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Find video protocols related to scientific articles indexed in Pubmed.
Asymmetric dose-volume optimization with smoothness control for rotating-shield brachytherapy.
Med Phys
PUBLISHED: 11-06-2014
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It is important to reduce fluence map complexity in rotating-shield brachytherapy (RSBT) inverse planning to improve delivery efficiency while maintaining plan quality. This study proposes an efficient and effective RSBT dose optimization method which enables to produce smooth fluence maps.
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Genome-wide discovery of drug-dependent human liver regulatory elements.
PLoS Genet.
PUBLISHED: 10-01-2014
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Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.
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Invasive reaction assisted strand-displacement signal amplification for sensitive DNA detection.
Chem. Commun. (Camb.)
PUBLISHED: 09-25-2014
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A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification. The detection limit was around 0.2 pM.
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Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions.
J. Bacteriol.
PUBLISHED: 09-08-2014
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Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi, does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNA-Seq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi.
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The Induction of Prolonged Myelopoietic Effects in Monkeys by GW003, A Recombinant Human Granulocyte Colony-Stimulating Factor Genetically Fused to Recombinant Human Albumin.
J Pharm Sci
PUBLISHED: 08-29-2014
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GW003, a genetic fusion protein of human serum albumin and granulocyte colony-stimulating factor (G-CSF), was developed based on a novel strategy for producing long-acting proteins. The purpose of this study was to evaluate the hematologic, pharmacokinetic, and toxicokinetic effects of GW003 on cynomolgus monkeys. We show that following a single subcutaneous administration of GW003, the absolute neutrophil count increased significantly compared with monkeys that received only the vehicle, and the magnitude of the neutrophilic response to GW003 was dose dependent. After an injection at equal molar dose, the clearance of GW003 in the monkeys was approximately fourfold slower, and the terminal half-life (T1/2 ) was fivefold longer than the corresponding values for recombinant methionyl human G-CSF. Interestingly, both the clearance and T1/2 decreased with increasing doses of GW003, and much faster elimination was observed after multidose exposure. In toxicokinetic studies, the serum concentration of GW003 after the eighth injection was much lower than it was after the first injection, and a neutralizing antibody against G-CSF was found to have a dose-dependent effect upon the treatment groups. Overall, the favorable pharmacokinetic and pharmacodynamic properties supported the selection and development of GW003 as a promising candidate for neutropenia therapy. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
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HrrF is the Fur-regulated small RNA in nontypeable Haemophilus influenzae.
PLoS ONE
PUBLISHED: 08-26-2014
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Nontypeable Haemophilus influenzae (NTHi) are Gram-negative commensal bacteria that reside in the nasopharynx. NTHi can also cause multiple upper and lower respiratory tract diseases that include sinusitis, conjunctivitis, bronchitis, and otitis media. In numerous bacterial species the ferric uptake regulator (Fur) acts as a global regulator of iron homeostasis by negatively regulating the expression of iron uptake systems. However in NTHi strain 86-028NP and numerous other bacterial species there are multiple instances where Fur positively affects gene expression. It is known that many instances of positive regulation by Fur occur indirectly through a small RNA intermediate. However, no examples of small RNAs have been described in NTHi. Therefore we used RNA-Seq analysis to analyze the transcriptome of NTHi strain 86-028NPrpsL and an isogenic 86-028NPrpsL?fur strain to identify Fur-regulated intergenic transcripts. From this analysis we identified HrrF, the first small RNA described in any Haemophilus species. Orthologues of this small RNA exist only among other Pasteurellaceae. Our analysis showed that HrrF is maximally expressed when iron levels are low. Additionally, Fur was shown to bind upstream of the hrrF promoter. RNA-Seq analysis was used to identify targets of HrrF which include genes whose products are involved in molybdate uptake, deoxyribonucleotide synthesis, and amino acid biosynthesis. The stability of HrrF is not dependent on the RNA chaperone Hfq. This study is the first step in an effort to investigate the role small RNAs play in altering gene expression in response to iron limitation in NTHi.
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Fluorinated graphene and hexagonal boron nitride as ALD seed layers for graphene-based van der Waals heterostructures.
Nanotechnology
PUBLISHED: 08-12-2014
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Ultrathin dielectric materials prepared by atomic-layer-deposition (ALD) technology are commonly used in graphene electronics. Using the first-principles density functional theory calculations with van der Waals (vdW) interactions included, we demonstrate that single-side fluorinated graphene (SFG) and hexagonal boron nitride (h-BN) exhibit large physical adsorption energy and strong electrostatic interactions with H2O-based ALD precursors, indicating their potential as the ALD seed layer for dielectric growth on graphene. In graphene-SFG vdW heterostructures, graphene is n-doped after ALD precursor adsorption on the SFG surface caused by vertical intrinsic polarization of SFG. However, graphene-h-BN vdW heterostructures help preserving the intrinsic characteristics of the underlying graphene due to in-plane intrinsic polarization of h-BN. By choosing SFG or BN as the ALD seed layer on the basis of actual device design needs, the graphene vdW heterostructures may find applications in low-dimensional electronics.
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Epigenetic targeting of ovarian cancer stem cells.
Cancer Res.
PUBLISHED: 07-17-2014
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Emerging results indicate that cancer stem-like cells contribute to chemoresistance and poor clinical outcomes in many cancers, including ovarian cancer. As epigenetic regulators play a major role in the control of normal stem cell differentiation, epigenetics may offer a useful arena to develop strategies to target cancer stem-like cells. Epigenetic aberrations, especially DNA methylation, silence tumor-suppressor and differentiation-associated genes that regulate the survival of ovarian cancer stem-like cells (OCSC). In this study, we tested the hypothesis that DNA-hypomethylating agents may be able to reset OCSC toward a differentiated phenotype by evaluating the effects of the new DNA methytransferase inhibitor SGI-110 on OCSC phenotype, as defined by expression of the cancer stem-like marker aldehyde dehydrogenase (ALDH). We demonstrated that ALDH(+) ovarian cancer cells possess multiple stem cell characteristics, were highly chemoresistant, and were enriched in xenografts residual after platinum therapy. Low-dose SGI-110 reduced the stem-like properties of ALDH(+) cells, including their tumor-initiating capacity, resensitized these OCSCs to platinum, and induced reexpression of differentiation-associated genes. Maintenance treatment with SGI-110 after carboplatin inhibited OCSC growth, causing global tumor hypomethylation and decreased tumor progression. Our work offers preclinical evidence that epigenome-targeting strategies have the potential to delay tumor progression by reprogramming residual cancer stem-like cells. Furthermore, the results suggest that SGI-110 might be administered in combination with platinum to prevent the development of recurrent and chemoresistant ovarian cancer.
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Correcting imbalanced reads coverage in bacterial transcriptome sequencing with extreme deep coverage.
Int J Comput Biol Drug Des
PUBLISHED: 05-28-2014
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High throughput bacterial RNA-Seq experiments can generate extremely high and imbalanced sequencing coverage. Over- or under-estimation of gene expression levels will hinder accurate gene differential expression analysis. Here we evaluated strategies to identify expression differences of genes with high coverage in bacterial transcriptome data using either raw sequence reads or unique reads with duplicate fragments removed. In addition, we proposed a generalised linear model (GLM) based approach to identify imbalance in read coverage based on sequence compositions. Our results show that analysis using raw reads identifies more differentially expressed genes with more accurate fold change than using unique reads. We also demonstrate the presence of sequence composition related biases that are independent of gene expression levels and experimental conditions. Finally, genes that still show strong coverage imbalance after correction were tagged using statistical approach.
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Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing.
J Nanosci Nanotechnol
PUBLISHED: 04-18-2014
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Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.
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Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank.
Breast Cancer Res.
PUBLISHED: 03-10-2014
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Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined.
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Anti-diabetic effects of CTB-APSL fusion protein in type 2 diabetic mice.
