Mutations in the ABCA4 (ABCR) Gene Are the Major Cause of Autosomal Recessive Cone-Rod Dystrophy

PubMed Central

Maugeri, Alessandra; Klevering, B. Jeroen; Rohrschneider, Klaus; Blankenagel, Anita; Brunner, Han G.; Deutman, August F.; Hoyng, Carel B.; Cremers, Frans P. M.

2000-01-01

The photoreceptor cell–specific ATP-binding cassette transporter gene (ABCA4; previously denoted “ABCR”) is mutated in most patients with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients with isolated CRD, all from Germany and The Netherlands . Single-strand conformation–polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans. PMID:10958761

Molecular and cellular characteristics of ABCA3 mutations associated with diffuse parenchymal lung diseases in children

PubMed Central

Flamein, Florence; Riffault, Laure; Muselet-Charlier, Céline; Pernelle, Julie; Feldmann, Delphine; Jonard, Laurence; Durand-Schneider, Anne-Marie; Coulomb, Aurore; Maurice, Michèle; Nogee, Lawrence M.; Inagaki, Nobuya; Amselem, Serge; Dubus, Jean Christophe; Rigourd, Virginie; Brémont, François; Marguet, Christophe; Brouard, Jacques; de Blic, Jacques; Clement, Annick; Epaud, Ralph; Guillot, Loïc

2012-01-01

ABCA3 (ATP-binding cassette subfamily A, member 3) is expressed in the lamellar bodies of alveolar type II cells and is crucial to pulmonary surfactant storage and homeostasis. ABCA3 gene mutations have been associated with neonatal respiratory distress (NRD) and pediatric interstitial lung disease (ILD). The objective of this study was to look for ABCA3 gene mutations in patients with severe NRD and/or ILD. The 30 ABCA3 coding exons were screened in 47 patients with severe NRD and/or ILD. ABCA3 mutations were identified in 10 out of 47 patients, including 2 homozygous, 5 compound heterozygous and 3 heterozygous patients. SP-B and SP-C expression patterns varied across patients. Among patients with ABCA3 mutations, five died shortly after birth and five developed ILD (including one without NRD). Functional studies of p.D253H and p.T1173R mutations revealed that p.D253H and p.T1173R induced abnormal lamellar bodies. Additionally, p.T1173R increased IL-8 secretion in vitro. In conclusion, we identified new ABCA3 mutations in patients with life-threatening NRD and/or ILD. Two mutations associated with ILD acted via different pathophysiological mechanisms despite similar clinical phenotypes. PMID:22068586

Regulation of Expression of abcA and Its Response to Environmental Conditions

PubMed Central

Villet, Regis A.; Truong-Bolduc, Que Chi; Wang, Yin; Estabrooks, Zoe; Medeiros, Heidi

2014-01-01

The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic β-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA. PMID:24509312

A Computational Approach From Gene to Structure Analysis of the Human ABCA4 Transporter Involved in Genetic Retinal Diseases.

PubMed

Trezza, Alfonso; Bernini, Andrea; Langella, Andrea; Ascher, David B; Pires, Douglas E V; Sodi, Andrea; Passerini, Ilaria; Pelo, Elisabetta; Rizzo, Stanislao; Niccolai, Neri; Spiga, Ottavia

2017-10-01

The aim of this article is to report the investigation of the structural features of ABCA4, a protein associated with a genetic retinal disease. A new database collecting knowledge of ABCA4 structure may facilitate predictions about the possible functional consequences of gene mutations observed in clinical practice. In order to correlate structural and functional effects of the observed mutations, the structure of mouse P-glycoprotein was used as a template for homology modeling. The obtained structural information and genetic data are the basis of our relational database (ABCA4Database). Sequence variability among all ABCA4-deposited entries was calculated and reported as Shannon entropy score at the residue level. The three-dimensional model of ABCA4 structure was used to locate the spatial distribution of the observed variable regions. Our predictions from structural in silico tools were able to accurately link the functional effects of mutations to phenotype. The development of the ABCA4Database gathers all the available genetic and structural information, yielding a global view of the molecular basis of some retinal diseases. ABCA4 modeled structure provides a molecular basis on which to analyze protein sequence mutations related to genetic retinal disease in order to predict the risk of retinal disease across all possible ABCA4 mutations. Additionally, our ABCA4 predicted structure is a good starting point for the creation of a new data analysis model, appropriate for precision medicine, in order to develop a deeper knowledge network of the disease and to improve the management of patients.

Allelic and Phenotypic Heterogeneity in ABCA4 mutations

PubMed Central

Burke, Tomas R; Tsang, Stephen H

2011-01-01

Since the discovery of the ABCA4 gene as the cause of autosomal recessive Stargardt disease/fundus flavimaculatus much has been written of the phenotypic variability in ABCA4 retinopathy. In this review the authors discuss the findings seen on examination and the disease features detected using various clinical tests. Important differential diagnoses are presented and unusual presentations of ABCA4 disease highlighted. PMID:21510770

A Comprehensive Survey of Sequence Variation in the ABCA4 (ABCR) Gene in Stargardt Disease and Age-Related Macular Degeneration

PubMed Central

Rivera, Andrea; White, Karen; Stöhr, Heidi; Steiner, Klaus; Hemmrich, Nadine; Grimm, Timo; Jurklies, Bernhard; Lorenz, Birgit; Scholl, Hendrik P. N.; Apfelstedt-Sylla, Eckhart; Weber, Bernhard H. F.

2000-01-01

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of ∼58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required. PMID:10958763

A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration.

PubMed

Rivera, A; White, K; Stöhr, H; Steiner, K; Hemmrich, N; Grimm, T; Jurklies, B; Lorenz, B; Scholl, H P; Apfelstedt-Sylla, E; Weber, B H

2000-10-01

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of approximately 58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required.

Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

PubMed Central

Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

PubMed

Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

PubMed

Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4*

PubMed Central

Biswas-Fiss, Esther E.; Affet, Stephanie; Ha, Malissa; Biswas, Subhasis B.

2012-01-01

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. PMID:23144455

Gene therapy for Stargardt disease associated with ABCA4 gene.

PubMed

Han, Zongchao; Conley, Shannon M; Naash, Muna I

2014-01-01

Mutations in the photoreceptor-specific flippase ABCA4 lead to accumulation of the toxic bisretinoid A2E, resulting in atrophy of the retinal pigment epithelium (RPE) and death of the photoreceptor cells. Many blinding diseases are associated with these mutations including Stargardt's disease (STGD1), cone-rod dystrophy, retinitis pigmentosa (RP), and increased susceptibility to age-related macular degeneration. There are no curative treatments for any of these dsystrophies. While the monogenic nature of many of these conditions makes them amenable to treatment with gene therapy, the ABCA4 cDNA is 6.8 kb and is thus too large for the AAV vectors which have been most successful for other ocular genes. Here we review approaches to ABCA4 gene therapy including treatment with novel AAV vectors, lentiviral vectors, and non-viral compacted DNA nanoparticles. Lentiviral and compacted DNA nanoparticles in particular have a large capacity and have been successful in improving disease phenotypes in the Abca4 (-/-) murine model. Excitingly, two Phase I/IIa clinical trials are underway to treat patients with ABCA4-associated Startgardt's disease (STGD1). As a result of the development of these novel technologies, effective therapies for ABCA4-associated diseases may finally be within reach.

Molecular diagnosis of putative Stargardt disease probands by exome sequencing

PubMed Central

2012-01-01

Background The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. Methods We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. Results Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. Conclusions Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques. PMID:22863181

ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

PubMed

Jonsson, Frida; Westin, Ida Maria; Österman, Lennart; Sandgren, Ola; Burstedt, Marie; Holmberg, Monica; Golovleva, Irina

2018-02-20

Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable. © 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol[S

PubMed Central

Zhu, Xuewei; Owen, John S.; Wilson, Martha D.; Li, Haitao; Griffiths, Gary L.; Thomas, Michael J.; Hiltbold, Elizabeth M.; Fessler, Michael B.; Parks, John S.

2010-01-01

We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1-M/-M macrophages. Abca1-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts. PMID:20650929

A Mouse Model of Harlequin Ichthyosis Delineates a Key Role for Abca12 in Lipid Homeostasis

PubMed Central

Smyth, Ian; Mukhamedova, Nigora; Meikle, Peter J.; Ellis, Sarah; Slattery, Keith; Collinge, Janelle E.; de Graaf, Carolyn A.; Bahlo, Melanie; Sviridov, Dmitri

2008-01-01

Harlequin Ichthyosis (HI) is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis. PMID:18802465

Novel Biomarker Candidates for Colorectal Cancer Metastasis: A Meta-analysis of In Vitro Studies

PubMed Central

Long, Nguyen Phuoc; Lee, Wun Jun; Huy, Nguyen Truong; Lee, Seul Ji; Park, Jeong Hill; Kwon, Sung Won

2016-01-01

Colorectal cancer (CRC) is one of the most common and lethal cancers. Although numerous studies have evaluated potential biomarkers for early diagnosis, current biomarkers have failed to reach an acceptable level of accuracy for distant metastasis. In this paper, we performed a gene set meta-analysis of in vitro microarray studies and combined the results from this study with previously published proteomic data to validate and suggest prognostic candidates for CRC metastasis. Two microarray data sets included found 21 significant genes. Of these significant genes, ALDOA, IL8 (CXCL8), and PARP4 had strong potential as prognostic candidates. LAMB2, MCM7, CXCL23A, SERPINA3, ABCA3, ALDH3A2, and POLR2I also have potential. Other candidates were more controversial, possibly because of the biologic heterogeneity of tumor cells, which is a major obstacle to predicting metastasis. In conclusion, we demonstrated a meta-analysis approach and successfully suggested ten biomarker candidates for future investigation. PMID:27688707

Novel Biomarker Candidates for Colorectal Cancer Metastasis: A Meta-analysis of In Vitro Studies.

PubMed

Long, Nguyen Phuoc; Lee, Wun Jun; Huy, Nguyen Truong; Lee, Seul Ji; Park, Jeong Hill; Kwon, Sung Won

2016-01-01

Colorectal cancer (CRC) is one of the most common and lethal cancers. Although numerous studies have evaluated potential biomarkers for early diagnosis, current biomarkers have failed to reach an acceptable level of accuracy for distant metastasis. In this paper, we performed a gene set meta-analysis of in vitro microarray studies and combined the results from this study with previously published proteomic data to validate and suggest prognostic candidates for CRC metastasis. Two microarray data sets included found 21 significant genes. Of these significant genes, ALDOA, IL8 (CXCL8), and PARP4 had strong potential as prognostic candidates. LAMB2, MCM7, CXCL23A, SERPINA3, ABCA3, ALDH3A2, and POLR2I also have potential. Other candidates were more controversial, possibly because of the biologic heterogeneity of tumor cells, which is a major obstacle to predicting metastasis. In conclusion, we demonstrated a meta-analysis approach and successfully suggested ten biomarker candidates for future investigation.

Extremely hypomorphic and severe deep intronic variants in the ABCA4 locus result in varying Stargardt disease phenotypes.

PubMed

Zernant, Jana; Lee, Winston; Nagasaki, Takayuki; Collison, Frederick T; Fishman, Gerald A; Bertelsen, Mette; Rosenberg, Thomas; Gouras, Peter; Tsang, Stephen H; Allikmets, Rando

2018-05-30

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of the ABCA4 locus in STGD1 patients identifies two expected disease-causing alleles in ~75% of patients and only one mutation in ~15% of patients. Recently, many possibly pathogenic variants in deep intronic sequences of ABCA4 have been identified in the latter group. We extended our analyses of deep intronic ABCA4 variants and determined that one of these, c.4253+43G>A (rs61754045), is present in 29/1155 (2.6%) of STGD1 patients. The variant is found at statistically significantly higher frequency in patients with only one pathogenic ABCA4 allele, 23/160 (14.38%), MAF=0.072, compared to MAF=0.013 in all STGD1 cases and MAF=0.006 in the matching general population (P<1x10-7). The variant, which is not predicted to have any effect on splicing, is the first reported intronic "extremely hypomorphic allele" in the ABCA4 locus; i.e., it is pathogenic only when in trans with a loss-of-function ABCA4 allele. It results in a distinct clinical phenotype characterized by late-onset of symptoms and foveal sparing. In ~70% of cases the variant was allelic with the c.6006-609T>A (rs575968112) variant, which was deemed non-pathogenic. Another rare deep intronic variant, c.5196+1056A>G (rs886044749), found in 5/834 (0.6%) of STGD1 cases is, conversely, a severe allele. This study determines pathogenicity for three non-coding variants in STGD1 patients of European descent accounting for ~3% of the disease. Defining disease-associated alleles in the non-coding sequences of the ABCA4 locus can be accomplished by integrated clinical and genetic analyses. Cold Spring Harbor Laboratory Press.

Deleterious ABCA7 mutations and transcript rescue mechanisms in early onset Alzheimer's disease.

PubMed

De Roeck, Arne; Van den Bossche, Tobi; van der Zee, Julie; Verheijen, Jan; De Coster, Wouter; Van Dongen, Jasper; Dillen, Lubina; Baradaran-Heravi, Yalda; Heeman, Bavo; Sanchez-Valle, Raquel; Lladó, Albert; Nacmias, Benedetta; Sorbi, Sandro; Gelpi, Ellen; Grau-Rivera, Oriol; Gómez-Tortosa, Estrella; Pastor, Pau; Ortega-Cubero, Sara; Pastor, Maria A; Graff, Caroline; Thonberg, Håkan; Benussi, Luisa; Ghidoni, Roberta; Binetti, Giuliano; de Mendonça, Alexandre; Martins, Madalena; Borroni, Barbara; Padovani, Alessandro; Almeida, Maria Rosário; Santana, Isabel; Diehl-Schmid, Janine; Alexopoulos, Panagiotis; Clarimon, Jordi; Lleó, Alberto; Fortea, Juan; Tsolaki, Magda; Koutroumani, Maria; Matěj, Radoslav; Rohan, Zdenek; De Deyn, Peter; Engelborghs, Sebastiaan; Cras, Patrick; Van Broeckhoven, Christine; Sleegers, Kristel

2017-09-01

Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer's disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)-control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5-41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may create opportunities for therapeutic interventions in AD.

Phenotype/genotype correlation in a case series of Stargardt's patients identifies novel mutations in the ABCA4 gene.

PubMed

Gemenetzi, M; Lotery, A J

2013-11-01

To investigate phenotypic variability in terms of best-corrected visual acuity (BCVA) in patients with Stargardt disease (STGD) and confirmed ABCA4 mutations. Entire coding region analysis of the ABCA4 gene by direct sequencing of seven patients with clinical findings of STGD seen in the Retina Clinics of Southampton Eye Unit between 2002 and 2011.Phenotypic variables recorded were BCVA, fluorescein angiographic appearance, electrophysiology, and visual fields. All patients had heterozygous amino acid-changing variants (missense mutations) in the ABCA4 gene. A splice sequence change was found in a 30-year-old patient with severly affected vision. Two novel sequence changes were identified: a missense mutation in a mildly affected 44-year-old patient and a frameshift mutation in a severly affected 34-year-old patient. The identified ABCA4 mutations were compatible with the resulting phenotypes in terms of BCVA. Higher BCVAs were recorded in patients with missense mutations. Sequence changes, predicted to have more deleterious effect on protein function, resulted in a more severe phenotype. This case series of STGD patients demonstrates novel genotype/phenotype correlations, which may be useful to counselling of patients. This information may prove useful in selection of candidates for clinical trials in ABCA4 disease.

Quantitative fundus autofluorescence in mice: correlation with HPLC quantitation of RPE lipofuscin and measurement of retina outer nuclear layer thickness.

PubMed

Sparrow, Janet R; Blonska, Anna; Flynn, Erin; Duncker, Tobias; Greenberg, Jonathan P; Secondi, Roberta; Ueda, Keiko; Delori, François C

2013-04-17

Our study was conducted to establish procedures and protocols for quantitative autofluorescence (qAF) measurements in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age. Fundus autofluorescence (AF) images (55° lens, 488 nm excitation) were acquired in albino Abca4(-/-), Abca4(+/-), and Abca4(+/+) mice (ages 2-12 months) with a confocal scanning laser ophthalmoscope (cSLO). Gray levels (GLs) in each image were calibrated to an internal fluorescence reference. The bisretinoid A2E was measured by quantitative high performance liquid chromatography (HPLC). Histometric analysis of ONL thicknesses was performed. The Bland-Altman coefficient of repeatability (95% confidence interval) was ±18% for between-session qAF measurements. Mean qAF values increased with age (2-12 months) in all groups of mice. qAF was approximately 2-fold higher in Abca4(-/-) mice than in Abca4(+/+) mice and approximately 20% higher in heterozygous mice. HPLC measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold difference between Abca4(-/-) and wild-type mice was more pronounced (approximately 3-4-fold) than measurable by qAF. Moreover, A2E levels declined after 8 months of age, a change not observed with qAF. The decline in A2E levels in the Abca4(-/-) mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age. The qAF method enables measurement of in vivo lipofuscin and the detection of genotype and age-associated differences. The use of this approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.

ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

PubMed

Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

2014-01-01

ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

ABCA1 in adipocytes regulates adipose tissue lipid content, glucose tolerance, and insulin sensitivity.

PubMed

de Haan, Willeke; Bhattacharjee, Alpana; Ruddle, Piers; Kang, Martin H; Hayden, Michael R

2014-03-01

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1(-ad/-ad)). When fed a high-fat, high-cholesterol diet, ABCA1(-ad/-ad) mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1(-ad/-ad) mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.

Mutations in ABCA12 Underlie the Severe Congenital Skin Disease Harlequin Ichthyosis

PubMed Central

Kelsell, David P.; Norgett, Elizabeth E.; Unsworth, Harriet; Teh, Muy-Teck; Cullup, Thomas; Mein, Charles A.; Dopping-Hepenstal, Patricia J.; Dale, Beverly A.; Tadini, Gianluca; Fleckman, Philip; Stephens, Karen G.; Sybert, Virginia P.; Mallory, Susan B.; North, Bernard V.; Witt, David R.; Sprecher, Eli; E. M. Taylor, Aileen; Ilchyshyn, Andrew; Kennedy, Cameron T.; Goodyear, Helen; Moss, Celia; Paige, David; Harper, John I.; Young, Bryan D.; Leigh, Irene M.; Eady, Robin A. J.; O’Toole, Edel A.

2005-01-01

Harlequin ichthyosis (HI) is the most severe and frequently lethal form of recessive congenital ichthyosis. Although defects in lipid transport, protein phosphatase activity, and differentiation have been described, the genetic basis underlying the clinical and cellular phenotypes of HI has yet to be determined. By use of single-nucleotide–polymorphism chip technology and homozygosity mapping, a common region of homozygosity was observed in five patients with HI in the chromosomal region 2q35. Sequencing of the ABCA12 gene, which maps within the minimal region defined by homozygosity mapping, revealed disease-associated mutations, including large intragenic deletions and frameshift deletions in 11 of the 12 screened individuals with HI. Since HI epidermis displays abnormal lamellar granule formation, ABCA12 may play a critical role in the formation of lamellar granules and the discharge of lipids into the intercellular spaces, which would explain the epidermal barrier defect seen in this disorder. This finding paves the way for early prenatal diagnosis. In addition, functional studies of ABCA12 will lead to a better understanding of epidermal differentiation and barrier formation. PMID:15756637

Identification of a Chrysanthemic Ester as an Apolipoprotein E Inducer in Astrocytes

PubMed Central

Zhao, Wenchen; Shimizu, Yoko; Pfeifer, Tom A.; Tak, Jun-Hyung; Isman, Murray B.; Van den Hoven, Bernard; Duggan, Mark E.; Wood, Michael W.; Wellington, Cheryl L.

2016-01-01

The apolipoprotein E (APOE) gene is the most highly associated susceptibility locus for late onset Alzheimer’s Disease (AD), and augmenting the beneficial physiological functions of apoE is a proposed therapeutic strategy. In a high throughput phenotypic screen for small molecules that enhance apoE secretion from human CCF-STTG1 astrocytoma cells, we show the chrysanthemic ester 82879 robustly increases expressed apoE up to 9.4-fold and secreted apoE up to 6-fold and is associated with increased total cholesterol in conditioned media. Compound 82879 is unique as structural analogues, including pyrethroid esters, show no effect on apoE expression or secretion. 82879 also stimulates liver x receptor (LXR) target genes including ATP binding cassette A1 (ABCA1), LXRα and inducible degrader of low density lipoprotein receptor (IDOL) at both mRNA and protein levels. In particular, the lipid transporter ABCA1 was increased by up to 10.6-fold upon 82879 treatment. The findings from CCF-STTG1 cells were confirmed in primary human astrocytes from three donors, where increased apoE and ABCA1 was observed along with elevated secretion of high-density lipoprotein (HDL)-like apoE particles. Nuclear receptor transactivation assays revealed modest direct LXR agonism by compound 82879, yet 10 μM of 82879 significantly upregulated apoE mRNA in mouse embryonic fibroblasts (MEFs) depleted of both LXRα and LXRβ, demonstrating that 82879 can also induce apoE expression independent of LXR transactivation. By contrast, deletion of LXRs in MEFs completely blocked mRNA changes in ABCA1 even at 10 μM of 82879, indicating the ability of 82879 to stimulate ABCA1 expression is entirely dependent on LXR transactivation. Taken together, compound 82879 is a novel chrysanthemic ester capable of modulating apoE secretion as well as apoE-associated lipid metabolic pathways in astrocytes, which is structurally and mechanistically distinct from known LXR agonists. PMID:27598782

ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

PubMed Central

Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

2014-01-01

ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters. PMID:25302608

ABCA1 in adipocytes regulates adipose tissue lipid content, glucose tolerance, and insulin sensitivity[S

PubMed Central

de Haan, Willeke; Bhattacharjee, Alpana; Ruddle, Piers; Kang, Martin H.; Hayden, Michael R.

2014-01-01

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1−ad/−ad). When fed a high-fat, high-cholesterol diet, ABCA1−ad/−ad mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1−ad/−ad mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis. PMID:24443560

Phenotypic spectrum of autosomal recessive cone-rod dystrophies caused by mutations in the ABCA4 (ABCR) gene.

PubMed

Klevering, B Jeroen; Blankenagel, Anita; Maugeri, Alessandra; Cremers, Frans P M; Hoyng, Carel B; Rohrschneider, Klaus

2002-06-01

To describe the phenotype of 12 patients with autosomal recessive or isolated cone-rod types of progressive retinal degeneration (CRD) caused by mutations in the ABCA4 gene. The charts of patients who had originally received a diagnosis of isolated or autosomal recessive CRD were reviewed after molecular analysis revealed mutations in the ABCA4 gene. In two of the patients both the photopic and scotopic electroretinogram were nonrecordable. In the remainder, the photopic cone b-wave amplitudes appeared to be more seriously affected than the scotopic rod b-wave amplitudes. Although the clinical presentation was heterogeneous, all patients experienced visual loss early in life, impaired color vision, and a central scotoma. Fundoscopy revealed evidence of early-onset maculopathy, sometimes accompanied by involvement of the retinal periphery in the later stages of the disease. Mutations in the ABCA4 gene are the pathologic cause of the CRD-like dystrophy in these patients, and the resultant clinical pictures are complex and heterogeneous. Given this wide clinical spectrum of CRD-like phenotypes associated with ABCA4 mutations, detailed clinical subclassifications are difficult and may not be very useful.

Fundus Autofluorescence in the Abca4−/− Mouse Model of Stargardt Disease—Correlation With Accumulation of A2E, Retinal Function, and Histology

PubMed Central

Charbel Issa, Peter; Barnard, Alun R.; Singh, Mandeep S.; Carter, Emma; Jiang, Zhichun; Radu, Roxana A.; Schraermeyer, Ulrich; MacLaren, Robert E.

2013-01-01

Purpose. To investigate fundus autofluorescence (AF) characteristics in the Abca4−/− mouse, an animal model for AMD and Stargardt disease, and to correlate findings with functional, structural, and biochemical assessments. Methods. Blue (488 nm) and near-infrared (790 nm) fundus AF images were quantitatively and qualitatively analyzed in pigmented Abca4−/− mice and wild type (WT) controls in vivo. Functional, structural, and biochemical assessments included electroretinography (ERG), light and electron microscopic analysis, and A2E quantification. All assessments were performed across age groups. Results. In Abca4−/− mice, lipofuscin-related 488 nm AF increased early in life with a ceiling effect after 6 months. This increase was first paralleled by an accumulation of typical lipofuscin granules in the retinal pigment epithelium (RPE). Later, lipofuscin and melanin granules decreased in number, whereas melanolipofuscin granules increased. This increase in melanolipofuscin granules paralleled an increase in melanin-related 790 nm AF. Old Abca4−/− mice revealed a flecked fundus AF pattern at both excitation wavelengths. The amount of A2E, a major lipofuscin component, increased 10- to 12-fold in 6- to 9-month-old Abca4−/− mice compared with controls, while 488 nm AF intensity only increased 2-fold. Despite pronounced lipofuscin accumulation in the RPE of Abca4−/− mice, ERG and histology showed a slow age-related thinning of the photoreceptor layer similar to WT controls up to 12 months. Conclusions. Fundus AF can be used to monitor lipofuscin accumulation and melanin-related changes in vivo in mouse models of retinal disease. High RPE lipofuscin may not adversely affect retinal structure or function over prolonged time intervals, and melanin-related changes (melanolipofuscin formation) may occur before the decline in retinal function. PMID:23761084

Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease

PubMed Central

Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V.; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L.; Polishchuk, Roman S.; Auricchio, Alberto

2015-01-01

Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4−/− mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5′-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4−/− mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1. PMID:26420842

Comprehensive analysis of Stargardt macular dystrophy patients reveals new genotype-phenotype correlations and unexpected diagnostic revisions

PubMed Central

Zaneveld, Jacques; Siddiqui, Sorath; Li, Huajin; Wang, Xia; Wang, Hui; Wang, Keqing; Li, Hui; Ren, Huanan; Lopez, Irma; Dorfman, Allison; Khan, Ayesha; Wang, Feng; Salvo, Jason; Gelowani, Violet; Li, Yumei; Sui, Ruifang; Koenekoop, Robert; Chen, Rui

2014-01-01

Purpose Stargardt macular dystrophy (STGD) results in early central vision loss. We sought to explain the genetic cause of STGD in a cohort of 88 patients from three different cultural backgrounds. Methods Next Generation Sequencing using a novel capture panel was used to search for disease causing mutations. Unsolved patients were clinically re-examined and tested for copy number variations (CNVs) as well as intronic mutations. Results We determined the cause of disease in 67% of our patients. Our analysis identified 35 novel ABCA4 alleles. Eleven patients had mutations in genes not previously reported to cause STGD. Finally, 45% of our unsolved patients had single deleterious mutations in ABCA4, a recessive disease gene. No likely pathogenic CNVs were identified. Conclusions This study expands our knowledge of STGD by identifying dozens of novel STGD causing alleles. The frequency of patients with single mutations in ABCA4 is higher than controls, indicating these mutations contribute to disease. Eleven patients were explained by mutations outside ABCA4 underlining the need to genotype all retinal disease genes to maximize genetic diagnostic rates. Few ABCA4 mutations were observed in our French Canadian patients. This population may contain an unidentified founder mutation. Our results indicate that CNVs are unlikely to be a major cause of STGD. PMID:25474345

Pharmacogenomic Characterization and Isobologram Analysis of the Combination of Ascorbic Acid and Curcumin-Two Main Metabolites of Curcuma longa-in Cancer Cells.

PubMed

Ooko, Edna; Kadioglu, Onat; Greten, Henry J; Efferth, Thomas

2017-01-01

Curcuma longa has long been used in China and India as anti-inflammatory agent to treat a wide variety of conditions and also as a spice for varied curry preparations. The chemoprofile of the Curcuma species exhibits the presence of varied phytochemicals with curcumin being present in all three species but AA only being shown in C. longa . This study explored the effect of a curcumin/AA combination on human cancer cell lines. The curcumin/AA combination was assessed by isobologram analysis using the Loewe additivity drug interaction model. The drug combination showed additive cytotoxicity toward CCRF-CEM and CEM/ADR5000 leukemia cell lines and HCT116p53 +/+ and HCT116p53 -/- colon cancer cell line, while the glioblastoma cell lines U87MG and U87MG.ΔEGFR showed additive to supra-additive cytotoxicity. Gene expression profiles predicting sensitivity and resistance of tumor cells to induction by curcumin and AA were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Numerous genes involved in transcription ( TFAM, TCERG1, RGS13, C11orf31 ), apoptosis-regulation ( CRADD, CDK7, CDK19, CD81, TOM1 ) signal transduction ( NR1D2, HMGN1, ABCA1, DE4ND4B, TRIM27 ) DNA repair ( TOPBP1, RPA2 ), mRNA metabolism ( RBBP4, HNRNPR, SRSF4, NR2F2, PDK1, TGM2 ), and transporter genes ( ABCA1 ) correlated with cellular responsiveness to curcumin and ascorbic acid. In conclusion, this study shows the effect of the curcumin/AA combination and identifies several candidate genes that may regulate the response of varied cancer cells to curcumin and AA.

Pharmacogenomic Characterization and Isobologram Analysis of the Combination of Ascorbic Acid and Curcumin—Two Main Metabolites of Curcuma longa—in Cancer Cells

PubMed Central

Ooko, Edna; Kadioglu, Onat; Greten, Henry J.; Efferth, Thomas

2017-01-01

Curcuma longa has long been used in China and India as anti-inflammatory agent to treat a wide variety of conditions and also as a spice for varied curry preparations. The chemoprofile of the Curcuma species exhibits the presence of varied phytochemicals with curcumin being present in all three species but AA only being shown in C. longa. This study explored the effect of a curcumin/AA combination on human cancer cell lines. The curcumin/AA combination was assessed by isobologram analysis using the Loewe additivity drug interaction model. The drug combination showed additive cytotoxicity toward CCRF-CEM and CEM/ADR5000 leukemia cell lines and HCT116p53+/+ and HCT116p53−/− colon cancer cell line, while the glioblastoma cell lines U87MG and U87MG.ΔEGFR showed additive to supra-additive cytotoxicity. Gene expression profiles predicting sensitivity and resistance of tumor cells to induction by curcumin and AA were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Numerous genes involved in transcription (TFAM, TCERG1, RGS13, C11orf31), apoptosis-regulation (CRADD, CDK7, CDK19, CD81, TOM1) signal transduction (NR1D2, HMGN1, ABCA1, DE4ND4B, TRIM27) DNA repair (TOPBP1, RPA2), mRNA metabolism (RBBP4, HNRNPR, SRSF4, NR2F2, PDK1, TGM2), and transporter genes (ABCA1) correlated with cellular responsiveness to curcumin and ascorbic acid. In conclusion, this study shows the effect of the curcumin/AA combination and identifies several candidate genes that may regulate the response of varied cancer cells to curcumin and AA. PMID:28210221

Macular Pigment and Lutein Supplementation in ABCA4-associated Retinal Degenerations

PubMed Central

Aleman, Tomas S.; Cideciyan, Artur V.; Windsor, Elizabeth A. M.; Schwartz, Sharon B.; Swider, Malgorzata; Chico, John D.; Sumaroka, Alexander; Pantelyat, Alexander Y.; Duncan, Keith G.; Gardner, Leigh M.; Emmons, Jessica M.; Steinberg, Janet D.; Stone, Edwin M.; Jacobson, Samuel G.

