New serine-derived gemini surfactants as gene delivery systems.

PubMed

Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; de Lima, Maria C Pedroso; Jurado, Amália S

2015-01-01

Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

NASA Astrophysics Data System (ADS)

Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

2013-12-01

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

PubMed

Erpen, L; Tavano, E C R; Harakava, R; Dutt, M; Grosser, J W; Piedade, S M S; Mendes, B M J; Mourão Filho, F A A

2018-05-23

Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

Loss of Activity-Induced Phosphorylation of MeCP2 Enhances Synaptogenesis, LTP, and Spatial Memory

PubMed Central

Li, Hongda; Zhong, Xiaofen; Chau, Kevin Fongching; Williams, Emily Cunningham; Chang, Qiang

2012-01-01

DNA methylation-dependent epigenetic mechanisms underlie the development and function of the mammalian brain. MeCP2 expresses highly in neurons, and functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Previous in vitro studies showed neuronal activity-induced phosphorylation (NAIP) of MeCP2 precedes its release from the Bdnf promoter and the ensuing Bdnf transcription. However, the in vivo function of this phosphorylation event remains elusive. We generated knockin mice that lack NAIP of MeCP2, and show here the Mecp2 phospho-mutant mice perform better in hippocampus-dependent memory tests, present enhanced LTP at two synapses in the hippocampus, and show increased excitatory synaptogenesis. At the molecular level, the phospho-mutant MeCP2 protein binds more tightly to several MeCP2 target gene promoters and alters the expression of these genes. Our results supply the first genetic evidence that NAIP of MeCP2 is required in modulating dynamic functions of the adult mouse brain. PMID:21765426

Loss of activity-induced phosphorylation of MeCP2 enhances synaptogenesis, LTP and spatial memory.

PubMed

Li, Hongda; Zhong, Xiaofen; Chau, Kevin Fongching; Williams, Emily Cunningham; Chang, Qiang

2011-07-17

DNA methylation-dependent epigenetic mechanisms underlie the development and function of the mammalian brain. MeCP2 is highly expressed in neurons and functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Previous in vitro studies have shown that neuronal activity-induced phosphorylation (NAIP) of methyl CpG-binding protein 2 (MeCP2) precedes its release from the Bdnf promoter and the ensuing Bdnf transcription. However, the in vivo function of this phosphorylation event remains elusive. We generated knock-in mice that lack NAIP of MeCP2 and found that they performed better in hippocampus-dependent memory tests, presented enhanced long-term potentiation at two synapses in the hippocampus and showed increased excitatory synaptogenesis. At the molecular level, the phospho-mutant MeCP2 protein bound more tightly to several MeCP2 target gene promoters and altered the expression of these genes. Our results suggest that NAIP of MeCP2 is required for modulating dynamic functions of the adult mouse brain.

Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

NASA Astrophysics Data System (ADS)

Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2017-11-01

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors.

PubMed

Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2017-11-17

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

Production of indole antibiotics induced by exogenous gene derived from sponge metagenomes.

PubMed

Takeshige, Yuya; Egami, Yoko; Wakimoto, Toshiyuki; Abe, Ikuro

2015-05-01

Sponge metagenomes are accessible genetic sources containing genes and gene clusters responsible for the biosynthesis of sponge-derived bioactive natural products. In this study, we obtained the clone pDC112, producing turbomycin A and 2,2-di(3-indolyl)-3-indolone, based on the functional screening of the metagenome library derived from the marine sponge Discodermia calyx. The subcloning experiment identified ORF 25, which is homologous to inosine 5'-monophosphate dehydrogenase and required for the production of 2,2-di(3-indolyl)-3-indolone in Escherichia coli.

Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ono, Hiromasa; Database Center for Life Science; Oki, Yoshinao

2011-04-15

Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed asmore » well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.« less

Transcriptome-Derived Tetranucleotide Microsatellites and Their Associated Genes from the Giant Panda (Ailuropoda melanoleuca).

PubMed

Song, Xuhao; Shen, Fujun; Huang, Jie; Huang, Yan; Du, Lianming; Wang, Chengdong; Fan, Zhenxin; Hou, Rong; Yue, Bisong; Zhang, Xiuyue

2016-09-01

Recently, an increasing number of microsatellites or simple sequence repeats (SSRs) have been found and characterized from transcriptomes. Such SSRs can be employed as putative functional markers to easily tag corresponding genes, which play an important role in biomedical studies and genetic analysis. However, the transcriptome-derived SSRs for giant panda (Ailuropoda melanoleuca) are not yet available. In this work, we identified and characterized 20 tetranucleotide microsatellite loci from a transcript database generated from the blood of giant panda. Furthermore, we assigned their predicted transcriptome locations: 16 loci were assigned to untranslated regions (UTRs) and 4 loci were assigned to coding regions (CDSs). Gene identities of 14 transcripts contained corresponding microsatellites were determined, which provide useful information to study the potential contribution of SSRs to gene regulation in giant panda. The polymorphic information content (PIC) values ranged from 0.293 to 0.789 with an average of 0.603 for the 16 UTRs-derived SSRs. Interestingly, 4 CDS-derived microsatellites developed in our study were also polymorphic, and the instability of these 4 CDS-derived SSRs was further validated by re-genotyping and sequencing. The genes containing these 4 CDS-derived SSRs were embedded with various types of repeat motifs. The interaction of all the length-changing SSRs might provide a way against coding region frameshift caused by microsatellite instability. We hope these newly gene-associated biomarkers will pave the way for genetic and biomedical studies for giant panda in the future. In sum, this set of transcriptome-derived markers complements the genetic resources available for giant panda. © The American Genetic Association. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Water-soluble fullerene (C60) derivatives as nonviral gene-delivery vectors.

PubMed

Sitharaman, Balaji; Zakharian, Tatiana Y; Saraf, Anita; Misra, Preeti; Ashcroft, Jared; Pan, Su; Pham, Quynh P; Mikos, Antonios G; Wilson, Lon J; Engler, David A

2008-01-01

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch-Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both

In vitro gene expression by cationized derivatives of an artificial protein with repeated RGD sequences, Pronectin.

PubMed

Hosseinkhani, Hossein; Tabata, Yasuhiko

2003-01-09

The objective of this study is to investigate the efficiency of a non-viral gene carrier with RGD sequences, Pronectin F(+) for gene transfection. The Pronectin F(+) was cationized by introducing ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) to the hydroxyl groups while the corresponding gelatin derivative was prepared similarly because gelatin also has one RGD sequence per molecule. The zeta potential and molecular size of Pronectin F(+) and gelatin derivatives were examined before and after polyion complexation with a plasmid DNA of luciferase. When complexed with the plasmid DNA at the Pronectin F(+)/plasmid DNA mixing ratio of 50, the complex exhibited a zeta potential of about 10 mV, which is similar to that of the gelatin derivative-plasmid DNA complex. Irrespective of the type of Pronectin F(+) and gelatin derivatives, their complexation enabled the apparent molecular size of plasmid DNA to reduce to about 200 nm, the size decreasing with the increased derivative/plasmid DNA weight mixing ratio. The rat gastric mucosal (RGM)-1 cells treated with both complexes exhibited significantly stronger luciferase activities than free plasmid DNA although the enhanced extent was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. Cell attachment was enhanced by the Pronectin F(+) derivative to a significant high extent compared with the gelatin derivative. The amount of plasmid DNA internalized into the cells was enhanced by the complexation with every Pronectin F(+) derivative compared with the gelatin derivative. For both of Pronectin F(+) and gelatin carriers, the buffering capacity of Sm derivatives was higher than that of Ed and Sd derivatives and comparable to that of polyethyleneimine. It is likely that the high efficiency of gene transfection for the Sm derivative is due to the superior buffering effect. We conclude that the Sm derivative of Pronectin F(+) is promising as a non-viral vector of gene transfection.

Identification of specific gene expression profiles in fibroblasts derived from middle ear cholesteatoma.

PubMed

Yoshikawa, Mamoru; Kojima, Hiromi; Wada, Kota; Tsukidate, Toshiharu; Okada, Naoko; Saito, Hirohisa; Moriyama, Hiroshi

2006-07-01

To investigate the role of fibroblasts in the pathogenesis of cholesteatoma. Tissue specimens were obtained from our patients. Middle ear cholesteatoma-derived fibroblasts (MECFs) and postauricular skin-derived fibroblasts (SFs) as controls were then cultured for a few weeks. These fibroblasts were stimulated with interleukin (IL) 1alpha and/or IL-1beta before gene expression assays. We used the human genome U133A probe array (GeneChip) and real-time polymerase chain reaction to examine and compare the gene expression profiles of the MECFs and SFs. Six patients who had undergone tympanoplasty. The IL-1alpha-regulated genes were classified into 4 distinct clusters on the basis of profiles differentially regulated by SF and MECF using a hierarchical clustering analysis. The messenger RNA expressions of LARC (liver and activation-regulated chemokine), GMCSF (granulocyte-macrophage colony-stimulating factor), epiregulin, ICAM1 (intercellular adhesion molecule 1), and TGFA (transforming growth factor alpha) were more strongly up-regulated by IL-1alpha and/or IL-1beta in MECF than in SF, suggesting that these fibroblasts derived from different tissues retained their typical gene expression profiles. Fibroblasts may play a role in hyperkeratosis of middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth. These fibroblasts may retain tissue-specific characteristics presumably controlled by epigenetic mechanisms.

Blood-Derived Smooth Muscle Cells as a Target for Gene Delivery

PubMed Central

Yang, Zhe; Shao, Hongwei; Tan, Yaohong; Eton, Darwin; Yu, Hong

2008-01-01

Objective To examine the feasibility of using blood-derived smooth muscle cells (BD-SMCs) as a target for to deliver therapeutic proteins. Materials and Methods Mononuclear cells (MNC) were isolated from peripheral blood. The outgrowth colonies from MNC culture were differentiated into BD-SMCs in media containing platelet-derived growth factor BB. Phenotypic characterization of BD-SMCs was assessed by immunocytochemistry. Cell proliferation, gene transfer efficiency with a retroviral vector, apoptosis, and the biological activity of the transduced gene product from the BD-SMCs were evaluated in vitro and in vivo in comparison with vascular derived SMC (VSMCs). Results BD-SMCs stained positive for SMC markers. No significant difference was observed between BD-SMCs and VSMCs in cell proliferation, migration, adhesiveness, and gene transfer efficiency. After BD-SMCs were transduced with a retroviral vector carrying the secreted alkaline phosphatase gene (SEAP), 174 ± 50 μg biologically active SEAP was produced per 106 cells over 24 hrs. After injecting 5×106 cells expressing SEAP intravenously into rabbits, SEAP concentration increased significantly in the circulation from 0.14 ± 0.04 μg/ml to 2.34 ± 0.16 μg/ml 3 days after cell injection (P<0.01, n=3). Circulating levels of SEAP decreased to 1.76 μg /ml one week later and remained at this level up to 8 weeks, then declined to pre-cell injection level at 12 weeks. VSMC in vivo gene expression data were equivalent. Conclusion BD-SMCs have similar characteristics to mature VSMCs, and can be used as a novel target for gene transfer to deliver a therapeutic protein. Clinical relevance Cell-based therapy strategies offer the potential to correct a wide spectrum of inherited and acquired human diseases. Translation to a clinical trial will require a detailed pre-clinical study to understand the characteristics of the isolated cells. BD-SMC are practical and effective targets for ex vivo genetic engineering. They are

An efficient nonviral gene-delivery vector based on hyperbranched cationic glycogen derivatives.

PubMed

Liang, Xuan; Ren, Xianyue; Liu, Zhenzhen; Liu, Yingliang; Wang, Jue; Wang, Jingnan; Zhang, Li-Ming; Deng, David Yb; Quan, Daping; Yang, Liqun

2014-01-01

The purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector. A series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino)-1-propylamine (DMAPA-Glyp) and 1-(2-aminoethyl) piperazine (AEPZ-Glyp) residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid-base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA) condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney) and the CNE2 (human nasopharyngeal carcinoma) cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley rats) was evaluated to identify the safety and transfection efficiency. The hyperbranched cationic glycogen derivatives conjugated with DMAPA and AEPZ residues were synthesized. They exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI). They were able to bind and condense pDNA to form the complexes of 100-250 nm in size. The transfection efficiency of the DMAPA-Glyp/pDNA complexes was higher than those of the AEPZ-Glyp/pDNA complexes in both the 293T and CNE2 cells, and almost equal to those of bPEI. Furthermore, pDNA could be more safely delivered to the blood vessels in brain tissue of Sprague Dawley rats

Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue.

PubMed

Brown, C M; Longhurst, C; Haynes, G; Plater-Zyberk, C; Malcolm, A; Maini, R N

1992-08-01

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38.5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3+) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovial-derived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5+ B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.

Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

PubMed Central

Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

2013-01-01

Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

Gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow

PubMed Central

Kim, Su-Hwan; Kim, Young-Sung; Lee, Su-Yeon; Kim, Kyoung-Hwa; Lee, Yong-Moo; Kim, Won-Kyung

2011-01-01

Purpose The aim of this study is to compare the gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow for characterization of dental stem cells. Methods We employed GeneChip analysis to the expression levels of approximately 32,321 kinds of transcripts in 5 samples of bone-marrow-derived mesenchymal stem cells (BMSCs) (n=1), periodontal ligament stem cells (PDLSCs) (n=2), and dental pulp stem cells (DPSCs) (n=2). Each cell was sorted by a FACS Vantage Sorter using immunocytochemical staining of the early mesenchymal stem cell surface marker STRO-1 before the microarray analysis. Results We identified 379 up-regulated and 133 down-regulated transcripts in BMSCs, 68 up-regulated and 64 down-regulated transcripts in PDLSCs, and 218 up-regulated and 231 down-regulated transcripts in DPSCs. In addition, anatomical structure development and anatomical structure morphogenesis gene ontology (GO) terms were over-represented in all three different mesenchymal stem cells and GO terms related to blood vessels, and neurons were over-represented only in DPSCs. Conclusions This study demonstrated the genome-wide gene expression patterns of STRO-1+ mesenchymal stem cells derived from dental tissues and bone marrow. The differences among the expression profiles of BMSCs, PDLSCs, and DPSCs were shown, and 999 candidate genes were found to be definitely up- or down-regulated. In addition, GOstat analyses of regulated gene products provided over-represented GO classes. These data provide a first step for discovering molecules key to the characteristics of dental stem cells. PMID:21954424

Ectopical expression of FABP4 gene can induce bovine muscle-derived stem cells adipogenesis.

PubMed

Zhang, Le; Zhao, Yanfang; Ning, Yue; Wang, Hongbao; Zan, Linsen

2017-01-08

Fatty acid binding protein 4 (FABP4) plays a key role in Fatty acid catabolism in mammals. Findings from our previous studies have indicated that FABP4 neither affect the differentiation of bovine preadipocytes nor does it change the expression of upstream genes. To investigate whether ectopically expressed FABP4 can induces Muscle-Derived Stem Cells (MDSCs) lipid synthesis and understand the regulatory mechanism behind it. In this study, adenoviruses infection is achieved to ectopically expressed FABP4 in bovine MDSCs, RNA-seq analyses at the very early stages of induction were performed to reveal gene expression level changes during MDSCs transdifferentiation. Results showed FABP4 can induce bovine Muscle-Derived Stem Cells transdifferentiation into adipocyte-like cells, 23 genes' expression levels changed after 24 h inducing although there is no significant change in cell phenotypes. Along with induction time, more differently expressed genes (256 genes changes after 48 h induction) were screened out. These genes should be at the downstream of signal pathways and be regulated by the 23 genes identified before. Our findings may provide a unique new model for studying the molecular control of cattle cross-talk between adipose and skeletal muscle. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of secondary organic aerosol from isoprene-derived hydroxyhydroperoxides on the expression of oxidative stress response genes in human bronchial epithelial cells.

PubMed

Arashiro, Maiko; Lin, Ying-Hsuan; Zhang, Zhenfa; Sexton, Kenneth G; Gold, Avram; Jaspers, Ilona; Fry, Rebecca C; Surratt, Jason D

2018-02-21

Isoprene-derived secondary organic aerosol (SOA), which comprise a large portion of atmospheric fine particulate matter (PM 2.5 ), can be formed through various gaseous precursors, including isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene hydroxyhydroperoxides (ISOPOOH). The composition of the isoprene-derived SOA affects its reactive oxygen species (ROS) generation potential and its ability to alter oxidative stress-related gene expression. In this study we assess effects of isoprene SOA derived solely from ISOPOOH oxidation on human bronchial epithelial cells by measuring the gene expression changes in 84 oxidative stress-related genes. In addition, the thiol reactivity of ISOPOOH-derived SOA was measured through the dithiothreitol (DTT) assay. Our findings show that ISOPOOH-derived SOA alter more oxidative-stress related genes than IEPOX-derived SOA but not as many as MAE-derived SOA on a mass basis exposure. More importantly, we found that the different types of SOA derived from the various gaseous precursors (MAE, IEPOX, and ISOPOOH) have unique contributions to changes in oxidative stress-related genes that do not total all gene expression changes seen in exposures to atmospherically relevant compositions of total isoprene-derived SOA mixtures. This study suggests that amongst the different types of known isoprene-derived SOA, MAE-derived SOA are the most potent inducer of oxidative stress-related gene changes but highlights the importance of considering isoprene-derived SOA as a total mixture for pollution controls and exposure studies.

HIV-derived vectors for gene therapy targeting dendritic cells.

PubMed

Rossetti, Maura; Cavarelli, Mariangela; Gregori, Silvia; Scarlatti, Gabriella

2013-01-01

Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) have the potential to mediate stable therapeutic gene transfer. However, similarly to other viral vectors, their benefit is compromised by the induction of an immune response toward transgene-expressing cells that closely mimics antiviral immunity. LV share with the parental HIV the ability to activate dendritic cells (DC), while lack the peculiar ability of subverting DC functions, which is responsible for HIV immune escape. Understanding the interaction between LV and DC, with plasmacytoid and myeloid DC playing fundamental and distinct roles, has paved the way to novel approaches aimed at regulating transgene-specific immune responses. Thanks to the ability to target either DC subsets LV might be a powerful tool to induce immunity (i.e., gene therapy of cancer), cell death (i.e., in HIV/AIDS infection), or tolerance (i.e., gene therapy strategies for monogenic diseases). In this chapter, similarities and differences between the LV-mediated and HIV-mediated induction of immune responses, with specific focus on their interactions with DC, are discussed.

Gene Expression Profiling in Human Lung Cells Exposed to Isoprene-Derived Secondary Organic Aerosol.

PubMed

Lin, Ying-Hsuan; Arashiro, Maiko; Clapp, Phillip W; Cui, Tianqu; Sexton, Kenneth G; Vizuete, William; Gold, Avram; Jaspers, Ilona; Fry, Rebecca C; Surratt, Jason D

2017-07-18

Secondary organic aerosol (SOA) derived from the photochemical oxidation of isoprene contributes a substantial mass fraction to atmospheric fine particulate matter (PM 2.5 ). The formation of isoprene SOA is influenced largely by anthropogenic emissions through multiphase chemistry of its multigenerational oxidation products. Considering the abundance of isoprene SOA in the troposphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. In this study, we assessed the effects of isoprene SOA on gene expression in human airway epithelial cells (BEAS-2B) through an air-liquid interface exposure. Gene expression profiling of 84 oxidative stress and 249 inflammation-associated human genes was performed. Our results show that the expression levels of 29 genes were significantly altered upon isoprene SOA exposure under noncytotoxic conditions (p < 0.05), with the majority (22/29) of genes passing a false discovery rate threshold of 0.3. The most significantly affected genes belong to the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor network. The Nrf2 function is confirmed through a reporter cell line. Together with detailed characterization of SOA constituents, this study reveals the impact of isoprene SOA exposure on lung responses and highlights the importance of further understanding its potential health outcomes.

Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

PubMed

Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

2015-07-01

Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression*

PubMed Central

Wang, Qian; Jia, Jingjing; Chikina, Maria; Pincas, Hanna; Dolios, Georgia; Sasaki, Kazuki; Wang, Rong; Minamino, Naoto; Sealfon, Stuart C.

2016-01-01

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gαs knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gαs knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gαs. In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs. PMID:27466366

GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells.

PubMed

Nazari, Fatemeh; Parham, Abbas; Maleki, Adham Fani

2015-01-01

Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. This study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

Differential reconstructed gene interaction networks for deriving toxicity threshold in chemical risk assessment.

PubMed

Yang, Yi; Maxwell, Andrew; Zhang, Xiaowei; Wang, Nan; Perkins, Edward J; Zhang, Chaoyang; Gong, Ping

2013-01-01

Pathway alterations reflected as changes in gene expression regulation and gene interaction can result from cellular exposure to toxicants. Such information is often used to elucidate toxicological modes of action. From a risk assessment perspective, alterations in biological pathways are a rich resource for setting toxicant thresholds, which may be more sensitive and mechanism-informed than traditional toxicity endpoints. Here we developed a novel differential networks (DNs) approach to connect pathway perturbation with toxicity threshold setting. Our DNs approach consists of 6 steps: time-series gene expression data collection, identification of altered genes, gene interaction network reconstruction, differential edge inference, mapping of genes with differential edges to pathways, and establishment of causal relationships between chemical concentration and perturbed pathways. A one-sample Gaussian process model and a linear regression model were used to identify genes that exhibited significant profile changes across an entire time course and between treatments, respectively. Interaction networks of differentially expressed (DE) genes were reconstructed for different treatments using a state space model and then compared to infer differential edges/interactions. DE genes possessing differential edges were mapped to biological pathways in databases such as KEGG pathways. Using the DNs approach, we analyzed a time-series Escherichia coli live cell gene expression dataset consisting of 4 treatments (control, 10, 100, 1000 mg/L naphthenic acids, NAs) and 18 time points. Through comparison of reconstructed networks and construction of differential networks, 80 genes were identified as DE genes with a significant number of differential edges, and 22 KEGG pathways were altered in a concentration-dependent manner. Some of these pathways were perturbed to a degree as high as 70% even at the lowest exposure concentration, implying a high sensitivity of our DNs approach

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

PubMed

Choi, Soon Gang; Wang, Qian; Jia, Jingjing; Chikina, Maria; Pincas, Hanna; Dolios, Georgia; Sasaki, Kazuki; Wang, Rong; Minamino, Naoto; Salton, Stephen R J; Sealfon, Stuart C

2016-09-30

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gα s knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gα s knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gα s In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Erythroid Kruppel-like factor (EKLF) is recruited to the γ-globin gene promoter as a co-activator and is required for γ-globin gene induction by short-chain fatty acid derivatives

PubMed Central

Perrine, Susan P.; Mankidy, Rishikesh; Boosalis, Michael S.; Bieker, James J.; Faller, Douglas V.

2011-01-01

Objectives The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for β-type globin gene switching, and specifically activates transcription of the adult β-globin gene promoter. We sought to determine if EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. Methods The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Results and conclusions Knockdown of EKLF prevents SCFA-induced expression of the γ-globin promoter in a stably expressed μLCRβprRlucAγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous γ-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for γ-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous γ-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. PMID:19220418

Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

PubMed

Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

2009-01-01

Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

Plant-derived isoprenoid sweeteners: recent progress in biosynthetic gene discovery and perspectives on microbial production.

PubMed

Seki, Hikaru; Tamura, Keita; Muranaka, Toshiya

2018-06-01

Increased public awareness of negative health effects associated with excess sugar consumption has triggered increasing interest in plant-derived natural sweeteners. Steviol glycosides are a group of highly sweet diterpene glycosides contained in the leaves of stevia (Stevia rebaudiana). Mogrosides, extracted from monk fruit (Siraitia grosvenorii), are a group of cucurbitane-type triterpenoid glycosides. Glycyrrhizin is an oleanane-type triterpenoid glycoside derived from the underground parts of Glycyrrhiza plants (licorice). This review focuses on the natural isoprenoid sweetening agents steviol glycosides, mogrosides, and glycyrrhizin, and describes recent progress in gene discovery and elucidation of the catalytic functions of their biosynthetic enzymes. Recently, remarkable progress has been made in engineering the production of various plant-specialized metabolites in microbial hosts such as Saccharomyces cerevisiae via the introduction of biosynthetic enzyme genes. Perspectives on the microbial production of plant-derived natural sweeteners are also discussed.

Small-interfering RNAs from natural antisense transcripts derived from a cellulose synthase gene modulate cell wall biosynthesis in barley

PubMed Central

Held, Michael A.; Penning, Bryan; Brandt, Amanda S.; Kessans, Sarah A.; Yong, Weidong; Scofield, Steven R.; Carpita, Nicholas C.

2008-01-01

Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3′-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends ≥1230 bp from the 3′ end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-14C-Glc into cellulose and into mixed-linkage (1 → 3),(1 → 4)-β-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis. PMID:19075248

Characterization of basic amino acids-conjugated PAMAM dendrimers as gene carriers for human adipose-derived mesenchymal stem cells.

PubMed

Bae, Yoonhee; Lee, Sunray; Green, Eric S; Park, Jung Hyun; Ko, Kyung Soo; Han, Jin; Choi, Joon Sig

2016-03-30

Since mesenchymal stem cells (MSCs) can self-renew and differentiate into multiple cell types, the delivery of genes to this type of cell can be an important tool in the emerging field of tissue regeneration and engineering. However, development of more efficient and safe nonviral vectors for gene delivery to stem cells in particular still remains a great challenge. In this study, we describe a group of nonviral gene delivery vectors, conjugated PAMAM derivatives (PAMAM-H-R, PAMAM-H-K, and PAMAM-H-O), displaying affinity toward human adipose-derived mesenchymal stem cells (AD-MSCs). Transfection efficiency using pDNA encoding for luciferase (Luc) and enhanced green fluorescent protein (EGFP), and cytotoxicity assays were performed in human AD-MSCs. The results show that transfection efficiencies of conjugated PAMAM derivatives are improved significantly compared to native PAMAM dendrimer, and that among PAMAM derivatives, cytotoxicity of PAMAM-H-K and PAMAM-H-O were very low. Also, treatment of human AD-MSCs to polyplex formation in conjugated PAMAM derivatives, their cellular uptake and localization were analyzed by flow cytometry and confocal microscopy. Copyright © 2016 Elsevier B.V. All rights reserved.

Intracellular high cholesterol content disorders the clock genes, apoptosis-related genes and fibrinolytic-related genes rhythmic expressions in human plaque-derived vascular smooth muscle cells.

PubMed

Lin, Changpo; Tang, Xiao; Xu, Lirong; Qian, Ruizhe; Shi, Zhenyu; Wang, Lixin; Cai, Tingting; Yan, Dong; Fu, Weiguo; Guo, Daqiao

2017-07-10

The clock genes are involved in regulating cardiovascular functions, and their expression disorders would lead to circadian rhythm disruptions of clock-controlled genes (CCGs), resulting in atherosclerotic plaque formation and rupture. Our previous study revealed the rhythmic expression of clock genes were attenuated in human plaque-derived vascular smooth muscle cells (PVSMCs), but failed to detect the downstream CCGs expressions and the underlying molecular mechanism. In this study, we examined the difference of CCGs rhythmic expression between human normal carotid VSMCs (NVSMCs) and PVSMCs. Furthermore, we compared the cholesterol and triglycerides levels between two groups and the link to clock genes and CCGs expressions. Seven health donors' normal carotids and 19 carotid plaques yielded viable cultured NVSMCs and PVSMCs. The expression levels of target genes were measured by quantitative real-time PCR and Western-blot. The intracellular cholesterol and triglycerides levels were measured by kits. The circadian expressions of apoptosis-related genes and fibrinolytic-related genes were disordered. Besides, the cholesterol levels were significant higher in PVSMCs. After treated with cholesterol or oxidized low density lipoprotein (ox-LDL), the expressions of clock genes were inhibited; and the rhythmic expressions of clock genes, apoptosis-related genes and fibrinolytic-related genes were disturbed in NVSMCs, which were similar to PVSMCs. The results suggested that intracellular high cholesterol content of PVSMCs would lead to the disorders of clock genes and CCGs rhythmic expressions. And further studies should be conducted to demonstrate the specific molecular mechanisms involved.

Modulation of Mycoplasma arthritidis-derived superantigen-induced cytokine gene expression by dexamethasone and interleukin-4.

PubMed Central

Mehindate, K; al-Daccak, R; Rink, L; Mecheri, S; Hébert, J; Mourad, W

1994-01-01

Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and the IL-1 receptor antagonist (IL-1Ra). In contrast, superantigens induce TNF-alpha and IL-1 beta but fail to affect IL-1Ra gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1 beta and TNF-alpha gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1 beta and TNF-alpha gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IL-4, but not DEX, induced a strong and rapid expression of IL-1Ra mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-1Ra gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX. Images PMID:7927746

Modulation of Mycoplasma arthritidis-derived superantigen-induced cytokine gene expression by dexamethasone and interleukin-4.

PubMed

Mehindate, K; al-Daccak, R; Rink, L; Mecheri, S; Hébert, J; Mourad, W

1994-11-01

Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and the IL-1 receptor antagonist (IL-1Ra). In contrast, superantigens induce TNF-alpha and IL-1 beta but fail to affect IL-1Ra gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1 beta and TNF-alpha gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1 beta and TNF-alpha gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IL-4, but not DEX, induced a strong and rapid expression of IL-1Ra mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-1Ra gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX.

MlNCD1: A novel Aegilops tauschii derived powdery mildew resistance gene identified in common wheat

USDA-ARS?s Scientific Manuscript database

Powdery mildew is a major fungal disease in wheat, especially in cool maritime climates. A novel Aegilops tauschii derived wheat powdery mildew resistance gene present in the germplasm line NC96BGTD1 was genetically characterized as a monogenic trait in field trials using F2 and F4-derived lines fr...

RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy.

PubMed

Tao, Wensi; Ayala-Haedo, Juan A; Field, Matthew G; Pelaez, Daniel; Wester, Sara T

2017-12-01

The purpose of this study was to characterize the intrinsic cellular properties of orbital adipose-derived stem cells (OASC) from patients with thyroid-associated orbitopathy (TAO) and healthy controls. Orbital adipose tissue was collected from a total of nine patients: four controls and five patients with TAO. Isolated OASC were characterized with mesenchymal stem cell-specific markers. Orbital adipose-derived stem cells were differentiated into three lineages: chondrocytes, osteocytes, and adipocytes. Reverse transcription PCR of genes involved in the adipogenesis, chondrogenesis, and osteogenesis pathways were selected to assay the differentiation capacities. RNA sequencing analysis (RNA-seq) was performed and results were compared to assess for differences in gene expression between TAO and controls. Selected top-ranked results were confirmed by RT-PCR. Orbital adipose-derived stem cells isolated from orbital fat expressed high levels of mesenchymal stem cell markers, but low levels of the pluripotent stem cell markers. Orbital adipose-derived stem cells isolated from TAO patients exhibited an increase in adipogenesis, and a decrease in chondrogenesis and osteogenesis. RNA-seq disclosed 54 differentially expressed genes. In TAO OASC, expression of early neural crest progenitor marker (WNT signaling, ZIC genes and MSX2) was lost. Meanwhile, ectopic expression of HOXB2 and HOXB3 was found in the OASC from TAO. Our results suggest that there are intrinsic genetic and cellular differences in the OASC populations derived from TAO patients. The upregulation in adipogenesis in OASC of TAO may be is consistent with the clinical phenotype. Downregulation of early neural crest markers and ectopic expression of HOXB2 and HOXB3 in TAO OASC demonstrate dysregulation of developmental and tissue patterning pathways.

RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy

PubMed Central

Tao, Wensi; Ayala-Haedo, Juan A.; Field, Matthew G.; Pelaez, Daniel; Wester, Sara T.

2017-01-01

Purpose The purpose of this study was to characterize the intrinsic cellular properties of orbital adipose-derived stem cells (OASC) from patients with thyroid-associated orbitopathy (TAO) and healthy controls. Methods Orbital adipose tissue was collected from a total of nine patients: four controls and five patients with TAO. Isolated OASC were characterized with mesenchymal stem cell–specific markers. Orbital adipose-derived stem cells were differentiated into three lineages: chondrocytes, osteocytes, and adipocytes. Reverse transcription PCR of genes involved in the adipogenesis, chondrogenesis, and osteogenesis pathways were selected to assay the differentiation capacities. RNA sequencing analysis (RNA-seq) was performed and results were compared to assess for differences in gene expression between TAO and controls. Selected top-ranked results were confirmed by RT-PCR. Results Orbital adipose-derived stem cells isolated from orbital fat expressed high levels of mesenchymal stem cell markers, but low levels of the pluripotent stem cell markers. Orbital adipose-derived stem cells isolated from TAO patients exhibited an increase in adipogenesis, and a decrease in chondrogenesis and osteogenesis. RNA-seq disclosed 54 differentially expressed genes. In TAO OASC, expression of early neural crest progenitor marker (WNT signaling, ZIC genes and MSX2) was lost. Meanwhile, ectopic expression of HOXB2 and HOXB3 was found in the OASC from TAO. Conclusion Our results suggest that there are intrinsic genetic and cellular differences in the OASC populations derived from TAO patients. The upregulation in adipogenesis in OASC of TAO may be is consistent with the clinical phenotype. Downregulation of early neural crest markers and ectopic expression of HOXB2 and HOXB3 in TAO OASC demonstrate dysregulation of developmental and tissue patterning pathways. PMID:29214313

Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

PubMed Central

Liu, Kehai; Wang, Xiaoyu; Fan, Wei; Zhu, Qing; Yang, Jingya; Gao, Jing; Gao, Shen

2012-01-01

Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy. PMID:22412301

The Effect of Silver Carp Skin-Derived Peptides on the Activities of VEGF and Hsp70.1 Gene Transcription Promoters.

PubMed

Wojtkowiak, Diana; Piechowicz, Janina; Grzenkowicz-Wydra, Jolanta; Wosiński, Stanisław; Dominiak, Marzena; Hadzik, Jakub; Frydrychowski, Andrzej F

2016-01-01

Studies conducted on human cell culture models have demonstrated that collagen-derived peptides can exert a beneficial effect in medicine. However, all these studies were conducted using animal collagen samples, most often originating from bovine or porcine skin. Currently attempts are being made to replace animal collagen with fish collagen. The aim of the study was to compare the effect of silver carp skin-derived peptide extract on the transcriptional activities of human VEGF and hsp70.1 gene promoters inserted into the plasmids with secreted alkaline phosphatase as a reporter gene. Changes in the activity of the promoters were investigated using a HEK293FT cell line transfected with pVEGF-SEAP or pHsp70-SEAP. The cells were cultured in dishes containing peptides separated using reverse-phase high performance liquid chromatography. The study demonstrated that the silver carp skin-derived peptide extract exerts both an inhibitory effect on the VEGF gene promoter and activating effect on the hsp70.1 gene promoter. Higher biological activity was recorded in the case of a freshly prepared peptide extract compared to one stored at 4°C for three months. The silver carp skin-derived collagen peptides influence VEGF and hsp70.1 gene promoters' transcriptional activity.

Novel pregnenolone derivatives modulate apoptosis via Bcl-2 family genes in hepatocellular carcinoma in vitro.

PubMed

Elhinnawi, Manar A; Mohareb, Rafat M; Rady, Hanaa M; Khalil, Wagdy K B; Abd Elhalim, Mervat M; Elmegeed, Gamal A

2018-06-10

A series of pregnenolone derivatives were synthesized and assessed for anti-cancer activity against hepatocellular carcinoma cell line (HepG2). The synthesized hetero-steroids (compounds 3, 4, 5, 6, 7, 8a and 8b) were evaluated for their cytotoxic activities using MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Apoptotic activity was assessed using dual acridine orange/ethidium bromide staining method and DNA fragmentation assay. Pro-apoptotic genes (Bax and Bak) and anti-apoptotic genes (Bcl-2 and Bcl-xL) were analyzed using quantitative real time PCR. The results revealed that compounds 4 and 6 displayed cytotoxic activity (IC 50s , 36.97 ± 2.18 and 18.46 ± 0.64 µM, respectively), while compounds 5 and 7 exhibited weak cytotoxic activity (IC 50s , 93.87 ± 8.30 µM and 93.48 ± 4.14 µM, respectively). All synthesized heterocyclic pregnenolone derivatives induced apoptosis through DNA fragmentation. Compounds 4 and 6 increased early and late apoptotic cell percentages while compounds 3, 5, 7 and 8b increased either early or late apoptotic cell percentage. Moreover, compounds 3, 6 and 8b up-regulated the expression level of Bak gene. On the other hand, compounds 4, 5, 7 and 8a down-regulated the Bcl-2 expression level, besides, compounds 5, 7 and 8a down-regulated the Bcl-xL expression level. Compounds 5, 7, 8a and 8b increased the Bak/Bcl-xL ratio, besides, compound 8a raised the Bax/Bcl-xL ratio whereas compound 5 elevated Bax/Bcl-2 and Bak/Bcl-2 ratios. The present work introduced novel pro-apoptotic pregnenolone derivatives that acted against HepG2 cells through DNA fragmentation, apoptotic morphological changes and were able to increase the pro-apoptotic/anti-apoptotic ratios of Bcl-2 family genes. This study particularly revealed that the cytotoxic compound 4 is the most promising pro-apoptotic compound among other synthesized derivatives where it induced apoptosis (late and early) through the down-regulation of

Ultrasound -Assisted Gene Transfer to Adipose Tissue-Derived Stem/Progenitor Cells (ASCs)

NASA Astrophysics Data System (ADS)

Miyamoto, Yoshitaka; Ueno, Hitomi; Hokari, Rei; Yuan, Wenji; Kuno, Shuichi; Kakimoto, Takashi; Enosawa, Shin; Negishi, Yoichi; Yoshinaka, Kiyoshi; Matsumoto, Yoichiro; Chiba, Toshio; Hayashi, Shuji

2011-09-01

In recent years, multilineage adipose tissue-derived stem cells (ASCs) have become increasingly attractive as a promising source for cell transplantation and regenerative medicine. Particular interest has been expressed in the potential to make tissue stem cells, such as ASCs and marrow stromal cells (MSCs), differentiate by gene transfection. Gene transfection using highly efficient viral vectors such as adeno- and sendai viruses have been developed for this purpose. Sonoporation, or ultrasound (US)-assisted gene transfer, is an alternative gene manipulation technique which employs the creation of a jet stream by ultrasonic microbubble cavitation. Sonoporation using non-viral vectors is expected to be a much safer, although less efficient, tool for prospective clinical gene therapy. In this report, we assessed the efficacy of the sonoporation technique for gene transfer to ASCs. We isolated and cultured adipocyets from mouse adipose tissue. ASCs that have the potential to differentiate with transformation into adipocytes or osteoblasts were obtained. Using the US-assisted system, plasmid DNA containing beta-galactosidase (beta-Gal) and green fluorescent protein (GFP) genes were transferred to the ASCs. For this purpose, a Sonopore 4000 (NEPAGENE Co.) and a Sonazoid (Daiichi Sankyo Co.) instrument were used in combination. ASCs were subjected to US (3.1 MHz, 50% duty cycle, burst rate 2.0 Hz, intensity 1.2 W/cm2, exposure time 30 sec). We observed that the gene was more efficiently transferred with increased concentrations of plasmid DNA (5-150 μg/mL). However, further optimization of the US parameters is required, as the gene transfer efficiency was still relatively low. In conclusion, we herein demonstrate that a gene can be transferred to ASCs using our US-assisted system. In regenerative medicine, this system might resolve the current issues surrounding the use of viral vectors for gene transfer.

Effect of systemic administration of lipopolysaccharides derived from Porphyromonas gingivalis on gene expression in mice kidney.

PubMed

Harada, Fumiya; Uehara, Osamu; Morikawa, Tetsuro; Hiraki, Daichi; Onishi, Aya; Toraya, Seiko; Adhikari, Bhoj Raj; Takai, Rie; Yoshida, Koki; Sato, Jun; Nishimura, Michiko; Chiba, Itsuo; Wu, Ching Zong; Abiko, Yoshihiro

2018-01-31

Although an association between periodontitis and chronic kidney disease (CKD) has been suggested, the mechanism involved remains unclear. Herein, we examined the global gene expression profile in a mouse model that showed no acute inflammation in the kidney following stimulation with lipopolysaccharides (LPS) derived from Porphyromonas gingivalis (PG-LPS). The mice were injected with PG-LPS at a concentration of 5 mg/kg intraperitoneally, every 3 days, for 1 month. Microarray analysis was used to identify 10 genes with the highest expression levels in the kidney stimulated with PG-LPS. Among them, the functions of five genes (Saa3, Ticam2, Reg3b, Ocxt2a, and Xcr1) were known. The upregulation of these genes was confirmed by quantitative polymerase chain reaction assay. Furthermore, we examined whether the expression of these upregulated genes were altered in endothelial cells derived from the kidney, in vitro. The mRNA expression levels of all five genes were significantly higher in the experimental group than in the controls (no LPS stimulation; *p < 0.05). In conclusion, the responses noted in the kidney may have arisen mainly from the endothelial cells. Moreover, upregulation of the expression levels of Saa3, Ticam2, Reg3b, Ocxt2a, and Xcr1 may be associated with the pathogenesis of CKD.

Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

PubMed

Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

2015-01-01

Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

A High Resolution Land Cover Data Product to Remove Urban Density Over-Estimation Bias for Coupled Urban-Vegetation-Atmosphere Interaction Studies

NASA Astrophysics Data System (ADS)

Shaffer, S. R.

2017-12-01

Coupled land-atmosphere interactions in urban settings modeled with the Weather Research and Forecasting model (WRF) derive urban land cover from 30-meter resolution National Land Cover Database (NLCD) products. However, within urban areas, the categorical NLCD lose information of non-urban classifications whenever the impervious cover within a grid cell is above 0%, and the current method to determine urban area over estimates the actual area, leading to a bias of urban contribution. To address this bias of urban contribution an investigation is conducted by employing a 1-meter resolution land cover data product derived from the National Agricultural Imagery Program (NAIP) dataset. Scenes during 2010 for the Central Arizona Phoenix Long Term Ecological Research (CAP-LTER) study area, roughly a 120 km x 100 km area containing metropolitan Phoenix, are adapted for use within WRF to determine the areal fraction and urban fraction of each WRF urban class. A method is shown for converting these NAIP data into classes corresponding to NLCD urban classes, and is evaluated in comparison with current WRF implementation using NLCD. Results are shown for comparisons of land cover products at the level of input data and aggregated to model resolution (1 km). The sensitivity of WRF short-term summertime pre-monsoon predictions within metropolitan Phoenix to different input data products of land cover, to method of aggregating these data to model grid scale (1 km), for the default and derived parameter values are examined with the Noah mosaic land surface scheme adapted for using these data. Issues with adapting these non-urban NAIP classes for use in the mosaic approach will also be discussed.

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

PubMed Central

Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Park, Ji-Sung; Lee, Seung-Chan; Baregundi Subbarao, Raghavendra; Lee, Sung-Lim; Park, Bong-Wook; King, William Allan; Rho, Gyu-Jin

2015-01-01

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs. PMID:25972899

Liver Cell-Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

PubMed Central

Witek, Rafal P.; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S.; Cheong, Yeiwon; Fearing, Caitlin M.; Agboola, Kolade M.; Chen, Wei; Diehl, Anna Mae

2013-01-01

Background & Aims Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). Methods MF-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Conclusions Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy. PMID:19013163

Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus.

PubMed

Kant, Ravi; Dasgupta, Indranil

2017-07-01

Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v /F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.

A New Class of SINEs with snRNA Gene-Derived Heads.

PubMed

Kojima, Kenji K

2015-05-27

Eukaryotic genomes are colonized by various transposons including short interspersed elements (SINEs). The 5' region (head) of the majority of SINEs is derived from one of the three types of RNA genes--7SL RNA, transfer RNA (tRNA), or 5S ribosomal RNA (rRNA)--and the internal promoter inside the head promotes the transcription of the entire SINEs. Here I report a new group of SINEs whose heads originate from either the U1 or U2 small nuclear RNA gene. These SINEs, named SINEU, are distributed among crocodilians and classified into three families. The structures of the SINEU-1 subfamilies indicate the recurrent addition of a U1- or U2-derived sequence onto the 5' end of SINEU-1 elements. SINEU-1 and SINEU-3 are ancient and shared among alligators, crocodiles, and gharials, while SINEU-2 is absent in the alligator genome. SINEU-2 is the only SINE family that was active after the split of crocodiles and gharials. All SINEU families, especially SINEU-3, are preferentially inserted into a family of Mariner DNA transposon, Mariner-N4_AMi. A group of Tx1 non-long terminal repeat retrotransposons designated Tx1-Mar also show target preference for Mariner-N4_AMi, indicating that SINEU was mobilized by Tx1-Mar. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Derived variants at six genes explain nearly half of size reduction in dog breeds.

PubMed

Rimbault, Maud; Beale, Holly C; Schoenebeck, Jeffrey J; Hoopes, Barbara C; Allen, Jeremy J; Kilroy-Glynn, Paul; Wayne, Robert K; Sutter, Nathan B; Ostrander, Elaine A

2013-12-01

Selective breeding of dogs by humans has generated extraordinary diversity in body size. A number of multibreed analyses have been undertaken to identify the genetic basis of this diversity. We analyzed four loci discovered in a previous genome-wide association study that used 60,968 SNPs to identify size-associated genomic intervals, which were too large to assign causative roles to genes. First, we performed fine-mapping to define critical intervals that included the candidate genes GHR, HMGA2, SMAD2, and STC2, identifying five highly associated markers at the four loci. We hypothesize that three of the variants are likely to be causative. We then genotyped each marker, together with previously reported size-associated variants in the IGF1 and IGF1R genes, on a panel of 500 domestic dogs from 93 breeds, and identified the ancestral allele by genotyping the same markers on 30 wild canids. We observed that the derived alleles at all markers correlated with reduced body size, and smaller dogs are more likely to carry derived alleles at multiple markers. However, breeds are not generally fixed at all markers; multiple combinations of genotypes are found within most breeds. Finally, we show that 46%-52.5% of the variance in body size of dog breeds can be explained by seven markers in proximity to exceptional candidate genes. Among breeds with standard weights <41 kg (90 lb), the genotypes accounted for 64.3% of variance in weight. This work advances our understanding of mammalian growth by describing genetic contributions to canine size determination in non-giant dog breeds.

Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

PubMed

Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

2016-12-30

This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

Gene targeting and cloning in pigs using fetal liver derived cells.

PubMed

Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

2011-12-01

Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative

PubMed Central

Wang, Yuqiang; Su, Jing; Cai, Wenwei; Lu, Ping; Yuan, Lifen; Jin, Tuo; Chen, Shuyan; Sheng, Jing

2013-01-01

Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79–100 nm and zeta potential was 6–15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53–65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery. PMID:23576866

Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone.

PubMed

Yang, Li; Yang, Jin Bo; Chen, Jia; Yu, Guang Yao; Zhou, Pei; Lei, Lei; Wang, Zhen Zhen; Cy Chang, Catherine; Yang, Xin Ying; Chang, Ta Yuan; Li, Bo Liang

2004-08-01

In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study, with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP-1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-1-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP-1-derived macrophages. The luciferase activity assay demonstrated that Dex could enhance the activity of human ACAT1 gene P1 promoter, a major factor leading to the ACAT1 activation, in a cell-specific manner. Further experimental evidences showed that a glucocorticoid response element (GRE) located within human ACAT1 gene P1 promoter to response to the elevation of human ACAT1 gene expression by Dex could be functionally bound with glucocorticoid receptor (GR) proteins. These data supported the hypothesis that the clinical treatment with Dex, which increased the incidence of atherosclerosis, may in part due to enhancing the ACAT1 expression to promote the accumulation of cholesteryl esters during the macrophage-derived foam cell formation, an early stage of atherosclerosis.

Derived variants at six genes explain nearly half of size reduction in dog breeds

PubMed Central

Rimbault, Maud; Beale, Holly C.; Schoenebeck, Jeffrey J.; Hoopes, Barbara C.; Allen, Jeremy J.; Kilroy-Glynn, Paul; Wayne, Robert K.; Sutter, Nathan B.; Ostrander, Elaine A.

2013-01-01

Selective breeding of dogs by humans has generated extraordinary diversity in body size. A number of multibreed analyses have been undertaken to identify the genetic basis of this diversity. We analyzed four loci discovered in a previous genome-wide association study that used 60,968 SNPs to identify size-associated genomic intervals, which were too large to assign causative roles to genes. First, we performed fine-mapping to define critical intervals that included the candidate genes GHR, HMGA2, SMAD2, and STC2, identifying five highly associated markers at the four loci. We hypothesize that three of the variants are likely to be causative. We then genotyped each marker, together with previously reported size-associated variants in the IGF1 and IGF1R genes, on a panel of 500 domestic dogs from 93 breeds, and identified the ancestral allele by genotyping the same markers on 30 wild canids. We observed that the derived alleles at all markers correlated with reduced body size, and smaller dogs are more likely to carry derived alleles at multiple markers. However, breeds are not generally fixed at all markers; multiple combinations of genotypes are found within most breeds. Finally, we show that 46%–52.5% of the variance in body size of dog breeds can be explained by seven markers in proximity to exceptional candidate genes. Among breeds with standard weights <41 kg (90 lb), the genotypes accounted for 64.3% of variance in weight. This work advances our understanding of mammalian growth by describing genetic contributions to canine size determination in non-giant dog breeds. PMID:24026177

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells.

PubMed

Joo, Jihoon E; Hiden, Ursula; Lassance, Luciana; Gordon, Lavinia; Martino, David J; Desoye, Gernot; Saffery, Richard

2013-07-15

The endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells. Placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase. Endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.

DNER, an epigenetically modulated gene, regulates glioblastoma-derived neurosphere cell differentiation and tumor propagation.