Mar Drugs
PUBLISHED: 02-19-2014
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To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-? (TNF-?) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.
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Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.
Exp Ther Med
PUBLISHED: 02-13-2014
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Bone marrow-derived mesenchymal stem cells (MSCs) are dominant seed cell sources for bone regeneration. Bone morphogenetic proteins (BMPs) initiate cartilage and bone formation in a sequential cascade. Vascular endothelial growth factor (VEGF) is an essential coordinator of extracellular matrix remodeling, angiogenesis and bone formation. In the present study, the effects of the vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP2) genes on bone regeneration were investigated by the lentivirus-mediated cotransfection of the two genes into rat bone marrow-derived MSCs. The successful co-expression of the two genes in the MSCs was confirmed using quantitative polymerase chain reaction (qPCR) and western blot analysis. The results of alizarin red and alkaline phosphatase (ALP) staining at 14 days subsequent to transfection showed that the area of staining in cells transfected with BMP2 alone was higher than that in cells transfected with BMP2 and VEGF165 or untransfected control cells, while the BMP2 + VEGF165 group showed significantly more staining than the untransfected control. This indicated that BMP2 alone exhibited a stronger effect in bone regeneration than BMP2 in combination with VEGF165. Similarly, in inducing culture medium, the ALP activity of the BMP2 + VEGF165 group was notably suppressed compared with that of the BMP2 group. The overexpression of VEGF165 inhibited BMP2-induced MSC differentiation and osteogenesis in vitro. Whether or not local VEGF gene therapy is likely to affect bone regeneration in vivo requires further investigation.
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Haemophilus ducreyi Hfq contributes to virulence gene regulation as cells enter stationary phase.
MBio
PUBLISHED: 02-13-2014
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To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of Haemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of hfq. Insertional inactivation of hfq altered the expression of ~16% of the H. ducreyi genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the hfq inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of hfq downregulated genes in the flp-tad and lspB-lspA2 operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked hfq deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of hfq downregulated Flp1 and impaired the ability of H. ducreyi to form microcolonies, downregulated DsrA and rendered H. ducreyi serum susceptible, and downregulated LspB and LspA2, which allow H. ducreyi to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of H. ducreyi stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog.
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Impact of human pathogenic micro-insertions and micro-deletions on post-transcriptional regulation.
Hum. Mol. Genet.
PUBLISHED: 01-16-2014
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Small insertions/deletions (INDELs) of ?21 bp comprise 18% of all recorded mutations causing human inherited disease and are evident in 24% of documented Mendelian diseases. INDELs affect gene function in multiple ways: for example, by introducing premature stop codons that either lead to the production of truncated proteins or affect transcriptional efficiency. However, the means by which they impact post-transcriptional regulation, including alternative splicing, have not been fully evaluated. In this study, we collate disease-causing INDELs from the Human Gene Mutation Database (HGMD) and neutral INDELs from the 1000 Genomes Project. The potential of these two types of INDELs to affect binding-site affinity of RNA-binding proteins (RBPs) was then evaluated. We identified several sequence features that can distinguish disease-causing INDELs from neutral INDELs. Moreover, we built a machine-learning predictor called PinPor (predicting pathogenic small insertions and deletions affecting post-transcriptional regulation, http://watson.compbio.iupui.edu/pinpor/) to ascertain which newly observed INDELs are likely to be pathogenic. Our results show that disease-causing INDELs are more likely to ablate RBP-binding sites and tend to affect more RBP-binding sites than neutral INDELs. Additionally, disease-causing INDELs give rise to greater deviations in binding affinity than neutral INDELs. We also demonstrated that disease-causing INDELs may be distinguished from neutral INDELs by several sequence features, such as their proximity to splice sites and their potential effects on RNA secondary structure. This predictor showed satisfactory performance in identifying numerous pathogenic INDELs, with a Matthews correlation coefficient (MCC) value of 0.51 and an accuracy of 0.75.
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Implications of genomic signatures in the differential vulnerability to fetal alcohol exposure in C57BL/6 and DBA/2 mice.
Front Genet
PUBLISHED: 01-01-2014
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Maternal alcohol consumption inflicts a multitude of phenotypic consequences that range from undetectable changes to severe dysmorphology. Using tightly controlled murine studies that deliver precise amounts of alcohol at discrete developmental stages, our group and other labs demonstrated in prior studies that the C57BL/6 and DBA/2 inbred mouse strains display differential susceptibility to the teratogenic effects of alcohol. Since the phenotypic diversity extends beyond the amount, dosage and timing of alcohol exposure, it is likely that an individual's genetic background contributes to the phenotypic spectrum. To identify the genomic signatures associated with these observed differences in alcohol-induced dysmorphology, we conducted a microarray-based transcriptome study that also interrogated the genomic signatures between these two lines based on genetic background and alcohol exposure. This approach is called a gene x environment (GxE) analysis; one example of a GxE interaction would be a gene whose expression level increases in C57BL/6, but decreases in DBA/2 embryos, following alcohol exposure. We identified 35 candidate genes exhibiting GxE interactions. To identify cis-acting factors that mediated these interactions, we interrogated the proximal promoters of these 35 candidates and found 241 single nucleotide variants (SNVs) in 16 promoters. Further investigation indicated that 186 SNVs (15 promoters) are predicted to alter transcription factor binding. In addition, 62 SNVs created, removed or altered the placement of a CpG dinucleotide in 13 of the proximal promoters, 53 of which overlapped putative transcription factor binding sites. These 53 SNVs are also our top candidates for future studies aimed at examining the effects of alcohol on epigenetic gene regulation.
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Dynamic rotating-shield brachytherapy.
Med Phys
PUBLISHED: 12-11-2013
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Purpose: To present dynamic rotating shield brachytherapy (D-RSBT), a novel form of high-dose-rate brachytherapy (HDR-BT) with electronic brachytherapy source, where the radiation shield is capable of changing emission angles during the radiation delivery process.Methods: A D-RSBT system uses two layers of independently rotating tungsten alloy shields, each with a 180° azimuthal emission angle. The D-RSBT planning is separated into two stages: anchor plan optimization and optimal sequencing. In the anchor plan optimization, anchor plans are generated by maximizing the D90 for the high-risk clinical-tumor-volume (HR-CTV) assuming a fixed azimuthal emission angle of 11.25°. In the optimal sequencing, treatment plans that most closely approximate the anchor plans under the delivery-time constraint will be efficiently computed. Treatment plans for five cervical cancer patients were generated for D-RSBT, single-shield RSBT (S-RSBT), and (192)Ir-based intracavitary brachytherapy with supplementary interstitial brachytherapy (IS + ICBT) assuming five treatment fractions. External beam radiotherapy doses of 45 Gy in 25 fractions of 1.8 Gy each were accounted for. The high-risk clinical target volume (HR-CTV) doses were escalated such that the D2cc of the rectum, sigmoid colon, or bladder reached its tolerance equivalent dose in 2 Gy fractions (EQD2 with ??? = 3 Gy) of 75 Gy, 75 Gy, or 90 Gy, respectively.Results: For the patients considered, IS + ICBT had an average total dwell time of 5.7 minutes?fraction (min?fx) assuming a 10 Ci(192)Ir source, and the average HR-CTV D90 was 78.9 Gy. In order to match the HR-CTV D90 of IS + ICBT, D-RSBT required an average of 10.1 min?fx more delivery time, and S-RSBT required 6.7 min?fx more. If an additional 20 min?fx of delivery time is allowed beyond that of the IS + ICBT case, D-RSBT and S-RSBT increased the HR-CTV D90 above IS + ICBT by an average of 16.3 Gy and 9.1 Gy, respectively.Conclusions: For cervical cancer patients, D-RSBT can boost HR-CTV D90 over IS + ICBT and S-RSBT without violating the tolerance doses to the bladder, rectum, or sigmoid. The D90 improvements from D-RSBT depend on the patient, the delivery time budget, and the applicator structure.