2008-01-01

PURPOSE To determine macular pigment (MP) optical density (OD) in patients with ABCA4-associated retinal degenerations (ABCA4-RD) and the response of MP and vision to supplementation with lutein. METHODS Stargardt disease or cone-rod dystrophy patients with foveal fixation and with known or suspected disease-causing mutations in the ABCA4 gene were included. MPOD profiles were measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity and retinal thickness were quantified. Changes in MPOD and central vision were determined in a subset of patients receiving oral supplementation with lutein for 6 months. RESULTS MPOD in patients ranged from normal to markedly abnormal. As a group, ABCA4-RD patients had reduced foveal MPOD and there was strong correlation with retinal thickness. Average foveal tissue concentration of MP, estimated by dividing MPOD by retinal thickness, was normal in patients whereas serum concentration of lutein and zeaxanthin was significantly lower than normal. After oral lutein supplementation for 6 months, 91% of the patients showed significant increases in serum lutein and 63% of the patient eyes showed a significant augmentation in MPOD. The retinal responders tended to be female, and have lower serum lutein and zeaxanthin, lower MPOD and greater retinal thickness at baseline. Responding eyes had significantly lower baseline MP concentration compared to non-responding eyes. Central vision was unchanged after the period of supplementation. CONCLUSIONS MP is strongly affected by the stage of ABCA4 disease leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in some patients. There was no change in central vision after 6 months of lutein supplementation. Long-term influences on the natural history of this supplement on macular degenerations require further study. PMID:17325179

Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease.

PubMed

Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L; Polishchuk, Roman S; Auricchio, Alberto

2015-12-01

Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1. © The Author 2015. Published by Oxford University Press.

Deducing the pathogenic contribution of recessive ABCA4 alleles in an outbred population.

PubMed

Schindler, Emily I; Nylen, Erik L; Ko, Audrey C; Affatigato, Louisa M; Heggen, Andrew C; Wang, Kai; Sheffield, Val C; Stone, Edwin M

2010-10-01

Accurate prediction of the pathogenic effects of specific genotypes is important for the design and execution of clinical trials as well as for meaningful counseling of individual patients. However, for many autosomal recessive diseases, it can be difficult to deduce the relative pathogenic contribution of individual alleles because relatively few affected individuals share the same two disease-causing variations. In this study, we used multiple regression analysis to estimate the pathogenicity of specific alleles of ABCA4 in patients with retinal phenotypes ranging from Stargardt disease to retinitis pigmentosa. This analysis revealed quantitative allelic effects on two aspects of the visual phenotype, visual acuity (P < 10(-3)) and visual field (P < 10(-7)). Discordance between visual acuity and visual field in individual patients suggests the existence of at least two non-ABCA4 modifying factors. The findings of this study will facilitate the discovery of factors that modify ABCA4 disease and will also aid in the optimal selection of subjects for clinical trials of new therapies.

Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

PubMed

Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

2015-11-01

The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ.

Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway[S

PubMed Central

Pamir, Nathalie; Hutchins, Patrick; Ronsein, Graziella; Vaisar, Tomas; Reardon, Catherine A.; Getz, Godfrey S.; Lusis, Aldons J.; Heinecke, Jay W.

2016-01-01

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages. PMID:26673204

Mutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration.

PubMed

Briggs, C E; Rucinski, D; Rosenfeld, P J; Hirose, T; Berson, E L; Dryja, T P

2001-09-01

To determine the spectrum of ABCR mutations associated with Stargardt macular degeneration and cone-rod degeneration (CRD). One hundred eighteen unrelated patients with recessive Stargardt macular degeneration and eight with recessive CRD were screened for mutations in ABCR (ABCA4) by single-strand conformation polymorphism analysis. Variants were characterized by direct genomic sequencing. Segregation analysis was performed on the families of 20 patients in whom at least two or more likely pathogenic sequence changes were identified. The authors found 77 sequence changes likely to be pathogenic: 21 null mutations (15 novel), 55 missense changes (26 novel), and one deletion of a consensus glycosylation site (also novel). Fifty-two patients with Stargardt macular degeneration (44% of those screened) and five with CRD each had two of these sequence changes or were homozygous for one of them. Segregation analyses in the families of 19 of these patients were informative and revealed that the index cases and all available affected siblings were compound heterozygotes or homozygotes. The authors found one instance of an apparently de novo mutation, Ile824Thr, in a patient. Thirty-seven (31%) of the 118 patients with Stargardt disease and one with CRD had only one likely pathogenic sequence change. Twenty-nine patients with Stargardt disease (25%) and two with CRD had no identified sequence changes. This report of 42 novel mutations brings the growing number of identified likely pathogenic sequence changes in ABCR to approximately 250.

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

PubMed

Chowdhury, Saiful M; Zhu, Xuewei; Aloor, Jim J; Azzam, Kathleen M; Gabor, Kristin A; Ge, William; Addo, Kezia A; Tomer, Kenneth B; Parks, John S; Fessler, Michael B

2015-07-01

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

PubMed Central

Chowdhury, Saiful M.; Zhu, Xuewei; Aloor, Jim J.; Azzam, Kathleen M.; Gabor, Kristin A.; Ge, William; Addo, Kezia A.; Tomer, Kenneth B.; Parks, John S.; Fessler, Michael B.

2015-01-01

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. PMID:25910759

Possible protective role of the ABCA4 gene c.1268A>G missense variant in Stargardt disease and syndromic retinitis pigmentosa in a Sicilian family: Preliminary data.

PubMed

D'Angelo, Rosalia; Donato, Luigi; Venza, Isabella; Scimone, Concetta; Aragona, Pasquale; Sidoti, Antonina

2017-04-01

In the wide horizon of ophthalmologically rare diseases among retinitis pigmentosa forms, Stargardt disease has gradually assumed a significant role due to its heterogeneity. In the present study, we aimed to support one of two opposite hypotheses concerning the causative or protective role of heterozygous c.1268A>G missense variant of the ABCA4 gene in Stargardt disease and in syndromic retinitis pigmentosa. This study was based on a family consisting of three members: proband, age 54, with high myopia, myopic chorioretinitis and retinal dystrophy; wife, age 65, with mild symptoms; daughter, age 29, asymptomatic. After genetic counseling, ABCA4 and RP1 gene analysis was performed. The results highlighted an important genetic picture. The proband was found to carry two variant RP1 SNPs, rs2293869 (c.2953A>T) and rs61739567 (c.6098G>A), and, a wild-type condition for four RP1 polymorphisms, rs444772 (c.2623G>A) and three SNPs in the 'hot-spot' region, exon 4. The proband's wife, instead, showed an opposite condition compared to her husband: a homozygous mutated condition for the first four SNPs analyzed, while the last two were wild-type. Regarding the ABCA4 gene, the proband evidenced a wild-type condition. Furthermore, the wife showed a heterozygous condition of ABCA4 rs3112831 (c.1268A>G). As expected, the daughter presented heterozygosity for all variants of both genes. In conclusion, even though the c.1268A>G missense variant of the ABCA4 gene has often been reported as causative of disease, and in other cases protective of disease, in our family case, the variant appears to reduce or delay the risk of onset of Stargardt disease.

ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease.

PubMed

Sangermano, Riccardo; Khan, Mubeen; Cornelis, Stéphanie S; Richelle, Valerie; Albert, Silvia; Garanto, Alejandro; Elmelik, Duaa; Qamar, Raheel; Lugtenberg, Dorien; van den Born, L Ingeborgh; Collin, Rob W J; Cremers, Frans P M

2018-01-01

Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes. © 2018 Sangermano et al.; Published by Cold Spring Harbor Laboratory Press.

The cholesterol transporter ABCA1 is expressed in stallion spermatozoa and reproductive tract tissues.

PubMed

Merkl, M; Ertl, R; Handschuh, S; Aurich, C; Schäfer-Somi, S

2016-04-01

In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen smears with different antibodies and counterstaining with Fluorescein isothiocyanate (FITC)-peanut agglutinin (PNA) and 4',6-Diamidin-2-phenylindol (DAPI). In a total of three samples, capacitation and acrosome reaction were induced by means of capacitation medium and progesterone substitution, respectively. Testicular and epididymal tissues were obtained from five prepubertal stallions aged 8 to 12 months and five adult stallions aged 4 to 9 years. For quantitative RT-PCR (qPCR), testicular and epididymal tissue of another seven adult (aged 1.5-14.5 years) and five prepupertal stallions (6-8 months) was used. For immunohistochemistry, sections from the caput, corpus, and cauda of the testes and epididymes were stained with the same specific antibodies as for immunocytochemistry. In stallion spermatozoa, strong immunosignal for ABCA1 was detected in the acrosomal area, the equatorial zone, and the principle piece of the flagellum but not in the caudal part of the head and the midpiece. In damaged or acrosome-reacted spermatozoa the FITC-PNA signal vanished together with the ABCA1 signal in most spermatozoa. In testicular tissue, strong immunostaining for ABCA1 was mainly visible in the heads and flagella of round spermatids and weaker signals in late spermatids and released spermatozoa. No staining was assessed in the Sertoli cells and spermatogonia of adult stallions, whereas strong signals in Leydig cells were present in prepubertal stallions. In prepubertal stallions, the ABCA1 messenger RNA level in testicular tissue was significantly higher than in adult stallions. We conclude that the ABCA1 transport molecule is present in adult and prepubertal stallion spermatozoa as well as testicular and epididymal tissue. ABCA1 is supposed to contribute to cholesterol transport and to support capacitation; however, this remains to be proven by functional studies. Species-specific differences concerning the localization inside the spermatozoa membrane are alike. Copyright © 2016 Elsevier Inc. All rights reserved.

Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease

PubMed Central

Braun, Terry A.; Mullins, Robert F.; Wagner, Alex H.; Andorf, Jeaneen L.; Johnston, Rebecca M.; Bakall, Benjamin B.; Deluca, Adam P.; Fishman, Gerald A.; Lam, Byron L.; Weleber, Richard G.; Cideciyan, Artur V.; Jacobson, Samuel G.; Sheffield, Val C.; Tucker, Budd A.; Stone, Edwin M.

2013-01-01

Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10−6). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing. PMID:23918662

Dual AAV Vectors for Stargardt Disease.

PubMed

Trapani, Ivana

2018-01-01

Stargardt disease (STGD1), due to mutations in the large ABCA4 gene, is the most common inherited macular degeneration in humans. Attempts at developing gene therapy approaches for treatment of STGD1 are currently ongoing. Among all the vectors available for gene therapy of inherited retinal diseases, those based on adeno-associated viruses (AAV) are the most promising given the efficacy shown in various animal models and their excellent safety profile in humans, as confirmed in many ongoing clinical trials. However, one of the main obstacles for the use of AAV is their limited effective packaging capacity of about 5 kb. Taking advantage of the AAV genome's ability to concatemerize , others and we have recently developed dual AAV vectors to overcome this limit. We tested dual AAV vectors for ABCA4 delivery, and found that they transduce efficiently both mouse and pig photoreceptors , and rescue the Abca4-/- mouse retinal phenotype, indicating their potential for gene therapy of STGD1. This chapter details how we designed dual AAV vectors for the delivery of the ABCA4 gene and describes the techniques that can be explored to evaluate dual AAV transduction efficiency in vitro and in the retina, and their efficacy in the mouse model of STGD1.

Omega-3 Fatty Acids Supplementation: Therapeutic Potential in a Mouse Model of Stargardt Disease.

PubMed

Prokopiou, Ekatherine; Kolovos, Panagiotis; Kalogerou, Maria; Neokleous, Anastasia; Nicolaou, Orthodoxia; Sokratous, Kleitos; Kyriacou, Kyriacos; Georgiou, Tassos

2018-06-01

To evaluate the therapeutic effects of omega-3 (ω3) fatty acids on retinal degeneration in the ABCA4-/- model of Stargardt disease when the blood level of arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is between 1 and 1.5. Eight-month-old mice were allocated to three groups: wild type (129S1), ABCA4-/- untreated, and ABCA4-/- ω3 treated. ω3 treatment lasted 3 months and comprised daily gavage administration of EPA and docosahexaenoic acid (DHA). Blood and retinal fatty acid analysis was performed using gas chromatography to adjust the blood AA/EPA ∼1 to 1.5. Eyecups were histologically examined using transmission electron microscopy and confocal microscopy to evaluate lipofuscin granules and the photoreceptor layer. Retinal N-retinylidene-N-retinylethanolamine (A2E), a major component of retinal pigment epithelium lipofuscin, was quantified using liquid chromatography and tandem mass spectrometry, in addition to retinal proteomic analysis to determine changes in inflammatory proteins. EPA levels increased and AA levels decreased in the blood and retinas of the treatment group. Significantly less A2E and lipofuscin granules were observed in the treatment group. The thickness of the outer nuclear layer was significantly greater in the treatment group (75.66 ± 4.80 μm) than in the wild-type (61.40 ± 1.84 μm) or untreated ABCA4-/- (56.50 ± 3.24 μm) groups. Proteomic analysis indicated lower levels of complement component 3 (C3) in the treatment group, indicative of lower complement-induced inflammatory response. Three months of ω3 supplementation (AA/EPA ∼1-1.5) reduces A2E levels, lipofuscin granules, and C3 levels in the ABCA4-/- mouse model of Stargardt disease, consistent with slowing of the disease.

Purification and ATPase activity of human ABCA1.

PubMed

Takahashi, Kei; Kimura, Yasuhisa; Kioka, Noriyuki; Matsuo, Michinori; Ueda, Kazumitsu

2006-04-21

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

PPAR{gamma} activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

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

Mogilenko, Denis A., E-mail: denis@iem.sp.ru; Department of Embryology, St. Petersburg State University, 199034 St. Petersburg; Shavva, Vladimir S.

Research highlights: {yields} PPAR{gamma} activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. {yields} Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1-LXR{beta} complex. {yields} Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex. {yields} Activation of PPAR{gamma} leads to increasing of the level of LXR{beta} associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPAR{gamma} is known as activator of ABCA1 expression, but details of PPAR{gamma}-mediatedmore » regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPAR{gamma} activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXR{beta} binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1/LXR{beta} complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex, but does not block PPAR{gamma}-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPAR{gamma} may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPAR{gamma}, LXR{beta} and MEK1/2 in regulation of ABCA1 mRNA and protein expression.« less

Pim-1L Protects Cell Surface-Resident ABCA1 From Lysosomal Degradation in Hepatocytes and Thereby Regulates Plasma High-Density Lipoprotein Level.

PubMed

Katsube, Akira; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

2016-12-01

ATP-binding cassette transporter A1 (ABCA1) exerts an atheroprotective action through the biogenesis of high-density lipoprotein in hepatocytes and prevents the formation of foam cells from macrophages. Controlling ABCA1 is a rational approach to improving atherosclerotic cardiovascular disease. Although much is known about the regulatory mechanism of ABCA1 synthesis, the molecular mechanism underpinning its degradation remains to be clearly described. ABCA1 possesses potential sites of phosphorylation by serine/threonine-protein kinase Pim-1 (Pim-1). Pim-1 depletion decreased the expression of cell surface-resident ABCA1 (csABCA1) and apolipoprotein A-I-mediated [ 3 H]cholesterol efflux in the human hepatoma cell line HepG2, but not in peritoneal macrophages from mice. In vitro kinase assay, immunoprecipitation, and immunocytochemistry suggested phosphorylation of csABCA1 by the long form of Pim-1 (Pim-1L). Cell surface biotinylation indicated that Pim-1L inhibited lysosomal degradation of csABCA1 involving the liver X receptor β, which interacts with csABCA1 and thereby protects it from ubiquitination and subsequent lysosomal degradation. Cell surface coimmunoprecipitation with COS-1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that Pim-1L-mediated phosphorylation of csABCA1 facilitated the interaction between csABCA1 and liver X receptor β and thereby stabilized the csABCA1-Pim-1L complex. Mice deficient in Pim-1 kinase activity showed lower expression of ABCA1 in liver plasma membranes and lower plasma high-density lipoprotein levels than control mice. Pim-1L protects hepatic csABCA1 from lysosomal degradation by facilitating the physical interaction between csABCA1 and liver X receptor β and subsequent stabilization of the csABCA1-Pim-1L complex and thereby regulates the circulating level of high-density lipoprotein. Our findings may aid the development of high-density lipoprotein-targeted therapy. © 2016 American Heart Association, Inc.

Association of ABCA1 with syntaxin 13 and flotillin-1 and enhanced phagocytosis in tangier cells.

PubMed

Bared, Salim Maa; Buechler, Christa; Boettcher, Alfred; Dayoub, Rania; Sigruener, Alexander; Grandl, Margot; Rudolph, Christian; Dada, Ashraf; Schmitz, Gerd

2004-12-01

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.

Association of ABCA1 with Syntaxin 13 and Flotillin-1 and Enhanced Phagocytosis in Tangier Cells

PubMed Central

Bared, Salim Maa; Buechler, Christa; Boettcher, Alfred; Dayoub, Rania; Sigruener, Alexander; Grandl, Margot; Rudolph, Christian; Dada, Ashraf; Schmitz, Gerd

2004-01-01

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I–dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX–insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains. PMID:15469992

Stargardt disease: towards developing a model to predict phenotype.

PubMed

Heathfield, Laura; Lacerda, Miguel; Nossek, Christel; Roberts, Lisa; Ramesar, Rajkumar S

2013-10-01

Stargardt disease is an ABCA4-associated retinopathy, which generally follows an autosomal recessive inheritance pattern and is a frequent cause of macular degeneration in childhood. ABCA4 displays significant allelic heterogeneity whereby different mutations can cause retinal diseases with varying severity and age of onset. A genotype-phenotype model has been proposed linking ABCA4 mutations, purported ABCA4 functional protein activity and severity of disease, as measured by degree of visual loss and the age of onset. It has, however, been difficult to verify this model statistically in observational studies, as the number of individuals sharing any particular mutation combination is typically low. Seven founder mutations have been identified in a large number of Caucasian Afrikaner patients in South Africa, making it possible to test the genotype-phenotype model. A generalised linear model was developed to predict and assess the relative pathogenic contribution of the seven mutations to the age of onset of Stargardt disease. It is shown that the pathogenicity of an individual mutation can differ significantly depending on the genetic context in which it occurs. The results reported here may be used to identify suitable candidates for inclusion in clinical trials, as well as guide the genetic counselling of affected individuals and families.

Stargardt Disease: towards developing a model to predict phenotype

PubMed Central

Heathfield, Laura; Lacerda, Miguel; Nossek, Christel; Roberts, Lisa; Ramesar, Rajkumar S

2013-01-01

Stargardt disease is an ABCA4-associated retinopathy, which generally follows an autosomal recessive inheritance pattern and is a frequent cause of macular degeneration in childhood. ABCA4 displays significant allelic heterogeneity whereby different mutations can cause retinal diseases with varying severity and age of onset. A genotype–phenotype model has been proposed linking ABCA4 mutations, purported ABCA4 functional protein activity and severity of disease, as measured by degree of visual loss and the age of onset. It has, however, been difficult to verify this model statistically in observational studies, as the number of individuals sharing any particular mutation combination is typically low. Seven founder mutations have been identified in a large number of Caucasian Afrikaner patients in South Africa, making it possible to test the genotype–phenotype model. A generalised linear model was developed to predict and assess the relative pathogenic contribution of the seven mutations to the age of onset of Stargardt disease. It is shown that the pathogenicity of an individual mutation can differ significantly depending on the genetic context in which it occurs. The results reported here may be used to identify suitable candidates for inclusion in clinical trials, as well as guide the genetic counselling of affected individuals and families. PMID:23695285

Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

PubMed

Mizuno, Tadahaya; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

2015-06-01

By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRβ inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRβ interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRβ and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system. © 2015 American Heart Association, Inc.

Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

PubMed Central

Jamieson, Sarra E.; de Roubaix, Lee-Anne; Cortina-Borja, Mario; Tan, Hooi Kuan; Mui, Ernest J.; Cordell, Heather J.; Kirisits, Michael J.; Miller, E. Nancy; Peacock, Christopher S.; Hargrave, Aubrey C.; Coyne, Jessica J.; Boyer, Kenneth; Bessieres, Marie-Hélène; Buffolano, Wilma; Ferret, Nicole; Franck, Jacqueline; Kieffer, François; Meier, Paul; Nowakowska, Dorota E.; Paul, Malgorzata; Peyron, François; Stray-Pedersen, Babill; Prusa, Andrea-Romana; Thulliez, Philippe; Wallon, Martine; Petersen, Eskild; McLeod, Rima; Gilbert, Ruth E.; Blackwell, Jenefer M.

2008-01-01

Background Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. Methods and Findings In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. Conclusions These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite. PMID:18523590

Double hyperautofluorescent ring on fundus autofluorescence in ABCA4.

PubMed

Abalem, Maria Fernanda; Qian, Cynthia X; Branham, Kari; Schlegel, Dana; Fahim, Abigail T; Khan, Naheed W; Heckenlively, John R; Jayasundera, K Thiran

2018-01-01

We report an unusual phenotype in a child with a clinical diagnosis of recessive Stargardt disease (STGD1) and two pathogenic variants in the ABCA4 gene. Typically, the diagnosis of early-onset STGD1 is challenging because children may present with a variety of fundus changes and a variable rate of progression. At the time of his initial visit, the 6-year-old boy presented with 20/200 OD (right eye) and 20/150 OS (left eye), symmetrical mild foveal atrophy without flecks on fundus exam, and foveal hypoautofluorescence surrounded by a homogeneous hyperautofluorescent background on wide-field fundus autofluorescence. Over 4 years of follow-up, the retinal atrophy continued to progress, resulting in two well-defined and concentric hyperautofluorescent rings: one ring located at the posterior pole and the other located around the peripapillary region. Visual acuity also deteriorated to counting fingers at 4ft OD and 20/500 OS. To the best of our knowledge, this phenotype has not been previously described with the ABCA4 gene.

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

Ye, Dan, E-mail: y.dan@lacdr.leidenuniv.nl; Meurs, Illiana; Ohigashi, Megumi

Objectives: To determine the role of macrophage ATP-binding cassette transporter A5 (ABCA5) in cellular cholesterol homeostasis and atherosclerotic lesion development. Methods and results: Chimeras with dysfunctional macrophage ABCA5 (ABCA5{sup -M/-M}) were generated by transplantation of bone marrow from ABCA5 knockout (ABCA5{sup -/-}) mice into irradiated LDLr{sup -/-} mice. In vitro, bone marrow-derived macrophages from ABCA5{sup -M/-M} chimeras exhibited a 29% (P < 0.001) decrease in cholesterol efflux to HDL, whereas a 21% (P = 0.07) increase in cholesterol efflux to apoA-I was observed. Interestingly, expression of ABCA1, but not ABCG1, was up-regulated in absence of functional ABCA5 in macrophages. Tomore » induce atherosclerosis, the transplanted LDLr{sup -/-} mice were fed a high-cholesterol Western-type diet (WTD) for 6, 10, or 18 weeks, allowing analysis of effects on initial as well as advanced lesion development. Atherosclerosis development was not affected in male ABCA5{sup -M/-M} chimeras after 6, 10, and 18 weeks WTD feeding. However, female ABCA5{sup -M/-M} chimeras did develop significantly (P < 0.05) larger aortic root lesions as compared with female controls after 6 and 10 weeks WTD feeding. Conclusions: ABCA5 influences macrophage cholesterol efflux, and selective disruption of ABCA5 in macrophages leads to increased atherosclerotic lesion development in female LDLr{sup -/-} mice.« less

Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

PubMed

Schwartz, S; Kohan, M; Pasion, R; Papenhausen, P R; Platt, L D

2018-02-01

Screening via noninvasive prenatal testing (NIPT) involving the analysis of cell-free DNA (cfDNA) from plasma has become readily available to screen for chromosomal and DNA aberrations through maternal blood. This report reviews a laboratory's experience with follow-up of positive NIPT screens for microdeletions. Patients that were screened positive by NIPT for a microdeletion involving 1p, 4p, 5p, 15q, or 22q who underwent diagnostic studies by either chorionic villus sampling or amniocentesis were evaluated. The overall positive predictive value for 349 patients was 9.2%. When a microdeletion was confirmed, 39.3% of the cases had additional abnormal microarray findings. Unrelated abnormal microarray findings were detected in 11.8% of the patients in whom the screen positive microdeletion was not confirmed. Stretches of homozygosity in the microdeletion were frequently associated with a false positive cfDNA microdeletion result. Overall, this report reveals that while cfDNA analysis will screen for microdeletions, the positive predictive value is low; in our series it is 9.2%. Therefore, the patient should be counseled accordingly. Confirmatory diagnostic microarray studies are imperative because of the high percentage of false positives and the frequent additional abnormalities not delineated by cfDNA analysis. © 2018 John Wiley & Sons, Ltd.

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein modulates ABCA1 trafficking and function

PubMed Central

Lin, Sisi; Zhou, Chun; Neufeld, Edward; Wang, Yu-Hua; Xu, Suo-Wen; Lu, Liang; Wang, Ying; Liu, Zhi-Ping; Li, Dong; Li, Cuixian; Chen, Shaorui; Le, Kang; Huang, Heqing; Liu, Peiqing; Moss, Joel; Vaughan, Martha; Shen, Xiaoyan

2013-01-01

Objective Cell surface localization and intracellular trafficking of ATP-binding cassette transporter A-1 (ABCA1) are essential for its function. However, regulation of these activities is still largely unknown. Brefeldin A (BFA), a uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism. Methods and Results By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells and inhibited by 60% cholesterol release. In contrast, BIG1 over-expression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through over-expression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 siRNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1 (ARF1). Conclusions BIG1, through its ability to activate ARF1, regulates cell surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action. PMID:23220274

Differences in ABCA1 R219K Polymorphisms and Serum Indexes in Alzheimer and Parkinson Diseases in Northern China

PubMed Central

Ya, Lagai; Lu, Zuneng

2017-01-01

Background ABCA1 R219K single-nucleotide polymorphisms (SNPs) was related to Alzheimer disease (AD) but not Parkinson disease (PD). Here, we analyzed the associations among ABCA1 R219K distribution, serum biomarkers, AD, and PD in a population in northern China. Material/Methods We used the Mini-Mental State Examination (MMSE) and the Hoehn and Yahr scale (H-Y) to evaluate AD and PD progression, separately. ABCA1 R219K was analyzed by matrix-assisted laser desorption ionization time of flight time mass spectrometry (MALDI-TOF-MS). Serum indexes were determined by enzyme-linked immunosorbent assay (ELISA). Results ABCA1 R219K RR+RK genotype frequency in AD and PD patients was lower than that in normal controls (NC), while ABCA1 R219K KK genotype frequency was significantly higher. ABCA1 R219K RR genotype frequency in AD patients and NC was lower than that in PD patients, while ABCA1 R219K RK+KK genotype frequency was significantly higher. ABCA1 R219K RR genotype was positively correlated to MMSE value in AD patients, while ABCA1 R219K KK genotype was negatively correlated to H-Y value in PD patients. Serum factors were significantly different among AD and PD patients and NC. Serum ABCA1, ApoA1, ApoA2, ApoB, HDL, TC, IL-1β, IL-6, and TNF-α were significantly different between AD and PD patients. Conclusions ABCA1 R219K R allele was the risk factor inducing abnormal serum levels of ApoA2, LDL, and TG in AD patients, and abnormal levels of serum ABCA1, HDL, IL-1β, IL-6, and TNF-α in PD patients, while ABCA1 R219K K allele was the risk factor inducing lower ABCA1 in AD patients. IL-1β, IL-6, and TNF-α were negatively correlated to MMSE in AD patients but positively correlated to H-Y in PD patients, while HDL was positively related to H-Y in PD patients. PMID:28943632

Differences in ABCA1 R219K Polymorphisms and Serum Indexes in Alzheimer and Parkinson Diseases in Northern China.

PubMed

Ya, Lagai; Lu, Zuneng

2017-09-25

BACKGROUND ABCA1 R219K single-nucleotide polymorphisms (SNPs) was related to Alzheimer disease (AD) but not Parkinson disease (PD). Here, we analyzed the associations among ABCA1 R219K distribution, serum biomarkers, AD, and PD in a population in northern China. MATERIAL AND METHODS We used the Mini-Mental State Examination (MMSE) and the Hoehn and Yahr scale (H-Y) to evaluate AD and PD progression, separately. ABCA1 R219K was analyzed by matrix-assisted laser desorption ionization time of flight time mass spectrometry (MALDI-TOF-MS). Serum indexes were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS ABCA1 R219K RR+RK genotype frequency in AD and PD patients was lower than that in normal controls (NC), while ABCA1 R219K KK genotype frequency was significantly higher. ABCA1 R219K RR genotype frequency in AD patients and NC was lower than that in PD patients, while ABCA1 R219K RK+KK genotype frequency was significantly higher. ABCA1 R219K RR genotype was positively correlated to MMSE value in AD patients, while ABCA1 R219K KK genotype was negatively correlated to H-Y value in PD patients. Serum factors were significantly different among AD and PD patients and NC. Serum ABCA1, ApoA1, ApoA2, ApoB, HDL, TC, IL-1β, IL-6, and TNF-α were significantly different between AD and PD patients. CONCLUSIONS ABCA1 R219K R allele was the risk factor inducing abnormal serum levels of ApoA2, LDL, and TG in AD patients, and abnormal levels of serum ABCA1, HDL, IL-1b, IL-6, and TNF-α in PD patients, while ABCA1 R219K K allele was the risk factor inducing lower ABCA1 in AD patients. IL-1β, IL-6, and TNF-α were negatively correlated to MMSE in AD patients but positively correlated to H-Y in PD patients, while HDL was positively related to H-Y in PD patients.

Mutations in GPR143/OA1 and ABCA4 Inform Interpretations of Short-Wavelength and Near-Infrared Fundus Autofluorescence

PubMed Central

Paavo, Maarjaliis; Zhao, Jin; Kim, Hye Jin; Lee, Winston; Zernant, Jana; Cai, Carolyn; Allikmets, Rando; Tsang, Stephen H.; Sparrow, Janet R.

2018-01-01

Purpose We sought to advance interpretations and quantification of short-wavelength fundus autofluorescence (SW-AF) emitted from bisretinoid lipofuscin and near-infrared autofluoresence (NIR-AF) originating from melanin. Methods Carriers of mutations in X-linked GPR143/OA1, a common form of ocular albinism; patients with confirmed mutations in ABCA4 conferring increased SW-AF; and subjects with healthy eyes were studied. SW-AF (488 nm excitation, 500–680 nm emission) and NIR-AF (excitation 787 nm, emission >830 nm) images were acquired with a confocal scanning laser ophthalmoscope. SW-AF images were analyzed for quantitative autofluoresence (qAF). Analogous methods of image acquisition and analysis were performed in albino and pigmented Abca4−/− mice and wild-type mice. Results Quantitation of SW-AF (qAF), construction of qAF color-coded maps, and examination of NIR-AF images from GPR143/OA1 carriers revealed mosaics in which patches of fundus exhibiting NIR-AF signal had qAF levels within normal limits whereas the hypopigmented areas in the NIR-AF image corresponded to foci of elevated qAF. qAF also was increased in albino versus pigmented mice. Although melanin contributes to fundus infrared reflectance, the latter appeared to be uniform in en face reflectance images of GPR143/OA1-carriers. In patients diagnosed with ABCA4-associated disease, NIR-AF increased in tandem with increased qAF originating in bisretinoid lipofuscin. Similarly in Abca4−/− mice having increased SW-AF, NIR-AF was more pronounced than in wild-type mice. Conclusions These studies corroborate RPE melanin as the major source of NIR-AF but also indicate that bisretinoid lipofuscin, when present at sufficient concentrations, contributes to the NIR-AF signal. Ocular melanin attenuates the SW-AF signal.

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition*

PubMed Central

Zhou, Jilin; Ueda, Keiko; Zhao, Jin; Sparrow, Janet R.