PubMed

Sun, Peng; Xia, Shuli; Lal, Bachchu; Eberhart, Charles G; Quinones-Hinojosa, Alfredo; Maciaczyk, Jarek; Matsui, William; Dimeco, Francesco; Piccirillo, Sara M; Vescovi, Angelo L; Laterra, John

2009-07-01

Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem-like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone-modifying agents to elucidate mechanisms by which the phenotype and tumor-promoting capacity of GBM-derived neoplastic stem-like cells are regulated. Using established GBM-derived neurosphere lines and low passage primary GBM-derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch-like epidermal growth factor-related receptor (DNER), inhibited the growth of GBM-derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor-promoting effects of canonical Notch signaling in brain cancer stem-like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem-like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies.

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

PubMed

Muller, Laurent; Mitsuhashi, Masato; Simms, Patricia; Gooding, William E; Whiteside, Theresa L

2016-02-04

Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4(+) Tconv, CD8(+) T or CD4(+) CD39(+) Treg were isolated from normal donors' peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24-27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4(+) Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes.

Characteristics of yak platelet derived growth factors-alpha gene and expression in brain tissues.

PubMed

Huang, Zhenhua; Pan, Yangyang; Liu, Penggang; Yu, Sijiu; Cui, Yan

2017-05-29

Platelet derived growth factors (PDGFs) are key components of autocrine and paracrine signaling, both of which play important roles in mammalian developmental processes. PDGF expression levels also relate to oxygen levels. The characteristics of yak PDGFs, which are indigenous to hypoxic environments, have not been clearly described until the current study. We amplified the open reading frame encoding yak (Bos grunniens) platelet derived growth factor-a (PDGFA) from a yak skin tissue cDNA library by reverse transcriptase polymerase chain reaction (PCR) using specific primers and Sanger dideoxy sequencing. Expression of PDGFA mRNA in different portions of yak brain tissue (cerebrum, cerebellum, hippocampus, and spinal cord) was detected by quantitative real-time PCR (qRT-PCR). PDGFA protein expression levels and its location in different portions of the yak brain were evaluated by western blot and immunohistochemistry. We obtained a yak PDGFA 755 bp cDNA gene fragment containing a 636 bp open reading frame, encoding 211 amino acids (GenBank: KU851801). Phylogenetic analysis shows yak PDGFA to be well conserved, having 98.1% DNA sequence identity to homologous Bubalus bubalus and Bos taurus PDGFA genes. However, eight nucleotides in the yak DNA sequence and four amino acids in the yak protein sequence differ from the other two species. PDGFA is widely expressed in yak brain tissue, and furthermore, PDGFA expression in the cerebrum and cerebellum are higher than in the hippocampus and spinal cord (p > 0.05). PDGFA was observed by immunohistochemistry in glial cells of the cerebrum, cerebellum, and hippocampus, as well as in pyramidal cells of the cerebrum, and Purkinje cell bodies of the hippocampus, but not in glial cells of the spinal cord. The PDGFA gene is well conserved in the animal kingdom; however, the yak PDGFA gene has unique characteristics and brain expression patterns specific to this high elevation species.

Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

PubMed

Mai, Kaijin; Zhang, Shanshan; Liang, Bing; Gao, Cong; Du, Wenjun; Zhang, Li-Ming

2015-06-05

To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector. Copyright © 2015 Elsevier Ltd. All rights reserved.

Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network.

PubMed

Qin, Tingting; Matmati, Nabil; Tsoi, Lam C; Mohanty, Bidyut K; Gao, Nan; Tang, Jijun; Lawson, Andrew B; Hannun, Yusuf A; Zheng, W Jim

2014-10-01

To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes' Ontology Fingerprints--a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms' corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities. We applied this method to the sphingolipid pathway in yeast and found that many top-ranked genes indeed displayed altered sphingolipid pathway functions, initially measured by their sensitivity to myriocin, an inhibitor of de novo sphingolipid biosynthesis. Further experiments confirmed the modulation of the sphingolipid pathway by one of these genes, PFA4, encoding a palmitoyl transferase. Comparative analysis showed that few of these novel genes could be discovered by other existing methods. Our novel gene network provides a unique and comprehensive resource to study pathway modulations and systems biology in general. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network

PubMed Central

Qin, Tingting; Matmati, Nabil; Tsoi, Lam C.; Mohanty, Bidyut K.; Gao, Nan; Tang, Jijun; Lawson, Andrew B.; Hannun, Yusuf A.; Zheng, W. Jim

2014-01-01

To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes’ Ontology Fingerprints—a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms’ corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities. We applied this method to the sphingolipid pathway in yeast and found that many top-ranked genes indeed displayed altered sphingolipid pathway functions, initially measured by their sensitivity to myriocin, an inhibitor of de novo sphingolipid biosynthesis. Further experiments confirmed the modulation of the sphingolipid pathway by one of these genes, PFA4, encoding a palmitoyl transferase. Comparative analysis showed that few of these novel genes could be discovered by other existing methods. Our novel gene network provides a unique and comprehensive resource to study pathway modulations and systems biology in general. PMID:25063300

Effect of Emdogain enamel matrix derivative and BMP-2 on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells.

PubMed

Fawzy El-Sayed, Karim M; Dörfer, Christof; Ungefroren, Hendrick; Kassem, Neemat; Wiltfang, Jörg; Paris, Sebastian

2014-07-01

The objective of this study was to evaluate the effect of Emdogain (Enamel Matrix Derivative, EMD) and Bone Morphogenetic Protein-2 (BMP-2), either solely or in combination, on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells. Stem/progenitor cells were isolated from human alveolar bone proper, magnetically sorted using STRO-1 antibodies, characterized flowcytometrically for their surface markers' expression, and examined for colony formation and multilineage differentiation potential. Subsequently, cells were treated over three weeks with 100 μg/ml Emdogain (EMD-Group), or 100 ng/ml BMP-2 (BMP-Group), or a combination of 100 ng/ml BMP-2 and 100 μg/ml Emdogain (BMP/EMD-Group). Unstimulated stem/progenitor cells (MACS(+)-Group) and osteoblasts (OB-Group) served as controls. Osteogenic gene expression was analyzed using RTq-PCR after 1, 2 and 3 weeks (N = 3/group). Mineralized nodule formation was evaluated by Alizarin-Red staining. BMP and EMD up-regulated the osteogenic gene expression. The BMP Group showed significantly higher expression of Collagen-I, III, and V, Alkaline phosphatase and Osteonectin compared to MACS(+)- and OB-Group (p < 0.05; Two-way ANOVA/Bonferroni) with no mineralized nodule formation. Under in-vitro conditions, Emdogain and BMP-2 up-regulate the osteogenic gene expression of stem/progenitor cells. The combination of BMP-2 and Emdogain showed no additive effect and would not be recommended for a combined clinical stimulation. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Prediction on the inhibition ratio of pyrrolidine derivatives on matrix metalloproteinase based on gene expression programming.

PubMed

Li, Yuqin; You, Guirong; Jia, Baoxiu; Si, Hongzong; Yao, Xiaojun

2014-01-01

Quantitative structure-activity relationships (QSAR) were developed to predict the inhibition ratio of pyrrolidine derivatives on matrix metalloproteinase via heuristic method (HM) and gene expression programming (GEP). The descriptors of 33 pyrrolidine derivatives were calculated by the software CODESSA, which can calculate quantum chemical, topological, geometrical, constitutional, and electrostatic descriptors. HM was also used for the preselection of 5 appropriate molecular descriptors. Linear and nonlinear QSAR models were developed based on the HM and GEP separately and two prediction models lead to a good correlation coefficient (R (2)) of 0.93 and 0.94. The two QSAR models are useful in predicting the inhibition ratio of pyrrolidine derivatives on matrix metalloproteinase during the discovery of new anticancer drugs and providing theory information for studying the new drugs.

Scoping of Flood Hazard Mapping Needs for Belknap County, New Hampshire

DTIC Science & Technology

2006-01-01

DEM Digital Elevation Model DFIRM Digital Flood Insurance Rate Map DOQ Digital Orthophoto Quadrangle DOQQ Digital Ortho Quarter Quadrangle DTM...Agriculture Imag- ery Program (NAIP) color Digital Orthophoto Quadrangles (DOQs)). Remote sensing, base map information, GIS data (for example, contour data...found on USGS topographic maps. More recently developed data were derived from digital orthophotos providing improved base map accuracy. NH GRANIT is

Cholesterol derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility.

PubMed

Ju, Jia; Huan, Meng-Lei; Wan, Ning; Hou, Yi-Lin; Ma, Xi-Xi; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2016-05-15

Cholesterol derivatives M1-M6 as synthetic cationic lipids were designed and the biological evaluation of the cationic liposomes based on them as non-viral gene delivery vectors were described. Plasmid pEGFP-N1, used as model gene, was transferred into 293T cells by cationic liposomes formed with M1-M6 and transfection efficiency and GFP expression were tested. Cationic liposomes prepared with cationic lipids M1-M6 exhibited good transfection activity, and the transfection activity was parallel (M2 and M4) or superior (M1 and M6) to that of DC-Chol derived from the same backbone. Among them, the transfection efficiency of cationic lipid M6 was parallel to that of the commercially available Lipofectamine2000. The optimal formulation of M1 and M6 were found to be at a mol ratio of 1:0.5 for cationic lipid/DOPE, and at a N/P charge mol ratio of 3:1 for liposome/DNA. Under optimized conditions, the efficiency of M1 and M6 is greater than that of all the tested commercial liposomes DC-Chol and Lipofectamine2000, even in the presence of serum. The results indicated that M1 and M6 exhibited low cytotoxicity, good serum compatibility and efficient transfection performance, having the potential of being excellent non-viral vectors for gene delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microsatellite mapping of a Triticum urartu Tum. derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.).

PubMed

Qiu, Y C; Zhou, R H; Kong, X Y; Zhang, S S; Jia, J Z

2005-11-01

A powdery mildew resistance gene from Triticum urartu Tum. accession UR206 was successfully transferred into hexaploid wheat (Triticum aestivum L.) through crossing and backcrossing. The F1 plants, which had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells (PMC), were obtained through embryos rescued owing to shriveling of endosperm in hybrid seed of cross Chinese Spring (CS) x UR206. Hybrid seeds were produced through backcrossing F1 with common wheat parents. The derivative lines had normal chromosome numbers and powdery mildew resistance similar to the donor UR206, indicating that the powdery mildew resistance gene originating from T. urartu accession UR206 was successfully transferred and expressed in a hexaploid wheat background. Genetic analysis indicated that a single dominant gene controlled the powdery mildew resistance at the seedling stage. To map and tag the powdery mildew resistance gene, 143 F2 individuals derived from a cross UR206 x UR203 were used to construct a linkage map. The resistant gene was mapped on the chromosome 7AL based on the mapped microsatellite makers. The map spanned 52.1 cM and the order of these microsatellite loci agreed well with the established microsatellite map of chromosome arm 7AL. The resistance gene was flanked by the microsatellite loci Xwmc273 and Xpsp3003, with the genetic distances of 2.2 cM and 3.8 cM, respectively. On the basis of the origin and chromosomal location of the gene, it was temporarily designated PmU.

Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder.

PubMed

Yasuda, Yuka; Hashimoto, Ryota; Yamamori, Hidenaga; Ohi, Kazutaka; Fukumoto, Motoyuki; Umeda-Yano, Satomi; Mohri, Ikuko; Ito, Akira; Taniike, Masako; Takeda, Masatoshi

2011-05-26

The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN) 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD. We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays. The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells. Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients.

Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder

PubMed Central

2011-01-01

Background The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN) 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD. Methods We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays. Results The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells. Conclusions Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients. PMID:21615902

Brain-Derived Neurotrophic Factor Gene Expression in Pediatric Bipolar Disorder: Effects of Treatment and Clinical Response

ERIC Educational Resources Information Center

Pandey, Ghanshyam N.; Rizavi, Hooriyah S.; Dwivedi, Yogesh; Pavuluri, Mani N.

2008-01-01

The study determines the gene expression of brain-derived neurotrophic factor (BDNF) in the lymphocytes of subjects with pediatric bipolar disorder (PBD) before and during treatment with mood stabilizers and in drug-free normal control subjects. Results indicate the potential of BDNF levels as a biomarker for PBD and as a treatment predictor and…

The Impact of the Brain-Derived Neurotrophic Factor Gene on Trauma and Spatial Processing.

PubMed

Miller, Jessica K; McDougall, Siné; Thomas, Sarah; Wiener, Jan

2017-11-27

The influence of genes and the environment on the development of Post-Traumatic Stress Disorder (PTSD) continues to motivate neuropsychological research, with one consistent focus being the Brain-Derived Neurotrophic Factor (BDNF) gene, given its impact on the integrity of the hippocampal memory system. Research into human navigation also considers the BDNF gene in relation to hippocampal dependent spatial processing. This speculative paper brings together trauma and spatial processing for the first time and presents exploratory research into their interactions with BDNF. We propose that quantifying the impact of BDNF on trauma and spatial processing is critical and may well explain individual differences in clinical trauma treatment outcomes and in navigation performance. Research has already shown that the BDNF gene influences PTSD severity and prevalence as well as navigation behaviour. However, more data are required to demonstrate the precise hippocampal dependent processing mechanisms behind these influences in different populations and environmental conditions. This paper provides insight from recent studies and calls for further research into the relationship between allocentric processing, trauma processing and BDNF. We argue that research into these neural mechanisms could transform PTSD clinical practice and professional support for individuals in trauma-exposing occupations such as emergency response, law enforcement and the military.

Inhibition of Super-Enhancer Activity in Autoinflammatory Site-Derived T Cells Reduces Disease-Associated Gene Expression.

PubMed

Peeters, Janneke G C; Vervoort, Stephin J; Tan, Sander C; Mijnheer, Gerdien; de Roock, Sytze; Vastert, Sebastiaan J; Nieuwenhuis, Edward E S; van Wijk, Femke; Prakken, Berent J; Creyghton, Menno P; Coffer, Paul J; Mokry, Michal; van Loosdregt, Jorg

2015-09-29

The underlying molecular mechanisms for many autoimmune diseases are poorly understood. Juvenile idiopathic arthritis (JIA) is an exceptionally well-suited model for studying autoimmune diseases due to its early onset and the possibility to analyze cells derived from the site of inflammation. Epigenetic profiling, utilizing primary JIA patient-derived cells, can contribute to the understanding of autoimmune diseases. With H3K27ac chromatin immunoprecipitation, we identified a disease-specific, inflammation-associated, typical enhancer and super-enhancer signature in JIA patient synovial-fluid-derived CD4(+) memory/effector T cells. RNA sequencing of autoinflammatory site-derived patient T cells revealed that BET inhibition, utilizing JQ1, inhibited immune-related super-enhancers and preferentially reduced disease-associated gene expression, including cytokine-related processes. Altogether, these results demonstrate the potential use of enhancer profiling to identify disease mediators and provide evidence for BET inhibition as a possible therapeutic approach for the treatment of autoimmune diseases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation.

PubMed

Kawata, Kazumi; Kubota, Satoshi; Eguchi, Takanori; Aoyama, Eriko; Moritani, Norifumi H; Oka, Morihiko; Kawaki, Harumi; Takigawa, Masaharu

2017-11-01

The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

New inter-correlated genes targeted by diatom-derived polyunsaturated aldehydes in the sea urchin Paracentrotus lividus.

PubMed

Ruocco, Nadia; Maria Fedele, Anna; Costantini, Susan; Romano, Giovanna; Ianora, Adrianna; Costantini, Maria

2017-08-01

The marine environment is continually subjected to the action of stressors (including natural toxins), which represent a constant danger for benthic communities. In the present work using network analysis we identified ten genes on the basis of associated functions (FOXA, FoxG, GFI-1, nodal, JNK, OneCut/Hnf6, TAK1, tcf4, TCF7, VEGF) in the sea urchin Paracentrotus lividus, having key roles in different processes, such as embryonic development and asymmetry, cell fate specification, cell differentiation and morphogenesis, and skeletogenesis. These genes are correlated with three HUB genes, Foxo, Jun and HIF1A. Real Time qPCR revealed that during sea urchin embryonic development the expression levels of these genes were modulated by three diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal. Our findings show how changes in gene expression levels may be used as an early indicator of stressful conditions in the marine environment. The identification of key genes and the molecular pathways in which they are involved represents a fundamental tool in understanding how marine organisms try to afford protection against toxicants, to avoid deleterious consequences and irreversible damages. The genes identified in this work as targets for PUAs can be considered as possible biomarkers to detect exposure to different environmental pollutants. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fasting-induced G0/G1 switch gene 2 and FGF21 expression in the liver are under regulation of adipose tissue derived fatty acids

PubMed Central

Jaeger, Doris; Schoiswohl, Gabriele; Hofer, Peter; Schreiber, Renate; Schweiger, Martina; Eichmann, Thomas O.; Pollak, Nina M.; Poecher, Nadja; Grabner, Gernot F.; Zierler, Kathrin A.; Eder, Sandra; Kolb, Dagmar; Radner, Franz P.W.; Preiss-Landl, Karina; Lass, Achim; Zechner, Rudolf; Kershaw, Erin E.; Haemmerle, Guenter

2015-01-01

Background & Aims Adipose tissue (AT)-derived fatty acids (FAs) are utilized for hepatic triacylglycerol (TG) generation upon fasting. However, their potential impact as signaling molecules is not established. Herein we examined the role of exogenous AT-derived FAs in the regulation of hepatic gene expression by investigating mice with a defect in AT-derived FA supply to the liver. Methods Plasma FA levels, tissue TG hydrolytic activities and lipid content were determined in mice lacking the lipase co-activator comparative gene identification-58 (CGI-58) selectively in AT (CGI-58-ATko) applying standard protocols. Hepatic expression of lipases, FA oxidative genes, transcription factors, ER stress markers, hormones and cytokines were determined by qRT-PCR, Western blotting and ELISA. Results Impaired AT-derived FA supply upon fasting of CGI-58-ATko mice causes a marked defect in liver PPARα-signaling and nuclear CREBH translocation. This severely reduced the expression of respective target genes such as the ATGL inhibitor G0/G1 switch gene-2 (G0S2) and the endocrine metabolic regulator FGF21. These changes could be reversed by lipid administration and raising plasma FA levels. Impaired AT-lipolysis failed to induce hepatic G0S2 expression in fasted CGI-58-ATko mice leading to enhanced ATGL-mediated TG-breakdown strongly reducing hepatic TG deposition. On high fat diet, impaired AT-lipolysis counteracts hepatic TG accumulation and liver stress linked to improved systemic insulin sensitivity. Conclusions AT-derived FAs are a critical regulator of hepatic fasting gene expression required for the induction of G0S2-expression in the liver to control hepatic TG-breakdown. Interfering with AT-lipolysis or hepatic G0S2 expression represents an effective strategy for the treatment of hepatic steatosis. PMID:25733154

Discovery and introgression of the wild sunflower-derived novel downy mildew resistance gene Pl 19 in confection sunflower (Helianthus annuus L.).

PubMed

Zhang, Z W; Ma, G J; Zhao, J; Markell, S G; Qi, L L

2017-01-01

A new downy mildew resistance gene, Pl 19 , was identified from wild Helianthus annuus accession PI 435414, introduced to confection sunflower, and genetically mapped to linkage group 4 of the sunflower genome. Wild Helianthus annuus accession PI 435414 exhibited resistance to downy mildew, which is one of the most destructive diseases to sunflower production globally. Evaluation of the 140 BC 1 F 2:3 families derived from the cross of CMS CONFSCLB1 and PI 435414 against Plasmopara halstedii race 734 revealed that a single dominant gene controls downy mildew resistance in the population. Bulked segregant analysis conducted in the BC 1 F 2 population with 860 simple sequence repeat (SSR) markers indicated that the resistance derived from wild H. annuus was associated with SSR markers located on linkage group (LG) 4 of the sunflower genome. To map and tag this resistance locus, designated Pl 19 , 140 BC 1 F 2 individuals were used to construct a linkage map of the gene region. Two SSR markers, ORS963 and HT298, were linked to Pl 19 within a distance of 4.7 cM. After screening 27 additional single nucleotide polymorphism (SNP) markers previously mapped to this region, two flanking SNP markers, NSA_003564 and NSA_006089, were identified as surrounding the Pl 19 gene at a distance of 0.6 cM from each side. Genetic analysis indicated that Pl 19 is different from Pl 17 , which had previously been mapped to LG4, but is closely linked to Pl 17 . This new gene is highly effective against the most predominant and virulent races of P. halstedii currently identified in North America and is the first downy mildew resistance gene that has been transferred to confection sunflower. The selected resistant germplasm derived from homozygous BC 2 F 3 progeny provides a novel gene for use in confection sunflower breeding programs.

Digital transcriptome profiling of normal and glioblastoma-derived neural stem cells identifies genes associated with patient survival

PubMed Central

2012-01-01

Background Glioblastoma multiforme, the most common type of primary brain tumor in adults, is driven by cells with neural stem (NS) cell characteristics. Using derivation methods developed for NS cells, it is possible to expand tumorigenic stem cells continuously in vitro. Although these glioblastoma-derived neural stem (GNS) cells are highly similar to normal NS cells, they harbor mutations typical of gliomas and initiate authentic tumors following orthotopic xenotransplantation. Here, we analyzed GNS and NS cell transcriptomes to identify gene expression alterations underlying the disease phenotype. Methods Sensitive measurements of gene expression were obtained by high-throughput sequencing of transcript tags (Tag-seq) on adherent GNS cell lines from three glioblastoma cases and two normal NS cell lines. Validation by quantitative real-time PCR was performed on 82 differentially expressed genes across a panel of 16 GNS and 6 NS cell lines. The molecular basis and prognostic relevance of expression differences were investigated by genetic characterization of GNS cells and comparison with public data for 867 glioma biopsies. Results Transcriptome analysis revealed major differences correlated with glioma histological grade, and identified misregulated genes of known significance in glioblastoma as well as novel candidates, including genes associated with other malignancies or glioma-related pathways. This analysis further detected several long non-coding RNAs with expression profiles similar to neighboring genes implicated in cancer. Quantitative PCR validation showed excellent agreement with Tag-seq data (median Pearson r = 0.91) and discerned a gene set robustly distinguishing GNS from NS cells across the 22 lines. These expression alterations include oncogene and tumor suppressor changes not detected by microarray profiling of tumor tissue samples, and facilitated the identification of a GNS expression signature strongly associated with patient survival (P = 1e

Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

PubMed

Kim, Dae Seong; Lee, Myoung Woo; Lee, Tae-Hee; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2017-03-01

The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm 2 . After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox ( Nanog ), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog ( c-Myc ), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc , were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

Production of rosamicin derivatives in Micromonospora rosaria by introduction of D-mycinose biosynthetic gene with PhiC31-derived integration vector pSET152.

PubMed

Anzai, Yojiro; Iizaka, Yohei; Li, Wei; Idemoto, Naoki; Tsukada, Shu-ichi; Koike, Kazuo; Kinoshita, Kenji; Kato, Fumio

2009-08-01

Some of the polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often impart specific biological activity to the molecule or enhance this activity. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. The D-mycinose biosynthesis genes, mycCI, mycCII, mycD, mycE, mycF, mydH, and mydI, present in the M. griseorubida A11725 chromosome were introduced into pSET152 under the regulation of the promoter of the apramycin-resistance gene aac(3)IV. The resulting plasmid pSETmycinose was introduced into Micromonospora rosaria IFO13697 cells, which produce the 16-membered macrolide antibiotic rosamicin containing a branched lactone and D-desosamine at the C-5 position. Although the M. rosaria TPMA0001 transconjugant exhibited low rosamicin productivity, two new compounds, IZI and IZII, were detected in the ethylacetate extract from the culture broth. IZI was identified as a mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (MW 741), which has previously been synthesized by a bioconversion technique. This is the first report on production of mycinosyl rosamicin-derivatives by a engineered biosynthesis approach. The integration site PhiC31attB was identified on M. rosaria IFO13697 chromosome, and the site lay within an ORF coding a pirin homolog protein. The pSETmycinose could be useful for stimulating the production of "unnatural" natural mycinosyl compounds by various actinomycete strains using the bacteriophage PhiC31 att/int system.

Next-generation text-mining mediated generation of chemical response-specific gene sets for interpretation of gene expression data.

PubMed

Hettne, Kristina M; Boorsma, André; van Dartel, Dorien A M; Goeman, Jelle J; de Jong, Esther; Piersma, Aldert H; Stierum, Rob H; Kleinjans, Jos C; Kors, Jan A

2013-01-29

Availability of chemical response-specific lists of genes (gene sets) for pharmacological and/or toxic effect prediction for compounds is limited. We hypothesize that more gene sets can be created by next-generation text mining (next-gen TM), and that these can be used with gene set analysis (GSA) methods for chemical treatment identification, for pharmacological mechanism elucidation, and for comparing compound toxicity profiles. We created 30,211 chemical response-specific gene sets for human and mouse by next-gen TM, and derived 1,189 (human) and 588 (mouse) gene sets from the Comparative Toxicogenomics Database (CTD). We tested for significant differential expression (SDE) (false discovery rate -corrected p-values < 0.05) of the next-gen TM-derived gene sets and the CTD-derived gene sets in gene expression (GE) data sets of five chemicals (from experimental models). We tested for SDE of gene sets for six fibrates in a peroxisome proliferator-activated receptor alpha (PPARA) knock-out GE dataset and compared to results from the Connectivity Map. We tested for SDE of 319 next-gen TM-derived gene sets for environmental toxicants in three GE data sets of triazoles, and tested for SDE of 442 gene sets associated with embryonic structures. We compared the gene sets to triazole effects seen in the Whole Embryo Culture (WEC), and used principal component analysis (PCA) to discriminate triazoles from other chemicals. Next-gen TM-derived gene sets matching the chemical treatment were significantly altered in three GE data sets, and the corresponding CTD-derived gene sets were significantly altered in five GE data sets. Six next-gen TM-derived and four CTD-derived fibrate gene sets were significantly altered in the PPARA knock-out GE dataset. None of the fibrate signatures in cMap scored significant against the PPARA GE signature. 33 environmental toxicant gene sets were significantly altered in the triazole GE data sets. 21 of these toxicants had a similar toxicity

Brain-derived neurotrophic factor as a model system for examining gene by environment interactions across development.

PubMed

Casey, B J; Glatt, C E; Tottenham, N; Soliman, F; Bath, K; Amso, D; Altemus, M; Pattwell, S; Jones, R; Levita, L; McEwen, B; Magariños, A M; Gunnar, M; Thomas, K M; Mezey, J; Clark, A G; Hempstead, B L; Lee, F S

2009-11-24

There has been a dramatic rise in gene x environment studies of human behavior over the past decade that have moved the field beyond simple nature versus nurture debates. These studies offer promise in accounting for more variability in behavioral and biological phenotypes than studies that focus on genetic or experiential factors alone. They also provide clues into mechanisms of modifying genetic risk or resilience in neurodevelopmental disorders. Yet, it is rare that these studies consider how these interactions change over the course of development. In this paper, we describe research that focuses on the impact of a polymorphism in a brain-derived neurotrophic factor (BDNF) gene, known to be involved in learning and development. Specifically we present findings that assess the effects of genotypic and environmental loadings on neuroanatomic and behavioral phenotypes across development. The findings illustrate the use of a genetic mouse model that mimics the human polymorphism, to constrain the interpretation of gene-environment interactions across development in humans.

Complete TCRα gene locus control region activity in T cells derived in vitro from embryonic stem cells

PubMed Central

Lahiji, Armin; Kučerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D.

2013-01-01

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering. PMID:23720809

Platelet-Derived Growth Factor-BB Stimulates Fibronectin Gene Expression in Fibroblasts Isolated from Rat Thoracic Aorta

DTIC Science & Technology

1994-06-13

MARYLAND 20814-4799 TEACHING HOSPITALS WALTER REED ARMY MEDtCA L CENTER APPROVAL SHEET NAVAL HOSPITAL. BETHESDA MALCOLM GROW AIR FORCE MEDICAL ...CENTER WILFORD HALL "IR FORCE MEDICAL CENTER Title of Dissertation: "Platelet-derived growth factor-BB stimulates fibronectin gene expression in...fascinating world of basic medical science. His dedication and pursuit of excellence in all facets of his work are standards by which I will guide my own

A New Class of SINEs with snRNA Gene-Derived Heads

PubMed Central

Kojima, Kenji K.

2015-01-01

Eukaryotic genomes are colonized by various transposons including short interspersed elements (SINEs). The 5′ region (head) of the majority of SINEs is derived from one of the three types of RNA genes—7SL RNA, transfer RNA (tRNA), or 5S ribosomal RNA (rRNA)—and the internal promoter inside the head promotes the transcription of the entire SINEs. Here I report a new group of SINEs whose heads originate from either the U1 or U2 small nuclear RNA gene. These SINEs, named SINEU, are distributed among crocodilians and classified into three families. The structures of the SINEU-1 subfamilies indicate the recurrent addition of a U1- or U2-derived sequence onto the 5′ end of SINEU-1 elements. SINEU-1 and SINEU-3 are ancient and shared among alligators, crocodiles, and gharials, while SINEU-2 is absent in the alligator genome. SINEU-2 is the only SINE family that was active after the split of crocodiles and gharials. All SINEU families, especially SINEU-3, are preferentially inserted into a family of Mariner DNA transposon, Mariner-N4_AMi. A group of Tx1 non-long terminal repeat retrotransposons designated Tx1-Mar also show target preference for Mariner-N4_AMi, indicating that SINEU was mobilized by Tx1-Mar. PMID:26019167

Exposure to Nickel, Chromium, or Cadmium Causes Distinct Changes in the Gene Expression Patterns of a Rat Liver Derived Cell Line

PubMed Central

Permenter, Matthew G.; Lewis, John A.; Jackson, David A.

2011-01-01

Many heavy metals, including nickel (Ni), cadmium (Cd), and chromium (Cr) are toxic industrial chemicals with an exposure risk in both occupational and environmental settings that may cause harmful outcomes. While these substances are known to produce adverse health effects leading to disease or health problems, the detailed mechanisms remain unclear. To elucidate the processes involved in the toxicity of nickel, cadmium, and chromium at the molecular level and to perform a comparative analysis, H4-II-E-C3 rat liver-derived cell lines were treated with soluble salts of each metal using concentrations derived from viability assays, and gene expression patterns were determined with DNA microarrays. We identified both common and unique biological responses to exposure to the three metals. Nickel, cadmium, chromium all induced oxidative stress with both similar and unique genes and pathways responding to this stress. Although all three metals are known to be genotoxic, evidence for DNA damage in our study only exists in response to chromium. Nickel induced a hypoxic response as well as inducing genes involved in chromatin structure, perhaps by replacing iron in key proteins. Cadmium distinctly perturbed genes related to endoplasmic reticulum stress and invoked the unfolded protein response leading to apoptosis. With these studies, we have completed the first gene expression comparative analysis of nickel, cadmium, and chromium in H4-II-E-C3 cells. PMID:22110744

Gene Transfer of Brain-derived Neurotrophic Factor (BDNF) Prevents Neurodegeneration Triggered by FXN Deficiency.

PubMed

Katsu-Jiménez, Yurika; Loría, Frida; Corona, Juan Carlos; Díaz-Nido, Javier

2016-05-01

Friedreich's ataxia is a predominantly neurodegenerative disease caused by recessive mutations that produce a deficiency of frataxin (FXN). Here, we have used a herpesviral amplicon vector carrying a gene encoding for brain-derived neurotrophic factor (BDNF) to drive its overexpression in neuronal cells and test for its effect on FXN-deficient neurons both in culture and in the mouse cerebellum in vivo. Gene transfer of BDNF to primary cultures of mouse neurons prevents the apoptosis which is triggered by the knockdown of FXN gene expression. This neuroprotective effect of BDNF is also observed in vivo in a viral vector-based knockdown mouse cerebellar model. The injection of a lentiviral vector carrying a minigene encoding for a FXN-specific short hairpin ribonucleic acid (shRNA) into the mouse cerebellar cortex triggers a FXN deficit which is accompanied by significant apoptosis of granule neurons as well as loss of calbindin in Purkinje cells. These pathological changes are accompanied by a loss of motor coordination of mice as assayed by the rota-rod test. Coinjection of a herpesviral vector encoding for BDNF efficiently prevents both the development of cerebellar neuropathology and the ataxic phenotype. These data demonstrate the potential therapeutic usefulness of neurotrophins like BDNF to protect FXN-deficient neurons from degeneration.

Next-generation text-mining mediated generation of chemical response-specific gene sets for interpretation of gene expression data

PubMed Central

2013-01-01

Background Availability of chemical response-specific lists of genes (gene sets) for pharmacological and/or toxic effect prediction for compounds is limited. We hypothesize that more gene sets can be created by next-generation text mining (next-gen TM), and that these can be used with gene set analysis (GSA) methods for chemical treatment identification, for pharmacological mechanism elucidation, and for comparing compound toxicity profiles. Methods We created 30,211 chemical response-specific gene sets for human and mouse by next-gen TM, and derived 1,189 (human) and 588 (mouse) gene sets from the Comparative Toxicogenomics Database (CTD). We tested for significant differential expression (SDE) (false discovery rate -corrected p-values < 0.05) of the next-gen TM-derived gene sets and the CTD-derived gene sets in gene expression (GE) data sets of five chemicals (from experimental models). We tested for SDE of gene sets for six fibrates in a peroxisome proliferator-activated receptor alpha (PPARA) knock-out GE dataset and compared to results from the Connectivity Map. We tested for SDE of 319 next-gen TM-derived gene sets for environmental toxicants in three GE data sets of triazoles, and tested for SDE of 442 gene sets associated with embryonic structures. We compared the gene sets to triazole effects seen in the Whole Embryo Culture (WEC), and used principal component analysis (PCA) to discriminate triazoles from other chemicals. Results Next-gen TM-derived gene sets matching the chemical treatment were significantly altered in three GE data sets, and the corresponding CTD-derived gene sets were significantly altered in five GE data sets. Six next-gen TM-derived and four CTD-derived fibrate gene sets were significantly altered in the PPARA knock-out GE dataset. None of the fibrate signatures in cMap scored significant against the PPARA GE signature. 33 environmental toxicant gene sets were significantly altered in the triazole GE data sets. 21 of these toxicants

The gene coding for glial cell line derived neurotrophic factor (GDNF) maps to chromosome 5p12-p13.1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schindelhauer, D.; Schuffenhauer, S.; Meitinger, T.

1995-08-10

The gene coding for glial cell line derived neurotrophic factor (GDNF) has biological properties that may have potential as a treatment for Parkinson`s and motoneuron diseases. Using the NIGMS Mapping Panel 2, we have localized the GDNF gene to human chromosome 5p12-p13.1. Large NruI and NotI fragments on chromosome 5 will facilitate the construction of a long-range map of the region. 26 refs., 1 fig., 1 tab.

Nanoparticle Based Galectin-1 Gene Silencing, Implications in Methamphetamine Regulation of HIV-1 Infection in Monocyte Derived Macrophages

PubMed Central

Law, Wing Cheung; Mahajan, Supriya D.; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E.; Yong, Ken-Tye; Hui, Rui; Prasad, Paras N.; Schwartz, Stanley A.

2012-01-01

Galectin-1, an adhesion molecule, is expressed in macrophages and implicated in human immunodeficiency virus (HIV-1) viral adsorption. In this study, we investigated the effects of methamphetamine on galectin-1 production in human monocyte derived macrophages (MDM) and the role of galectin-1 in methamphetamine potentiation of HIV-1 infection. Herein we show that levels of galectin-1 gene and protein expression are significantly increased by meth-amphetamine. Furthermore, concomitant incubation of MDM with galectin-1 and methamphetamine facilitates HIV-1 infection compared to galectin-1 alone or methamphetamine alone. We utilized a nanotechnology approach that uses gold nanorod (GNR)-galectin-1 siRNA complexes (nanoplexes) to inhibit gene expression for galectin-1. Nanoplexes significantly silenced gene expression for galectin-1 and reversed the effects of methamphetamine on galectin-1 gene expression. Moreover, the effects of methamphetamine on HIV-1 infection were attenuated in the presence of the nanoplex in MDM. PMID:22689223

Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.)

PubMed Central

Asha, Srinivasan; Soniya, Eppurath V.

2016-01-01

Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5′tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5′tRFs in the infected leaf and root. The abundance of 5′tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5′AlaCGC tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5′Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper. PMID:27313593

Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

PubMed

Asha, Srinivasan; Soniya, Eppurath V

2016-01-01

Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

Histone Deacetylase Inhibitor Induces the Expression of Select Epithelial Genes in Mouse Utricle Sensory Epithelia-Derived Progenitor Cells

PubMed Central

Wang, Jue

2014-01-01

Abstract Mouse utricle sensory epithelial cell–derived progenitor cells (MUCs), which have hair cell progenitor and mesenchymal features via epithelial-to-mesenchymal transition (EMT) as previously described, provide a potential approach for hair cell regeneration via cell transplantation. In this study, we treated MUCs with trichostatin A (TSA) to determine whether histone deacetylase inhibitor is able to stimulate the expression of epithelial genes in MUCs, an essential step for guiding mesenchymal-like MUCs to become sensory epithelial cells. After 72 h of TSA treatment, MUCs acquired epithelial-like features, which were indicated by increased expression of epithelial markers such as Cdh1, Krt18, and Dsp. Additionally, TSA decreased the expression of mesenchymal markers, including Zeb1, Zeb2, Snai1, and Snai2, and prosensory genes Lfng, Six1, and Dlx5. Moreover, the expression of the hair cell genes Atoh1 and Myo6 was increased in TSA-treated MUCs. We also observed significantly decreased expression of Hdac2 and Hdac3 in TSA-treated MUCs. However, no remarkable change was detected in protein expression using immunofluorescence, indicating that TSA-induced HDAC inhibition may contribute to the initial stage of the mesenchymal-to-epithelial phenotypic change. In the future, more work is needed to induce hair cell regeneration using inner ear tissue–derived progenitors to achieve an entire mesenchymal-to-epithelial transition. PMID:24945595

A Histologically Distinctive Interstitial Pneumonia Induced by Overexpression of the Interleukin 6, Transforming Growth Factor β1, or Platelet-Derived Growth Factor B Gene

NASA Astrophysics Data System (ADS)

Yoshida, Mitsuhiro; Sakuma, Junko; Hayashi, Seiji; Abe, Kin'ya; Saito, Izumu; Harada, Shizuko; Sakatani, Mitsunoir; Yamamoto, Satoru; Matsumoto, Norinao; Kaneda, Yasufumi; Kishmoto, Tadamitsu

1995-10-01

Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor β1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor β1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

Regulation of human bone sialoprotein gene transcription by platelet-derived growth factor-BB.

PubMed

Mezawa, Masaru; Araki, Shouta; Takai, Hideki; Sasaki, Yoko; Wang, Shuang; Li, Xinyue; Kim, Dong-Soon; Nakayama, Youhei; Ogata, Yorimasa

2009-04-15

Platelet-derived growth factor (PDGF) is produced by mesenchymal cells and released by platelets following aggregation and is synthesized by osteoblasts. In bone, PDGF stimulates proliferation and differentiation of osteoblasts. PDGF also increases bone resorption, most likely by increasing the number of osteoclasts. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, selectively expressed by differentiated osteoblast. To determine the molecular mechanisms PDGF regulation of human BSP gene transcription, we have analyzed the effects of PDGF-BB on osteoblast-like Saos2 and ROS17/2.8 cells. PDGF-BB (5 ng/ml) increased BSP mRNA and protein levels at 12 h in Saos2 cells, and induced BSP mRNA expression at 3 h, reached maximal at 12 h in ROS17/2.8 cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of Saos2 cells with PDGF-BB (5 ng/ml, 12 h) increased luciferase activities of all constructs between -184LUC to -2672LUC including the human BSP gene promoter. Effects of PDGF-BB abrogated in constructs included 2 bp mutations in the two cAMP response elements (CRE1 and CRE2), activator protein 1(3) (AP1(3)) and shear stress response element 1 (SSRE1). Luciferase activities induced by PDGF-BB were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel mobility shift analyses showed that PDGF-BB increased binding of CRE1, CRE2, AP1(3) and SSRE1 elements. CRE1- and CRE2-protein complexes were supershifted by CREB1 and phospho-CREB1 antibodies. Notably, AP1(3)-protein complexes were supershifted by c-Fos and JunD, and disrupted by CREB1, phospho-CREB1, c-Jun and Fra2 antibodies. These studies, therefore, demonstrate that PDGF-BB stimulates human BSP transcription by targeting the CRE1, CRE2, AP1(3) and SSRE1 elements in the human BSP gene promoter.

A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis

PubMed Central

Fitzpatrick, David A; Logue, Mary E; Stajich, Jason E; Butler, Geraldine

2006-01-01

Background To date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available. Results A dataset of 345,829 genes was extracted from 42 publicly available fungal genomes. Supertree methods were employed to derive phylogenies from 4,805 single gene families. We found that the average consensus supertree method may suffer from long-branch attraction artifacts, while matrix representation with parsimony (MRP) appears to be immune from these. A genome phylogeny was also reconstructed from a concatenated alignment of 153 universally distributed orthologs. Our MRP supertree and concatenated phylogeny are highly congruent. Within the Ascomycota, the sub-phyla Pezizomycotina and Saccharomycotina were resolved. Both phylogenies infer that the Leotiomycetes are the closest sister group to the Sordariomycetes. There is some ambiguity regarding the placement of Stagonospora nodurum, the sole member of the class Dothideomycetes present in the dataset. Within the Saccharomycotina, a monophyletic clade containing organisms that translate CTG as serine instead of leucine is evident. There is also strong support for two groups within the CTG clade, one containing the fully sexual species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus. The second major clade within the Saccharomycotina contains species whose genomes have undergone a whole genome duplication (WGD), and their close relatives. We could not

Mapping and characterization of the new adult plant leaf rust resistance gene Lr77 derived from Santa Fe winter wheat.

PubMed

Kolmer, James A; Su, Zhenqi; Bernardo, Amy; Bai, Guihua; Chao, Shiaoman

2018-07-01

A new gene for adult plant leaf rust resistance in wheat was mapped to chromosome 3BL. This gene was designated as Lr77. 'Santa Fe' is a hard red winter cultivar that has had long-lasting resistance to the leaf rust fungus, Puccinia triticina. The objective of this study was to determine the chromosome location of the adult plant leaf rust resistance in Santa Fe wheat. A partial backcross line of 'Thatcher' (Tc) wheat with adult plant leaf rust resistance derived from Santa Fe was crossed with Thatcher to develop a Thatcher//Tc*2/Santa Fe F 6 recombinant inbred line (RIL) population. The RIL population and parental lines were evaluated for segregation of leaf rust resistance in three field plot tests and in an adult plant greenhouse test. A genetic map of the RIL population was constructed using 90,000 single-nucleotide polymorphism (SNP) markers with the Illumina Infinium iSelect 90K wheat bead array. A significant quantitative trait locus for reduction of leaf rust severity in all four tests was found on chromosome 3BL that segregated as a single adult plant resistance gene. The RILs with the allele from the resistant parent for SNP marker IWB10344 had lower leaf rust severity and a moderately resistant to moderately susceptible response compared to the susceptible RILs and Thatcher. The gene derived from Santa Fe on chromosome 3BL was designated as Lr77. Kompetitive allele-specific polymerase chain reaction assay markers linked to Lr77 on 3BL should be useful for selection of wheat germplasm with this gene.

Diverse and Abundant Secondary Metabolism Biosynthetic Gene Clusters in the Genomes of Marine Sponge Derived Streptomyces spp. Isolates.

PubMed

Jackson, Stephen A; Crossman, Lisa; Almeida, Eduardo L; Margassery, Lekha Menon; Kennedy, Jonathan; Dobson, Alan D W

2018-02-20

The genus Streptomyces produces secondary metabolic compounds that are rich in biological activity. Many of these compounds are genetically encoded by large secondary metabolism biosynthetic gene clusters (smBGCs) such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) which are modular and can be highly repetitive. Due to the repeats, these gene clusters can be difficult to resolve using short read next generation datasets and are often quite poorly predicted using standard approaches. We have sequenced the genomes of 13 Streptomyces spp. strains isolated from shallow water and deep-sea sponges that display antimicrobial activities against a number of clinically relevant bacterial and yeast species. Draft genomes have been assembled and smBGCs have been identified using the antiSMASH (antibiotics and Secondary Metabolite Analysis Shell) web platform. We have compared the smBGCs amongst strains in the search for novel sequences conferring the potential to produce novel bioactive secondary metabolites. The strains in this study recruit to four distinct clades within the genus Streptomyces . The marine strains host abundant smBGCs which encode polyketides, NRPS, siderophores, bacteriocins and lantipeptides. The deep-sea strains appear to be enriched with gene clusters encoding NRPS. Marine adaptations are evident in the sponge-derived strains which are enriched for genes involved in the biosynthesis and transport of compatible solutes and for heat-shock proteins. Streptomyces spp. from marine environments are a promising source of novel bioactive secondary metabolites as the abundance and diversity of smBGCs show high degrees of novelty. Sponge derived Streptomyces spp. isolates appear to display genomic adaptations to marine living when compared to terrestrial strains.

The influence of aging on the methylation status of brain-derived neurotrophic factor gene in blood.

PubMed

Ihara, Kazushige; Fuchikami, Manabu; Hashizume, Masahiro; Okada, Satoshi; Kawai, Hisashi; Obuchi, Shuichi; Hirano, Hirohiko; Fujiwara, Yoshinori; Hachisu, Mitsugu; Hongyong, Kim; Morinobu, Shigeru

2018-06-28

Brain-derived neurotrophic factor (BDNF) is involved in the pathophysiology of psychiatric disorders in adults and elderly individuals, and as a result, the DNA methylation (DNAm) of the BDNF gene in peripheral tissues including blood has been extensively examined to develop a useful biomarker for psychiatric disorders. However, studies to date have not previously investigated the effect of age on DNAm of the BDNF gene in blood. In this context, we measured DNAm of 39 CpG units in the CpG island at the promoter of exon I of the BDNF gene. We analyzed genomic DNA from peripheral blood of 105 health Japanese women 20 to 80 years of age to identify aging-associated change in DNAm of the BDNF gene. In addition, we examined the relationship between total MMSE scores, numbers of stressful life events, and serum BDNF levels on DNAm of the BDNF gene. The DNAm rate at each CpG unit was measured using a MassArray ® system (Agena Bioscience), and serum BDNF levels were measured by ELISA. There was a significant correlation between DNAm and age in 13 CpGs. However, there was no significant correlation between DNAm and total MMSE scores, numbers of life events, or serum BDNF levels. Despite the small number of subjects and the inclusion of only female subjects, our results suggest that DNAm of 13 CpGs of the BDNF gene may be an appropriate biomarker for aging and useful for predicting increased susceptibility to age-related psychiatric disorders. © 2018 John Wiley & Sons, Ltd.

Chitosan derivatives for gene transfer: effect of phosphorylcholine and diethylaminoethyl grafts on the in vitro transfection efficiency.

PubMed

Picola, Isadora Pfeifer Dalla; Shi, Qin; Fernandes, Júlio Cesar; Petrônio, Maicon Segalla; Lima, Aline Margarete Furuyama; de Oliveira Tiera, Vera Aparecida; Tiera, Marcio José

2016-11-01

The purpose of this work was to improve the functional properties of chitosan for gene transfer by inserting phosphorylcholine (PC) and diethylaminoethyl (DEAE) groups into the main chain. A series of derivatives containing increasing contents of DEAE and a fixed content of PC groups were synthesized and characterized, aiming to evaluate the effect of these groups on the nanoparticles' properties and the in vitro transfection efficiency. The derivatives were soluble at physiological pH levels and all derivatives were less cytotoxic than the control, the lipid lipofectamine. The obtained derivatives complexed pDNA into nanoparticles with smaller sizes and higher zeta potentials than plain chitosan. The in vitro transfection was performed with nanoparticles prepared at pH 6.3 and 7.4 and the results showed that nanoparticles prepared with derivatives containing 20% of PC groups (PC18-CH) and high degrees of substitution by DEAE (PC20-CH-DEAE100, CH-DEAE80, CH-DEAE100) displayed the better transfection efficiencies in HeLa cells, reaching relative values comparable to lipofectamine. The most effective derivative, PC18CH, was selected for complexation with siRNA and its complexes demonstrated an in vitro knockdown efficiency highly dependent on the N/P ratio. Our combined results indicated that, by means of controlled modifications, the limitations of chitosan can be overcome to obtain more effective carriers based on chitosan, and the derivatives here studied hold potential for in vivo studies.

Exposure to Nickel, Chromium, or Cadmium Causes Distinct Changes in the Gene Expression Patterns of a Rat Liver Derived Cell Line

DTIC Science & Technology

2011-11-16

protein A (Rpa2), the minichromosome maintenance complex component genes which encode helicases, DNA ligase (Lig1), DNA polymerase e ( Pole and Pole2...and DNA polymerase d ( Pold1 and Pold2 ) are all up-regulated as a result of exposure to chromium (Figure 6), suggesting that there is an increase in...Exposure to Nickel, Chromium, or Cadmium Causes Distinct Changes in the Gene Expression Patterns of a Rat Liver Derived Cell Line Matthew G

Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.