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New aQTL SNPs for the CYP2D6 identified by a novel mediation analysis of genome-wide SNP arrays, gene expression arrays, and CYP2D6 activity.
Biomed Res Int
PUBLISHED: 08-30-2013
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The genome-wide association studies (GWAS) have been successful during the last few years. A key challenge is that the interpretation of the results is not straightforward, especially for transacting SNPs. Integration of transcriptome data into GWAS may provide clues elucidating the mechanisms by which a genetic variant leads to a disease.
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Characterizing the heterogeneity of triple-negative breast cancers using microdissected normal ductal epithelium and RNA-sequencing.
Breast Cancer Res. Treat.
PUBLISHED: 08-23-2013
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Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90 % of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.
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Regulation of microRNA expression by rifampin in human hepatocytes.
Drug Metab. Dispos.
PUBLISHED: 08-09-2013
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Rifampin causes drug interactions by altering hepatic drug metabolism. Because microRNAs (miRNAs) have been shown to regulate genes involved in drug metabolism, we determined the effect of rifampin on the expression of hepatic miRNAs. Primary human hepatocytes from seven subjects were treated with rifampin, and the expression of miRNA and cytochrome P450 (P450) mRNAs was measured by TaqMan assays and RNA-seq, respectively. Rifampin induced the expression of 10 clinically important and 13 additional P450 genes and repressed the expression of 9 other P450 genes (P < 0.05). Rifampin induced the expression of 33 miRNAs and repressed the expression of 35 miRNAs (P < 0.05). Several of these changes were highly negatively correlated with the rifampin-induced changes in the expression of their predicted target P450 mRNAs, supporting the possibility of miRNA-induced regulation of P450 mRNA expression. In addition, several other miRNA changes were positively correlated with the changes in P450 mRNA expression, suggesting similar regulatory mechanisms. Despite the interindividual variability in the rifampin effects on miRNA expression, principal components analysis clearly separated the rifampin-treated samples from the controls. In conclusion, rifampin treatment alters miRNA expression patterns in human hepatocytes, and some of the changes were correlated with the rifampin-induced changes in expression of the P450 mRNAs they are predicted to target.
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Comprehensive genomic profiling in diabetic nephropathy reveals the predominance of proinflammatory pathways.
Physiol. Genomics
PUBLISHED: 06-11-2013
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Despite advances in the treatment of diabetic nephropathy (DN), currently available therapies have not prevented the epidemic of progressive chronic kidney disease (CKD). The morbidity of CKD, and the inexorable increase in the prevalence of end-stage renal disease, demands more effective approaches to prevent and treat progressive CKD. We undertook next-generation sequencing in a rat model of diabetic nephropathy to study in depth the pathogenic alterations involved in DN with progressive CKD. We employed the obese, diabetic ZS rat, a model that develops diabetic nephropathy, characterized by progressive CKD, inflammation, and fibrosis, the hallmarks of human disease. We then used RNA-seq to examine the combined effects of renal cells and infiltrating inflammatory cells acting as a pathophysiological unit. The comprehensive systems biology analysis of progressive CKD revealed multiple interactions of altered genes that were integrated into morbid networks. These pathological gene assemblies lead to renal inflammation and promote apoptosis and cell cycle arrest in progressive CKD. Moreover, in what is clearly a major therapeutic challenge, multiple and redundant pathways were found to be linked to renal fibrosis, a major cause of kidney loss. We conclude that systems biology applied to progressive CKD in DN can be used to develop novel therapeutic strategies directed to restore critical anomalies in affected gene networks.
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Numerical simulation of bubble dynamics in an elastic vessel.
Eur Phys J E Soft Matter
PUBLISHED: 06-08-2013
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The nonlinear evolution of a gas bubble in the middle of an elastic vessel is investigated numerically. The fluids inside and outside the vessel are assumed to be incompressible and potential. A boundary element method (BEM) is adopted to solve the Laplace equation for the velocity potential. The gas inside the bubble is described by the Boyle Law. The fluid outside the vessel is assumed to contain the elasticity. The dynamic boundary condition on the vessel interface i is obtained from the derived Bernoulli Equation. This numerical model is validated through the comparisons with both the analytic solution of Rayleigh-Plesset equation and spark bubble experiment. The bubble dynamic behavior with different elastic parameters and vessel inner radius is further discussed.
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Activation of CpxRA in Haemophilus ducreyi primarily inhibits the expression of its targets, including major virulence determinants.
J. Bacteriol.
PUBLISHED: 05-31-2013
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Haemophilus ducreyi causes chancroid, a genital ulcer disease that facilitates the transmission of human immunodeficiency virus type 1. In humans, H. ducreyi is surrounded by phagocytes and must adapt to a hostile environment to survive. To sense and respond to environmental cues, bacteria frequently use two-component signal transduction (2CST) systems. The only obvious 2CST system in H. ducreyi is CpxRA; CpxR is a response regulator, and CpxA is a sensor kinase. Previous studies by Hansen and coworkers showed that CpxR directly represses the expression of dsrA, the lspB-lspA2 operon, and the flp operon, which are required for virulence in humans. They further showed that CpxA functions predominantly as a phosphatase in vitro to maintain the expression of virulence determinants. Since a cpxA mutant is avirulent while a cpxR mutant is fully virulent in humans, CpxA also likely functions predominantly as a phosphatase in vivo. To better understand the role of H. ducreyi CpxRA in controlling virulence determinants, here we defined genes potentially regulated by CpxRA by using RNA-Seq. Activation of CpxR by deletion of cpxA repressed nearly 70% of its targets, including seven established virulence determinants. Inactivation of CpxR by deletion of cpxR differentially regulated few genes and increased the expression of one virulence determinant. We identified a CpxR binding motif that was enriched in downregulated but not upregulated targets. These data reinforce the hypothesis that CpxA phosphatase activity plays a critical role in controlling H. ducreyi virulence in vivo. Characterization of the downregulated genes may offer new insights into pathogenesis.
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Rotating-shield brachytherapy for cervical cancer.
Phys Med Biol
PUBLISHED: 05-16-2013
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In this treatment planning study, the potential benefits of a rotating shield brachytherapy (RSBT) technique based on a partially-shielded electronic brachytherapy source were assessed for treating cervical cancer. Conventional intracavitary brachytherapy (ICBT), intracavitary plus supplementary interstitial (IS+ICBT), and RSBT treatment plans for azimuthal emission angles of 180° (RSBT-180) and 45° (RSBT-45) were generated for five patients. For each patient, high-risk clinical target volume (HR-CTV) equivalent dose in 2 Gy fractions (EQD2) (?/? = 10 Gy) was escalated until bladder, rectum, or sigmoid colon tolerance EQD2 values were reached. External beam radiotherapy dose (1.8 Gy × 25) was accounted for, and brachytherapy was assumed to have been delivered in 5 fractions. IS+ICBT provided a greater HR-CTV D90 (minimum EQD2 to the hottest 90%) than ICBT. D90 was greater for RSBT-45 than IS+ICBT for all five patients, and greater for RSBT-180 than IS+ICBT for two patients. When the RSBT-45/180 plan with the lowest HR-CTV D90 that was greater than the D90 the ICBT or IS+ICBT plan was selected, the average (range) of D90 increases for RSBT over ICBT and IS+ICBT were 16.2 (6.3-27.2)and 8.5 (0.03-20.16) Gy, respectively. The average (range) treatment time increase per fraction of RSBT was 34.56 (3.68-70.41) min over ICBT and 34.59 (3.57-70.13) min over IS+ICBT. RSBT can increase D90 over ICBT and IS+ICBT without compromising organ-at-risk sparing. The D90 and treatment time improvements from RSBT depend on the patient and shield emission angle.
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Rapid emission angle selection for rotating-shield brachytherapy.