2015-01-01

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch's membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch's membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4−/− and Rdh8−/−/Abca4−/− mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4−/− but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4−/− mice revealed that carbonyl adduct deposition in Bruch's membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4−/− mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch's membrane that can confer risk of age-related macular degeneration. PMID:26400086

Intestinal ABCA1 directly contributes to HDL biogenesis in vivo

PubMed Central

Brunham, Liam R.; Kruit, Janine K.; Iqbal, Jahangir; Fievet, Catherine; Timmins, Jenelle M.; Pape, Terry D.; Coburn, Bryan A.; Bissada, Nagat; Staels, Bart; Groen, Albert K.; Hussain, M. Mahmood; Parks, John S.; Kuipers, Folkert; Hayden, Michael R.

2006-01-01

Plasma HDL cholesterol levels are inversely related to risk for atherosclerosis. The ATP-binding cassette, subfamily A, member 1 (ABCA1) mediates the rate-controlling step in HDL particle formation, the assembly of free cholesterol and phospholipids with apoA-I. ABCA1 is expressed in many tissues; however, the physiological functions of ABCA1 in specific tissues and organs are still elusive. The liver is known to be the major source of plasma HDL, but it is likely that there are other important sites of HDL biogenesis. To assess the contribution of intestinal ABCA1 to plasma HDL levels in vivo, we generated mice that specifically lack ABCA1 in the intestine. Our results indicate that approximately 30% of the steady-state plasma HDL pool is contributed by intestinal ABCA1 in mice. In addition, our data suggest that HDL derived from intestinal ABCA1 is secreted directly into the circulation and that HDL in lymph is predominantly derived from the plasma compartment. These data establish a critical role for intestinal ABCA1 in plasma HDL biogenesis in vivo. PMID:16543947

Amphipathic Polyproline Peptides Stimulate Cholesterol Efflux by the ABCA1 Transporter

PubMed Central

Sviridov, D.O.; Drake, S.K.; Freeman, L.A.; Remaley, A.T.

2016-01-01

ApoA-I mimetics are short synthetic peptides that contain an amphipathic αα-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenol group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop - Prog - Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-αhelical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p<0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides. PMID:26879139

Insulin-like Growth Factor 1 Regulates the Expression of ATP-Binding Cassette Transporter A1 in Pancreatic Beta Cells.

PubMed

Lyu, J; Imachi, H; Iwama, H; Zhang, H; Murao, K

2016-05-01

ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and cholesterol homeostasis. The present study investigates whether insulin-like growth factor 1 (IGF-1), which mediates stimulation of ABCA1 gene expression, could also interfere with the phosphatidylinositol 3-kinase (PI3-K) cascade.ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with IGF-1. The binding of forkhead box O1 (FoxO1) protein to the ABCA1 promoter was assessed by a chromatin immunoprecipitation (ChIP) assay. ABCA1 protein levels increased in response to rising concentrations of IGF-1. Real-time PCR analysis showed a significant increase in ABCA1 mRNA expression. However, both effects were suppressed after silencing the IGF-1 receptor. In parallel with its effect on endogenous ABCA1 mRNA levels, IGF-1 induced the activity of a reporter construct containing the ABCA1 promoter, while it was abrogated by LY294002, a specific inhibitor of PI3-K. Constitutively active Akt stimulated activity of the ABCA1 promoter, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element in the ABCA1 promoter abolished the ability of IGF-1 to stimulate promoter activity. A ChIP assay showed that FoxO1 mediated its transcriptional activity by directly binding to the ABCA1 promoter region. The knockdown of FoxO1 disrupted the effect of IGF-1 on ABCA1 expression. Furthermore, IGF-1 promoted cholesterol efflux and reduced the pancreatic lipotoxicity. These results demonstrate that the PI3-K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF-1 stimulation. © Georg Thieme Verlag KG Stuttgart · New York.

Retinal phenotypic characterization of patients with ABCA4 retinopathydue to the homozygous p.Ala1773Val mutation

PubMed Central

López-Rubio, Salvador; Chacon-Camacho, Oscar F.; Matsui, Rodrigo; Guadarrama-Vallejo, Dalia; Astiazarán, Mirena C.

2018-01-01

Purpose To describe the retinal clinical features of a group of Mexican patients with Stargardt disease carrying the uncommon p.Ala1773Val founder mutation in ABCA4. Methods Ten patients carrying the p.Ala1773Val mutation, nine of them homozygously, were included. Visual function studies included best-corrected visual acuity, electroretinography, Goldmann kinetic visual fields, and full-field electroretinography (ERG). In addition, imaging studies, such as optical coherence tomography (OCT), short-wave autofluorescence imaging, and quantitative analyses of hypofluorescence, were performed in each patient. Results Best-corrected visual acuities ranged from 20/200 to 4/200. The median age of the patients at diagnosis was 23.3 years. The majority of the patients had photophobia and nyctalopia, and were classified as Fishman stage 4 (widespread choriocapillaris atrophy, resorption of flecks, and greatly reduced ERG amplitudes). An atypical retinal pigmentation pattern was observed in the patients, and the majority showed cone-rod dystrophy on full-field ERG. In vivo retinal microstructure assessment with OCT demonstrated central retinal thinning, variable loss of photoreceptors, and three different patterns of structural retinal degeneration. Two dissimilar patterns of abnormal autofluorescence were observed. No apparent age-related differences in the pattern of retinal degeneration were observed. Conclusions The results indicate that this particular mutation in ABCA4 is associated with a severe retinal phenotype and thus, could be classified as null. Careful phenotyping of patients carrying specific mutations in ABCA4 is essential to enhance our understanding of disease expression linked to particular mutations and the resulting genotype–phenotype correlations. PMID:29422768

Targeted inactivation of the murine Abca3 gene leads to respiratory failure in newborns with defective lamellar bodies

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

Hammel, Markus; Michel, Geert; Hoefer, Christina

2007-08-10

Mutations in the human ABCA3 gene, encoding an ABC-transporter, are associated with respiratory failure in newborns and pediatric interstitial lung disease. In order to study disease mechanisms, a transgenic mouse model with a disrupted Abca3 gene was generated by targeting embryonic stem cells. While heterozygous animals developed normally and were fertile, individuals homozygous for the altered allele (Abca3-/-) died within one hour after birth from respiratory failure, ABCA3 protein being undetectable. Abca3-/- newborns showed atelectasis of the lung in comparison to a normal gas content in unaffected or heterozygous littermates. Electron microscopy demonstrated the absence of normal lamellar bodies inmore » type II pneumocytes. Instead, condensed structures with apparent absence of lipid content were found. We conclude that ABCA3 is required for the formation of lamellar bodies and lung surfactant function. The phenotype of respiratory failure immediately after birth corresponds to the clinical course of severe ABCA3 mutations in human newborns.« less

Interaction of Extracellular Domain 2 of the Human Retina-specific ATP-binding Cassette Transporter (ABCA4) with All-trans-retinal*

PubMed Central

Biswas-Fiss, Esther E.; Kurpad, Deepa S.; Joshi, Kinjalben; Biswas, Subhasis B.

2010-01-01

The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 ± 3% α-helix, 20 ± 3% β-pleated sheet, and 53 ± 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal. PMID:20404325

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum*♦

PubMed Central

Yamauchi, Yoshio; Iwamoto, Noriyuki; Rogers, Maximillian A.; Abe-Dohmae, Sumiko; Fujimoto, Toyoshi; Chang, Catherine C. Y.; Ishigami, Masato; Kishimoto, Takuma; Kobayashi, Toshihide; Ueda, Kazumitsu; Furukawa, Koichi; Chang, Ta-Yuan; Yokoyama, Shinji

2015-01-01

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process. PMID:26198636

A novel compound inhibits rHDL assembly and blocks nascent HDL biogenesis downstream of apoAI binding to ABCA1 expressing cells

PubMed Central

Lyssenko, Nicholas N.; Brubaker, Gregory; Smith, Bradley D.; Smith, Jonathan D.

2011-01-01

Objective Nascent high-density lipoprotein (HDL) particles form from cellular lipids and extracellular lipid-free apolipoprotein AI (apoAI) in a process mediated by ATP-binding cassette transporter A1 (ABCA1). We have sought out compounds that inhibit nascent HDL biogenesis without affecting ABCA1 activity. Methods and Results Reconstituted HDL (rHDL) formation and cellular cholesterol efflux assays were used to show that two compounds that bond via hydrogen with phospholipids inhibit rHDL and nascent HDL production. In rHDL formation assays, the inhibitory effect of compound 1 (methyl 3α-acetoxy-7α,12α-di[(phenylaminocarbonyl)amino]-5β-cholan-24-oate), the more active of the two, depended on its ability to associate with phospholipids. In cell assays, compound 1 suppressed ABCA1-mediated cholesterol efflux to apoAI, the 18A peptide, and taurocholate with high specificity, without affecting ABCA1-independent cellular cholesterol efflux to HDL and endocytosis of acetylated low-density lipoprotein (AcLDL) and transferrin. Furthermore, compound 1 did not affect ABCA1 activity adversely, as ABCA1-mediated shedding of microparticles proceeded unabated and apoAI binding to ABCA1-expressing cells increased in its presence. Conclusions The inhibitory effects of compound 1 support a three-step model of nascent HDL biogenesis: plasma membrane remodeling by ABCA1, apoAI binding to ABCA1, and lipoprotein particle assembly. The compound inhibits the final step, causing accumulation of apoAI in ABCA1-expressing cells. PMID:21836073

Increase in HDL-C concentration by a dietary portfolio with soy protein and soluble fiber is associated with the presence of the ABCA1R230C variant in hyperlipidemic Mexican subjects.

PubMed

Guevara-Cruz, Martha; Tovar, Armando R; Larrieta, Elena; Canizales-Quinteros, Samuel; Torres, Nimbe

2010-01-01

A dietary portfolio has been used to reduce blood lipids in hyperlipidemic subjects. To increase the effectiveness of these dietary treatments in specific populations, it is important to study the genetic variability associated with the development of certain types of hyperlipidemias. Low plasma high-density lipoprotein cholesterol (HDL-C) levels are the most common dyslipidemia in Mexican adults and are coupled with the presence of the ABCA1 R230C genotype. Therefore, the aim of this study was to assess the response of HDL-C concentration to a dietary portfolio in a group of Mexican hyperlipidemic subjects with ABCA1R230C (rs9282541) and R219K (rs2230806) polymorphisms. Forty-three hyperlipidemic subjects (20 men and 23 women) were given a low saturated fat (LSF) diet for one month, followed by a LSF diet that included 25g of soy protein and 15g of soluble fiber daily for 2months. We analyzed two ABCA1 polymorphisms and studied their association with serum lipids before and after treatment. Hyperlipidemic subjects with the ABCA1 R230C genotype showed lower HDL-C concentrations at the beginning of the study and were better responders to the dietary treatment than subjects with the ABCA1 R230R genotype (+4.6% vs. +14.6%) (p=.05). According to gender and the presence of the R230C genotype, women responded more significantly to the dietary treatment, reflected by an increase of 21.9% in HDL concentration (p=.022), than women with R230R genotype who only experienced an increase of 2.7% in HDL-C concentration. There was no association between the presence of the ABCA1 R219K variant (p=.544) and HDL concentration. Hyperlipidemic Mexican subjects with the ABCA1 R230C genotype showed lower HDL-concentrations and were better responders to dietary portfolio treatments for increasing HDL-C concentrations than subjects with the R230R genotype. Copyright © 2010 Elsevier Inc. All rights reserved.

Sandwich ELISA Microarrays: Generating Reliable and Reproducible Assays for High-Throughput Screens

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

Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

The sandwich ELISA microarray is a powerful screening tool in biomarker discovery and validation due to its ability to simultaneously probe for multiple proteins in a miniaturized assay. The technical challenges of generating and processing the arrays are numerous. However, careful attention to possible pitfalls in the development of your antibody microarray assay can overcome these challenges. In this chapter, we describe in detail the steps that are involved in generating a reliable and reproducible sandwich ELISA microarray assay.

Predicting Progression of ABCA4-Associated Retinal Degenerations Based on Longitudinal Measurements of the Leading Disease Front

PubMed Central

Cideciyan, Artur V.; Swider, Malgorzata; Schwartz, Sharon B.; Stone, Edwin M.; Jacobson, Samuel G.

2015-01-01

Purpose To evaluate the progression of the earliest stage of disease in ABCA4-associated retinal degenerations (RDs). Methods Near-infrared excited reduced-illuminance autofluorescence imaging was acquired across the retina up to 80 degrees eccentricity in 44 patients with two ABCA4 alleles. The eccentricity of the leading disease front (LDF) corresponding to the earliest stage of disease was measured along the four meridians. A mathematical model describing the expansion of the LDF was developed based on 6 years of longitudinal follow-up. Results The extent of LDF along the superior, inferior, and temporal meridians showed a wide spectrum from 3.5 to 70 degrees. In patients with longitudinal data, the average centrifugal expansion rate was 2 degrees per year. The nasal extent of LDF between the fovea and ONH ranged from 4.3 to 16.5 degrees and expanded at 0.35 degrees per year. The extent of LDF beyond ONH ranged from 19 to 75 degrees and expanded on average at 2 degrees per year. A mathematical model fit well to the longitudinal data describing the expansion of the LDF. Conclusions The eccentricity of the LDF in ABCA4-RD shows a continuum from parafovea to far periphery along all four meridians consistent with a wide spectrum of severity observed clinically. The model of progression may provide a quantitative prediction of the LDF expansion based on the age and eccentricity of the LDF at a baseline visit, and thus contribute significantly to the enrollment of candidates appropriate for clinical trials planning specific interventions, efficacy outcomes, and durations. PMID:26377081

MicroRNA-20a/b regulates cholesterol efflux through post-transcriptional repression of ATP-binding cassette transporter A1.

PubMed

Liang, Bin; Wang, Xin; Song, Xiaosu; Bai, Rui; Yang, Huiyu; Yang, Zhiming; Xiao, Chuanshi; Bian, Yunfei

2017-09-01

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in reverse cholesterol transport and exhibits anti-atherosclerosis effects. Some microRNAs (miRs) regulate ABCA1 expression, and recent studies have shown that miR-20a/b might play a critical role in atherosclerotic diseases. Here, we attempted to clarify the potential contribution of miR-20a/b in post-transcriptional regulation of ABCA1, cholesterol efflux, and atherosclerosis. We performed bioinformatics analysis and found that miR-20a/b was highly conserved and directly bound to ABCA1 mRNA with low binding free energy. Luciferase-reporter assay also confirmed that miR-20a/b significantly reduced luciferase activity associated with the ABCA1 3' untranslated region reporter construct. Additionally, miR-20a/b decreased ABCA1 expression, which, in turn, decreased cholesterol efflux and increased cholesterol content in THP-1 and RAW 264.7 macrophage-derived foam cells. In contrast, miR-20a/b inhibitors increased ABCA1 expression and cholesterol efflux, decreased cholesterol content, and inhibited foam-cell formation. Consistent with our in vitro results, miR-20a/b-treated ApoE -/- mice showed decreased ABCA1expression in the liver and reductions of reverse cholesterol transport in vivo. Furthermore, miR-20a/b regulated the formation of nascent high-density lipoprotein and promoted atherosclerotic development, whereas miR-20a/b knockdown attenuated atherosclerotic formation. miR-20 is a new miRNA capable of targeting ABCA1 and regulating ABCA1 expression. Therefore, miR-20 inhibition constitutes a new strategy for ABCA1-based treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

IL-8 negatively regulates ABCA1 expression and cholesterol efflux via upregulating miR-183 in THP-1 macrophage-derived foam cells.

PubMed

Tang, Xiao-Er; Li, Heng; Chen, Ling-Yan; Xia, Xiao-Dan; Zhao, Zhen-Wang; Zheng, Xi-Long; Zhao, Guo-Jun; Tang, Chao-Ke

2018-04-24

Previous studies suggest that IL-8 has an important role in the regulation of cholesterol efflux, but whether miRNAs are involved in this process is still unknown. The purpose of this study is to explore whether IL-8 promotes cholesterol accumulation by enhancing miR-183 expression in macrophages and its underlying mechanism. Treatment of THP-1 macrophage-derived foam cells with IL-8 decreased ABCA1 expression and cholesterol efflux. Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-183 was highly conserved during evolution and directly inhibited ABCA1 protein and mRNA expression by targeting ABCA1 3'UTR. MiR-183 directly regulated endogenous ABCA1 expression levels. Furthermore, IL-8 enhanced the expression of miR-183 and decrease ABCA1 expression. Cholesterol transport assays confirmed that IL-8 dramatically inhibited apolipoprotein AI-mediated ABCA1-dependent cholesterol efflux by increasing miR-183 expression. In contrast, treatment with anti-IL-8 antibody reversed these effects. IL-8 enhances the expression of miR-183, which then inhibits ABCA1 expression and cholesterol efflux. Our studies suggest that the IL-8-miR-183-ABCA1 axis may play an intermediary role in the development of atherosclerosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

PubMed Central

Jacob, Daria; Hunegnaw, Ruth; Sabyrzyanova, Tatyana A.; Pushkarsky, Tatiana; Chekhov, Vladimir O.; Adzhubei, Alexei A.; Kalebina, Tatyana S.; Bukrinsky, Michael

2014-01-01

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using coimmunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope. PMID:24406162

Role of APOE Isforms in the Pathogenesis of TBI Induced Alzheimer’s Disease

DTIC Science & Technology

2014-10-01

the inheritance of APOe4 is the only proven genetic risk factor for sporadic Alzheimer disease (AD). Importantly, TBI is a risk factor for the...mice on human APOE genetic background were exceptionally difficult to generate. We are considering changes in the genotype of those particular groups...mediated through ABCA1. 2 Keywords Traumatic brain injury, APOE isoforms, ABCA1, Alzheimer disease, APPmice, amyloid beta, axonal injury, inflamma

The ABCA4 2588G>C Stargardt mutation: single origin and increasing frequency from South-West to North-East Europe.

PubMed

Maugeri, Alessandra; Flothmann, Kris; Hemmrich, Nadine; Ingvast, Sofie; Jorge, Paula; Paloma, Eva; Patel, Reshma; Rozet, Jean-Michel; Tammur, Jaana; Testa, Francesco; Balcells, Susana; Bird, Alan C; Brunner, Han G; Hoyng, Carel B; Metspalu, Andres; Simonelli, Francesca; Allikmets, Rando; Bhattacharya, Shomi S; D'Urso, Michele; Gonzàlez-Duarte, Roser; Kaplan, Josseline; te Meerman, Gerard J; Santos, Rosário; Schwartz, Marianne; Van Camp, Guy; Wadelius, Claes; Weber, Bernhard H F; Cremers, Frans P M

2002-03-01

Inherited retinal dystrophies represent the most important cause of vision impairment in adolescence, affecting approximately 1 out of 3000 individuals. Mutations of the photoreceptor-specific gene ABCA4 (ABCR) are a common cause of retinal dystrophy. A number of mutations have been repeatedly reported for this gene, notably the 2588G>C mutation which is frequent in both patients and controls. Here we ascertained the frequency of the 2588G>C mutation in a total of 2343 unrelated random control individuals from 11 European countries and 241 control individuals from the US, as well as in 614 patients with STGD both from Europe and the US. We found an overall carrier frequency of 1 out of 54 in Europe, compared with 1 out of 121 in the US, confirming that the 2588G>C ABCA4 mutation is one of the most frequent autosomal recessive mutations in the European population. Carrier frequencies show an increasing gradient in Europe from South-West to North-East. The lowest carrier frequency, 0 out of 199 (0%), was found in Portugal; the highest, 11 out of 197 (5.5%), was found in Sweden. Haplotype analysis in 16 families segregating the 2588G>C mutation showed four intragenic polymorphisms invariably present in all 16 disease chromosomes and sharing of the same allele for several markers flanking the ABCA4 locus in most of the disease chromosomes. These results indicate a single origin of the 2588G>C mutation which, to our best estimate, occurred between 2400 and 3000 years ago.

A Novel Plasmid-Based Microarray Screen Identifies Suppressors of rrp6Δ in Saccharomyces cerevisiae▿†

PubMed Central

Abruzzi, Katharine; Denome, Sylvia; Olsen, Jens Raabjerg; Assenholt, Jannie; Haaning, Line Lindegaard; Jensen, Torben Heick; Rosbash, Michael

2007-01-01

Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Δ temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Δ strains at 37°C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6Δ strains. Microarray analyses of gene expression in rrp6Δ strains and a number of suppressor strains support this hypothesis. PMID:17101774

Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

PubMed

Fournier, Natalie; Tardivel, Sylviane; Benoist, Jean-François; Vedie, Benoît; Rousseau-Ralliard, Delphine; Nowak, Maxime; Allaoui, Fatima; Paul, Jean-Louis

2016-04-01

A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages. Copyright © 2016 Elsevier B.V. All rights reserved.

Phenotypic characteristics of Alzheimer patients carrying an ABCA7 mutation.

PubMed

Van den Bossche, Tobi; Sleegers, Kristel; Cuyvers, Elise; Engelborghs, Sebastiaan; Sieben, Anne; De Roeck, Arne; Van Cauwenberghe, Caroline; Vermeulen, Steven; Van den Broeck, Marleen; Laureys, Annelies; Peeters, Karin; Mattheijssens, Maria; Vandenbulcke, Mathieu; Vandenberghe, Rik; Martin, Jean-Jacques; De Deyn, Peter P; Cras, Patrick; Van Broeckhoven, Christine

2016-06-07

To generate a clinical and pathologic phenotype of patients carrying rare loss-of-function mutations in ABCA7, identified in a Belgian Alzheimer patient cohort and in an autosomal dominant family. We performed a retrospective review of available data records, medical records, results of CSF analyses and neuroimaging studies, and neuropathology data. The mean onset age of the mutation carriers (n = 22) was 73.4 ± 8.4 years with a wide age range of 36 (54-90) years, which was independent of APOE genotype and cerebrovascular disease. The mean disease duration was 5.7 ± 3.0 years (range 2-12 years). A positive family history was recorded for 10 carriers (45.5%). All patient carriers except one presented with memory complaints. The 4 autopsied brains showed typical immunohistochemical changes of late-onset Alzheimer disease. All patients carrying a loss-of-function mutation in ABCA7 exhibited a classical Alzheimer disease phenotype, though with a striking wide onset age range, suggesting the influence of unknown modifying factors. © 2016 American Academy of Neurology.

Phenotypic characteristics of Alzheimer patients carrying an ABCA7 mutation

PubMed Central

Van den Bossche, Tobi; Sleegers, Kristel; Cuyvers, Elise; Engelborghs, Sebastiaan; Sieben, Anne; De Roeck, Arne; Van Cauwenberghe, Caroline; Vermeulen, Steven; Van den Broeck, Marleen; Laureys, Annelies; Peeters, Karin; Mattheijssens, Maria; Vandenbulcke, Mathieu; Vandenberghe, Rik; Martin, Jean-Jacques; De Deyn, Peter P.; Cras, Patrick

2016-01-01

Objective: To generate a clinical and pathologic phenotype of patients carrying rare loss-of-function mutations in ABCA7, identified in a Belgian Alzheimer patient cohort and in an autosomal dominant family. Methods: We performed a retrospective review of available data records, medical records, results of CSF analyses and neuroimaging studies, and neuropathology data. Results: The mean onset age of the mutation carriers (n = 22) was 73.4 ± 8.4 years with a wide age range of 36 (54–90) years, which was independent of APOE genotype and cerebrovascular disease. The mean disease duration was 5.7 ± 3.0 years (range 2–12 years). A positive family history was recorded for 10 carriers (45.5%). All patient carriers except one presented with memory complaints. The 4 autopsied brains showed typical immunohistochemical changes of late-onset Alzheimer disease. Conclusions: All patients carrying a loss-of-function mutation in ABCA7 exhibited a classical Alzheimer disease phenotype, though with a striking wide onset age range, suggesting the influence of unknown modifying factors. PMID:27037232

Evaluating concentration estimation errors in ELISA microarray experiments

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

Daly, Don S.; White, Amanda M.; Varnum, Susan M.

Enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay to predict a protein concentration in a sample. Deploying ELISA in a microarray format permits simultaneous prediction of the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Evaluating prediction error is critical to interpreting biological significance and improving the ELISA microarray process. Evaluating prediction error must be automated to realize a reliable high-throughput ELISA microarray system. Methods: In this paper, we present a statistical method based on propagation of error to evaluate prediction errors in the ELISA microarray process. Althoughmore » propagation of error is central to this method, it is effective only when comparable data are available. Therefore, we briefly discuss the roles of experimental design, data screening, normalization and statistical diagnostics when evaluating ELISA microarray prediction errors. We use an ELISA microarray investigation of breast cancer biomarkers to illustrate the evaluation of prediction errors. The illustration begins with a description of the design and resulting data, followed by a brief discussion of data screening and normalization. In our illustration, we fit a standard curve to the screened and normalized data, review the modeling diagnostics, and apply propagation of error.« less

Lipocalin 2 Plays an Important Role in Regulating Inflammation in Retinal Degeneration.

PubMed

Parmar, Tanu; Parmar, Vipul M; Perusek, Lindsay; Georges, Anouk; Takahashi, Masayo; Crabb, John W; Maeda, Akiko

2018-05-01

It has become increasingly important to understand how retinal inflammation is regulated because inflammation plays a role in retinal degenerative diseases. Lipocalin 2 (LCN2), an acute stress response protein with multiple innate immune functions, is increased in ATP-binding cassette subfamily A member 4 ( Abca4 ) -/- retinol dehydrogenase 8 ( Rdh8 ) -/- double-knockout mice, an animal model for Stargardt disease and age-related macular degeneration (AMD). To examine roles of LCN2 in retinal inflammation and degeneration, Lcn2 -/- Abca4 -/- Rdh8 -/- triple-knockout mice were generated. Exacerbated inflammation following light exposure was observed in Lcn2 -/- Abca4 -/- Rdh8 -/- mice as compared with Abca4 -/- Rdh8 -/- mice, with upregulation of proinflammatory genes and microglial activation. RNA array analyses revealed an increase in immune response molecules such as Ccl8 , Ccl2 , and Cxcl10 To further probe a possible regulatory role for LCN2 in retinal inflammation, we examined the in vitro effects of LCN2 on NF-κB signaling in human retinal pigmented epithelial (RPE) cells differentiated from induced pluripotent stem cells derived from healthy donors. We found that LCN2 induced expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 2 in these RPE cells and could inhibit the cytotoxic effects of H 2 O 2 and LPS. ELISA revealed increased LCN2 levels in plasma of patients with Stargardt disease, retinitis pigmentosa, and age-related macular degeneration as compared with healthy controls. Finally, overexpression of LCN2 in RPE cells displayed protection from cell death. Overall these results suggest that LCN2 is involved in prosurvival responses during cell stress and plays an important role in regulating inflammation during retinal degeneration. Copyright © 2018 by The American Association of Immunologists, Inc.

Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis

PubMed Central

Wapner, Ronald J.; Martin, Christa Lese; Levy, Brynn; Ballif, Blake C.; Eng, Christine M.; Zachary, Julia M.; Savage, Melissa; Platt, Lawrence D.; Saltzman, Daniel; Grobman, William A.; Klugman, Susan; Scholl, Thomas; Simpson, Joe Leigh; McCall, Kimberly; Aggarwal, Vimla S.; Bunke, Brian; Nahum, Odelia; Patel, Ankita; Lamb, Allen N.; Thom, Elizabeth A.; Beaudet, Arthur L.; Ledbetter, David H.; Shaffer, Lisa G.; Jackson, Laird

2013-01-01

Background Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. We aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. Methods Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. Results We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down’s syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. Conclusions In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.) PMID:23215555

TiO2 Nanolayer-Enhanced Fluorescence for Simultaneous Multiplex Mycotoxin Detection by Aptamer Microarrays on a Porous Silicon Surface.

PubMed

Liu, Rui; Li, Wei; Cai, Tingting; Deng, Yang; Ding, Zhi; Liu, Yan; Zhu, Xuerui; Wang, Xin; Liu, Jie; Liang, Baowen; Zheng, Tiesong; Li, Jianlin

2018-05-02

A new aptamer microarray method on the TiO 2 -porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO 2 nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO 2 -PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B 1 (AFB 1 ), and 0.001-10 ng/mL for fumonisin B 1 (FB 1 ) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB 1 , and FB 1 , respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

Single-Nucleotide Polymorphism-Microarray Ploidy Analysis of Paraffin-Embedded Products of Conception in Recurrent Pregnancy Loss Evaluations.

PubMed

Maslow, Bat-Sheva L; Budinetz, Tara; Sueldo, Carolina; Anspach, Erica; Engmann, Lawrence; Benadiva, Claudio; Nulsen, John C

2015-07-01

To compare the analysis of chromosome number from paraffin-embedded products of conception using single-nucleotide polymorphism (SNP) microarray with the recommended screening for the evaluation of couples presenting with recurrent pregnancy loss who do not have previous fetal cytogenetic data. We performed a retrospective cohort study including all women who presented for a new evaluation of recurrent pregnancy loss over a 2-year period (January 1, 2012, to December 31, 2013). All participants had at least two documented first-trimester losses and both the recommended screening tests and SNP microarray performed on at least one paraffin-embedded products of conception sample. Single-nucleotide polymorphism microarray identifies all 24 chromosomes (22 autosomes, X, and Y). Forty-two women with a total of 178 losses were included in the study. Paraffin-embedded products of conception from 62 losses were sent for SNP microarray. Single-nucleotide polymorphism microarray successfully diagnosed fetal chromosome number in 71% (44/62) of samples, of which 43% (19/44) were euploid and 57% (25/44) were noneuploid. Seven of 42 (17%) participants had abnormalities on recurrent pregnancy loss screening. The per-person detection rate for a cause of pregnancy loss was significantly higher in the SNP microarray (0.50; 95% confidence interval [CI] 0.36-0.64) compared with recurrent pregnancy loss evaluation (0.17; 95% CI 0.08-0.31) (P=.002). Participants with one or more euploid loss identified on paraffin-embedded products of conception were significantly more likely to have an abnormality on recurrent pregnancy loss screening than those with only noneuploid results (P=.028). The significance remained when controlling for age, number of losses, number of samples, and total pregnancies. These results suggest that SNP microarray testing of paraffin-embedded products of conception is a valuable tool for the evaluation of recurrent pregnancy loss in patients without prior fetal cytogenetic results. Recommended recurrent pregnancy loss screening was unnecessary in almost half the patients in our study. II.

ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.

PubMed

Quazi, Faraz; Molday, Robert S

2014-04-01

The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from photoreceptors following photoexcitation. How rod and cone cells prevent the accumulation of 11-cis-retinal in photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-trans-retinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration.

Rod outer segment retinol formation is independent of Abca4, arrestin, rhodopsin kinase, and rhodopsin palmitylation.

PubMed

Blakeley, Lorie R; Chen, Chunhe; Chen, Ching-Kang; Chen, Jeannie; Crouch, Rosalie K; Travis, Gabriel H; Koutalos, Yiannis

2011-06-01

The reactive aldehyde all-trans retinal is released in rod photoreceptor outer segments by photoactivated rhodopsin and is eliminated through reduction to all-trans retinol. This study was undertaken to determine whether all-trans retinol formation depends on Abca4, arrestin, rhodopsin kinase, and the palmitylation of rhodopsin, all of which are factors that affect the release and sequestration of all-trans retinal. Experiments were performed in isolated retinas and single living rods derived from 129/sv wild-type mice and Abca4-, arrestin-, and rhodopsin kinase-deficient mice and in genetically modified mice containing unpalmitylated rhodopsin. Formation of all-trans retinol was measured by imaging its fluorescence and by HPLC of retina extracts. The release of all-trans retinal from photoactivated rhodopsin was measured in purified rod outer segment membranes according to the increase in tryptophan fluorescence. All experiments were performed at 37°C. The kinetics of all-trans retinol formation in the different types of genetically modified mice are in reasonable agreement with those in wild-type animals. The kinetics of all-trans retinol formation in 129/sv mice are similar to those in C57BL/6, although the latter are known to regenerate rhodopsin much more slowly. The release of all-trans retinal from rhodopsin in purified membranes is significantly faster than the formation of all-trans retinol in intact cells and is independent of the presence of the palmitate groups. The regeneration of rhodopsin and the recycling of its chromophore are not strongly coupled. Neither the activities of Abca4, rhodopsin kinase, and arrestin, nor the palmitylation of rhodopsin affects the formation of all-trans retinol.