PubMed Central

Mérida, A; Flores, E; Florencio, F J

1992-01-01

The glnA gene from Synechocystis sp. strain PCC 6803 was cloned by hybridization with the glnA gene from Anabaena sp. strain PCC 7120, and a deletion-insertion mutation of the Synechocystis gene was generated in vitro. A strain derived from Synechocystis sp. strain PCC 6803 which contained integrated into the chromosome, in addition to its own glnA gene, the Anabaena glnA gene was constructed. From that strain, a Synechocystis sp. glnA mutant could be obtained by transformation with the inactivated Synechocystis glnA gene; this mutant grew by using Anabaena glutamine synthetase and was not a glutamine auxotroph. A Synechocystis sp. glnA mutant could not be obtained, however, from the wild-type Synechocystis sp. The Anabaena glutamine synthetase enzyme was subject to ammonium-promoted inactivation when expressed in the Synechocystis strain but not in the Anabaena strain itself. Images PMID:1345914

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

PubMed Central

Oliveira, L M A; Falomir-Lockhart, L J; Botelho, M G; Lin, K-H; Wales, P; Koch, J C; Gerhardt, E; Taschenberger, H; Outeiro, T F; Lingor, P; Schüle, B; Arndt-Jovin, D J; Jovin, T M

2015-01-01

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction. PMID:26610207

Recently-Derived Variants of Brain-Size Genes "ASPM", "MCPH1", "CDK5RAP" and "BRCA1" Not Associated with General Cognition, Reading or Language

ERIC Educational Resources Information Center

Bates, Timothy C.; Luciano, Michelle; Lind, Penelope A.; Wright, Margaret J.; Montgomery, Grant W.; Martin, Nicholas G.

2008-01-01

Derived changes in genes associated with primary microcephaly (MCPH) have been suggested to be "currently sweeping to fixation" i.e., increasing in frequency in most populations, with the likely outcome that the derived allele will completely displace the ancestral allele over time. Possible causes for this sweep include effects on human reasoning…

Infrequent detectable somatic mutations of the RET and glial cell line-derived neurotrophic factor (GDNF) genes in human pituitary adenomas.

PubMed

Yoshimoto, K; Tanaka, C; Moritani, M; Shimizu, E; Yamaoka, T; Yamada, S; Sano, T; Itakura, M

1999-02-01

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and tumors. RET is activated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF receptor-alpha (GDNFR-alpha). Activating mutations of the RET proto-oncogene were found in multiple endocrine neoplasia (MEN) 2 and in sporadic medullary thyroid carcinoma and pheochromocytoma of neuroendocrine origin. Mutations of the RET proto-oncogene and the glial cell line-derived neurotrophic factor (GDNF) gene were examined in human pituitary tumors. No mutations of the RET proto-oncogene including the cysteine-rich region or codon 768 and 918 in the tyrosine kinase domain were detected in 172 human pituitary adenomas either by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) or by PCR-restriction fragment length polymorphism (RFLP). Further, somatic mutations of the GDNF gene in 33 human pituitary adenomas were not detected by PCR-SSCP. One polymorphism of the GDNF gene at codon 145 of TGC or TGT was observed in a prolactinoma. The RET proto-oncogene message was detected in a normal human pituitary gland or 4 of 4 human pituitary adenomas with reverse transcription (RT)-PCR, and in rodent pituitary tumor cell lines with Western blotting. The expression of GDNF gene was detected in 1 of 4 human somatotroph adenomas, 1 of 2 corticotroph adenomas, and 2 of 6 rodent pituitary tumor cell lines with RT-PCR. Based on these, it is concluded that somatic mutations of the RET proto-oncogene or the GDNF gene do not appear to play a major role in the pituitary tumorigenesis in examined tumors.

Identification of stable reference genes for gene expression analysis of three-dimensional cultivated human bone marrow-derived mesenchymal stromal cells for bone tissue engineering.

PubMed

Rauh, Juliane; Jacobi, Angela; Stiehler, Maik

2015-02-01

The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues. For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n=31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan(®) assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BM-MSCs. Notably, genes that are routinely used as RGs, for example, beta actin

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

PubMed Central

Rauh, Juliane; Jacobi, Angela

2015-01-01

The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues. For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n=31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan® assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BM-MSCs. Notably, genes that are routinely used as RGs, for example, beta actin

Characterization and engineering of the biosynthesis gene cluster for antitumor macrolides PM100117 and PM100118 from a marine actinobacteria: generation of a novel improved derivative.

PubMed

Salcedo, Raúl García; Olano, Carlos; Gómez, Cristina; Fernández, Rogelio; Braña, Alfredo F; Méndez, Carmen; de la Calle, Fernando; Salas, José A

2016-02-22

PM100117 and PM100118 are glycosylated polyketides with remarkable antitumor activity, which derive from the marine symbiotic actinobacteria Streptomyces caniferus GUA-06-05-006A. Structurally, PM100117 and PM100118 are composed of a macrocyclic lactone, three deoxysugar units and a naphthoquinone (NQ) chromophore that shows a clear structural similarity to menaquinone. Whole-genome sequencing of S. caniferus GUA-06-05-006A has enabled the identification of PM100117 and PM100118 biosynthesis gene cluster, which has been characterized on the basis of bioinformatics and genetic engineering data. The product of four genes shows high identity to proteins involved in the biosynthesis of menaquinone via futalosine. Deletion of one of these genes led to a decay in PM100117 and PM100118 production, and to the accumulation of several derivatives lacking NQ. Likewise, five additional genes have been genetically characterized to be involved in the biosynthesis of this moiety. Moreover, the generation of a mutant in a gene coding for a putative cytochrome P450 has led to the production of PM100117 and PM100118 structural analogues showing an enhanced in vitro cytotoxic activity relative to the parental products. Although a number of compounds structurally related to PM100117 and PM100118 has been discovered, this is, to our knowledge, the first insight reported into their biosynthesis. The structural resemblance of the NQ moiety to menaquinone, and the presence in the cluster of four putative menaquinone biosynthetic genes, suggests a connection between the biosynthesis pathways of both compounds. The availability of the PM100117 and PM100118 biosynthetic gene cluster will surely pave a way to the combinatorial engineering of more derivatives.

Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.

PubMed

Manira, Maarof; Khairul Anuar, Khairoji; Seet, Wan Tai; Ahmad Irfan, Abd Wahab; Ng, Min Hwei; Chua, Kien Hui; Mohd Heikal, Mohd Yunus; Aminuddin, Bin Saim; Ruszymah, Bt Hj Idrus

2014-03-01

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

Thinopyrum 7Ai-1-derived small chromatin with Barley Yellow Dwarf Virus (BYDV) resistance gene integrated into the wheat genome with retrotransposon.

PubMed

Ma, Y-Z; Tomita, M

2013-01-01

Thinopyrum intermedium is a useful source of resistance genes for Barley Yellow Dwarf Virus (BYDV), one of the most damaging wheat diseases. In this study, wheat/Th. intermedium translocation lines with a BYDV resistance gene were developed using the Th. intermedium 7Ai- 1 chromosome. Genomic in situ hybridization (GISH), using a Th. intermedium total genomic DNA probe, enabled detection of 7Ai-1-derived small chromatins containing a BYDV resistance gene, which were translocated onto the end of wheat chromosomes in the lines Y95011 and Y960843. Random amplified polymorphic DNA (RAPD) analyses using 120 random 10-mer primers were conducted to compare the BYDV-resistant translocation lines with susceptible lines. Two primers amplified the DNA fragments specific to the resistant line that would be useful as molecular markers to identify 7Ai-1-derived BYDV resistance chromatin in the wheat genome. Additionally, the isolated Th. intermedium-specific retrotransposon-like sequence pTi28 can be used to identify Th. intermedium chromatin transferred to the wheat genome.

Transformation of apatite phosphorus and non-apatite inorganic phosphorus during incineration of sewage sludge.

PubMed

Li, Rundong; Zhang, Ziheng; Li, Yanlong; Teng, Wenchao; Wang, Weiyun; Yang, Tianhua

2015-12-01

The recovery of phosphorus from incinerated sewage sludge ash (SSA) is assumed to be economical. Transformation from non-apatite inorganic phosphorus (NAIP) to apatite phosphorus (AP), which has a higher bioavailability and more extensive industrial applications, was studied at 750-950°C by sewage sludge incineration and model compound incineration with a calcium oxide (CaO) additive. Thermogravimetric differential scanning calorimetry analysis and X-ray diffraction measurements were used to analyze the reactions between NAIP with CaO and crystallized phases in SSA. High temperatures stimulated the volatilization of NAIP instead of AP. Sewage sludge incineration with CaO transformed NAIP into AP, and the percentage of AP from the total phosphorus reached 99% at 950°C. Aluminum phosphate reacted with CaO, forming Ca2P2O7 and Ca3(PO4)2 at 750-950°C. Reactions between iron phosphate and CaO occurred at lower temperatures, forming Ca(PO3)2 before reaching 850°C. Copyright © 2015 Elsevier Ltd. All rights reserved.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

2015-09-14

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression.

PubMed

Darvishi, Marzieh; Tiraihi, Taki; Mesbah-Namin, Seyed A; Delshad, AliReza; Taheri, Taher

2017-03-01

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

Mammalian-specific genomic functions: Newly acquired traits generated by genomic imprinting and LTR retrotransposon-derived genes in mammals.

PubMed

Kaneko-Ishino, Tomoko; Ishino, Fumitoshi

2015-01-01

Mammals, including human beings, have evolved a unique viviparous reproductive system and a highly developed central nervous system. How did these unique characteristics emerge in mammalian evolution, and what kinds of changes did occur in the mammalian genomes as evolution proceeded? A key conceptual term in approaching these issues is "mammalian-specific genomic functions", a concept covering both mammalian-specific epigenetics and genetics. Genomic imprinting and LTR retrotransposon-derived genes are reviewed as the representative, mammalian-specific genomic functions that are essential not only for the current mammalian developmental system, but also mammalian evolution itself. First, the essential roles of genomic imprinting in mammalian development, especially related to viviparous reproduction via placental function, as well as the emergence of genomic imprinting in mammalian evolution, are discussed. Second, we introduce the novel concept of "mammalian-specific traits generated by mammalian-specific genes from LTR retrotransposons", based on the finding that LTR retrotransposons served as a critical driving force in the mammalian evolution via generating mammalian-specific genes.

Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

PubMed Central

Perkins, Archibald S.; Kirschmeier, Paul T.; Gattoni-Celli, Sebastiano; Weinstein, I. Bernard

1983-01-01

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique PstI site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt+ colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR-gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt+ phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells. Images PMID:6308426

Statistical correction of lidar-derived digital elevation models with multispectral airborne imagery in tidal marshes

USGS Publications Warehouse

Buffington, Kevin J.; Dugger, Bruce D.; Thorne, Karen M.; Takekawa, John Y.

2016-01-01

Airborne light detection and ranging (lidar) is a valuable tool for collecting large amounts of elevation data across large areas; however, the limited ability to penetrate dense vegetation with lidar hinders its usefulness for measuring tidal marsh platforms. Methods to correct lidar elevation data are available, but a reliable method that requires limited field work and maintains spatial resolution is lacking. We present a novel method, the Lidar Elevation Adjustment with NDVI (LEAN), to correct lidar digital elevation models (DEMs) with vegetation indices from readily available multispectral airborne imagery (NAIP) and RTK-GPS surveys. Using 17 study sites along the Pacific coast of the U.S., we achieved an average root mean squared error (RMSE) of 0.072 m, with a 40–75% improvement in accuracy from the lidar bare earth DEM. Results from our method compared favorably with results from three other methods (minimum-bin gridding, mean error correction, and vegetation correction factors), and a power analysis applying our extensive RTK-GPS dataset showed that on average 118 points were necessary to calibrate a site-specific correction model for tidal marshes along the Pacific coast. By using available imagery and with minimal field surveys, we showed that lidar-derived DEMs can be adjusted for greater accuracy while maintaining high (1 m) resolution.

In vitro exposure to isoprene-derived secondary organic aerosol by direct deposition and its effects on COX-2 and IL-8 gene expression

NASA Astrophysics Data System (ADS)

Arashiro, Maiko; Lin, Ying-Hsuan; Sexton, Kenneth G.; Zhang, Zhenfa; Jaspers, Ilona; Fry, Rebecca C.; Vizuete, William G.; Gold, Avram; Surratt, Jason D.

2016-11-01

Atmospheric oxidation of isoprene, the most abundant non-methane hydrocarbon emitted into Earth's atmosphere primarily from terrestrial vegetation, is now recognized as a major contributor to the global secondary organic aerosol (SOA) burden. Anthropogenic pollutants significantly enhance isoprene SOA formation through acid-catalyzed heterogeneous chemistry of epoxide products. Since isoprene SOA formation as a source of fine aerosol is a relatively recent discovery, research is lacking on evaluating its potential adverse effects on human health. The objective of this study was to examine the effect of isoprene-derived SOA on inflammation-associated gene expression in human lung cells using a direct deposition exposure method. We assessed altered expression of inflammation-related genes in human bronchial epithelial cells (BEAS-2B) exposed to isoprene-derived SOA generated in an outdoor chamber facility. Measurements of gene expression of known inflammatory biomarkers interleukin 8 (IL-8) and cyclooxygenase 2 (COX-2) in exposed cells, together with complementary chemical measurements, showed that a dose of 0.067 µg cm-2 of SOA from isoprene photooxidation leads to statistically significant increases in IL-8 and COX-2 mRNA levels. Resuspension exposures using aerosol filter extracts corroborated these findings, supporting the conclusion that isoprene-derived SOA constituents induce the observed changes in mRNA levels. The present study is an attempt to examine the early biological responses of isoprene SOA exposure in human lung cells.

Analysis of Societal Response in Urban Landscape Irrigation due to the Recent California Drought Utilizing Remotely Sensed Satellite and High Resolution Aerial NAIP Imagery

NASA Astrophysics Data System (ADS)

Munoz, V.; Mendoza, J.; Ha, J. J.; Pagan, B. R.; Margulis, S. A.; Pal, J. S.

2016-12-01

California is currently amidst the most severe multi-year drought on record. Severe strains on water supply throughout the state have resulted in calls from water agencies and the Governor of California for extensive conservation. In Southern California, as much as 65% of daily water usage in single family homes goes towards outdoor use, primarily for grass lawns and landscaping. Therefore, the majority of the water conservation messaging has been targeted at turf removal and limiting outdoor irrigation. However, quantification of the effectiveness of these conservation campaigns and subsequent impacts on outdoor irrigation in large urban areas is difficult due to a lack of readily available in situ metering data separating indoor and outdoor use. Here we use a suite of high-resolution remotely sensed products to examine the impacts of the drought on irrigated vegetation over the Greater Los Angeles County. One-meter National Agriculture Imagery Program (NAIP) data from the United States Department of Agriculture and 30-meter Landsat data over Los Angeles is used to calculate the Normalized Difference Vegetation Index (NDVI). NDVI is compared between non-drought (2005, 2009, 2010) and drought years (2012, 2014). Declines in NDVI are typically manifested as browning of vegetation due to reduced irrigation, or the replacement of water intensive grass lawns with more native vegetation. Results suggest that changes in NDVI during the drought may be dependent on societal responses to water conservation campaigns. Our analysis suggests that disparities in plant greenness, a proxy of plant health, and total vegetated area exist between wealthy and poor communities, both prior to and during the drought. This disparity is likely to increase during drought periods, assuming wealthier communities are less likely to respond to increasing water rates and more stringent watering restrictions.

Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagao, Kenji; Ohta, Takayuki; Hinohara, Atsushi

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferationmore » of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.« less

The chemokine receptor CCR1 is identified in mast cell-derived exosomes

PubMed Central

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses. PMID:29511430

The chemokine receptor CCR1 is identified in mast cell-derived exosomes.

PubMed

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses.

Influence of Light and Temperature on Gene Expression Leading to Accumulation of Specific Flavonol Glycosides and Hydroxycinnamic Acid Derivatives in Kale (Brassica oleracea var. sabellica).

PubMed

Neugart, Susanne; Krumbein, Angelika; Zrenner, Rita

2016-01-01

Light intensity and temperature are very important signals for the regulation of plant growth and development. Plants subjected to less favorable light or temperature conditions often respond with accumulation of secondary metabolites. Some of these metabolites have been identified as bioactive compounds, considered to exert positive effects on human health when consumed regularly. In order to test a typical range of growth parameters for the winter crop Brassica oleracea var. sabellica, plants were grown either at 400 μmol m(-2) s(-1) or 100 μmol m(-2) s(-1) at 10°C, or at 400 μmol m(-2) s(-1) with 5 or 15°C. The higher light intensity overall increased flavonol content of leaves, favoring the main quercetin glycosides, a caffeic acid monoacylated kaempferol triglycoside, and disinapoyl-gentiobiose. The higher temperature mainly increased the hydroxycinnamic acid derivative disinapoyl-gentiobiose, while at lower temperature synthesis is in favor of very complex sinapic acid acylated flavonol tetraglycosides such as kaempferol-3-O-sinapoyl-sophoroside-7-O-diglucoside. A global analysis of light and temperature dependent alterations of gene expression in B. oleracea var. sabellica leaves was performed with the most comprehensive Brassica microarray. When compared to the light experiment much less genes were differentially expressed in kale leaves grown at 5 or 15°C. A structured evaluation of differentially expressed genes revealed the expected enrichment in the functional categories of e.g. protein degradation at different light intensities or phytohormone metabolism at different temperature. Genes of the secondary metabolism namely phenylpropanoids are significantly enriched with both treatments. Thus, the genome of B. oleracea was screened for predicted genes putatively involved in the biosynthesis of flavonoids and hydroxycinnamic acid derivatives. All identified B. oleracea genes were analyzed for their most specific 60-mer oligonucleotides present on the

Influence of Light and Temperature on Gene Expression Leading to Accumulation of Specific Flavonol Glycosides and Hydroxycinnamic Acid Derivatives in Kale (Brassica oleracea var. sabellica)

PubMed Central

Neugart, Susanne; Krumbein, Angelika; Zrenner, Rita

2016-01-01

Light intensity and temperature are very important signals for the regulation of plant growth and development. Plants subjected to less favorable light or temperature conditions often respond with accumulation of secondary metabolites. Some of these metabolites have been identified as bioactive compounds, considered to exert positive effects on human health when consumed regularly. In order to test a typical range of growth parameters for the winter crop Brassica oleracea var. sabellica, plants were grown either at 400 μmol m−2 s−1 or 100 μmol m−2 s−1 at 10°C, or at 400 μmol m−2 s−1 with 5 or 15°C. The higher light intensity overall increased flavonol content of leaves, favoring the main quercetin glycosides, a caffeic acid monoacylated kaempferol triglycoside, and disinapoyl-gentiobiose. The higher temperature mainly increased the hydroxycinnamic acid derivative disinapoyl-gentiobiose, while at lower temperature synthesis is in favor of very complex sinapic acid acylated flavonol tetraglycosides such as kaempferol-3-O-sinapoyl-sophoroside-7-O-diglucoside. A global analysis of light and temperature dependent alterations of gene expression in B. oleracea var. sabellica leaves was performed with the most comprehensive Brassica microarray. When compared to the light experiment much less genes were differentially expressed in kale leaves grown at 5 or 15°C. A structured evaluation of differentially expressed genes revealed the expected enrichment in the functional categories of e.g. protein degradation at different light intensities or phytohormone metabolism at different temperature. Genes of the secondary metabolism namely phenylpropanoids are significantly enriched with both treatments. Thus, the genome of B. oleracea was screened for predicted genes putatively involved in the biosynthesis of flavonoids and hydroxycinnamic acid derivatives. All identified B. oleracea genes were analyzed for their most specific 60-mer oligonucleotides present on the

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

PubMed

Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

2016-06-01

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer's disease.

PubMed

Yamamoto, Satoshi; Ooshima, Yuki; Nakata, Mitsugu; Yano, Takashi; Matsuoka, Kunio; Watanabe, Sayuri; Maeda, Ryouta; Takahashi, Hideki; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

2013-06-01

Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

Genotypic characterization of CRF01_AE env genes derived from human immunodeficiency virus type 1-infected patients residing in central Thailand.

PubMed

Utachee, Piraporn; Jinnopat, Piyamat; Isarangkura-Na-Ayuthaya, Panasda; de Silva, Udayanga Chandimal; Nakamura, Shota; Siripanyaphinyo, Uamporn; Wichukchinda, Nuanjun; Tokunaga, Kenzo; Yasunaga, Teruo; Sawanpanyalert, Pathom; Ikuta, Kazuyoshi; Auwanit, Wattana; Kameoka, Masanori

2009-02-01

CRF01_AE is a major subtype of human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia, including Thailand. HIV-1 env genes were amplified by polymerase chain reaction from blood samples of HIV-1-infected patients residing in Thailand in 2006, and cloned into the pNL4-3-derived reporter viral construct. Generated envelope protein (Env)-recombinant virus was examined for its infectivity, and then 35 infectious CRF01_AE Env-recombinant viruses were selected. Sequencing analysis revealed that the interclone variation of the deduced amino acid sequences was higher in CRF01_AE env genes isolated in 2006 than in those isolated in the early 1990s, suggesting that env gene variation has been increasing gradually among CRF01_AE viruses prevalent in Thailand. We also examined the characteristics of the deduced amino acid sequences of 35 CRF01_AE env genes. Our results may provide useful information to help in better understanding the genotype of env genes of CRF01_AE viruses currently circulating in Thailand.

Mammalian-specific genomic functions: Newly acquired traits generated by genomic imprinting and LTR retrotransposon-derived genes in mammals

PubMed Central

KANEKO-ISHINO, Tomoko; ISHINO, Fumitoshi

2015-01-01

Mammals, including human beings, have evolved a unique viviparous reproductive system and a highly developed central nervous system. How did these unique characteristics emerge in mammalian evolution, and what kinds of changes did occur in the mammalian genomes as evolution proceeded? A key conceptual term in approaching these issues is “mammalian-specific genomic functions”, a concept covering both mammalian-specific epigenetics and genetics. Genomic imprinting and LTR retrotransposon-derived genes are reviewed as the representative, mammalian-specific genomic functions that are essential not only for the current mammalian developmental system, but also mammalian evolution itself. First, the essential roles of genomic imprinting in mammalian development, especially related to viviparous reproduction via placental function, as well as the emergence of genomic imprinting in mammalian evolution, are discussed. Second, we introduce the novel concept of “mammalian-specific traits generated by mammalian-specific genes from LTR retrotransposons”, based on the finding that LTR retrotransposons served as a critical driving force in the mammalian evolution via generating mammalian-specific genes. PMID:26666304

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Jung-Hwa; Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113; Son, Mi-Young

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10–200 μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24 h. The results showed that Ag NPs evoked significant toxicity in hESC-derivedmore » NPCs at 24 h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. - Highlights: • This system served as a suitable model for developmental neurotoxicity testing. • Ag NPs induce the apoptosis in human neural cells by ROS generation. • Genes for development of neurons were dysregulated in response to Ag NPs. • Molecular events during early developmental neurotoxicity were proposed.« less

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

PubMed

Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

2012-05-01

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. Copyright © 2012 AlphaMed Press.

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation

PubMed Central

Kantor, Gal; Cheishvili, David; Even, Aviel; Birger, Anastasya; Turetsky, Tikva; Gil, Yaniv; Even-Ram, Sharona; Aizenman, Einat; Bashir, Nibal; Maayan, Channa; Razin, Aharon; Reubinoff, Benjamim E.; Weil, Miguel

2015-01-01

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD. PMID:26437462

The powdery mildew resistance gene Pm8 derived from rye is suppressed by its wheat ortholog Pm3.

PubMed

Hurni, Severine; Brunner, Susanne; Stirnweis, Daniel; Herren, Gerhard; Peditto, David; McIntosh, Robert A; Keller, Beat

2014-09-01

The powdery mildew resistance gene Pm8 derived from rye is located on a 1BL.1RS chromosome translocation in wheat. However, some wheat lines with this translocation do not show resistance to isolates of the wheat powdery mildew pathogen avirulent to Pm8 due to an unknown genetically dominant suppression mechanism. Here we show that lines with suppressed Pm8 activity contain an intact and expressed Pm8 gene. Therefore, the absence of Pm8 function in certain 1BL.1RS-containing wheat lines is not the result of gene loss or mutation but is based on suppression. The wheat gene Pm3, an ortholog of rye Pm8, suppressed Pm8-mediated powdery mildew resistance in lines containing Pm8 in a transient single-cell expression assay. This result was further confirmed in transgenic lines with combined Pm8 and Pm3 transgenes. Expression analysis revealed that suppression is not the result of gene silencing, either in wheat 1BL.1RS translocation lines carrying Pm8 or in transgenic genotypes with both Pm8 and Pm3 alleles. In addition, a similar abundance of the PM8 and PM3 proteins in single or double homozygous transgenic lines suggested that a post-translational mechanism is involved in suppression of Pm8. Co-expression of Pm8 and Pm3 genes in Nicotiana benthamiana leaves followed by co-immunoprecipitation analysis showed that the two proteins interact. Therefore, the formation of a heteromeric protein complex might result in inefficient or absent signal transmission for the defense reaction. These data provide a molecular explanation for the suppression of resistance genes in certain genetic backgrounds and suggest ways to circumvent it in future plant breeding. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Global gene expression and systems biology analysis of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis.

PubMed

Magee, David A; Taraktsoglou, Maria; Killick, Kate E; Nalpas, Nicolas C; Browne, John A; Park, Stephen D E; Conlon, Kevin M; Lynn, David J; Hokamp, Karsten; Gordon, Stephen V; Gormley, Eamonn; MacHugh, David E

2012-01-01

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array. Comparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤ 0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis. The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.

Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate

PubMed Central

2011-01-01

Background Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, e.g. mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate. Results Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization. Conclusions Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of in vitro mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate

Single Cell Gene Expression Profiling of Skeletal Muscle-Derived Cells.

PubMed

Gatto, Sole; Puri, Pier Lorenzo; Malecova, Barbora

2017-01-01

Single cell gene expression profiling is a fundamental tool for studying the heterogeneity of a cell population by addressing the phenotypic and functional characteristics of each cell. Technological advances that have coupled microfluidic technologies with high-throughput quantitative RT-PCR analyses have enabled detailed analyses of single cells in various biological contexts. In this chapter, we describe the procedure for isolating the skeletal muscle interstitial cells termed Fibro-Adipogenic Progenitors (FAPs ) and their gene expression profiling at the single cell level. Moreover, we accompany our bench protocol with bioinformatics analysis designed to process raw data as well as to visualize single cell gene expression data. Single cell gene expression profiling is therefore a useful tool in the investigation of FAPs heterogeneity and their contribution to muscle homeostasis.

Gene correction of induced pluripotent stem cells derived from a murine model of X-linked chronic granulomatous disorder.

PubMed

Mukherjee, Sayandip; Thrasher, Adrian J

2014-01-01

Gene therapy presents an attractive alternative to allogeneic haematopoietic stem cell transplantation (HSCT) for treating patients suffering from primary immunodeficiency disorder (PID). The conceptual advantage of gene correcting a patient's autologous HSCs lies in minimizing or completely avoiding immunological complications arising from allogeneic transplantation while conferring the same benefits of immune reconstitution upon long-term engraftment. Clinical trials targeting X-linked chronic granulomatous disorder (X-CGD) have shown promising results in this context. However, long-term clinical benefits in these patients have been limited by issues of poor engraftment of gene-transduced cells coupled with transgene silencing and vector induced clonal proliferation. Novel vectors incorporating safety features such as self-inactivating (SIN) mutations in the long terminal repeats (LTRs) along with synthetic promoters driving lineage-restricted sustainable expression of the gp91phox transgene are expected to resolve the current pitfalls and require rigorous preclinical testing. In this chapter, we have outlined a protocol in which X-CGD mouse model derived induced pluripotent stem cells (iPSCs) have been utilized to develop a platform for investigating the efficacy and safety profiles of novel vectors prior to clinical evaluation.

Gene expression analysis of human adipose tissue-derived stem cells during the initial steps of in vitro osteogenesis.

PubMed

Robert, Anny Waloski; Angulski, Addeli Bez Batti; Spangenberg, Lucia; Shigunov, Patrícia; Pereira, Isabela Tiemy; Bettes, Paulo Sergio Loiacono; Naya, Hugo; Correa, Alejandro; Dallagiovanna, Bruno; Stimamiglio, Marco Augusto

2018-03-16

Mesenchymal stem cells (MSCs) have been widely studied with regard to their potential use in cell therapy protocols and regenerative medicine. However, a better comprehension about the factors and molecular mechanisms driving cell differentiation is now mandatory to improve our chance to manipulate MSC behavior and to benefit future applications. In this work, we aimed to study gene regulatory networks at an early step of osteogenic differentiation. Therefore, we analyzed both the total mRNA and the mRNA fraction associated with polysomes on human adipose tissue-derived stem cells (hASCs) at 24 h of osteogenesis induction. The RNA-seq results evidenced that hASC fate is not compromised with osteogenesis at this time and that 21 days of continuous cell culture stimuli are necessary for full osteogenic differentiation of hASCs. Furthermore, early stages of osteogenesis induction involved gene regulation that was linked to the management of cell behavior in culture, such as the control of cell adhesion and proliferation. In conclusion, although discrete initial gene regulation related to osteogenesis occur, the first 24 h of induction is not sufficient to trigger and drive in vitro osteogenic differentiation of hASCs.

DNA methylation in schizophrenia in different patient-derived cell types.

PubMed

Vitale, Alejandra M; Matigian, Nicholas A; Cristino, Alexandre S; Nones, Katia; Ravishankar, Sugandha; Bellette, Bernadette; Fan, Yongjun; Wood, Stephen A; Wolvetang, Ernst; Mackay-Sim, Alan

2017-01-01

DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a "ground state" upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells.

PubMed

Barbet, Romain; Peiffer, Isabelle; Hatzfeld, Antoinette; Charbord, Pierre; Hatzfeld, Jacques A

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

Discovery of SCORs: Anciently derived, highly conserved gene-associated repeats in stony corals.

PubMed

Qiu, Huan; Zelzion, Ehud; Putnam, Hollie M; Gates, Ruth D; Wagner, Nicole E; Adams, Diane K; Bhattacharya, Debashish

2017-10-01

Stony coral (Scleractinia) genomes are still poorly explored and many questions remain about their evolution and contribution to the success and longevity of reefs. We analyzed transcriptome and genome data from Montipora capitata, Acropora digitifera, and transcriptome data from 20 other coral species. To our surprise, we found highly conserved, anciently derived, Scleractinia COral-specific Repeat families (SCORs) that are abundant in all the studied lineages. SCORs form complex secondary structures and are located in untranslated regions and introns, but most abundant in intergenic DNA. These repeat families have undergone frequent duplication and degradation, suggesting a 'boom and bust' cycle of invasion and loss. We speculate that due to their surprisingly high sequence identities across deeply diverged corals, physical association with genes, and dynamic evolution, SCORs might have adaptive functions in corals that need to be explored using population genomic and function-based approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

PubMed

Peng, Yingmei; Cai, Jing; Wang, Wen; Su, Bing

2012-01-01

Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

The In Vitro Differentiation of GDNF Gene-Engineered Amniotic Fluid-Derived Stem Cells into Renal Tubular Epithelial-Like Cells.

PubMed

Lu, Ying; Wang, Zhuojun; Chen, Lu; Wang, Jia; Li, Shulin; Liu, Caixia; Sun, Dong

2018-05-01

Amniotic fluid is an alternative source of stem cells, and human amniotic fluid-derived stem cells (AFSCs) obtained from a small amount of amniotic fluid collected during the second trimester represent a novel source for use in regenerative medicine. These AFSCs are characterized by lower diversity, a higher proliferation rate, and a wider differentiation capability than adult mesenchymal stem cells. AFSCs are selected based on the cell surface marker c-kit receptor (CD117) using immunomagnetic sorting. Glial cell line-derived neurotrophic factor (GDNF) is expressed during early kidney development and regulates the proliferation and differentiation of stem cells in vitro. In this study, c-kit-sorted AFSCs were induced toward osteogenic or adipogenic differentiation. AFSCs engineered via the insertion of GDNF were cocultured with mouse renal tubular epithelial cells (mRTECs), which were preconditioned by hypoxia-reoxygenation in vitro. After coculture for 8 days, AFSCs differentiation into epithelial-like cells was evaluated by performing immunofluorescence, flow cytometry, and quantitative real-time polymerase chain reaction to identify cells expressing the renal epithelial markers, cytokeratin 18 (CK18), E-cadherin, aquaporin-1 (AQP1), and paired box 2 gene (Pax2). The GDNF gene enhanced AFSCs differentiation into RTECs. AFSCs possess self-renewal ability and multiple differentiation potential and thus represent a new source of stem cells.

Pigment epithelial-derived factor gene loaded novel COOH-PEG-PLGA-COOH nanoparticles promoted tumor suppression by systemic administration.

PubMed

Yu, Ting; Xu, Bei; He, Lili; Xia, Shan; Chen, Yan; Zeng, Jun; Liu, Yongmei; Li, Shuangzhi; Tan, Xiaoyue; Ren, Ke; Yao, Shaohua; Song, Xiangrong

2016-01-01

Anti-angiogenesis has been proposed as an effective therapeutic strategy for cancer treatment. Pigment epithelium-derived factor (PEDF) is one of the most powerful endogenous anti-angiogenic reagents discovered to date and PEDF gene therapy has been recognized as a promising treatment option for various tumors. There is an urgent need to develop a safe and valid vector for its systemic delivery. Herein, a novel gene delivery system based on the newly synthesized copolymer COOH-PEG-PLGA-COOH (CPPC) was developed in this study, which was probably capable of overcoming the disadvantages of viral vectors and cationic lipids/polymers-based nonviral carriers. PEDF gene loaded CPPC nanoparticles (D-NPs) were fabricated by a modified double-emulsion water-in-oil-in-water (W/O/W) solvent evaporation method. D-NPs with uniform spherical shape had relatively high drug loading (~1.6%), probably because the introduced carboxyl group in poly (D,L-lactide-co-glycolide) terminal enhanced the interaction of copolymer with the PEDF gene complexes. An excellent in vitro antitumor effect was found in both C26 and A549 cells treated by D-NPs, in which PEDF levels were dramatically elevated due to the successful transfection of PEDF gene. D-NPs also showed a strong inhibitory effect on proliferation of human umbilical vein endothelial cells in vitro and inhibited the tumor-induced angiogenesis in vivo by an alginate-encapsulated tumor cell assay. Further in vivo antitumor investigation, carried out in a C26 subcutaneous tumor model by intravenous injection, demonstrated that D-NPs could achieve a significant antitumor activity with sharply reduced microvessel density and significantly promoted tumor cell apoptosis. Additionally, the in vitro hemolysis analysis and in vivo serological and biochemical analysis revealed that D-NPs had no obvious toxicity. All the data indicated that the novel CPPC nanoparticles were ideal vectors for the systemic delivery of PEDF gene and might be widely

Cationic carbon quantum dots derived from alginate for gene delivery: One-step synthesis and cellular uptake.

PubMed

Zhou, Jie; Deng, Wenwen; Wang, Yan; Cao, Xia; Chen, Jingjing; Wang, Qiang; Xu, Wenqian; Du, Pan; Yu, Qingtong; Chen, Jiaxin; Spector, Myron; Yu, Jiangnan; Xu, Ximing

2016-09-15

Carbon quantum dots (CQDs), unlike semiconductor quantum dots, possess fine biocompatibility, excellent upconversion properties, high photostability and low toxicity. Here, we report multifunctional CQDs which were developed using alginate, 3% hydrogen peroxide and double distilled water through a facile, eco-friendly and inexpensive one-step hydrothermal carbonization route. In this reaction, the alginate served as both the carbon source and the cationization agent. The resulting CQDs exhibited strong and stable fluorescence with water-dispersible and positively-charged properties which could serve as an excellent DNA condensation. As non-viral gene vector being used for the first time, the CQDs showed considerably high transfection efficiency (comparable to Lipofectamine2000 and significantly higher than PEI, p<0.05) and negligible toxicity. The photoluminescence properties of CQDs also permitted easy tracking of the cellular-uptake. The findings showed that both caveolae- and clathrin-mediated endocytosis pathways were involved in the internalization process of CQDs/pDNA complexes. Taken together, the alginate-derived photoluminescent CQDs hold great potential in biomedical applications due to their dual role as efficient non-viral gene vectors and bioimaging probes. This manuscript describes a facile and simple one-step hydrothermal carbonization route for preparing optically tunable photoluminescent carbon quantum dots (CQDs) from a novel raw material, alginate. These CQDs enjoy low cytotoxicity, positive zeta potential, excellent ability to condense macromolecular DNA, and most importantly, notably high transfection efficiency. The interesting finding is that the negatively-charged alginate can convert into positively charged CQDs without adding any cationic reagents. The significance of this study is that the cationic carbon quantum dots play dual roles as both non-viral gene vectors and bioimaging probes at the same time, which are most desirable in many

hTERT gene immortalized human adipose-derived stem cells and its multiple differentiations: a preliminary investigation.

PubMed

Wang, L; Song, K; Qu, X; Wang, H; Zhu, H; Xu, X; Zhang, M; Tang, Y; Yang, X

2013-03-01

Human adipose-derived adult stem cells (hADSCs) can express human telomerase reverse transcriptase phenotypes under an appropriate culture condition. Because adipose tissue is abundant and easily accessible, hADSCs offer a promising source of stem cells for tissue engineering application and other cell-based therapies. However, the shortage of cells number and the difficulty to proliferate, known as the "Hayflick limit" in vitro, limit their further clinical application. Here, hADSCs were transfected with human telomerase reverse transcriptase (hTERT) gene by the lentiviral vector to prolong the lifespan of stem cells and even immortalize them. Following to this, the cellular properties and functionalities of the transfected cell lines were assayed. The results demonstrated that hADSCs had been successfully transfected with hTERT gene (hTERT-ADSCs). Then, hTERT-ADSCs were initially selected by G418 and subsequently expanded over 20 passages in vitro. Moreover, the qualitative and quantitative differentiation criteria for 20 passages of hTERT-ADSCs also demonstrated that hTERT-ADSCs could differentiate into osteogenesis, chondrogenesis, and adipogenesis phenotypes in lineage-specific differentiation media. These findings confirmed that this transfection could prolong the lifespan of hADSCs.

Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics.

PubMed

Jones, Anya C; Troy, Niamh M; White, Elisha; Hollams, Elysia M; Gout, Alexander M; Ling, Kak-Ming; Kicic, Anthony; Stick, Stephen M; Sly, Peter D; Holt, Patrick G; Hall, Graham L; Bosco, Anthony

2018-01-24

Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDM S ) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDM S -wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics.

New Rimocidin/CE-108 Derivatives Obtained by a Crotonyl-CoA Carboxylase/Reductase Gene Disruption in Streptomyces diastaticus var. 108: Substrates for the Polyene Carboxamide Synthase PcsA

PubMed Central

Escudero, Leticia; Al-Refai, Mahmoud; Nieto, Cristina; Laatsch, Hartmut; Malpartida, Francisco; Seco, Elena M.

2015-01-01

The rimJ gene, which codes for a crotonyl-CoA carboxylase/reductase, lies within the biosynthetic gene cluster for two polyketides belonging to the polyene macrolide group (CE-108 and rimocidin) produced by Streptomyces diastaticus var. 108. Disruption of rimJ by insertional inactivation gave rise to a recombinant strain overproducing new polyene derivatives besides the parental CE-108 (2a) and rimocidin (4a). The structure elucidation of one of them, CE-108D (3a), confirmed the incorporation of an alternative extender unit for elongation step 13. Other compounds were also overproduced in the fermentation broth of rimJ disruptant. The new compounds are in vivo substrates for the previously described polyene carboxamide synthase PcsA. The rimJ disruptant strain, constitutively expressing the pcsA gene, allowed the overproduction of CE-108E (3b), the corresponding carboxamide derivative of CE-108D (3a), with improved pharmacological properties. PMID:26284936

Concerted transcription of auxin and carbohydrate homeostasis-related genes underlies improved adventitious rooting of microcuttings derived from far-red treated Eucalyptus globulus Labill mother plants.

PubMed

Ruedell, Carolina Michels; de Almeida, Márcia Rodrigues; Fett-Neto, Arthur Germano

2015-12-01

Economically important plant species, such as Eucalyptus globulus, are often rooting recalcitrant. We have previously shown that far-red light enrichment applied to E. globulus donor-plants improved microcutting rooting competence and increased rooting zone/shoot carbohydrate ratio. To better understand this developmental response, the relative expression profiles of genes involved in auxin signaling (ARF6, ARF8, AGO1), biosynthesis (YUC3) and transport (AUX1, PIN1, PIN2); sucrose cleavage (SUS1, CWINV1), transport (SUC5), hexose phosphorylation (HXK1, FLN1) and starch biosynthesis (SS3) were quantified during adventitious rooting of E. globulus microcuttings derived from donor plants exposed to far-red or white light. Expression of auxin transport-related genes increased in the first days of root induction. Far-red enrichment of donor plants induced ARF6, ARF8 and AGO1 in microcuttings. The first two gene products could activate GH3 and other rooting related genes, whereas AGO1 deregulation of the repressor ARF17 may relief rooting inhibition. Increased sink strength at the basal stem with sucrose unloading in root tissue mediated by SUC and subsequent hydrolysis by SUS1 were also supported by gene expression profile. Fructose phosphorylation and starch biosynthesis could also contribute to proper carbon allocation at the site of rooting, as evidenced by increased expression of related genes. These data are in good agreement with increased contents of hexoses and starch at the cutting base severed from far-red exposed donor plants. To sum up, pathways integrating auxin and carbohydrate metabolism were activated in microcuttings derived from donor plants exposed to far red light enrichment, thereby improving rooting response in E. globulus. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Evidence of associations between brain-derived neurotrophic factor (BDNF) serum levels and gene polymorphisms with tinnitus.

PubMed

Coskunoglu, Aysun; Orenay-Boyacioglu, Seda; Deveci, Artuner; Bayam, Mustafa; Onur, Ece; Onan, Arzu; Cam, Fethi S

2017-01-01

Brain-derived neurotrophic factor (BDNF) gene polymorphisms are associated with abnormalities in regulation of BDNF secretion. Studies also linked BDNF polymorphisms with changes in brainstem auditory-evoked response test results. Furthermore, BDNF levels are reduced in tinnitus, psychiatric disorders, depression, dysthymic disorder that may be associated with stress, conversion disorder, and suicide attempts due to crises of life. For this purpose, we investigated whether there is any role of BDNF changes in the pathophysiology of tinnitus. In this study, we examined the possible effects of BDNF variants in individuals diagnosed with tinnitus for more than 3 months. Fifty-two tinnitus subjects between the ages of 18 and 55, and 42 years healthy control subjects in the same age group, who were free of any otorhinolaryngology and systemic disease, were selected for examination. The intensity of tinnitus and depression was measured using the tinnitus handicap inventory, and the differential diagnosis of psychiatric diagnoses made using the Structured Clinical Interview for Fourth Edition of Mental Disorders. BDNF gene polymorphism was analyzed in the genomic deoxyribonucleic acid (DNA) samples extracted from the venous blood, and the serum levels of BDNF were measured. One-way analysis of variance and Chi-squared tests were applied. Serum BDNF level was found lower in the tinnitus patients than controls, and it appeared that there is no correlation between BDNF gene polymorphism and tinnitus. This study suggests neurotrophic factors such as BDNF may have a role in tinnitus etiology. Future studies with larger sample size may be required to further confirm our results.

Phylogenomic Analysis of Lactobacillus curvatus Reveals Two Lineages Distinguished by Genes for Fermenting Plant-Derived Carbohydrates.

PubMed

Terán, Lucrecia C; Coeuret, Gwendoline; Raya, Raúl; Zagorec, Monique; Champomier-Vergès, Marie-Christine; Chaillou, Stéphane

2018-06-01

Lactobacillus curvatus is a lactic acid bacterium encountered in many different types of fermented food (meat, seafood, vegetables, and cereals). Although this species plays an important role in the preservation of these foods, few attempts have been made to assess its genomic diversity. This study uses comparative analyses of 13 published genomes (complete or draft) to better understand the evolutionary processes acting on the genome of this species. Phylogenomic analysis, based on a coalescent model of evolution, revealed that the 6,742 sites of single nucleotide polymorphism within the L. curvatus core genome delineate two major groups, with lineage 1 represented by the newly sequenced strain FLEC03, and lineage 2 represented by the type-strain DSM20019. The two lineages could also be distinguished by the content of their accessory genome, which sheds light on a long-term evolutionary process of lineage-dependent genetic acquisition and the possibility of population structure. Interestingly, one clade from lineage 2 shared more accessory genes with strains of lineage 1 than with other strains of lineage 2, indicating recent convergence in carbohydrate catabolism. Both lineages had a wide repertoire of accessory genes involved in the fermentation of plant-derived carbohydrates that are released from polymers of α/β-glucans, α/β-fructans, and N-acetylglucosan. Other gene clusters were distributed among strains according to the type of food from which the strains were isolated. These results give new insight into the ecological niches in which L. curvatus may naturally thrive (such as silage or compost heaps) in addition to fermented food.

Evidence of associations between brain-derived neurotrophic factor (BDNF) serum levels and gene polymorphisms with tinnitus

PubMed Central

Coskunoglu, Aysun; Orenay-Boyacioglu, Seda; Deveci, Artuner; Bayam, Mustafa; Onur, Ece; Onan, Arzu; Cam, Fethi S.

2017-01-01

Background: Brain-derived neurotrophic factor (BDNF) gene polymorphisms are associated with abnormalities in regulation of BDNF secretion. Studies also linked BDNF polymorphisms with changes in brainstem auditory-evoked response test results. Furthermore, BDNF levels are reduced in tinnitus, psychiatric disorders, depression, dysthymic disorder that may be associated with stress, conversion disorder, and suicide attempts due to crises of life. For this purpose, we investigated whether there is any role of BDNF changes in the pathophysiology of tinnitus. Materials and Methods: In this study, we examined the possible effects of BDNF variants in individuals diagnosed with tinnitus for more than 3 months. Fifty-two tinnitus subjects between the ages of 18 and 55, and 42 years healthy control subjects in the same age group, who were free of any otorhinolaryngology and systemic disease, were selected for examination. The intensity of tinnitus and depression was measured using the tinnitus handicap inventory, and the differential diagnosis of psychiatric diagnoses made using the Structured Clinical Interview for Fourth Edition of Mental Disorders. BDNF gene polymorphism was analyzed in the genomic deoxyribonucleic acid (DNA) samples extracted from the venous blood, and the serum levels of BDNF were measured. One-way analysis of variance and Chi-squared tests were applied. Results: Serum BDNF level was found lower in the tinnitus patients than controls, and it appeared that there is no correlation between BDNF gene polymorphism and tinnitus. Conclusions: This study suggests neurotrophic factors such as BDNF may have a role in tinnitus etiology. Future studies with larger sample size may be required to further confirm our results. PMID:28615544

Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway

PubMed Central

Ardiani, Andressa; Gameiro, Sofia R.; Kwilas, Anna R.; Donahue, Renee N.; Hodge, James W.

2014-01-01

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT. PMID:25344864

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration.

PubMed

Ramos-Solano, Moisés; Meza-Canales, Ivan D; Torres-Reyes, Luis A; Alvarez-Zavala, Monserrat; Alvarado-Ruíz, Liliana; Rincon-Orozco, Bladimiro; Garcia-Chagollan, Mariel; Ochoa-Hernández, Alejandra B; Ortiz-Lazareno, Pablo C; Rösl, Frank; Gariglio, Patricio; Jave-Suárez, Luis F; Aguilar-Lemarroy, Adriana

2015-07-01

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. Copyright © 2015 Elsevier Inc. All rights reserved.

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations

PubMed Central

Gasparis, Sebastian; Kała, Maciej; Przyborowski, Mateusz; Orczyk, Waclaw; Nadolska-Orczyk, Anna

2017-01-01

Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence Puroindoline a (Pina) and Puroindoline b (Pinb) hardness genes in wheat and their orthologues Secaloindoline a (Sina) and Secaloindoline b (Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of Pina/Sina or Pinb/Sinb genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the Pinb-derived amiR vector for silencing of Pinb or Sinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the Pina-derived amiR cassette, despite large numbers of embryos attempted. Silencing of Pinb in wheat and Sinb in triticale was highly efficient in the T1 generation. The transcript level of Pinb in wheat was reduced up to 92% and Sinb in triticale was reduced up to 98%. Moreover, intended silencing of Pinb/Sinb with Pinb-derived amiR cassette was highly correlated with simultaneous silencing of Pina/Sina in the same transgenic plants. High downregulation of Pinb/Pina genes in T1 plants of wheat and Sinb/Sina genes in T1 plants of triticale was associated with strong expression of Pinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the Pinb-derived amiR cassette was stably inherited in the T2 generation of wheat and

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2

PubMed Central

Ma, Tianzhong; Su, Zhongjing; Chen, Ling; Liu, Shuyan; Zhu, Ningxia; Wen, Lifeng; Yuan, Yan; Lv, Leili; Chen, Xiancai; Huang, Jianmin; Chen, Haibin

2012-01-01

Background and Objectives Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study. PMID:22655088

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients.

PubMed

Wang, Li-jia; Bai, Yu; Bao, Zhao-shi; Chen, Yan; Yan, Zhuo-hong; Zhang, Wei; Zhang, Quan-geng

2013-01-01

Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets. We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors. Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013). Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.

Are genetic variants in the platelet-derived growth factor [beta] gene associated with chronic pancreatitis?