Med Phys
PUBLISHED: 05-03-2013
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The authors present a rapid emission angle selection (REAS) method that enables the efficient selection of the azimuthal shield angle for rotating shield brachytherapy (RSBT). The REAS method produces a Pareto curve from which a potential RSBT user can select a treatment plan that balances the tradeoff between delivery time and tumor dose conformity.
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Broadband optical non-linearity induced by charge-transfer excitons in type-II CdSe/ZnTe nanocrystals.
Adv. Mater. Weinheim
PUBLISHED: 04-09-2013
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Benefiting from excitonic charge-transfer states, an efficient non-linear optical response is observed in type-II nanocrystals by four-wave mixing when the incident photon energy lies below the bandgaps of constituent semiconductors. The non-linear optical properties in nanocrystals can be manipulated by the band alignment of the core-shell components, which serves as a promising platform to design broadband non-linear optical devices.
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A Pd(II)-catalyzed asymmetric approach toward chiral [3.3.1]-bicyclic ketals using 2-hydroxyphenylboronic acid as a pro-bis(nucleophile).
Chem. Commun. (Camb.)
PUBLISHED: 03-19-2013
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Enantioselective Pd(II)-catalyzed construction of [3.3.1]-bicyclic ketals from 2-hydroxyphenylboronic acid and enone is reported, yielding enantioenriched [3.3.1]-bicyclic ketals in up to 97% yields and 98% ee.
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DDIG-in: discriminating between disease-associated and neutral non-frameshifting micro-indels.
Genome Biol.
PUBLISHED: 03-13-2013
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Micro-indels (insertions or deletions shorter than 21 bps) constitute the second most frequent class of human gene mutation after single nucleotide variants. Despite the relative abundance of non-frameshifting indels, their damaging effect on protein structure and function has gone largely unstudied. We have developed a support vector machine-based method named DDIG-in (Detecting disease-causing genetic variations due to indels) to prioritize non-frameshifting indels by comparing disease-associated mutations with putatively neutral mutations from the 1,000 Genomes Project. The final model gives good discrimination for indels and is robust against annotation errors. A webserver implementing DDIG-in is available at http://sparks-lab.org/ddig.
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Amplification of distant estrogen response elements deregulates target genes associated with tamoxifen resistance in breast cancer.
Cancer Cell
PUBLISHED: 03-12-2013
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A causal role of gene amplification in tumorigenesis is well known, whereas amplification of DNA regulatory elements as an oncogenic driver remains unclear. In this study, we integrated next-generation sequencing approaches to map distant estrogen response elements (DEREs) that remotely control the transcription of target genes through chromatin proximity. Two densely mapped DERE regions located on chromosomes 17q23 and 20q13 were frequently amplified in estrogen receptor-?-positive luminal breast cancer. These aberrantly amplified DEREs deregulated target gene expression potentially linked to cancer development and tamoxifen resistance. Progressive accumulation of DERE copies was observed in normal breast progenitor cells chronically exposed to estrogenic chemicals. These findings may extend to other DNA regulatory elements, the amplification of which can profoundly alter target transcriptome during tumorigenesis.
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Fast switching thyristor applied in nanosecond-pulse high-voltage generator with closed transformer core.
Rev Sci Instrum
PUBLISHED: 03-08-2013
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For a compact and reliable nanosecond-pulse high-voltage generator (NPHVG), the specification parameter selection and potential usage of fast controllable state-solid switches have an important bearing on the optimal design. The NPHVG with closed transformer core and fast switching thyristor (FST) was studied in this paper. According to the analysis of T-type circuit, the expressions for the voltages and currents of the primary and secondary windings on the transformer core of NPHVG were deduced, and the theoretical maximum analysis was performed. For NPHVG, the rise-rate of turn-on current (di/dt) across a FST may exceed its transient rating. Both mean and maximum values of di/dt were determined by the leakage inductances of the transformer, and the difference is 1.57 times. The optimum winding ratio is helpful to getting higher voltage output with lower specification FST, especially when the primary and secondary capacitances have been established. The oscillation period analysis can be effectively used to estimate the equivalent leakage inductance. When the core saturation effect was considered, the maximum di/dt estimated from the oscillating period of the primary current is more accurate than one from the oscillating period of the secondary voltage. Although increasing the leakage inductance of NPHVG can decrease di/dt across FST, it may reduce the output peak voltage of the NPHVG.
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Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR.
Analyst
PUBLISHED: 03-07-2013
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Allelic ratio of an SNP has been used for prenatal diagnosis of fetal trisomy 21 by MALDI-TOF mass spectrometry (MS). Because MALDI-TOF MS is challenging in quantification performance, pyrosequencing was proposed to replace MS by better quantification of allelic ratios. To achieve a simple and rapid clinical diagnostic, PCR with "HpH Buffer" (a buffer with a high pH) was developed to directly amplify amniotic fluid. By the established assay, 114 samples of amniotic fluid were analyzed by pyrosequencing five SNPs of each sample; the allelic ratios of euploid heterozygotes were thus calculated to determine the cut-off values for prenatal diagnosis of trisomy 21. The panel of five SNPs were high in heterozygosity so that at least one heterozygote was found in each sample, and 86% of the samples had at least two heterozygotes, giving a nearly 100% sensitivity (population coverage) of the assay. By using the cut-off values of each SNP, 20 pre-diagnosed clinical samples were detected as trisomy 21 carriers with a confidence level over 99%, indicating that our method and karyotyping analysis were consistent in results. In conclusion, this pyrosequencing-based approach, coupled with direct amplification of amniotic fluid, is accurate in quantitative genotyping and simple in operation. We believe that the approach could be a promising alternative to karyotyping analysis in prenatal diagnosis.
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A shark liver gene-derived active peptide expressed in the silkworm, Bombyx mori: preliminary studies for oral administration of the recombinant protein.
Mar Drugs
PUBLISHED: 02-12-2013
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Active peptide from shark liver (APSL) is a cytokine from Chiloscyllium plagiosum that can stimulate liver regeneration and protects the pancreas. To study the effect of orally administered recombinant APSL (rAPSL) on an animal model of type 2 diabetes mellitus, the APSL gene was cloned, and APSL was expressed in Bombyx mori N cells (BmN cells), silkworm larvae and silkworm pupae using the silkworm baculovirus expression vector system (BEVS). It was demonstrated that rAPSL was able to significantly reduce the blood glucose level in mice with type 2 diabetes induced by streptozotocin. The analysis of paraffin sections of mouse pancreatic tissues revealed that rAPSL could effectively protect mouse islets from streptozotocin-induced lesions. Compared with the powder prepared from normal silkworm pupae, the powder prepared from pupae expressing rAPSL exhibited greater protective effects, and these results suggest that rAPSL has potential uses as an oral drug for the treatment of diabetes mellitus in the future.
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Potential roles of microRNAs in regulating long intergenic noncoding RNAs.
BMC Med Genomics
PUBLISHED: 01-23-2013
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Over 10,000 long intergenic non-coding RNAs (lincRNAs) have been identified in the human genome. Some have been well characterized and known to participate in various stages of gene regulation. In the post-transcriptional process, another class of well-known small non-coding RNA, or microRNA (miRNA), is very active in inhibiting mRNA. Though similar features between mRNA and lincRNA have been revealed in several recent studies, and a few isolated miRNA-lincRNA relationships have been observed. Despite these advances, the comprehensive miRNA regulation pattern of lincRNA has not been clarified.
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Identification of regulatory regions of bidirectional genes in cervical cancer.
BMC Med Genomics
PUBLISHED: 01-23-2013
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Bidirectional promoters are shared promoter sequences between divergent gene pair (genes proximal to each other on opposite strands), and can regulate the genes in both directions. In the human genome, > 10% of protein-coding genes are arranged head-to-head on opposite strands, with transcription start sites that are separated by < 1,000 base pairs. Many transcription factor binding sites occur in the bidirectional promoters that influence the expression of 2 opposite genes. Recently, RNA polymerase II (RPol II) ChIP-seq data are used to identify the promoters of coding genes and non-coding RNAs. However, a bidirectional promoter with RPol II ChIP-Seq data has not been found.
