, and the compact layer was placed against the lateral side. The results showed that the transplantation of neural stem cells in a double-layer collagen membrane with unequal pore sizes promoted the differentiation of neural stem cells, attenuated the pathological lesion, and significantly improved the motor function of the rats with incomplete spinal cord injuries. These experimental findings suggest that the transplantation of neural stem cells in a double-layer collagen membrane with unequal pore sizes is an effective therapeutic strategy to repair an injured spinal cord.
PMCID: PMC4146296  PMID: 25206753
nerve regeneration; spinal cord injury; collagen; scaffolds; neural stem cells; cell transplantation; nerve repair; neural regeneration
24.  Endoscopic papillary large balloon dilation vs endoscopic sphincterotomy for retrieval of common bile duct stones: A meta-analysis 
AIM: To compare the efficacy and safety of endoscopic papillary large balloon dilation (EPLBD) with endoscopic sphincterotomy (EST) in retrieval of common bile duct stones (≥ 10 mm).
METHODS: PubMed, Web of Knowledge, EBSCO, the Cochrane Library, and EMBASE were searched for eligible studies. Randomized controlled trials (RCTs) that compared EPLBD with EST were identified. Data extraction and quality assessment were performed by two independent reviewers using the same criteria. Any disagreement was discussed with a third reviewer until a final consensus was reached. Pooled outcomes of complete bile duct stone clearance, stone clearance in one session, requirement for mechanical lithotripsy, and overall complication rate were determined using relative risk and 95%CI. The separate post-endoscopic retrograde cholangiopancreatography complications were pooled and determined with the Peto odds ratio and 95%CI because of the small number of events. Heterogeneity was evaluated with the chi-squared test with P ≤ 0.1 and I2 with a cutoff of ≥ 50%. A fixed effects model was used primarily. A random effects model was applied when significant heterogeneity was detected. Sensitivity analysis was applied to explore the potential bias.
RESULTS: Five randomized controlled trials with 621 participants were included. EPLBD compared with EST had similar outcomes with regard to complete stone removal rate (93.7% vs 92.5%, P = 0.54) and complete duct clearance in one session (82.2% vs 77.7%, P = 0.17). Mechanical lithotripsy was performed less in EPLBD in the retrieval of whole stones (15.5% vs 25.2%, P = 0.003), as well as in the stratified subgroup of stones larger than 15 mm (24.2% vs 40%, P = 0.001). There was no statistically significant difference in the incidence of overall adverse events (7.9% vs 10.7%, P = 0.25), post-ERCP pancreatitis (4.0% vs 5.0%, P = 0.54), hemorrhage (1.7% vs 2.8%, P = 0.32), perforation (0.3% vs 0.9%, P = 0.35) or acute cholangitis (1.3% vs 1.3%, P = 0.92).
CONCLUSION: EPLBD could be advocated as an alternative to EST in the retrieval of large common bile duct stones.
PMCID: PMC4017071  PMID: 24833886
Endoscopic papillary large balloon dilation; Endoscopic sphincterotomy; Mechanical lithotripsy; Common bile duct stones; Meta analysis.

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