PubMed

Muddana, Venkata; Park, James; Lamb, Janette; Yadav, Dhiraj; Papachristou, Georgios I; Hawes, Robert H; Brand, Randall; Slivka, Adam; Whitcomb, David C

2010-11-01

Platelet-derived growth factor [beta] (PDGF-[beta]) is a major signal in proliferation and matrix synthesis through activated pancreatic stellate cells, leading to fibrosis of the pancreas. Recurrent acute pancreatitis (RAP) seems to predispose to chronic pancreatitis (CP) in some patients but not others. We tested the hypothesis that 2 known PDGF-[beta] polymorphisms are associated with progression from RAP to CP. We also tested the hypothesis that PDGF-[beta] polymorphisms in combination with environmental risk factors such as alcohol and smoking are associated with CP. Three hundred eighty-two patients with CP (n = 176) and RAP (n = 206) and 251 controls were evaluated. Platelet-derived growth factor [beta] polymorphisms +286 A/G (rs#1800818) seen in 5'-UTR and +1135 A/C (rs#1800817) in first intron were genotyped using single-nucleotide polymorphism polymerase chain reaction approach and confirmed by DNA sequencing. The genotypic frequencies for PDGF-[beta] polymorphisms in positions +286 and +1135 were found to be similar in controls and patients with RAP and CP. There was no difference in genotypic frequencies among RAP, CP, and controls in subjects in the alcohol and smoking subgroups. Known variations in the PDGF-[beta] gene do not have a significant effect on promoting or preventing fibrogenesis in pancreatitis. Further evaluation of this important pathway is warranted.

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles.

PubMed

Oh, Jung-Hwa; Son, Mi-Young; Choi, Mi-Sun; Kim, Soojin; Choi, A-Young; Lee, Hyang-Ae; Kim, Ki-Suk; Kim, Janghwan; Song, Chang Woo; Yoon, Seokjoo

2016-05-15

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10-200μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24h. The results showed that Ag NPs evoked significant toxicity in hESC-derived NPCs at 24h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

GeneSigDB—a curated database of gene expression signatures

PubMed Central

Culhane, Aedín C.; Schwarzl, Thomas; Sultana, Razvan; Picard, Kermshlise C.; Picard, Shaita C.; Lu, Tim H.; Franklin, Katherine R.; French, Simon J.; Papenhausen, Gerald; Correll, Mick; Quackenbush, John

2010-01-01

The primary objective of most gene expression studies is the identification of one or more gene signatures; lists of genes whose transcriptional levels are uniquely associated with a specific biological phenotype. Whilst thousands of experimentally derived gene signatures are published, their potential value to the community is limited by their computational inaccessibility. Gene signatures are embedded in published article figures, tables or in supplementary materials, and are frequently presented using non-standard gene or probeset nomenclature. We present GeneSigDB (http://compbio.dfci.harvard.edu/genesigdb) a manually curated database of gene expression signatures. GeneSigDB release 1.0 focuses on cancer and stem cells gene signatures and was constructed from more than 850 publications from which we manually transcribed 575 gene signatures. Most gene signatures (n = 560) were successfully mapped to the genome to extract standardized lists of EnsEMBL gene identifiers. GeneSigDB provides the original gene signature, the standardized gene list and a fully traceable gene mapping history for each gene from the original transcribed data table through to the standardized list of genes. The GeneSigDB web portal is easy to search, allows users to compare their own gene list to those in the database, and download gene signatures in most common gene identifier formats. PMID:19934259

The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

PubMed Central

Broadbent, Andrew J.; Santos, Celia P.; Godbout, Rachel A.

2014-01-01

ABSTRACT Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (≥100-fold) replication at 39°C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple-reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine viruses. The VN04ts sig and CA07ts sig viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39°C, but the replication of WSNts sig was restricted ≥100-fold compared to that at 33°C. Reassortant CA07ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication ≥100-fold at 39°C. In ferrets, the CA07ts sig virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. IMPORTANCE Live attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our

The temperature-sensitive and attenuation phenotypes conferred by mutations in the influenza virus PB2, PB1, and NP genes are influenced by the species of origin of the PB2 gene in reassortant viruses derived from influenza A/California/07/2009 and A/WSN/33 viruses.

PubMed

Broadbent, Andrew J; Santos, Celia P; Godbout, Rachel A; Subbarao, Kanta

2014-11-01

Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (≥ 100-fold) replication at 39 °C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391 E, 581 G, and 661 T), PB2 (265 S), and NP (34 G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple-reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine viruses. The VN04(ts sig) and CA07(ts sig) viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39 °C, but the replication of WSN(ts sig) was restricted ≥ 100-fold compared to that at 33 °C. Reassortant CA07(ts sig) viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication ≥ 100-fold at 39 °C. In ferrets, the CA07(ts sig) virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07(ts sig) virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. Live attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

PubMed

Seoane-Zonjic, Pedro; Cañas, Rafael A; Bautista, Rocío; Gómez-Maldonado, Josefa; Arrillaga, Isabel; Fernández-Pozo, Noé; Claros, M Gonzalo; Cánovas, Francisco M; Ávila, Concepción

2016-02-27

In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a

Benzoylsalicylic acid derivatives as defense activators in tobacco and Arabidopsis.

PubMed

Kamatham, Samuel; Pallu, Reddanna; Pasupulati, Anil Kumar; Singh, Surya Satyanarayana; Gudipalli, Padmaja

2017-11-01

Systemic acquired resistance (SAR) is a long lasting inducible whole plant immunity often induced by either pathogens or chemical elicitors. Salicylic acid (SA) is a known SAR signal against a broad spectrum of pathogens in plants. In a recent study, we have reported that benzoylsalicylic acid (BzSA) is a SAR inducer in tobacco and Arabidopsis plants. Here, we have synthesized BzSA derivatives using SA and benzoyl chlorides of various moieties as substrates. The chemical structures of BzSA derivatives were elucidated using Infrared spectroscopy (IR), Nuclear magnetic spectroscopy (NMR) and High-resolution mass spectrometer (HRMS) analysis. The bioefficacy of BzSA derivatives in inducing defense response against tobacco mosaic virus (TMV) was investigated in tobacco and SA abolished transgenic NahG Arabidopsis plants. Interestingly, pre-treatment of local leaves of tobacco with BzSA derivatives enhanced the expression of SAR genes such as NPR1 [Non-expressor of pathogenesis-related (PR) genes 1], PR and other defense marker genes (HSR203, SIPK, WIPK) in systemic leaves. Pre-treatment of BzSA derivatives reduced the spread of TMV infection to uninfected areas by restricting lesion number and diameter both in local and systemic leaves of tobacco in a dose-dependent manner. Furthermore, pre-treatment of BzSA derivatives in local leaves of SA deficient Arabidopsis NahG plants induced SAR through AtPR1 and AtPR5 gene expression and reduced leaf necrosis and curling symptoms in systemic leaves as compared to BzSA. These results suggest that BzSA derivatives are potent SAR inducers against TMV in tobacco and Arabidopsis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Experimentally-Derived Fibroblast Gene Signatures Identify Molecular Pathways Associated with Distinct Subsets of Systemic Sclerosis Patients in Three Independent Cohorts

PubMed Central

Johnson, Michael E.; Mahoney, J. Matthew; Taroni, Jaclyn; Sargent, Jennifer L.; Marmarelis, Eleni; Wu, Ming-Ru; Varga, John; Hinchcliff, Monique E.; Whitfield, Michael L.

2015-01-01

Genome-wide expression profiling in systemic sclerosis (SSc) has identified four ‘intrinsic’ subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. Here we examine experimentally derived gene expression signatures in dermal fibroblasts for thirteen different signaling pathways implicated in SSc pathogenesis. These data show distinct and overlapping sets of genes induced by each pathway, allowing for a better understanding of the molecular relationship between profibrotic and immune signaling networks. Pathway-specific gene signatures were analyzed across a compendium of microarray datasets consisting of skin biopsies from three independent cohorts representing 80 SSc patients, 4 morphea, and 26 controls. IFNα signaling showed a strong association with early disease, while TGFβ signaling spanned the fibroproliferative and inflammatory subsets, was associated with worse MRSS, and was higher in lesional than non-lesional skin. The fibroproliferative subset was most strongly associated with PDGF signaling, while the inflammatory subset demonstrated strong activation of innate immune pathways including TLR signaling upstream of NF-κB. The limited and normal-like subsets did not show associations with fibrotic and inflammatory mediators such as TGFβ and TNFα. The normal-like subset showed high expression of genes associated with lipid signaling, which was absent in the inflammatory and limited subsets. Together, these data suggest a model by which IFNα is involved in early disease pathology, and disease severity is associated with active TGFβ signaling. PMID:25607805

EnviroAtlas -- Memphis, TN (2012) -- One Meter Resolution Urban Land Cover Data Web Service

EPA Pesticide Factsheets

This EnviroAtlas web service supports research and online mapping activities related to EnviroAtlas (https://www.epa.gov/enviroatlas ). The Memphis, TN EnviroAtlas One Meter-scale Urban Land Cover (MULC) dataset comprises 2,733 km2 around the city of Memphis, surrounding towns, and rural areas. These leaf-on LC data and maps were derived from 1-m pixel, four-band (red, green, blue, and near-infrared) aerial photography acquired from the United States Department of Agriculture (USDA) National Agriculture Imagery Program (NAIP) on four dates in 2012: June 15, June 18, June 21 and June 23, and one date in 2013: July 12. Three separate LiDAR (Light Detection and Ranging) data sets collected on February 19, 2009 00e2?? August 2, 2010, December 1-2, 2011 and January 23-24, 2012 were integrated for Shelby Co., TN, Crittenden Co., AR, and DeSoto Co, MS. Five MULC classes were mapped directly from the NAIP and LiDAR data: Water, Impervious, Soil, Trees, and Grass/Herbaceous. Agriculture was derived from USDA Common Land Unit (CLU) data. Woody and emergent wetlands were copied from existing National Wetlands Inventory (NWI) data. Analysis of a random sampling of 612 photo-interpreted land cover reference points yielded an overall users accuracy of 86.9%. This dataset was produced by the US EPA to support research and online mapping activities related to EnviroAtlas. EnviroAtlas (https://www.epa.gov/enviroatlas) allows the user to interact with a web-based, easy-to-u

EnviroAtlas -- Memphis, TN (2012) -- One Meter Resolution Urban Land Cover Data

EPA Pesticide Factsheets

The Memphis, TN EnviroAtlas One Meter-scale Urban Land Cover (MULC) dataset comprises 2,733 km2 around the city of Memphis, surrounding towns, and rural areas. These leaf-on LC data and maps were derived from 1-m pixel, four-band (red, green, blue, and near-infrared) aerial photography acquired from the United States Department of Agriculture (USDA) National Agriculture Imagery Program (NAIP) on four dates in 2012: June 15, June 18, June 21 and June 23, and one date in 2013: July 12. Three separate LiDAR (Light Detection and Ranging) data sets collected on February 19, 2009 00e2?? August 2, 2010, December 1-2, 2011 and January 23-24, 2012 were integrated for Shelby Co., TN, Crittenden Co., AR, and DeSoto Co, MS. Five MULC classes were mapped directly from the NAIP and LiDAR data: Water, Impervious, Soil, Trees, and Grass/Herbaceous. Agriculture was derived from USDA Common Land Unit (CLU) data. Woody and emergent wetlands were copied from existing National Wetlands Inventory (NWI) data. Analysis of a random sampling of 612 photo-interpreted land cover reference points yielded an overall users accuracy of 86.9%. This dataset was produced by the US EPA to support research and online mapping activities related to EnviroAtlas. EnviroAtlas (https://www.epa.gov/enviroatlas) allows the user to interact with a web-based, easy-to-use, mapping application to view and analyze multiple ecosystem services for the contiguous United States. The dataset is available as do

The important role of von Willebrand factor in platelet-derived FVIII gene therapy for murine hemophilia A in the presence of inhibitory antibodies.

PubMed

Shi, Q; Schroeder, J A; Kuether, E L; Montgomery, R R

2015-07-01

Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with von Willebrand factor (VWF) in platelet α-granules and that platelet-derived FVIII (2bF8) corrects the murine hemophilia A phenotype even in the presence of high-titer anti-FVIII inhibitory antibodies (inhibitors). To explore how VWF has an impact on platelet gene therapy for hemophilia A with inhibitors. 2bF8 transgenic mice in the FVIII(-/-) background (2bF8(tg+/-) F8(-/-) ) with varying VWF phenotypes were used in this study. Animals were analyzed by VWF ELISA, FVIII activity assay, Bethesda assay and tail clip survival test. Only 18% of 2bF8(tg+/-) F8(-/-) VWF(-/-) animals, in which VWF was deficient, survived the tail clip challenge with inhibitor titers of 3-8000 BU mL(-1) . In contrast, 82% of 2bF8(tg+/-) F8(-/-) VWF(+/+) mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10-50,000 BU mL(-1) . All 2bF8(tg+/-) F8(-/-) VWF(-/-) mice without inhibitors survived tail clipping and no VWF(-/-) F8(-/-) mice survived this challenge. Because VWF is synthesized by endothelial cells and megakaryocytes and is distributed in both plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF on platelet-FVIII gene therapy for hemophilia A with inhibitors. In the presence of inhibitors, 42% of animals survived tail clipping in the group with plasma-VWF and 50% survived in the platelet-VWF group. VWF is essential for platelet gene therapy for hemophilia A with inhibitors. Both platelet-VWF and plasma-VWF are required for optimal platelet-derived FVIII gene therapy for hemophilia A in the presence of inhibitors. © 2015 International Society on Thrombosis and Haemostasis.

Digital representation of oil and natural gas well pad scars in southwest Wyoming

USGS Publications Warehouse

Garman, Steven L.; McBeth, Jamie L.

2014-01-01

The recent proliferation of oil and natural gas energy development in southwest Wyoming has stimulated the need to understand wildlife responses to this development. Central to many wildlife assessments is the use of geospatial methods that rely on digital representation of energy infrastructure. Surface disturbance of the well pad scars associated with oil and natural gas extraction has been an important but unavailable infrastructure layer. To provide a digital baseline of this surface disturbance, we extracted visible oil and gas well pad scars from 1-meter National Agriculture Imagery Program imagery (NAIP) acquired in 2009 for a 7.7 million-hectare region of southwest Wyoming. Scars include the pad area where wellheads, pumps, and storage facilities reside, and the surrounding area that was scraped and denuded of vegetation during the establishment of the pad. Scars containing tanks, compressors, and the storage of oil and gas related equipment, and produced-water ponds were also collected on occasion. Our extraction method was a two-step process starting with automated extraction followed by manual inspection and clean up. We used available well-point information to guide manual clean up and to derive estimates of year of origin and duration of activity on a pad scar. We also derived estimates of the proportion of non-vegetated area on a scar using a Normalized Difference Vegetation Index derived using 1-meter NAIP imagery. We extracted 16,973 pad scars of which 15,318 were oil and gas well pads. Digital representation of pad scars along with time-stamps of activity and estimates of non-vegetated area provides important baseline (circa 2009) data for assessments of wildlife responses, land-use trends, and disturbance-mediated pattern assessments.

Microarray expression profiles of genes in lung tissues of rats subjected to focal cerebral ischemia-induced lung injury following bone marrow-derived mesenchymal stem cell transplantation.

PubMed

Hu, Yue; Xiong, Liu-Lin; Zhang, Piao; Wang, Ting-Hua

2017-01-01

Ischemia-induced stroke is the most common disease of the nervous system and is associated with a high mortality rate worldwide. Cerebral ischemia may lead to remote organ dysfunction, particular in the lungs, resulting in lung injury. Nowadays, bone marrow-derived mesenchymal stem cells (BMSCs) are widely studied in clinical trials as they may provide an effective solution to the treatment of neurological and cardiac diseases; however, the underlying molecular mechanisms remain unknown. In this study, a model of permanent focal cerebral ischemia-induced lung injury was successfully established and confirmed by neurological evaluation and lung injury scores. We demonstrated that the transplantation of BMSCs (passage 3) via the tail vein into the lung tissues attenuated lung injury. In order to elucidate the underlying molecular mechanisms, we analyzed the gene expression profiles in lung tissues from the rats with focal cerebral ischemia and transplanted with BMSCs using a Gene microarray. Moreover, the Gene Ontology database was employed to determine gene function. We found that the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) were downregulated in the BMSC transplantation groups, compared with the control group. These results suggested that BMSC transplantation may attenuate lung injury following focal cerebral ischemia and that this effect is associated with the downregulation of TGF-β, PDGF and the PI3K-AKT pathway.

Novel cancer gene variants and gene fusions of triple-negative breast cancers (TNBCs) reveal their molecular diversity conserved in the patient-derived xenograft (PDX) model.

PubMed

Jung, Jaeyun; Jang, Kiwon; Ju, Jung Min; Lee, Eunji; Lee, Jong Won; Kim, Hee Jung; Kim, Jisun; Lee, Sae Byul; Ko, Beom Seok; Son, Byung Ho; Lee, Hee Jin; Gong, Gyungyup; Ahn, Sei Yeon; Choi, Jung Kyoon; Singh, Shree Ram; Chang, Suhwan

2018-08-01

Despite the improved 5-year survival rate of breast cancer, triple-negative breast cancer (TNBC) remains a challenge due to lack of effective targeted therapy and higher recurrence and metastasis than other subtypes. To identify novel druggable targets and to understand its unique biology, we tried to implement 24 patient-derived xenografts (PDXs) of TNBC. The overall success rate of PDX implantation was 45%, much higher than estrogen receptor (ER)-positive cases. Immunohistochemical analysis revealed conserved ER/PR/Her2 negativity (with two exceptions) between the original and PDX tumors. Genomic analysis of 10 primary tumor-PDX pairs with Ion AmpliSeq CCP revealed high degree of variant conservation (85.0%-96.9%) between primary and PDXs. Further analysis showed 44 rare variants with a predicted high impact in 36 genes including Trp53, Pten, Notch1, and Col1a1. Among them, we confirmed frequent Notch1 variant. Furthermore, RNA-seq analysis of 24 PDXs revealed 594 gene fusions, of which 163 were in-frame, including AZGP1-GJC3 and NF1-AARSD1. Finally, western blot analysis of oncogenic signaling proteins supporting molecular diversity of TNBC PDXs. Overall, our report provides a molecular basis for the usefulness of the TNBC PDX model in preclinical study. Published by Elsevier B.V.

Comparative Transcriptome Analyses Reveal Core Parasitism Genes and Suggest Gene Duplication and Repurposing as Sources of Structural Novelty

PubMed Central

Yang, Zhenzhen; Wafula, Eric K.; Honaas, Loren A.; Zhang, Huiting; Das, Malay; Fernandez-Aparicio, Monica; Huang, Kan; Bandaranayake, Pradeepa C.G.; Wu, Biao; Der, Joshua P.; Clarke, Christopher R.; Ralph, Paula E.; Landherr, Lena; Altman, Naomi S.; Timko, Michael P.; Yoder, John I.; Westwood, James H.; dePamphilis, Claude W.

2015-01-01

The origin of novel traits is recognized as an important process underlying many major evolutionary radiations. We studied the genetic basis for the evolution of haustoria, the novel feeding organs of parasitic flowering plants, using comparative transcriptome sequencing in three species of Orobanchaceae. Around 180 genes are upregulated during haustorial development following host attachment in at least two species, and these are enriched in proteases, cell wall modifying enzymes, and extracellular secretion proteins. Additionally, about 100 shared genes are upregulated in response to haustorium inducing factors prior to host attachment. Collectively, we refer to these newly identified genes as putative “parasitism genes.” Most of these parasitism genes are derived from gene duplications in a common ancestor of Orobanchaceae and Mimulus guttatus, a related nonparasitic plant. Additionally, the signature of relaxed purifying selection and/or adaptive evolution at specific sites was detected in many haustorial genes, and may play an important role in parasite evolution. Comparative analysis of gene expression patterns in parasitic and nonparasitic angiosperms suggests that parasitism genes are derived primarily from root and floral tissues, but with some genes co-opted from other tissues. Gene duplication, often taking place in a nonparasitic ancestor of Orobanchaceae, followed by regulatory neofunctionalization, was an important process in the origin of parasitic haustoria. PMID:25534030

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology

PubMed Central

Ahn, So Yoon; Sung, Se In; Yoo, Hye Soo; Choi, Soo Jin; Kim, Soo Yoon; Park, Won Soon

2015-01-01

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (IT) versus intravenous (IV) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the IT (5×105) or IV (2×106) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both IT and IV transplantations. However, IT administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to IV administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the IT group, but not in the IV group. Although the IT group received only one fourth of the number of MSCs that the IV group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the IT group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the IT group, but not in the IV group. Thus, local IT MSC transplantation was more effective

Characterization of glycol chitosan grafted with low molecular weight polyethylenimine as a gene carrier for human adipose-derived mesenchymal stem cells.

PubMed

Bae, Yoonhee; Lee, Young Hwa; Lee, Sunray; Han, Jin; Ko, Kyung Soo; Choi, Joon Sig

2016-11-20

Mesenchymal stem cells (MSCs) have a great capacity for self-renewal while still maintaining their multipotency, and can differentiate into a variety of cell types. The delivery of genes to a site of injury is a current and interesting field of gene therapy. In the present study, we describe a nonviral gene delivery carrier, glycol chitosan-methyl acrylate-polyethylenimine (GMP) polymer targeted towards human adipose-derived mesenchymal stem cells (AD-MSCs). Transfection efficiency, using luciferase (Luc) and a pDNA encoding enhanced green fluorescent protein (EGFP), along with cytotoxicity assays, were performed in human AD-MSCs. The results show that the transfection efficiency of the GMP polymer was similar to that of PEI25kD, and the cytotoxicity was lower. Moreover, human AD-MSCs were treated with the GMP polymer/pDNA polyplex and its cellular uptake and distribution were analyzed by flow cytometry and confocal microscopy. Furthermore, we performed endosomal escape analysis using LysoTracker Red, and found that the conjugated GMP polymer could escape from the endosome to the cytosol. Human AD-MSCs treated with the GMP polymer maintained their potential for osteogenic differentiation and phenotypic expression of human AD-MSCs based on flow cytometry analysis. The present study demonstrates that the GMP polymer can be used as a potential targeted-delivery carrier for effective gene delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

IDENTIFICATION OF BIOLOGICALLY RELEVANT GENES USING A DATABASE OF RAT LIVER AND KIDNEY BASELINE GENE EXPRESSION

EPA Science Inventory

Microarray data from independent labs and studies can be compared to potentially identify toxicologically and biologically relevant genes. The Baseline Animal Database working group of HESI was formed to assess baseline gene expression from microarray data derived from control or...

Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

DOEpatents

Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

2015-12-08

This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

X-linked gene expression in the Virginia opossum: differences between the paternally derived Gpd and Pgk-A loci

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samollow, P.B.; Ford, A.L.; VandeBerg, J.L.

1987-01-01

Expression of X-linked glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase-A (PGK-A) in the Virginia opossum (Didelphis virginiana) was studied electrophoretically in animals from natural populations and those produced through controlled laboratory crosses. Blood from most of the wild animals exhibited a common single-banded phenotype for both enzymes. Rare variant animals, regardless of sex, exhibited single-banded phenotypes different in mobility from the common mobility class of the respective enzyme. The laboratory crosses confirmed the allelic basis for the common and rare phenotypes. Transmission of PGK-A phenotypes followed the pattern of determinate (nonrandom) inactivation of the paternally derived Pgk-A allele, and transmission ofmore » G6PD also was consistent with this pattern. A survey of tissue-specific expression of G6PD phenotypes of heterozygous females revealed, in almost all tissues, three-banded patterns skewed in favor of the allele that was expressed in blood cells. Three-banded patterns were never observed in males or in putatively homozygous females. These patterns suggest simultaneous, but unequal, expression of the maternally and paternally derived Gpd alleles within individual cells. The absence of such partial expression was noted in a parallel survey of females heterozygous at the Pgd-A locus. Thus, it appears that Gpd and Pgk-A are X-linked in D. virginiana and subject to preferential paternal allele inactivation, but that dosage compensation may not be complete for all paternally derived X-linked genes.« less

TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation.

PubMed Central

Taylor, J P; Pomerantz, R; Bagasra, O; Chowdhury, M; Rappaport, J; Khalili, K; Amini, S

1992-01-01

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1505523

MR Imaging-derived Oxygen Metabolism and Neovascularization Characterization for Grading and IDH Gene Mutation Detection of Gliomas.

PubMed

Stadlbauer, Andreas; Zimmermann, Max; Kitzwögerer, Melitta; Oberndorfer, Stefan; Rössler, Karl; Dörfler, Arnd; Buchfelder, Michael; Heinz, Gertraud

2017-06-01

Purpose To explore the diagnostic performance of physiological magnetic resonance (MR) imaging of oxygen metabolism and neovascularization activity for grading and characterization of isocitrate dehydrogenase (IDH) gene mutation status of gliomas. Materials and Methods This retrospective study had institutional review board approval; written informed consent was obtained from all patients. Eighty-three patients with histopathologically proven glioma (World Health Organization [WHO] grade II-IV) were examined with quantitative blood oxygen level-dependent imaging and vascular architecture mapping. Biomarker maps of neovascularization activity (microvessel radius, microvessel density, and microvessel type indicator [MTI]) and oxygen metabolism (oxygen extraction fraction [OEF] and cerebral metabolic rate of oxygen [CMRO 2 ]) were calculated. Receiver operating characteristic analysis was used to determine diagnostic performance for grading and detection of IDH gene mutation status. Results Low-grade (WHO grade II) glioma showed areas with increased OEF (+18%, P < .001, n = 20), whereas anaplastic glioma (WHO grade III) and glioblastoma (WHO grade IV) showed decreased OEF when compared with normal brain tissue (-54% [P < .001, n = 21] and -49% [P < .001, n = 41], respectively). This allowed clear differentiation between low- and high-grade glioma (area under the receiver operating characteristic curve [AUC], 1) for the patient cohort. MTI had the highest diagnostic performance (AUC, 0.782) for differentiation between gliomas of grades III and IV among all biomarkers. CMRO 2 was decreased (P = .037) in low-grade glioma with a mutated IDH gene, and MTI was significantly increased in glioma grade III with IDH mutation (P = .013) when compared with the IDH wild-type counterparts. CMRO 2 showed the highest diagnostic performance for IDH gene mutation detection in low-grade glioma (AUC, 0.818) and MTI in high-grade glioma (AUC, 0.854) and for all WHO grades (AUC, 0

Platelet-derived Growth Factor-mediated Induction of the Synaptic Plasticity Gene Arc/Arg3.1*

PubMed Central

Peng, Fuwang; Yao, Honghong; Bai, Xuetao; Zhu, Xuhui; Reiner, Benjamin C.; Beazely, Michael; Funa, Keiko; Xiong, Huangui; Buch, Shilpa

2010-01-01

Platelet-derived growth factor (PDGF) is a pleiotropic protein with critical roles in both developmental as well as pathogenic processes. In the central nervous system specifically, PDGF is critical for neuronal proliferation and differentiation and has also been implicated as a neuroprotective agent. Whether PDGF also plays a role in synaptic plasticity, however, remains poorly understood. In the present study we demonstrated that in the rat hippocampal neurons PDGF regulated the expression of Arc/Arg3.1 gene that has been implicated in both synapse plasticity and long term potentiation. Relevance of these findings was further confirmed in vivo by injecting mice with intracerebral inoculations of PDGF, which resulted in a rapid induction of Arc in the hippocampus of the injected mice. PDGF induced long term potentiation in rat hippocampal slices, which was abolished by PDGF receptor-tyrosine kinase inhibitor STI-571. We also present evidence that PDGF-mediated induction of Arc/Arg3.1 involved activation of the MAPK/ERK (MEK) pathway. Additionally, induction of Arc/Arg3.1 also involved the upstream release of intracellular calcium stores, an effect that could be blocked by thapsigargin but not by EGTA. Pharmacological approach using inhibitors specific for either MAPK/ERK phosphorylation or calcium release demonstrated that the two pathways converged downstream at a common point involving activation of the immediate early gene Egr-1. Chromatin immunoprecipitation assays demonstrated the binding of Egr-1, but not Egr-3, to the Arc promoter. These findings for the first time, thus, suggest an additional role of PDGF, that of induction of Arc. PMID:20452974

Gene expression profiling analysis of the effects of low-intensity pulsed ultrasound on induced pluripotent stem cell-derived neural crest stem cells.

PubMed

Xia, Bin; Zou, Yang; Xu, Zhiling; Lv, Yonggang

2017-11-01

Low-intensity pulsed ultrasound (LIPUS) is a noninvasive technique that has been shown to affect cell proliferation, migration, and differentiation and promote the regeneration of damaged peripheral nerve. Our previous studies had proved that LIPUS can significantly promote the neural differentiation of induced pluripotent stem cell-derived neural crest stem cells (iPSCs-NCSCs) and enhance the repair of rat-transected sciatic nerve. To further explore the underlying mechanisms of LIPUS treatment of iPSCs-NCSCs, this study reported the gene expression profiling analysis of iPSCs-NCSCs before and after LIPUS treatment using the RNA-sequencing (RNA-Seq) method. It was found that expression of 76 genes of iPSCs-NCSCs cultured in a serum-free neural induction medium and expression of 21 genes of iPSCs-NCSCs cultured in a neuronal differentiation medium were significantly changed by LIPUS treatment. The differentially expressed genes are related to angiogenesis, nervous system activity and functions, cell activities, and so on. The RNA-seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). High correlation was observed between the results obtained from qRT-PCR and RNA-Seq. This study presented new information on the global gene expression patterns of iPSCs-NCSCs after LIPUS treatment and may expand the understanding of the complex molecular mechanism of LIPUS treatment of iPSCs-NCSCs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells

PubMed Central

Bavassano, Carlo; Eigentler, Andreas; Stanika, Ruslan; Obermair, Gerald J.; Boesch, Sylvia; Dechant, Georg

2017-01-01

Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the α1A subunit of the P/Q type voltage-gated Ca2+ channel CaV2.1 but also the α1ACT protein, a 75 kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the α1A subunit. Isoforms of both proteins contain the polyglutamine (polyQ) domain that is expanded in SCA6 patients. Although certain SCA6 phenotypes appear to be specific for Purkinje neurons, other pathogenic effects of the SCA6 polyQ mutation can affect a broad spectrum of central nervous system (CNS) neuronal subtypes. We investigated the expression and function of CACNA1A gene products in human neurons derived from induced pluripotent stem cells from two SCA6 patients. Expression levels of CACNA1A encoding α1A subunit were similar between SCA6 and control neurons, and no differences were found in the subcellular distribution of CaV2.1 channel protein. The α1ACT immunoreactivity was detected in the majority of cell nuclei of SCA6 and control neurons. Although no SCA6 genotype-dependent differences in CaV2.1 channel function were observed, they were found in the expression levels of the α1ACT target gene Granulin (GRN) and in glutamate-induced cell vulnerability. PMID:28946818

Gene-gene interactions and gene polymorphisms of VEGFA and EG-VEGF gene systems in recurrent pregnancy loss.

PubMed

Su, Mei-Tsz; Lin, Sheng-Hsiang; Chen, Yi-Chi; Kuo, Pao-Lin

2014-06-01

Both vascular endothelial growth factor A (VEGFA) and endocrine gland-derived vascular endothelial growth factor (EG-VEGF) systems play major roles in angiogenesis. A body of evidence suggests VEGFs regulate critical processes during pregnancy and have been associated with recurrent pregnancy loss (RPL). However, little information is available regarding the interaction of these two major major angiogenesis-related systems in early human pregnancy. This study was conducted to investigate the association of gene polymorphisms and gene-gene interaction among genes in VEGFA and EG-VEGF systems and idiopathic RPL. A total of 98 women with history of idiopathic RPL and 142 controls were included, and 5 functional SNPs selected from VEGFA, KDR, EG-VEGF (PROK1), PROKR1 and PROKR2 were genotyped. We used multifactor dimensionality reduction (MDR) analysis to choose a best model and evaluate gene-gene interactions. Ingenuity pathways analysis (IPA) was introduced to explore possible complex interactions. Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL (P<0.01). The MDR test revealed that the KDR (Q472H) polymorphism was the best loci to be associated with RPL (P=0.02). IPA revealed EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3 signaling pathways. Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL. EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3.

[Detection of bcr/abl fusion gene and its derivative chromosome 9 deletions in CML by using home-made bcr/abl extra-signal probe].

PubMed

Lai, Yue-Yun; Feng, Lin; Wang, Zheng; Lü, Shan; Dang, Hui; Shi, Yan; He, Qi; Huang, Xiao-Jun

2010-02-01

This study was aimed to verify the efficacy of home-made LSI bcr/abl ES probe for detection of bcr/abl fusion gene and derivative chromosome 9 deletions in chronic myeloid leukemia (CML). Fluorescence in situ hybridization (FISH) was carried out with dual color bcr/abl extra signal (ES) probe in 97 cases of CML based on morphology and cytogenetic karyotype and 129 cases of non-hematological malignancies/non-myeloproliferative diseases with normal cytogenetic karyotype. For the patients with signals of 1R1G1F indicating der(9) deletions, FISH were done using ASS DNA probe. The results showed that 91 cases with standard t(9;22) and 6 cases with variant translocation of t(9;22) were detected by conventional G banding technique. All of the 97 patients displayed bcr/abl fusion gene by ES-FISH, including 16 cases with signal patterns of 1R1G1F showing der(9) deletions. Among the 16 cases with der(9) deletions, 13 cases were detected to have deletions of ASS gene. Meanwhile, none of the 129 cases of negative control showed bcr/abl fusion gene by ES-FISH. It is concluded that home-made LSI bcr/abl ES probe is effective to identify the bcr/abl fusion gene and der(9) deletions in CML, and the ES-FISH results are consistent with conventional cytogenetic karyotype.

Rooting gene trees without outgroups: EP rooting.

PubMed

Sinsheimer, Janet S; Little, Roderick J A; Lake, James A

2012-01-01

Gene sequences are routinely used to determine the topologies of unrooted phylogenetic trees, but many of the most important questions in evolution require knowing both the topologies and the roots of trees. However, general algorithms for calculating rooted trees from gene and genomic sequences in the absence of gene paralogs are few. Using the principles of evolutionary parsimony (EP) (Lake JA. 1987a. A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony. Mol Biol Evol. 4:167-181) and its extensions (Cavender, J. 1989. Mechanized derivation of linear invariants. Mol Biol Evol. 6:301-316; Nguyen T, Speed TP. 1992. A derivation of all linear invariants for a nonbalanced transversion model. J Mol Evol. 35:60-76), we explicitly enumerate all linear invariants that solely contain rooting information and derive algorithms for rooting gene trees directly from gene and genomic sequences. These new EP linear rooting invariants allow one to determine rooted trees, even in the complete absence of outgroups and gene paralogs. EP rooting invariants are explicitly derived for three taxon trees, and rules for their extension to four or more taxa are provided. The method is demonstrated using 18S ribosomal DNA to illustrate how the new animal phylogeny (Aguinaldo AMA et al. 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387:489-493; Lake JA. 1990. Origin of the metazoa. Proc Natl Acad Sci USA 87:763-766) may be rooted directly from sequences, even when they are short and paralogs are unavailable. These results are consistent with the current root (Philippe H et al. 2011. Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 470:255-260).

Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling

PubMed Central

Angelastro, James M.; Klimaschewski, Lars; Tang, Song; Vitolo, Ottavio V.; Weissman, Tamily A.; Donlin, Laura T.; Shelanski, Michael L.; Greene, Lloyd A.

2000-01-01

Neurotrophic factors such as nerve growth factor (NGF) promote a wide variety of responses in neurons, including differentiation, survival, plasticity, and repair. Such actions often require changes in gene expression. To identify the regulated genes and thereby to more fully understand the NGF mechanism, we carried out serial analysis of gene expression (SAGE) profiling of transcripts derived from rat PC12 cells before and after NGF-promoted neuronal differentiation. Multiple criteria supported the reliability of the profile. Approximately 157,000 SAGE tags were analyzed, representing at least 21,000 unique transcripts. Of these, nearly 800 were regulated by 6-fold or more in response to NGF. Approximately 150 of the regulated transcripts have been matched to named genes, the majority of which were not previously known to be NGF-responsive. Functional categorization of the regulated genes provides insight into the complex, integrated mechanism by which NGF promotes its multiple actions. It is anticipated that as genomic sequence information accrues the data derived here will continue to provide information about neurotrophic factor mechanisms. PMID:10984536

The association between brain-derived neurotrophic factor gene polymorphism and migraine: a meta-analysis.

PubMed

Cai, Xiaoying; Shi, Xiaolei; Zhang, Ximeng; Zhang, Aiwu; Zheng, Minying; Fang, Yannan

2017-12-01

Migraine is a recurrent headache disease related to genetic variants. The brain-derived neurotrophic factor (BDNF) gene rs6265 (Val66Met) and rs2049046 polymorphism has been found to be associated with migraine. However, their roles in this disorder are not well established. Then we conduct this meta-analysis to address this issue. PubMed, Web of Science and Cochrane databases were systematically searched to identify all relevant studies. Odds ratio (OR) with corresponding 95% confidence interval (CI) was used to estimate the strength of association between BDNF gene rs6265 and rs2049046 polymorphism and migraine. Four studies with 1598 cases and 1585 controls, fulfilling the inclusion criteria were included in our meta-analysis. Overall data showed significant association between rs6265 polymorphism and migraine in allele model (OR = 0.86, 95%CI: 0.76-0.99, p = 0.03), recessive model (OR = 0.84, 95%CI: 0.72-0.98, p = 0.03) and additive model (GG vs GA: OR = 0.85, 95%CI: 0.72-1.00, p = 0.04), respectively. We also found significant association between rs2049046(A/T) polymorphism and migraine in allele model (OR = 0.88, 95%CI: 0.79-0.98, p = 0.02), recessive model (OR = 0.80, 95%CI: 0.67-0.96, p = 0.02) and additive model (AA vs TT: OR = 0.72, 95%CI: 0.57-0.92, p = 0.008; AA vs AT: OR = 0.81, 95%CI: 0.67-0.99, p = 0.03), respectively. Our meta-analysis suggested that BDNF rs6265 and rs2049046 polymorphism were associated with common migraine in Caucasian population. Further studies are awaited to update this finding in Asian population and other types of migraine.

The managed clearing: An overlooked land-cover type in urbanizing regions?

PubMed Central

Madden, Marguerite; Gray, Josh; Meentemeyer, Ross K.

2018-01-01

Urban ecosystem assessments increasingly rely on widely available map products, such as the U.S. Geological Service (USGS) National Land Cover Database (NLCD), and datasets that use generic classification schemes to detect and model large-scale impacts of land-cover change. However, utilizing existing map products or schemes without identifying relevant urban class types such as semi-natural, yet managed land areas that account for differences in ecological functions due to their pervious surfaces may severely constrain assessments. To address this gap, we introduce the managed clearings land-cover type–semi-natural, vegetated land surfaces with varying degrees of management practices–for urbanizing landscapes. We explore the extent to which managed clearings are common and spatially distributed in three rapidly urbanizing areas of the Charlanta megaregion, USA. We visually interpreted and mapped fine-scale land cover with special attention to managed clearings using 2012 U.S. Department of Agriculture (USDA) National Agriculture Imagery Program (NAIP) images within 150 randomly selected 1-km2 blocks in the cities of Atlanta, Charlotte, and Raleigh, and compared our maps with National Land Cover Database (NLCD) data. We estimated the abundance of managed clearings relative to other land use and land cover types, and the proportion of land-cover types in the NLCD that are similar to managed clearings. Our study reveals that managed clearings are the most common land cover type in these cities, covering 28% of the total sampled land area– 6.2% higher than the total area of impervious surfaces. Managed clearings, when combined with forest cover, constitutes 69% of pervious surfaces in the sampled region. We observed variability in area estimates of managed clearings between the NAIP-derived and NLCD data. This suggests using high-resolution remote sensing imagery (e.g., NAIP) instead of modifying NLCD data for improved representation of spatial heterogeneity and

The managed clearing: An overlooked land-cover type in urbanizing regions?

PubMed

Singh, Kunwar K; Madden, Marguerite; Gray, Josh; Meentemeyer, Ross K

2018-01-01

Urban ecosystem assessments increasingly rely on widely available map products, such as the U.S. Geological Service (USGS) National Land Cover Database (NLCD), and datasets that use generic classification schemes to detect and model large-scale impacts of land-cover change. However, utilizing existing map products or schemes without identifying relevant urban class types such as semi-natural, yet managed land areas that account for differences in ecological functions due to their pervious surfaces may severely constrain assessments. To address this gap, we introduce the managed clearings land-cover type-semi-natural, vegetated land surfaces with varying degrees of management practices-for urbanizing landscapes. We explore the extent to which managed clearings are common and spatially distributed in three rapidly urbanizing areas of the Charlanta megaregion, USA. We visually interpreted and mapped fine-scale land cover with special attention to managed clearings using 2012 U.S. Department of Agriculture (USDA) National Agriculture Imagery Program (NAIP) images within 150 randomly selected 1-km2 blocks in the cities of Atlanta, Charlotte, and Raleigh, and compared our maps with National Land Cover Database (NLCD) data. We estimated the abundance of managed clearings relative to other land use and land cover types, and the proportion of land-cover types in the NLCD that are similar to managed clearings. Our study reveals that managed clearings are the most common land cover type in these cities, covering 28% of the total sampled land area- 6.2% higher than the total area of impervious surfaces. Managed clearings, when combined with forest cover, constitutes 69% of pervious surfaces in the sampled region. We observed variability in area estimates of managed clearings between the NAIP-derived and NLCD data. This suggests using high-resolution remote sensing imagery (e.g., NAIP) instead of modifying NLCD data for improved representation of spatial heterogeneity and

Locations and attributes of wind turbines in Colorado, 2011

USGS Publications Warehouse

Carr, Natasha B.; Diffendorfer, James E.; Fancher, Tammy; Hawkins, Sarah J.; Latysh, Natalie; Leib, Kenneth J.; Matherne, Anne Marie

2013-01-01

This dataset represents an update to U.S. Geological Survey Data Series 597. Locations and attributes of wind turbines in Colorado, 2009 (available at http://pubs.usgs.gov/ds/597/). This updated Colorado wind turbine Data Series provides geospatial data for all 1,204 wind turbines established within the State of Colorado as of September 2011, an increase of 297 wind turbines from 2009. Attributes specific to each turbine include: turbine location, manufacturer and model, rotor diameter, hub height, rotor height, potential megawatt output, land ownership, county, and development status of the wind turbine. Wind energy facility data for each turbine include: facility name, facility power capacity, number of turbines associated with each facility to date, facility developer, facility ownership, and year the facility went online. The locations of turbines are derived from 1-meter true-color aerial photographs produced by the National Agriculture Imagery Program (NAIP); the photographs have a positional accuracy of about ±5 meters. Locations of turbines constructed during or prior to August 2009 are based on August 2009 NAIP imagery and turbine locations constructed after August 2009 were based on September 2011 NAIP imagery. The location of turbines under construction during September 2011 likely will be less accurate than the location of existing turbines. This data series contributes to an Online Interactive Energy Atlas developed by the U.S. Geological Survey (http://my.usgs.gov/eerma/). The Energy Atlas synthesizes data on existing and potential energy development in Colorado and New Mexico and includes additional natural resource data layers. This information may be used by decisionmakers to evaluate and compare the potential benefits and tradeoffs associated with different energy development strategies or scenarios. Interactive maps, downloadable data layers, comprehensive metadata, and decision-support tools also are included in the Energy Atlas. The format of

Locations and attributes of wind turbines in New Mexico, 2011

USGS Publications Warehouse

Carr, Natasha B.; Diffendorfer, James B.; Fancher, Tammy; Hawkins, Sarah J.; Latysh, Natalie; Leib, Kenneth J.; Matherne, Anne Marie

2013-01-01

This dataset represents an update to U.S. Geological Survey Data Series 596. Locations and attributes of wind turbines in New Mexico, 2009 (available at http://pubs.usgs.gov/ds/596/).This updated New Mexico wind turbine Data Series provides geospatial data for all 562 wind turbines established within the State of New Mexico as of June 2011, an increase of 155 wind turbines from 2009. Attributes specific to each turbine include: turbine location, manufacturer and model, rotor diameter, hub height, rotor height, potential megawatt output, land ownership, county, and development status of wind turbine. Wind energy facility data for each turbine include: facility name, facility power capacity, number of turbines associated with each facility to date, facility developer, facility ownership, and year the facility went online. The locations of turbines are derived from 1-meter true-color aerial photographs produced by the National Agriculture Imagery Program (NAIP); the photographs have a positional accuracy of about ±5 meters. The locations of turbines constructed during or prior to August 2009 are based on August 2009 NAIP imagery and turbine locations constructed after August 2009 were based June 2011 NAIP imagery. The location of turbines under construction during June 2011 likely will be less accurate than the location of existing turbines. This data series contributes to an Online Interactive Energy Atlas developed by the U.S. Geological Survey (http://my.usgs.gov/eerma/). The Energy Atlas synthesizes data on existing and potential energy development in Colorado and New Mexico and includes additional natural resource data layers. This information may be used by decisionmakers to evaluate and compare the potential benefits and tradeoffs associated with different energy development strategies or scenarios. Interactive maps, downloadable data layers, comprehensive metadata, and decision-support tools also are included in the Energy Atlas. The format of the Energy

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

PubMed

van den Eede, G; Aarts, H; Buhk, H-J; Corthier, G; Flint, H J; Hammes, W; Jacobsen, B; Midtvedt, T; van der Vossen, J; von Wright, A; Wackernagel, W; Wilcks, A

2004-07-01

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms. Copryright 2004 Elsevier Ltd.

Gene expression profiling for human iPS-derived motor neurons from sporadic ALS patients reveals a strong association between mitochondrial functions and neurodegeneration

PubMed Central

Alves, Chrystian J.; Dariolli, Rafael; Jorge, Frederico M.; Monteiro, Matheus R.; Maximino, Jessica R.; Martins, Roberto S.; Strauss, Bryan E.; Krieger, José E.; Callegaro, Dagoberto; Chadi, Gerson

2015-01-01

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that leads to widespread motor neuron death, general palsy and respiratory failure. The most prevalent sporadic ALS form is not genetically inherited. Attempts to translate therapeutic strategies have failed because the described mechanisms of disease are based on animal models carrying specific gene mutations and thus do not address sporadic ALS. In order to achieve a better approach to study the human disease, human induced pluripotent stem cell (hiPSC)-differentiated motor neurons were obtained from motor nerve fibroblasts of sporadic ALS and non-ALS subjects using the STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus system and submitted to microarray analyses using a whole human genome platform. DAVID analyses of differentially expressed genes identified molecular function and biological process-related genes through Gene Ontology. REVIGO highlighted the related functions mRNA and DNA binding, GTP binding, transcription (co)-repressor activity, lipoprotein receptor binding, synapse organization, intracellular transport, mitotic cell cycle and cell death. KEGG showed pathways associated with Parkinson's disease and oxidative phosphorylation, highlighting iron homeostasis, neurotrophic functions, endosomal trafficking and ERK signaling. The analysis of most dysregulated genes and those representative of the majority of categorized genes indicates a strong association between mitochondrial function and cellular processes possibly related to motor neuron degeneration. In conclusion, iPSC-derived motor neurons from motor nerve fibroblasts of sporadic ALS patients may recapitulate key mechanisms of neurodegeneration and may offer an opportunity for translational investigation of sporadic ALS. Large gene profiling of differentiated motor neurons from sporadic ALS patients highlights mitochondrial participation in the establishment of autonomous mechanisms associated with sporadic ALS

Testing Gene-Gene Interactions in the Case-Parents Design

PubMed Central

Yu, Zhaoxia

2011-01-01

The case-parents design has been widely used to detect genetic associations as it can prevent spurious association that could occur in population-based designs. When examining the effect of an individual genetic locus on a disease, logistic regressions developed by conditioning on parental genotypes provide complete protection from spurious association caused by population stratification. However, when testing gene-gene interactions, it is unknown whether conditional logistic regressions are still robust. Here we evaluate the robustness and efficiency of several gene-gene interaction tests that are derived from conditional logistic regressions. We found that in the presence of SNP genotype correlation due to population stratification or linkage disequilibrium, tests with incorrectly specified main-genetic-effect models can lead to inflated type I error rates. We also found that a test with fully flexible main genetic effects always maintains correct test size and its robustness can be achieved with negligible sacrifice of its power. When testing gene-gene interactions is the focus, the test allowing fully flexible main effects is recommended to be used. PMID:21778736

Plant-derived nanostructures: types and applications

EPA Science Inventory

Plant-derived nanostructures and nanoparticles (NPs) have functional applications in numerous disciplines such as health care, food and feed, cosmetics, biomedical science, energy science, drug-gene delivery, environmental health, and so on. Consequently, it is imperative for res...

Pediatric Glioblastoma Therapies Based on Patient-Derived Stem Cell Resources

DTIC Science & Technology

2013-10-01

stem cell lines have been successfully isolated from adults, in this proposal we aim to isolate and characterize GSC populations from pediatric patients. In the past two years we have successfully derived and cultured eight patient-derived pediatric glioma stem cell lines. In the past year we have continued molecular and phenotypic characterization of these lines. This characterization included analysis of gene expression and patient-specific gene mutations, and also proof-of-concept shRNA screens. In addition we have begun to identify candidate

PubNet: a flexible system for visualizing literature derived networks

PubMed Central

Douglas, Shawn M; Montelione, Gaetano T; Gerstein, Mark

2005-01-01

We have developed PubNet, a web-based tool that extracts several types of relationships returned by PubMed queries and maps them into networks, allowing for graphical visualization, textual navigation, and topological analysis. PubNet supports the creation of complex networks derived from the contents of individual citations, such as genes, proteins, Protein Data Bank (PDB) IDs, Medical Subject Headings (MeSH) terms, and authors. This feature allows one to, for example, examine a literature derived network of genes based on functional similarity. PMID:16168087

K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hattori, Yutaka; Odagiri, Hiroki; Nakatani, Hiroshi

1990-08-01

DNA fragments amplified in a stomach cancer-derived cell line, KATO-III, were previously identified by the in-gel DNA renaturation method, and a 0.2-kilobase-pair fragment of the amplified sequence was subsequently cloned. By genomic walking, a portion of the exon of the gene flanking this 0.2-kilobase-pair fragment was cloned, and the gene was designated as K-sam ({und K}ATO-III cell-derived {und s}tomach cancer {und am}plified gene). The K-sam cDNAs, corresponding to the 3.5-kilobase K-sam mRNA, were cloned from the KATO-III cells. Sequence analysis revealed that this gene coded for 682 amino acid residues that satisfied the characteristics of the receptor tyrosine kinase. Themore » K-sam gene had significant homologies with bek, FLG, and chicken basic fibroblast growth factor receptor gene. The K-sam gene was amplified in KATO-III cells with the major transcript of 3.5-kilobases in size. This gene was also expressed in some other stomach cancer cells, a small cell lung cancer, and germ cell tumors.« less

An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio.