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NGSUtils: a software suite for analyzing and manipulating next-generation sequencing datasets.
Bioinformatics
PUBLISHED: 01-12-2013
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NGSUtils is a suite of software tools for manipulating data common to next-generation sequencing experiments, such as FASTQ, BED and BAM format files. These tools provide a stable and modular platform for data management and analysis.
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The identification of microRNAs in the whitespotted bamboo shark (Chiloscyllium plagiosum) liver by Illumina sequencing.
Gene
PUBLISHED: 01-10-2013
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MicroRNAs are indispensable players in the regulation of a broad range of biological processes. Here, we report the first deep sequencing of the whitespotted bamboo shark (Chiloscyllium plagiosum) liver. We mapped 91 miRNAs in the Callorhinchus milii genome that have previously been described in the Danio rerio, Fugu rubripes, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Homo sapiens, and Mus musculus. In addition, 156 new putative candidate (PC) C. plagiosum miRNAs were identified. From these 247 miRNAs, 39 miRNA clusters were identified, and the expression of these clustered miRNAs was observed to vary significantly. A total of 7 candidate miRNAs were selected for expression confirmation by stem-loop RT-PCR. This study resulted in the addition of a significant number of novel miRNA sequences to GenBank and laid the foundation for further understanding of the function of miRNAs in the regulation of C. plagiosum liver development.
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Novel specific microRNA biomarkers in idiopathic inflammatory bowel disease unrelated to disease activity.
Mod. Pathol.
PUBLISHED: 01-04-2013
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The diagnosis of idiopathic inflammatory bowel disease can be challenging. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein synthesis through post-transcriptional suppression. This study is to identify new miRNA markers in inflammatory bowel disease, and to examine whether miRNA biomarkers might assist in the diagnosis of inflammatory bowel disease. Illumina small RNA sequencing was performed on non-dysplastic fresh-frozen colonic mucosa samples of the distalmost colectomy tissue from 19 patients with inflammatory bowel disease (10 ulcerative colitis and 9 Crohn disease) and 18 patients with diverticular disease serving as controls. To determine differentially expressed miRNAs, the USeq software package identified 44 miRNAs with altered expression (fold change ?2 and false discovery rate ?0.10) compared with the controls. Among them, a panel of nine miRNAs was aberrantly expressed in both ulcerative colitis and Crohn disease. Validation assays performed using quantitative reverse transcription PCR (qRT-PCR) on additional frozen tissue from ulcerative colitis, Crohn disease, and control groups confirmed specific differential expression in inflammatory bowel disease for miR-31, miR-206, miR-424, and miR-146a (P<0.05). The expression of these four miRNAs was further evaluated on formalin-fixed, paraffin-embedded tissue of the distalmost colectomy mucosa from cohorts of diverticular disease controls (n=29), ulcerative colitis (n=36), Crohn disease (n=26), and the other diseases mimicking inflammatory bowel disease including infectious colitis (n=12) and chronic ischemic colitis (n=19), again confirming increased expression specific to inflammatory bowel disease (P<0.05). In summary, we demonstrate that miR-31, miR-206, miR-424, and miR-146a are novel specific biomarkers of inflammatory bowel disease. Furthermore, miR-31 is universally expressed in both ulcerative colitis and Crohn disease not only in fresh-frozen but also in formalin-fixed, paraffin-embedded tissues.Modern Pathology advance online publication, 20 September 2013; doi:10.1038/modpathol.2013.152.
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A new method for stranded whole transcriptome RNA-seq.
Methods
PUBLISHED: 01-04-2013
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This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).
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From genes to networks: in systematic points of view.
BMC Syst Biol
PUBLISHED: 12-23-2011
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We present a report of the BIOCOMP10 - The 2010 International Conference on Bioinformatics & Computational Biology and other related work in the area of systems biology.
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Changes in predicted protein disorder tendency may contribute to disease risk.
BMC Genomics
PUBLISHED: 12-23-2011
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Recent studies suggest that many proteins or regions of proteins lack 3D structure. Defined as intrinsically disordered proteins, these proteins/peptides are functionally important. Recent advances in next generation sequencing technologies enable genome-wide identification of novel nucleotide variations in a specific population or cohort.
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Predicting sequence and structural specificities of RNA binding regions recognized by splicing factor SRSF1.
BMC Genomics
PUBLISHED: 12-23-2011
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RNA-binding proteins (RBPs) play diverse roles in eukaryotic RNA processing. Despite their pervasive functions in coding and noncoding RNA biogenesis and regulation, elucidating the sequence specificities that define protein-RNA interactions remains a major challenge. Recently, CLIP-seq (Cross-linking immunoprecipitation followed by high-throughput sequencing) has been successfully implemented to study the transcriptome-wide binding patterns of SRSF1, PTBP1, NOVA and fox2 proteins. These studies either adopted traditional methods like Multiple EM for Motif Elicitation (MEME) to discover the sequence consensus of RBPs binding sites or used Z-score statistics to search for the overrepresented nucleotides of a certain size. We argue that most of these methods are not well-suited for RNA motif identification, as they are unable to incorporate the RNA structural context of protein-RNA interactions, which may affect to binding specificity. Here, we describe a novel model-based approach--RNAMotifModeler to identify the consensus of protein-RNA binding regions by integrating sequence features and RNA secondary structures.
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Analysis of exome sequences with and without incorporating prior biological knowledge.
Genet. Epidemiol.
PUBLISHED: 12-01-2011
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Next-generation sequencing technology provides new opportunities and challenges in the search for genetic variants that underlie complex traits. It will also presumably uncover many new rare variants, but exactly how these variants should be incorporated into the data analysis remains a question. Several papers in our group from Genetic Analysis Workshop 17 evaluated different methods of rare variant analysis, including single-variant, gene-based, and pathway-based analyses and analyses that incorporated biological information. Although the performance of some of these methods strongly depends on the underlying disease model, integration of known biological information is helpful in detecting causal genes. Two work groups demonstrated that use of a Bayesian network and a collapsing receiver operating characteristic curve approach improves risk prediction when a disease is caused by many rare variants. Another work group suggested that modeling local rather than global ancestry may be beneficial when controlling the effect of population structure in rare variant association analysis.
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Identification of multiple rare variants associated with a disease.
BMC Proc
PUBLISHED: 11-29-2011
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Identifying rare variants that are responsible for complex disease has been promoted by advances in sequencing technologies. However, statistical methods that can handle the vast amount of data generated and that can interpret the complicated relationship between disease and these variants have lagged. We apply a zero-inflated Poisson regression model to take into account the excess of zeros caused by the extremely low frequency of the 24,487 exonic variants in the Genetic Analysis Workshop 17 data. We grouped the 697 subjects in the data set as Europeans, Asians, and Africans based on principal components analysis and found the total number of rare variants per gene for each individual. We then analyzed these collapsed variants based on the assumption that rare variants are enriched in a group of people affected by a disease compared to a group of unaffected people. We also tested the hypothesis with quantitative traits Q1, Q2, and Q4. Analyses performed on the combined 697 individuals and on each ethnic group yielded different results. For the combined population analysis, we found that UGT1A1, which was not part of the simulation model, was associated with disease liability and that FLT1, which was a causal locus in the simulation model, was associated with Q1. Of the causal loci in the simulation models, FLT1 and KDR were associated with Q1 and VNN1 was correlated with Q2. No significant genes were associated with Q4. These results show the feasibility and capability of our new statistical model to detect multiple rare variants influencing disease risk.
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Prioritizing single-nucleotide variations that potentially regulate alternative splicing.