Lipid Absorption Defects in Intestine-specific Microsomal Triglyceride Transfer Protein and ATP-binding Cassette Transporter A1-deficient Mice*

PubMed Central

Iqbal, Jahangir; Parks, John S.; Hussain, M. Mahmood

2013-01-01

We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92–95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations. PMID:24019513

Exon Microarray Analysis of Human Dorsolateral Prefrontal Cortex in Alcoholism

PubMed Central

Manzardo, Ann M.; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G.

2014-01-01

Background Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Methods Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC, Brodmann area 9) of 7 adult Alcoholic (6 males, 1 female, mean age 48 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST Array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using qRT-PCR, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Results Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN) and signaling (e.g., RASGRP, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease, and development including cellular assembly and organization impacting on psychological disorders. Conclusions Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation and signaling that targets white matter of the brain. PMID:24890784

Exon microarray analysis of human dorsolateral prefrontal cortex in alcoholism.

PubMed

Manzardo, Ann M; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G

2014-06-01

Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function, and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC; Brodmann area 9) of 7 adult alcoholic (6 males, 1 female, mean age 49 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using quantitative reverse transcription polymerase chain reaction, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN), and signaling (e.g., RASGRP3, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease and development including cellular assembly and organization impacting on psychological disorders. Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation, and signaling that targets white matter of the brain. Copyright © 2014 by the Research Society on Alcoholism.

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

Wang, Yan; Wu, Jian-Feng; Tang, Yan-Yan

Highlights: • U II reduces cholesterol efflux in THP-1 macrophages. • U II decreases the expression of ABCA1. • Inhibition of the ERK/NF-κB pathway reduces U II effects on ABCA1 expression and cholesterol efflux. - Abstract: Objective: Foam cell formation in the arterial wall plays a key role in the development of atherosclerosis. Recent studies showed that Urotensin II (U II) is involved in the pathogenesis of atherosclerosis. Here we examined the effects of human U II on ATP-binding cassette transporter A1 (ABCA1) expression and the underlying mechanism in THP-1 macrophages. Methods and results: Cultured THP-1 macrophages were treated withmore » U II, followed by measuring the intracellular lipid contents, cholesterol efflux and ABCA1 levels. The results showed that U II dramatically decreased ABCA1 levels and impaired cholesterol efflux. However, the effects of U II on ABCA1 protein expression and cellular cholesterol efflux were partially reversed by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa B (NF-κB) activity, suggesting the potential roles of ERK1/2 and NF-κB in ABCA1 expression, respectively. Conclusion: Our current data indicate that U II may have promoting effects on the progression of atherosclerosis, likely through suppressing ABCA1 expression via activation of the ERK/NF-κB pathway and reducing cholesterol efflux to promote macrophage foam cell formation.« less

Activation of Liver X Receptor Decreases Atherosclerosis in Ldlr−/− mice in the Absence of ABCA1 and ABCG1 in Myeloid Cells

PubMed Central

Kappus, Mojdeh S.; Murphy, Andrew J.; Abramowicz, Sandra; Ntonga, Vusisizwe; Welch, Carrie L.; Tall, Alan R.; Westerterp, Marit

2014-01-01

Objective Liver X Receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly up-regulate genes involved in reverse cholesterol transport (RCT) and (2) exert anti-inflammatory effects mediated by transrepression of NFκB target genes. We investigated whether myeloid cell deficiency of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), principal targets of LXR that promote macrophage cholesterol efflux and initiate RCT, would abolish the beneficial effects of LXR activation on atherosclerosis. Approach and Results LXR activator T0901317 (T0) substantially reduced inflammatory gene expression in macrophages lacking ABCA1/G1. Ldlr−/− mice were transplanted with Abca1−/−Abcg1−/− or wild-type bone marrow (BM) and fed a Western-type diet (WTD) for 6 weeks with or without T0 supplementation. Abca1/g1 BM deficiency increased atherosclerotic lesion complexity and inflammatory cell infiltration into the adventitia and myocardium. T0 markedly decreased lesion area, complexity and inflammatory cell infiltration in the Abca1−/−Abcg1−/− BM transplanted mice. To investigate whether this was due to macrophage Abca1/g1 deficiency, Ldlr−/− mice were transplanted with LysmCreAbca1fl/flAbcg1fl/fl or Abca1fl/flAbcg1fl/fl BM and fed WTD with or without the more specific LXR agonist GW3965 for 12 weeks. GW3965 decreased lesion size in both groups and the decrease was more prominent in the LysmCreAbca1fl/flAbcg1fl/fl group. Conclusions The results suggest that anti-inflammatory effects of LXR activators are of key importance to their anti-atherosclerotic effects in vivo independent of cholesterol efflux pathways mediated by macrophage ABCA1/G1. This has implications for the development of LXR activators that lack adverse effects on lipogenic genes while maintaining the ability to trans-repress inflammatory genes. PMID:24311381

Differential regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick type C1 in murine hepatocytes and macrophages.

PubMed

Wang, Ming-Dong; Franklin, Vivian; Sundaram, Meenakshi; Kiss, Robert S; Ho, Kenneth; Gallant, Michel; Marcel, Yves L

2007-08-03

Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr alpha mRNA is not changed, and Lxr alpha target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [(35)S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate.

Living Cell Microarrays: An Overview of Concepts

PubMed Central

Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

2016-01-01

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

Activation of liver X receptor decreases atherosclerosis in Ldlr⁻/⁻ mice in the absence of ATP-binding cassette transporters A1 and G1 in myeloid cells.

PubMed

Kappus, Mojdeh S; Murphy, Andrew J; Abramowicz, Sandra; Ntonga, Vusisizwe; Welch, Carrie L; Tall, Alan R; Westerterp, Marit

2014-02-01

Liver X receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly upregulate genes involved in reverse cholesterol transport and (2) exert anti-inflammatory effects mediated by transrepression of nuclear factor-κB target genes. We investigated whether myeloid cell deficiency of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), principal targets of LXR that promote macrophage cholesterol efflux and initiate reverse cholesterol transport, would abolish the beneficial effects of LXR activation on atherosclerosis. LXR activator T0901317 substantially reduced inflammatory gene expression in macrophages lacking ABCA1/G1. Ldlr(-/-) mice were transplanted with Abca1(-/-)Abcg1(-/-) or wild-type bone marrow (BM) and fed a Western-type diet for 6 weeks with or without T0901317 supplementation. Abca1/g1 BM deficiency increased atherosclerotic lesion complexity and inflammatory cell infiltration into the adventitia and myocardium. T0901317 markedly decreased lesion area, complexity, and inflammatory cell infiltration in the Abca1(-/-)Abcg1(-/-) BM-transplanted mice. To investigate whether this was because of macrophage Abca1/g1 deficiency, Ldlr(-/-) mice were transplanted with LysmCreAbca1(fl/fl)Abcg1(fl/fl) or Abca1(fl/fl)Abcg1(fl/fl) BM and fed Western-type diet with or without the more specific LXR agonist GW3965 for 12 weeks. GW3965 decreased lesion size in both groups, and the decrease was more prominent in the LysmCreAbca1(fl/fl)Abcg1(fl/fl) group. The results suggest that anti-inflammatory effects of LXR activators are of key importance to their antiatherosclerotic effects in vivo independent of cholesterol efflux pathways mediated by macrophage ABCA1/G1. This has implications for the development of LXR activators that lack adverse effects on lipogenic genes while maintaining the ability to transrepress inflammatory genes.

The detection of autoantibodies to ATP-binding cassette transporter A1 and its role in the pathogenesis of atherosclerosis in patients with systemic lupus erythematosus.

PubMed

Zeng, Ting; Li, Shu-Jie; Ao, Wen; Zheng, Hui; Wu, Feng-Xia; Chen, Yi; Yang, Cheng-De

2012-11-01

To investigate the prevalence of autoantibodies against ATP-binding cassette transporter A1 (ABCA1) in SLE patients, and evaluate the association between anti-ABCA1 autoantibodies and atherosclerosis in SLE. The sera of 75 SLE patients and 75 healthy controls were tested by immunoblotting. Then, we examined the effect of anti-ABCA1 autoantibodies on cholesterol efflux in vitro. The prevalence of anti-ABCA1 antibodies in SLE patients was significantly higher than the controls (p<0.05). The prevalence in the SLE-plaque group was higher than that in the SLE-non-plaque group (p<0.05). The IgG purified from anti-ABCA1-antibody positive sera can inhibit cellular cholesterol efflux from THP-1 cells in vitro with a significantly higher inhibition ratio than that of the healthy controls. Our observations suggest that anti-ABCA1 autoantibodies are involved in the pathogenesis of lupus atherosclerosis and that autoantibodies against ABCA1 may act as biomarkers for atherosclerosis in SLE. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Reduced-illuminance autofluorescence imaging in ABCA4-associated retinal degenerations

NASA Astrophysics Data System (ADS)

Cideciyan, Artur V.; Swider, Malgorzata; Aleman, Tomas S.; Roman, Marisa I.; Sumaroka, Alexander; Schwartz, Sharon B.; Stone, Edwin M.; Jacobson, Samuel G.

2007-05-01

The health of the retinal pigment epithelium (RPE) can be estimated with autofluorescence (AF) imaging of lipofuscin, which accumulates as a byproduct of retinal exposure to light. Lipofuscin may be toxic to the RPE, and its toxicity may be enhanced by short-wavelength (SW) illumination. The high-intensity and SW excitation light used in conventional AF imaging could, at least in principle, increase the rate of lipofuscin accumulation and/or increase its toxicity. We considered two reduced-illuminance AF imaging (RAFI) methods as alternatives to conventional AF imaging. RAFI methods use either near-infrared (NIR) light or reduced-radiance SW illumination for excitation of fluorophores. We quantified the distribution of RAFI signals in relation to retinal structure and function in patients with the prototypical lipofuscin accumulation disease caused by mutations in ABCA4. There was evidence for two subclinical stages of macular ABCA4 disease involving hyperautofluorescence of both SW- and NIR-RAFI with and without associated loss of visual function. Use of RAFI methods and microperimetry in future clinical trials involving lipofuscinopathies should allow quantification of subclinical disease expression and progression without subjecting the diseased retina/RPE to undue light exposure.

Next generation sequencing in the diagnosis of Stargardt's disease.

PubMed

Jimenez-Rolando, B; Noval, S; Rosa-Perez, I; Mata Diaz, E; Del Pozo, A; Ibañez, C; Silla, J C; Montaño, V E F; Martin-Arenas, R; Vallespin, E

2018-03-01

Stargardt's disease is the most frequent form of inherited macular dystrophy in children and adults. It is a genetic eye disorder caused by mutations in ABCA4 gene with an autosomal recessive inheritance. ABCA4 is a very polymorphic and large gene containing 50 exons. The development of next generation sequencing (NGS) can be used for the genetic diagnosis of this disease. A report is presented on two patients with a clinical diagnosis of Stargardt's disease whose genetic confirmation was performed by a NGS panel of 298 genes. Clinically, the patients showed bull's eye maculopathy and absence of flecks, and genetically they shared the Gly1961Glu mutation that could explain their common phenotype, together with c.C3056T:p.T1019M for case 1, and c.287del:p.Asn96Thrfs*19 for case 2. NGS is particularly useful in the diagnosis of Stargardt's disease as ABCA4 is a large gene with a high allelic heterogeneity that causes a wide range of clinical manifestations. Copyright © 2017 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

PubMed

Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

PubMed Central

Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide. PMID:20210464

Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages: IMPACT ON INTRACELLULAR SURVIVAL OF LEISHMANIA (VIANNIA) PANAMENSIS.

PubMed

Dohmen, Luuk C T; Navas, Adriana; Vargas, Deninson Alejandro; Gregory, David J; Kip, Anke; Dorlo, Thomas P C; Gomez, Maria Adelaida

2016-04-29

Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3(KD) macrophages. ABCA3(KD) resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3(KD) reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MicroRNA 302a is a novel modulator of cholesterol homeostasis and atherosclerosis

PubMed Central

Meiler, Svenja; Baumer, Yvonne; Toulmin, Emma; Seng, Kosal; Boisvert, William A.

2014-01-01

Objective Macrophage foam cell formation is a key feature of atherosclerosis. Recent studies have shown that specific microRNAs (miRs) are regulated in modified low-density lipoprotein (LDL)- treated macrophages, which can affect the cellular cholesterol homeostasis. Undertaking a genome-wide screen of microRNAs regulated in primary macrophages by modified LDL, miR-302a emerged as a potential candidate that may play a key role in macrophage cholesterol homeostasis. Approach and Results The objective of this study was to assess the involvement of miR-302a in macrophage lipid homeostasis and if it can influence circulating lipid levels and atherosclerotic development when it is inhibited in a murine atherosclerosis model. We found that transfection of primary macrophages with either miR-302a or anti-miR-302a regulated the expression of ATP-binding cassette (ABC) transporter ABCA1 mRNA and protein. Luciferase reporter assays showed that miR-302a repressed the 3′UTR activity of mouse Abca1 by 48% and human ABCA1 by 45%. Additionally, transfection of murine macrophages with miR-302a attenuated cholesterol efflux to apolipoprotein A-1 (apoA-1) by 38%. Long-term in vivo administration of anti-miR-302a to mice with LDL receptor deficiency (Ldlr−/−) fed an atherogenic diet led to an increase in ABCA1 in the liver and aorta as well as an increase in circulating plasma HDL levels by 35% compared with that of control mice. The anti-miR-302a-treated mice also displayed reduced atherosclerotic plaque size by approximately 25% as well as a more stable plaque morphology with reduced signs of inflammation. Conclusions These studies identify miR-302a as a novel modulator of cholesterol efflux and a potential therapeutic target for suppressing atherosclerosis. PMID:25524771

MicroRNA 302a is a novel modulator of cholesterol homeostasis and atherosclerosis.

PubMed

Meiler, Svenja; Baumer, Yvonne; Toulmin, Emma; Seng, Kosal; Boisvert, William A

2015-02-01

Macrophage foam cell formation is a key feature of atherosclerosis. Recent studies have shown that specific microRNAs (miRs) are regulated in modified low-density lipoprotein-treated macrophages, which can affect the cellular cholesterol homeostasis. Undertaking a genome-wide screen of miRs regulated in primary macrophages by modified low-density lipoprotein, miR-302a emerged as a potential candidate that may play a key role in macrophage cholesterol homeostasis. The objective of this study was to assess the involvement of miR-302a in macrophage lipid homeostasis and if it can influence circulating lipid levels and atherosclerotic development when it is inhibited in a murine atherosclerosis model. We found that transfection of primary macrophages with either miR-302a or anti-miR-302a regulated the expression of ATP-binding cassette (ABC) transporter ABCA1 mRNA and protein. Luciferase reporter assays showed that miR-302a repressed the 3' untranslated regions (UTR) activity of mouse Abca1 by 48% and human ABCA1 by 45%. In addition, transfection of murine macrophages with miR-302a attenuated cholesterol efflux to apolipoprotein A-1 (apoA-1) by 38%. Long-term in vivo administration of anti-miR-302a to mice with low-density lipoprotein receptor deficiency (Ldlr(-/-)) fed an atherogenic diet led to an increase in ABCA1 in the liver and aorta as well as an increase in circulating plasma high-density lipoprotein levels by 35% compared with that of control mice. The anti-miR-302a-treated mice also displayed reduced atherosclerotic plaque size by ≈25% and a more stable plaque morphology with reduced signs of inflammation. These studies identify miR-302a as a novel modulator of cholesterol efflux and a potential therapeutic target for suppressing atherosclerosis. © 2014 American Heart Association, Inc.

Genome of the Netherlands population-specific imputations identify an ABCA6 variant associated with cholesterol levels

PubMed Central

van Leeuwen, Elisabeth M.; Karssen, Lennart C.; Deelen, Joris; Isaacs, Aaron; Medina-Gomez, Carolina; Mbarek, Hamdi; Kanterakis, Alexandros; Trompet, Stella; Postmus, Iris; Verweij, Niek; van Enckevort, David J.; Huffman, Jennifer E.; White, Charles C.; Feitosa, Mary F.; Bartz, Traci M.; Manichaikul, Ani; Joshi, Peter K.; Peloso, Gina M.; Deelen, Patrick; van Dijk, Freerk; Willemsen, Gonneke; de Geus, Eco J.; Milaneschi, Yuri; Penninx, Brenda W.J.H.; Francioli, Laurent C.; Menelaou, Androniki; Pulit, Sara L.; Rivadeneira, Fernando; Hofman, Albert; Oostra, Ben A.; Franco, Oscar H.; Leach, Irene Mateo; Beekman, Marian; de Craen, Anton J.M.; Uh, Hae-Won; Trochet, Holly; Hocking, Lynne J.; Porteous, David J.; Sattar, Naveed; Packard, Chris J.; Buckley, Brendan M.; Brody, Jennifer A.; Bis, Joshua C.; Rotter, Jerome I.; Mychaleckyj, Josyf C.; Campbell, Harry; Duan, Qing; Lange, Leslie A.; Wilson, James F.; Hayward, Caroline; Polasek, Ozren; Vitart, Veronique; Rudan, Igor; Wright, Alan F.; Rich, Stephen S.; Psaty, Bruce M.; Borecki, Ingrid B.; Kearney, Patricia M.; Stott, David J.; Adrienne Cupples, L.; Neerincx, Pieter B.T.; Elbers, Clara C.; Francesco Palamara, Pier; Pe'er, Itsik; Abdellaoui, Abdel; Kloosterman, Wigard P.; van Oven, Mannis; Vermaat, Martijn; Li, Mingkun; Laros, Jeroen F.J.; Stoneking, Mark; de Knijff, Peter; Kayser, Manfred; Veldink, Jan H.; van den Berg, Leonard H.; Byelas, Heorhiy; den Dunnen, Johan T.; Dijkstra, Martijn; Amin, Najaf; Joeri van der Velde, K.; van Setten, Jessica; Kattenberg, Mathijs; van Schaik, Barbera D.C.; Bot, Jan; Nijman, Isaäc J.; Mei, Hailiang; Koval, Vyacheslav; Ye, Kai; Lameijer, Eric-Wubbo; Moed, Matthijs H.; Hehir-Kwa, Jayne Y.; Handsaker, Robert E.; Sunyaev, Shamil R.; Sohail, Mashaal; Hormozdiari, Fereydoun; Marschall, Tobias; Schönhuth, Alexander; Guryev, Victor; Suchiman, H. Eka D.; Wolffenbuttel, Bruce H.; Platteel, Mathieu; Pitts, Steven J.; Potluri, Shobha; Cox, David R.; Li, Qibin; Li, Yingrui; Du, Yuanping; Chen, Ruoyan; Cao, Hongzhi; Li, Ning; Cao, Sujie; Wang, Jun; Bovenberg, Jasper A.; Jukema, J. Wouter; van der Harst, Pim; Sijbrands, Eric J.; Hottenga, Jouke-Jan; Uitterlinden, Andre G.; Swertz, Morris A.; van Ommen, Gert-Jan B.; de Bakker, Paul I.W.; Eline Slagboom, P.; Boomsma, Dorret I.; Wijmenga, Cisca; van Duijn, Cornelia M.

2015-01-01

Variants associated with blood lipid levels may be population-specific. To identify low-frequency variants associated with this phenotype, population-specific reference panels may be used. Here we impute nine large Dutch biobanks (~35,000 samples) with the population-specific reference panel created by the Genome of the Netherlands Project and perform association testing with blood lipid levels. We report the discovery of five novel associations at four loci (P value <6.61 × 10−4), including a rare missense variant in ABCA6 (rs77542162, p.Cys1359Arg, frequency 0.034), which is predicted to be deleterious. The frequency of this ABCA6 variant is 3.65-fold increased in the Dutch and its effect (βLDL-C=0.135, βTC=0.140) is estimated to be very similar to those observed for single variants in well-known lipid genes, such as LDLR. PMID:25751400

Modulation of ABCA1 by an LXR Agonist Reduces Beta-Amyloid Levels and Improves Outcome after Traumatic Brain Injury

PubMed Central

Loane, David J.; Washington, Patricia M.; Vardanian, Lilit; Pocivavsek, Ana; Hoe, Hyang-Sook; Duff, Karen E.; Cernak, Ibolja; Rebeck, G. William; Faden, Alan I.

2011-01-01

Abstract Traumatic brain injury (TBI) increases brain beta-amyloid (Aβ) in humans and animals. Although the role of Aβ in the injury cascade is unknown, multiple preclinical studies have demonstrated a correlation between reduced Aβ and improved outcome. Therefore, therapeutic strategies that enhance Aβ clearance may be beneficial after TBI. Increased levels of ATP-binding cassette A1 (ABCA1) transporters can enhance Aβ clearance through an apolipoprotein E (apoE)-mediated pathway. By measuring Aβ and ABCA1 after experimental TBI in C57BL/6J mice, we found that Aβ peaked early after injury (1–3 days), whereas ABCA1 had a delayed response (beginning at 3 days). As ABCA1 levels increased, Aβ levels returned to baseline levels—consistent with the known role of ABCA1 in Aβ clearance. To test if enhancing ABCA1 levels could block TBI-induced Aβ, we treated TBI mice with the liver X-receptor (LXR) agonist T0901317. Pre- and post-injury treatment increased ABCA1 levels at 24 h post-injury, and reduced the TBI-induced increase in Aβ. This reduction in Aβ was not due to decreased amyloid precursor protein processing, or a shift in the solubility of Aβ, indicating enhanced clearance. T0901317 also limited motor coordination deficits in injured mice and reduced brain lesion volume. These data indicate that activation of LXR can reduce Aβ accumulation after TBI, and is accompanied by improved functional recovery. PMID:21175399

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

PubMed

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-11-19

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

PubMed Central

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-01-01

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides. PMID:28952593

Development and application of a DNA microarray-based yeast two-hybrid system

PubMed Central

Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

2013-01-01

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

PubMed Central

Kim, Min-Sik; Kuppireddy, Sarada V.; Sakamuri, Sruthi; Singal, Mukul; Getnet, Derese; Harsha, H. C.; Goel, Renu; Balakrishnan, Lavanya; Jacob, Harrys K. C.; Kashyap, Manoj K.; Tankala, Shantal G.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Jaffee, Elizabeth; Goggins, Michael G.; Velculescu, Victor E.; Hruban, Ralph H.; Pandey, Akhilesh

2013-01-01

Tissue microarrays have become a valuable tool for high-throughput analysis using immunohistochemical labeling. However, the large majority of biochemical studies are carried out in cell lines to further characterize candidate biomarkers or therapeutic targets with subsequent studies in animals or using primary tissues. Thus, cell line-based microarrays could be a useful screening tool in some situations. Here, we constructed a cell microarray (CMA) containing a panel of 40 pancreatic cancer cell lines available from American Type Culture Collection in addition to those locally available at Johns Hopkins. As proof of principle, we performed immunocytochemical labeling of an epithelial cell adhesion molecule (Ep-CAM), a molecule generally expressed in the epithelium, on this pancreatic cancer CMA. In addition, selected molecules that have been previously shown to be differentially expressed in pancreatic cancer in the literature were validated. For example, we observed strong labeling of CA19-9 antigen, a prognostic and predictive marker for pancreatic cancer. We also carried out a bioinformatics analysis of a literature curated catalog of pancreatic cancer biomarkers developed previously by our group and identified two candidate biomarkers, HLA class I and transmembrane protease, serine 4 (TMPRSS4), and examined their expression in the cell lines represented on the pancreatic cancer CMAs. Our results demonstrate the utility of CMAs as a useful resource for rapid screening of molecules of interest and suggest that CMAs can become a universal standard platform in cancer research. PMID:22985314

Retinoic Acid Isomers Facilitate Apolipoprotein E Production and Lipidation in Astrocytes through the Retinoid X Receptor/Retinoic Acid Receptor Pathway*

PubMed Central

Zhao, Jing; Fu, Yuan; Liu, Chia-Chen; Shinohara, Mitsuru; Nielsen, Henrietta M.; Dong, Qiang; Kanekiyo, Takahisa; Bu, Guojun

2014-01-01

Apolipoprotein E (apoE) is the major cholesterol transport protein in the brain. Among the three human APOE alleles (APOE2, APOE3, and APOE4), APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease (AD). The accumulation of amyloid-β (Aβ) is a central event in AD pathogenesis. Increasing evidence demonstrates that apoE isoforms differentially regulate AD-related pathways through both Aβ-dependent and -independent mechanisms; therefore, modulating apoE secretion, lipidation, and function might be an attractive approach for AD therapy. We performed a drug screen for compounds that modulate apoE production in immortalized astrocytes derived from apoE3-targeted replacement mice. Here, we report that retinoic acid (RA) isomers, including all-trans-RA, 9-cis-RA, and 13-cis-RA, significantly increase apoE secretion to ∼4-fold of control through retinoid X receptor (RXR) and RA receptor. These effects on modulating apoE are comparable with the effects recently reported for the RXR agonist bexarotene. Furthermore, all of these compounds increased the expression of the cholesterol transporter ABCA1 and ABCG1 levels and decreased cellular uptake of Aβ in an apoE-dependent manner. Both bexarotene and 9-cis-RA promote the lipidation status of apoE, in which 9-cis-RA promotes a stronger effect and exhibits less cytotoxicity compared with bexarotene. Importantly, we showed that oral administration of bexarotene and 9-cis-RA significantly increases apoE, ABCA1, and ABCG1 levels in mouse brains. Taken together, our results demonstrate that RXR/RA receptor agonists, including several RA isomers, are effective modulators of apoE secretion and lipidation and may be explored as potential drugs for AD therapy. PMID:24599963

A Report on Molecular Diagnostic Testing for Inherited Retinal Dystrophies by Targeted Genetic Analyses.

PubMed

Ramkumar, Hema L; Gudiseva, Harini V; Kishaba, Kameron T; Suk, John J; Verma, Rohan; Tadimeti, Keerti; Thorson, John A; Ayyagari, Radha

2017-02-01

To test the utility of targeted sequencing as a method of clinical molecular testing in patients diagnosed with inherited retinal degeneration (IRD). After genetic counseling, peripheral blood was drawn from 188 probands and 36 carriers of IRD. Single gene testing was performed on each patient in a Clinical Laboratory Improvement Amendment (CLIA) certified laboratory. DNA was isolated, and all exons in the gene of interest were analyzed along with 20 base pairs of flanking intronic sequence. Genetic testing was most often performed on ABCA4, CTRP5, ELOV4, BEST1, CRB1, and PRPH2. Pathogenicity of novel sequence changes was predicted by PolyPhen2 and sorting intolerant from tolerant (SIFT). Of the 225 genetic tests performed, 150 were for recessive IRD, and 75 were for dominant IRD. A positive molecular diagnosis was made in 70 (59%) of probands with recessive IRD and 19 (26%) probands with dominant IRD. Analysis confirmed 12 (34%) of individuals as carriers of familial mutations associated with IRD. Thirty-two novel variants were identified; among these, 17 sequence changes in four genes were predicted to be possibly or probably damaging including: ABCA4 (14), BEST1 (2), PRPH2 (1), and TIMP3 (1). Targeted analysis of clinically suspected genes in 225 subjects resulted in a positive molecular diagnosis in 26% of patients with dominant IRD and 59% of patients with recessive IRD. Novel damaging mutations were identified in four genes. Single gene screening is not an ideal method for diagnostic testing given the phenotypic and genetic heterogeneity among IRD cases. High-throughput sequencing of all genes associated with retinal degeneration may be more efficient for molecular diagnosis.

A longitudinal study of Stargardt disease: quantitative assessment of fundus autofluorescence, progression, and genotype correlations.

PubMed

Fujinami, Kaoru; Lois, Noemi; Mukherjee, Rajarshi; McBain, Vikki A; Tsunoda, Kazushige; Tsubota, Kazuo; Stone, Edwin M; Fitzke, Fred W; Bunce, Catey; Moore, Anthony T; Webster, Andrew R; Michaelides, Michel

2013-12-17

We characterized subtypes of fundus autofluorescence (AF) and the progression of retinal atrophy, and correlated these findings with genotype in Stargardt disease. Full clinical examination and AF imaging was undertaken in 68 patients with Stargardt disease. The baseline data were compared to those at follow-up. Patients were classified into three AF subtypes: type 1 had a localized low signal at the fovea surrounded by a homogeneous background, type 2 had a localized low signal at the macula surrounded by a heterogeneous background with numerous foci of abnormal signal, and type 3 had multiple low signal areas at the posterior pole with a heterogeneous background. At baseline, there were 19 patients with type 1, 41 with type 2, and 8 with type 3 disease. The areas of reduced AF signal were measured and rate of atrophy enlargement (RAE) was calculated as the difference of the atrophy size over time (mm²) divided by the follow-up interval (years). Molecular screening of ABCA4 was undertaken. The mean follow-up interval was 9.1 years. A total of 42% cases with type 1 disease progressed to type 2, and 12% with type 2 progressed to type 3. The RAE (mm²/y) based upon baseline AF subtypes was significantly different; 0.06 in type 1, 0.67 in type 2, and 4.37 in type 3. ABCA4 variants were identified in 57 patients. There was a significant association between AF subtype and genotype. The AF pattern at baseline influences the enlargement of atrophy over time and has genetic correlates. These data are likely to assist in the provision of counseling on prognosis in Stargardt disease and be valuable for future clinical trials.

Inhibition of soluble epoxide hydrolase in mice promotes reverse cholesterol transport and regression of atherosclerosis.

PubMed

Shen, Li; Peng, Hongchun; Peng, Ran; Fan, Qingsong; Zhao, Shuiping; Xu, Danyan; Morisseau, Christophe; Chiamvimonvat, Nipavan; Hammock, Bruce D

2015-04-01

Adipose tissue is the body largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), which maintains plasma high-density lipoprotein (HDL) levels. HDLs have a protective role in atherosclerosis by mediating reverse cholesterol transport (RCT). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition has various beneficial effects on cardiovascular disease. The sEH is highly expressed in adipocytes, and it converts epoxyeicosatrienoic acids (EETs) into less bioactive dihydroxyeicosatrienoic acids. We previously showed that increasing EETs levels with a sEH inhibitor (sEHI) (t-AUCB) resulted in elevated ABCA1 expression and promoted ABCA1-mediated cholesterol efflux from 3T3-L1 adipocytes. The present study investigates the impacts of t-AUCB in mice deficient for the low density lipoprotein (LDL) receptor (Ldlr(-/-) mice) with established atherosclerotic plaques. The sEH inhibitor delivered in vivo for 4 weeks decreased the activity of sEH in adipose tissue, enhanced ABCA1 expression and cholesterol efflux from adipose depots, and consequently increased HDL levels. Furthermore, t-AUCB enhanced RCT to the plasma, liver, bile and feces. It also showed the reduction of plasma LDL-C levels. Consistently, t-AUCB-treated mice showed reductions in the size of atherosclerotic plaques. These studies establish that raising adipose ABCA1 expression, cholesterol efflux, and plasma HDL levels with t-AUCB treatment promotes RCT, decreasing LDL-C and atherosclerosis regression, suggesting that sEH inhibition may be a promising strategy to treat atherosclerotic vascular disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Inhibition of cholesterol absorption associated with a PPAR alpha-dependent increase in ABC binding cassette transporter A1 in mice.

PubMed

Knight, Brian L; Patel, Dilip D; Humphreys, Sandy M; Wiggins, David; Gibbons, Geoffrey F

2003-11-01

Dietary supplementation with the peroxisome proliferator-activated receptor alpha (PPAR alpha) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPAR alpha-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor alpha (LXR alpha). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPAR alpha influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXR alpha.