PubMed

Parichy, D M; Ransom, D G; Paw, B; Zon, L I; Johnson, S L

2000-07-01

Developmental mechanisms underlying traits expressed in larval and adult vertebrates remain largely unknown. Pigment patterns of fishes provide an opportunity to identify genes and cell behaviors required for postembryonic morphogenesis and differentiation. In the zebrafish, Danio rerio, pigment patterns reflect the spatial arrangements of three classes of neural crest-derived pigment cells: black melanocytes, yellow xanthophores and silver iridophores. We show that the D. rerio pigment pattern mutant panther ablates xanthophores in embryos and adults and has defects in the development of the adult pattern of melanocyte stripes. We find that panther corresponds to an orthologue of the c-fms gene, which encodes a type III receptor tyrosine kinase and is the closest known homologue of the previously identified pigment pattern gene, kit. In mouse, fms is essential for the development of macrophage and osteoclast lineages and has not been implicated in neural crest or pigment cell development. In contrast, our analyses demonstrate that fms is expressed and required by D. rerio xanthophore precursors and that fms promotes the normal patterning of melanocyte death and migration during adult stripe formation. Finally, we show that fms is required for the appearance of a late developing, kit-independent subpopulation of adult melanocytes. These findings reveal an unexpected role for fms in pigment pattern development and demonstrate that parallel neural crest-derived pigment cell populations depend on the activities of two essentially paralogous genes, kit and fms.

Egg ovotransferrin-derived ACE inhibitory peptide IRW increases ACE2 but decreases proinflammatory genes expression in mesenteric artery of spontaneously hypertensive rats.

PubMed

Majumder, Kaustav; Liang, Guanxiang; Chen, Yanhong; Guan, LeLuo; Davidge, Sandra T; Wu, Jianping

2015-09-01

Egg ovotransferrin-derived angiotensin converting enzyme (ACE) inhibitory peptide IRW was previously shown to reduce blood pressure in spontaneously hypertensive rats through reduced vascular inflammation and increased nitric oxide-mediated vasorelaxation. The main objective of the present study was to investigate the molecular mechanism of this peptide through transcriptome analysis by RNAseq technique. Total RNA was extracted from kidney and mesenteric arteries; the RNAseq libraries (from untreated and IRW-treated groups) were constructed and subjected to sequence using HiSeq 2000 system (Illumina) system. A total of 12 764 and 13 352 genes were detected in kidney and mesenteric arteries, respectively. The differentially expressed (DE) genes between untreated and IRW-treated groups were identified and the functional analysis through ingenuity pathway analysis revealed a greater role of DE genes identified from mesenteric arteries than that of kidney in modulating various cardiovascular functions. Subsequent qPCR analysis further confirmed that IRW significantly increased the expression of ACE-2, ABCB-1, IRF-8, and CDH-1 while significantly decreased the expression ICAM-1 and VCAM-1 in mesenteric arteries. Our research showed for the first time that ACE inhibitory peptide IRW could contribute to its antihypertensive activity through increased ACE2 and decreased proinflammatory genes expression. © 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

PubMed

Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng

2017-01-01

The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

Prevalence of Antibiotic Resistance Genes among Human Gut-Derived Bifidobacteria.

PubMed

Duranti, Sabrina; Lugli, Gabriele Andrea; Mancabelli, Leonardo; Turroni, Francesca; Milani, Christian; Mangifesta, Marta; Ferrario, Chiara; Anzalone, Rosaria; Viappiani, Alice; van Sinderen, Douwe; Ventura, Marco

2017-02-01

The microbiota of the human gastrointestinal tract (GIT) may regularly be exposed to antibiotics, which are used to prevent and treat infectious diseases caused by bacteria and fungi. Bacterial communities of the gut retain a reservoir of antibiotic resistance (AR) genes, and antibiotic therapy thus positively selects for those microorganisms that harbor such genetic features, causing microbiota modulation. During the first months following birth, bifidobacteria represent some of the most dominant components of the human gut microbiota, although little is known about their AR gene complement (or resistome). In the current study, we assessed the resistome of the Bifidobacterium genus based on phenotypic and genotypic data of members that represent all currently recognized bifidobacterial (sub)species. Moreover, a comparison between the bifidobacterial resistome and gut metagenome data sets from adults and infants shows that the bifidobacterial community present at the first week following birth possesses a reduced AR arsenal compared to that present in the infant bifidobacterial population in subsequent weeks of the first year of life. Our findings reinforce the concept that the early infant gut microbiota is more susceptible to disturbances by antibiotic treatment than the gut microbiota developed at a later life stage. The spread of resistance to antibiotics among bacterial communities has represented a major concern since their discovery in the last century. The risk of genetic transfer of resistance genes between microorganisms has been extensively investigated due to its relevance to human health. In contrast, there is only limited information available on antibiotic resistance among human gut commensal microorganisms such as bifidobacteria, which are widely exploited by the food industry as health-promoting microorganisms or probiotic ingredients. In the current study, we explored the occurrence of antibiotic resistance genes in the genomes of bifidobacteria

Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

PubMed

Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

2006-08-01

Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

High-Throughput Screening Using iPSC-Derived Neuronal Progenitors to Identify Compounds Counteracting Epigenetic Gene Silencing in Fragile X Syndrome.

PubMed

Kaufmann, Markus; Schuffenhauer, Ansgar; Fruh, Isabelle; Klein, Jessica; Thiemeyer, Anke; Rigo, Pierre; Gomez-Mancilla, Baltazar; Heidinger-Millot, Valerie; Bouwmeester, Tewis; Schopfer, Ulrich; Mueller, Matthias; Fodor, Barna D; Cobos-Correa, Amanda

2015-10-01

Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused in most of cases by epigenetic silencing of the Fmr1 gene. Today, no specific therapy exists for FXS, and current treatments are only directed to improve behavioral symptoms. Neuronal progenitors derived from FXS patient induced pluripotent stem cells (iPSCs) represent a unique model to study the disease and develop assays for large-scale drug discovery screens since they conserve the Fmr1 gene silenced within the disease context. We have established a high-content imaging assay to run a large-scale phenotypic screen aimed to identify compounds that reactivate the silenced Fmr1 gene. A set of 50,000 compounds was tested, including modulators of several epigenetic targets. We describe an integrated drug discovery model comprising iPSC generation, culture scale-up, and quality control and screening with a very sensitive high-content imaging assay assisted by single-cell image analysis and multiparametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of fragile X mental retardation protein (FMRP) and thus sets the basis for further large-scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention. © 2015 Society for Laboratory Automation and Screening.

Rooting Gene Trees without Outgroups: EP Rooting

PubMed Central

Sinsheimer, Janet S.; Little, Roderick J. A.; Lake, James A.

2012-01-01

Gene sequences are routinely used to determine the topologies of unrooted phylogenetic trees, but many of the most important questions in evolution require knowing both the topologies and the roots of trees. However, general algorithms for calculating rooted trees from gene and genomic sequences in the absence of gene paralogs are few. Using the principles of evolutionary parsimony (EP) (Lake JA. 1987a. A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony. Mol Biol Evol. 4:167–181) and its extensions (Cavender, J. 1989. Mechanized derivation of linear invariants. Mol Biol Evol. 6:301–316; Nguyen T, Speed TP. 1992. A derivation of all linear invariants for a nonbalanced transversion model. J Mol Evol. 35:60–76), we explicitly enumerate all linear invariants that solely contain rooting information and derive algorithms for rooting gene trees directly from gene and genomic sequences. These new EP linear rooting invariants allow one to determine rooted trees, even in the complete absence of outgroups and gene paralogs. EP rooting invariants are explicitly derived for three taxon trees, and rules for their extension to four or more taxa are provided. The method is demonstrated using 18S ribosomal DNA to illustrate how the new animal phylogeny (Aguinaldo AMA et al. 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387:489–493; Lake JA. 1990. Origin of the metazoa. Proc Natl Acad Sci USA 87:763–766) may be rooted directly from sequences, even when they are short and paralogs are unavailable. These results are consistent with the current root (Philippe H et al. 2011. Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 470:255–260). PMID:22593551

Is the Val66Met polymorphism of the brain-derived neurotrophic factor gene associated with panic disorder? A meta-analysis.

PubMed

Chen, Kaiyuan; Wang, Na; Zhang, Jie; Hong, Xiaohong; Xu, Haiyun; Zhao, Xiaofeng; Huang, Qingjun

2017-06-01

Although emerging evidence has suggested an association between the Val66Met (rs6265) polymorphisms in brain-derived neurotrophic factor (BDNF) gene and the panic disorder, the conclusion is inclusive given the mixed results. This meta-analysis reviewed and analyzed the recent studies addressing the potential association between the Val66Met polymorphisms and panic disorder susceptibility. Related case-control studies were retrieved by database searching and selected according to established inclusion criteria. Six articles were identified, which explored the association between the BDNF Val66Met polymorphism and panic disorder. Statistical analyses revealed no association for the allele contrast and the dominant model. However, the recessive model showed a significant association between the BDNF Val66Met polymorphism and panic disorder (odds ratio = 1.26, 95% confidence interval = 1.04-1.52, z = 2.39, P = 0.02). Despite of some limitations, this meta-analysis suggests that the Val66Met polymorphism of BDNF gene is a susceptibility factor for panic disorder. © 2015 Wiley Publishing Asia Pty Ltd.

Observed parenting behaviors interact with a polymorphism of the brain-derived neurotrophic factor gene to predict the emergence of oppositional defiant and callous-unemotional behaviors at age 3 years.

PubMed

Willoughby, Michael T; Mills-Koonce, Roger; Propper, Cathi B; Waschbusch, Daniel A

2013-11-01

Using the Durham Child Health and Development Study, this study (N = 171) tested whether observed parenting behaviors in infancy (6 and 12 months) and toddlerhood/preschool (24 and 36 months) interacted with a child polymorphism of the brain-derived neurotrophic factor gene to predict oppositional defiant disorder (ODD) and callous-unemotional (CU) behaviors at age 3 years. Child genotype interacted with observed harsh and intrusive (but not sensitive) parenting to predict ODD and CU behaviors. Harsh-intrusive parenting was more strongly associated with ODD and CU for children with a methionine allele of the brain-derived neurotrophic factor gene. CU behaviors were uniquely predicted by harsh-intrusive parenting in infancy, whereas ODD behaviors were predicted by harsh-intrusive parenting in both infancy and toddlerhood/preschool. The results are discussed from the perspective of the contributions of caregiving behaviors as contributing to distinct aspects of early onset disruptive behavior.

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology.

PubMed

Sung, Dong Kyung; Chang, Yun Sil; Ahn, So Yoon; Sung, Se In; Yoo, Hye Soo; Choi, Soo Jin; Kim, Soo Yoon; Park, Won Soon

2015-01-01

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (i.t.) versus intravenous (i.v.) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the i.t. (5×10(5)) or i.v. (2×10(6)) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both i.t. and i.v. transplantations. However, i.t. administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to i.v. administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the i.t. group, but not in the i.v. group. Although the i.t. group received only one fourth of the number of MSCs that the i.v. group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the i.t. group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the i.t. group, but not in the i.v. group. Thus, local i.t. MSC

Mice Deficient for Glucagon Gene-Derived Peptides Display Normoglycemia and Hyperplasia of Islet α-Cells But Not of Intestinal L-Cells

PubMed Central

Hayashi, Yoshitaka; Yamamoto, Michiyo; Mizoguchi, Hiroyuki; Watanabe, Chika; Ito, Ryoichi; Yamamoto, Shiori; Sun, Xiao-yang; Murata, Yoshiharu

2009-01-01

Multiple bioactive peptides, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, are derived from the glucagon gene (Gcg). In the present study, we disrupted Gcg by introduction of GFP cDNA and established a knock-in mouse line. Gcggfp/gfp mice that lack most, if not all, of Gcg-derived peptides were born in an expected Mendelian ratio without gross abnormalities. Gcggfp/gfp mice showed lower blood glucose levels at 2 wk of age, but those in adult Gcggfp/gfp mice were not significantly different from those in Gcg+/+ and Gcggfp/+ mice, even after starvation for 16 h. Serum insulin levels in Gcggfp/gfp mice were lower than in Gcg+/+ and Gcggfp/+ on ad libitum feeding, but no significant differences were observed on starvation. Islet α-cells and intestinal L-cells were readily visualized in Gcggfp/gfp and Gcggfp/+ mice under fluorescence. The Gcggfp/gfp postnatally developed hyperplasia of islet α-cells, whereas the population of intestinal L-cells was not increased. In the Gcggfp/gfp, expression of Aristaless-related homeobox (Arx) was markedly increased in pancreas but not in intestine and suggested involvement of Arx in differential regulation of proliferation of Gcg-expressing cells. These results illustrated that Gcg-derived peptides are dispensable for survival and maintaining normoglycemia in adult mice and that Gcg-derived peptides differentially regulate proliferation/differentiation of α-cells and L-cells. The present model is useful for analyzing glucose/energy metabolism in the absence of Gcg-derived peptides. It is useful also for analysis of the development, differentiation, and function of Gcg-expressing cells, because such cells are readily visualized by fluorescence in this model. PMID:19819987

Primetime for Learning Genes.

PubMed

Keifer, Joyce

2017-02-11

Learning genes in mature neurons are uniquely suited to respond rapidly to specific environmental stimuli. Expression of individual learning genes, therefore, requires regulatory mechanisms that have the flexibility to respond with transcriptional activation or repression to select appropriate physiological and behavioral responses. Among the mechanisms that equip genes to respond adaptively are bivalent domains. These are specific histone modifications localized to gene promoters that are characteristic of both gene activation and repression, and have been studied primarily for developmental genes in embryonic stem cells. In this review, studies of the epigenetic regulation of learning genes in neurons, particularly the brain-derived neurotrophic factor gene ( BDNF ), by methylation/demethylation and chromatin modifications in the context of learning and memory will be highlighted. Because of the unique function of learning genes in the mature brain, it is proposed that bivalent domains are a characteristic feature of the chromatin landscape surrounding their promoters. This allows them to be "poised" for rapid response to activate or repress gene expression depending on environmental stimuli.

Gene-breaking: A new paradigm for human retrotransposon-mediated gene evolution

PubMed Central

Wheelan, Sarah J.; Aizawa, Yasunori; Han, Jeffrey S.; Boeke, Jef D.

2005-01-01

The L1 retrotransposon is the most highly successful autonomous retrotransposon in mammals. This prolific genome parasite may on occasion benefit its host through genome rearrangements or adjustments of host gene expression. In examining possible effects of L1 elements on host gene expression, we investigated whether a full-length L1 element inserted in the antisense orientation into an intron of a cellular gene may actually split the gene's transcript into two smaller transcripts: (1) a transcript containing the upstream exons and terminating in the major antisense polyadenylation site (MAPS) of the L1, and (2) a transcript derived from the L1 antisense promoter (ASP) that includes the downstream exons of the gene. Bioinformatic analysis and experimental follow-up provide evidence for this L1 “gene-breaking” hypothesis. We identified three human genes apparently “broken” by L1 elements, as well as 12 more candidate genes. Most of the inserted L1 elements in our 15 candidate genes predate the human/chimp divergence. If indeed split, the transcripts of these genes may in at least one case encode potentially interacting proteins, and in another case may encode novel proteins. Gene-breaking represents a new mechanism through which L1 elements remodel mammalian genomes. PMID:16024818

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

PubMed

Kao, Tim; Labonne, Tanya; Niclis, Jonathan C; Chaurasia, Ritu; Lokmic, Zerina; Qian, Elizabeth; Bruveris, Freya F; Howden, Sara E; Motazedian, Ali; Schiesser, Jacqueline V; Costa, Magdaline; Sourris, Koula; Ng, Elizabeth; Anderson, David; Giudice, Antonietta; Farlie, Peter; Cheung, Michael; Lamande, Shireen R; Penington, Anthony J; Parish, Clare L; Thomson, Lachlan H; Rafii, Arash; Elliott, David A; Elefanty, Andrew G; Stanley, Edouard G

2016-09-13

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Fine mapping of a quantitative resistance gene for gray leaf spot of maize (Zea mays L.) derived from teosinte (Z. mays ssp. parviglumis).

PubMed

Zhang, Xinye; Yang, Qin; Rucker, Elizabeth; Thomason, Wade; Balint-Kurti, Peter

2017-06-01

In this study we mapped the QTL Qgls8 for gray leaf spot (GLS) resistance in maize to a ~130 kb region on chromosome 8 including five predicted genes. In previous work, using near isogenic line (NIL) populations in which segments of the teosinte (Zea mays ssp. parviglumis) genome had been introgressed into the background of the maize line B73, we had identified a QTL on chromosome 8, here called Qgls8, for gray leaf spot (GLS) resistance. We identified alternate teosinte alleles at this QTL, one conferring increased GLS resistance and one increased susceptibility relative to the B73 allele. Using segregating populations derived from NIL parents carrying these contrasting alleles, we were able to delimit the QTL region to a ~130 kb (based on the B73 genome) which encompassed five predicted genes.

I.V. infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat.

PubMed

Nomura, T; Honmou, O; Harada, K; Houkin, K; Hamada, H; Kocsis, J D

2005-01-01

I.V. delivery of mesenchymal stem cells prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischemia models. Administration of the brain-derived neurotrophic factor to the infarction site has also been demonstrated to be neuroprotective. To test the hypothesis that brain-derived neurotrophic factor contributes to the therapeutic benefits of mesenchymal stem cell delivery, we compared the efficacy of systemic delivery of human mesenchymal stem cells and human mesenchymal stem cells transfected with a fiber-mutant F/RGD adenovirus vector with a brain-derived neurotrophic factor gene (brain-derived neurotrophic factor-human mesenchymal stem cells). A permanent middle cerebral artery occlusion was induced by intraluminal vascular occlusion with a microfilament. Human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells were i.v. injected into the rats 6 h after middle cerebral artery occlusion. Lesion size was assessed at 6 h, 1, 3 and 7 days using MR imaging, and histological methods. Functional outcome was assessed using the treadmill stress test. Both human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells reduced lesion volume and elicited functional improvement compared with the control sham group, but the effect was greater in the brain-derived neurotrophic factor-human mesenchymal stem cell group. ELISA analysis of the infarcted hemisphere revealed an increase in brain-derived neurotrophic factor in the human mesenchymal stem cell groups, but a greater increase in the brain-derived neurotrophic factor-human mesenchymal stem cell group. These data support the hypothesis that brain-derived neurotrophic factor contributes to neuroprotection in cerebral ischemia and cellular delivery of brain-derived neurotrophic factor can be achieved by i.v. delivery of human mesenchymal stem cells.

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed Central

Torrent, C; Gabus, C; Darlix, J L

1994-01-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer. Images PMID:8289369

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed

Torrent, C; Gabus, C; Darlix, J L

1994-02-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae): Unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer

PubMed Central

Brouard, Jean-Simon; Otis, Christian; Lemieux, Claude; Turmel, Monique

2008-01-01

Background To gain insight into the branching order of the five main lineages currently recognized in the green algal class Chlorophyceae and to expand our understanding of chloroplast genome evolution, we have undertaken the sequencing of chloroplast DNA (cpDNA) from representative taxa. The complete cpDNA sequences previously reported for Chlamydomonas (Chlamydomonadales), Scenedesmus (Sphaeropleales), and Stigeoclonium (Chaetophorales) revealed tremendous variability in their architecture, the retention of only few ancestral gene clusters, and derived clusters shared by Chlamydomonas and Scenedesmus. Unexpectedly, our recent phylogenies inferred from these cpDNAs and the partial sequences of three other chlorophycean cpDNAs disclosed two major clades, one uniting the Chlamydomonadales and Sphaeropleales (CS clade) and the other uniting the Oedogoniales, Chaetophorales and Chaetopeltidales (OCC clade). Although molecular signatures provided strong support for this dichotomy and for the branching of the Oedogoniales as the earliest-diverging lineage of the OCC clade, more data are required to validate these phylogenies. We describe here the complete cpDNA sequence of Oedogonium cardiacum (Oedogoniales). Results Like its three chlorophycean homologues, the 196,547-bp Oedogonium chloroplast genome displays a distinctive architecture. This genome is one of the most compact among photosynthetic chlorophytes. It has an atypical quadripartite structure, is intron-rich (17 group I and 4 group II introns), and displays 99 different conserved genes and four long open reading frames (ORFs), three of which are clustered in the spacious inverted repeat of 35,493 bp. Intriguingly, two of these ORFs (int and dpoB) revealed high similarities to genes not usually found in cpDNA. At the gene content and gene order levels, the Oedogonium genome most closely resembles its Stigeoclonium counterpart. Characters shared by these chlorophyceans but missing in members of the CS clade

Ancient gene transfer from algae to animals: Mechanisms and evolutionary significance

PubMed Central

2012-01-01

Background Horizontal gene transfer (HGT) is traditionally considered to be rare in multicellular eukaryotes such as animals. Recently, many genes of miscellaneous algal origins were discovered in choanoflagellates. Considering that choanoflagellates are the existing closest relatives of animals, we speculated that ancient HGT might have occurred in the unicellular ancestor of animals and affected the long-term evolution of animals. Results Through genome screening, phylogenetic and domain analyses, we identified 14 gene families, including 92 genes, in the tunicate Ciona intestinalis that are likely derived from miscellaneous photosynthetic eukaryotes. Almost all of these gene families are distributed in diverse animals, suggesting that they were mostly acquired by the common ancestor of animals. Their miscellaneous origins also suggest that these genes are not derived from a particular algal endosymbiont. In addition, most genes identified in our analyses are functionally related to molecule transport, cellular regulation and methylation signaling, suggesting that the acquisition of these genes might have facilitated the intercellular communication in the ancestral animal. Conclusions Our findings provide additional evidence that algal genes in aplastidic eukaryotes are not exclusively derived from historical plastids and thus important for interpreting the evolution of eukaryotic photosynthesis. Most importantly, our data represent the first evidence that more anciently acquired genes might exist in animals and that ancient HGT events have played an important role in animal evolution. PMID:22690978

Macrophage Activation Mechanisms in Human Monocytic Cell Line-derived Macrophages.

PubMed

Sumiya, Yu; Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

2015-08-01

Although the mechanisms of macrophage activation are important for cancer immunotherapy, they are poorly understood. Recently, easy and robust assay systems for assessing the macrophage-activating factor (MAF) using monocytic cell line-derived macrophages were established. Gene-expression profiles of U937- and THP-1-derived macrophages were compared using gene expression microarray analysis and their responses against several MAFs were examined by in vitro experiments. Activated states of these macrophages could not be assigned to a specific sub-type but showed, however, different unique characteristics. The unique of monocytic cell line-derived macrophages could provide clues to understand the activation mechanism of macrophages and, therefore, help to develop effective cancer immunotherapy with MAFs. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Intervertebral disc-derived stem cells: implications for regenerative medicine and neural repair.

PubMed

Erwin, W Mark; Islam, Diana; Eftekarpour, Eftekhar; Inman, Robert D; Karim, Muhammad Zia; Fehlings, Michael G

2013-02-01

An in vitro and in vivo evaluation of intervertebral disc (IVD)-derived stem/progenitor cells. To determine the chondrogenic, adipogenic, osteogenic, and neurogenic differentiation capacity of disc-derived stem/progenitor cells in vitro and neurogenic differentiation in vivo. Tissue repair strategies require a source of appropriate cells that could be used to replace dead or damaged cells and tissues such as stem cells. Here we examined the potential use of IVD-derived stem cells in regenerative medicine approaches and neural repair. Nonchondrodystrophic canine IVD nucleus pulposus (NP) cells were used to generate stem/progenitor cells (NP progenitor cells [NPPCs]) and the NPPCs were differentiated in vitro into chondrogenic, adipogenic, and neurogenic lineages and in vivo into the neurogenic lineage. NPPCs were compared with bone marrow-derived mesenchymal (stromal) stem cells in terms of the expression of stemness genes. The expression of the neural crest marker protein 0 and the Brachyury gene were evaluated in NP cells and NPPCs. NPPCs contain stem/progenitor cells and express "stemness" genes such as Sox2, Oct3/4, Nanog, CD133, Nestin, and neural cell adhesion molecule but differ from mesenchymal (stromal) stem cells in the higher expression of the Nanog gene by NPPCs. NPPCs do not express protein 0 or the Brachyury gene both of which are expressed by the totality of IVD NP cells. The percentage of NPPCs within the IVD is 1% of the total as derived by colony-forming assay. NPPCs are capable of differentiating along chondrogenic, adipogenic, and neurogenic lineages in vitro and into oligodendrocyte, neuron, and astroglial specific precursor cells in vivo within the compact myelin-deficient shiverer mouse. We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.

The platelet-activating factor acetylhydrolase gene derived from Trichoderma harzianum induces maize resistance to Curvularia lunata through the jasmonic acid signaling pathway.

PubMed

Yu, Chuanjin; Fan, Lili; Gao, Jinxin; Wang, Meng; Wu, Qiong; Tang, Jun; Li, Yaqian; Chen, Jie

2015-01-01

Platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum was upregulated by the interaction of T. harzianum with maize roots or the foliar pathogen Curvularia lunata. PAF-AH was associated with chitinase and cellulase expressions, but especially with chitinase, because its activity in the KO40 transformant (PAF-AH disruption transformant) was lower, compared with the wild-type strain T28. The result demonstrated that the colonization of maize roots by T. harzianum induced systemic protection of leaves inoculated with C. lunata. Such protection was associated with the expression of inducible jasmonic acid pathway-related genes. Moreover, the data from liquid chromatography-mass spectrometry confirmed that the concentration of jasmonic acid in maize leaves was associated with the expression level of defense-related genes, suggesting that PAF-AH induced resistance to the foliar pathogen. Our findings showed that PAF-AH had an important function in inducing systemic resistance to maize leaf spot pathogen.

Lipopolysaccharide and Concanavalin A Differentially Induce the Expression of Immune Response Genes in Caprine Monocyte Derived Macrophages.

PubMed

Walia, Vishakh; Kumar, Rohit; Mitra, Abhijit

2015-01-01

Monocyte derived macrophages (MDMs), as an in vitro model in pathogen challenge studies, are generally induced with lipopolysaccharide (LPS) and concanavalin A (ConA) to assay cellular immunity. General immune responses to LPS and ConA have been studied in a wide range of species, but similar studies are limited to goats. In the present study, caprine MDMs were induced with LPS and ConA and the expression profile of immune response (IR) genes, namely, Tumor Necrosis Factor Alpha (TNFA), Interferon Gamma (IFNG), Interleukin 2 (IL2), Granulocyte Macrophage Colony Stimulating Factor (GMCSF), Interleukin 10 (IL10), Transforming Growth Factor Beta (TGFB), Natural Resistance-Associated Macrophage Protein-1 (NRAMP1), inducible nitric oxide synthase (NOS2), and caspase1 (CASP1) were studied to compare the potential of LPS and ConA in initiating immune responses in goat macrophages. Real Time quantitative PCR (RT-qPCR) analysis revealed that both LPS and ConA caused an upregulation (p < 0.05) of GMCSF, TGFB1, IL10, and IFNG and down-regulation of NRAMP1. TNFA and IL2, and NOS2 were upregulated (p < 0.05) by ConA and LPS, respectively. Whereas, the expression of CASP1 remain unaltered. Comparatively, the effect of ConA was more pronounced (p < 0.05) in regulating the expression of IR genes suggesting its suitability for studying the general immune responses in caprine MDM.

Generation of Human Induced Pluripotent Stem Cell‐Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy

PubMed Central

Meneghini, Vasco; Sala, Davide; De Cicco, Silvia; Luciani, Marco; Cavazzin, Chiara; Paulis, Marianna; Mentzen, Wieslawa; Morena, Francesco; Giannelli, Serena; Sanvito, Francesca; Villa, Anna; Bulfone, Alessandro; Broccoli, Vania; Martino, Sabata

2016-01-01

Abstract Allogeneic fetal‐derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient‐specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene‐therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC‐derived neural stem cells (NSCs) showing a reliable “NSC signature” is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS‐NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a “gold standard” in a side‐by‐side comparison when validating the phenotype of hiPS‐NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS‐NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA‐overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA‐overexpressing iPSC‐derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient‐specific ARSA‐overexpressing hiPS‐NSCs may be used in autologous ex vivo gene therapy protocols to provide long‐lasting enzymatic

Tumor targeting of gene expression through metal-coordinated conjugation with dextran.

PubMed

Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

2003-03-07

Tumor targeting of plasmid DNA was achieved through the conjugation of dextran derivatives with chelate residues based on metal coordination. Diethylenetriamine pentaacetic acid (DTPA), spermidine (Sd), and spermine (Sm) were chemically introduced to the hydroxyl groups of dextran to obtain dextran-DTPA, dextran-Sd and dextran-Sm derivatives. Conjugation of the dextran derivative by Zn(2+) coordination decreased the apparent size of the plasmid DNA, depending on the derivative type. The negative zeta potential of plasmid DNA became almost 0 mV after Zn(2+)-coordinated conjugation with dextran-Sm. When the dextran derivative-plasmid DNA conjugates with Zn(2+) coordination were intravenously injected subcutaneously into mice bearing Meth-AR-1 fibrosarcoma, the dextran-Sm-plasmid DNA conjugate significantly enhanced the level of gene expression in the tumor, in contrast to the conjugate of other dextran derivatives and free plasmid DNA. The enhanced gene expression produced by the Zn(2+)-coordinated dextran-Sm-plasmid DNA conjugate was specific to the tumor, whereas a simple mixture of dextran-Sm and plasmid DNA was not effective. The level of gene expression depended on the percentage of chelate residues introduced, the mixing weight ratio of the plasmid DNA/Sm residue used for conjugate preparation, and the plasmid DNA dose. A fluorescent microscopic study revealed that localization of plasmid DNA in the tumor tissue was observed only after injection of the dextran-Sm-plasmid DNA conjugate with Zn(2+) coordination. In addition, the gene expression induced by the conjugate lasted for more than 10 days after the injection. We conclude that Zn(2+)-coordinated dextran-Sm conjugation is a promising way to enable plasmid DNA to target the tumor in gene expression as well as to prolong the duration of gene expression.

Gene-based SSR markers for common bean (Phaseolus vulgaris L.) derived from root and leaf tissue ESTs: an integration of the BMc series.

PubMed

Blair, Matthew W; Hurtado, Natalia; Chavarro, Carolina M; Muñoz-Torres, Monica C; Giraldo, Martha C; Pedraza, Fabio; Tomkins, Jeff; Wing, Rod

2011-03-22

Sequencing of cDNA libraries for the development of expressed sequence tags (ESTs) as well as for the discovery of simple sequence repeats (SSRs) has been a common method of developing microsatellites or SSR-based markers. In this research, our objective was to further sequence and develop common bean microsatellites from leaf and root cDNA libraries derived from the Andean gene pool accession G19833 and the Mesoamerican gene pool accession DOR364, mapping parents of a commonly used reference map. The root libraries were made from high and low phosphorus treated plants. A total of 3,123 EST sequences from leaf and root cDNA libraries were screened and used for direct simple sequence repeat discovery. From these EST sequences we found 184 microsatellites; the majority containing tri-nucleotide motifs, many of which were GC rich (ACC, AGC and AGG in particular). Di-nucleotide motif microsatellites were about half as common as the tri-nucleotide motif microsatellites but most of these were AGn microsatellites with a moderate number of ATn microsatellites in root ESTs followed by few ACn and no GCn microsatellites. Out of the 184 new SSR loci, 120 new microsatellite markers were developed in the BMc (Bean Microsatellites from cDNAs) series and these were evaluated for their capacity to distinguish bean diversity in a germplasm panel of 18 genotypes. We developed a database with images of the microsatellites and their polymorphism information content (PIC), which averaged 0.310 for polymorphic markers. The present study produced information about microsatellite frequency in root and leaf tissues of two important genotypes for common bean genomics: namely G19833, the Andean genotype selected for whole genome shotgun sequencing from race Peru, and DOR364 a race Mesoamerica subgroup 2 genotype that is a small-red seeded, released variety in Central America. Both race Peru and Mesoamerica subgroup 2 (small red beans) have been understudied in comparison to race Nueva

Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction.

PubMed

Hasegawa, Daigo; Ochiai-Shino, Hiromi; Onodera, Shoko; Nakamura, Takashi; Saito, Akiko; Onda, Takeshi; Watanabe, Katsuhito; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Kosaki, Kenjiro; Yamaguchi, Akira; Shibahara, Takahiko; Azuma, Toshifumi

2017-01-01

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype-phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are

Immune gene expression for diverse haemocytes derived from pacific white shrimp, Litopenaeus vannamei.

PubMed

Yang, Chih-Chiu; Lu, Chung-Lun; Chen, Sherwin; Liao, Wen-Liang; Chen, Shiu-Nan

2015-05-01

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-β-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to β-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evolution and Variation of Renin Genes in Mice

PubMed Central

Dickinson, Douglas P.; Gross, Kenneth W.; Piccini, Nina; Wilson, Carol M.

1984-01-01

Inbred strains of mice carry Ren-1, a gene encoding the thermostable Renin-1 isozyme. Ren-1 is expressed at relatively low levels in mouse submandibular gland and kidney. Some strains also carry Ren-2, a gene encoding the thermolabile Renin-2 isozyme. Ren-2 is expressed at high levels in the mouse submandibular gland and at very low levels, if at all, in the kidney. Ren-1 and Ren-2 are closely linked on mouse chromosome 1, show extensive homology in coding and noncoding regions and provide a model for studying the regulation of gene expression. An investigation of renin genes and enzymatic activity in wild-derived mice identified several restriction site polymorphisms as well as putative variants in renin gene expression and protein structure. The number of renin genes carried by different subpopulations of wild-derived mice is consistent with the occurrence of a gene duplication event prior to the divergence of M. spretus (2.75–5.5 million yr ago). This conclusion is in agreement with a prior estimate based upon comparative sequence analysis of Ren-1 and Ren-2 from inbred laboratory mice. PMID:6389258

Creatine Enhances Transdifferentiation of Bone Marrow Stromal Cell-Derived Neural Stem Cell Into GABAergic Neuron-Like Cells Characterized With Differential Gene Expression.

PubMed

Darabi, Shahram; Tiraihi, Taki; Delshad, AliReza; Sadeghizadeh, Majid; Taheri, Taher; Hassoun, Hayder K

2017-04-01

Creatine was reported to induce bone marrow stromal cells (BMSC) into GABAergic neuron-like cells (GNLC). In a previous study, creatine was used as a single inducer for BMSC into GNLC with low yield. In this study, BMSC-derived neurospheres (NS) have been used in generating GABAergic phenotype. The BMSC were isolated from adult rats and used in generating neurospheres and used for producing neural stem cells (NSC). A combination of all-trans-retinoic acid (RA), the ciliary neurotrophic factor (CNTF), and creatine was used in order to improve the yield of GNLC. We also used other protocols for the transdifferentiation including RA alone; RA and creatine; RA and CNTF; and RA, CNTF, and creatine. The BMSC, NSC, and GNLC were characterized by specific markers. The activity of the GNLC was evaluated using FM1-43. The isolated BMSC expressed Oct4, fibronectin, and CD44. The NS were immunoreactive to nestin and SOX2, the NSC were immunoreactive to nestin, NF68 and NF160, while the GNLC were immunoreactive to GAD1/2, VGAT, GABA, and synaptophysin. Oct4 and c-MYC, pluripotency genes, were expressed in the BMSC, while SOX2 and c-MYC were expressed in the NSC. The activity of GNLC indicates that the synaptic vesicles were released upon stimulation. The conclusion is that the combination of RA, CNTF, and creatine induced differentiation of neurosphere-derived NSC into GNLC within 1 week. This protocol gives higher yield than the other protocols used in this study. The mechanism of induction was clearly associated with several differential pluripotent genes.

Regulated expression of the Ren-2 gene in transgenic mice derived from parental strains carrying only the Ren-1 gene.

PubMed Central

Tronik, D; Dreyfus, M; Babinet, C; Rougeon, F

1987-01-01

The Ren-2 gene encoding the mouse submaxillary gland (SMG) renin was microinjected into the pronuclei of fertilized eggs from mice carrying only the Ren-1 gene. In addition to the whole transcription unit, the injected DNA contained 2.5 and 3 kb of upstream and downstream flanking sequences, respectively. Three independent transgenic mice lines were obtained; two of them had integrated one copy of the Ren-2 gene, the last one had integrated five and eleven copies at two independent sites. Independently of the number of Ren-2 copies integrated, the pattern of Ren-2 gene expression in all the transgenic mice was identical to that observed in wild-type animals in which Ren-1 and Ren-2 are closely linked on chromosome 1. In particular, the exogenous Ren-2 gene was only transcribed in the kidney and in the SMG. In the kidney, Ren-1 and Ren-2 mRNAs were present at a comparable level, whereas in the SMG Ren-2 mRNA was at least 100-fold more abundant than Ren-1 mRNA. Moreover, Ren-2 expression in the SMG was positively regulated by androgens. Only one difference between transgenic mice and wild-type mice carrying the Ren-2 gene has been observed: the basal level of Ren-2 transcription in the SMG of transgenic females was lower than in two-gene strain females. Androgen treatment of transgenic females induced SMG renin mRNA to a level identical to that of transgenic males. This suggests that the basal level of SMG renin mRNA is dependent upon cis-acting elements which are not present in the microinjected fragment. Images Fig. 1. Fig. 2. Fig. 3. PMID:3297677

Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

PubMed Central

Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

2012-01-01

Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

Patient-derived Hormone-naive Prostate Cancer Xenograft Models Reveal Growth Factor Receptor Bound Protein 10 as an Androgen Receptor-repressed Gene Driving the Development of Castration-resistant Prostate Cancer.

PubMed

Hao, Jun; Ci, Xinpei; Xue, Hui; Wu, Rebecca; Dong, Xin; Choi, Stephen Yiu Chuen; He, Haiqing; Wang, Yu; Zhang, Fang; Qu, Sifeng; Zhang, Fan; Haegert, Anne M; Gout, Peter W; Zoubeidi, Amina; Collins, Colin; Gleave, Martin E; Lin, Dong; Wang, Yuzhuo

2018-06-01

Although androgen deprivation therapy is initially effective in controlling growth of hormone-naive prostate cancers (HNPCs) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops. To identify CRPC driver genes that may provide new targets to enhance CRPC therapy. Patient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers. The functionality of a potential CRPC driver was validated via its knockdown in cultured prostate cancer cells; its clinical relevance was established using data from prostate cancer patient databases. Eighty genes were found to be significantly upregulated at the CRPC stage, while seven of them also showed elevated expression prior to CRPC development. Among the latter, growth factor receptor bound protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, elevated GRB10 expression in clinical prostate cancer samples correlated with more aggressive tumor types and poorer patient treatment outcome. GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. A positive correlation between AKT activity and GRB10 expression was also found in clinical cohorts. GRB10 acts as a driver of CRPC and sensitizes androgen receptor pathway inhibitors, and hence GRB10 targeting provides a novel therapeutic strategy for the disease. Development of castration-resistant prostate cancer (CRPC) is a major problem in the management of the disease. Using state-of-the-art patient-derived hormone-naive prostate cancer xenograft models, we found and validated the growth factor receptor bound protein 10 gene as a driver of CRPC, indicating that it may be used as a

Clustering change patterns using Fourier transformation with time-course gene expression data.

PubMed

Kim, Jaehee

2011-01-01

To understand the behavior of genes, it is important to explore how the patterns of gene expression change over a period of time because biologically related gene groups can share the same change patterns. In this study, the problem of finding similar change patterns is induced to clustering with the derivative Fourier coefficients. This work is aimed at discovering gene groups with similar change patterns which share similar biological properties. We developed a statistical model using derivative Fourier coefficients to identify similar change patterns of gene expression. We used a model-based method to cluster the Fourier series estimation of derivatives. We applied our model to cluster change patterns of yeast cell cycle microarray expression data with alpha-factor synchronization. It showed that, as the method clusters with the probability-neighboring data, the model-based clustering with our proposed model yielded biologically interpretable results. We expect that our proposed Fourier analysis with suitably chosen smoothing parameters could serve as a useful tool in classifying genes and interpreting possible biological change patterns.

Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

PubMed

Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

2015-10-01

Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration. © 2015 International Federation for Cell Biology.

Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

PubMed Central

Kurth, Elizabeth G.; Peremyslov, Valera V.; Prokhnevsky, Alexey I.; Kasschau, Kristin D.; Miller, Marilyn; Carrington, James C.

2012-01-01

The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests. PMID:22438553

Cytogenetic analysis and mapping of leaf rust resistance in Aegilops speltoides Tausch derived bread wheat line Selection2427 carrying putative gametocidal gene(s).

PubMed

Niranjana, M; Vinod; Sharma, J B; Mallick, Niharika; Tomar, S M S; Jha, S K

2017-12-01

Leaf rust (Puccinia triticina) is a major biotic stress affecting wheat yields worldwide. Host-plant resistance is the best method for controlling leaf rust. Aegilops speltoides is a good source of resistance against wheat rusts. To date, five Lr genes, Lr28, Lr35, Lr36, Lr47, and Lr51, have been transferred from Ae. speltoides to bread wheat. In Selection2427, a bread wheat introgresed line with Ae. speltoides as the donor parent, a dominant gene for leaf rust resistance was mapped to the long arm of chromosome 3B (LrS2427). None of the Lr genes introgressed from Ae. speltoides have been mapped to chromosome 3B. Since none of the designated seedling leaf rust resistance genes have been located on chromosome 3B, LrS2427 seems to be a novel gene. Selection2427 showed a unique property typical of gametocidal genes, that when crossed to other bread wheat cultivars, the F 1 showed partial pollen sterility and poor seed setting, whilst Selection2427 showed reasonable male and female fertility. Accidental co-transfer of gametocidal genes with LrS2427 may have occurred in Selection2427. Though LrS2427 did not show any segregation distortion and assorted independently of putative gametocidal gene(s), its utilization will be difficult due to the selfish behavior of gametocidal genes.

Gene screening of Wharton's jelly derived stem cells.

PubMed

Mechiche Alami, S; Velard, F; Draux, F; Siu Paredes, F; Josse, J; Lemaire, F; Gangloff, S C; Graesslin, O; Laurent-Maquin, D; Kerdjoudj, H

2014-01-01

Stem cells are the most powerful candidate for the treatment of various diseases. Suitable stem cell source should be harvested with minimal invasive procedure, found in great quantity, and transplanted with no risk of immune response and tumor formation. Fetal derived stem cells have been introduced as an excellent alternative to adult and embryonic stem cells use, but unfortunately, their degree of "stemness" and molecular characterization is still unclear. Several studies have been performed deciphering whether fetal stem cells meet the needs of regenerative medicine. We believe that a transcriptomic screening of Wharton's jelly stem cells will bring insights on cell population features.

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD).

PubMed

Meade, Kieran G; Gormley, Eamonn; Park, Stephen D E; Fitzsimons, Tara; Rosa, Guilherme J M; Costello, Eamon; Keane, Joseph; Coussens, Paul M; MacHugh, David E

2006-09-15

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (approximately 17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course (P<0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold (P<0.01) revealed 35 genes (approximately 3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-gamma gene (IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated

Generating gene knockout rats by homologous recombination in embryonic stem cells

PubMed Central

Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

2013-01-01

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

Cadherin juxtamembrane region derived peptides inhibit TGFβ1 induced gene expression

PubMed Central

Stavropoulos, Ilias; Golla, Kalyan; Moran, Niamh; Martin, Finian; Shields, Denis C

2014-01-01

Bioactive peptides in the juxtamembrane regions of proteins are involved in many signaling events. The juxtamembrane regions of cadherins were examined for the identification of bioactive regions. Several peptides spanning the cytoplasmic juxtamembrane regions of E- and N-cadherin were synthesized and assessed for the ability to influence TGFβ responses in epithelial cells at the gene expression and protein levels. Peptides from regions closer to the membrane appeared more potent inhibitors of TGFβ signaling, blocking Smad3 phosphorylation. Thus inhibiting nuclear translocation of phosphorylated Smad complexes and subsequent transcriptional activation of TGFβ signal propagating genes. The peptides demonstrated a peptide-specific potential to inhibit other TGFβ superfamily members, such as BMP4. PMID:25108297

Investigation of brain-derived neurotrophic factor (BDNF) gene expression in hypothalamus of obese rats: Modulation by omega-3 fatty acids.

PubMed

Abdel-Maksoud, Sahar M; Hassanein, Sally I; Gohar, Neveen A; Attia, Saad M M; Gad, Mohamed Z

2017-10-01

The aim of this study was investigating the effect of omega-3 fatty acids (ω-3 FAs) on brain-derived neurotrophic factor (BDNF) gene expression, using in vivo and in vitro models, to unravel the potential mechanisms of polyunsaturated fatty acids use in obesity. Twenty-nine Sprague-Dawley rats were divided into three groups; lean controls fed normal chow diet for 14 weeks, obese controls fed 60% of their diet as saturated fats for 14 weeks, and ω-3 FAs-treated rats fed 60% saturated fat diet for 14 weeks with concomitant oral administration of 400 mg/kg/day ω-3 FAs, mainly docosahexaenoic acid and EPA, from week 12 to week 14. For the in vitro experiment, hypothalamic cells from six obese rats were cultured in the presence of different concentrations of ω-3 FAs to determine its direct effect on BDNF expression. In vivo results showed that obesity has negative effect on BDNF gene expression in rat hypothalamus that was reversed by administration of ω-3 FAs. Obese rats showed hypercholesterolemia, hypertriglyceridemia, normoinsulinemia, hyperglycemia and hyperleptinemia. Treatment with ω-3 FAs showed significant decrease in serum total cholesterol and TAG. Also serum glucose level and HOMA index were decreased significantly. In vitro results demonstrated the increase in BDNF expression by ω-3 FAs in a dose-dependent manner. Obesity causes down-regulation of BDNF gene expression that can be reversed by ω-3 FAs treatment, making them an interesting treatment approach for obesity and metabolic disease.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

PubMed

Weber, Marlen; Apostolova, Galina; Widera, Darius; Mittelbronn, Michel; Dechant, Georg; Kaltschmidt, Barbara; Rohrer, Hermann

2015-02-01

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage. © 2014 AlphaMed Press.

Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery.

PubMed

Nam, Kihoon; Jung, Simhyun; Nam, Joung-Pyo; Kim, Sung Wan

2015-12-28

Branched poly(ethylenimine) (PEI) 25 kDa is an efficient gene delivery vector with outstanding gene condensation ability and great endosome escape activity. However, it also induces higher cytotoxicity. Transfection efficiency and toxicity of PEI are highly dependent upon their molecular weight and structure. We developed a bioreducible poly(ethylenimine) (PEI (-s-s-)) derived from low molecular weight PEI (1.8 kDa) for efficient gene delivery. Bioreducible core molecule is expected to increase molecular weight and reduce the cytotoxicity of the copolymer. PEI (-s-s-) polyplexes showed higher transfection efficiency and lower cytotoxicity compared to branched PEI 25 kDa, Lipofectamine® 2000 and, FuGENE® 6. In addition, PEI (-s-s-) derivative (16 kDa) formed stable polyplexes with a zeta-potential value of +34 mV and polyplex size of 61 nm. PEI (-s-s-) derivative (16 kDa) showed excellent transfection efficiency: 3.6 times higher than branched PEI 25 kDa in HeLa cells and 7.4 times higher than Lipofectamine® 2000 in H9C2 cell. The derivatives also showed lower cytotoxicity compared with Lipofectamine® 2000 and PEI 25 kDa in various cell types. In addition, newly synthesized PEI (-s-s-) derivatives have high reproducibility. Copyright © 2015 Elsevier B.V. All rights reserved.

Revealing Less Derived Nature of Cartilaginous Fish Genomes with Their Evolutionary Time Scale Inferred with Nuclear Genes

PubMed Central

Renz, Adina J.; Meyer, Axel; Kuraku, Shigehiro

2013-01-01

Cartilaginous fishes, divided into Holocephali (chimaeras) and Elasmoblanchii (sharks, rays and skates), occupy a key phylogenetic position among extant vertebrates in reconstructing their evolutionary processes. Their accurate evolutionary time scale is indispensable for better understanding of the relationship between phenotypic and molecular evolution of cartilaginous fishes. However, our current knowledge on the time scale of cartilaginous fish evolution largely relies on estimates using mitochondrial DNA sequences. In this study, making the best use of the still partial, but large-scale sequencing data of cartilaginous fish species, we estimate the divergence times between the major cartilaginous fish lineages employing nuclear genes. By rigorous orthology assessment based on available genomic and transcriptomic sequence resources for cartilaginous fishes, we selected 20 protein-coding genes in the nuclear genome, spanning 2973 amino acid residues. Our analysis based on the Bayesian inference resulted in the mean divergence time of 421 Ma, the late Silurian, for the Holocephali-Elasmobranchii split, and 306 Ma, the late Carboniferous, for the split between sharks and rays/skates. By applying these results and other documented divergence times, we measured the relative evolutionary rate of the Hox A cluster sequences in the cartilaginous fish lineages, which resulted in a lower substitution rate with a factor of at least 2.4 in comparison to tetrapod lineages. The obtained time scale enables mapping phenotypic and molecular changes in a quantitative framework. It is of great interest to corroborate the less derived nature of cartilaginous fish at the molecular level as a genome-wide phenomenon. PMID:23825540

Revealing less derived nature of cartilaginous fish genomes with their evolutionary time scale inferred with nuclear genes.