BMC Proc
PUBLISHED: 11-29-2011
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Recent evidence suggests that many complex diseases are caused by genetic variations that play regulatory roles in controlling gene expression. Most genetic studies focus on nonsynonymous variations that can alter the amino acid composition of a protein and are therefore believed to have the highest impact on phenotype. Synonymous variations, however, can also play important roles in disease pathogenesis by regulating pre-mRNA processing and translational control. In this study, we systematically survey the effects of single-nucleotide variations (SNVs) on binding affinity of RNA-binding proteins (RBPs). Among the 10,113 synonymous SNVs identified in 697 individuals in the 1,000 Genomes Project and distributed by Genetic Analysis Workshop 17 (GAW17), we identified 182 variations located in alternatively spliced exons that can significantly change the binding affinity of nine RBPs whose binding preferences on 7-mer RNA sequences were previously reported. We found that the minor allele frequencies of these variations are similar to those of nonsynonymous SNVs, suggesting that they are in fact functional. We propose a workflow to identify phenotype-associated regulatory SNVs that might affect alternative splicing from exome-sequencing-derived genetic variations. Based on the affecting SNVs on the quantitative traits simulated in GAW17, we further identified two and four functional SNVs that are predicted to be involved in alternative splicing regulation in traits Q1 and Q2, respectively.
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Ratiometric fluorescence detection of cyanide based on a hybrid coumarin-hemicyanine dye: the large emission shift and the high selectivity.
Chem. Commun. (Camb.)
PUBLISHED: 11-02-2011
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A new ratiometric fluorescent cyanide probe was developed based on the nucleophilic attack of CN(-) toward the indolium group of a hybrid coumarin-hemicyanine dye, by which high selectivity as well as large emission shift could be achieved.
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Tbx20 transcription factor is a downstream mediator for bone morphogenetic protein-10 in regulating cardiac ventricular wall development and function.
J. Biol. Chem.
PUBLISHED: 09-02-2011
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Bone morphogenetic protein 10 (BMP10) belongs to the TGF?-superfamily. Previously, we had demonstrated that BMP10 is a key regulator for ventricular chamber formation, growth, and maturation. Ablation of BMP10 leads to hypoplastic ventricular wall formation, and elevated levels of BMP10 are associated with abnormal ventricular trabeculation/compaction and wall maturation. However, the molecular mechanism(s) by which BMP10 regulates ventricle wall growth and maturation is still largely unknown. In this study, we sought to identify the specific transcriptional network that is potentially mediated by BMP10. We analyzed and compared the gene expression profiles between ?-myosin heavy chain (?MHC)-BMP10 transgenic hearts and nontransgenic littermate controls using Affymetrix mouse exon arrays. T-box 20 (Tbx20), a cardiac transcription factor, was significantly up-regulated in ?MHC-BMP10 transgenic hearts, which was validated by quantitative RT-PCR and in situ hybridization. Ablation of BMP10 reduced Tbx20 expression specifically in the BMP10-expressing region of the developing ventricle. In vitro promoter analysis demonstrated that BMP10 was able to induce Tbx20 promoter activity through a conserved Smad binding site in the Tbx20 promoter proximal region. Furthermore, overexpression of Tbx20 in myocardium led to dilated cardiomyopathy that exhibited ventricular hypertrabeculation and an abnormal muscular septum, which phenocopied genetically modified mice with elevated BMP10 levels. Taken together, our findings demonstrate that the BMP10-Tbx20 signaling cascade is important for ventricular wall development and maturation.
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A ratiometric fluorescent probe for cyanide based on FRET.
Org. Biomol. Chem.
PUBLISHED: 05-25-2011
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On the basis of FRET from 4-(N,N-dimethylamino)benzamide to fluorescein, a new ratiometric fluorescence probe bearing a hydrazone binding unit was developed for highly selective and sensitive detection of CN(-) in aqueous solution.
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A modulated empirical Bayes model for identifying topological and temporal estrogen receptor ? regulatory networks in breast cancer.
BMC Syst Biol
PUBLISHED: 05-09-2011
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Estrogens regulate diverse physiological processes in various tissues through genomic and non-genomic mechanisms that result in activation or repression of gene expression. Transcription regulation upon estrogen stimulation is a critical biological process underlying the onset and progress of the majority of breast cancer. Dynamic gene expression changes have been shown to characterize the breast cancer cell response to estrogens, the every molecular mechanism of which is still not well understood.
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Hepatic FoxOs regulate lipid metabolism via modulation of expression of the nicotinamide phosphoribosyltransferase gene.
J. Biol. Chem.
PUBLISHED: 03-08-2011
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FoxO transcription factors have been implicated in lipid metabolism; however, the underlying mechanisms are not well understood. Here, in an effort to elucidate such mechanisms, we examined the phenotypic consequences of liver-specific deletion of three members of the FoxO family: FoxO1, FoxO3, and FoxO4. These liver-specific triply null mice, designated LTKO, exhibited elevated triglycerides in the liver on regular chow diet. More remarkably, LTKO mice developed severe hepatic steatosis following placement on a high fat diet. Further analyses revealed that hepatic NAD(+) levels and Sirt1 activity were decreased in the liver of the LTKO mice relative to controls. At the mechanistic level, expression profile analyses showed that LTKO livers had significantly down-regulated expression of the nicotinamide phosphoribosyltransferase (Nampt) gene encoding the rate-limiting enzyme in the salvage pathway of NAD(+) biosynthesis. Luciferase reporter assays and chromatin immunoprecipitation analyses demonstrated that Nampt is a transcriptional target gene of FoxOs. Significantly, overexpression of Nampt gene reduced, whereas knockdown increased, hepatic triglyceride levels in vitro and in vivo. Thus, FoxOs control the Nampt gene expression and the NAD(+) signaling in the regulation of hepatic triglyceride homeostasis.
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Alteration of gene expression by alcohol exposure at early neurulation.
BMC Genomics
PUBLISHED: 02-21-2011
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We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables.
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Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients.
Breast Cancer Res.
PUBLISHED: 02-14-2011
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Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease.
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An 2-(2-aminophenyl)benzoxazole-based OFF-ON fluorescent chemosensor for Zn2+ in aqueous solution.
Org. Biomol. Chem.
PUBLISHED: 02-11-2011
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A water-soluble fluorescent sensor, 1, based on the "receptor-spacer-fluorophore" [2-(2-aminophenyl)benzoxazole-amide-2-picolylamine] sensor platform, demonstrates the high sensitivity for Zn(2+) with a 25-fold fluorescence enhancement upon chelation to Zn(2+) and also exhibits high selectivity to Zn(2+) over other metal ions. X-ray crystal structure of Zn(2+) complex reveals that the amide oxygen (O2) cooperates with 2-picolylamine unit (N3, N4) as a receptor bind Zn(2+).
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Genomic and proteomic analysis of the impact of mitotic quiescence on the engraftment of human CD34+ cells.
PLoS ONE
PUBLISHED: 02-07-2011
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It is well established that in adults, long-term repopulating hematopoietic stem cells (HSC) are mitotically quiescent cells that reside in specialized bone marrow (BM) niches that maintain the dormancy of HSC. Our laboratory demonstrated that the engraftment potential of human HSC (CD34(+) cells) from BM and mobilized peripheral blood (MPB) is restricted to cells in the G0 phase of cell cycle but that in the case of umbilical cord blood (UCB) -derived CD34(+) cells, cell cycle status is not a determining factor in the ability of these cells to engraft and sustain hematopoiesis. We used this distinct in vivo behavior of CD34(+) cells from these tissues to identify genes associated with the engraftment potential of human HSC. CD34(+) cells from BM, MPB, and UCB were fractionated into G0 and G1 phases of cell cycle and subjected in parallel to microarray and proteomic analyses. A total of 484 target genes were identified to be associated with engraftment potential of HSC. System biology modeling indicated that the top four signaling pathways associated with these genes are Integrin signaling, p53 signaling, cytotoxic T lymphocyte-mediated apoptosis, and Myc mediated apoptosis signaling. Our data suggest that a continuum of functions of hematopoietic cells directly associated with cell cycle progression may play a major role in governing the engraftment potential of stem cells. While proteomic analysis identified a total of 646 proteins in analyzed samples, a very limited overlap between genomic and proteomic data was observed. These data provide a new insight into the genetic control of engraftment of human HSC from distinct tissues and suggest that mitotic quiescence may not be the requisite characteristic of engrafting stem cells, but instead may be the physiologic status conducive to the expression of genetic elements favoring engraftment.