[Differentially expressed genes of cell signal transduction associated with benzene poisoning by cDNA microarray].

PubMed

Wang, Hong; Bi, Yongyi; Tao, Ning; Wang, Chunhong

2005-08-01

To detect the differential expression of cell signal transduction genes associated with benzene poisoning, and to explore the pathogenic mechanisms of blood system damage induced by benzene. Peripheral white blood cell gene expression profile of 7 benzene poisoning patients, including one aplastic anemia, was determined by cDNA microarray. Seven chips from normal workers were served as controls. Cluster analysis of gene expression profile was performed. Among the 4265 target genes, 176 genes associated with cell signal transduction were differentially expressed. 35 up-regulated genes including PTPRC, STAT4, IFITM1 etc were found in at least 6 pieces of microarray; 45 down-regulated genes including ARHB, PPP3CB, CDC37 etc were found in at least 5 pieces of microarray. cDNA microarray technology is an effective technique for screening the differentially expressed genes of cell signal transduction. Disorder in cell signal transduction may play certain role in the pathogenic mechanism of benzene poisoning.

Synonymous ABCA3 Variants Do Not Increase Risk for Neonatal Respiratory Distress Syndrome

PubMed Central

Wambach, Jennifer A.; Wegner, Daniel J.; Heins, Hillary B.; Druley, Todd E.; Mitra, Robi D.; Hamvas, Aaron; Cole, F. Sessions

2014-01-01

Objective To determine whether synonymous variants in the adenosine triphosphate-binding cassette A3 transporter (ABCA3) gene increase the risk for neonatal respiratory distress syndrome (RDS) in term and late preterm infants of European and African descent. Study design Using next-generation pooled sequencing of race-stratified DNA samples from infants of European and African descent at $34 weeks gestation with and without RDS (n = 503), we scanned all exons of ABCA3, validated each synonymous variant with an independent genotyping platform, and evaluated race-stratified disease risk associated with common synonymous variants and collapsed frequencies of rare synonymous variants. Results The synonymous ABCA3 variant frequency spectrum differs between infants of European descent and those of African descent. Using in silico prediction programs and statistical strategies, we found no potentially disruptive synonymous ABCA3 variants or evidence of selection pressure. Individual common synonymous variants and collapsed frequencies of rare synonymous variants did not increase disease risk in term and late-preterm infants of European or African descent. Conclusion In contrast to rare, nonsynonymous ABCA3 mutations, synonymous ABCA3 variants do not increase the risk for neonatal RDS among term and late-preterm infants of European or African descent. PMID:24657120

Cellular cholesterol regulates ubiquitination and degradation of the cholesterol export proteins ABCA1 and ABCG1.

PubMed

Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C; Brown, Andrew J; Sandoval, Cecilia; Hallab, Jeannette C; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-03-14

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

PubMed Central

Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-01-01

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

Hydrogen Sulfide Up-Regulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARα.

PubMed

Li, Dong; Xiong, Qinghui; Peng, Jin; Hu, Bin; Li, Wanzhen; Zhu, Yizhun; Shen, Xiaoyan

2016-04-29

ATP binding cassette transporter A1 (ABCA1) plays a key role in atherogenesis. Hydrogen sulfide (H₂S), a gasotransmitter, has been reported to play an anti-atherosclerotic role. However, the underlying mechanisms are largely unknown. In this study we examined whether and how H₂S regulates ABCA1 expression. The effect of H₂S on ABCA1 expression and lipid metabolism were assessed in vitro by cultured human hepatoma cell line HepG2, and in vivo by ApoE(-/-) mice with a high-cholesterol diet. NaHS (an exogenous H₂S donor) treatment significantly increased the expression of ABCA1, ApoA1, and ApoA2 and ameliorated intracellular lipid accumulation in HepG2 cells. Depletion of the endogenous H₂S generator cystathionine γ-lyase (CSE) by small RNA interference (siRNA) significantly decreased the expression of ABCA1 and resulted in the accumulation of lipids in HepG2 cells. In vivo NaHS treatment significantly reduced the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL), diminished atherosclerotic plaque size, and increased hepatic ABCA1 expression in fat-fed ApoE(-/-) mice. Further study revealed that NaHS upregulated ABCA1 expression by promoting peroxisome proliferator-activated receptor α (PPARα) nuclear translocation. H₂S up-regulates the expression of ABCA1 by promoting the nuclear translocation of PPARα, providing a fundamental mechanism for the anti-atherogenic activity of H₂S. H₂S may be a promising potential drug candidate for the treatment of atherosclerosis.

Screening hybridomas for anabolic androgenic steroids by steroid analog antigen microarray.

PubMed

Du, Hongwu; Chen, Guangyu; Bian, Yongzhong; Xing, Cenzan; Ding, Xue; Zhu, Mengliang; Xun, Yiping; Chen, Peng; Zhou, Yabin; Li, Shaoxu

2015-01-01

Currently, dozens of anabolic androgenic steroids (AAS) are forbidden in the World Anti-Doping Agency Prohibited List, however, despite extensive investigation, there are still lots of AAS without corresponding monoclonal antibodies. A steroid analog antigen microarray made up of ten AAS was fabricated to screen the hybridoma and it was found an original unsuccessful clone turned out to be a candidate anti-boldenone antibody, without any cross-reactions with endogenous AAS or 44 different AAS standard reference materials tested. Our findings suggested that steroid analog antigen microarray could be a promising tool to screen and characterize new applications of antibodies for structure analogs, and this also exhibits the potential to fast identify effective epitopes of hybridomas in a single assay.

Interaction between FTO rs9939609 and the Native American-origin ABCA1 rs9282541 affects BMI in the admixed Mexican population.

PubMed

Villalobos-Comparán, Marisela; Antuna-Puente, Bárbara; Villarreal-Molina, María Teresa; Canizales-Quinteros, Samuel; Velázquez-Cruz, Rafael; León-Mimila, Paola; Villamil-Ramírez, Hugo; González-Barrios, Juan Antonio; Merino-García, José Luis; Thompson-Bonilla, María Rocío; Jarquin, Diego; Sánchez-Hernández, Osvaldo Erik; Rodríguez-Arellano, Martha Eunice; Posadas-Romero, Carlos; Vargas-Alarcón, Gilberto; Campos-Pérez, Francisco; Quiterio, Manuel; Salmerón-Castro, Jorge; Carnevale, Alessandra; Romero-Hidalgo, Sandra

2017-05-02

The aim of this study was to explore whether interactions between FTO rs9939609 and ABCA1 rs9282541 affect BMI and waist circumference (WC), and could explain previously reported population differences in FTO-obesity and FTO-BMI associations in the Mexican and European populations. A total of 3938 adults and 636 school-aged children from Central Mexico were genotyped for both polymorphisms. Subcutaneous and visceral adipose tissue biopsies from 22 class III obesity patients were analyzed for FTO and ABCA1 mRNA expression. Generalized linear models were used to test for associations and gene-gene interactions affecting BMI, WC and FTO expression. FTO and ABCA1 risk alleles were not individually associated with higher BMI or WC. However, in the absence of the ABCA1 risk allele, the FTO risk variant was significantly associated with higher BMI (P = 0.043) and marginally associated with higher WC (P = 0.067), as reported in Europeans. The gene-gene interaction affecting BMI and WC was statistically significant only in adults. FTO mRNA expression in subcutaneous abdominal adipose tissue according to ABCA1 genotype was consistent with these findings. This is the first report showing evidence of FTO and ABCA1 gene variant interactions affecting BMI, which may explain previously reported population differences. Further studies are needed to confirm this interaction.

High-throughput screening in two dimensions: binding intensity and off-rate on a peptide microarray.

PubMed

Greving, Matthew P; Belcher, Paul E; Cox, Conor D; Daniel, Douglas; Diehnelt, Chris W; Woodbury, Neal W

2010-07-01

We report a high-throughput two-dimensional microarray-based screen, incorporating both target binding intensity and off-rate, which can be used to analyze thousands of compounds in a single binding assay. Relative binding intensities and time-resolved dissociation are measured for labeled tumor necrosis factor alpha (TNF-alpha) bound to a peptide microarray. The time-resolved dissociation is fitted to a one-component exponential decay model, from which relative dissociation rates are determined for all peptides with binding intensities above background. We show that most peptides with the slowest off-rates on the microarray also have the slowest off-rates when measured by surface plasmon resonance (SPR). 2010 Elsevier Inc. All rights reserved.

Hydrogen Sulfide Up-Regulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARα

PubMed Central

Li, Dong; Xiong, Qinghui; Peng, Jin; Hu, Bin; Li, Wanzhen; Zhu, Yizhun; Shen, Xiaoyan

2016-01-01

ATP binding cassette transporter A1 (ABCA1) plays a key role in atherogenesis. Hydrogen sulfide (H2S), a gasotransmitter, has been reported to play an anti-atherosclerotic role. However, the underlying mechanisms are largely unknown. In this study we examined whether and how H2S regulates ABCA1 expression. The effect of H2S on ABCA1 expression and lipid metabolism were assessed in vitro by cultured human hepatoma cell line HepG2, and in vivo by ApoE−/− mice with a high-cholesterol diet. NaHS (an exogenous H2S donor) treatment significantly increased the expression of ABCA1, ApoA1, and ApoA2 and ameliorated intracellular lipid accumulation in HepG2 cells. Depletion of the endogenous H2S generator cystathionine γ-lyase (CSE) by small RNA interference (siRNA) significantly decreased the expression of ABCA1 and resulted in the accumulation of lipids in HepG2 cells. In vivo NaHS treatment significantly reduced the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL), diminished atherosclerotic plaque size, and increased hepatic ABCA1 expression in fat-fed ApoE−/− mice. Further study revealed that NaHS upregulated ABCA1 expression by promoting peroxisome proliferator-activated receptor α (PPARα) nuclear translocation. H2S up-regulates the expression of ABCA1 by promoting the nuclear translocation of PPARα, providing a fundamental mechanism for the anti-atherogenic activity of H2S. H2S may be a promising potential drug candidate for the treatment of atherosclerosis. PMID:27136542

Myeloid-specific genetic ablation of ATP-binding cassette transporter ABCA1 is protective against cancer

PubMed Central

Zamanian-Daryoush, Maryam; Lindner, Daniel J.; DiDonato, Joseph A.; Wagner, Matthew; Buffa, Jennifer; Rayman, Patricia; Parks, John S.; Westerterp, Marit; Tall, Alan R.; Hazen, Stanley L.

2017-01-01

Increased circulating levels of apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), by genetic manipulation or infusion, protects against melanoma growth and metastasis. Herein, we explored potential roles in melanoma tumorigenesis for host scavenger receptor class B, type 1 (SR-B1), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), all mediators of apoA-I and HDL sterol and lipid transport function. In a syngeneic murine melanoma tumor model, B16F10, mice with global deletion of SR-B1 expression exhibited increased plasma HDL cholesterol (HDLc) levels and decreased tumor volume, indicating host SR-B1 does not directly contribute to HDL-associated anti-tumor activity. In mice with myeloid-specific loss of ABCA1 (Abca1−M/−M; A1−M/−M), tumor growth was inhibited by ∼4.8-fold relative to wild type (WT) animals. Abcg1−M/−M (G1−M/−M) animals were also protected by 2.5-fold relative to WT, with no further inhibition of tumor growth in Abca1/Abcg1 myeloid-specific double knockout animals (DKO). Analyses of tumor-infiltrating immune cells revealed a correlation between tumor protection and decreased presence of the immune suppressive myeloid-derived suppressor cell (MDSC) subsets, Ly-6G+Ly-6CLo and Ly-6GnegLy-6CHi cells. The growth of the syngeneic MB49 murine bladder cancer cells was also inhibited in A1−M/−M mice. Collectively, our studies provide further evidence for an immune modulatory role for cholesterol homeostasis pathways in cancer. PMID:29069761

See what you eat--broad GMO screening with microarrays.

PubMed

von Götz, Franz

2010-03-01

Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative.

Microarrays Made Simple: "DNA Chips" Paper Activity

ERIC Educational Resources Information Center

Barnard, Betsy

2006-01-01

DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

Mutations in the Cholesterol Transporter Gene ABCA5 Are Associated with Excessive Hair Overgrowth

PubMed Central

DeStefano, Gina M.; Kurban, Mazen; Anyane-Yeboa, Kwame; Dall'Armi, Claudia; Di Paolo, Gilbert; Feenstra, Heather; Silverberg, Nanette; Rohena, Luis; López-Cepeda, Larissa D.; Jobanputra, Vaidehi; Fantauzzo, Katherine A.; Kiuru, Maija; Tadin-Strapps, Marija; Sobrino, Antonio; Vitebsky, Anna; Warburton, Dorothy; Levy, Brynn; Salas-Alanis, Julio C.; Christiano, Angela M.

2014-01-01

Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole-exome sequencing. We identified a single base pair substitution in the 5′ donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles. The homozygous recessive disruption of ABCA5 leads to reduced lysosome function, which results in an accumulation of autophagosomes, autophagosomal cargos as well as increased endolysosomal cholesterol in CGHT keratinocytes. In an unrelated sporadic case of CGHT, we identified a 1.3 Mb cryptic deletion of chr17q24.2-q24.3 encompassing ABCA5 and found that ABCA5 levels are dramatically reduced throughout patient hair follicles. Collectively, our findings support ABCA5 as a gene underlying the CGHT phenotype and suggest a novel, previously unrecognized role for this gene in regulating hair growth. PMID:24831815

An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux

PubMed Central

Gillespie, Mark A; Gold, Elizabeth S; Ramsey, Stephen A; Podolsky, Irina; Aderem, Alan; Ranish, Jeffrey A

2015-01-01

LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment-quantitative mass spectrometry (PE-QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand- and binding-dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll-like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro-inflammatory and anti-inflammatory pathways that results in repression of macrophage cholesterol efflux. PMID:25755249

Microarray-based screening of heat shock protein inhibitors.

PubMed

Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.

PubMed

Storti, Federica; Raphael, Gabriele; Griesser, Vera; Klee, Katrin; Drawnel, Faye; Willburger, Carolin; Scholz, Rebecca; Langmann, Thomas; von Eckardstein, Arnold; Fingerle, Jürgen; Grimm, Christian; Maugeais, Cyrille

2017-12-01

Genetic studies have linked age-related macular degeneration (AMD) to genes involved in high-density lipoprotein (HDL) metabolism, including ATP-binding cassette transporter A1 (ABCA1). The retinal pigment epithelium (RPE) handles large amounts of lipids, among others cholesterol, partially derived from internalized photoreceptor outer segments (OS) and lipids physiologically accumulate in the aging eye. To analyze the potential function of ABCA1 in the eye, we measured cholesterol efflux, the first step of HDL generation, in RPE cells. We show the expression of selected genes related to HDL metabolism in mouse and human eyecups as well as in ARPE-19 and human primary RPE cells. Immunofluorescence staining revealed localization of ABCA1 on both sides of polarized RPE cells. This was functionally confirmed by directional efflux to apolipoprotein AI (ApoA-I) of 3 H-labeled cholesterol given to the cells via serum or via OS. ABCA1 expression and activity was modulated using a liver-X-receptor (LXR) agonist and an ABCA1 neutralizing antibody, demonstrating that the efflux was ABCA1-dependent. We concluded that the ABCA1-mediated lipid efflux pathway, and hence HDL biosynthesis, is functional in RPE cells towards both the basal (choroidal) and apical (subretinal) space. Impaired activity of the pathway might cause age-related perturbations of lipid homeostasis in the outer retina and thus may contribute to disease development and/or progression. Copyright © 2017 Elsevier Ltd. All rights reserved.

[A novel compound heterozygous mutation in ABCA3 gene in a child with diffuse parenchymal lung disease].

PubMed

Bao, Y M; Liu, X L; Liu, X L; Chen, J H; Zheng, Y J

2017-11-02

Objective: To summarize the clinical characteristics of the diffuse parenchymal lung diseases in a child caused by a novel compound heterozygous ABCA3 mutation and explore the association between the phenotype and ABCA3 mutation. Method: The clinical material of a patient diagnosed with diffuse parenchymal lung disease with ABCA3 mutation in December 2016 in Shenzhen Children's Hospital was analyzed. The information about ABCA3 gene mutation updated before April, 2017 was searched and collected from the gene databases (including 1000Genomes, HGMD, EXAC) and the literatures (including Wanfang Chinese database and Pubmed). Result: The girl was one year and nine months old. She presented with chronic cough, tachypnea, cyanosis and failure to thrive since she was one year and three months old. Her condition gradually deteriorated after she was empirically treated. Physical examination showed malnutrition, tachypnea and clubbed-fingers. Her high resolution computed tomography (HRCT) revealed diffused ground-glass opacities, thickened interlobular septum, and multiple subpleural small air-filled lung cysts. The second generation sequencing study identified a novel compound heterozygous mutation (c.1755delC+c.2890G>A) in her ABCA3 gene, which derived respectively from her parents and has not been reported in the database and the literatures mentioned above. Conclusion: c.1755delC+c.2890G>A is a new kind of compound heterozygous mutation in ABCA3, which can cause children's diffuse parenchymal lung disease. Its phenotype is related to its genotype.

7-ketocholesteryl-9-carboxynonanoate enhances ATP binding cassette transporter A1 expression mediated by PPARγ in THP-1 macrophages.

PubMed

Chi, Yan; Wang, Le; Liu, Yuanyuan; Ma, Yanhua; Wang, Renjun; Han, Xiaofei; Qiao, Hui; Lin, Jiabin; Matsuura, Eiji; Liu, Shuqian; Liu, Qingping

2014-06-01

ATP binding cassette transporter A1 (ABCA1) is a member of the ATP-binding cassette transporter family. It plays an essential role in mediating the efflux of excess cholesterol. It is known that peroxisome proliferator-activated receptor gamma (PPARγ) promoted ABCA1 expression. We previously found 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) upregulated ABCA1 partially through CD36 mediated signals. In the present study, we intended to test if PPARγ signally is involved in the upregulation mediated by oxLig-1. First, we docked oxLig-1 and the ligand-binding domain (LBD) of PPARγ by using AutoDock 3.05 and subsequently confirmed the binding by ELISA assay. Western blotting analyses showed that oxLig-1 induces liver X receptor alpha (LXRα), PPARγ and consequently ABCA1 expression. Furthermore, oxLig-1 significantly enhanced ApoA-I-mediated cholesterol efflux. Pretreatment with an inhibitor for PPARγ (GW9662) or/and LXRα (GGPP) attenuated oxLig-1-induced ABCA1 expression. Under PPARγ knockdown by using PPARγ-shRNA, oxLig-1-induced ABCA1 expression and cholesterol efflux in THP-1 macrophages was blocked by 62% and 25% respectively. These observations suggest that oxLig-1 is a novel PPARγ agonist, promoting ApoA-I-mediated cholesterol efflux from THP-1 macrophages by increasing ABCA1 expression via induction of PPARγ. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A Protein Microarray for the Rapid Screening of Patients Suspected of Infection with Various Food-Borne Helminthiases

PubMed Central

Ai, Lin; Chen, Jun-Hu; Chen, Shao-Hong; Zhang, Yong-Nian; Cai, Yu-Chun; Zhu, Xing-Quan; Zhou, Xiao-Nong

2012-01-01

Background Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. Methodology/Principal Findings In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. Conclusions/Significance The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening. PMID:23209851

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

PubMed

Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

2012-05-01

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

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

Wang, Jing-Min, E-mail: wjm730222@163.com; Wang, Dong, E-mail: 8888dd@163.com; Tan, Yu-Yan, E-mail: tyytyz@sina.com

Highlights: • Cholesterosis is a metabolic disease characterized by excessive lipid droplets. • Lipid droplet efflux is mediated by the ABCA1 transporter. • 22(R)-hydroxycholesterol can activate LXRα and up-regulate ABCA1. • Pioglitazone up-regulates ABCA1 in a PPARγ–LXRα–ABCA1-dependent manner. • 22(R)-hydroxycholesterol and pioglitazone synergistically decrease lipid droplets. - Abstract: Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis andmore » the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid droplets associated with cholesterosis in GBECs. In conclusion, the present findings indicate that the anti-lipid deposition action of 22(R)-hydroxycholesterol combined with pioglitazone involves the activation of the PPARγ–LXRα–ABCA1 pathway, increased ABCA1 expression and the efflux of cholesterol from GBECs. Thus, 22(R)-hydroxycholesterol synergistically combined with pioglitazone to produce a remarkable effect on lipid deposition in cholesterosis GBECs.« less

Peripheral Visual Fields in ABCA4 Stargardt Disease and Correlation With Disease Extent on Ultra-widefield Fundus Autofluorescence.

PubMed

Abalem, Maria Fernanda; Otte, Benjamin; Andrews, Chris; Joltikov, Katherine A; Branham, Kari; Fahim, Abigail T; Schlegel, Dana; Qian, Cynthia X; Heckenlively, John R; Jayasundera, Thiran

2017-12-01

To evaluate the disease extent on ultra-widefield fundus autofluorescence (UWF-FAF) in patients with ABCA4 Stargardt disease (STGD) and correlate these data with functional outcome measures. Retrospective cross-sectional study. Setting: Kellogg Eye Center, University of Michigan. Sixty-five patients with clinical diagnosis and proven pathogenic variants in the ABCA4 gene. Observational Procedures: The UWF-FAF images were obtained using Optos (200 degrees) and classified into 3 types. Functional testing included kinetic widefield perimetry, full-field electroretinogram (ffERG), and visual acuity (VA). All results were evaluated with respect to UWF-FAF classification. Classification of UWF-FAF; area comprising the I4e, III4e, and IV4e isopters; ffERG patterns; and VA. For UWF-FAF, 27 subjects (41.5%) were classified as type I, 17 (26.2%) as type II, and 21 (32.4%) as type III. The area of each isopter correlated inversely with the extent of the disease and all isopters were able to detect differences among UWF-FAF types (IV4e, P = .0013; III4e, P = .0003; I4e, P < .0001 = 3.93e -8 ). ffERG patterns and VA were also different among the 3 UWF-FAF types (P < .001 = 6.61e- 6 and P < .001 = 7.3e -5 , respectively). Patients with widespread disease presented with more constriction of peripheral visual fields and had more dysfunction on ffERG and worse VA compared to patients with disease confined to the macula. UWF-FAF images may provide information for estimating peripheral and central visual function in STGD. Copyright © 2017. Published by Elsevier Inc.

Increased Risk of Interstitial Lung Disease in Children with a Single R288K Variant of ABCA3

PubMed Central

Wittmann, Thomas; Frixel, Sabrina; Höppner, Stefanie; Schindlbeck, Ulrike; Schams, Andrea; Kappler, Matthias; Hegermann, Jan; Wrede, Christoph; Liebisch, Gerhard; Vierzig, Anne; Zacharasiewicz, Angela; Kopp, Matthias Volkmar; Poets, Christian F; Baden, Winfried; Hartl, Dominik; van Kaam, Anton H; Lohse, Peter; Aslanidis, Charalampos; Zarbock, Ralf; Griese, Matthias

2016-01-01

The ABCA3 gene encodes a lipid transporter in type II pneumocytes critical for survival and normal respiratory function. The frequent ABCA3 variant R288K increases the risk for neonatal respiratory distress syndrome among term and late preterm neonates, but its role in children’s interstitial lung disease has not been studied in detail. In a retrospective cohort study of 228 children with interstitial lung disease related to the alveolar surfactant system, the frequency of R288K was assessed and the phenotype of patients carrying a single R288K variant further characterized by clinical course, lung histology, computed tomography and bronchoalveolar lavage phosphatidylcholine PC 32:0. Cell lines stably transfected with ABCA3-R288K were analyzed for intracellular transcription, processing and targeting of the protein. ABCA3 function was assessed by detoxification assay of doxorubicin, and the induction and volume of lamellar bodies. We found nine children with interstitial lung disease carrying a heterozygous R288K variant, a frequency significantly higher than in the general Caucasian population. All identified patients had neonatal respiratory insufficiency, recovered and developed chronic interstitial lung disease with intermittent exacerbations during early childhood. In vitro analysis showed normal transcription, processing, and targeting of ABCA3-R288K, but impaired detoxification function and smaller lamellar bodies. We propose that the R288K variant can underlie interstitial lung disease in childhood due to reduced function of ABCA3, demonstrated by decelerated detoxification of doxorubicin, reduced PC 32:0 content and decreased lamellar body volume. PMID:26928390

Investigation of ABCA1 C69T polymorphism in patients with type 2 diabetes mellitus.

PubMed

Ergen, H Arzu; Zeybek, Umit; Gök, Ozlem; Karaali, Z Ermis

2012-01-01

Non insulin dependent diabetes mellitus is the most common type of diabetes. Genetic factors, lipid profiles, hypertension are potential risk factors for diabetes mellitus. Adenosine binding cassette transporter proteins 1 (ABCA1) plays a role in cholesterol metabolism, especially high density lipoprotein (HDL-cholesterol). There are multiple mechanisms by which HDL-cholesterol can be atheroprotective, it is clear that the relative activity of ABCA1 plays a major role. We aimed to investigate association of ABCA1 C69T gene polymorphism with lipid levels in Turkish type 2 diabetic patients. After isolation of DNA by ethanol precipitation we determined ABCA1 gene polymorphism by using polimerase chain reaction--restriction fragment lenght polymorphism (PCR-RFLP) method in 107 type 2 diabetic patients and 50 healthy controls. We have observed that the frequency of TT genotype is significantly higher in healthy controls compared to patients (14% vs. 3%; P = 0.008). Also frequency of T allele was higher in controls than in patients (34% vs. 21%; P = 0.020; OR (95% CI) = 0.52 (0.30-0.88)). There was no association of lipid levels and ABCA1 C69T polymorphism subgroups. We have found significantly higher frequency of both T allele and genotype in control group when compared to patients that made us think that T allele may be a protective factor against diabetes mellitus. But, we could not find a relationship between genotypes and lipid concentrations in our two groups. Larger studies will help us to understand the relationship between ABCA1 C69T genotype and lipid parameters in diabetes mellitus.

Loss of Peripapillary Sparing in non-Group I Stargardt Disease

PubMed Central

Burke, Tomas R; Allikmets, Rando; Smith, R. Theodore; Gouras, Peter; Tsang, Stephen H

2010-01-01

The aim of this study was to assess peripapillary sparing in patients with non-group I Stargardt disease. We suggest this as a useful clinical sign for formulating disease severity. Patients with a diagnosis of Stargardt disease were grouped by electroretinogram (ERG). Fundus autofluorescence was used to assess the peripapillary area for involvement in the Stargardt disease process. From a cohort of 32 patients (64 eyes), 17 patients (33 eyes) demonstrated loss of peripapillary sparing. One of 15 patients in Group I, six of 7 patients in group II and 9 of 10 patients in group III demonstrated peripapillary atrophy. One patient in group II had peripapillary flecks. All patients had at least one mutation detected in the ABCA4 gene. Both mutations were detected in 21 patients. Patients in groups II and III had the earliest ages of onset and the poorest visual acuities. Two novel disease causing mutation in the ABCA4 gene were detected. Our data supports the observation that peripapillary sparing is not universal finding for Stargardt disease and peripapillary atrophy is a useful clinical sign for identifying patients with Stargardt disease who fall into the more severe ERG groups, i.e. groups II and III. The presence of atrophy suggests a continuum of disease between groups II and III. Loss of peripapillary sparing is likely associated with the more deleterious mutations of the ABCA4 gene. PMID:20696155

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

PubMed

Ohtsuki, Sumio; Ikeda, Chiemi; Uchida, Yasuo; Sakamoto, Yumi; Miller, Florence; Glacial, Fabienne; Decleves, Xavier; Scherrmann, Jean-Michel; Couraud, Pierre-Olivier; Kubo, Yoshiyuki; Tachikawa, Masanori; Terasaki, Tetsuya

2013-01-07

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Application of chemical arrays in screening elastase inhibitors.

PubMed

Gao, Feng; Du, Guan-Hua

2006-06-01

Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).

Identification of regulatory targets of tissue-specific transcription factors: application to retina-specific gene regulation

PubMed Central

Qian, Jiang; Esumi, Noriko; Chen, Yangjian; Wang, Qingliang; Chowers, Itay; Zack, Donald J.

2005-01-01

Identification of tissue-specific gene regulatory networks can yield insights into the molecular basis of a tissue's development, function and pathology. Here, we present a computational approach designed to identify potential regulatory target genes of photoreceptor cell-specific transcription factors (TFs). The approach is based on the hypothesis that genes related to the retina in terms of expression, disease and/or function are more likely to be the targets of retina-specific TFs than other genes. A list of genes that are preferentially expressed in retina was obtained by integrating expressed sequence tag, SAGE and microarray datasets. The regulatory targets of retina-specific TFs are enriched in this set of retina-related genes. A Bayesian approach was employed to integrate information about binding site location relative to a gene's transcription start site. Our method was applied to three retina-specific TFs, CRX, NRL and NR2E3, and a number of potential targets were predicted. To experimentally assess the validity of the bioinformatic predictions, mobility shift, transient transfection and chromatin immunoprecipitation assays were performed with five predicted CRX targets, and the results were suggestive of CRX regulation in 5/5, 3/5 and 4/5 cases, respectively. Together, these experiments strongly suggest that RP1, GUCY2D, ABCA4 are novel targets of CRX. PMID:15967807

Fundus Autofluorescence and Photoreceptor Cell Rosettes in Mouse Models

PubMed Central

Flynn, Erin; Ueda, Keiko; Auran, Emily; Sullivan, Jack M.; Sparrow, Janet R.

2014-01-01

Purpose. This study was conducted to study correlations among fundus autofluorescence (AF), RPE lipofuscin accumulation, and photoreceptor cell degeneration and to investigate the structural basis of fundus AF spots. Methods. Fundus AF images (55° lens; 488-nm excitation) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired in pigmented Rdh8−/−/Abca4−/− mice (ages 1–9 months) with a confocal scanning laser ophthalmoscope (cSLO). For quantitative fundus AF (qAF), gray levels (GLs) were calibrated to an internal fluorescence reference. Retinal bisretinoids were measured by quantitative HPLC. Histometric analysis of outer nuclear layer (ONL) thicknesses was performed, and cryostat sections of retina were examined by fluorescence microscopy. Results. Quantified A2E and qAF intensities increased until age 4 months in the Rdh8−/−/Abca4−/− mice. The A2E levels declined after 4 months of age, but qAF intensity values continued to rise. The decline in A2E levels in the Rdh8−/−/Abca4−/− mice paralleled reduced photoreceptor cell viability as reflected in ONL thinning. Hyperautofluorescent puncta in fundus AF images corresponded to photoreceptor cell rosettes in SD-OCT images and histological sections stained with hematoxylin and eosin. The inner segment/outer segment–containing core of the rosette emitted an autofluorescence detected by fluorescence microscopy. Conclusions. When neural retina is disordered, AF from photoreceptor cells can contribute to noninvasive fundus AF images. Hyperautofluorescent puncta in fundus AF images are attributable, in at least some cases, to photoreceptor cell rosettes. PMID:25015357

Identification of the Key Genes and Pathways in Esophageal Carcinoma.