PubMed

Renz, Adina J; Meyer, Axel; Kuraku, Shigehiro

2013-01-01

Cartilaginous fishes, divided into Holocephali (chimaeras) and Elasmoblanchii (sharks, rays and skates), occupy a key phylogenetic position among extant vertebrates in reconstructing their evolutionary processes. Their accurate evolutionary time scale is indispensable for better understanding of the relationship between phenotypic and molecular evolution of cartilaginous fishes. However, our current knowledge on the time scale of cartilaginous fish evolution largely relies on estimates using mitochondrial DNA sequences. In this study, making the best use of the still partial, but large-scale sequencing data of cartilaginous fish species, we estimate the divergence times between the major cartilaginous fish lineages employing nuclear genes. By rigorous orthology assessment based on available genomic and transcriptomic sequence resources for cartilaginous fishes, we selected 20 protein-coding genes in the nuclear genome, spanning 2973 amino acid residues. Our analysis based on the Bayesian inference resulted in the mean divergence time of 421 Ma, the late Silurian, for the Holocephali-Elasmobranchii split, and 306 Ma, the late Carboniferous, for the split between sharks and rays/skates. By applying these results and other documented divergence times, we measured the relative evolutionary rate of the Hox A cluster sequences in the cartilaginous fish lineages, which resulted in a lower substitution rate with a factor of at least 2.4 in comparison to tetrapod lineages. The obtained time scale enables mapping phenotypic and molecular changes in a quantitative framework. It is of great interest to corroborate the less derived nature of cartilaginous fish at the molecular level as a genome-wide phenomenon.

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

PubMed Central

Dodds, Peter N.; Lawrence, Gregory J.; Catanzariti, Ann-Maree; Teh, Trazel; Wang, Ching-I. A.; Ayliffe, Michael A.; Kobe, Bostjan; Ellis, Jeffrey G.

2006-01-01

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R–Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R–Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R–Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant–pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes. PMID:16731621

Molecular Evolution of the Nuclear Factor (Erythroid-Derived 2)-Like 2 Gene Nrf2 in Old World Fruit Bats (Chiroptera: Pteropodidae).

PubMed

Yin, Qiuyuan; Zhu, Lei; Liu, Di; Irwin, David M; Zhang, Shuyi; Pan, Yi-Hsuan

2016-01-01

Mammals developed antioxidant systems to defend against oxidative damage in their daily life. Enzymatic antioxidants and low molecular weight antioxidants (LMWAs) constitute major parts of the antioxidant systems. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2, encoded by the Nrf2 gene) is a central transcriptional regulator, regulating transcription, of many antioxidant enzymes. Frugivorous bats eat large amounts of fruits that contain high levels of LMWAs such as vitamin C, thus, a reliance on LMWAs might greatly reduce the need for antioxidant enzymes in comparison to insectivorous bats. Therefore, it is possible that frugivorous bats have a reduced need for Nrf2 function due to their substantial intake of diet-antioxidants. To test whether the Nrf2 gene has undergone relaxed evolution in fruit-eating bats, we obtained Nrf2 sequences from 16 species of bats, including four Old World fruit bats (Pteropodidae) and one New World fruit bat (Phyllostomidae). Our molecular evolutionary analyses revealed changes in the selection pressure acting on Nrf2 gene and identified seven specific amino acid substitutions that occurred on the ancestral lineage leading to Old World fruit bats. Biochemical experiments were conducted to examine Nrf2 in Old World fruit bats and showed that the amount of catalase, which is regulated by Nrf2, was significantly lower in the brain, heart and liver of Old World fruit bats despite higher levels of Nrf2 protein in Old World fruit bats. Computational predictions suggest that three of these seven amino acid replacements might be deleterious to Nrf2 function. Therefore, the results suggest that Nrf2 gene might have experienced relaxed constraint in Old World fruit bats, however, we cannot rule out the possibility of positive selection. Our study provides the first data on the molecular adaptation of Nrf2 gene in frugivorous bats in compensation to the increased levels of LWMAs from their fruit-diet.

Molecular Evolution of the Nuclear Factor (Erythroid-Derived 2)-Like 2 Gene Nrf2 in Old World Fruit Bats (Chiroptera: Pteropodidae)

PubMed Central

Liu, Di; Irwin, David M.; Zhang, Shuyi; Pan, Yi-Hsuan

2016-01-01

Mammals developed antioxidant systems to defend against oxidative damage in their daily life. Enzymatic antioxidants and low molecular weight antioxidants (LMWAs) constitute major parts of the antioxidant systems. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2, encoded by the Nrf2 gene) is a central transcriptional regulator, regulating transcription, of many antioxidant enzymes. Frugivorous bats eat large amounts of fruits that contain high levels of LMWAs such as vitamin C, thus, a reliance on LMWAs might greatly reduce the need for antioxidant enzymes in comparison to insectivorous bats. Therefore, it is possible that frugivorous bats have a reduced need for Nrf2 function due to their substantial intake of diet-antioxidants. To test whether the Nrf2 gene has undergone relaxed evolution in fruit-eating bats, we obtained Nrf2 sequences from 16 species of bats, including four Old World fruit bats (Pteropodidae) and one New World fruit bat (Phyllostomidae). Our molecular evolutionary analyses revealed changes in the selection pressure acting on Nrf2 gene and identified seven specific amino acid substitutions that occurred on the ancestral lineage leading to Old World fruit bats. Biochemical experiments were conducted to examine Nrf2 in Old World fruit bats and showed that the amount of catalase, which is regulated by Nrf2, was significantly lower in the brain, heart and liver of Old World fruit bats despite higher levels of Nrf2 protein in Old World fruit bats. Computational predictions suggest that three of these seven amino acid replacements might be deleterious to Nrf2 function. Therefore, the results suggest that Nrf2 gene might have experienced relaxed constraint in Old World fruit bats, however, we cannot rule out the possibility of positive selection. Our study provides the first data on the molecular adaptation of Nrf2 gene in frugivorous bats in compensation to the increased levels of LWMAs from their fruit-diet. PMID:26735303

Uncertainties in mapping forest carbon in urban ecosystems.

PubMed

Chen, Gang; Ozelkan, Emre; Singh, Kunwar K; Zhou, Jun; Brown, Marilyn R; Meentemeyer, Ross K

2017-02-01

Spatially explicit urban forest carbon estimation provides a baseline map for understanding the variation in forest vertical structure, informing sustainable forest management and urban planning. While high-resolution remote sensing has proven promising for carbon mapping in highly fragmented urban landscapes, data cost and availability are the major obstacle prohibiting accurate, consistent, and repeated measurement of forest carbon pools in cities. This study aims to evaluate the uncertainties of forest carbon estimation in response to the combined impacts of remote sensing data resolution and neighborhood spatial patterns in Charlotte, North Carolina. The remote sensing data for carbon mapping were resampled to a range of resolutions, i.e., LiDAR point cloud density - 5.8, 4.6, 2.3, and 1.2 pt s/m 2 , aerial optical NAIP (National Agricultural Imagery Program) imagery - 1, 5, 10, and 20 m. Urban spatial patterns were extracted to represent area, shape complexity, dispersion/interspersion, diversity, and connectivity of landscape patches across the residential neighborhoods with built-up densities from low, medium-low, medium-high, to high. Through statistical analyses, we found that changing remote sensing data resolution introduced noticeable uncertainties (variation) in forest carbon estimation at the neighborhood level. Higher uncertainties were caused by the change of LiDAR point density (causing 8.7-11.0% of variation) than changing NAIP image resolution (causing 6.2-8.6% of variation). For both LiDAR and NAIP, urban neighborhoods with a higher degree of anthropogenic disturbance unveiled a higher level of uncertainty in carbon mapping. However, LiDAR-based results were more likely to be affected by landscape patch connectivity, and the NAIP-based estimation was found to be significantly influenced by the complexity of patch shape. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

PubMed Central

Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

2003-01-01

Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

Production of SV40-derived vectors.

PubMed

Strayer, David S; Mitchell, Christine; Maier, Dawn A; Nichols, Carmen N

2010-06-01

Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Here we describe a protocol for production and purification of these vectors. These procedures require only packaging cells (e.g., COS-7) and circular vector genome DNA. Amplification involves repeated infection of packaging cells with vector produced by transfection. Cotransfection is not required in any step. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients and resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. These approaches are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. These vectors are best applied to long-term expression of proteins normally encoded by mammalian cells or by viruses that infect mammalian cells, or of untranslated RNAs (e.g., RNA interference). The preparative approaches described facilitate application of these vectors and allow almost any laboratory to exploit their strengths for diverse gene delivery applications.

Pathogenic infection of Rhesus macaques by an evolving SIV-HIV derived from CCR5-using envelope genes of acute HIV-1 infections

PubMed Central

Asmal, Mohammed; Lane, Sophie; Tian, Meijuan; Nickle, Gabrielle; Venner, Colin; Dirk, Brennan; Dikeakos, Jimmy; Luedemann, Corinne; Mach, Linh; Balachandran, Harikrishnan; Buzby, Adam; Rao, Srinivas; Letvin, Norman; Gao, Yong; Arts, Eric J.

2016-01-01

For studies on vaccines and therapies for HIV disease, SIV-HIV chimeric viruses harboring the HIV-1 env gene (SHIVenv) remain the best virus in non-human primate models. However, there are still very few SHIVenv viruses that can cause AIDS in non-CD8-depleted animals. In the present study, a recently created CCR5-using SHIVenv_B3 virus with env gene derived from acute/early HIV-1 infections (AHI) successfully established pathogenic infection in macaques. Through a series of investigations on the evolution, mutational profile, and phenotype of the virus and the resultant humoral immune response in infected rhesus macaques, we found that the E32K mutation in the Env C1 domain was associated with macaque pathogenesis, and that the electrostatic interactions in Env may favor E32K at the gp120 N terminus and “lock” the binding to heptad repeat 1 of gp41 in the trimer and produce a SHIVenv with increased fitness and pathogenesis during macaque infections. PMID:27723488

Identification and function analysis of contrary genes in Dupuytren's contracture.

PubMed

Ji, Xianglu; Tian, Feng; Tian, Lijie

2015-07-01

The present study aimed to analyze the expression of genes involved in Dupuytren's contracture (DC), using bioinformatic methods. The profile of GSE21221 was downloaded from the gene expression ominibus, which included six samples, derived from fibroblasts and six healthy control samples, derived from carpal-tunnel fibroblasts. A Distributed Intrusion Detection System was used in order to identify differentially expressed genes. The term contrary genes is proposed. Contrary genes were the genes that exhibited opposite expression patterns in the positive and negative groups, and likely exhibited opposite functions. These were identified using Coexpress software. Gene ontology (GO) function analysis was conducted for the contrary genes. A network of GO terms was constructed using the reduce and visualize gene ontology database. Significantly expressed genes (801) and contrary genes (98) were screened. A significant association was observed between Chitinase-3-like protein 1 and ten genes in the positive gene set. Positive regulation of transcription and the activation of nuclear factor-κB (NF-κB)-inducing kinase activity exhibited the highest degree values in the network of GO terms. In the present study, the expression of genes involved in the development of DC was analyzed, and the concept of contrary genes proposed. The genes identified in the present study are involved in the positive regulation of transcription and activation of NF-κB-inducing kinase activity. The contrary genes and GO terms identified in the present study may potentially be used for DC diagnosis and treatment.

Expression and characterization of constitutive heat shock protein 70.1 (HSPA-1A) gene in in vitro produced and in vivo-derived buffalo (Bubalus bubalis) embryos.

PubMed

Sharma, G T; Nath, A; Prasad, S; Singhal, S; Singh, N; Gade, N E; Dubey, P K; Saikumar, G

2012-12-01

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA-1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA-1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo-derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA-1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA-1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8-16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA-1A revealed that it shares 91-98% identity with other mammalian sequences. It can be concluded that higher level of HSPA-1A mRNA in IVP embryos in comparison with in vivo-derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA-1A gene could be used as a stress biomarker during pre-implantation development. © 2012 Blackwell Verlag GmbH.

Novel genomic island modifies DNA with 7-deazaguanine derivatives

PubMed Central

Thiaville, Jennifer J.; Kellner, Stefanie M.; Yuan, Yifeng; Hutinet, Geoffrey; Thiaville, Patrick C.; Jumpathong, Watthanachai; Mohapatra, Susovan; Brochier-Armanet, Celine; Letarov, Andrey V.; Hillebrand, Roman; Malik, Chanchal K.; Rizzo, Carmelo J.; Dedon, Peter C.; de Crécy-Lagard, Valérie

2016-01-01

The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ0) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2’-deoxy-preQ0 and 2’-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ0 derivative, 2’-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism. PMID:26929322

Receptor protein kinase gene encoded at the self-incompatibility locus

DOEpatents

Nasrallah, June B.; Nasrallah, Mikhail E.; Stein, Joshua

1996-01-01

Described herein is a S receptor kinase gene (SRK), derived from the S locus in Brassica oleracea, having a extracellular domain highly similar to the secreted product of the S-locus glycoprotein gene.

Comparative mapping of powdery mildew resistance gene Pm21 and functional characterization of resistance-related genes in wheat.

PubMed

He, Huagang; Zhu, Shanying; Jiang, Zhengning; Ji, Yaoyong; Wang, Feng; Zhao, Renhui; Bie, Tongde

2016-04-01

The powdery mildew resistance gene Pm21 was physically and comparatively mapped by newly developed markers. Seven candidate genes were verified to be required for Pm21 -mediated resistance to wheat powdery mildew. Pm21, a gene derived from wheat wild relative Dasypyrum villosum, has been transferred into common wheat and widely utilized in wheat resistance breeding for powdery mildew. Previously, Pm21 has been located to the bin FL0.45-0.58 of 6VS by using deletion stocks. However, its fine mapping is still a hard work. In the present study, 30 gene-derived 6VS-specific markers were obtained based on the collinearity among genomes of Brachypodium distachyon, Oryza and Triticeae, and then physically and comparatively mapped in the bin FL0.45-0.58 and its nearby chromosome region. According to the maps, the bin FL0.45-0.58 carrying Pm21 was closely flanked by the markers 6VS-03 and 6VS-23, which further narrowed the orthologous regions to 1.06 Mb in Brachypodium and 1.38 Mb in rice, respectively. Among the conserved genes shared by Brachypodium and rice, four serine/threonine protein kinase genes (DvMPK1, DvMLPK, DvUPK and DvPSYR1), one protein phosphatase gene (DvPP2C) and two transcription factor genes (DvGATA and DvWHY) were confirmed to be required for Pm21-mediated resistance to wheat powdery mildew by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) and transcriptional pattern analyses. In summary, this study gives new insights into the genetic basis of the Pm21 locus and the disease resistance pathways mediated by Pm21.

Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Huiwu; Health and Science Center, SIBS CAS and SSMU, 225 South Chongqing Road, Shanghai 200025; Dai Kerong

2007-05-18

In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a {beta}-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified bymore » BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals.« less

The therapeutic potential of plant-derived vaccines and antibodies.

PubMed

Rodgers, P B; Hamilton, W D; Adair, J R

1999-03-01

The production of recombinant proteins in plants is reviewed with a particular focus on plant-derived vaccines and antibodies for human healthcare. Issues relating to foreign gene expression, such as protein yield, localisation and glycosylation are also considered. Emphasis is placed on reporting progress with preclinical and clinical evaluation of plant-derived vaccines and antibodies. An assessment is made of the likely future direction of research and development in this area.

Remodeling of Hepatic Metabolism and Hyperaminoacidemia in Mice Deficient in Proglucagon-Derived Peptides

PubMed Central

Watanabe, Chika; Seino, Yusuke; Miyahira, Hiroki; Yamamoto, Michiyo; Fukami, Ayako; Ozaki, Nobuaki; Takagishi, Yoshiko; Sato, Jun; Fukuwatari, Tsutomu; Shibata, Katsumi; Oiso, Yutaka; Murata, Yoshiharu; Hayashi, Yoshitaka

2012-01-01

Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon–green fluorescent protein (gfp) knock-in mice (Gcggfp/gfp: GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action. PMID:22187375

A universal reference sample derived from clone vector for improved detection of differential gene expression

PubMed Central

Khan, Rishi L; Gonye, Gregory E; Gao, Guang; Schwaber, James S

2006-01-01

Background Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs™. Results We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. Conclusion Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in

Gene therapy strategies for urological dysfunction.

PubMed

Chancellor, M B; Yoshimura, N; Pruchnic, R; Huard, J

2001-07-01

Novel molecular techniques such as conventional and ex vivo gene therapy, and tissue engineering have only recently been introduced to the field of urology. The lower urinary tract is ideally suited for minimally invasive therapy, and also ex vivo approaches would limit the risk of systemic side effects. Muscle-derived stem cells have been used successfully to treat stress incontinence, and rats with diabetic bladder dysfunction benefited from nerve growth factor (NGF)-based gene therapy. Nitric oxide synthase and capase-7 might provide suitable gene therapy targets for erectile dysfunction and benign prostatic hyperplasia, respectively.

Reconstructing directed gene regulatory network by only gene expression data.

PubMed

Zhang, Lu; Feng, Xi Kang; Ng, Yen Kaow; Li, Shuai Cheng

2016-08-18

Accurately identifying gene regulatory network is an important task in understanding in vivo biological activities. The inference of such networks is often accomplished through the use of gene expression data. Many methods have been developed to evaluate gene expression dependencies between transcription factor and its target genes, and some methods also eliminate transitive interactions. The regulatory (or edge) direction is undetermined if the target gene is also a transcription factor. Some methods predict the regulatory directions in the gene regulatory networks by locating the eQTL single nucleotide polymorphism, or by observing the gene expression changes when knocking out/down the candidate transcript factors; regrettably, these additional data are usually unavailable, especially for the samples deriving from human tissues. In this study, we propose the Context Based Dependency Network (CBDN), a method that is able to infer gene regulatory networks with the regulatory directions from gene expression data only. To determine the regulatory direction, CBDN computes the influence of source to target by evaluating the magnitude changes of expression dependencies between the target gene and the others with conditioning on the source gene. CBDN extends the data processing inequality by involving the dependency direction to distinguish between direct and transitive relationship between genes. We also define two types of important regulators which can influence a majority of the genes in the network directly or indirectly. CBDN can detect both of these two types of important regulators by averaging the influence functions of candidate regulator to the other genes. In our experiments with simulated and real data, even with the regulatory direction taken into account, CBDN outperforms the state-of-the-art approaches for inferring gene regulatory network. CBDN identifies the important regulators in the predicted network: 1. TYROBP influences a batch of genes that are

Gene Suppression of Mouse Testis In Vivo Using Small Interfering RNA Derived from Plasmid Vectors

PubMed Central

Takizawa, Takami; Ishikawa, Tomoko; Kosuge, Takuji; Mizuguchi, Yoshiaki; Sato, Yoko; Koji, Takehiko; Araki, Yoshihiko; Takizawa, Toshihiro

2012-01-01

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules. PMID:22489107

CAGEd-oPOSSUM: motif enrichment analysis from CAGE-derived TSSs.

PubMed

Arenillas, David J; Forrest, Alistair R R; Kawaji, Hideya; Lassmann, Timo; Wasserman, Wyeth W; Mathelier, Anthony

2016-09-15

With the emergence of large-scale Cap Analysis of Gene Expression (CAGE) datasets from individual labs and the FANTOM consortium, one can now analyze the cis-regulatory regions associated with gene transcription at an unprecedented level of refinement. By coupling transcription factor binding site (TFBS) enrichment analysis with CAGE-derived genomic regions, CAGEd-oPOSSUM can identify TFs that act as key regulators of genes involved in specific mammalian cell and tissue types. The webtool allows for the analysis of CAGE-derived transcription start sites (TSSs) either provided by the user or selected from ∼1300 mammalian samples from the FANTOM5 project with pre-computed TFBS predicted with JASPAR TF binding profiles. The tool helps power insights into the regulation of genes through the study of the specific usage of TSSs within specific cell types and/or under specific conditions. The CAGEd-oPOSUM web tool is implemented in Perl, MySQL and Apache and is available at http://cagedop.cmmt.ubc.ca/CAGEd_oPOSSUM CONTACTS: anthony.mathelier@ncmm.uio.no or wyeth@cmmt.ubc.ca Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

CAGEd-oPOSSUM: motif enrichment analysis from CAGE-derived TSSs

PubMed Central

Arenillas, David J.; Forrest, Alistair R. R.; Kawaji, Hideya; Lassmann, Timo; Wasserman, Wyeth W.; Mathelier, Anthony

2016-01-01

With the emergence of large-scale Cap Analysis of Gene Expression (CAGE) datasets from individual labs and the FANTOM consortium, one can now analyze the cis-regulatory regions associated with gene transcription at an unprecedented level of refinement. By coupling transcription factor binding site (TFBS) enrichment analysis with CAGE-derived genomic regions, CAGEd-oPOSSUM can identify TFs that act as key regulators of genes involved in specific mammalian cell and tissue types. The webtool allows for the analysis of CAGE-derived transcription start sites (TSSs) either provided by the user or selected from ∼1300 mammalian samples from the FANTOM5 project with pre-computed TFBS predicted with JASPAR TF binding profiles. The tool helps power insights into the regulation of genes through the study of the specific usage of TSSs within specific cell types and/or under specific conditions. Availability and Implementation: The CAGEd-oPOSUM web tool is implemented in Perl, MySQL and Apache and is available at http://cagedop.cmmt.ubc.ca/CAGEd_oPOSSUM. Contacts: anthony.mathelier@ncmm.uio.no or wyeth@cmmt.ubc.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27334471

[Multifunctional nano-vector for gene delivery into human adipose derived mesenchymal stem cells and in vitro cellular magnetic resonance imaging].

PubMed

Pang, Pengfei; Li, Bing; Hu, Xiaojun; Kang, Zhuang; Guan, Shouhai; Gong, Faming; Meng, Xiaochun; Li, Dan; Huang, Mingsheng; Shan, Hong

2014-04-08

To examine the feasibility and efficacy of using superparamagnetic iron oxide nanoparticles coated with polyethylene glycol-grafted polyethylenimine (PEG-g-PEI-SPION) as a carrier for gene delivery into human adipose derived mesenchymal stem cells (hADMSCs) and in vitro cellular magnetic resonance imaging (MRI). PEG-g-PEI-SPION was synthesized as previously reported. Gel electrophoresis was performed to assess the pDNA condensation capacity of PEG-g-PEI-SPION. The particle size and zeta potential of PEG-g-PEI-SPION/pDNA complexes were determined by dynamic light scattering. Cytotoxicity of PEG-g-PEI-SPION was evaluated by CCK-8 assay with hADMSCs. Gene transfection efficiency of PEG-g-PEI-SPION in hADMSCs was quantified by flow cytometry. The cellular internalization of PEG-g-PEI-SPION/pDNA nanocomplexes was studied by confocal laser scanning microscopy and Prussian blue staining. MRI function of PEG-g-PEI-SPION was studied by in vitro cellular MRI scanning. PEG-g-PEI-SPION condensed pDNA to form stable complexes of 80-100 nm in diameter and showed low cytotoxicity in hADMSCs. At the optimal N/P ratio of 20, PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of 22.8% ± 3.6% in hADMSCs. And it was higher than that obtained with lipofectamine 11.2% ± 2.6% (P < 0.05). Furthermore, hADMSCs labeled with PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T2-weighted images in vitro. PEG-g-PEI-SPION is an efficient and MRI-visible nano-vector for gene delivery into hADMSCs.

A functional brain-derived neurotrophic factor (BDNF) gene variant increases the risk of moderate-to-severe allergic rhinitis.

PubMed

Jin, Peng; Andiappan, Anand Kumar; Quek, Jia Min; Lee, Bernett; Au, Bijin; Sio, Yang Yie; Irwanto, Astrid; Schurmann, Claudia; Grabe, Hans Jörgen; Suri, Bani Kaur; Matta, Sri Anusha; Westra, Harm-Jan; Franke, Lude; Esko, Tonu; Sun, Liangdan; Zhang, Xuejun; Liu, Hong; Zhang, Furen; Larbi, Anis; Xu, Xin; Poidinger, Michael; Liu, Jianjun; Chew, Fook Tim; Rotzschke, Olaf; Shi, Li; Wang, De Yun

2015-06-01

Brain-derived neurotrophic factor (BDNF) is a secretory protein that has been implicated in the pathogenesis of allergic rhinitis (AR), atopic asthma, and eczema, but it is currently unknown whether BDNF polymorphisms influence susceptibility to moderate-to-severe AR. We sought to identify disease associations and the functional effect of BDNF genetic variants in patients with moderate-to-severe AR. Tagging single nucleotide polymorphisms (SNPs) spanning the BDNF gene were selected from the human HapMap Han Chinese from Beijing (CHB) data set, and associations with moderate-to-severe AR were assessed in 2 independent cohorts of Chinese patients (2216 from Shandong province and 1239 living in Singapore). The functional effects of the BDNF genetic variants were determined by using both in vitro and ex vivo assays. The tagging SNP rs10767664 was significantly associated with the risk of moderate-to-severe AR in both Singapore Chinese (P = .0017; odds ratio, 1.324) and Shandong Chinese populations (P = .039; odds ratio, 1.180). The coding nonsynonymous SNP rs6265 was in perfect linkage with rs10767664 and conferred increased BDNF protein secretion by a human cell line in vitro. Subjects bearing the AA genotype of rs10767664 exhibited increased risk of moderate-to-severe AR and displayed increased BDNF protein and total IgE levels in plasma. Using a large-scale expression quantitative trait locus study, we demonstrated that BDNF SNPs are significantly associated with altered BDNF concentrations in peripheral blood. A common genetic variant of the BDNF gene is associated with increased risk of moderate-to-severe AR, and the AA genotype is associated with increased BDNF mRNA levels in peripheral blood. Together, these data indicate that functional BDNF gene variants increase the risk of moderate-to-severe AR. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Detection of drought tolerant genes within seedling apple rootstocks in Syria

USDA-ARS?s Scientific Manuscript database

This investigation was conducted to detect the drought tolerant genes (four genes) within seedling apple rootstocks derived from five apple genotypes, including Syrian apple cultivars. The results showed that the gene MdPepPro (a cyclophilin) was found in all studied genotypes and their progenies e...

Eukaryotic genes of archaebacterial origin are more important than the more numerous eubacterial genes, irrespective of function.

PubMed

Cotton, James A; McInerney, James O

2010-10-05

The traditional tree of life shows eukaryotes as a distinct lineage of living things, but many studies have suggested that the first eukaryotic cells were chimeric, descended from both Eubacteria (through the mitochondrion) and Archaebacteria. Eukaryote nuclei thus contain genes of both eubacterial and archaebacterial origins, and these genes have different functions within eukaryotic cells. Here we report that archaebacterium-derived genes are significantly more likely to be essential to yeast viability, are more highly expressed, and are significantly more highly connected and more central in the yeast protein interaction network. These findings hold irrespective of whether the genes have an informational or operational function, so that many features of eukaryotic genes with prokaryotic homologs can be explained by their origin, rather than their function. Taken together, our results show that genes of archaebacterial origin are in some senses more important to yeast metabolism than genes of eubacterial origin. This importance reflects these genes' origin as the ancestral nuclear component of the eukaryotic genome.

Gene annotation from scientific literature using mappings between keyword systems.

PubMed

Pérez, Antonio J; Perez-Iratxeta, Carolina; Bork, Peer; Thode, Guillermo; Andrade, Miguel A

2004-09-01

The description of genes in databases by keywords helps the non-specialist to quickly grasp the properties of a gene and increases the efficiency of computational tools that are applied to gene data (e.g. searching a gene database for sequences related to a particular biological process). However, the association of keywords to genes or protein sequences is a difficult process that ultimately implies examination of the literature related to a gene. To support this task, we present a procedure to derive keywords from the set of scientific abstracts related to a gene. Our system is based on the automated extraction of mappings between related terms from different databases using a model of fuzzy associations that can be applied with all generality to any pair of linked databases. We tested the system by annotating genes of the SWISS-PROT database with keywords derived from the abstracts linked to their entries (stored in the MEDLINE database of scientific references). The performance of the annotation procedure was much better for SWISS-PROT keywords (recall of 47%, precision of 68%) than for Gene Ontology terms (recall of 8%, precision of 67%). The algorithm can be publicly accessed and used for the annotation of sequences through a web server at http://www.bork.embl.de/kat

GeneRIF indexing: sentence selection based on machine learning.

PubMed

Jimeno-Yepes, Antonio J; Sticco, J Caitlin; Mork, James G; Aronson, Alan R

2013-05-31

A Gene Reference Into Function (GeneRIF) describes novel functionality of genes. GeneRIFs are available from the National Center for Biotechnology Information (NCBI) Gene database. GeneRIF indexing is performed manually, and the intention of our work is to provide methods to support creating the GeneRIF entries. The creation of GeneRIF entries involves the identification of the genes mentioned in MEDLINE®; citations and the sentences describing a novel function. We have compared several learning algorithms and several features extracted or derived from MEDLINE sentences to determine if a sentence should be selected for GeneRIF indexing. Features are derived from the sentences or using mechanisms to augment the information provided by them: assigning a discourse label using a previously trained model, for example. We show that machine learning approaches with specific feature combinations achieve results close to one of the annotators. We have evaluated different feature sets and learning algorithms. In particular, Naïve Bayes achieves better performance with a selection of features similar to one used in related work, which considers the location of the sentence, the discourse of the sentence and the functional terminology in it. The current performance is at a level similar to human annotation and it shows that machine learning can be used to automate the task of sentence selection for GeneRIF annotation. The current experiments are limited to the human species. We would like to see how the methodology can be extended to other species, specifically the normalization of gene mentions in other species.

An efficient annotation and gene-expression derivation tool for Illumina Solexa datasets.

PubMed

Hosseini, Parsa; Tremblay, Arianne; Matthews, Benjamin F; Alkharouf, Nadim W

2010-07-02

The data produced by an Illumina flow cell with all eight lanes occupied, produces well over a terabyte worth of images with gigabytes of reads following sequence alignment. The ability to translate such reads into meaningful annotation is therefore of great concern and importance. Very easily, one can get flooded with such a great volume of textual, unannotated data irrespective of read quality or size. CASAVA, a optional analysis tool for Illumina sequencing experiments, enables the ability to understand INDEL detection, SNP information, and allele calling. To not only extract from such analysis, a measure of gene expression in the form of tag-counts, but furthermore to annotate such reads is therefore of significant value. We developed TASE (Tag counting and Analysis of Solexa Experiments), a rapid tag-counting and annotation software tool specifically designed for Illumina CASAVA sequencing datasets. Developed in Java and deployed using jTDS JDBC driver and a SQL Server backend, TASE provides an extremely fast means of calculating gene expression through tag-counts while annotating sequenced reads with the gene's presumed function, from any given CASAVA-build. Such a build is generated for both DNA and RNA sequencing. Analysis is broken into two distinct components: DNA sequence or read concatenation, followed by tag-counting and annotation. The end result produces output containing the homology-based functional annotation and respective gene expression measure signifying how many times sequenced reads were found within the genomic ranges of functional annotations. TASE is a powerful tool to facilitate the process of annotating a given Illumina Solexa sequencing dataset. Our results indicate that both homology-based annotation and tag-count analysis are achieved in very efficient times, providing researchers to delve deep in a given CASAVA-build and maximize information extraction from a sequencing dataset. TASE is specially designed to translate sequence data

An efficient annotation and gene-expression derivation tool for Illumina Solexa datasets

PubMed Central

2010-01-01

Background The data produced by an Illumina flow cell with all eight lanes occupied, produces well over a terabyte worth of images with gigabytes of reads following sequence alignment. The ability to translate such reads into meaningful annotation is therefore of great concern and importance. Very easily, one can get flooded with such a great volume of textual, unannotated data irrespective of read quality or size. CASAVA, a optional analysis tool for Illumina sequencing experiments, enables the ability to understand INDEL detection, SNP information, and allele calling. To not only extract from such analysis, a measure of gene expression in the form of tag-counts, but furthermore to annotate such reads is therefore of significant value. Findings We developed TASE (Tag counting and Analysis of Solexa Experiments), a rapid tag-counting and annotation software tool specifically designed for Illumina CASAVA sequencing datasets. Developed in Java and deployed using jTDS JDBC driver and a SQL Server backend, TASE provides an extremely fast means of calculating gene expression through tag-counts while annotating sequenced reads with the gene's presumed function, from any given CASAVA-build. Such a build is generated for both DNA and RNA sequencing. Analysis is broken into two distinct components: DNA sequence or read concatenation, followed by tag-counting and annotation. The end result produces output containing the homology-based functional annotation and respective gene expression measure signifying how many times sequenced reads were found within the genomic ranges of functional annotations. Conclusions TASE is a powerful tool to facilitate the process of annotating a given Illumina Solexa sequencing dataset. Our results indicate that both homology-based annotation and tag-count analysis are achieved in very efficient times, providing researchers to delve deep in a given CASAVA-build and maximize information extraction from a sequencing dataset. TASE is specially

Hyperphagia, Severe Obesity, Impaired Cognitive Function, and Hyperactivity Associated With Functional Loss of One Copy of the Brain-Derived Neurotrophic Factor (BDNF) Gene

PubMed Central

Gray, Juliette; Yeo, Giles S.H.; Cox, James J.; Morton, Jenny; Adlam, Anna-Lynne R.; Keogh, Julia M.; Yanovski, Jack A.; El Gharbawy, Areeg; Han, Joan C.; Tung, Y.C. Loraine; Hodges, John R.; Raymond, F. Lucy; O’Rahilly, Stephen; Farooqi, I. Sadaf

2008-01-01

The neurotrophin brain-derived neurotrophic factor (BDNF) inhibits food intake, and rodent models of BDNF disruption all exhibit increased food intake and obesity, as well as hyperactivity. We report an 8-year-old girl with hyperphagia and severe obesity, impaired cognitive function, and hyperactivity who harbored a de novo chromosomal inversion, 46,XX,inv(11)(p13p15.3), a region encompassing the BDNF gene. We have identified the proximal inversion breakpoint that lies 850 kb telomeric of the 5′ end of the BDNF gene. The patient’s genomic DNA was heterozygous for a common coding polymorphism in BDNF, but monoallelic expression was seen in peripheral lymphocytes. Serum concentration of BDNF protein was reduced compared with age- and BMI-matched subjects. Haploinsufficiency for BDNF was associated with increased ad libitum food intake, severe early-onset obesity, hyper-activity, and cognitive impairment. These findings provide direct evidence for the role of the neurotrophin BDNF in human energy homeostasis, as well as in cognitive function, memory, and behavior. PMID:17130481

Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

PubMed

Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

2016-02-01

Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular Structure-Based Large-Scale Prediction of Chemical-Induced Gene Expression Changes.

PubMed

Liu, Ruifeng; AbdulHameed, Mohamed Diwan M; Wallqvist, Anders

2017-09-25

The quantitative structure-activity relationship (QSAR) approach has been used to model a wide range of chemical-induced biological responses. However, it had not been utilized to model chemical-induced genomewide gene expression changes until very recently, owing to the complexity of training and evaluating a very large number of models. To address this issue, we examined the performance of a variable nearest neighbor (v-NN) method that uses information on near neighbors conforming to the principle that similar structures have similar activities. Using a data set of gene expression signatures of 13 150 compounds derived from cell-based measurements in the NIH Library of Integrated Network-based Cellular Signatures program, we were able to make predictions for 62% of the compounds in a 10-fold cross validation test, with a correlation coefficient of 0.61 between the predicted and experimentally derived signatures-a reproducibility rivaling that of high-throughput gene expression measurements. To evaluate the utility of the predicted gene expression signatures, we compared the predicted and experimentally derived signatures in their ability to identify drugs known to cause specific liver, kidney, and heart injuries. Overall, the predicted and experimentally derived signatures had similar receiver operating characteristics, whose areas under the curve ranged from 0.71 to 0.77 and 0.70 to 0.73, respectively, across the three organ injury models. However, detailed analyses of enrichment curves indicate that signatures predicted from multiple near neighbors outperformed those derived from experiments, suggesting that averaging information from near neighbors may help improve the signal from gene expression measurements. Our results demonstrate that the v-NN method can serve as a practical approach for modeling large-scale, genomewide, chemical-induced, gene expression changes.

Gene transfer to the cerebellum.

PubMed

Louboutin, Jean-Pierre; Reyes, Beverly A S; Van Bockstaele, Elisabeth J; Strayer, David S

2010-12-01

There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4 weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.

Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.

PubMed

Chen, Jian; Lin, Mingyan; Foxe, John J; Pedrosa, Erika; Hrabovsky, Anastasia; Carroll, Reed; Zheng, Deyou; Lachman, Herbert M

2013-01-01

Induced pluripotent stem cell (iPSC) technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD) and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs) and fibroblasts (F-iPSCs). This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (p<0.05), of which 63 remained significant after correcting for genome-wide analysis (FDR <0.05). The most striking difference was the lower level of expression detected for numerous members of the all four HOX gene families in neurons derived from T-iPSCs. In addition, an increased level of expression was seen for several transcription factors expressed in the developing forebrain (FOXP2, OTX1, and LHX2, for example). Overall, pathway analysis revealed that differentially expressed genes that showed higher levels of expression in neurons derived from T-iPSCs were enriched for genes implicated in schizophrenia (SZ). The findings suggest that neurons derived from T-iPSCs are suitable for disease-modeling neuropsychiatric

Polyploidization altered gene functions in cotton (Gossypium spp.)

USDA-ARS?s Scientific Manuscript database

Cotton fibers are seed trichomes derived from individual cells of the epidermal layer of the seed coat. It has been known for a long time that a large set of genes determine the development of cotton fiber, and more recently it has been determined that these genes are distributed across the At and ...

Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents1[OPEN

PubMed Central

2015-01-01

Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays. The increases of Asp, Lys, and Met in ak-hsdh2 were also observed in ak1-1, ak2-1, ak3-1, and ak-hsdh1-1. However, the Thr increase in ak-hsdh2 was observed in ak-hsdh1-1 but not in ak1-1, ak2-1, or ak3-1. Activity assays showed that AK2 and AK-HSDH1 are the major contributors to overall AK and HSDH activities, respectively. Pairwise correlation analysis revealed positive correlations between the amount of AK transcripts and Lys-sensitive AK activity and between the amount of AK-HSDH transcripts and both Thr-sensitive AK activity and total HSDH activity. In addition, the ratio of total AK activity to total HSDH activity negatively correlates with the ratio of Lys to the total amount of Met, Thr, and isoleucine. These data led to the hypothesis that the balance between Lys-sensitive AKs and Thr-sensitive AK-HSDHs is important for maintaining the amounts and ratios of Asp-derived amino acids. PMID:26063505

Identification of a novel HLA-A*02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene

PubMed Central

YOSHIMURA, MAYUKO; TADA, YOSHITAKA; OFUZI, KAZUYA; YAMAMOTO, MASAKAZU; NAKATSURA, TETSUYA

2014-01-01

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A*02:01- and HLA-A*24:02-restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK-derived candidate peptides for the induction of tumor-reactive CTLs. Nine EML4-ALK-derived peptides were selected by a computer algorithm based on a permissive HLA-A*02:01 or HLA-A*24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide-specific CTL clone. This CTL clone specifically recognized peptide-pulsed T2 cells and H2228 cells expressing HLA-A*02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA-class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A*02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4-ALK-positive cancers. PMID:24842630

Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene.

PubMed

Yoshimura, Mayuko; Tada, Yoshitaka; Ofuzi, Kazuya; Yamamoto, Masakazu; Nakatsura, Tetsuya

2014-07-01

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A 02:01- and HLA-A 24:02‑restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK‑derived candidate peptides for the induction of tumor‑reactive CTLs. Nine EML4-ALK‑derived peptides were selected by a computer algorithm based on a permissive HLA-A 02:01 or HLA-A 24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide‑specific CTL clone. This CTL clone specifically recognized peptide‑pulsed T2 cells and H2228 cells expressing HLA-A 02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA‑class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A 02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4‑ALK-positive cancers.

Induction of the plasticity-associated immediate early gene Arc by stress and hallucinogens: role of brain-derived neurotrophic factor.

PubMed

Benekareddy, Madhurima; Nair, Amrita R; Dias, Brian G; Suri, Deepika; Autry, Anita E; Monteggia, Lisa M; Vaidya, Vidita A

2013-03-01

Exposure to stress and hallucinogens in adulthood evokes persistent alterations in neurocircuitry and emotional behaviour. The structural and functional changes induced by stress and hallucinogen exposure are thought to involve transcriptional alterations in specific effector immediate early genes. The immediate early gene, activity regulated cytoskeletal-associated protein (Arc), is important for both activity and experience dependent plasticity. We sought to examine whether trophic factor signalling through brain-derived neurotrophic factor (BDNF) contributes to the neocortical regulation of Arc mRNA in response to distinct stimuli such as immobilization stress and the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI). Acute exposure to either immobilization stress or DOI induced Arc mRNA levels within the neocortex. BDNF infusion into the neocortex led to a robust up-regulation of local Arc transcript expression. Further, baseline Arc mRNA expression in the neocortex was significantly decreased in inducible BDNF knockout mice with an adult-onset, forebrain specific BDNF loss. The induction of Arc mRNA levels in response to both acute immobilization stress or a single administration of DOI was significantly attenuated in the inducible BDNF knockout mice. Taken together, our results implicate trophic factor signalling through BDNF in the regulation of cortical Arc mRNA expression, both under baseline conditions and following stress and hallucinogen exposure. These findings suggest the possibility that the regulation of Arc expression via BDNF provides a molecular substrate for the structural and synaptic plasticity observed following stimuli such as stress and hallucinogens.

Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue.

PubMed

Synnergren, Jane; Améen, Caroline; Jansson, Andreas; Sartipy, Peter

2012-02-27

It is now well documented that human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes. These cells constitute a promising source of material for use in drug development, toxicity testing, and regenerative medicine. To assess their utility as replacement or complement to existing models, extensive phenotypic characterization of the cells is required. In the present study, we used microarrays and analyzed the global transcription of hESC-derived cardiomyocyte clusters (CMCs) and determined similarities as well as differences compared with reference samples from fetal and adult heart tissue. In addition, we performed a focused analysis of the expression of cardiac ion channels and genes involved in the Ca(2+)-handling machinery, which in previous studies have been shown to be immature in stem cell-derived cardiomyocytes. Our results show that hESC-derived CMCs, on a global level, have a highly similar gene expression profile compared with human heart tissue, and their transcriptional phenotype was more similar to fetal than to adult heart. Despite the high similarity to heart tissue, a number of significantly differentially expressed genes were identified, providing some clues toward understanding the molecular difference between in vivo sourced tissue and stem cell derivatives generated in vitro. Interestingly, some of the cardiac-related ion channels and Ca(2+)-handling genes showed differential expression between the CMCs and heart tissues. These genes may represent candidates for future genetic engineering to create hESC-derived CMCs that better mimic the phenotype of the cardiomyocytes present in the adult human heart.

Not so bad after all: retroviruses and long terminal repeat retrotransposons as a source of new genes in vertebrates.

PubMed

Naville, M; Warren, I A; Haftek-Terreau, Z; Chalopin, D; Brunet, F; Levin, P; Galiana, D; Volff, J-N

2016-04-01

Viruses and transposable elements, once considered as purely junk and selfish sequences, have repeatedly been used as a source of novel protein-coding genes during the evolution of most eukaryotic lineages, a phenomenon called 'molecular domestication'. This is exemplified perfectly in mammals and other vertebrates, where many genes derived from long terminal repeat (LTR) retroelements (retroviruses and LTR retrotransposons) have been identified through comparative genomics and functional analyses. In particular, genes derived from gag structural protein and envelope (env) genes, as well as from the integrase-coding and protease-coding sequences, have been identified in humans and other vertebrates. Retroelement-derived genes are involved in many important biological processes including placenta formation, cognitive functions in the brain and immunity against retroelements, as well as in cell proliferation, apoptosis and cancer. These observations support an important role of retroelement-derived genes in the evolution and diversification of the vertebrate lineage. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

In Utero Fine Particle Air Pollution and Placental Expression of Genes in the Brain-Derived Neurotrophic Factor Signaling Pathway: An ENVIRONAGE Birth Cohort Study.

PubMed

Saenen, Nelly D; Plusquin, Michelle; Bijnens, Esmée; Janssen, Bram G; Gyselaers, Wilfried; Cox, Bianca; Fierens, Frans; Molenberghs, Geert; Penders, Joris; Vrijens, Karen; De Boever, Patrick; Nawrot, Tim S

2015-08-01

Developmental processes in the placenta and the fetal brain are shaped by the same biological signals. Recent evidence suggests that adaptive responses of the placenta to the maternal environment may influence central nervous system development. We studied the association between in utero exposure to fine particle air pollution with a diameter ≤ 2.5 μm (PM2.5) and placental expression of genes implicated in neural development. Expression of 10 target genes in the brain-derived neurotrophic factor (BDNF) signaling pathway were quantified in placental tissue of 90 mother-infant pairs from the ENVIRONAGE birth cohort using quantitative real-time polymerase chain reaction. Trimester-specific PM2.5 exposure levels were estimated for each mother's home address using a spatiotemporal model. Mixed-effects models were used to evaluate the association between the target genes and PM2.5 exposure measured in different time windows of pregnancy. A 5-μg/m3 increase in residential PM2.5 exposure during the first trimester of pregnancy was associated with a 15.9% decrease [95% confidence interval (CI): -28.7, -3.2%, p = 0.015] in expression of placental BDNF at birth. The corresponding estimate for synapsin 1 (SYN1) was a 24.3% decrease (95% CI: -42.8, -5.8%, p = 0.011). Placental expression of BDNF and SYN1, two genes implicated in normal neurodevelopmental trajectories, decreased with increasing in utero exposure to PM2.5. Future studies are needed to confirm our findings and evaluate the potential relevance of associations between PM2.5 and placental expression of BDNF and SYN1 on neurodevelopment. We provide the first molecular epidemiological evidence concerning associations between in utero fine particle air pollution exposure and the expression of genes that may influence neurodevelopmental processes.

rs10767664 Gene Variant in Brain-Derived Neurotrophic Factor Is Associated with Diabetes Mellitus Type 2 in Caucasian Females with Obesity.

PubMed

de Luis, Daniel Antonio; Aller, Rocío; Izaola, Olatz; Primo, David; Romero, Enrique

2017-01-01

The role of brain-derived neurotrophic factor (BDNF) variants on diabetes prevalence, basal adipokine levels, body weight, and cardiovascular risk factors remains unclear in obese patients. This study is aimed at analyzing the effects of rs10767664 BDNF gene polymorphism on diabetes mellitus prevalence, body weight, cardiovascular risk factors, and serum adipokine levels in obese female patients. A total of 507 obese women were enrolled in a prospective way. Biochemical evaluation and anthropometric measures were recorded. The frequency of diabetes mellitus in the group of patients with non-T allele was 20.1 and 28.3% in T-allele carriers. Logistic regression showed a risk of diabetes mellitus of 1.33 (95% CI 1.17-2.08) in subjects with T allele adjusted by age and body mass index (BMI). T-allele carriers with diabetes mellitus have a higher weight, BMI, waist circumference, blood pressure, glucose, homeostasis model assessment insulin resistance (HOMA-IR), insulin, and C-reactive protein (CRP) levels than non-T-allele carriers. rs10767664 polymorphism of BDNF gene is associated with prevalence of diabetes mellitus in obese female patients. T-allele carriers with diabetes mellitus have a higher weight, fat mass, blood pressure, level of insulin, glucose, HOMA-IR, and CRP than non-T-allele carriers. © 2017 S. Karger AG, Basel.

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells.

PubMed

Matsunaga, Yusuke; Fukuma, Daiki; Hirata, Shinya; Fukushima, Satoshi; Haruta, Miwa; Ikeda, Tokunori; Negishi, Izumi; Nishimura, Yasuharu; Senju, Satoru

2008-11-01

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

Mapping fusiform rust resistance genes within a complex mating design of loblolly pine

Treesearch

Tania Quesada; Marcio F.R. Resende Jr.; Patricio Munoz; Jill L. Wegrzyn; David B. Neale; Matias Kirst; Gary F. Peter; Salvador A. Gezan; C.Dana Nelson; John M. Davis

2014-01-01

Fusiform rust resistance can involve gene-for-gene interactions where resistance (Fr) genes in the host interact with corresponding avirulence genes in the pathogen, Cronartium quercuum f.sp. fusiforme (Cqf). Here, we identify trees with Fr genes in a loblolly pine population derived from a complex mating design challenged with two Cqf inocula (one gall and 10 gall...

In vitro transfection of plasmid DNA by amine derivatives of gelatin accompanied with ultrasound irradiation.