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Alcohol alters DNA methylation patterns and inhibits neural stem cell differentiation.
Alcohol. Clin. Exp. Res.
PUBLISHED: 01-11-2011
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Potential epigenetic mechanisms underlying fetal alcohol syndrome (FAS) include alcohol-induced alterations of methyl metabolism, resulting in aberrant patterns of DNA methylation and gene expression during development. Having previously demonstrated an essential role for epigenetics in neural stem cell (NSC) development and that inhibiting DNA methylation prevents NSC differentiation, here we investigated the effect of alcohol exposure on genome-wide DNA methylation patterns and NSC differentiation.
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2K09 and thereafter : the coming era of integrative bioinformatics, systems biology and intelligent computing for functional genomics and personalized medicine research.
BMC Genomics
PUBLISHED: 12-01-2010
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Significant interest exists in establishing synergistic research in bioinformatics, systems biology and intelligent computing. Supported by the United States National Science Foundation (NSF), International Society of Intelligent Biological Medicine (http://www.ISIBM.org), International Journal of Computational Biology and Drug Design (IJCBDD) and International Journal of Functional Informatics and Personalized Medicine, the ISIBM International Joint Conferences on Bioinformatics, Systems Biology and Intelligent Computing (ISIBM IJCBS 2009) attracted more than 300 papers and 400 researchers and medical doctors world-wide. It was the only inter/multidisciplinary conference aimed to promote synergistic research and education in bioinformatics, systems biology and intelligent computing. The conference committee was very grateful for the valuable advice and suggestions from honorary chairs, steering committee members and scientific leaders including Dr. Michael S. Waterman (USC, Member of United States National Academy of Sciences), Dr. Chih-Ming Ho (UCLA, Member of United States National Academy of Engineering and Academician of Academia Sinica), Dr. Wing H. Wong (Stanford, Member of United States National Academy of Sciences), Dr. Ruzena Bajcsy (UC Berkeley, Member of United States National Academy of Engineering and Member of United States Institute of Medicine of the National Academies), Dr. Mary Qu Yang (United States National Institutes of Health and Oak Ridge, DOE), Dr. Andrzej Niemierko (Harvard), Dr. A. Keith Dunker (Indiana), Dr. Brian D. Athey (Michigan), Dr. Weida Tong (FDA, United States Department of Health and Human Services), Dr. Cathy H. Wu (Georgetown), Dr. Dong Xu (Missouri), Drs. Arif Ghafoor and Okan K Ersoy (Purdue), Dr. Mark Borodovsky (Georgia Tech, President of ISIBM), Dr. Hamid R. Arabnia (UGA, Vice-President of ISIBM), and other scientific leaders. The committee presented the 2009 ISIBM Outstanding Achievement Awards to Dr. Joydeep Ghosh (UT Austin), Dr. Aidong Zhang (Buffalo) and Dr. Zhi-Hua Zhou (Nanjing) for their significant contributions to the field of intelligent biological medicine.
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Highly sensitive and selective turn-on fluorescent and chromogenic probe for Cu2+ and ClO- based on a N-picolinyl rhodamine B-hydrazide derivative.
Org. Biomol. Chem.
PUBLISHED: 10-28-2010
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A new rhodamine B-based dual-function chromo- and fluorogenic probe for Cu(2+) and ClO(-) has been designed, synthesized, and characterized. The probe comprises a spectroscopic unit of rhodamine B and a Cu(2+)-specific chelating unit of pyridinecarboxamide as well as a ClO(-)-specific reactive moiety of diacylhydrazine, and is a highly selective and extremely sensitive fluorescent and colorimetric sensor for Cu(2+) and ClO(-) in different pH conditions. Compared with the reported probes for Cu(2+) or ClO(-), this is the first chemosensor based on a small molecule that can detect both Cu(2+) and ClO(-), respectively, at 1 nM level.
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Rhodamine-based chemosensor for Hg(2+) in aqueous solution with a broad pH range and its application in live cell imaging.
Org. Biomol. Chem.
PUBLISHED: 07-23-2010
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A new fluorescent probe, rhodamine B derivative (1) bearing an 8-hydroxyquinoline group, was synthesized and displayed highly selective and sensitive Hg(2+)-amplified absorbance and fluorescence emission above 500 nm in aqueous solution with a broad pH range 4-9. It was found that mercury ions coordinate reversibly to 1 and the spirolactam ring of 1 was opened, forming a 1 : 1 metal-ligand complex. Furthermore, this sensor was applied for in vivo imaging in HeLa cells to confirm that 1 can be used as a fluorescent probe for monitoring Hg(2+) in living cells.
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A molecular signature of normal breast epithelial and stromal cells from Li-Fraumeni syndrome mutation carriers.
Oncotarget
PUBLISHED: 07-07-2010
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Specific changes in gene expression during cancer initiation should enable discovery of biomarkers for risk assessment, early detection and targets for chemoprevention. It has been previously demonstrated that altered mRNA and proteome signatures of morphologically normal cells bearing a single inherited "hit" in a tumor suppressor gene parallel many changes observed in the corresponding sporadic cancer. Here, we report on the global gene expression profile of morphologically normal, cultured primary breast epithelial and stromal cells from Li-Fraumeni syndrome (LFS) TP53 mutation carriers. Our analyses identified multiple changes in gene expression in both morphologically normal breast epithelial and stromal cells associated with TP53 haploinsufficiency, as well as interlocking pathways. Notably, a dysregulated p53 signaling pathway was readily detectable. Pharmacological intervention with the p53 rescue compounds CP-31398 and PRIMA-1 provided further evidence in support of the central role of p53 in affecting these changes in LFS cells and treatment for this cancer. Because loss of signaling mediated by TP53 is associated with the development and survival of many human tumors, identification of gene expression profiles in morphologically normal cells that carry "one-hit" p53 mutations may reveal novel biomarkers, enabling the discovery of potential targets for chemoprevention of sporadic tumors as well.
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An empirical Bayes model for gene expression and methylation profiles in antiestrogen resistant breast cancer.
BMC Med Genomics
PUBLISHED: 06-22-2010
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The nuclear transcription factor estrogen receptor alpha (ER-alpha) is the target of several antiestrogen therapeutic agents for breast cancer. However, many ER-alpha positive patients do not respond to these treatments from the beginning, or stop responding after being treated for a period of time. Because of the association of gene transcription alteration and drug resistance and the emerging evidence on the role of DNA methylation on transcription regulation, understanding of these relationships can facilitate development of approaches to re-sensitize breast cancer cells to treatment by restoring DNA methylation patterns.
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Alternative splicing in bone following mechanical loading.