PubMed

Su, Peng; Wen, Shiwang; Zhang, Yuefeng; Li, Yong; Xu, Yanzhao; Zhu, Yonggang; Lv, Huilai; Zhang, Fan; Wang, Mingbo; Tian, Ziqiang

2016-01-01

Objective . Esophageal carcinoma (EC) is a frequently common malignancy of gastrointestinal cancer in the world. This study aims to screen key genes and pathways in EC and elucidate the mechanism of it. Methods . 5 microarray datasets of EC were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened by bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction were performed to obtain the biological roles of DEGs in EC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression level of DEGs in EC. Results . A total of 1955 genes were filtered as DEGs in EC. The upregulated genes were significantly enriched in cell cycle and the downregulated genes significantly enriched in Endocytosis. PPI network displayed CDK4 and CCT3 were hub proteins in the network. The expression level of 8 dysregulated DEGs including CDK4, CCT3, THSD4, SIM2, MYBL2, CENPF, CDCA3, and CDKN3 was validated in EC compared to adjacent nontumor tissues and the results were matched with the microarray analysis. Conclusion . The significantly DEGs including CDK4, CCT3, THSD4, and SIM2 may play key roles in tumorigenesis and development of EC involved in cell cycle and Endocytosis.

Overlap between Parkinson disease and Alzheimer disease in ABCA7 functional variants

PubMed Central

Nuytemans, Karen; Maldonado, Lizmarie; Ali, Aleena; John-Williams, Krista; Beecham, Gary W.; Martin, Eden; Scott, William K.

2016-01-01

Objective: Given their reported function in phagocytosis and clearance of protein aggregates in Alzheimer disease (AD), we hypothesized that variants in ATP-binding cassette transporter A7 (ABCA7) might be involved in Parkinson disease (PD). Methods: ABCA7 variants were identified using whole-exome sequencing (WES) on 396 unrelated patients with PD and 222 healthy controls. In addition, we used the publicly available WES data from the Parkinson's Progression Markers Initiative (444 patients and 153 healthy controls) as a second, independent data set. Results: We observed a higher frequency of loss-of-function (LOF) variants and rare putative highly functional variants (Combined Annotation Dependent Depletion [CADD] >20) in clinically diagnosed patients with PD than in healthy controls in both data sets. Overall, we identified LOF variants in 11 patients and 1 healthy control (odds ratio [OR] 4.94, Fisher exact p = 0.07). Four of these variants have been previously implicated in AD risk (p.E709AfsX86, p.W1214X, p.L1403RfsX7, and rs113809142). In addition, rare variants with CADD >20 were observed in 19 patients vs 3 healthy controls (OR 2.85, Fisher exact p = 0.06). Conclusion: The presence of ABCA7 LOF variants in clinically defined PD suggests that they might be risk factors for neurodegeneration in general, especially those variants hallmarked by protein aggregation. More studies will be needed to evaluate the overall impact of this transporter in neurodegenerative disease. PMID:27066581

ABC Transporter Genes and Risk of Type 2 Diabetes

PubMed Central

Schou, Jesper; Tybjærg-Hansen, Anne; Møller, Holger J.; Nordestgaard, Børge G.; Frikke-Schmidt, Ruth

2012-01-01

OBJECTIVE Alterations of pancreatic β-cell cholesterol content may contribute to β-cell dysfunction. Two important determinants of intracellular cholesterol content are the ATP-binding cassette (ABC) transporters A1 (ABCA1) and -G1 (ABCG1). Whether genetic variation in ABCA1 and ABCG1 predicts risk of type 2 diabetes in the general population is unknown. RESEARCH DESIGN AND METHODS We tested whether genetic variation in the promoter and coding regions of ABCA1 and ABCG1 predicted risk of type 2 diabetes in the general population. Twenty-seven variants, identified by previous resequencing of both genes, were genotyped in the Copenhagen City Heart Study (CCHS) (n = 10,185). Two loss-of-function mutations (ABCA1 N1800H and ABCG1 g.-376C>T) (n = 322) and a common variant (ABCG1 g.-530A>G) were further genotyped in the Copenhagen General Population Study (CGPS) (n = 30,415). RESULTS Only one of the variants examined, ABCG1 g.-530A>G, predicted a decreased risk of type 2 diabetes in the CCHS (P for trend = 0.05). Furthermore, when validated in the CGPS or in the CCHS and CGPS combined (n = 40,600), neither the two loss-of-function mutations (ABCA1 N1800H, ABCG1 g.-376C>T) nor ABCG1 g.-530A>G were associated with type 2 diabetes (P values >0.57 and >0.30, respectively). CONCLUSIONS Genetic variations in ABCA1 and ABCG1 were not associated with increased risk of type 2 diabetes in the general population. These data were obtained in general population samples harboring the largest number of heterozygotes for loss-of-function mutations in ABCA1 and ABCG1. PMID:23139370

Tangshen Formula Attenuates Diabetic Nephropathy by Promoting ABCA1-Mediated Renal Cholesterol Efflux in db/db Mice.

PubMed

Liu, Peng; Peng, Liang; Zhang, Haojun; Tang, Patrick Ming-Kuen; Zhao, Tingting; Yan, Meihua; Zhao, Hailing; Huang, Xiaoru; Lan, Huiyao; Li, Ping

2018-01-01

The commonly prescribed Tangshen Formula (TSF) is a traditional Chinese formulation that has been shown to reduce plasma lipid metabolism and proteinuria and improve the estimated glomerular filtration rate (eGFR) in patients with diabetic kidney disease. This study investigated the underlying mechanism whereby TSF regulates renal lipid accumulation and ameliorates diabetic renal injuries in spontaneous diabetic db/db mice and in vitro in sodium palmitate (PA)-stimulated and Abca1-SiRNA-transfected mouse tubular epithelial cells (mTECs). The results revealed that TSF treatment significantly ameliorated the renal injuries by lowering urinary albumin excretion and improving renal tissue injuries in diabetic (db/db) mice. Interestingly, the treatment with TSF also resulted in decreased cholesterol levels in the renal tissues of db/db mice, which was associated with increased expression of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), the Liver X receptors (LXR), and ATP-binding cassette subfamily A member 1 (ABCA1), suggesting that TSF might attenuate diabetic kidney injury via a mechanism associated with improving cholesterol efflux in the diabetic kidney. This was investigated in vitro in mTECs, and the results showed that TSF reduced the PA-stimulated cholesterol accumulation in mTECs. Mechanistically, the addition of TSF was capable of reversing PA-induced downregulation of PGC-1α, LXR, and ABCA1 expression and cholesterol accumulation in mTECs, suggesting that TSF might act the protection via the PGC-1α-LXR-ABCA1 pathway to improve the cholesterol efflux in the renal tissues of db/db mice. This was further confirmed by silencing ABCA1 to block the promotive effect of TSF on cholesterol efflux in vitro . In conclusion, TSF might ameliorate diabetic kidney injuries by promoting ABCA1-mediated renal cholesterol efflux.

Effect Of G2706A and G1051A polymorphisms of the ABCA1 gene on the lipid, oxidative stress and homocystein levels in Turkish patients with polycystıc ovary syndrome

PubMed Central

2011-01-01

Background Obesity, insulin resistance and hyperandrogenism, crucial parameters of Polycystic ovary syndrome (PCOS) play significant pathophysiological roles in lipidemic aberrations associated within the syndrome. Parts of the metabolic syndrome (low HDL and insulin resistance) appeared to facilitate the association between PCOS and coronary artery disease, independently of obesity. ABCA1 gene polymorphism may be altered this components in PCOS patients. In this study, we studied 98 PCOS patients and 93 healthy controls. All subjects underwent venous blood drawing for complete hormonal assays, lipid profile, glucose, insulin, malondialdehyde, nitric oxide, disulfide levels and ABCA genetic study. Results In PCOS group fasting glucose, DHEAS, 17-OHP, free testosterone, total-cholesterol, triglyceride, LDL-cholesterol and fibrinogen were significantly different compare to controls. The genotype ABCA G2706A distribution differed between the control group (GG 60.7%, GA 32.1%, AA 7.1%) and the PCOS patients (GG 8.7%, GA 8.7%, AA 76.8%). The frequency of the A allele (ABCAG2706A) was higher in PCOS patients than control group with 13,0% and 23,2%, respectively. In this study, the homocystein and insulin levels were significantly higher in PCOS patients with ABCA G1051A mutant genotype than those with heterozygote and wild genotypes. Conclusions We found higher percentage of AA genotype and A allele of ABCA G2706A in PCOS patients compare to controls. The fasting insulin and homocystein levels were significantly higher in PCOS patients with ABCA G1051A mutant genotype than those with heterozygote and wild genotypes. PMID:22035022

Recurrent amplification of RTEL1 and ABCA13 and its synergistic effect associated with clinicopathological data of gastric adenocarcinoma.

PubMed

Araújo, T M; Seabra, A D; Lima, E M; Assumpção, P P; Montenegro, R C; Demachki, S; Burbano, R M; Khayat, A S

2016-01-01

Despite progression in treatment of gastric cancer, prognosis of patients remains poor, in part due to the low rate of diagnosis during its early stages. This paradigm implies the necessity to identify molecular biomarkers for early gastric cancer diagnosis, as well as for disease monitoring, thus contributing to the development of new therapeutic approaches. In a previous study, performed by array-Comparative Genomic Hybridization, we described for the first time in literature recurrent amplification of RTEL1 and ABCA13 genes in gastric cancer. Thus, the aim of the present study was to validate recurrent amplification of RTEL1 and ABCA13 genes and associate CNV status with clinicopathological data. Results showed RTEL1 and ABCA13 amplification in 38 % of samples. Statistical analysis demonstrated that RTEL amplification is more common in older patients and more associated with intestinal type and ABCA13 amplification increases the risk of lymph node metastasis and is more common in men. Co-amplification of these genes showed a significant association with advanced staging. aCGH is a very useful tool for investigating novel genes associated with carcinogenesis and RTEL1 amplification may be important for the development of gastric cancer in older patients, besides being a probable event contributing for chromosomal instability in intestinal gastric carcinogenesis. ABCA13 amplification may have age-specific function and could be considered a useful marker for predicting lymph node metastasis in resected gastric cancer patients in early stage. Lastly, RTEL1 and ABCA13 synergistic effect may be considered as a putative marker for advanced staging in gastric cancer patients.

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells.

PubMed

Zhang, Huijie; Oellers, Tobias; Feng, Wenqian; Abdulazim, Tarik; Saw, En Ning; Ludwig, Alfred; Levkin, Pavel A; Plumeré, Nicolas

2017-06-06

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

Peptides having reduced toxicity that stimulate cholesterol efflux

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

Bielicki, John K.; Johansson, Jan; Danho, Waleed

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Puerarin promotes ABCA1-mediated cholesterol efflux and decreases cellular lipid accumulation in THP-1 macrophages.

PubMed

Li, Cong-Hui; Gong, Duo; Chen, Ling-Yan; Zhang, Min; Xia, Xiao-Dan; Cheng, Hai-Peng; Huang, Chong; Zhao, Zhen-Wang; Zheng, Xi-Long; Tang, Xiao-Er; Tang, Chao-Ke

2017-09-15

It was reported that puerarin decreases the total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and increases high-density lipoprotein cholesterol (HDL-C) level, but the underlying mechanism is unclear. This study was designed to determine whether puerarin decreased lipid accumulation via up-regulation of ABCA1-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. Our results showed that puerarin significantly promoted the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via the AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor-alpha (LXR-α) pathway and decreased cellular lipid accumulation in human THP-1 macrophage-derived foam cells. The miR-7 directly targeted 3' untranslated region of STK11 (Serine/Threonine Kinase 11), which activated the AMPK pathway. Transfection with miR-7 mimic significantly reduced STK11 expression in puerarin-treated macrophages, decreased the phosphorylation of AMPK, down-regulated the expression of the PPARγ-LXR-α-ABCA1 expression. Additionally, treatment with miR-7 decreased cholesterol efflux and increased cholesterol levels in THP-1 macrophage-derived foam cells. Our study demonstrates that puerarin promotes ABCA1-mediated cholesterol efflux and decreases intracellular cholesterol levels through the pathway involving miR-7, STK11, and the AMPK-PPARγ-LXR-α-ABCA1 cascade. Copyright © 2017 Elsevier B.V. All rights reserved.

Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

PubMed Central

Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

2008-01-01

We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promising drug candidates. These molecules, in conjunction with triplex forming oligonucleotides, could potentially be used to achieve control of gene expression by interfering with transcription factors that bind to DNA. Therefore, the ability to screen for these molecules in a high-throughput fashion could dramatically improve the drug screening process. The assay reported here provides excellent discrimination between strong, intermediate, and weak duplex and triplex DNA binders in a high-throughput fashion. PMID:17614366

Novel Mutation in the ATP-Binding Cassette Transporter A3 (ABCA3) Encoding Gene Causes Respiratory Distress Syndrome in A Term Newborn in Southwest Iran

PubMed Central

Rezaei, Farideh; Shafiei, Mohammad; Shariati, Gholamreza; Dehdashtian, Ali; Mohebbi, Maryam; Galehdari, Hamid

2016-01-01

Introduction ABCA3 glycoprotein belongs to the ATP-binding cassette (ABC) superfamily of transporters, which utilize the energy derived from hydrolysis of ATP for the translocation of a wide variety of substrates across the plasma membrane. Mutations in the ABCA3 gene are knowingly causative for fatal surfactant deficiency, particularly respiratory distress syndrome (RDS) in term babies. Case Presentation In this study, Sanger sequencing of the whole ABCA3 gene (NCBI NM_001089) was performed in a neonatal boy with severe RDS. A homozygous mutation has been identified in the patient. Parents were heterozygous for the same missense mutation GGA > AGA at position 202 in exon 6 of the ABCA3 gene (c.604G > A; p.G202R). Furthermore, 70 normal individuals have been analyzed for the mentioned change with negative results. Conclusions Regarding Human Genome Mutation Database (HGMD) and other literature recherche, the detected change is a novel mutation and has not been reported before. Bioinformatics mutation predicting tools prefer it as pathogenic. PMID:27437095

Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

PubMed

Schäfer-Somi, S; Palme, N

2016-04-01

The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings. © 2016 Blackwell Verlag GmbH.

A new functional membrane protein microarray based on tethered phospholipid bilayers.

PubMed

Chadli, Meriem; Maniti, Ofelia; Marquette, Christophe; Tillier, Bruno; Cortès, Sandra; Girard-Egrot, Agnès

2018-04-30

A new prototype of a membrane protein biochip is presented in this article. This biochip was created by the combination of novel technologies of peptide-tethered bilayer lipid membrane (pep-tBLM) formation and solid support micropatterning. Pep-tBLMs integrating a membrane protein were obtained in the form of microarrays on a gold chip. The formation of the microspots was visualized in real-time by surface plasmon resonance imaging (SPRi) and the functionality of a GPCR (CXCR4), reinserted locally into microwells, was assessed by ligand binding studies. In brief, to achieve micropatterning, P19-4H, a 4 histidine-possessing peptide spacer, was spotted inside microwells obtained on polystyrene-coated gold, and Ni-chelating proteoliposomes were injected into the reaction chamber. Proteoliposome binding to the peptide was based on metal-chelate interaction. The peptide-tethered lipid bilayer was finally obtained by addition of a fusogenic peptide (AH peptide) to promote proteoliposome fusion. The CXCR4 pep-tBLM microarray was characterized by surface plasmon resonance imaging (SPRi) throughout the building-up process. This new generation of membrane protein biochip represents a promising method of developing a screening tool for drug discovery.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Presenting Papers at ABCA Conferences: Opinions and Recommendations.

ERIC Educational Resources Information Center

Mier, Denise; Myers, Robert

1981-01-01

Offers guidelines for preparing and presenting papers at American Business Communication Association (ABCA) conferences. Topics covered include: (1) types of delivery, (2) extemporaneous speaking, and (3) manuscript speaking. (FL)

High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components.

PubMed

San Segundo-Acosta, Pablo; Garranzo-Asensio, María; Oeo-Santos, Carmen; Montero-Calle, Ana; Quiralte, Joaquín; Cuesta-Herranz, Javier; Villalba, Mayte; Barderas, Rodrigo

2018-05-01

Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning. The screening of the phage microarrays with individual sera from allergic patients enabled the identification of 10 and 9 IgE-reactive unique amino acid sequences from olive pollen and mustard seeds, respectively. Five immunoreactive amino acid sequences displayed on phages were selected for their expression as His6-GST tag fusion proteins and validation. After immunological characterization, we assessed the IgE-reactivity of the constructs. Our results show that protein microarrays printed with T7 phages displaying peptides from allergenic sources might be used to identify allergenic components -peptides, proteins or mimotopes- through their screening with specific IgE antibodies from allergic patients. Copyright © 2018 Elsevier B.V. All rights reserved.

ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma

PubMed Central

2013-01-01

Background Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death. Methods Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place. Results Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome. Conclusions ABC transporter inhibitors are already being trialed clinically, with the aim of decreasing chemotherapy resistance. Our findings suggest that the inhibition of ABC transporters could also increase the efficacy of radiation treatment for medulloblastoma patients. Additionally, the finding that certain family members are associated with particular molecular subtypes (most notably high ABCA8 and ABCB4 expression in Sonic Hedgehog pathway driven tumors), along with cell membrane location, suggests ABC transporters are worthy of consideration for the diagnostic classification of medulloblastoma. PMID:24219920

Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

PubMed

Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Novel apo E-derived ABCA1 agonist peptide (CS-6253) promotes reverse cholesterol transport and induces formation of preβ-1 HDL in vitro

DOE PAGES

Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; ...

2015-07-24

Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from themore » carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic.« less

Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro

PubMed Central

Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; Genest, Jacques

2015-01-01

Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic. PMID:26207756

Genetic Variants Associated with Lipid Profiles in Chinese Patients with Type 2 Diabetes

PubMed Central

Xing, Xiaoyan; Zhang, Bo; Zhang, Xuelian; Hong, Jing; Yang, Wenying

2015-01-01

Dyslipidemia is a strong risk factor for cardiovascular disease among patients with type 2 diabetes (T2D). The aim of this study was to identify lipid-related genetic variants in T2D patients of Han Chinese ancestry. Among 4,908 Chinese T2D patients who were not taking lipid-lowering medications, single nucleotide polymorphisms (SNPs) in seven genes previously found to be associated with lipid traits in genome-wide association studies conducted in populations of European ancestry (ABCA1, GCKR, BAZ1B, TOMM40, DOCK7, HNF1A, and HNF4A) were genotyped. After adjusting for multiple covariates, SNPs in ABCA1, GCKR, BAZ1B, TOMM40, and HNF1A were identified as significantly associated with triglyceride levels in T2D patients (P < 0.05). The associations between the SNPs in ABCA1 (rs3890182), GCKR (rs780094), and BAZ1B (rs2240466) remained significant even after correction for multiple testing (P = 8.85×10−3, 7.88×10−7, and 2.03×10−6, respectively). BAZ1B (rs2240466) also was associated with the total cholesterol level (P = 4.75×10−2). In addition, SNP rs157580 in TOMM40 was associated with the low-density lipoprotein cholesterol level (P = 6.94×10−3). Our findings confirm that lipid-related genetic loci are associated with lipid profiles in Chinese patients with type 2 diabetes. PMID:26252223

Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

PubMed

De Silva, Samantha R; Charbel Issa, Peter; Singh, Mandeep S; Lipinski, Daniel M; Barnea-Cramer, Alona O; Walker, Nathan J; Barnard, Alun R; Hankins, Mark W; MacLaren, Robert E

2016-11-01

Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4 -/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4 -/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages

PubMed Central

Wang, Limei; Rotter, Susanne; Ladurner, Angela; Heiss, Elke H.; Oberlies, Nicholas H.; Dirsch, Verena M.; Atanasov, Atanas G.

2016-01-01

Silymarin is a hepatoprotective mixture of flavonolignans and flavonoids extracted from the seeds of milk thistle (Silybum marianum L. Gaertn). This study investigates the effect of major bioactive constituents from silymarin, silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, isosilychristin, and taxifolin, on the expression of ABCA1, an important cholesterol efflux transporter, in THP-1-derived macrophages. Four of the studied compounds, isosilybin A, silybin B, silychristin and isosilychristin, were found to significantly induce ABCA1 protein expression without affecting cell viability. Moreover, isosilybin A, a partial PPARγ agonist, was found to promote cholesterol efflux from THP-1 macrophages in a concentration-dependent manner. These findings first show ABCA1 protein up-regulating activity of active constituents of silymarin and provide new avenues for their further study in the context of cardiovascular disease. PMID:26729088

Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages.

PubMed

Wang, Limei; Rotter, Susanne; Ladurner, Angela; Heiss, Elke H; Oberlies, Nicholas H; Dirsch, Verena M; Atanasov, Atanas G

2015-12-31

Silymarin is a hepatoprotective mixture of flavonolignans and flavonoids extracted from the seeds of milk thistle (Silybum marianum L. Gaertn). This study investigates the effect of major bioactive constituents from silymarin, silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, isosilychristin, and taxifolin, on the expression of ABCA1, an important cholesterol efflux transporter, in THP-1-derived macrophages. Four of the studied compounds, isosilybin A, silybin B, silychristin and isosilychristin, were found to significantly induce ABCA1 protein expression without affecting cell viability. Moreover, isosilybin A, a partial PPARγ agonist, was found to promote cholesterol efflux from THP-1 macrophages in a concentration-dependent manner. These findings first show ABCA1 protein up-regulating activity of active constituents of silymarin and provide new avenues for their further study in the context of cardiovascular disease.

Piperine inhibits ABCA1 degradation and promotes cholesterol efflux from THP‐1‐derived macrophages

PubMed Central

Wang, Limei; Palme, Veronika; Rotter, Susanne; Schilcher, Nicole; Cukaj, Malsor; Wang, Dongdong; Ladurner, Angela; Heiss, Elke H.; Stangl, Herbert; Dirsch, Verena M.

2016-01-01

1 Scope Increased macrophage cholesterol efflux (ChE) is considered to have anti‐atherosclerotic effect counteracting cardiovascular disease. The principle pungent ingredient of the fruits of Piper nigrum, piperine, is identified in this study as a ChE inducer in THP‐1‐derived macrophages, and mechanisms underlying this effect are explored. 2 Methods and results Without affecting cell viability, piperine concentration‐dependently enhances ChE in THP‐1‐derived macrophages from 25 to 100 μM. The expression level of the key cholesterol transporter protein ATP‐binding cassette transporter A1 (ABCA1) is significantly upregulated by piperine, as revealed by western blot analyses. However, two other ChE‐mediating transporter proteins, ATP‐binding cassette transporter G1 (ABCG1) and scavenger receptor class B member 1 (SR‐B1), remain unaffected. Piperine exerts no influence on ABCA1 mRNA levels, but significantly inhibits the degradation of ABCA1, as evident by an increased half‐life of the protein in the presence of cycloheximide. Furthermore, it is found that piperine likely interferes with the calpain‐mediated ABCA1 degradation pathway and exhibits significant inhibition of calpain activity. 3 Conclusion Our findings suggest that piperine promotes ChE in THP‐1‐derived macrophages by upregulation of ABCA1, which might be mediated by inhibition of calpain activity. This novel bioactivity makes the dietary constituent piperine a good candidate to be further explored for therapeutic or preventive applications in the context of atherosclerosis. PMID:27862930

The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams in Staphylococcus aureus▿

PubMed Central

Truong-Bolduc, Que Chi; Hooper, David C.

2007-01-01

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four β-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA. PMID:17277059

Microelectroporation device for genomic screening

DOEpatents

Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

2014-09-09

We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages

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

Ohara, Kazuaki, E-mail: Kazuaki_Ohara@kirin.co.jp; Wakabayashi, Hideyuki; Taniguchi, Yoshimasa

Highlights: •The major circulating quercetin metabolite (Q3GA) activated LXRα. •Q3GA induced ABCA1 via LXRα activation in macrophages. •Nelumbo nucifera leaf extracts contained quercetin glycosides. •N. nucifera leaf extract feeding elevated HDLC in mice. -- Abstract: Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found thatmore » quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.« less

Cholesterol Accumulation in Dendritic Cells Links the Inflammasome to Acquired Immunity.

PubMed

Westerterp, Marit; Gautier, Emmanuel L; Ganda, Anjali; Molusky, Matthew M; Wang, Wei; Fotakis, Panagiotis; Wang, Nan; Randolph, Gwendalyn J; D'Agati, Vivette D; Yvan-Charvet, Laurent; Tall, Alan R

2017-06-06

Autoimmune diseases such as systemic lupus erythematosus (SLE) are associated with increased cardiovascular disease and reduced plasma high-density lipoprotein (HDL) levels. HDL mediates cholesterol efflux from immune cells via the ATP binding cassette transporters A1 and G1 (ABCA1/G1). The significance of impaired cholesterol efflux pathways in autoimmunity is unknown. We observed that Abca1/g1-deficient mice develop enlarged lymph nodes (LNs) and glomerulonephritis suggestive of SLE. This lupus-like phenotype was recapitulated in mice with knockouts of Abca1/g1 in dendritic cells (DCs), but not in macrophages or T cells. DC-Abca1/g1 deficiency increased LN and splenic CD11b + DCs, which displayed cholesterol accumulation and inflammasome activation, increased cell surface levels of the granulocyte macrophage-colony stimulating factor receptor, and enhanced inflammatory cytokine secretion. Consequently, DC-Abca1/g1 deficiency enhanced T cell activation and T h 1 and T h 17 cell polarization. Nlrp3 inflammasome deficiency diminished the enlarged LNs and enhanced T h 1 cell polarization. These findings identify an essential role of DC cholesterol efflux pathways in maintaining immune tolerance. Copyright © 2017 Elsevier Inc. All rights reserved.

APPLICATION OF CDNA MICROARRAY TECHNOLOGY TO IN VITRO TOXICOLOGY AND THE SELECTION OF GENES FOR A REAL TIME RT-PCR-BASED SCREEN FOR OXIDATIVE STRESS IN HEP-G2 CELLS

EPA Science Inventory

Large-scale analysis of gene expression using cDNA microarrays promises the
rapid detection of the mode of toxicity for drugs and other chemicals. cDNA
microarrays were used to examine chemically-induced alterations of gene
expression in HepG2 cells exposed to oxidative ...

Controlling false-negative errors in microarray differential expression analysis: a PRIM approach.

PubMed

Cole, Steve W; Galic, Zoran; Zack, Jerome A

2003-09-22

Theoretical considerations suggest that current microarray screening algorithms may fail to detect many true differences in gene expression (Type II analytic errors). We assessed 'false negative' error rates in differential expression analyses by conventional linear statistical models (e.g. t-test), microarray-adapted variants (e.g. SAM, Cyber-T), and a novel strategy based on hold-out cross-validation. The latter approach employs the machine-learning algorithm Patient Rule Induction Method (PRIM) to infer minimum thresholds for reliable change in gene expression from Boolean conjunctions of fold-induction and raw fluorescence measurements. Monte Carlo analyses based on four empirical data sets show that conventional statistical models and their microarray-adapted variants overlook more than 50% of genes showing significant up-regulation. Conjoint PRIM prediction rules recover approximately twice as many differentially expressed transcripts while maintaining strong control over false-positive (Type I) errors. As a result, experimental replication rates increase and total analytic error rates decline. RT-PCR studies confirm that gene inductions detected by PRIM but overlooked by other methods represent true changes in mRNA levels. PRIM-based conjoint inference rules thus represent an improved strategy for high-sensitivity screening of DNA microarrays. Freestanding JAVA application at http://microarray.crump.ucla.edu/focus

Microarray platform affords improved product analysis in mammalian cell growth studies

PubMed Central

Li, Lingyun; Migliore, Nicole; Schaefer, Eugene; Sharfstein, Susan T.; Dordick, Jonathan S.; Linhardt, Robert J.

2014-01-01

High throughput (HT) platforms serve as cost-efficient and rapid screening method for evaluating the effect of cell culture conditions and screening of chemicals. The aim of the current study was to develop a high-throughput cell-based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/MSX CHO cell line, which produces a therapeutic monoclonal antibody, was examined using microarray system in conjunction with conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60 nl spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. Results show the non-proprietary medium developed allows cell growth, production and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base media results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the high-throughput microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as, cell growth, metabolism and productivity. PMID:24227746

Associations Between Genetic Variants in 19p13 and 19q13 Regions and Susceptibility to Alzheimer Disease: A Meta-Analysis.

PubMed

Bao, Jie; Wang, Xiao-jie; Mao, Zong-fu

2016-01-22

Alzheimer disease (AD) has become an epidemic within the growing elderly population and effective therapies of AD have not been discovered. Genetic factors accounted for over 70% of the incidence of AD and the disease-related polymorphisms are located on chromosome 19, which is one of several prominent chromosomes related to the development of AD. Many inconsistent associations between polymorphisms in ABCA7, CD33, and TOMM40 genes and the susceptibility to AD have been suggested by several independent studies. A comprehensive literature search for studies involving the association between gene polymorphisms and AD was performed, and we finally selected 3 genes (4 polymorphisms) for the meta-analysis: ABCA7 (rs3764650), CD33 (rs3865444), and TOMM40 (rs157580, rs2075650). A total of 25 articles investigating 3 genes (4 polymorphisms) were included in the meta-analysis. The pooled results of 4 polymorphisms were all significantly associated with the susceptibility to AD. The pooled effect of ABCA7 rs3764605 allele G was significantly associated with an increased the risk of AD (OR=1.20, 95% CI: 1.14-1.26, P value <0.001). Similarly, our evidence suggested that allele A of TOMM40 rs2075650 polymorphism was a risk factor for AD (OR=2.87, 95% CI: 2.46-3.34, P value <0.001). Alleles A of CD33 rs3865444 and A of TOMM40 rs157580 were both protective factors for AD onset (OR=0.94, 95% CI: 0.90-0.98, P value=0.003; OR=0.62, 95% CI: 0.57-0.66, P value <0.001). CONCLUSIONS" Results from the meta-analysis revealed that the pooled ABCA7 rs376465, CD33 rs3865444, TOMM40 rs157580, and rs2075650 variants were significantly associated with the susceptibility to AD. However, the association differed significantly between Asian and Caucasian groups for SNPs of CD33 rs3865444, TOMM40 rs157580, and rs2075650.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications.

PubMed

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-09-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

PubMed Central

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

PubMed

Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

2017-09-05

Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

PubMed

Aleidi, Shereen M; Yang, Alryel; Sharpe, Laura J; Rao, Geetha; Cochran, Blake J; Rye, Kerry-Anne; Kockx, Maaike; Brown, Andrew J; Gelissen, Ingrid C

2018-04-01

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated. Copyright © 2018 Elsevier B.V. All rights reserved.

ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells.

PubMed

Reddy, Srinivasa T; Hama, Susan; Ng, Carey; Grijalva, Victor; Navab, Mohamad; Fogelman, Alan M

2002-11-01

We have previously reported that products of the lipoxygenase pathway, hydroperoxyoctadecadienoic acid and hydroperoxyeicosatetraenoic acid, as well as cholesterol linoleate hydroperoxides, collectively termed seeding molecules, are removed by apolipoprotein A-I (apoA-I) from the artery wall cells and render low density lipoprotein (LDL) resistant to oxidation by human artery wall cells. The mechanisms by which oxidized lipids are transported and/or transferred to lipoproteins and the pathways by which apoA-I facilitates their removal remain unclear. ATP-binding cassette transporter 1 (ABCA1) is known to facilitate the release of cellular phospholipids and cholesterol from the plasma membrane to apoA-I and high density lipoprotein. Therefore, we evaluated whether ABCA1 participates in LDL oxidation. In this report, we show that (1) chemical inhibitors of ABCA1 function, glyburide and DIDS, block artery wall cell-mediated oxidative modification of LDL, (2) inhibition of ABCA1 with the use of antisense (but not sense) oligonucleotides prevents LDL-induced lipid hydroperoxide formation and LDL-induced monocyte chemotactic activity by the artery wall cells, and (3) oxysterols that induce ABCA1 expression, such as 22(R)hydroxycholesterol, enhance cell-mediated LDL oxidation. Furthermore, we also show that 22(R)hydroxycholesterol induces the production of reactive oxygen species in the artery wall cells, which can be removed by incubating the artery wall cells with apoA-I. Our data suggest that ABCA1 plays an important role in artery wall cell-mediated modification/oxidation of LDL by modulating the release of reactive oxygen species from artery wall cells that are necessary for LDL oxidation.