PubMed

Hosseinkhani, Hossein; Aoyama, Ternyoshi; Yamamoto, Shingo; Ogawa, Osamu; Tabata, Yasuhiko

2002-10-01

The purpose of this study is to examine the ultrasound (US)-enhanced gene expression by the complexes of a plasmid DNA with gelatin derivatives of aminization. Gelatin derivatives with different introduced extents of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were prepared with a water-soluble carbodiimide. The molecular size and zeta potential of the gelatin derivatives before and after complexation with the plasmid DNA were examined. After incubation with the complexes with or without US exposure, the DNA expression of rat gastric mucosal cells was measured to evaluate the effect of the type of gelatin derivatives on their gene expression. The cell uptake of the complexes, the cell viability, and the buffering effect of gelatin derivatives were examined. The apparent molecular size and zeta potential of gelatin derivatives became larger as their aminization extent increased although the Sm gelatin derivative of higher aminization showed a larger value than other corresponding derivatives. Irrespective of the type of gelatin derivatives, the apparent molecular size of plasmid DNA was reduced by increasing the gelatin-DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin-DNA complexes showed the zeta potential of 10-15 mV. The cells incubated with the complex exhibited significantly stronger luciferase activities than free plasmid DNA, and the activity was further enhanced by US irradiation. The enhancement was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. The amount of plasmid DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative, whereas US irradiation did not significantly increase the DNA internalization. US irradiation had no effect on the viability of cells incubated with every gelatin derivative-plasmid DNA complex, although the viability was decreased by the complex incubation. The buffering capacity of Sm derivative

Narrowing down the region of the Vf locus for scab resistance in apple using AFLP-derived SCARs.

PubMed

Huaracha, E; Xu, M; Korban, S S

2004-01-01

A narrow-down strategy to restrict the Vf region, which controls resistance to the fungal disease apple scab in apple, to a genetic distance of 0.4 cM is presented. Using 11 AFLP-derived SCARs and three RAPD-derived SCARs, all linked to the Vf gene, we subjected 1,412 scab-resistant individuals from 16 mapping populations to genotype analysis. Eleven recombinant individuals were identified within a genetic distance of 0.9 cM around the Vf gene. Using these 11 recombinants, we achieved fine-resolution of several AFLP-derived SCAR markers surrounding the Vf gene, resulting in the following genetic linkage map: ACS-6 and ACS are located left of the Vf gene at genetic distances of 0.2 cM and 0.1 cM, respectively; ACS-7 and ACS-9 are inseparable from the Vf gene; ACS-8, ACS-10, and ACS-4 are located to the right of the Vf gene at genetic distances of 0.1 cM, 0.4 cM, and 0.5 cM, respectively; the remaining five SCARs-ACS-11, ACS-5, ACS-2, ACS-1, and AL07-are inseparable and are located right of the Vf gene at a genetic distance of 0.7 cM. By integrating this linkage data with our previous physical map, we generated a revised map of the narrowed-down region of Vf.

Nutrient-gene interactions in early pregnancy: a vascular hypothesis.

PubMed

Steegers-Theunissen, R P M; Steegers, E A P

2003-02-10

It is hypothesized that the following periconceptional and early pregnancy nutrient-gene interactions link vascular-related reproductive complications and cardiovascular diseases in adulthood: (1) Maternal and paternal genetically controlled nutrient status affects the quality of gametes and fertilization capacity; (2) The embryonic genetic constitution, derived from both parents, and the maternal genetically controlled nutrient environment determine embryogenesis and fetal growth; (3) Trophoblast invasion of decidua and spiral arteries is driven by genes derived from both parents as well as by maternal nutritional factors; (4) Angiogenesis, vasculogenesis and vascular function are dependent on the genetic constitution of the embryo, derived from both parents, and the maternal genetically controlled nutritional environment.Early intra-uterine programming of vessels may concern the same (in)dependent determinants of vascular-related complications during pregnancy and cardiovascular diseases in later life.

"Bad genes" & criminal responsibility.

PubMed

González-Tapia, María Isabel; Obsuth, Ingrid

2015-01-01

The genetics of the accused is trying to break into the courts. To date several candidate genes have been put forward and their links to antisocial behavior have been examined and documented with some consistency. In this paper, we focus on the so called "warrior gene", or the low-activity allele of the MAOA gene, which has been most consistently related to human behavior and specifically to violence and antisocial behavior. In preparing this paper we had two objectives. First, to summarize and analyze the current scientific evidence, in order to gain an in depth understanding of the state of the issue and determine whether a dominant line of generally accepted scientific knowledge in this field can be asserted. Second, to derive conclusions and put forward recommendations related to the use of genetic information, specifically the presence of the low-activity genotype of the MAOA gene, in modulation of criminal responsibility in European and US courts. Copyright © 2015 Elsevier Ltd. All rights reserved.

The low-recombining pericentromeric region of barley restricts gene diversity and evolution but not gene expression

PubMed Central

Baker, Katie; Bayer, Micha; Cook, Nicola; Dreißig, Steven; Dhillon, Taniya; Russell, Joanne; Hedley, Pete E; Morris, Jenny; Ramsay, Luke; Colas, Isabelle; Waugh, Robbie; Steffenson, Brian; Milne, Iain; Stephen, Gordon; Marshall, David; Flavell, Andrew J

2014-01-01

The low-recombining pericentromeric region of the barley genome contains roughly a quarter of the genes of the species, embedded in low-recombining DNA that is rich in repeats and repressive chromatin signatures. We have investigated the effects of pericentromeric region residency upon the expression, diversity and evolution of these genes. We observe no significant difference in average transcript level or developmental RNA specificity between the barley pericentromeric region and the rest of the genome. In contrast, all of the evolutionary parameters studied here show evidence of compromised gene evolution in this region. First, genes within the pericentromeric region of wild barley show reduced diversity and significantly weakened purifying selection compared with the rest of the genome. Second, gene duplicates (ohnolog pairs) derived from the cereal whole-genome duplication event ca. 60MYa have been completely eliminated from the barley pericentromeric region. Third, local gene duplication in the pericentromeric region is reduced by 29% relative to the rest of the genome. Thus, the pericentromeric region of barley is a permissive environment for gene expression but has restricted gene evolution in a sizeable fraction of barley's genes. PMID:24947331

Modeling leaderless transcription and atypical genes results in more accurate gene prediction in prokaryotes.

PubMed

Lomsadze, Alexandre; Gemayel, Karl; Tang, Shiyuyun; Borodovsky, Mark

2018-05-17

In a conventional view of the prokaryotic genome organization, promoters precede operons and ribosome binding sites (RBSs) with Shine-Dalgarno consensus precede genes. However, recent experimental research suggesting a more diverse view motivated us to develop an algorithm with improved gene-finding accuracy. We describe GeneMarkS-2, an ab initio algorithm that uses a model derived by self-training for finding species-specific (native) genes, along with an array of precomputed "heuristic" models designed to identify harder-to-detect genes (likely horizontally transferred). Importantly, we designed GeneMarkS-2 to identify several types of distinct sequence patterns (signals) involved in gene expression control, among them the patterns characteristic for leaderless transcription as well as noncanonical RBS patterns. To assess the accuracy of GeneMarkS-2, we used genes validated by COG (Clusters of Orthologous Groups) annotation, proteomics experiments, and N-terminal protein sequencing. We observed that GeneMarkS-2 performed better on average in all accuracy measures when compared with the current state-of-the-art gene prediction tools. Furthermore, the screening of ∼5000 representative prokaryotic genomes made by GeneMarkS-2 predicted frequent leaderless transcription in both archaea and bacteria. We also observed that the RBS sites in some species with leadered transcription did not necessarily exhibit the Shine-Dalgarno consensus. The modeling of different types of sequence motifs regulating gene expression prompted a division of prokaryotic genomes into five categories with distinct sequence patterns around the gene starts. © 2018 Lomsadze et al.; Published by Cold Spring Harbor Laboratory Press.

Responses of human cells to ZnO nanoparticles: a gene transcription study†

PubMed Central

Moos, Philip J.; Olszewski, Kyle; Honeggar, Matthew; Cassidy, Pamela; Leachman, Sancy; Woessner, David; Cutler, N. Shane; Veranth, John M.

2013-01-01

The gene transcript profile responses to metal oxide nanoparticles was studied using human cell lines derived from the colon and skin tumors. Much of the research on nanoparticle toxicology has focused on models of inhalation and intact skin exposure, and effects of ingestion exposure and application to diseased skin are relatively unknown. Powders of nominally nanosized SiO2, TiO2, ZnO and Fe2O3 were chosen because these substances are widely used in consumer products. The four oxides were evaluated using colon-derived cell lines, RKO and CaCo-2, and ZnO and TiO2 were evaluated further using skin-derived cell lines HaCaT and SK Mel-28. ZnO induced the most notable gene transcription changes, even though this material was applied at the lowest concentration. Nano-sized and conventional ZnO induced similar responses suggesting common mechanisms of action. The results showed neither a non-specific response pattern common to all substances nor synergy of the particles with TNF-α cotreatment. The response to ZnO was not consistent with a pronounced proinflammatory signature, but involved changes in metal metabolism, chaperonin proteins, and protein folding genes. This response was observed in all cell lines when ZnO was in contact with the human cells. When the cells were exposed to soluble Zn, the genes involved in metal metabolism were induced but the genes involved in protein refoldling were unaffected. This provides some of the first data on the effects of commercial metal oxide nanoparticles on human colon-derived and skin-derived cells. PMID:21769377

Quantitative comparison of microarray experiments with published leukemia related gene expression signatures.

PubMed

Klein, Hans-Ulrich; Ruckert, Christian; Kohlmann, Alexander; Bullinger, Lars; Thiede, Christian; Haferlach, Torsten; Dugas, Martin

2009-12-15

Multiple gene expression signatures derived from microarray experiments have been published in the field of leukemia research. A comparison of these signatures with results from new experiments is useful for verification as well as for interpretation of the results obtained. Currently, the percentage of overlapping genes is frequently used to compare published gene signatures against a signature derived from a new experiment. However, it has been shown that the percentage of overlapping genes is of limited use for comparing two experiments due to the variability of gene signatures caused by different array platforms or assay-specific influencing parameters. Here, we present a robust approach for a systematic and quantitative comparison of published gene expression signatures with an exemplary query dataset. A database storing 138 leukemia-related published gene signatures was designed. Each gene signature was manually annotated with terms according to a leukemia-specific taxonomy. Two analysis steps are implemented to compare a new microarray dataset with the results from previous experiments stored and curated in the database. First, the global test method is applied to assess gene signatures and to constitute a ranking among them. In a subsequent analysis step, the focus is shifted from single gene signatures to chromosomal aberrations or molecular mutations as modeled in the taxonomy. Potentially interesting disease characteristics are detected based on the ranking of gene signatures associated with these aberrations stored in the database. Two example analyses are presented. An implementation of the approach is freely available as web-based application. The presented approach helps researchers to systematically integrate the knowledge derived from numerous microarray experiments into the analysis of a new dataset. By means of example leukemia datasets we demonstrate that this approach detects related experiments as well as related molecular mutations and may

Differential gene expression in small and large rainbow trout derived from two seasonal spawning groups

PubMed Central

2014-01-01

Background Growth in fishes is regulated via many environmental and physiological factors and is shaped by the genetic background of each individual. Previous microarray studies of salmonid growth have examined fish experiencing either muscle wastage or accelerated growth patterns following refeeding, or the influence of growth hormone and transgenesis. This study determines the gene expression profiles of genetically unmanipulated large and small fish from a domesticated salmonid strain reared on a typical feeding regime. Gene expression profiles of white muscle and liver from rainbow trout (Oncorhynchus mykiss) from two seasonal spawning groups (September and December lots) within a single strain were examined when the fish were 15 months of age to assess the influence of season (late fall vs. onset of spring) and body size (large vs. small). Results Although IGFBP1 gene expression was up-regulated in the livers of small fish in both seasonal lots, few expression differences were detected in the liver overall. Faster growing Dec. fish showed a greater number of differences in white muscle expression compared to Sept. fish. Significant differences in the GO Generic Level 3 categories ‘response to external stimulus’, ‘establishment of localization’, and ‘response to stress’ were detected in white muscle tissue between large and small fish. Larger fish showed up-regulation of cytoskeletal component genes while many genes related to myofibril components of muscle tissue were up-regulated in small fish. Most of the genes up-regulated in large fish within the ‘response to stress’ category are involved in immunity while in small fish most of these gene functions are related to apoptosis. Conclusions A higher proportion of genes in white muscle compared to liver showed similar patterns of up- or down-regulation within the same size class across seasons supporting their utility as biomarkers for growth in rainbow trout. Differences between large and small

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

PubMed Central

Gori, Jennifer L.; Tian, Xinghui; Swanson, Debra; Gunther, Roland; Shultz, Leonard D.; McIvor, R. Scott; Kaufman, Dan S.

2009-01-01

SUMMARY Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase engraftment of gene-modified hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR – GFP expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR-cells in vivo, and provides a novel approach for combined human cell and gene therapy. PMID:19829316

Gene network reconstruction from transcriptional dynamics under kinetic model uncertainty: a case for the second derivative

PubMed Central

Bickel, David R.; Montazeri, Zahra; Hsieh, Pei-Chun; Beatty, Mary; Lawit, Shai J.; Bate, Nicholas J.

2009-01-01

Motivation: Measurements of gene expression over time enable the reconstruction of transcriptional networks. However, Bayesian networks and many other current reconstruction methods rely on assumptions that conflict with the differential equations that describe transcriptional kinetics. Practical approximations of kinetic models would enable inferring causal relationships between genes from expression data of microarray, tag-based and conventional platforms, but conclusions are sensitive to the assumptions made. Results: The representation of a sufficiently large portion of genome enables computation of an upper bound on how much confidence one may place in influences between genes on the basis of expression data. Information about which genes encode transcription factors is not necessary but may be incorporated if available. The methodology is generalized to cover cases in which expression measurements are missing for many of the genes that might control the transcription of the genes of interest. The assumption that the gene expression level is roughly proportional to the rate of translation led to better empirical performance than did either the assumption that the gene expression level is roughly proportional to the protein level or the Bayesian model average of both assumptions. Availability: http://www.oisb.ca points to R code implementing the methods (R Development Core Team 2004). Contact: dbickel@uottawa.ca Supplementary information: http://www.davidbickel.com PMID:19218351

Identification of Primary Transcriptional Regulation of Cell Cycle-Regulated Genes upon DNA Damage

PubMed Central

Zhou, Tong; Chou, Jeff; Mullen, Thomas E.; Elkon, Rani; Zhou, Yingchun; Simpson, Dennis A.; Bushel, Pierre R.; Paules, Richard S.; Lobenhofer, Edward K.; Hurban, Patrick; Kaufmann, William K.

2007-01-01

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. PMID:17404513

Gene expression analysis of pancreatic cell lines reveals genes overexpressed in pancreatic cancer.

PubMed

Alldinger, Ingo; Dittert, Dag; Peiper, Matthias; Fusco, Alberto; Chiappetta, Gennaro; Staub, Eike; Lohr, Matthias; Jesnowski, Ralf; Baretton, Gustavo; Ockert, Detlef; Saeger, Hans-Detlev; Grützmann, Robert; Pilarsky, Christian

2005-01-01

Pancreatic cancer is one of the leading causes of cancer-related death. Using DNA gene expression analysis based on a custom made Affymetrix cancer array, we investigated the expression pattern of both primary and established pancreatic carcinoma cell lines. We analyzed the gene expression of 5 established pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2 and HPAF II) and 5 primary isolates, 1 of them derived from benign pancreatic duct cells. Out of 1,540 genes which were expressed in at least 3 experiments, we found 122 genes upregulated and 18 downregulated in tumor cell lines compared to benign cells with a fold change >3. Several of the upregulated genes (like Prefoldin 5, ADAM9 and E-cadherin) have been associated with pancreatic cancer before. The other differentially regulated genes, however, play a so far unknown role in the course of human pancreatic carcinoma. By means of immunohistochemistry we could show that thymosin beta-10 (TMSB10), upregulated in tumor cell lines, is expressed in human pancreatic carcinoma, but not in non-neoplastic pancreatic tissue, suggesting a role for TMSB10 in the carcinogenesis of pancreatic carcinoma. Using gene expression profiling of pancreatic cell lines we were able to identify genes differentially expressed in pancreatic adenocarcinoma, which might contribute to pancreatic cancer development. Copyright 2005 S. Karger AG, Basel.

The Drosophila gene collection: Identification of putative full-length cDNAs for 70 percent of D. melanogaster genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stapleton, Mark; Liao, Guochun; Brokstein, Peter

2002-08-12

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5prime expressed sequence tags (EST) from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to {approx}40 percent of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remainingmore » genes, we have generated an additional 157,835 5prime ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22hr embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70 percent of the predicted genes in Drosophila.« less

Topological Constraints on Transvection between White Genes within the Transposing Element Te35b in Drosophila Melanogaster

PubMed Central

Gubb, D.; Roote, J.; Trenear, J.; Coulson, D.; Ashburner, M.

1997-01-01

The transposable element TE35B carries two copies of the white (w) gene at 35B1.2 on the second chromosome. These w genes are suppressed in a zeste-1 (z(1)) mutant background in a synapsis-dependent manner. Single-copy derivatives of the original TE35B stock give red eyes when heterozygous, but zeste eyes when homozygous. TE35B derivatives carrying single, double or triple copies of w were crossed to generate flies carrying from two to five ectopic w genes. Within this range, z(1)-mediated suppression is insensitive to copynumber and does not distinguish between w genes that are in cis or in trans. Suppression does not require the juxtaposition of even numbers of w genes, but is extremely sensitive to chromosomal topology. When arranged in a tight cluster, in triple-copy TE derivatives, w genes are nonsuppressible. Breakpoints falling within TE35B and separating two functional w genes act as partial suppressors of z(1). Similarly, breakpoints immediately proximal or distal to both w genes give partial suppression. This transvection-dependent downregulation of w genes may result from mis-activation of the X-chromosome dosage compensation mechanism. PMID:9215897

Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates.

PubMed

Burki, Fabien; Imanian, Behzad; Hehenberger, Elisabeth; Hirakawa, Yoshihisa; Maruyama, Shinichiro; Keeling, Patrick J

2014-02-01

Plastid establishment involves the transfer of endosymbiotic genes to the host nucleus, a process known as endosymbiotic gene transfer (EGT). Large amounts of EGT have been shown in several photosynthetic lineages but also in present-day plastid-lacking organisms, supporting the notion that endosymbiotic genes leave a substantial genetic footprint in the host nucleus. Yet the extent of this genetic relocation remains debated, largely because the long period that has passed since most plastids originated has erased many of the clues to how this process unfolded. Among the dinoflagellates, however, the ancestral peridinin-containing plastid has been replaced by tertiary plastids on several more recent occasions, giving us a less ancient window to examine plastid origins. In this study, we evaluated the endosymbiotic contribution to the host genome in two dinoflagellate lineages with tertiary plastids. We generated the first nuclear transcriptome data sets for the "dinotoms," which harbor diatom-derived plastids, and analyzed these data in combination with the available transcriptomes for kareniaceans, which harbor haptophyte-derived plastids. We found low level of detectable EGT in both dinoflagellate lineages, with only 9 genes and 90 genes of possible tertiary endosymbiotic origin in dinotoms and kareniaceans, respectively, suggesting that tertiary endosymbioses did not heavily impact the host dinoflagellate genomes.

CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons.

PubMed

Zhou, Yifan; Kumari, Daman; Sciascia, Nicholas; Usdin, Karen

2016-01-01

Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different

Exogenous application of pectin-derived oligosaccharides to grape berries modifies anthocyanin accumulation, composition and gene expression.

PubMed

Villegas, Daniel; Handford, Michael; Alcalde, José Antonio; Perez-Donoso, Alonso

2016-07-01

Anthocyanins are secondary metabolites synthesized in grape berry skins via the phenylpropanoid pathway, with functions ranging from skin coloration to protection against pathogens or UV light. Accumulation of these compounds is highly variable depending on genetics, environmental factors and viticultural practices. Besides their biological functions, anthocyanins improve wine quality, as a high anthocyanin content in berries has a positive impact on the color, total phenolic concentration and, ultimately, the price of wine. The present work studies the effect of the pre-veraison application of pectin derived oligosaccharides (PDO) on the synthesis and accumulation of these compounds, and associates the changes observed with the expression of key genes in the phenylpropanoid pathways. To this end, pre-veraison Cabernet Sauvignon bunches were treated with PDO to subsequently determine total anthocyanin content, the anthocyanin profile (by HPLC-DAD) and gene expression (by qRT-PCR), using Ethrel and water treatments for comparison. The results show that PDO were as efficient as Ethrel in generating a significant rise in total anthocyanin content at 30 days after treatment (dat), compared with water treatments (1.32, 1.48 and 1.02 mg e.Mv-3G/g FW respectively) without any undesirable effect on berry size, soluble solids, tartaric acid concentration or pH. In addition, a significant alteration in the anthocyanin profile was observed. Specifically, a significant increase in the relative concentration of malvidin was observed for both PDO and Ethrel treatments, compared with water controls (52.8; 55.0 and 48.3%, respectively), with a significant rise in tri-hydroxylated forms and a fall in di-hydroxylated anthocyanins. The results of gene expression analyses suggest that the increment in total anthocyanin content is related to a short term increase in phenylalanine ammonia-lyase (PAL) expression, mediated by a decrease in MYB4A expression. A longer term increase in UDP

Assessing Greater Sage-Grouse Selection of Brood-Rearing Habitat Using Remotely-Sensed Imagery: Can Readily Available High-Resolution Imagery Be Used to Identify Brood-Rearing Habitat Across a Broad Landscape?

PubMed

Westover, Matthew; Baxter, Jared; Baxter, Rick; Day, Casey; Jensen, Ryan; Petersen, Steve; Larsen, Randy

2016-01-01

Greater sage-grouse populations have decreased steadily since European settlement in western North America. Reduced availability of brood-rearing habitat has been identified as a limiting factor for many populations. We used radio-telemetry to acquire locations of sage-grouse broods from 1998 to 2012 in Strawberry Valley, Utah. Using these locations and remotely-sensed NAIP (National Agricultural Imagery Program) imagery, we 1) determined which characteristics of brood-rearing habitat could be used in widely available, high resolution imagery 2) assessed the spatial extent at which sage-grouse selected brood-rearing habitat, and 3) created a predictive habitat model to identify areas of preferred brood-rearing habitat. We used AIC model selection to evaluate support for a list of variables derived from remotely-sensed imagery. We examined the relationship of these explanatory variables at three spatial extents (45, 200, and 795 meter radii). Our top model included 10 variables (percent shrub, percent grass, percent tree, percent paved road, percent riparian, meters of sage/tree edge, meters of riparian/tree edge, distance to tree, distance to transmission lines, and distance to permanent structures). Variables from each spatial extent were represented in our top model with the majority being associated with the larger (795 meter) spatial extent. When applied to our study area, our top model predicted 75% of naïve brood locations suggesting reasonable success using this method and widely available NAIP imagery. We encourage application of our methodology to other sage-grouse populations and species of conservation concern.

Validation of osteogenic properties of Cytochalasin D by high-resolution RNA-sequencing in mesenchymal stem cells derived from bone marrow and adipose tissues.

PubMed

Samsonraj, Rebekah; Paradise, Christopher R; Dudakovic, Amel; Sen, Buer; Nair, Asha A; Dietz, Allan B; Deyle, David R; Cool, Simon M; Rubin, Janet; van Wijnen, Andre

2018-06-08

Differentiation of mesenchymal stromal/stem cells (MSCs) involves a series of molecular signals and gene transcription events required for attaining cell lineage commitment. Modulation of the actin cytoskeleton using cytochalasin D (CytoD) drives osteogenesis at early time points in bone marrow-derived MSCs, and also initiates a robust osteogenic differentiation program in adipose-derived MSCs. To understand the molecular basis for these pronounced effects on osteogenic differentiation, we investigated global changes in gene expression in CytoD-treated murine and human MSCs by high-resolution RNA-sequencing (RNA-seq) analysis. A three-way bioinformatic comparison between human adipose-derived, human bone marrow-derived and mouse bone marrow-derived MSCs revealed significant upregulation of genes linked to extracellular matrix organization, cell adhesion and bone metabolism. As anticipated, the activation of these differentiation related genes is accompanied by a downregulation of nuclear and cell cycle-related genes presumably reflecting cytostatic effects of CytoD. We also identified eight novel CytoD activated genes - VGLL4, ARHGAP24, KLHL24, RCBTB2, BDH2, SCARF2, ACAD10, HEPH - which are commonly upregulated across the two species and tissue sources of our MSC samples. We selected the Hippo-pathway related VGLL4 gene, which encodes the transcriptional co-factor Vestigial-like 4, for further study because this pathway is linked to osteogenesis. VGLL4 siRNA depletion reduces mineralization of adipose-derived MSCs during CytoD-induced osteogenic differentiation. Together, our RNA-seq analyses suggest that while the stimulatory effects of CytoD on osteogenesis are pleiotropic and depend on the biological state of the cell type, a small group of genes including VGLL4 may contribute to MSC commitment towards the bone lineage.

Complementary Information Derived from CRISPR Cas9 Mediated Gene Deletion and Suppression. | Office of Cancer Genomics

Cancer.gov

CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes.

A Generalized Approach for Measuring Relationships Among Genes.

PubMed

Wang, Lijun; Ahsan, Md Asif; Chen, Ming

2017-07-21

Several methods for identifying relationships among pairs of genes have been developed. In this article, we present a generalized approach for measuring relationships between any pairs of genes, which is based on statistical prediction. We derive two particular versions of the generalized approach, least squares estimation (LSE) and nearest neighbors prediction (NNP). According to mathematical proof, LSE is equivalent to the methods based on correlation; and NNP is approximate to one popular method called the maximal information coefficient (MIC) according to the performances in simulations and real dataset. Moreover, the approach based on statistical prediction can be extended from two-genes relationships to multi-genes relationships. This application would help to identify relationships among multi-genes.

Specification, testing, and interpretation of gene-by-measured-environment interaction models in the presence of gene-environment correlation

PubMed Central

Rathouz, Paul J.; Van Hulle, Carol A.; Lee Rodgers, Joseph; Waldman, Irwin D.; Lahey, Benjamin B.

2009-01-01

Purcell (2002) proposed a bivariate biometric model for testing and quantifying the interaction between latent genetic influences and measured environments in the presence of gene-environment correlation. Purcell’s model extends the Cholesky model to include gene-environment interaction. We examine a number of closely-related alternative models that do not involve gene-environment interaction but which may fit the data as well Purcell’s model. Because failure to consider these alternatives could lead to spurious detection of gene-environment interaction, we propose alternative models for testing gene-environment interaction in the presence of gene-environment correlation, including one based on the correlated factors model. In addition, we note mathematical errors in the calculation of effect size via variance components in Purcell’s model. We propose a statistical method for deriving and interpreting variance decompositions that are true to the fitted model. PMID:18293078

Characterization and expression of amphioxus ApoD gene encoding an archetype of vertebrate ApoD proteins.

PubMed

Wang, Lei; Zhang, Shicui; Liu, Zhenhui; Li, Hongyan; Wang, Yongjun; Jiang, Shengjuan

2007-01-01

Here we report a homologue of the apolipoprotein D gene (AmphiApoD) in amphioxus, Branchiostoma belcheri tsingtauense, the first such finding in a basal chordate cephalochordate. The main features of the protein predicted from AmphiApoD are characteristic of the apolipoprotein D. Phylogenetic analysis places AmphiApoD at the base of the phylogenetic tree, suggesting that AmphiApoD is the archetype of the vertebrate ApoD genes. Both whole mount in situ hybridization and Northern blotting and RT-PCR as well as in situ hybridization histochemistry reveal that AmphiApoD is expressed in tissues derived from mesoderm and endoderm including notochord and hind-gut, which contrasts with the strong expression patterns of ApoD genes in the ectodermal derivatives in mammals and birds. The expression profiles of the ApoD gene may have been changed to be expressed in the endo-mesodermal derivatives in amphioxus after the vertebrate and cephalochordate lineages diverged; alternatively, the ApoD gene may first have been expressed in the endo-mesoderm during embryogenesis in the last common ancestor of all chordates, and subsequently came to be expressed in the ectodermal derivatives of vertebrates including mammals and birds.

[Analysis of horizontal transfer gene of Bombyx mori NPV].

PubMed

Duan, Hai-Rong; Qiu, De-Bin; Gong, Cheng-Liang; Huang, Mo-Li

2011-06-01

For research on genetic characters and evolutionary origin of the genome of baculoviruses, a comprehensive homology search and phylogenetic analysis of the complete genomes of Bombyx mori NPV and Bombyx mori were used. Three horizontally transferred genes (inhibitor of apoptosis, chitinase, and UDP-glucosyltransferase) were identified, and there was evidence that all of these genes were derived from the insect host. The results of analysis showed lots of differences between the features of horizontal transferred genes and the ones of whole genomic genes, such as nucleotide composition, codon usagebias and selection pressure. These results reconfirmed that the horizontally transferred genes are exogenous. The analysis of gene function suggested that horizontally transferred genes acquired from an ancestral host insect can increase the efficiency of baculoviruses transmission.

Identification and proteomic analysis of osteoblast-derived exosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ge, Min; Ke, Ronghu; Cai, Tianyi

Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786more » proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.« less

Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells.

PubMed

Bassuk, Alexander G; Zheng, Andrew; Li, Yao; Tsang, Stephen H; Mahajan, Vinit B

2016-01-27

Induced pluripotent stem cells (iPSCs) generated from patient fibroblasts could potentially be used as a source of autologous cells for transplantation in retinal disease. Patient-derived iPSCs, however, would still harbor disease-causing mutations. To generate healthy patient-derived cells, mutations might be repaired with new gene-editing technology based on the bacterial system of clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9, thereby yielding grafts that require no patient immunosuppression. We tested whether CRISPR/Cas9 could be used in patient-specific iPSCs to precisely repair an RPGR point mutation that causes X-linked retinitis pigmentosa (XLRP). Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce iPSCs carrying the patient's c.3070G > T mutation. The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene's repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele. This is the first report using CRISPR to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This important proof-of-concept finding supports the development of personalized iPSC-based transplantation therapies for retinal disease.

Positive Selection and Multiple Losses of the LINE-1-Derived L1TD1 Gene in Mammals Suggest a Dual Role in Genome Defense and Pluripotency

PubMed Central

Yang, Lei; Neme, Rafik; Wichman, Holly A.; Malik, Harmit S.

2014-01-01

Mammalian genomes comprise many active and fossilized retroelements. The obligate requirement for retroelement integration affords host genomes an opportunity to ‘domesticate’ retroelement genes for their own purpose, leading to important innovations in genome defense and placentation. While many such exaptations involve retroviruses, the L1TD1 gene is the only known domesticated gene whose protein-coding sequence is almost entirely derived from a LINE-1 (L1) retroelement. Human L1TD1 has been shown to play an important role in pluripotency maintenance. To investigate how this role was acquired, we traced the origin and evolution of L1TD1. We find that L1TD1 originated in the common ancestor of eutherian mammals, but was lost or pseudogenized multiple times during mammalian evolution. We also find that L1TD1 has evolved under positive selection during primate and mouse evolution, and that one prosimian L1TD1 has ‘replenished’ itself with a more recent L1 ORF1 from the prosimian genome. These data suggest that L1TD1 has been recurrently selected for functional novelty, perhaps for a role in genome defense. L1TD1 loss is associated with L1 extinction in several megabat lineages, but not in sigmodontine rodents. We hypothesize that L1TD1 could have originally evolved for genome defense against L1 elements. Later, L1TD1 may have become incorporated into pluripotency maintenance in some lineages. Our study highlights the role of retroelement gene domestication in fundamental aspects of mammalian biology, and that such domesticated genes can adopt different functions in different lineages. PMID:25211013

Identification of pathogenic genes related to rheumatoid arthritis through integrated analysis of DNA methylation and gene expression profiling.

PubMed

Zhang, Lei; Ma, Shiyun; Wang, Huailiang; Su, Hang; Su, Ke; Li, Longjie

2017-11-15

The purpose of our study was to identify new pathogenic genes used for exploring the pathogenesis of rheumatoid arthritis (RA). To screen pathogenic genes of RA, an integrated analysis was performed by using the microarray datasets in RA derived from the Gene Expression Omnibus (GEO) database. The functional annotation and potential pathways of differentially expressed genes (DEGs) were further discovered by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Afterwards, the integrated analysis of DNA methylation and gene expression profiling was used to screen crucial genes. In addition, we used RT-PCR and MSP to verify the expression levels and methylation status of these crucial genes in 20 synovial biopsy samples obtained from 10 RA model mice and 10 normal mice. BCL11B, CCDC88C, FCRLA and APOL6 were both up-regulated and hypomethylated in RA according to integrated analysis, RT-PCR and MSP verification. Four crucial genes (BCL11B, CCDC88C, FCRLA and APOL6) identified and analyzed in this study might be closely connected with the pathogenesis of RA. Copyright © 2017. Published by Elsevier B.V.

4'-Acetoamido-4-hydroxychalcone, a chalcone derivative, inhibits glioma growth and invasion through regulation of the tropomyosin 1 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ku, Bo Mi; Ryu, Hyung Won; Lee, Yeon Kyung

2010-11-19

Research highlights: {yields} 4'-Acetoamido-4-hydroxychalcone (AHC) has anti-cancer property for glioma. {yields} 4'-Acetoamido-4-hydroxychalcone (AHC) increased tropomyosin expreesion through activattion of PKA signaling. {yields} 4'-Acetoamido-4-hydroxychalcone (AHC) inhibits glioma cell migration and invasion. {yields} In vivo administration of 4'-acetoamido-4-hydroxychalcone (AHC) reduced tumor growth. -- Abstract: Chalcones are precursors of flavonoids and have been shown to have anti-cancer activity. Here, we identify the synthetic chalcone derivative 4'-acetoamido-4-hydroxychalcone (AHC) as a potential therapeutic agent for the treatment of glioma. Treatment with AHC reduced glioma cell invasion, migration, and colony formation in a concentration-dependent manner. In addition, AHC inhibited vascular endothelial growth factor-induced migration, invasion, andmore » tube formation in HUVECs. To determine the mechanism underlying the inhibitory effect of AHC on glioma cell invasion and migration, we investigated the effect of AHC on the gene expression change and found that AHC affects actin dynamics in U87MG glioma cells. In actin cytoskeleton regulating system, AHC increased tropomyosin expression and stress fiber formation, probably through activation of PKA. Suppression of tropomyosin expression by siRNA or treatment with the PKA inhibitor H89 reduced the inhibitory effects of AHC on glioma cell invasion and migration. In vivo experiments also showed that AHC inhibited tumor growth in a xenograft mouse tumor model. Together, these data suggest that the synthetic chalcone derivative AHC has potent anti-cancer activity through inhibition of glioma proliferation, invasion, and angiogenesis and is therefore a potential chemotherapeutic candidate for the treatment of glioma.« less

Activation and comparative analysis of cryptic xiamycin gene cluster from marine-derived Streptomyces sp. FXJ 7.388.

PubMed

Uhong Lü, Yuhong; Liu, Xiaoli; Wang, Miao; Li, Yuanyuan; Liu, Ning; Bao, Yuxin; Liu, Minghao; Li, Xiaoqian; Wang, Yinyin; Qian, Shenyan; Yue, Changwu; Huang, Ying

2016-09-01

In order to obtain the natural products synthesized by the three putative xiamycin biosynthesis gene clusters which were predicted via antiSMASH during the genome mining of marine Streptomyces sp. FXJ 7.388, Streptomyces sp. FXJ 8.012, and Streptomyces olivaceus FXJ 7.023. Sixteen genes involved in xiamycin assembly, modification, and regulation with higher identity than the newest reported xiamycin biosynthetic gene cluster from marine Streptomyces sp. SCSIO 02999, Streptomyces sp. HKI0576, and Streptomyces sp. FXJ 7.388 were discovered via gene cluster comparative analysis. A ribosome engineering strategy was adopted to activate such cryptic gene clusters with different final concentrations antibiotics that act on the ribosome, and two indolosesquiterpenes were isolated from idlethaldose streptomycin-resistant Streptomyces sp. FXJ 7.388 strains. However, no such product was detected in Streptomyces sp. FXJ 8.012 and Streptomyces olivaceus FXJ 7.023 under the same treatment. This result suggested that these genes might hold the least gene content for xiamycin biosynthesis.

Using the TIGR gene index databases for biological discovery.

PubMed

Lee, Yuandan; Quackenbush, John

2003-11-01

The TIGR Gene Index web pages provide access to analyses of ESTs and gene sequences for nearly 60 species, as well as a number of resources derived from these. Each species-specific database is presented using a common format with a homepage. A variety of methods exist that allow users to search each species-specific database. Methods implemented currently include nucleotide or protein sequence queries using WU-BLAST, text-based searches using various sequence identifiers, searches by gene, tissue and library name, and searches using functional classes through Gene Ontology assignments. This protocol provides guidance for using the Gene Index Databases to extract information.

Comparative genome analysis of PHB gene family reveals deep evolutionary origins and diverse gene function.

PubMed

Di, Chao; Xu, Wenying; Su, Zhen; Yuan, Joshua S

2010-10-07

PHB (Prohibitin) gene family is involved in a variety of functions important for different biological processes. PHB genes are ubiquitously present in divergent species from prokaryotes to eukaryotes. Human PHB genes have been found to be associated with various diseases. Recent studies by our group and others have shown diverse function of PHB genes in plants for development, senescence, defence, and others. Despite the importance of the PHB gene family, no comprehensive gene family analysis has been carried to evaluate the relatedness of PHB genes across different species. In order to better guide the gene function analysis and understand the evolution of the PHB gene family, we therefore carried out the comparative genome analysis of the PHB genes across different kingdoms. The relatedness, motif distribution, and intron/exon distribution all indicated that PHB genes is a relatively conserved gene family. The PHB genes can be classified into 5 classes and each class have a very deep evolutionary origin. The PHB genes within the class maintained the same motif patterns during the evolution. With Arabidopsis as the model species, we found that PHB gene intron/exon structure and domains are also conserved during the evolution. Despite being a conserved gene family, various gene duplication events led to the expansion of the PHB genes. Both segmental and tandem gene duplication were involved in Arabidopsis PHB gene family expansion. However, segmental duplication is predominant in Arabidopsis. Moreover, most of the duplicated genes experienced neofunctionalization. The results highlighted that PHB genes might be involved in important functions so that the duplicated genes are under the evolutionary pressure to derive new function. PHB gene family is a conserved gene family and accounts for diverse but important biological functions based on the similar molecular mechanisms. The highly diverse biological function indicated that more research needs to be carried out

Genome-Wide Analysis of Gene-Gene and Gene-Environment Interactions Using Closed-Form Wald Tests.

PubMed

Yu, Zhaoxia; Demetriou, Michael; Gillen, Daniel L

2015-09-01

Despite the successful discovery of hundreds of variants for complex human traits using genome-wide association studies, the degree to which genes and environmental risk factors jointly affect disease risk is largely unknown. One obstacle toward this goal is that the computational effort required for testing gene-gene and gene-environment interactions is enormous. As a result, numerous computationally efficient tests were recently proposed. However, the validity of these methods often relies on unrealistic assumptions such as additive main effects, main effects at only one variable, no linkage disequilibrium between the two single-nucleotide polymorphisms (SNPs) in a pair or gene-environment independence. Here, we derive closed-form and consistent estimates for interaction parameters and propose to use Wald tests for testing interactions. The Wald tests are asymptotically equivalent to the likelihood ratio tests (LRTs), largely considered to be the gold standard tests but generally too computationally demanding for genome-wide interaction analysis. Simulation studies show that the proposed Wald tests have very similar performances with the LRTs but are much more computationally efficient. Applying the proposed tests to a genome-wide study of multiple sclerosis, we identify interactions within the major histocompatibility complex region. In this application, we find that (1) focusing on pairs where both SNPs are marginally significant leads to more significant interactions when compared to focusing on pairs where at least one SNP is marginally significant; and (2) parsimonious parameterization of interaction effects might decrease, rather than increase, statistical power. © 2015 WILEY PERIODICALS, INC.

Meta-Analysis of Transcriptome Data Related to Hippocampus Biopsies and iPSC-Derived Neuronal Cells from Alzheimer's Disease Patients Reveals an Association with FOXA1 and FOXA2 Gene Regulatory Networks.

PubMed

Wruck, Wasco; Schröter, Friederike; Adjaye, James

2016-01-01

Although the incidence of Alzheimer's disease (AD) is continuously increasing in the aging population worldwide, effective therapies are not available. The interplay between causative genetic and environmental factors is partially understood. Meta-analyses have been performed on aspects such as polymorphisms, cytokines, and cognitive training. Here, we propose a meta-analysis approach based on hierarchical clustering analysis of a reliable training set of hippocampus biopsies, which is condensed to a gene expression signature. This gene expression signature was applied to various test sets of brain biopsies and iPSC-derived neuronal cell models to demonstrate its ability to distinguish AD samples from control. Thus, our identified AD-gene signature may form the basis for determination of biomarkers that are urgently needed to overcome current diagnostic shortfalls. Intriguingly, the well-described AD-related genes APP and APOE are not within the signature because their gene expression profiles show a lower correlation to the disease phenotype than genes from the signature. This is in line with the differing characteristics of the disease as early-/late-onset or with/without genetic predisposition. To investigate the gene signature's systemic role(s), signaling pathways, gene ontologies, and transcription factors were analyzed which revealed over-representation of response to stress, regulation of cellular metabolic processes, and reactive oxygen species. Additionally, our results clearly point to an important role of FOXA1 and FOXA2 gene regulatory networks in the etiology of AD. This finding is in corroboration with the recently reported major role of the dopaminergic system in the development of AD and its regulation by FOXA1 and FOXA2.

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

PubMed Central

Chambouvet, Aurélie; Milner, David S.; Attah, Victoria; Terrado, Ramón; Lovejoy, Connie; Moreau, Hervé; Derelle, Évelyne; Richards, Thomas A.

2017-01-01

Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer. PMID:28827361

A unique mosaic Turner syndrome patient with androgen receptor gene derived marker chromosome.

PubMed

Kalkan, Rasime; Özdağ, Nermin; Bundak, Rüveyde; Çirakoğlu, Ayşe; Serakinci, Nedime

2016-01-01

Patients with Turner syndrome are generally characterized by having short stature with no secondary sexual characteristics. Some abnormalities, such as webbed neck, renal malformations (>50%) and cardiac defects (10%) are less common. The intelligence of these patients is considered normal. Non-mosaic monosomy X is observed in approximately 45% of postnatal patients with Turner syndrome and the rest of the patients have structural abnormalities or mosaicism involving 46,X,i(Xq), 45,X/46,XX, 45,X and other variants. The phenotype of 45,X/46,X,+mar individuals varies by the genetic continent and degree of the mosaicism. The gene content of the marker chromosome is the most important when correlating the phenotype with the genotype. Here we present an 11-year-old female who was referred for evaluation of her short stature and learning disabilities. Conventional cytogenetic investigation showed a mosaic 45,X/46,X,+mar karyotype. Fluorescence in situ hybridization showed that the marker chromosome originated from the X chromosome within the androgen receptor (AR) and X-inactive specific transcript (XIST) genes. Therefore, it is possible that aberrant activation of the marker chromosome, compromising the AR and XIST genes, may modify the Turner syndrome phenotype.

GeneTopics - interpretation of gene sets via literature-driven topic models

PubMed Central

2013-01-01

Background Annotation of a set of genes is often accomplished through comparison to a library of labelled gene sets such as biological processes or canonical pathways. However, this approach might fail if the employed libraries are not up to date with the latest research, don't capture relevant biological themes or are curated at a different level of granularity than is required to appropriately analyze the input gene set. At the same time, the vast biomedical literature offers an unstructured repository of the latest research findings that can be tapped to provide thematic sub-groupings for any input gene set. Methods Our proposed method relies on a gene-specific text corpus and extracts commonalities between documents in an unsupervised manner using a topic model approach. We automatically determine the number of topics summarizing the corpus and calculate a gene relevancy score for each topic allowing us to eliminate non-specific topics. As a result we obtain a set of literature topics in which each topic is associated with a subset of the input genes providing directly interpretable keywords and corresponding documents for literature research. Results We validate our method based on labelled gene sets from the KEGG metabolic pathway collection and the genetic association database (GAD) and show that the approach is able to detect topics consistent with the labelled annotation. Furthermore, we discuss the results on three different types of experimentally derived gene sets, (1) differentially expressed genes from a cardiac hypertrophy experiment in mice, (2) altered transcript abundance in human pancreatic beta cells, and (3) genes implicated by GWA studies to be associated with metabolite levels in a healthy population. In all three cases, we are able to replicate findings from the original papers in a quick and semi-automated manner. Conclusions Our approach provides a novel way of automatically generating meaningful annotations for gene sets that are directly

Cancer Cell Signaling Pathways Targeted by Spice-Derived Nutraceuticals

PubMed Central

Sung, Bokyung; Prasad, Sahdeo; Yadav, Vivek R.; Aggarwal, Bharat B.

2012-01-01

Extensive research within the last half a century has revealed that cancer is caused by dysregulation of as many as 500 different gene products. Most natural products target multiple gene products and thus are ideally suited for prevention and treatment of various chronic diseases, including cancer. Dietary agents such as spices have been used extensively in the Eastern world for a variety of ailments for millennia, and five centuries ago they took a golden journey to the Western world. Various spice-derived nutraceuticals, including 1′-acetoxychavicol acetate, anethole, capsaicin, car-damonin, curcumin, dibenzoylmethane, diosgenin, eugenol, gambogic acid, gingerol, thymoquinone, ursolic acid, xanthohumol, and zerumbone derived from galangal, anise, red chili, black cardamom, turmeric, licorice, fenugreek, clove, kokum, ginger, black cumin, rosemary, hop, and pinecone ginger, respectively, are the focus of this review. The modulation of various transcription factors, growth factors, protein kinases, and inflammatory mediators by these spice-derived nutraceuticals are described. The anticancer potential through the modulation of various targets is also the subject of this review. Although they have always been used to improve taste and color and as a preservative, they are now also used for prevention and treatment of a wide variety of chronic inflammatory diseases, including cancer. PMID:22149093

Characteristics of siRNAs derived from Southern rice black-streaked dwarf virus in infected rice and their potential role in host gene regulation.

PubMed

Xu, Donglin; Zhou, Guohui

2017-02-10

Virus-derived siRNAs (vsiRNAs)-mediated RNA silencing plays important roles in interaction between plant viruses and their hosts. Southern rice black-streaked dwarf virus (SRBSDV) is a newly emerged devastating rice reovirus with ten dsRNA genomic segments. The characteristics of SRBSDV-derived siRNAs and their biological implications in SRBSDV-rice interaction remain unexplored. VsiRNAs profiling from SRBSDV-infected rice samples was done via small RNA deep sequencing. The putative rice targets of abundantly expressed vsiRNAs were bioinformatically predicted and subjected to functional annotation. Differential expression analysis of rice targets and RNA silencing components between infected and healthy samples was done using RT-qPCR. The vsiRNA was barely detectable at 14 days post infection (dpi) but abundantly present along with elevated expression level of the viral genome at 28 dpi. From the 28-dpi sample, 70,878 reads of 18 ~ 30-nt vsiRNAs were recognized (which mostly were 21-nt and 22-nt), covering 75 ~ 91% of the length of the ten genomic segments respectively. 86% of the vsiRNAs had a <50% GC content and 79% of them were 5'-uridylated or adenylated. The production of vsiRNAs had no strand polarity but varied among segment origins. Each segment had a few hotspot regions where vsiRNAs of high abundance were produced. 151 most abundant vsiRNAs were predicted to target 844 rice genes, including several types of host resistance or pathogenesis related genes encoding F-box/LRR proteins, receptor-like protein kinases, universal stress proteins, tobamovirus multiplication proteins, and RNA silencing components OsDCL2a and OsAGO17 respectively, some of which showed down regulation in infected plants in RT-qPCR. GO and KEGG classification showed that a majority of the predicted targets were related to cell parts and cellular processes and involved in carbohydrate metabolism, translation, and signal transduction. The silencing component genes OsDCL2a, Os

The human somatostatin receptor type 2 as an imaging and suicide reporter gene for pluripotent stem cell-derived therapy of myocardial infarction

PubMed Central

Neyrinck, Katrien; Breuls, Natacha; Holvoet, Bryan; Oosterlinck, Wouter; Wolfs, Esther; Vanbilloen, Hubert; Gheysens, Olivier; Duelen, Robin; Gsell, Willy; Lambrichts, Ivo; Himmelreich, Uwe; Verfaillie, Catherine M.; Sampaolesi, Maurilio; Deroose, Christophe M.

2018-01-01

magnetic resonance imaging. Results: Teratoma growth of both hNIS+ and hSSTr2+ ESCs could be followed noninvasively over time by both PET and BLI. After 177Lu-DOTATATE administration, successful cell killing of the hSSTr2+ ESCs was achieved both in vitro and in vivo, indicated by reductions in total tracer lesion uptake, BLI signal and teratoma volume. As undifferentiated hSSTr2+ ESCs are not therapeutically relevant, they were differentiated towards CMs and injected in immune-deficient mice with a MI. Long-term cell survival could be monitored without uncontrolled cell proliferation. However, no improvement in the left ventricular ejection fraction was observed. Conclusion: We developed isogenic hSSTr2-expressing ESCs that allow noninvasive cell monitoring in the context of PSC-derived regenerative therapy. Furthermore, we are the first to use the hSSTr2 not only as an imaging reporter gene, but also as a suicide mechanism for radionuclide therapy in the setting of PSC-derived cell treatment. PMID:29774076

The human somatostatin receptor type 2 as an imaging and suicide reporter gene for pluripotent stem cell-derived therapy of myocardial infarction.