Bone
PUBLISHED: 06-21-2010
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It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to evaluate the extent of alternative splicing in a bone subjected to mechanical loading and subsequent bone formation. We used the rat forelimb loading model, in which the right forelimb was loaded axially for 3 min, while the left forearm served as a non-loaded control. Animals were subjected to loading sessions every day, with 24 h between sessions. Ulnae were sampled at 11 time points, from 4 h to 32days after beginning loading. RNA was isolated and mRNA abundance was measured at each time point using Affymetrix exon arrays (GeneChip® Rat Exon 1.0 ST Arrays). An ANOVA model was used to identify potential alternatively spliced genes across the time course, and five alternatively spliced genes were validated with qPCR: Akap12, Fn1, Pcolce, Sfrp4, and Tpm1. The number of alternatively spliced genes varied with time, ranging from a low of 68 at 12h to a high of 992 at 16d. We identified genes across the time course that encoded proteins with known functions in bone formation, including collagens, matrix proteins, and components of the Wnt/?-catenin and TGF-? signaling pathways. We also identified alternatively spliced genes encoding cytokines, ion channels, muscle-related genes, and solute carriers that do not have a known function in bone formation and represent potentially novel findings. In addition, a functional characterization was performed to categorize the global functions of the alternatively spliced genes in our data set. In conclusion, mechanical loading induces alternative splicing in bone, which may play an important role in the response of bone to mechanical loading.
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Prioritization of disease microRNAs through a human phenome-microRNAome network.
BMC Syst Biol
PUBLISHED: 05-28-2010
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The identification of disease-related microRNAs is vital for understanding the pathogenesis of diseases at the molecular level, and is critical for designing specific molecular tools for diagnosis, treatment and prevention. Experimental identification of disease-related microRNAs poses considerable difficulties. Computational analysis of microRNA-disease associations is an important complementary means for prioritizing microRNAs for further experimental examination.
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MicroRNA cluster 221-222 and estrogen receptor alpha interactions in breast cancer.
J. Natl. Cancer Inst.
PUBLISHED: 04-13-2010
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Several lines of evidence have suggested that estrogen receptor alpha (ERalpha)-negative breast tumors, which are highly aggressive and nonresponsive to hormonal therapy, arise from ERalpha-positive precursors through different molecular pathways. Because microRNAs (miRNAs) modulate gene expression, we hypothesized that they may have a role in ER-negative tumor formation.
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Microarray studies on effects of Pneumocystis carinii infection on global gene expression in alveolar macrophages.
BMC Microbiol.
PUBLISHED: 04-08-2010
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Pneumocystis pneumonia is a common opportunistic disease in AIDS patients. The alveolar macrophage is an important effector cell in the clearance of Pneumocystis organisms by phagocytosis. However, both the number and phagocytic activity of alveolar macrophages are decreased in Pneumocystis infected hosts. To understand how Pneumocystis inactivates alveolar macrophages, Affymetrix GeneChip RG-U34A DNA microarrays were used to study the difference in global gene expression in alveolar macrophages from uninfected and Pneumocystis carinii-infected Sprague-Dawley rats.
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Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys.
J Pharm Biomed Anal
PUBLISHED: 04-05-2010
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A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K(d) values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5-32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1-8.8% and (3.0-9.0)%, respectively with the inter-assay precision and accuracy were 5.7-11.5% and (10.7-9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32-320 ?g/kg).
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Identification of genes influencing skeletal phenotypes in congenic P/NP rats.
J. Bone Miner. Res.
PUBLISHED: 03-05-2010
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We previously showed that alcohol-preferring (P) rats have higher bone density than alcohol-nonpreferring (NP) rats. Genetic mapping in P and NP rats identified a major quantitative trait locus (QTL) between 4q22 and 4q34 for alcohol preference. At the same location, several QTLs linked to bone density and structure were detected in Fischer 344 (F344) and Lewis (LEW) rats, suggesting that bone mass and strength genes might cosegregate with genes that regulate alcohol preference. The aim of this study was to identify the genes segregating for skeletal phenotypes in congenic P and NP rats. Transfer of the NP chromosome 4 QTL into the P background (P.NP) significantly decreased areal bone mineral density (aBMD) and volumetric bone mineral density (vBMD) at several skeletal sites, whereas transfer of the P chromosome 4 QTL into the NP background (NP.P) significantly increased bone mineral content (BMC) and aBMD in the same skeletal sites. Microarray analysis from the femurs using Affymetrix Rat Genome arrays revealed 53 genes that were differentially expressed among the rat strains with a false discovery rate (FDR) of less than 10%. Nine candidate genes were found to be strongly correlated (r(2) > 0.50) with bone mass at multiple skeletal sites. The top three candidate genes, neuropeptide Y (Npy), alpha synuclein (Snca), and sepiapterin reductase (Spr), were confirmed using real-time quantitative PCR (qPCR). Ingenuity pathway analysis revealed relationships among the candidate genes related to bone metabolism involving beta-estradiol, interferon-gamma, and a voltage-gated calcium channel. We identified several candidate genes, including some novel genes on chromosome 4 segregating for skeletal phenotypes in reciprocal congenic P and NP rats.
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RNA polymerase II binding patterns reveal genomic regions involved in microRNA gene regulation.
PLoS ONE
PUBLISHED: 02-10-2010
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MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression. Due to the poor annotation of primary microRNA (pri-microRNA) transcripts, the precise location of promoter regions driving expression of many microRNA genes is enigmatic. This deficiency hinders our understanding of microRNA-mediated regulatory networks. In this study, we develop a computational approach to identify the promoter region and transcription start site (TSS) of pri-microRNAs actively transcribed using genome-wide RNA Polymerase II (RPol II) binding patterns derived from ChIP-seq data. Based upon the assumption that the distribution of RPol II binding patterns around the TSS of microRNA and protein coding genes are similar, we designed a statistical model to mimic RPol II binding patterns around the TSS of highly expressed, well-annotated promoter regions of protein coding genes. We used this model to systematically scan the regions upstream of all intergenic microRNAs for RPol II binding patterns similar to those of TSS from protein coding genes. We validated our findings by examining the conservation, CpG content, and activating histone marks in the identified promoter regions. We applied our model to assess changes in microRNA transcription in steroid hormone-treated breast cancer cells. The results demonstrate many microRNA genes have lost hormone-dependent regulation in tamoxifen-resistant breast cancer cells. MicroRNA promoter identification based upon RPol II binding patterns provides important temporal and spatial measurements regarding the initiation of transcription, and therefore allows comparison of transcription activities between different conditions, such as normal and disease states.
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Signal transducers and activators of transcription-1 (STAT1) regulates microRNA transcription in interferon gamma-stimulated HeLa cells.
PLoS ONE
PUBLISHED: 01-11-2010
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Constructing and modeling the gene regulatory network is one of the central themes of systems biology. With the growing understanding of the mechanism of microRNA biogenesis and its biological function, establishing a microRNA-mediated gene regulatory network is not only desirable but also achievable.
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Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation.
Epigenetics
PUBLISHED: 12-17-2009
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Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10, and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p<0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in gene expression, which together may contribute to the observed abnormal fetal development.
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Laboratory methods for high-throughput genotyping.
Cold Spring Harb Protoc
PUBLISHED: 10-15-2009
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The genetics of complex diseases has been given a tremendous boost in recent years by the introduction of high-throughput laboratory methods that make it possible to approach larger questions in larger populations and to cover the genome more comprehensively. The ability to determine genotypes of many individuals accurately and efficiently has allowed genetic studies that cover more of the variation within individual genes, instead of focusing only on one or a few coding variants, and to do so in study samples of reasonable power. Chip-based genotyping assays, combined with knowledge of the patterns of coinheritance of markers (linkage disequilibrium [LD]), have stimulated genome-wide association studies (GWAS) of complex diseases. Recent successes of GWAS in identifying specific genes that affect risk for common diseases are dramatic illustrations of how improved technology can lead to scientific breakthroughs. A key issue in high-throughput genotyping is to choose the appropriate technology for your goals and for the stage of your experiment, being cognizant of your sample numbers and resources. This article introduces some of the commonly used methods of high-throughput single-nucleotide polymorphism (SNP) genotyping for different stages of genetic studies and briefly reviews some of the high-throughput sequencing methods just coming into use. It also mentions some recent developments in "next-generation" sequencing that will enable other kinds of studies. This article is not intended to be comprehensive, and because technology in this area is rapidly changing, our comments should be taken as a starting point for further investigation.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.