Relationship of students' conceptual representations and problem-solving abilities in acid-base chemistry

NASA Astrophysics Data System (ADS)

Powers, Angela R.

2000-10-01

This study explored the relationship between secondary chemistry students' conceptual representations of acid-base chemistry, as shown in student-constructed concept maps, and their ability to solve acid-base problems, represented by their score on an 18-item paper and pencil test, the Acid-Base Concept Assessment (ABCA). The ABCA, consisting of both multiple-choice and short-answer items, was originally designed using a question-type by subtopic matrix, validated by a panel of experts, and refined through pilot studies and factor analysis to create the final instrument. The concept map task included a short introduction to concept mapping, a prototype concept map, a practice concept-mapping activity, and the instructions for the acid-base concept map task. The instruments were administered to chemistry students at two high schools; 108 subjects completed both instruments for this study. Factor analysis of ABCA results indicated that the test was unifactorial for these students, despite the intention to create an instrument with multiple "question-type" scales. Concept maps were scored both holistically and by counting valid concepts. The two approaches were highly correlated (r = 0.75). The correlation between ABCA score and concept-map score was 0.29 for holistically-scored concept maps and 0.33 for counted-concept maps. Although both correlations were significant, they accounted for only 8.8 and 10.2% of variance in ABCA scores, respectively. However, when the reliability of the instruments used is considered, more than 20% of the variance in ABCA scores may be explained by concept map scores. MANOVAs for ABCA and concept map scores by instructor, student gender, and year in school showed significant differences for both holistic and counted concept-map scores. Discriminant analysis revealed that the source of these differences was the instruction variable. Significant differences between classes receiving different instruction were found in the frequency of concepts listed by students for 9 of 10 concepts evaluated. Mean ABCA scores did not differ significantly between the two instruction groups. The results of this study failed to provide evidence of conceptual distinctions among different "types" of problem-solving items. The results suggested that several factors influence success in chemistry problem solving, including concept knowledge and organization. Further research into the nature of chemistry problems and problem solving is recommended.

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

PubMed Central

Halder, Sujata; Cotmore, Susan; Heimburg-Molinaro, Jamie; Smith, David F.; Cummings, Richard D.; Chen, Xi; Trollope, Alana J.; North, Simon J.; Haslam, Stuart M.; Dell, Anne; Tattersall, Peter; McKenna, Robert; Agbandje-McKenna, Mavis

2014-01-01

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen. PMID:24475195

A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

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

Varnum, Susan M.

An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

Analysis of the ABCR (ABCA4) gene in 4-aminoquinoline retinopathy: is retinal toxicity by chloroquine and hydroxychloroquine related to Stargardt disease?

PubMed

Shroyer, N F; Lewis, R A; Lupski, J R

2001-06-01

To determine if mutations in ABCR (ABCA4) are associated with chloroquine/hydroxychloroquine retinopathy. DNA from eight patients with chloroquine or hydroxychloroquine retinopathy was studied. Controls were 80 individuals over age 65 years with normal retinal examinations. Ophthalmoscopy, color vision testing, visual fields, retinal photography, and fluorescein angiography were performed on the eight patients. Direct DNA sequencing of the exons and flanking intronic regions of the ABCR gene was completed for all patients. Clinical evaluation confirmed the diagnosis of chloroquine/hydroxychloroquine retinopathy and excluded Stargardt disease in each patient. Two patients had heterozygous ABCR missense mutations previously associated with Stargardt disease. None of the controls had these missense mutations. Three other patients had other missense polymorphisms. Some individuals who have ABCR mutations may be predisposed to develop retinal toxicity when exposed to chloroquine/hydroxychloroquine. We urge further study of a larger cohort of patients with chloroquine/hydroxychloroquine retinopathy.

Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

PubMed

Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

2016-10-01

High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH 2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Association of Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 with pathological diagnosis of Alzheimer disease.

PubMed

Yu, Lei; Chibnik, Lori B; Srivastava, Gyan P; Pochet, Nathalie; Yang, Jingyun; Xu, Jishu; Kozubek, James; Obholzer, Nikolaus; Leurgans, Sue E; Schneider, Julie A; Meissner, Alexander; De Jager, Philip L; Bennett, David A

2015-01-01

Recent large-scale genome-wide association studies have discovered several genetic variants associated with Alzheimer disease (AD); however, the extent to which DNA methylation in these AD loci contributes to the disease susceptibility remains unknown. To examine the association of brain DNA methylation in 28 reported AD loci with AD pathologies. Ongoing community-based clinical pathological cohort studies of aging and dementia (the Religious Orders Study and the Rush Memory and Aging Project) among 740 autopsied participants 66.0 to 108.3 years old. DNA methylation levels at individual CpG sites generated from dorsolateral prefrontal cortex tissue using a bead assay. Pathological diagnosis of AD by National Institute on Aging-Reagan criteria following a standard postmortem examination. Overall, 447 participants (60.4%) met the criteria for pathological diagnosis of AD. Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 was associated with pathological AD. The association was robustly retained after replacing the binary trait of pathological AD with 2 quantitative and molecular specific hallmarks of AD, namely, Aβ load and paired helical filament tau tangle density. Furthermore, RNA expression of transcripts of SORL1 and ABCA7 was associated with paired helical filament tau tangle density, and the expression of BIN1 was associated with Aβ load. Brain DNA methylation in multiple AD loci is associated with AD pathologies. The results provide further evidence that disruption of DNA methylation is involved in the pathological process of AD.

Spectrum of ABCA4 (ABCR) gene mutations in Spanish patients with autosomal recessive macular dystrophies.

PubMed

Paloma, E; Martínez-Mir, A; Vilageliu, L; Gonzàlez-Duarte, R; Balcells, S

2001-06-01

The ABCA4 gene has been involved in several forms of inherited macular dystrophy. In order to further characterize the complex genotype-phenotype relationships involving this gene, we have performed a mutation analysis of ABCA4 in 14 Spanish patients comprising eight STGD (Stargardt), four FFM (fundus flavimaculatus), and two CRD (Cone-rod dystrophy) patients. SSCP (single-strand conformation polymorphism) analysis and DNA sequencing of the coding and 5' upstream regions of this gene allowed the identification of 16 putatively pathogenic alterations, nine of which are novel. Most of these were missense changes, and no patient was found to carry two null alleles. Overall, the new data agree with a working model relating the different pathogenic phenotypes to the severity of the mutations. When considering the information presented here together with that of previous reports, a picture of the geographic distribution of three particular mutations emerges. The R212C change has been found in French, Italian, Dutch, German, and Spanish but not in British patients. In the Spanish collection, R212C was found in a CRD patient, indicating that it may be a rather severe change. In contrast, c.2588G>C, a very common mild allele in the Dutch population, is rarely found in Southern Europe. Interestingly, the c.2588G>C mutation has been found in a double mutant allele together with the missense R1055W. Finally, the newly described L1940P was found in two unrelated Spanish patients, and may be a moderate to severe allele. Copyright 2001 Wiley-Liss, Inc.

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

PubMed Central

Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

2016-01-01

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. PMID:26497474

A survey of ABCA1 sequence variation confirms association with dementia

PubMed Central

Reynolds, Chandra A.; Hong, Mun-Gwan; Eriksson, Ulrika K.; Blennow, Kaj; Bennet, Anna M.; Johansson, Boo; Malmberg, Bo; Berg, Stig; Wiklund, Fredrik; Gatz, Margaret; Pedersen, Nancy L.; Prince, Jonathan A.

2009-01-01

We and others have conducted targeted genetic association analyses of ABCA1 in relation to Alzheimer disease risk with a resultant mixture of both support and refutation, but all previous studies have been based upon only a few markers. Here, a detailed survey of genetic variation in the ABCA1 region has been performed in a total of 1567 Swedish dementia cases (including 1275 with Alzheimer disease) and 2203 controls, providing evidence of association with maximum significance at marker rs2230805 (OR = 1.39; 95% CI 1.23–1.57, P = 7.7 × 10−8). Haplotype-based tests confirmed association of this genomic region after excluding rs2230805, and imputation did not reveal additional markers with greater support. Significantly associating markers reside in two distinct linkage disequilibrium blocks with maxima near the promoter and in the terminal exon of a truncated ABCA1 splice-form. The putative risk allele of rs2230805 was also found to be associated with reduced cerebrospinal fluid levels of β-amyloid. The strongest evidence of association was obtained when all forms of dementia were considered together, but effect sizes were similar when only confirmed Alzheimer disease cases were assessed. Results further implicate ABCA1 in dementia, reinforcing the putative involvement of lipid transport in neurodegenerative disease. PMID:19606474

ABCA1 gene variants regulate postprandial lipid metabolism in healthy men

PubMed Central

Delgado-Lista, Javier; Perez-Martinez, Pablo; Perez-Jimenez, Francisco; Garcia-Rios, Antonio; Fuentes, Francisco; Marin, Carmen; Gómez-Luna, Purificación; Camargo, Antonio; Parnell, Laurence D; Ordovas, Jose Maria; Lopez-Miranda, Jose

2010-01-01

Objective Genetic variants of ABCA1, an ATP-binding cassette (ABC) transporter, have been linked to altered atherosclerosis progression and fasting lipid concentration, mainly high density lipoproteins (HDL) and Apolipoprotein A1 (APOA1), but results from different studies have been inconsistent. Methods and results In order to further characterize the effects of ABCA1 variants in human postprandial lipid metabolism, we studied the influence of three single nucleotide polymorphisms (SNPs) [i27943 (rs2575875); i48168 (rs4149272); R219K (rs2230806)] in the postprandial lipemia of 88 normolipidemic young men, who were given a fatty meal. For i27943 and i48168 SNPs, fasting and postprandial values of APOA1 were higher, and postprandial lipemia was much lower in homozygotes for the major alleles, for total triglycerides in plasma, and large-triglyceride rich lipoproteins (TRL) triglycerides. These persons also showed higher APOA1/APOB ratio. Major allele homozygotes for i48168 and i27943 showed additionally higher HDL and lower postprandial Apolipoprotein B (ApoB). Conclusions Our work shows that major allele homozygotes for ABCA1 SNPs i27943 and i48168 have a lower postprandial response as compared to minor allele carriers. This finding may further characterize the role of ABCA1 in lipid metabolism. PMID:20185793

Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent.

PubMed

Conklin, Chris M J; Craddock, Kenneth J; Have, Cherry; Laskin, Janessa; Couture, Christian; Ionescu, Diana N

2013-01-01

Fluorescence in situ hybridization (FISH) is the standard procedure for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangement in non-small-cell lung carcinoma (NSCLC) but is expensive and time consuming. We tested three antibodies to ALK, using various detection systems, and hypothesized that ALK immunohistochemistry (IHC) may represent a cost-effective and efficient means of screening for ALK rearrangement in NSCLC. We screened 377 stage I or II NSCLC cases in a tissue microarray by FISH and IHC (5A4 [Leica Biosystems Newcastle Ltd, Newcastle upon Tyne, UYnited Kingdom] by Nichirei's N-Histofine ALK detection kit [Nichirei Biosciences inc., Tokyo, Japan], 5A4 by Novocastra with ADVANCE [Dako Canada inc., Burlington, Ontario, Canada], D5F3 by Cell Signaling Technology with ADVANCE [Cell Signalling Technologies inc., Danvers, MA], and DAKO clone ALK1 with FLEX [Dako Canada inc., Burlington, Ontario, Canada] and ADVANCE). IHC was scored as 0, 1+, 2+, or 3+. Possibly positive or positive cases were further analyzed by IHC and FISH on whole section. Tissue microarray results were available on 377 cases by IHC and 273 cases by FISH. Eleven cases were positive or possibly positive by either IHC or FISH, and three cases were positive or possibly positive by both methods. Three cases were ALK-positive by FISH on whole section validation. There was no correlation between semiquantitative IHC score (1+, 2+, 3+) and ALK rearrangement by FISH. D5F3 (Cell Signaling by ADVANCE) and 5A4 (Novocastra by ADVANCE) showed the greatest combination of sensitivity (100%) and specificity (87.5% for 5A4 by Novocastra and 75% for D5F3 by Cell Signaling), and produced no false-negative results. IHC is a reliable screening tool for identification of ALK rearrangement in NSCLC and is antibody dependent. D5F3 (Cell Signaling) and 5A4 (Novocastra) can be used with FISH for identification of IHC-positive cases to reduce screening costs.

Effect of sulfonylurea agents on reverse cholesterol transport in vitro and vivo.

PubMed

Terao, Yoshio; Ayaori, Makoto; Ogura, Masatsune; Yakushiji, Emi; Uto-Kondo, Harumi; Hisada, Tetsuya; Ozasa, Hideki; Takiguchi, Shunichi; Nakaya, Kazuhiro; Sasaki, Makoto; Komatsu, Tomohiro; Iizuka, Maki; Horii, Shunpei; Mochizuki, Seibu; Yoshimura, Michihiro; Ikewaki, Katsunori

2011-01-01

Reverse cholesterol transport (RCT) is a critical mechanism for the anti-atherogenic property of HDL. The inhibitory effect of the sulfonylurea agent (SUA) glibenclamide on ATP binding-cassette transporter (ABC) A1 may decrease HDL function but it remains unclear whether it attenuates RCT in vivo. We therefore investigated how the SUAs glibenclamide and glimepiride affected the functionality of ABCA1/ABCG1 and scavenger receptor class B type I (SR-BI) expression in macrophages in vitro and overall RCT in vivo. RAW264.7, HEK293 and BHK-21 cells were used for in vitro studies. To investigate RCT in vivo, 3H-cholesterol-labeled and acetyl LDL-loaded RAW264.7 cells were injected into mice. High dose (500µM) of glibenclamide inhibited ABCA1 function and apolipoprotein A-I (apoA-I)-mediated cholesterol efflux, and attenuated ABCA1 expression. Although glimepiride maintained apoA-I-mediated cholesterol efflux from RAW264.7 cells, like glibenclamide, it inhibited ABCA1-mediated cholesterol efflux from transfected HEK293 cells. Similarly, the SUAs inhibited SR-BI-mediated cholesterol efflux from transfected BHK-21 cells. High doses of SUAs increased ABCG1 expression in RAW264.7 cells, promoting HDL-mediated cholesterol efflux in an ABCG1-independent manner. Low doses (0.1-100 µM) of SUAs did not affect cholesterol efflux from macrophages despite dose-dependent increases in ABCA1/G1 expression. Furthermore, they did not change RCT or plasma lipid levels in mice. High doses of SUAs inhibited the functionality of ABCA1/SR-BI, but not ABCG1. At lower doses, they had no unfavorable effects on cholesterol efflux or overall RCT in vivo. These results indicate that SUAs do not have adverse effects on atherosclerosis contrary to previous findings for glibenclamide.

Neopterin negatively regulates expression of ABCA1 and ABCG1 by the LXRα signaling pathway in THP-1 macrophage-derived foam cells.

PubMed

Yan, Jin-quan; Tan, Chun-zhi; Wu, Jin-hua; Zhang, Dong-cui; Chen, Ji-ling; Zeng, Bin-yuan; Jiang, Yu-ping; Nie, Jin; Liu, Wei; Liu, Qin; Dai, Hao

2013-07-01

To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis.

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

Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.

Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from themore » carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic.« less

MicroRNA-19b promotes macrophage cholesterol accumulation and aortic atherosclerosis by targeting ATP-binding cassette transporter A1.

PubMed

Lv, Yun-Cheng; Tang, Yan-Yan; Peng, Juan; Zhao, Guo-Jun; Yang, Jing; Yao, Feng; Ouyang, Xin-Ping; He, Ping-Ping; Xie, Wei; Tan, Yu-Lin; Zhang, Min; Liu, Dan; Tang, Deng-Pei; Cayabyab, Francisco S; Zheng, Xi-Long; Zhang, Da-Wei; Tian, Guo-Ping; Tang, Chao-Ke

2014-09-01

Macrophage accumulation of cholesterol leads to foam cell formation which is a major pathological event of atherosclerosis. Recent studies have shown that microRNA (miR)-19b might play an important role in cholesterol metabolism and atherosclerotic diseases. Here, we have identified miR-19b binding to the 3'UTR of ATP-binding cassette transporter A1 (ABCA1) transporters, and further determined the potential roles of this novel interaction in atherogenesis. To investigate the molecular mechanisms involved in a miR-19b promotion of macrophage cholesterol accumulation and the development of aortic atherosclerosis. We performed bioinformatics analysis using online websites, and found that miR-19b was highly conserved during evolution and directly bound to ABCA1 mRNA with very low binding free energy. Luciferase reporter assay confirmed that miR-19b bound to 3110-3116 sites within ABCA1 3'UTR. MiR-19b directly regulated the expression levels of endogenous ABCA1 in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by qRT-PCR and western blot. Cholesterol transport assays revealed that miR-19b dramatically suppressed apolipoprotein AI-mediated ABCA1-dependent cholesterol efflux, resulting in the increased levels of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) as revealed by HPLC. The excretion of (3)H-cholesterol originating from cholesterol-laden MPMs into feces was decreased in mice overexpressing miR-19b. Finally, we evaluated the proatherosclerotic role of miR-19b in apolipoprotein E deficient (apoE(-/-)) mice. Treatment with miR-19b precursor reduced plasma high-density lipoprotein (HDL) levels, but increased plasma low-density lipoprotein (LDL) levels. Consistently, miR-19b precursor treatment increased aortic plaque size and lipid content, but reduced collagen content and ABCA1 expression. In contrast, treatment with the inhibitory miR-19b antisense oligonucleotides (ASO) prevented or reversed these effects. MiR-19b promotes macrophage cholesterol accumulation, foam cell formation and aortic atherosclerotic development by targeting ABCA1. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Galectins are human milk glycan receptors

PubMed Central

Noll, Alexander J; Gourdine, Jean-Philippe; Yu, Ying; Lasanajak, Yi; Smith, David F; Cummings, Richard D

2016-01-01

The biological recognition of human milk glycans (HMGs) is poorly understood. Because HMGs are rich in galactose we explored whether they might interact with human galectins, which bind galactose-containing glycans and are highly expressed in epithelial cells and other cell types. We screened a number of human galectins for their binding to HMGs on a shotgun glycan microarray consisting of 247 HMGs derived from human milk, as well as to a defined HMG microarray. Recombinant human galectins (hGal)-1, -3, -4, -7, -8 and -9 bound selectively to glycans, with each galectin recognizing a relatively unique binding motif; by contrast hGal-2 did not recognize HMGs, but did bind to the human blood group A Type 2 determinants on other microarrays. Unlike other galectins, hGal-7 preferentially bound to glycans expressing a terminal Type 1 (Galβ1-3GlcNAc) sequence, a motif that had eluded detection on non-HMG glycan microarrays. Interactions with HMGs were confirmed in a solution setting by isothermal titration microcalorimetry and hapten inhibition experiments. These results demonstrate that galectins selectively bind to HMGs and suggest the possibility that galectin–HMG interactions may play a role in infant immunity. PMID:26747425

Droplet Microarray Based on Patterned Superhydrophobic Surfaces Prevents Stem Cell Differentiation and Enables High-Throughput Stem Cell Screening.

PubMed

Tronser, Tina; Popova, Anna A; Jaggy, Mona; Bastmeyer, Martin; Levkin, Pavel A

2017-12-01

Over the past decades, stem cells have attracted growing interest in fundamental biological and biomedical research as well as in regenerative medicine, due to their unique ability to self-renew and differentiate into various cell types. Long-term maintenance of the self-renewal ability and inhibition of spontaneous differentiation, however, still remain challenging and are not fully understood. Uncontrolled spontaneous differentiation of stem cells makes high-throughput screening of stem cells also difficult. This further hinders investigation of the underlying mechanisms of stem cell differentiation and the factors that might affect it. In this work, a dual functionality of nanoporous superhydrophobic-hydrophilic micropatterns is demonstrated in their ability to inhibit differentiation of mouse embryonic stem cells (mESCs) and at the same time enable formation of arrays of microdroplets (droplet microarray) via the effect of discontinuous dewetting. Such combination makes high-throughput screening of undifferentiated mouse embryonic stem cells possible. The droplet microarray is used to investigate the development, differentiation, and maintenance of stemness of mESC, revealing the dependence of stem cell behavior on droplet volume in nano- and microliter scale. The inhibition of spontaneous differentiation of mESCs cultured on the droplet microarray for up to 72 h is observed. In addition, up to fourfold increased cell growth rate of mESCs cultured on our platform has been observed. The difference in the behavior of mESCs is attributed to the porosity and roughness of the polymer surface. This work demonstrates that the droplet microarray possesses the potential for the screening of mESCs under conditions of prolonged inhibition of stem cells' spontaneous differentiation. Such a platform can be useful for applications in the field of stem cell research, pharmacological testing of drug efficacy and toxicity, biomedical research as well as in the field of regenerative medicine and tissue engineering. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Screening Mammalian Cells on a Hydrogel: Functionalized Small Molecule Microarray.

PubMed

Zhu, Biwei; Jiang, Bo; Na, Zhenkun; Yao, Shao Q

2017-01-01

Mammalian cell-based microarray technology has gained wide attention, for its plethora of promising applications. The platform is able to provide simultaneous information on multiple parameters for a given target, or even multiple target proteins, in a complex biological system. Here we describe the preparation of mammalian cell-based microarrays using selectively captured of human prostate cancer cells (PC-3). This platform was then used in controlled drug release and measuring the associated drug effects on these cancer cells.

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis.

PubMed

Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

2016-01-01

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

A low density microarray method for the identification of human papillomavirus type 18 variants.

PubMed

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C

2013-09-26

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.

A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

PubMed Central

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

2013-01-01

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

Deciphering the glycosaminoglycan code with the help of microarrays.

PubMed

de Paz, Jose L; Seeberger, Peter H

2008-07-01

Carbohydrate microarrays have become a powerful tool to elucidate the biological role of complex sugars. Microarrays are particularly useful for the study of glycosaminoglycans (GAGs), a key class of carbohydrates. The high-throughput chip format enables rapid screening of large numbers of potential GAG sequences produced via a complex biosynthesis while consuming very little sample. Here, we briefly highlight the most recent advances involving GAG microarrays built with synthetic or naturally derived oligosaccharides. These chips are powerful tools for characterizing GAG-protein interactions and determining structure-activity relationships for specific sequences. Thereby, they contribute to decoding the information contained in specific GAG sequences.

Using Microarrays to Facilitate Positional Cloning: Identification of Tomosyn as an Inhibitor of Neurosecretion

PubMed Central

Dybbs, Michael; Ngai, John; Kaplan, Joshua M

2005-01-01

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning. PMID:16103915

Cone Photoreceptor Abnormalities Correlate with Vision Loss in Patients with Stargardt Disease

PubMed Central

Chen, Yingming; Ratnam, Kavitha; Sundquist, Sanna M.; Lujan, Brandon; Ayyagari, Radha; Gudiseva, V. Harini; Roorda, Austin

2011-01-01

Purpose. To study the relationship between macular cone structure, fundus autofluorescence (AF), and visual function in patients with Stargardt disease (STGD). Methods. High-resolution images of the macula were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography in 12 patients with STGD and 27 age-matched healthy subjects. Measures of retinal structure and AF were correlated with visual function, including best-corrected visual acuity, color vision, kinetic and static perimetry, fundus-guided microperimetry, and full-field electroretinography. Mutation analysis of the ABCA4 gene was completed in all patients. Results. Patients were 15 to 55 years old, and visual acuity ranged from 20/25–20/320. Central scotomas were present in all patients, although the fovea was spared in three patients. The earliest cone spacing abnormalities were observed in regions of homogeneous AF, normal visual function, and normal outer retinal structure. Outer retinal structure and AF were most normal near the optic disc. Longitudinal studies showed progressive increases in AF followed by reduced AF associated with losses of visual sensitivity, outer retinal layers, and cones. At least one disease-causing mutation in the ABCA4 gene was identified in 11 of 12 patients studied; 1 of 12 patients showed no disease-causing ABCA4 mutations. Conclusions. AOSLO imaging demonstrated abnormal cone spacing in regions of abnormal fundus AF and reduced visual function. These findings provide support for a model of disease progression in which lipofuscin accumulation results in homogeneously increased AF with cone spacing abnormalities, followed by heterogeneously increased AF with cone loss, then reduced AF with cone and RPE cell death. (ClinicalTrials.gov number, NCT00254605.) PMID:21296825

Heart ABCA1 and PPAR- α Genes Expression Responses in Male rats: Effects of High Intensity Treadmill Running Training and Aqueous Extraction of Black Crataegus-Pentaegyna.

PubMed

Ghanbari-Niaki, Abbass; Ghanbari-Abarghooi, Safieyh; Rahbarizadeh, Fatemeh; Zare-Kookandeh, Navabeh; Gholizadeh, Monireh; Roudbari, Fatemeh; Zare-Kookandeh, Asghar

2013-11-01

Heart as a high metabolic and aerobic tissue is consuming lipid as a fuel for its energy provision at rest during light and moderate exercise, except when lactate level is higher in blood circulation. It has been shown that any type of regular exercise and crataegus species would improve cardiovascular function and minimizes several risk factors via stimulating lipid metabolism by acting on enzymes and genes expression such as ABCA1 and PPAR α which are involving in this process. Twenty Wistar male rats (4-6 weeks old, 140-173 g weight) were used. Animals were randomly classified into training (n = 10) and control (n = 10) groups and then divided into saline-control (SC), saline-training (ST), Crataegus-Pentaegyna -control (CPC), and Crataegus-Pentaegyna -training (CPT) groups. Training groups have performed a high-intensity running program (at 34 m/min (0% grade), 60 min/day, 5 days/week) on a motor-driven treadmill for eight weeks. Animals were orally fed with Crataegus-Pentaegyna extraction (500mg/kg) and saline solution for six weeks. Seventy- two hours after the last training session, rats were sacrificed, hearts were excised, cleaned and immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Plasma also was collected for plasma variable measurements. Statistical analysis was performed using a two way analysis of variance, and significance was accepted at P < 0.05. A non-significant (P < 0.4, P < 0.79, respectively) increase in ABCA1 and PPAR α genes expression was accompanied by a significant (P < 0.01, P < 0.04, P < 0.04, respectively) reduction in TC, TG, and VLDL-C levels in Crataegus-Pentaegyna groups. Our findings show that a high intensity treadmill running was able to express ABCA1 and PPAR α in rat heart. Data also possibly indicate that the Crataeguse-Pentaegyna supplementation solely could mimic training effect on the mentioned genes and lipid profiles via different mechanism(s).

Heart ABCA1 and PPAR- α Genes Expression Responses in Male rats: Effects of High Intensity Treadmill Running Training and Aqueous Extraction of Black Crataegus-Pentaegyna

PubMed Central

Ghanbari-Niaki, Abbass; Ghanbari-Abarghooi, Safieyh; Rahbarizadeh, Fatemeh; Zare-Kookandeh, Navabeh; Gholizadeh, Monireh; Roudbari, Fatemeh; Zare-Kookandeh, Asghar

2013-01-01

Introduction: Heart as a high metabolic and aerobic tissue is consuming lipid as a fuel for its energy provision at rest during light and moderate exercise, except when lactate level is higher in blood circulation. It has been shown that any type of regular exercise and crataegus species would improve cardiovascular function and minimizes several risk factors via stimulating lipid metabolism by acting on enzymes and genes expression such as ABCA1 and PPAR α which are involving in this process. Materials and Methods: Twenty Wistar male rats (4-6 weeks old, 140-173 g weight) were used. Animals were randomly classified into training (n = 10) and control (n = 10) groups and then divided into saline-control (SC), saline-training (ST), Crataegus-Pentaegyna -control (CPC), and Crataegus-Pentaegyna -training (CPT) groups. Training groups have performed a high-intensity running program (at 34 m/min (0% grade), 60 min/day, 5 days/week) on a motor-driven treadmill for eight weeks. Animals were orally fed with Crataegus-Pentaegyna extraction (500mg/kg) and saline solution for six weeks. Seventy- two hours after the last training session, rats were sacrificed, hearts were excised, cleaned and immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Plasma also was collected for plasma variable measurements. Statistical analysis was performed using a two way analysis of variance, and significance was accepted at P < 0.05. Results: A non-significant (P < 0.4, P < 0.79, respectively) increase in ABCA1 and PPAR α genes expression was accompanied by a significant (P < 0.01, P < 0.04, P < 0.04, respectively) reduction in TC, TG, and VLDL-C levels in Crataegus-Pentaegyna groups. Conclusions: Our findings show that a high intensity treadmill running was able to express ABCA1 and PPAR α in rat heart. Data also possibly indicate that the Crataeguse-Pentaegyna supplementation solely could mimic training effect on the mentioned genes and lipid profiles via different mechanism(s). PMID:25478513

cDNA Microarray Screening in Food Safety

PubMed Central

ROY, SASHWATI; SEN, CHANDAN K

2009-01-01

The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests. PMID:16466843

Screening individual hybridomas by microengraving to discover monoclonal antibodies

PubMed Central

Ogunniyi, Adebola O; Story, Craig M; Papa, Eliseo; Guillen, Eduardo; Love, J Christopher

2014-01-01

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells). PMID:19528952

Rapid and Facile Microwave-Assisted Surface Chemistry for Functionalized Microarray Slides

PubMed Central

Lee, Jeong Heon; Hyun, Hoon; Cross, Conor J.; Henary, Maged; Nasr, Khaled A.; Oketokoun, Rafiou; Choi, Hak Soo; Frangioni, John V.

2011-01-01

We describe a rapid and facile method for surface functionalization and ligand patterning of glass slides based on microwave-assisted synthesis and a microarraying robot. Our optimized reaction enables surface modification 42-times faster than conventional techniques and includes a carboxylated self-assembled monolayer, polyethylene glycol linkers of varying length, and stable amide bonds to small molecule, peptide, or protein ligands to be screened for binding to living cells. We also describe customized slide racks that permit functionalization of 100 slides at a time to produce a cost-efficient, highly reproducible batch process. Ligand spots can be positioned on the glass slides precisely using a microarraying robot, and spot size adjusted for any desired application. Using this system, we demonstrate live cell binding to a variety of ligands and optimize PEG linker length. Taken together, the technology we describe should enable high-throughput screening of disease-specific ligands that bind to living cells. PMID:23467787

[Clinical Tests Testing New Therapies for Stargardt Disease].

PubMed

Kousal, B; Ďuďáková, Ľ; Hlavatá, L; Lišková, P

2016-02-01

To provide information on currently ongoing clinical trials for Stargardt disease. We have searched the clinical trial register (www.clinicaltrials.gov) for the keyword "Stargardt" and list active ongoing studies. There are currently eight registered clinical trials enrolling patients with Stargardt disease; all in phase I or II aiming at four mechanisms of action: inhibition of the production of vitamin A toxic dimers, gene therapy restoring wild type transcription of the ABCA4 gene, neuroprotection preventing retinal cells from oxidative damage, and replacement of the damaged retinal pigment epithelium using stem cell therapy. The basic prerequisite for enrolment in the vast majority of clinical trials is confirmation of the clinical diagnosis by mutational analysis. The wide variety of therapies that are registered as clinical trials for Stargardt disease significantly raises the possibility that effective treatments will be available in the near future for this currently incurable condition and that molecular genetic testing should be increasingly considered. Stargardt disease, clinical trial, ABCA4, mutation.

Microarray as a First Genetic Test in Global Developmental Delay: A Cost-Effectiveness Analysis

ERIC Educational Resources Information Center

Trakadis, Yannis; Shevell, Michael

2011-01-01

Aim: Microarray technology has a significantly higher clinical yield than karyotyping in individuals with global developmental delay (GDD). Despite this, it has not yet been routinely implemented as a screening test owing to the perception that this approach is more expensive. We aimed to evaluate the effect that replacing karyotype with…

ABCA1 and biogenesis of HDL.