PubMed

Neyrinck, Katrien; Breuls, Natacha; Holvoet, Bryan; Oosterlinck, Wouter; Wolfs, Esther; Vanbilloen, Hubert; Gheysens, Olivier; Duelen, Robin; Gsell, Willy; Lambrichts, Ivo; Himmelreich, Uwe; Verfaillie, Catherine M; Sampaolesi, Maurilio; Deroose, Christophe M

2018-01-01

cardiac magnetic resonance imaging. Results: Teratoma growth of both hNIS + and hSSTr2 + ESCs could be followed noninvasively over time by both PET and BLI. After 177 Lu-DOTATATE administration, successful cell killing of the hSSTr2 + ESCs was achieved both in vitro and in vivo , indicated by reductions in total tracer lesion uptake, BLI signal and teratoma volume. As undifferentiated hSSTr2 + ESCs are not therapeutically relevant, they were differentiated towards CMs and injected in immune-deficient mice with a MI. Long-term cell survival could be monitored without uncontrolled cell proliferation. However, no improvement in the left ventricular ejection fraction was observed. Conclusion: We developed isogenic hSSTr2-expressing ESCs that allow noninvasive cell monitoring in the context of PSC-derived regenerative therapy. Furthermore, we are the first to use the hSSTr2 not only as an imaging reporter gene, but also as a suicide mechanism for radionuclide therapy in the setting of PSC-derived cell treatment.

GeneStoryTeller: a mobile app for quick and comprehensive information retrieval of human genes

PubMed Central

Eleftheriou, Stergiani V.; Bourdakou, Marilena M.; Athanasiadis, Emmanouil I.; Spyrou, George M.

2015-01-01

In the last few years, mobile devices such as smartphones and tablets have become an integral part of everyday life, due to their software/hardware rapid development, as well as the increased portability they offer. Nevertheless, up to now, only few Apps have been developed in the field of bioinformatics, capable to perform fast and robust access to services. We have developed the GeneStoryTeller, a mobile application for Android platforms, where users are able to instantly retrieve information regarding any recorded human gene, derived from eight publicly available databases, as a summary story. Complementary information regarding gene–drugs interactions, functional annotation and disease associations for each selected gene is also provided in the gene story. The most challenging part during the development of the GeneStoryTeller was to keep balance between storing data locally within the app and obtaining the updated content dynamically via a network connection. This was accomplished with the implementation of an administrative site where data are curated and synchronized with the application requiring a minimum human intervention. Database URL: http://bioserver-3.bioacademy.gr/Bioserver/GeneStoryTeller/. PMID:26055097

GeneStoryTeller: a mobile app for quick and comprehensive information retrieval of human genes.

PubMed

Eleftheriou, Stergiani V; Bourdakou, Marilena M; Athanasiadis, Emmanouil I; Spyrou, George M

2015-01-01

In the last few years, mobile devices such as smartphones and tablets have become an integral part of everyday life, due to their software/hardware rapid development, as well as the increased portability they offer. Nevertheless, up to now, only few Apps have been developed in the field of bioinformatics, capable to perform fast and robust access to services. We have developed the GeneStoryTeller, a mobile application for Android platforms, where users are able to instantly retrieve information regarding any recorded human gene, derived from eight publicly available databases, as a summary story. Complementary information regarding gene-drugs interactions, functional annotation and disease associations for each selected gene is also provided in the gene story. The most challenging part during the development of the GeneStoryTeller was to keep balance between storing data locally within the app and obtaining the updated content dynamically via a network connection. This was accomplished with the implementation of an administrative site where data are curated and synchronized with the application requiring a minimum human intervention. © The Author(s) 2015. Published by Oxford University Press.

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

PubMed

Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

MicroRNA-derived network analysis of differentially methylated genes in schizophrenia, implicating GABA receptor B1 [GABBR1] and protein kinase B [AKT1].

PubMed

Gumerov, Vadim; Hegyi, Hedi

2015-10-08

While hundreds of genes have been implicated already in the etiology of schizophrenia, the exact cause is not known or the disease is considered multigenic in origin. Recent discoveries of new types of RNAs and the gradual elimination of the "junk DNA" hypothesis refocused the attention on the noncoding part of the human genome. Here we re-analyzed a recent dataset of differentially methylated genes from schizophrenic patients and cross-tabulated them with cis regulatory and repetitive elements and microRNAs known to be involved in schizophrenia. We found that the number of schizophrenia-related (SZ) microRNA targets follows a scale-free distribution with several microRNA hubs and that schizophrenia-related microRNAs with shared targets form a small-world network. The top ten microRNAs with the highest number of SZ gene targets regulate approximately 80 % of all microRNA-regulated genes whereas the top two microRNAs regulate 40-52 % of all such genes. We also found that genes that are regulated by the same microRNAs tend to have more protein-protein interactions than randomly selected schizophrenia genes. This highlights the role microRNAs possibly play in coordinating the abundance of interacting proteins, an important function that has not been sufficiently explored before. The analysis revealed that GABBR1 is regulated by both of the top two microRNAs and acts as a hub by interacting with many schizophrenia-related genes and sharing several types of transcription-binding sites with its interactors. We also found that differentially methylated repetitive elements are significantly more methylated in schizophrenia, pointing out their potential role in the disease. We find that GABBR1 has a central importance in schizophrenia, even if no direct cause and effect have been shown for it for the time. In addition to being a hub in microRNA-derived regulatory pathways and protein-protein interactions, its centrality is also supported by the high number of cis regulatory

Promoter polymorphisms in the ATP binding cassette transporter gene influence production of cell-derived microparticles and are highly associated with susceptibility to severe malaria in humans.

PubMed

Sahu, Upasana; Mohapatra, Biranchi N; Kar, Shantanu K; Ranjit, Manoranjan

2013-04-01

Microparticle (MP) efflux is known to be mediated by the ABCA1 protein, and the plasma level of these cell-derived MPs is elevated considerably during human malarial infection. Therefore, two polymorphisms at positions -477 and -320 in the promoter of the ABCA1 gene were genotyped and tested for association with the plasma MP level in four groups of malaria patients segregated according to the clinical severity, i.e., cerebral malaria (CM), multiorgan dysfunction (MOD), noncerebral severe malaria, and uncomplicated malaria (UM). The TruCount tube-based flow cytometric method was used for the exact quantification of different cell-derived MPs in patients. Polymorphisms in the ABCA1 gene promoter were analyzed by use of the PCR/two-primer-pair method, followed by restriction fragment length polymorphism, in 428 malaria patients. The level of circulating plasma MPs was significantly higher in febrile patients with Plasmodium falciparum infection, especially in CM patients compared to healthy individuals. The homozygous wild-type -477 and -320 genotype was observed to be significantly higher in patients with severe malaria. These patients also showed marked increases in the plasma MP numbers compared to UM patients. We report here for the first time an association of ABCA1 promoter polymorphisms with susceptibility to severe malaria, especially to CM and MOD, indicating the protective effect of the mutant variant of the polymorphism. We hypothesize that the -477T and -320G polymorphisms affect the downregulation of MP efflux and may be a predictor of organ complication during P. falciparum malarial infections.

Pandoraviruses are highly derived phycodnaviruses

PubMed Central

2013-01-01

The recently discovered Pandoraviruses are by far the largest viruses known, with their 2 megabase genomes exceeding in size the genomes of numerous bacteria and archaea. Pandoraviruses show a distant relationship with other nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes, lack some of the NCLDV core genes and in particular do not appear to be specifically related to the other, better characterized family of giant viruses, the Mimiviridae. Here we report phylogenetic analysis of 6 core NCLDV genes that confidently places Pandoraviruses within the family Phycodnaviridae, with an apparent specific affinity with Coccolithoviruses. We conclude that, despite their many unusual characteristics, Pandoraviruses are highly derived phycodnaviruses. These findings imply that giant viruses have independently evolved from smaller NCLDV on at least two occasions. This article was reviewed by Patrick Forterre and Lakshminarayan Iyer. For the full reviews, see the Reviewers’ reports section. PMID:24148757

Evolution and Expression Patterns of CYC/TB1 Genes in Anacyclus: Phylogenetic Insights for Floral Symmetry Genes in Asteraceae

PubMed Central

Bello, María A.; Cubas, Pilar; Álvarez, Inés; Sanjuanbenito, Guillermo; Fuertes-Aguilar, Javier

2017-01-01

Homologs of the CYC/TB1 gene family have been independently recruited many times across the eudicots to control aspects of floral symmetry The family Asteraceae exhibits the largest known diversification in this gene paralog family accompanied by a parallel morphological floral richness in its specialized head-like inflorescence. In Asteraceae, whether or not CYC/TB1 gene floral symmetry function is preserved along organismic and gene lineages is unknown. In this study, we used phylogenetic, structural and expression analyses focused on the highly derived genus Anacyclus (tribe Anthemidae) to address this question. Phylogenetic reconstruction recovered eight main gene lineages present in Asteraceae: two from CYC1, four from CYC2 and two from CYC3-like genes. The species phylogeny was recovered in most of the gene lineages, allowing the delimitation of orthologous sets of CYC/TB1 genes in Asteraceae. Quantitative real-time PCR analysis indicated that in Anacyclus three of the four isolated CYC2 genes are more highly expressed in ray flowers. The expression of the four AcCYC2 genes overlaps in several organs including the ligule of ray flowers, as well as in anthers and ovules throughout development. PMID:28487706

Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades.

PubMed

Hering, Lars; Bouameur, Jamal-Eddine; Reichelt, Julian; Magin, Thomas M; Mayer, Georg

2016-02-03

Intermediate filament (IF) proteins, including nuclear lamins and cytoplasmic IF proteins, are essential cytoskeletal components of bilaterian cells. Despite their important role in protecting tissues against mechanical force, no cytoplasmic IF proteins have been convincingly identified in arthropods. Here we show that the ancestral cytoplasmic IF protein gene was lost in the entire panarthropod (onychophoran + tardigrade + arthropod) rather than arthropod lineage and that nuclear, lamin-derived proteins instead acquired new cytoplasmic roles at least three times independently in collembolans, copepods, and tardigrades. Transcriptomic and genomic data revealed three IF protein genes in the tardigrade Hypsibius dujardini, one of which (cytotardin) occurs exclusively in the cytoplasm of epidermal and foregut epithelia, where it forms belt-like filaments around each epithelial cell. These results suggest that a lamin derivative has been co-opted to enhance tissue stability in tardigrades, a function otherwise served by cytoplasmic IF proteins in all other bilaterians.

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

PubMed

Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

2015-01-01

In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

PubMed Central

Rittelmeyer, Ina; Sharma, Amar Deep; Sgodda, Malte; Zaehres, Holm; Bleidißel, Martina; Greber, Boris; Gentile, Luca; Han, Dong Wook; Rudolph, Cornelia; Steinemann, Doris; Schambach, Axel; Ott, Michael; Schöler, Hans R.; Cantz, Tobias

2011-01-01

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH −/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH −/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH −/− iPS cell lines, we aggregated FAH −/−-iPS cells with tetraploid embryos and obtained entirely FAH −/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH −/− mice. Then, we transduced FAH cDNA into the FAH −/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models. PMID:21765802

Histone-derived piRNA biogenesis depends on the ping-pong partners Piwi5 and Ago3 in Aedes aegypti

PubMed Central

Girardi, Erika; Miesen, Pascal; Pennings, Bas; Frangeul, Lionel; Saleh, Maria-Carla

2017-01-01

Abstract The piRNA pathway is of key importance in controlling transposable elements in most animal species. In the vector mosquito Aedes aegypti, the presence of eight PIWI proteins and the accumulation of viral piRNAs upon arbovirus infection suggest additional functions of the piRNA pathway beyond genome defense. To better understand the regulatory potential of this pathway, we analyzed in detail host-derived piRNAs in A. aegypti Aag2 cells. We show that a large repertoire of protein-coding genes and non-retroviral integrated RNA virus elements are processed into genic piRNAs by different combinations of PIWI proteins. Among these, we identify a class of genes that produces piRNAs from coding sequences in an Ago3- and Piwi5-dependent fashion. We demonstrate that the replication-dependent histone gene family is a genic source of ping-pong dependent piRNAs and that histone-derived piRNAs are dynamically expressed throughout the cell cycle, suggesting a role for the piRNA pathway in the regulation of histone gene expression. Moreover, our results establish the Aag2 cell line as an accessible experimental model to study gene-derived piRNAs. PMID:28115625

Adenovirus vector-mediated ex vivo gene transfer of brain-derived neurotrophic factor to bone marrow stromal cells promotes axonal regeneration after transplantation in completely transected adult rat spinal cord

PubMed Central

Kamada, Takahito; Hashimoto, Masayuki; Murakami, Masazumi; Shirasawa, Hiroshi; Sakao, Seiichiro; Ino, Hidetoshi; Yoshinaga, Katsunori; Koshizuka, Shuhei; Moriya, Hideshige; Yamazaki, Masashi

2007-01-01

The aim of this study was to evaluate the efficacy in adult rat completely transected spinal cord of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to bone marrow stromal cells (BMSC). BMSC were infected with adenovirus vectors carrying β-galactosidase (AxCALacZ) or BDNF (AxCABDNF) genes. The T8 segment of spinal cord was removed and replaced by graft containing Matrigel alone (MG group) or Matrigel and BMSC infected by AxCALacZ (BMSC-LacZ group) or AxCABDNF (BMSC-BDNF group). Axons in the graft were evaluated by immunohistochemistry and functional recovery was assessed with BBB locomotor scale. In the BMSC-BDNF group, the number of fibers positive for growth associated protein-43, tyrosine hydroxylase, and calcitonin gene-related peptide was significantly larger than numbers found for the MG and BMSC-LacZ groups. Rats from BMSC-BDNF and BMSC-LacZ groups showed significant recovery of hind limb function compared with MG rats; however, there was no significant difference between groups in degree of functional recovery. These findings demonstrate that adenovirus vector-mediated ex vivo gene transfer of BDNF enhances the capacity of BMSC to promote axonal regeneration in this completely transected spinal cord model; however, BDNF failed to enhance hind limb functional recovery. Further investigation is needed to establish an optimal combination of cell therapy and neurotrophin gene transfer for cases of spinal cord injury. PMID:17885772

GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

PubMed Central

Di, Yanming; Schafer, Daniel W.; Wilhelm, Larry J.; Fox, Samuel E.; Sullivan, Christopher M.; Curzon, Aron D.; Carrington, James C.; Mockler, Todd C.; Chang, Jeff H.

2011-01-01

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts. PMID:21998647

Applicability of the chymopapain gene used as endogenous reference gene for transgenic huanong no. 1 papaya detection.

PubMed

Guo, Jinchao; Yang, Litao; Liu, Xin; Zhang, Haibo; Qian, Bingjun; Zhang, Dabing

2009-08-12

The virus-resistant papaya (Carica papaya L.), Huanong no. 1, was the genetically modified (GM) fruit approved for growing in China in 2006. To implement the labeling regulation of GM papaya and its derivates, the development of papaya endogenous reference gene is very necessary for GM papaya detection. Herein, we reported one papaya specific gene, Chymopapain (CHY), as one suitable endogenous reference gene, used for GM papaya identification. Thereafter, we established the conventional and real-time quantitative PCR assays of the CHY gene. In the CHY conventional PCR assay, the limit of detection (LOD) was 25 copies of haploid papaya genome. In the CHY real-time quantitative PCR assay, both the LOD and the limit of quantification (LOQ) were as low as 12.5 copies of haploid papaya genome. Furthermore, we revealed the construct-specific sequence of Chinese GM papaya Huanong no. 1 and developed its conventional and quantitative PCR systems employing the CHY gene as endogenous reference gene. This work is useful for papaya specific identification and GM papaya detection.

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

PubMed

Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

2016-04-01

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study. © 2016 Japanese Society of Developmental Biologists.

Transcriptional enhancer from milk protein genes

DOEpatents

Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

1999-01-01

The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

Gene encoding acetyl-coenzyme A carboxylase

DOEpatents

Roessler, Paul G.; Ohlrogge, John B.

1996-01-01

A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

Membrane-derived second messenger regulates x-ray-mediated tumor necrosis factor alpha gene induction.

PubMed Central

Hallahan, D E; Virudachalam, S; Kuchibhotla, J; Kufe, D W; Weichselbaum, R R

1994-01-01

Cells adapt to adverse environmental conditions through a wide range of responses that are conserved throughout evolution. Physical agents such as ionizing radiation are known to initiate a stress response that is triggered by the recognition of DNA damage. We have identified a signaling pathway involving the activation of phospholipase A2 and protein kinase C in human cells that confers x-ray induction of the tumor necrosis factor alpha gene. Treatment of human cells with ionizing radiation or H2O2 was associated with the production of arachidonic acid. Inhibition of phospholipase A2 abolished radiation-mediated arachidonate production as well as the subsequent activation of protein kinase C and tumor necrosis factor alpha gene expression. These findings demonstrate that ionizing radiation-mediated gene expression in human cells is regulated in part by extranuclear signal transduction. One practical application of phospholipase A2 inhibitors is to ameliorate the adverse effects of radiotherapy associated with tumor necrosis factor alpha production. Images PMID:8197153

A formal theory of the selfish gene.

PubMed

Gardner, A; Welch, J J

2011-08-01

Adaptation is conventionally regarded as occurring at the level of the individual organism. In contrast, the theory of the selfish gene proposes that it is more correct to view adaptation as occurring at the level of the gene. This view has received much popular attention, yet has enjoyed only limited uptake in the primary research literature. Indeed, the idea of ascribing goals and strategies to genes has been highly controversial. Here, we develop a formal theory of the selfish gene, using optimization theory to capture the analogy of 'gene as fitness-maximizing agent' in mathematical terms. We provide formal justification for this view of adaptation by deriving mathematical correspondences that translate the optimization formalism into dynamical population genetics. We show that in the context of social interactions between genes, it is the gene's inclusive fitness that provides the appropriate maximand. Hence, genic selection can drive the evolution of altruistic genes. Finally, we use the formalism to assess the various criticisms that have been levelled at the theory of the selfish gene, dispelling some and strengthening others. © 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.

New genes in the evolution of the neural crest differentiation program

PubMed Central

2007-01-01

Background Development of the vertebrate head depends on the multipotency and migratory behavior of neural crest derivatives. This cell population is considered a vertebrate innovation and, accordingly, chordate ancestors lacked neural crest counterparts. The identification of neural crest specification genes expressed in the neural plate of basal chordates, in addition to the discovery of pigmented migratory cells in ascidians, has challenged this hypothesis. These new findings revive the debate on what is new and what is ancient in the genetic program that controls neural crest formation. Results To determine the origin of neural crest genes, we analyzed Phenotype Ontology annotations to select genes that control the development of this tissue. Using a sequential blast pipeline, we phylogenetically classified these genes, as well as those associated with other tissues, in order to define tissue-specific profiles of gene emergence. Of neural crest genes, 9% are vertebrate innovations. Our comparative analyses show that, among different tissues, the neural crest exhibits a particularly high rate of gene emergence during vertebrate evolution. A remarkable proportion of the new neural crest genes encode soluble ligands that control neural crest precursor specification into each cell lineage, including pigmented, neural, glial, and skeletal derivatives. Conclusion We propose that the evolution of the neural crest is linked not only to the recruitment of ancestral regulatory genes but also to the emergence of signaling peptides that control the increasingly complex lineage diversification of this plastic cell population. PMID:17352807

Identification and Characterization of Novel Immunomodulatory Bursal-derived Pentapeptide-II (BPP-II)*

PubMed Central

Feng, Xiu-Li; Liu, Qing-Tao; Cao, Rui-Bing; Zhou, Bin; Ma, Zhi-Yong; Deng, Wen-Lei; Wei, Jian-Chao; Qiu, Ya-Feng; Wang, Fang-Quan; Gu, Jin-Yan; Wang, Feng-Juan; Zheng, Qi-Sheng; Ishag, Hassan; Chen, Pu-Yan

2012-01-01

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement. PMID:22184121

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

PubMed

Feng, Xiu-Li; Liu, Qing-Tao; Cao, Rui-Bing; Zhou, Bin; Ma, Zhi-Yong; Deng, Wen-Lei; Wei, Jian-Chao; Qiu, Ya-Feng; Wang, Fang-Quan; Gu, Jin-Yan; Wang, Feng-Juan; Zheng, Qi-Sheng; Ishag, Hassan; Chen, Pu-Yan

2012-02-03

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

Human adipose-derived mesenchymal stromal cell pigment epithelium-derived factor cytotherapy modifies genetic and epigenetic profiles of prostate cancer cells.

PubMed

Zolochevska, Olga; Shearer, Joseph; Ellis, Jayne; Fokina, Valentina; Shah, Forum; Gimble, Jeffrey M; Figueiredo, Marxa L

2014-03-01

Adipose-derived mesenchymal stromal cells (ASCs) are promising tools for delivery of cytotherapy against cancer. However, ASCs can exert profound effects on biological behavior of tumor cells. Our study aimed to examine the influence of ASCs on gene expression and epigenetic methylation profiles of prostate cancer cells as well as the impact of expressing a therapeutic gene on modifying the interaction between ASCs and prostate cancer cells. ASCs were modified by lentiviral transduction to express either green fluorescent protein as a control or pigment epithelium-derived factor (PEDF) as a therapeutic molecule. PC3 prostate cancer cells were cultured in the presence of ASC culture-conditioned media (CCM), and effects on PC3 or DU145. Ras cells were examined by means of real-time quantitative polymerase chain reaction, EpiTect methyl prostate cancer-focused real-time quantitative polymerase chain reaction arrays, and luciferase reporter assays. ASCs transduced with lentiviral vectors were able to mediate expression of several tumor-inhibitory genes, some of which correlated with epigenetic methylation changes on cocultured PC3 prostate cancer cells. When PC3 cells were cultured with ASC-PEDF CCM, we observed a shift in the balance of gene expression toward tumor inhibition, which suggests that PEDF reduces the potential tumor-promoting activity of unmodified ASCs. These results suggest that ASC-PEDF CCM can promote reprogramming of tumor cells in a paracrine manner. An improved understanding of genetic and epigenetic events in prostate cancer growth in response to PEDF paracrine therapy would enable a more effective use of ASC-PEDF, with the goal of achieving safer yet more potent anti-tumor effects. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics

PubMed Central

Handel, Adam E.; Chintawar, Satyan; Lalic, Tatjana; Whiteley, Emma; Vowles, Jane; Giustacchini, Alice; Argoud, Karene; Sopp, Paul; Nakanishi, Mahito; Bowden, Rory; Cowley, Sally; Newey, Sarah; Akerman, Colin; Ponting, Chris P.; Cader, M. Zameel

2016-01-01

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells. PMID:26740550

GeneMesh: a web-based microarray analysis tool for relating differentially expressed genes to MeSH terms.

PubMed

Jani, Saurin D; Argraves, Gary L; Barth, Jeremy L; Argraves, W Scott

2010-04-01

An important objective of DNA microarray-based gene expression experimentation is determining inter-relationships that exist between differentially expressed genes and biological processes, molecular functions, cellular components, signaling pathways, physiologic processes and diseases. Here we describe GeneMesh, a web-based program that facilitates analysis of DNA microarray gene expression data. GeneMesh relates genes in a query set to categories available in the Medical Subject Headings (MeSH) hierarchical index. The interface enables hypothesis driven relational analysis to a specific MeSH subcategory (e.g., Cardiovascular System, Genetic Processes, Immune System Diseases etc.) or unbiased relational analysis to broader MeSH categories (e.g., Anatomy, Biological Sciences, Disease etc.). Genes found associated with a given MeSH category are dynamically linked to facilitate tabular and graphical depiction of Entrez Gene information, Gene Ontology information, KEGG metabolic pathway diagrams and intermolecular interaction information. Expression intensity values of groups of genes that cluster in relation to a given MeSH category, gene ontology or pathway can be displayed as heat maps of Z score-normalized values. GeneMesh operates on gene expression data derived from a number of commercial microarray platforms including Affymetrix, Agilent and Illumina. GeneMesh is a versatile web-based tool for testing and developing new hypotheses through relating genes in a query set (e.g., differentially expressed genes from a DNA microarray experiment) to descriptors making up the hierarchical structure of the National Library of Medicine controlled vocabulary thesaurus, MeSH. The system further enhances the discovery process by providing links between sets of genes associated with a given MeSH category to a rich set of html linked tabular and graphic information including Entrez Gene summaries, gene ontologies, intermolecular interactions, overlays of genes onto KEGG

Methods for Gene Transfer to the Central Nervous System

PubMed Central

Kantor, Boris; Bailey, Rachel M.; Wimberly, Keon; Kalburgi, Sahana N.; Gray, Steven J.

2015-01-01

Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed. PMID:25311922

Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells.

PubMed

Boensch, C; Huang, S S; Connolly, D T; Huang, J S

1999-04-09

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.

Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

PubMed

Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

2014-05-09

The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A common polymorphism in the brain-derived neurotrophic factor gene (BDNF) modulates human cortical plasticity and the response to rTMS.

PubMed

Cheeran, Binith; Talelli, Penelope; Mori, Francesco; Koch, Giacomo; Suppa, Antonio; Edwards, Mark; Houlden, Henry; Bhatia, Kailash; Greenwood, Richard; Rothwell, John C

2008-12-01

The brain-derived neurotrophic factor gene (BDNF) is one of many genes thought to influence synaptic plasticity in the adult brain and shows a common single nucleotide polymorphism (BDNF Val66Met) in the normal population that is associated with differences in hippocampal volume and episodic memory. It is also thought to influence possible synaptic changes in motor cortex following a simple motor learning task. Here we extend these studies by using new non-invasive transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (TDCS) techniques that directly test the excitability and plasticity of neuronal circuits in human motor cortex in subjects at rest. We investigated whether the susceptibility to TMS probes of plasticity is significantly influenced by the BDNF polymorphism. Val66Met carriers were matched with Val66Val individuals and tested on the following protocols: continuous and intermittent theta burst TMS; median nerve paired associative stimulation; and homeostatic plasticity in the TDCS/1 Hz rTMS model. The response of Met allele carriers differed significantly in all protocols compared with the response of Val66Val individuals. We suggest that this is due to the effect of BNDF on the susceptibility of synapses to undergo LTP/LTD. The circuits tested here are implicated in the pathophysiology of movement disorders such as dystonia and are being assessed as potential new targets in the treatment of stroke. Thus the polymorphism may be one factor that influences the natural response of the brain to injury and disease.

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade

PubMed Central

Boothby, Thomas C.; Tenlen, Jennifer R.; Smith, Frank W.; Wang, Jeremy R.; Patanella, Kiera A.; Osborne Nishimura, Erin; Tintori, Sophia C.; Li, Qing; Jones, Corbin D.; Yandell, Mark; Glasscock, Jarret; Goldstein, Bob

2015-01-01

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes. PMID:26598659

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.

PubMed

Boothby, Thomas C; Tenlen, Jennifer R; Smith, Frank W; Wang, Jeremy R; Patanella, Kiera A; Nishimura, Erin Osborne; Tintori, Sophia C; Li, Qing; Jones, Corbin D; Yandell, Mark; Messina, David N; Glasscock, Jarret; Goldstein, Bob

2015-12-29

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tschaplinski, Timothy J; Tsai, Chung-Jui; Harding, Scott A

Salicin-based phenolic glycosides, hydroxycinnamate derivatives and flavonoid-derived condensed tannins comprise up to one-third of Populus leaf dry mass. Genes regulating the abundance and chemical diversity of these substances have not been comprehensively analysed in tree species exhibiting this metabolically demanding level of phenolic metabolism. Here, shikimate-phenylpropanoid pathway genes thought to give rise to these phenolic products were annotated from the Populus genome, their expression assessed by semiquantitative or quantitative reverse transcription polymerase chain reaction (PCR), and metabolic evidence for function presented. Unlike Arabidopsis, Populus leaves accumulate an array of hydroxycinnamoyl-quinate esters, which is consistent with broadened function of the expandedmore » hydroxycinnamoyl-CoA transferase gene family. Greater flavonoid pathway diversity is also represented, and flavonoid gene families are larger. Consistent with expanded pathway function, most of these genes were upregulated during wound-stimulated condensed tannin synthesis in leaves. The suite of Populus genes regulating phenylpropanoid product accumulation should have important application in managing phenolic carbon pools in relation to climate change and global carbon cycling.« less

[Association of Val66Met polymorphism of brain-derived neurotrophic factor gene with cognitive impairment and clinical symptoms in first episode schizophrenia].

PubMed

Zhang, Chengcheng; Gu, Xiaochu; Wang, Qiang; Li, Mingli; Deng, Wei; Guo, Wanjun; Zhao, Liansheng; Ma, Xiaohong; Li, Tao

2017-08-10

To assess the association of cognitive impairment and clinical symptoms in first-episode schizophrenia with the Val66Met (rs6265) polymorphism of brain-derived neurotrophic factor (BDNF) gene. For 87 patients with first-episode schizophrenia and 76 healthy controls, the Val66Met polymorphism was determined with a Taqman Assay-on-Demand method. Wechsler intelligence test was carried out for all participants. Correlation of cognitive impairment with clinical severity was also analyzed. The patients were significantly lower in total IQ, verbal IQ and performance IQ compared to the controls. The lower total IQ (F=4.59, P= 0.01) and verbal IQ (F=4.44, P=0.01) were influenced by genetic factors and diagnostic interaction. The vertal IQ of Val/Val patients was significantly lower than those of Val/Met and Met/Met carriers. For the control group, the verbal IQ of Met/Met carriers was lower than that of Val/Met carriers, and the total IQ of Met/Met carriers was lower than those of Val/Met and Val/Val carriers. For the patient group, the total IQ of Val/Val carriers was negatively correlated with positive symptoms (r=-0.65, P=0.03) and thought disorders (r=-0.61, P=0.02). Cognitive impairment in first-episode schizophrenic patients is associated with the Val66Met polymorphism of the BDNF gene, and has an important clinical relevance.

Gene encoding acetyl-coenzyme A carboxylase

DOEpatents

Roessler, P.G.; Ohlrogge, J.B.

1996-09-24

A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

Inverse gene-for-gene interactions contribute additively to tan spot susceptibility in wheat.

PubMed

Liu, Zhaohui; Zurn, Jason D; Kariyawasam, Gayan; Faris, Justin D; Shi, Gongjun; Hansen, Jana; Rasmussen, Jack B; Acevedo, Maricelis

2017-06-01

Tan spot susceptibility is conferred by multiple interactions of necrotrophic effector and host sensitivity genes. Tan spot of wheat, caused by Pyrenophora tritici-repentis, is an important disease in almost all wheat-growing areas of the world. The disease system is known to involve at least three fungal-produced necrotrophic effectors (NEs) that interact with the corresponding host sensitivity (S) genes in an inverse gene-for-gene manner to induce disease. However, it is unknown if the effects of these NE-S gene interactions contribute additively to the development of tan spot. In this work, we conducted disease evaluations using different races and quantitative trait loci (QTL) analysis in a wheat recombinant inbred line (RIL) population derived from a cross between two susceptible genotypes, LMPG-6 and PI 626573. The two parental lines each harbored a single known NE sensitivity gene with LMPG-6 having the Ptr ToxC sensitivity gene Tsc1 and PI 626573 having the Ptr ToxA sensitivity gene Tsn1. Transgressive segregation was observed in the population for all races. QTL mapping revealed that both loci (Tsn1 and Tsc1) were significantly associated with susceptibility to race 1 isolates, which produce both Ptr ToxA and Ptr ToxC, and the two genes contributed additively to tan spot susceptibility. For isolates of races 2 and 3, which produce only Ptr ToxA and Ptr ToxC, only Tsn1 and Tsc1 were associated with tan spot susceptibility, respectively. This work clearly demonstrates that tan spot susceptibility in this population is due primarily to two NE-S interactions. Breeders should remove both sensitivity genes from wheat lines to obtain high levels of tan spot resistance.

The mosaic accessory gene structures of the SXT/R391-like integrative and conjugative elements derived from Vibrio spp. isolated from aquatic products and environment in the Yangtze River estuary, China

PubMed Central

2013-01-01

Background The emergence, resurgence and spread of human food-borne pathogenic Vibrios are one of the major contributors to disease burden and mortality particularly in developing countries with disputable sanitary conditions. Previous research on pathogenic Vibrio cholerae and Vibrio parahaemolitycus derived from clinical samples has proposed links between acquisition of virulence and multiple drug resistance traits and intercellular transmissibility of mobile genetic elements in the environment. To date, very few information is available on environmental Vibrio isolates. In this study, we characterized eleven Vibrio strains bearing the SXT/R391-like integrative and conjugative elements (ICEs) derived from aquatic products and environment in the Yangtze River Estuary, China. Results The eleven Vibrio strains were isolated in 2010 to 2011, and taxonomically identified, which included six Vibrio cholerae, three Vibrio parahaemolyticus, one Vibrio alginolyticus and one Vibrio natriegens. Most of the strains displayed strong resistance phenotypes to ampicillin, mercury and chromium. The majority of their ICEs, which belong to S and R exclusion system groups, contain ICEs-chromosome junction sequences and highly conserved core-genes required for ICE transfer. However, comparative sequence analysis uncovered interesting diversity in their mosaic accessory gene structures, which carry many novel genes that have not been described in any known ICEs to date. In addition, antibiotic resistance was transmitted by ICEVchChn6 and ICEVpaChn1 from V. cholerae, V. parahaemolyticus to E. coli MG1655 via conjugation, respectively. Our data also revealed that the ICEs characterized in this study are phylogenetically distant from most of the SXT/R391 ICEs reported previously, which may represent a novel cluster likely shaped by the ecological environment in the Yangtze River Estuary, China. Conclusions This study constitutes the first investigation of ICEs-positive Vibrio spp. in the

Novel recurrently mutated genes in African American colon cancers.

PubMed

Guda, Kishore; Veigl, Martina L; Varadan, Vinay; Nosrati, Arman; Ravi, Lakshmeswari; Lutterbaugh, James; Beard, Lydia; Willson, James K V; Sedwick, W David; Wang, Zhenghe John; Molyneaux, Neil; Miron, Alexander; Adams, Mark D; Elston, Robert C; Markowitz, Sanford D; Willis, Joseph E

2015-01-27

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.

Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome.

PubMed

Albers, Sonja-Verena; Driessen, Arnold J M

2008-12-01

The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.

Using iPSC-derived human DA neurons from opioid-dependent subjects to study dopamine dynamics.

PubMed

Sheng, Yang; Filichia, Emily; Shick, Elizabeth; Preston, Kenzie L; Phillips, Karran A; Cooperman, Leslie; Lin, Zhicheng; Tesar, Paul; Hoffer, Barry; Luo, Yu

2016-08-01

The dopaminergic (DA) system plays important roles in addiction. However, human DA neurons from drug-dependent subjects were not available for study until recent development in inducible pluripotent stem cells (iPSCs) technology. In this study, we produced DA neurons differentiated using iPSCs derived from opioid-dependent and control subjects carrying different 3' VNTR (variable number tandem repeat) polymorphism in the human dopamine transporter (DAT or SLC6A3). In addition, the effects of valproic acid (VPA) exposures on iPSC-derived human DA neurons are also examined. We present the first evidence suggesting that the 3' VNTR polymorphism in the hDAT gene affects DAT expression level in iPSC-derived human DA neurons. In human DA neurons, which provide an appropriate cellular milieu, VPA treatment alters the expression of several genes important for dopaminergic neuron function including DAT, Nurr1, and TH; this might partly explain its action in regulating addictive behaviors. VPA treatment also significantly increased DA D2 receptor (Drd2) expression, especially in the opioid-dependent iPSC cell lines. Our data suggest that human iPSC-derived DA neurons may be useful in in vitro experimental model to examine the effects of genetic variation in gene regulation, to examine the underlying mechanisms in neurological disorders including drug addiction, and to serve as a platform for therapeutic development.

Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades

PubMed Central

Hering, Lars; Bouameur, Jamal-Eddine; Reichelt, Julian; Magin, Thomas M; Mayer, Georg

2016-01-01

Intermediate filament (IF) proteins, including nuclear lamins and cytoplasmic IF proteins, are essential cytoskeletal components of bilaterian cells. Despite their important role in protecting tissues against mechanical force, no cytoplasmic IF proteins have been convincingly identified in arthropods. Here we show that the ancestral cytoplasmic IF protein gene was lost in the entire panarthropod (onychophoran + tardigrade + arthropod) rather than arthropod lineage and that nuclear, lamin-derived proteins instead acquired new cytoplasmic roles at least three times independently in collembolans, copepods, and tardigrades. Transcriptomic and genomic data revealed three IF protein genes in the tardigrade Hypsibius dujardini, one of which (cytotardin) occurs exclusively in the cytoplasm of epidermal and foregut epithelia, where it forms belt-like filaments around each epithelial cell. These results suggest that a lamin derivative has been co-opted to enhance tissue stability in tardigrades, a function otherwise served by cytoplasmic IF proteins in all other bilaterians. DOI: http://dx.doi.org/10.7554/eLife.11117.001 PMID:26840051

Gene variant linked to lung cancer risk

Cancer.gov

A variation of the gene NFKB1, called rs4648127, is associated with an estimated 44 percent reduction in lung cancer risk. When this information, derived from samples obtained as part of a large NCI-sponsored prevention clinical trial, was compared with d

Asymmetric histone modifications between the original and derived loci of human segmental duplications

PubMed Central

Zheng, Deyou

2008-01-01

Background Sequencing and annotation of several mammalian genomes have revealed that segmental duplications are a common architectural feature of primate genomes; in fact, about 5% of the human genome is composed of large blocks of interspersed segmental duplications. These segmental duplications have been implicated in genomic copy-number variation, gene novelty, and various genomic disorders. However, the molecular processes involved in the evolution and regulation of duplicated sequences remain largely unexplored. Results In this study, the profile of about 20 histone modifications within human segmental duplications was characterized using high-resolution, genome-wide data derived from a ChIP-Seq study. The analysis demonstrates that derivative loci of segmental duplications often differ significantly from the original with respect to many histone methylations. Further investigation showed that genes are present three times more frequently in the original than in the derivative, whereas pseudogenes exhibit the opposite trend. These asymmetries tend to increase with the age of segmental duplications. The uneven distribution of genes and pseudogenes does not, however, fully account for the asymmetry in the profile of histone modifications. Conclusion The first systematic analysis of histone modifications between segmental duplications demonstrates that two seemingly 'identical' genomic copies are distinct in their epigenomic properties. Results here suggest that local chromatin environments may be implicated in the discrimination of derived copies of segmental duplications from their originals, leading to a biased pseudogenization of the new duplicates. The data also indicate that further exploration of the interactions between histone modification and sequence degeneration is necessary in order to understand the divergence of duplicated sequences. PMID:18598352

Differentiation and Application of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

PubMed

Maguire, Eithne Margaret; Xiao, Qingzhong; Xu, Qingbo

2017-11-01

Vascular smooth muscle cells (VSMCs) play a role in the development of vascular disease, for example, neointimal formation, arterial aneurysm, and Marfan syndrome caused by genetic mutations in VSMCs, but little is known about the mechanisms of the disease process. Advances in induced pluripotent stem cell technology have now made it possible to derive VSMCs from several different somatic cells using a selection of protocols. As such, researchers have set out to delineate key signaling processes involved in triggering VSMC gene expression to grasp the extent of gene regulatory networks involved in phenotype commitment. This technology has also paved the way for investigations into diseases affecting VSMC behavior and function, which may be treatable once an identifiable culprit molecule or gene has been repaired. Moreover, induced pluripotent stem cell-derived VSMCs are also being considered for their use in tissue-engineered blood vessels as they may prove more beneficial than using autologous vessels. Finally, while several issues remains to be clarified before induced pluripotent stem cell-derived VSMCs can become used in regenerative medicine, they do offer both clinicians and researchers hope for both treating and understanding vascular disease. In this review, we aim to update the recent progress on VSMC generation from stem cells and the underlying molecular mechanisms of VSMC differentiation. We will also explore how the use of induced pluripotent stem cell-derived VSMCs has changed the game for regenerative medicine by offering new therapeutic avenues to clinicians, as well as providing researchers with a new platform for modeling of vascular disease. © 2017 American Heart Association, Inc.

Virus-Based RNA Silencing Agents and Virus-Derived Expression Vectors as Gene Therapy Vehicles.

PubMed

Venkataraman, Srividhya; Ahmad, Tauqeer; AbouHaidar, Mounir G; Hefferon, Kathleen L

2017-01-01

In consideration of recent developments in understanding the genomics and proteomics of viruses, the use of viral DNA / RNA sequences as well as their gene expression schemes, have found new in-roads towards the prognosis and therapy of diseases. Correspondingly, the sphere of the patenting scenario has expanded significantly. The current review addresses patented inventions concerning the use of virus sequences as gene silencing machineries and inventions concerning the generation and application of viral sequences as expression vectors. Furthermore, this review also discusses the employment of these patents for clinical, agricultural and biotechnological applications. Considering these objectives, the Delphion Research Intellectual Property Network database was searched using keywords such as "gene silencing", "engineered viruses" and "expression vectors" and descriptions of recent patents on the said topics were discussed. Despite several recent advances in the use of viruses as disease therapy vehicles and biotechnological vectors, these developments have yet to be proven effective in practice, in clinical and field trials. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Discovery and validation of a glioblastoma co-expressed gene module

PubMed Central

Dunwoodie, Leland J.; Poehlman, William L.; Ficklin, Stephen P.; Feltus, Frank Alexander

2018-01-01

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network. PMID:29541392

Discovery and validation of a glioblastoma co-expressed gene module.

PubMed

Dunwoodie, Leland J; Poehlman, William L; Ficklin, Stephen P; Feltus, Frank Alexander

2018-02-16

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network.

The WRKY Transcription Factor Genes in Lotus japonicus.

PubMed

Song, Hui; Wang, Pengfei; Nan, Zhibiao; Wang, Xingjun

2014-01-01

WRKY transcription factor genes play critical roles in plant growth and development, as well as stress responses. WRKY genes have been examined in various higher plants, but they have not been characterized in Lotus japonicus. The recent release of the L. japonicus whole genome sequence provides an opportunity for a genome wide analysis of WRKY genes in this species. In this study, we identified 61 WRKY genes in the L. japonicus genome. Based on the WRKY protein structure, L. japonicus WRKY (LjWRKY) genes can be classified into three groups (I-III). Investigations of gene copy number and gene clusters indicate that only one gene duplication event occurred on chromosome 4 and no clustered genes were detected on chromosomes 3 or 6. Researchers previously believed that group II and III WRKY domains were derived from the C-terminal WRKY domain of group I. Our results suggest that some WRKY genes in group II originated from the N-terminal domain of group I WRKY genes. Additional evidence to support this hypothesis was obtained by Medicago truncatula WRKY (MtWRKY) protein motif analysis. We found that LjWRKY and MtWRKY group III genes are under purifying selection, suggesting that WRKY genes will become increasingly structured and functionally conserved.

The WRKY Transcription Factor Genes in Lotus japonicus

PubMed Central

Wang, Pengfei; Wang, Xingjun

2014-01-01

WRKY transcription factor genes play critical roles in plant growth and development, as well as stress responses. WRKY genes have been examined in various higher plants, but they have not been characterized in Lotus japonicus. The recent release of the L. japonicus whole genome sequence provides an opportunity for a genome wide analysis of WRKY genes in this species. In this study, we identified 61 WRKY genes in the L. japonicus genome. Based on the WRKY protein structure, L. japonicus WRKY (LjWRKY) genes can be classified into three groups (I–III). Investigations of gene copy number and gene clusters indicate that only one gene duplication event occurred on chromosome 4 and no clustered genes were detected on chromosomes 3 or 6. Researchers previously believed that group II and III WRKY domains were derived from the C-terminal WRKY domain of group I. Our results suggest that some WRKY genes in group II originated from the N-terminal domain of group I WRKY genes. Additional evidence to support this hypothesis was obtained by Medicago truncatula WRKY (MtWRKY) protein motif analysis. We found that LjWRKY and MtWRKY group III genes are under purifying selection, suggesting that WRKY genes will become increasingly structured and functionally conserved. PMID:24745006

The Early ANTP Gene Repertoire: Insights from the Placozoan Genome

PubMed Central

Schierwater, Bernd; Kamm, Kai; Srivastava, Mansi; Rokhsar, Daniel; Rosengarten, Rafael D.; Dellaporta, Stephen L.

2008-01-01

The evolution of ANTP genes in the Metazoa has been the subject of conflicting hypotheses derived from full or partial gene sequences and genomic organization in higher animals. Whole genome sequences have recently filled in some crucial gaps for the basal metazoan phyla Cnidaria and Porifera. Here we analyze the complete genome of Trichoplax adhaerens, representing the basal metazoan phylum Placozoa, for its set of ANTP class genes. The Trichoplax genome encodes representatives of Hox/ParaHox-like, NKL, and extended Hox genes. This repertoire possibly mirrors the condition of a hypothetical cnidarian-bilaterian ancestor. The evolution of the cnidarian and bilaterian ANTP gene repertoires can be deduced by a limited number of cis-duplications of NKL and “extended Hox” genes and the presence of a single ancestral “ProtoHox” gene. PMID:18716659

Gene Structures, Evolution and Transcriptional Profiling of the WRKY Gene Family in Castor Bean (Ricinus communis L.).

PubMed

Zou, Zhi; Yang, Lifu; Wang, Danhua; Huang, Qixing; Mo, Yeyong; Xie, Guishui

2016-01-01

WRKY proteins comprise one of the largest transcription factor families in plants and form key regulators of many plant processes. This study presents the characterization of 58 WRKY genes from the castor bean (Ricinus communis L., Euphorbiaceae) genome. Compared with the automatic genome annotation, one more WRKY-encoding locus was identified and 20 out of the 57 predicted gene models were manually corrected. All RcWRKY genes were shown to contain at least one intron in their coding sequences. According to the structural features of the present WRKY domains, the identified RcWRKY genes were assigned to three previously defined groups (I-III). Although castor bean underwent no recent whole-genome duplication event like physic nut (Jatropha curcas L., Euphorbiaceae), comparative genomics analysis indicated that one gene loss, one intron loss and one recent proximal duplication occurred in the RcWRKY gene family. The expression of all 58 RcWRKY genes was supported by ESTs and/or RNA sequencing reads derived from roots, leaves, flowers, seeds and endosperms. Further global expression profiles with RNA sequencing data revealed diverse expression patterns among various tissues. Results obtained from this study not only provide valuable information for future functional analysis and utilization of the castor bean WRKY genes, but also provide a useful reference to investigate the gene family expansion and evolution in Euphorbiaceus plants.

A global evolutionary and metabolic analysis of human obesity gene risk variants.

PubMed

Castillo, Joseph J; Hazlett, Zachary S; Orlando, Robert A; Garver, William S

2017-09-05

It is generally accepted that the selection of gene variants during human evolution optimized energy metabolism that now interacts with our obesogenic environment to increase the prevalence of obesity. The purpose of this study was to perform a global evolutionary and metabolic analysis of human obesity gene risk variants (110 human obesity genes with 127 nearest gene risk variants) identified using genome-wide association studies (GWAS) to enhance our knowledge of early and late genotypes. As a result of determining the mean frequency of these obesity gene risk variants in 13 available populations from around the world our results provide evidence for the early selection of ancestral risk variants (defined as selection before migration from Africa) and late selection of derived risk variants (defined as selection after migration from Africa). Our results also provide novel information for association of these obesity genes or encoded proteins with diverse metabolic pathways and other human diseases. The overall results indicate a significant differential evolutionary pattern for the selection of obesity gene ancestral and derived risk variants proposed to optimize energy metabolism in varying global environments and complex association with metabolic pathways and other human diseases. These results are consistent with obesity genes that encode proteins possessing a fundamental role in maintaining energy metabolism and survival during the course of human evolution. Copyright © 2017. Published by Elsevier B.V.

Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes.

PubMed

Bergmann, D J; Arciero, D M; Hooper, A B

1994-06-01

The organization of genes for three proteins involved in ammonia oxidation in Nitrosomonas europaea has been investigated. The amino acid sequence of the N-terminal region and four heme-containing peptides produced by proteolysis of the tetraheme cytochrome c554 of N. europaea were determined by Edman degradation. The gene (cycA) encoding this cytochrome is present in three copies per genome (H. McTavish, F. LaQuier, D. Arciero, M. Logan, G. Mundfrom, J.A. Fuchs, and A. B. Hooper, J. Bacteriol. 175:2445-2447, 1993). Three clones, representing at least two copies of cycA, were isolated and sequenced by the dideoxy-chain termination procedure. In both copies, the sequences of 211 amino acids derived from the gene sequence are identical and include all amino acids predicted by the proteolytic peptides. In two copies, the cycA open reading frame (ORF) is followed closely (three bases in one copy) by a second ORF predicted to encode a 28-kDa tetraheme c cytochrome not previously characterized but similar to the nirT gene product of Pseudomonas stutzeri. In one copy of the cycA gene cluster, the second ORF is absent.

Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy

Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the averagemore » nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.« less

CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

PubMed

Zhang, Hanrui; Shi, Jianting; Hachet, Melanie A; Xue, Chenyi; Bauer, Robert C; Jiang, Hongfeng; Li, Wenjun; Tohyama, Junichiro; Millar, John; Billheimer, Jeffrey; Phillips, Michael C; Razani, Babak; Rader, Daniel J; Reilly, Muredach P

2017-11-01

To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA ( LIPA -/- ) had barely detectable LAL enzymatic activity. Control and LIPA -/- IPSDM were loaded with [ 3 H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [ 3 H]-cholesterol to apolipoprotein A-I was abolished in LIPA -/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [ 3 H]-cholesterol-labeled AcLDL, [ 3 H]-cholesterol efflux was, however, not different between control and LIPA -/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA -/- IPSDM. In nonlipid loaded state, LIPA -/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA -/- IPSDM. LIPA -/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B , IL6 , and CCL5. CONCLUSIONS: LIPA -/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human

Epidermal Growth Factor and Interleukin-1β Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells1

PubMed Central

Ackerman, William E.; Rovin, Brad H.; Kniss, Douglas A.

2006-01-01

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intra-uterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1β-driven PGE2 production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1β might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1β resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1β-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFκB) regulatory elements alone, but was evident when a fragment (−891/+9) of the COX-2 gene 5′-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1β-driven NFκB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1β appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1β employs the NFκB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1β and EGF does not rely on augmented NFκB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter. PMID:15329330