PubMed

Yokoyama, Shinji

2006-02-01

Mammalian somatic cells do not catabolize cholesterol and therefore export it for sterol homeostasis at cell and whole body levels. This mechanism may reduce intracellularly accumulated excess cholesterol, and thereby would contribute to the prevention or cure of the initial stage of atherosclerotic vascular lesion. High-density lipoprotein (HDL) plays a central role in this reaction by removing cholesterol from cells and transporting it to the liver, the major cholesterol catabolic site. Two independent mechanisms have been identified for cellular cholesterol release. The first is non-specific diffusion-mediated cholesterol "efflux" from the cell surface, in which cholesterol is trapped by various extracellular acceptors including lipoproteins. Extracellular cholesterol esterification of HDL provides a driving force for the net removal of cell cholesterol by this pathway, and some cellular factors may enhance this reaction. The other mechanism is an apolipoprotein-mediated process to generate new HDL particles by removing cellular phospholipid and cholesterol. This reaction is mediated by a membrane protein ATP-binding cassette transporter A1 (ABCA1), and lipid-free or lipid-poor helical apolipoproteins recruit cellular phospholipid and cholesterol to assemble HDL particles. The reaction is composed of two elements: the assembly of HDL particles with phospholipid by apolipoprotein, and cholesterol enrichment in HDL. ABCA1 is essential for the former step and the latter requires further intracellular events. ABCA1 is a rate-limiting factor of HDL assembly and is regulated by transcriptional and post-transcriptional factors. Post-transcriptional regulation of ABCA1 involves modulation of its calpain-mediated degradation.

Assembly of high density lipoprotein by the ABCA1/apolipoprotein pathway.

PubMed

Yokoyama, Shinji

2005-06-01

Mammalian somatic cells do not catabolize cholesterol and therefore need to export it for sterol homeostasis at the levels of cells and whole bodies. This mechanism may reduce intracellularly accumulated cholesterol in excess, and thereby would contribute to the prevention or cure of the initial stage of atherosclerotic vascular lesions. HDL is thought to play a main role in this reaction on the basis of epidemiological evidence and in-vitro experimental data. Two independent mechanisms have been identified for this reaction. One is non-specific diffusion-mediated cholesterol 'efflux' from the cell surface, and cholesterol is trapped by various extracellular acceptors including lipoproteins. Extracellular cholesterol esterification on HDL provides a driving force for the net removal of cell cholesterol, and some cellular factors may enhance this reaction. The other mechanism is an apolipoprotein-mediated process to generate HDL by removing cellular phospholipid and cholesterol. This reaction is mediated by a membrane protein ABCA1, and lipid-free or lipid-poor helical apolipoproteins recruit cellular phospholipid and cholesterol to assemble HDL particles. The reaction is composed of two elements: the assembly of HDL particles with phospholipid by apolipoprotein, and cholesterol enrichment in HDL. ABCA1 is essential for the former step, and the latter step requires further intracellular events. ABCA1 is a rate-limiting factor of HDL assembly and is regulated by transcriptional factors and posttranscriptional factors. Posttranscriptional regulation of ABCA1 involves the modulation of its calpain-mediated degradation.

Gender-specific association of ATP-binding cassette transporter 1 (ABCA1) polymorphisms with the risk of late-onset Alzheimer's disease.

PubMed

Sundar, Purnima Desai; Feingold, Eleanor; Minster, Ryan L; DeKosky, Steven T; Kamboh, M Ilyas

2007-06-01

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder caused by a complex interaction of genetic and environmental factors. Increasing evidence highlights a potential role for cholesterol in the pathophysiology of AD. The ABCA1 gene, located in close vicinity to the 9q linkage peaks identified by genome-wide AD linkage studies, plays an important role in cellular cholesterol efflux, and is likely a good candidate gene. However, results from published genetic association studies between ABCA1 and AD are ambiguous. In the present study, we examined the role of two ABCA1 polymorphisms, R219K (rs2230806) and G-17C (rs2740483) in modifying the risk of late-onset AD (LOAD) in a large American white cohort of 992 AD cases and 699 controls. We observed significant gender x R219K interaction (p=0.00008). Female carriers of the 219K allele showed a 1.75-fold increased risk of developing AD compared to non-219K carrier females (95% CI 1.34-2.29; p=0.00004). The overall two-site haplotype distribution was also significant between female AD cases and controls (p=0.017). The risk associated with the R219K polymorphism was independent of the recently reported significant association in the ubiquilin (UBQLN1) gene in this region on chromosome 9q. Our data suggest a gender-specific and APOE and UBQLN1 independent association between the ABCA1/R219K polymorphism and LOAD.

The expression of cholesterol metabolism genes in monocytes from HIV-infected subjects suggests intracellular cholesterol accumulation.

PubMed

Feeney, Eoin R; McAuley, Nuala; O'Halloran, Jane A; Rock, Clare; Low, Justin; Satchell, Claudette S; Lambert, John S; Sheehan, Gerald J; Mallon, Patrick W G

2013-02-15

Human immunodeficiency virus (HIV) infection is associated with increased cardiovascular risk and reduced high-density lipoprotein cholesterol (HDL-c). In vitro, HIV impairs monocyte-macrophage cholesterol efflux, a major determinant of circulating HDL-c, by increasing ABCA1 degradation, with compensatory upregulation of ABCA1 messenger RNA (mRNA). We examined expression of genes involved in cholesterol uptake, metabolism, and efflux in monocytes from 22 HIV-positive subjects on antiretroviral therapy (ART-Treated), 30 untreated HIV-positive subjects (ART-Naive), and 22 HIV-negative controls (HIV-Neg). HDL-c was lower and expression of ABCA1 mRNA was higher in ART-Naive subjects than in both ART-Treated and HIV-Neg subjects (both P < .01), with HDL-c inversely correlated with HIV RNA (ρ = -0.52; P < .01). Expression of genes involved in cholesterol uptake (LDLR, CD36), synthesis (HMGCR), and regulation (SREBP2, LXRA) was significantly lower in both ART-Treated and ART-Naive subjects than in HIV-Neg controls. In vivo, increased monocyte ABCA1 expression in untreated HIV-infected patients and normalization of ABCA1 expression with virological suppression by ART supports direct HIV-induced impairment of cholesterol efflux previously demonstrated in vitro. However, decreased expression of cholesterol sensing, uptake, and synthesis genes in both untreated and treated HIV infection suggests that both HIV and ART affect monocyte cholesterol metabolism in a pattern consistent with accumulation of intramonocyte cholesterol.

Allicin induces the upregulation of ABCA1 expression via PPARγ/LXRα signaling in THP-1 macrophage-derived foam cells

PubMed Central

Lin, Xiao-Long; Hu, Hui-Jun; Liu, Yuan-Bo; Hu, Xue-Mei; Fan, Xiao-Juan; Zou, Wei-Wen; Pan, Yong-Quan; Zhou, Wen-Quan; Peng, Min-Wen; Gu, Cai-Hong

2017-01-01

Allicin is considered anti-atherosclerotic due to its antioxidant and anti-inflammatory effects, which makes it an important drug for the prevention and treatment of atherosclerosis. However, the effects of allicin on foam cells are unclear. Thus, in this study, we examined the effects of allicin on lipid accumulation via peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) in THP-1 macrophage-derived foam cells. THP-1 cells were exposed to 100 nM phorbol myristate acetate (PMA) for 24 h, and then to oxydized low-density lipoprotein (ox-LDL; 50 mg/ml) to induce foam cell formation. The results of Oil Red O staining and high-performance liquid chromatography (HPLC) revealed showed that pre-treatment of the foam cells with allicin decreased total cholesterol, free cholesterol (FC) and cholesterol ester levels in cells, and also decreased lipid accumulation. Moreover, allicin upregulated ATP binding cassette transporter A1 (ABCA1) expression and promoted cholesterol efflux. However, these effects were significantly abolished by transfection with siRNA targeting ABCA1. Furthermore, PPARγ/LXRα signaling was activated by allicin treatment. The allicin-induced upregulation of ABCA1 expression was also abolished by PPARγ inhibitor (GW9662) and siRNA or LXRα siRNA co-treatment. Overall, our data demonstrate that the allicin-induced upregulation of ABCA1 promotes cholesterol efflux and reduces lipid accumulation via PPARγ/LXRα signaling in THP-1 macrophage-derived foam cells. PMID:28440421

Kimchi methanol extract and the kimchi active compound, 3'-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid, downregulate CD36 in THP-1 macrophages stimulated by oxLDL.

PubMed

Yun, Ye-Rang; Kim, Hyun-Ju; Song, Yeong-Ok

2014-08-01

Macrophage foam cell formation by oxidized low-density lipoprotein (oxLDL) is a key step in the progression of atherosclerosis, which is involved in cholesterol influx and efflux in macrophages mediated by related proteins such as peroxisome proliferator-activated receptor γ (PPARγ), CD36, PPARα, liver-X receptor α (LXRα), and ATP-binding cassette transporter A1 (ABCA1). The aim of this study was to investigate the beneficial effects of kimchi methanol extract (KME) and a kimchi active compound, 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA) on cholesterol flux in THP-1-derived macrophages treated with oxLDL. The effects of KME and HDMPPA on cell viability and lipid peroxidation were determined. Furthermore, the protein expression of PPARγ, CD36, PPARα, LXRα, and ABCA1 was examined. OxLDL strongly induced cell death and lipid peroxidation in THP-1-derived macrophages. However, KME and HDMPPA significantly improved cell viability and inhibited lipid peroxidation induced by oxLDL in THP-1-derived macrophages (P<.05). Moreover, KME and HDMPPA suppressed CD36 and PPARγ expressions, both of which participate in cholesterol influx. In contrast, KME and HDMPPA augmented LXRα, PPARα, and ABCA1 expression, which are associated with cholesterol efflux. Consequently, KME and HDMPPA suppressed lipid accumulation. These results indicate that KME and HDMPPA may inhibit lipid accumulation, in part, by regulating cholesterol influx- and efflux-related proteins. These findings will thus be useful for future prevention strategies against atherosclerosis.

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

Emergent FDA biodefense issues for microarray technology: process analytical technology.

PubMed

Weinberg, Sandy

2004-11-01

A successful biodefense strategy relies upon any combination of four approaches. A nation can protect its troops and citizenry first by advanced mass vaccination, second, by responsive ring vaccination, and third, by post-exposure therapeutic treatment (including vaccine therapies). Finally, protection can be achieved by rapid detection followed by exposure limitation (suites and air filters) or immediate treatment (e.g., antibiotics, rapid vaccines and iodine pills). All of these strategies rely upon or are enhanced by microarray technologies. Microarrays can be used to screen, engineer and test vaccines. They are also used to construct early detection tools. While effective biodefense utilizes a variety of tactical tools, microarray technology is a valuable arrow in that quiver.

HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation

PubMed Central

Tardy, Claudine; Goffinet, Marine; Boubekeur, Nadia; Cholez, Guy; Ackermann, Rose; Sy, Gavin; Keyserling, Constance; Lalwani, Narendra; Paolini, John F.; Dasseux, Jean-Louis; Barbaras, Ronald; Baron, Rudi

2015-01-01

Objective CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3). Methods and Results CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL3 compared to non-lipidated apoA-I. In vivo, in an apoE-/- mouse “flow cessation model,” in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively. Conclusions These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both in vitro and in vivo. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression. PMID:26335690

HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation.

PubMed

Tardy, Claudine; Goffinet, Marine; Boubekeur, Nadia; Cholez, Guy; Ackermann, Rose; Sy, Gavin; Keyserling, Constance; Lalwani, Narendra; Paolini, John F; Dasseux, Jean-Louis; Barbaras, Ronald; Baron, Rudi

2015-01-01

CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3). CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL3 compared to non-lipidated apoA-I. In vivo, in an apoE-/- mouse "flow cessation model," in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively. These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both in vitro and in vivo. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression.

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

ELISA microarray technology as a high-throughput system for cancer biomarker validation

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

Zangar, Richard C.; Daly, Don S.; White, Amanda M.

A large gap currently exists between the ability to discover potential biomarkers and the ability to assess the real value of these proteins for cancer screening. One major challenge in biomarker validation is the inherent variability in biomarker levels. This variability stems from the diversity across the human population and the considerable molecular heterogeneity between individual tumors, even those that originate from a single tissue. Another major challenge with cancer screening is that most cancers are rare in the general population, meaning that the specificity of an assay must be very high if the number of false positive is notmore » going to be much greater than the number of true positives. Because of these challenges with biomarker validation, it is necessary to analysis of thousands of samples before a clear idea of the utility of a screening assay can be determined. Enzyme-linked immunosorbent assay (ELISA) microarray technology can simultaneously quantify levels of multiple proteins and has the potential to accelerate biomarker validation. In this review, we discuss current ELISA microarray technology and the enabling advances needed to achieve the reproducibility and throughput that are required to evaluate cancer biomarkers.« less

Discovering ligands for a microRNA precursor with peptoid microarrays

PubMed Central

Chirayil, Sara; Chirayil, Rachel; Luebke, Kevin J.

2009-01-01

We have screened peptoid microarrays to identify specific ligands for the RNA hairpin precursor of miR-21, a microRNA involved in cancer and heart disease. Microarrays were printed by spotting a library of 7680 N-substituted oligoglycines (peptoids) onto glass slides. Two compounds on the array specifically bind RNA having the sequence and predicted secondary structure of the miR-21 precursor hairpin and have specific affinity for the target in solution. Their binding induces a conformational change around the hairpin loop, and the most specific compound recognizes the loop sequence and a bulged uridine in the proximal duplex. Functional groups contributing affinity and specificity were identified, and by varying a critical methylpyridine group, a compound with a dissociation constant of 1.9 μM for the miR-21 precursor hairpin and a 20-fold discrimination against a closely-related hairpin was created. This work describes a systematic approach to discovery of ligands for specific pre-defined novel RNA structures. It demonstrates discovery of new ligands for an RNA for which no specific lead compounds were previously known by screening a microarray of small molecules. PMID:19561197

A Reverse-phase Protein Microarray-based Screen Identifies Host Signaling Dynamics upon Burkholderia spp. Infection

DTIC Science & Technology

2015-07-27

University, Manassas, VA, USA, 4 PerkinElmer, Inc., Waltham, MA, USA Burkholderia is a diverse genus of gram-negative bacteria that causes high...Burkholderia spp. infections. Materials and Methods Bacterial Strains Bm ATCC 23344, NCTC 10247, NCTC 10229, NCTC 3708, NCTC 3709, 2002721278 (Burtnick...macrophage cell line RAW264.7 was obtained from ATCC (Manassas, VA). Cells were cultured in DMEM (Life Technologies) supplemented with 10% fetal

Targeting TMPRSS2-ERG in Prostate Cancer

DTIC Science & Technology

2015-09-01

small molecule microarrays ( SMM ) with lysates overexpressing ERG (months 1-12) 3a. Request compounds from compound management that scored from...preliminary SMM screen using 293T lysates overexpressing ERG (months 1-2 – completed November 2013) 3b. Reprint slides with compounds from 3a for...counter SMM assays (months 3-4 – completed December 2013) 3c. Perform counter SMM assay with 293T lysates expressing unrelated protein (months 5-10

Targeting TMPRSS2-ERG in Prostate Cancer

DTIC Science & Technology

2014-09-01

microarrays ( SMM ) with lysates overexpressing ERG (months 1-12) 3a. Request compounds from compound management that scored from preliminary SMM ...screen using 293T lysates overexpressing ERG (months 1-2 – completed November 2013) 3b. Reprint slides with compounds from 3a for counter SMM ...assays (months 3-4 – completed December 2013) 3c. Perform counter SMM assay with 293T lysates expressing unrelated protein (months 5-10 – completed

Screening of biomarkers for prediction of response to and prognosis after chemotherapy for breast cancers.

PubMed

Bing, Feng; Zhao, Yu

2016-01-01

To screen the biomarkers having the ability to predict prognosis after chemotherapy for breast cancers. Three microarray data of breast cancer patients undergoing chemotherapy were collected from Gene Expression Omnibus database. After preprocessing, data in GSE41112 were analyzed using significance analysis of microarrays to screen the differentially expressed genes (DEGs). The DEGs were further analyzed by Differentially Coexpressed Genes and Links to construct a function module, the prognosis efficacy of which was verified by the other two datasets (GSE22226 and GSE58644) using Kaplan-Meier plots. The involved genes in function module were subjected to a univariate Cox regression analysis to confirm whether the expression of each prognostic gene was associated with survival. A total of 511 DEGs between breast cancer patients who received chemotherapy or not were obtained, consisting of 421 upregulated and 90 downregulated genes. Using the Differentially Coexpressed Genes and Links package, 1,244 differentially coexpressed genes (DCGs) were identified, among which 36 DCGs were regulated by the transcription factor complex NFY (NFYA, NFYB, NFYC). These 39 genes constructed a gene module to classify the samples in GSE22226 and GSE58644 into three subtypes and these subtypes exhibited significantly different survival rates. Furthermore, several genes of the 39 DCGs were shown to be significantly associated with good (such as CDC20) and poor (such as ARID4A) prognoses following chemotherapy. Our present study provided a serial of biomarkers for predicting the prognosis of chemotherapy or targets for development of alternative treatment (ie, CDC20 and ARID4A) in breast cancer patients.

MicroRNA-326 and microRNA-200c: Two novel biomarkers for diagnosis and prognosis of pediatric acute lymphoblastic leukemia.

PubMed

Ghodousi, Elaheh S; Rahgozar, Soheila

2018-04-06

Multidrug resistance (MDR) is considered as the major obstacle for treating pediatric acute lymphoblastic leukemia (ALL). MicroRNAs (miRNAs) are small non coding RNAs which may potentially regulate response to chemotherapy. In this study, total RNA was isolated from bone marrow samples of 46 children with de novo ALL and 16 controls. Quantitative reverse transcriptase polymerase chain reaction was used to investigate the expression profile of the predicted miRNAs; miR-326 and miR-200c, and their predicted targets ABCA2, and ABCA3 transporters. The presence of minimal residual disease was studied using PCR-SSCP (single-strand conformation polymorphism) 1 year after treatment. The association between the miRNA expression and drug resistance was analyzed statistically. Results showed a significant down-regulation of both miR-326 and miR-200c expressions in ALL patients compared with non-cancer controls (P = 0.0002, AUC = 0.813 and P = 0.035, AUC = 0.79, respectively). A considerable negative association between miR-326 expression and MDR was identified which could raise the risk of chemoresistance by 4.8- fold. The expression profiles of miR-326 and ABCA2 transporter were inversely correlated. Data revealed, a novel diagnostic role for miR-326 and miR-200c as potential biomarkers of pediatric ALL. Down-regulation of miR-326 was introduced, for the first time, as a prognostic factor for drug resistance in childhood ALL. To the best of our knowledge, this is the first time that ABCA2 transporter is proposed as a target gene for miR-326, through which it can exert its impact on drug resistance. These data may provide novel approaches to new therapeutics and diagnostics. © 2018 Wiley Periodicals, Inc.

Retinoic Acid Isomers Up-Regulate ATP Binding Cassette A1 and G1 and Cholesterol Efflux in Rat Astrocytes: Implications for Their Therapeutic and Teratogenic Effects

PubMed Central

Chen, Jing; Costa, Lucio G.

2011-01-01

Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (±0.25), 3.6- (±0.42), 4.1- (±0.5), and 1.75- (±0.43) fold, respectively, and Abcg1 by 2.1- (±0.26), 2.2- (±0.33), 2.5- (±0.23), and 2.2- (±0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions. PMID:21628419

Regulation of ATP-binding Cassette Transporters and Cholesterol Efflux by Glucose in Primary Human Monocytes and Murine Bone Marrow-derived Macrophages

PubMed Central

Spartano, N. L.; Lamon-Fava, S.; Matthan, N. R.; Ronxhi, J.; Greenberg, A. S.; Obin, M. S.; Lichtenstein, A. H.

2014-01-01

Purpose Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM). Methods 10 subjects (4 F/6 M, 50–85 years, BMI 25–35 kg/m2) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5 mmol/L D-glucose (control) or additional 20 mmol/L D-or L-glucose and 25 ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol efflux and ABC-transporter (ABCA1 and ABCG1) expression were determined. Results Baseline ABCA1and ABCG1 expression was lower (> 50 %) in human monocytes and PBMC than leukocytes (p < 0.05). 1 h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37 % and 30 %, respectively (p < 0.05), and began to return to baseline thereafter. There was no significant change in monocyte ABC-transporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol efflux (10 %; p < 0.01) without significantly affecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression. Conclusions Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol efflux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se, suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol efflux may potentially contribute to diabetes-associated atherosclerosis. PMID:24838154

Using pathway modules as targets for assay development in xenobiotic screening

EPA Science Inventory

Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological syst...

Evaluation of the Association Between Common Genetic Variants Near the ABCA1 Gene and Primary Angle Closure Glaucoma in a Han Chinese Population.

PubMed

Luo, Huaichao; Chen, Yuhong; Ye, Zimeng; Sun, Xinghuai; Shi, Yi; Luo, Qian; Gong, Bo; Shuai, Ping; Yang, Jiyun; Zhou, Yu; Liu, Xiaoqi; Zhang, Kaijiong; Tan, Chang; Li, Yuanfeng; Lin, Ying; Yang, Zhenglin

2015-10-01

Recently, three large genome-wide association studies have identified multiple variants associated with primary open angle glaucoma (POAG) near the ABCA1 gene. Considering that POAG and primary angle closure glaucoma (PACG) share many similar clinical manifestations, the present study was conducted to investigate whether these genetic variants were also associated with PACG in a Han Chinese population. A case-control association study of 1122 cases (PACG/PAC) and 1311 normal, matched controls was undertaken. Seven single-nucleotide polymorphisms (SNPs) near the ABCA1 gene, including rs2422493, rs2487042, rs2472496, rs2472493, rs2487032, rs2472459, and rs2472519, were genotyped. Genotype and allele frequencies were assessed using χ² tests. Linkage disequilibrium (LD) structure was analyzed by computer software. Among the SNPs genotyped, no association was observed between these SNPs and PACG. However, we discovered that two haplotypes, CATTTAC (corrected P = 0.048) and CGCCCGC (corrected P = 0.048), remained significantly associated with PACG/PAC after Bonferroni correction. Subjects with the CATTTAC haplotype have a 1.71-fold increased possibility of having PACG/PAC, whereas subjects with the CGCCCGC haplotype have 0.47-fold decreased possibility of developing PACG. Our findings suggest that the genetic backgrounds of PACG and POAG might be different. However, whether or not ABCA1 plays a role in the development of PACG is still not made certain by this study. Thus, further research is needed to find the role of ABCA1 in the progress of PACG.

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

Xu, Xiaolin; Li, Qian; Pang, Liewen

Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-densitymore » lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.« less

Chemokine Receptor CXCR4 Expression in Patients With Melanoma and Colorectal Cancer Liver Metastases and the Association With Disease Outcome

PubMed Central

Kim, Joseph; Mori, Takuji; Chen, Steven L.; Amersi, Farin F.; Martinez, Steve R.; Kuo, Christine; Turner, Roderick R.; Ye, Xing; Bilchik, Anton J.; Morton, Donald L.; Hoon, Dave S. B.

2006-01-01

Objective: To determine the role of chemokine receptor (CR) expression in patients with melanoma and colorectal cancer (CRC) liver metastases. Summary Background Data: Murine and in vitro models have identified CR as potential factors in organ-specific metastasis of multiple cancers. Chemokines via their respective receptors have been shown to promote cell migration to distant organs. Methods: Patients who underwent hepatic surgery for melanoma or CRC liver metastases were assessed. Screening cDNA microarrays of melanoma/CRC cell lines and tumor specimens were analyzed to identify CR. Microarray data were validated by quantitative real-time RT-PCR (qRT) in paraffin-embedded liver metastases. Migration assays and immunohistochemistry were performed to verify CR function and confirm CR expression, respectively. Results: Microarray analysis identified CXCR4 as the most common CR expressed by both cancers. qRT demonstrated CXCR4 expression in 24 of 27 (89%) melanoma and 28 of 29 (97%) CRC liver metastases. In vitro treatment of melanoma or CRC cells with CXCL12, the ligand for CXCR4, significantly increased cell migration (P < 0.001). Low versus high CXCR4 expression in CRC liver metastases correlated with a significant difference in overall survival (median 27 months vs. 10 months, respectively; P = 0.036). In melanoma, low versus high CXCR4 expression in liver metastases demonstrated no difference in overall survival (median 11 months vs. 8 months, respectively; P = not significant). Conclusions: CXCR4 is expressed and functional on melanoma and CRC cells. The ligand for CXCR4 is highly expressed in liver and may specifically attract melanoma and CRC CXCR4 (+) cells. Quantitative analysis of CXCR4 gene expression in patients with liver metastases has prognostic significance for disease outcome. PMID:16794396

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

PubMed Central

Grubaugh, Nathan D.; Petz, Lawrence N.; Melanson, Vanessa R.; McMenamy, Scott S.; Turell, Michael J.; Long, Lewis S.; Pisarcik, Sarah E.; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L.; Lee, John S.

2013-01-01

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors. PMID:23249687

Ligand, receptor, and cell type-dependent regulation of ABCA1 and ABCG1 mRNA in prostate cancer epithelial cells

USDA-ARS?s Scientific Manuscript database

Recent evidence suggests that the liver X receptor (LXR) is a potential anti-cancer target in prostate carcinoma. There is little characterization, however, of how the two major isoforms LXRa or LXRß regulate the LXR-responsive genes ATP-binding cassette sub-family A 1 (ABCA1) and sub-family member ...

The multidimensional perturbation value: a single metric to measure similarity and activity of treatments in high-throughput multidimensional screens.

PubMed

Hutz, Janna E; Nelson, Thomas; Wu, Hua; McAllister, Gregory; Moutsatsos, Ioannis; Jaeger, Savina A; Bandyopadhyay, Somnath; Nigsch, Florian; Cornett, Ben; Jenkins, Jeremy L; Selinger, Douglas W

2013-04-01

Screens using high-throughput, information-rich technologies such as microarrays, high-content screening (HCS), and next-generation sequencing (NGS) have become increasingly widespread. Compared with single-readout assays, these methods produce a more comprehensive picture of the effects of screened treatments. However, interpreting such multidimensional readouts is challenging. Univariate statistics such as t-tests and Z-factors cannot easily be applied to multidimensional profiles, leaving no obvious way to answer common screening questions such as "Is treatment X active in this assay?" and "Is treatment X different from (or equivalent to) treatment Y?" We have developed a simple, straightforward metric, the multidimensional perturbation value (mp-value), which can be used to answer these questions. Here, we demonstrate application of the mp-value to three data sets: a multiplexed gene expression screen of compounds and genomic reagents, a microarray-based gene expression screen of compounds, and an HCS compound screen. In all data sets, active treatments were successfully identified using the mp-value, and simulations and follow-up analyses supported the mp-value's statistical and biological validity. We believe the mp-value represents a promising way to simplify the analysis of multidimensional data while taking full advantage of its richness.

Genotyping microarray: Mutation screening in Spanish families with autosomal dominant retinitis pigmentosa

PubMed Central

García-Hoyos, María; Cortón, Marta; Ávila-Fernández, Almudena; Riveiro-Álvarez, Rosa; Giménez, Ascensión; Hernan, Inma; Carballo, Miguel; Ayuso, Carmen

2012-01-01

Purpose Presently, 22 genes have been described in association with autosomal dominant retinitis pigmentosa (adRP); however, they explain only 50% of all cases, making genetic diagnosis of this disease difficult and costly. The aim of this study was to evaluate a specific genotyping microarray for its application to the molecular diagnosis of adRP in Spanish patients. Methods We analyzed 139 unrelated Spanish families with adRP. Samples were studied by using a genotyping microarray (adRP). All mutations found were further confirmed with automatic sequencing. Rhodopsin (RHO) sequencing was performed in all negative samples for the genotyping microarray. Results The adRP genotyping microarray detected the mutation associated with the disease in 20 of the 139 families with adRP. As in other populations, RHO was found to be the most frequently mutated gene in these families (7.9% of the microarray genotyped families). The rate of false positives (microarray results not confirmed with sequencing) and false negatives (mutations in RHO detected with sequencing but not with the genotyping microarray) were established, and high levels of analytical sensitivity (95%) and specificity (100%) were found. Diagnostic accuracy was 15.1%. Conclusions The adRP genotyping microarray is a quick, cost-efficient first step in the molecular diagnosis of Spanish patients with adRP. PMID:22736939

A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

PubMed Central

Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

2015-01-01

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

A New Way to Introduce Microarray Technology in a Lecture/Laboratory Setting by Studying the Evolution of This Modern Technology

ERIC Educational Resources Information Center

Rowland-Goldsmith, Melissa

2009-01-01

DNA microarray is an ordered grid containing known sequences of DNA, which represent many of the genes in a particular organism. Each DNA sequence is unique to a specific gene. This technology enables the researcher to screen many genes from cells or tissue grown in different conditions. We developed an undergraduate lecture and laboratory…

A Cytogenetic Abnormality and Rare Coding Variants Identify ABCA13 as a Candidate Gene in Schizophrenia, Bipolar Disorder, and Depression

PubMed Central

Knight, Helen M.; Pickard, Benjamin S.; Maclean, Alan; Malloy, Mary P.; Soares, Dinesh C.; McRae, Allan F.; Condie, Alison; White, Angela; Hawkins, William; McGhee, Kevin; van Beck, Margaret; MacIntyre, Donald J.; Starr, John M.; Deary, Ian J.; Visscher, Peter M.; Porteous, David J.; Cannon, Ronald E.; St Clair, David; Muir, Walter J.; Blackwood, Douglas H.R.

2009-01-01

Schizophrenia and bipolar disorder are leading causes of morbidity across all populations, with heritability estimates of ∼80% indicating a substantial genetic component. Population genetics and genome-wide association studies suggest an overlap of genetic risk factors between these illnesses but it is unclear how this genetic component is divided between common gene polymorphisms, rare genomic copy number variants, and rare gene sequence mutations. We report evidence that the lipid transporter gene ABCA13 is a susceptibility factor for both schizophrenia and bipolar disorder. After the initial discovery of its disruption by a chromosome abnormality in a person with schizophrenia, we resequenced ABCA13 exons in 100 cases with schizophrenia and 100 controls. Multiple rare coding variants were identified including one nonsense and nine missense mutations and compound heterozygosity/homozygosity in six cases. Variants were genotyped in additional schizophrenia, bipolar, depression (n > 1600), and control (n > 950) cohorts and the frequency of all rare variants combined was greater than controls in schizophrenia (OR = 1.93, p = 0.0057) and bipolar disorder (OR = 2.71, p = 0.00007). The population attributable risk of these mutations was 2.2% for schizophrenia and 4.0% for bipolar disorder. In a study of 21 families of mutation carriers, we genotyped affected and unaffected relatives and found significant linkage (LOD = 4.3) of rare variants with a phenotype including schizophrenia, bipolar disorder, and major depression. These data identify a candidate gene, highlight the genetic overlap between schizophrenia, bipolar disorder, and depression, and suggest that rare coding variants may contribute significantly to risk of these disorders. PMID:19944402

Variants in the ATP-binding cassette transporter (ABCA7), apolipoprotein E ϵ4,and the risk of late-onset Alzheimer disease in African Americans.

PubMed

Reitz, Christiane; Jun, Gyungah; Naj, Adam; Rajbhandary, Ruchita; Vardarajan, Badri Narayan; Wang, Li-San; Valladares, Otto; Lin, Chiao-Feng; Larson, Eric B; Graff-Radford, Neill R; Evans, Denis; De Jager, Philip L; Crane, Paul K; Buxbaum, Joseph D; Murrell, Jill R; Raj, Towfique; Ertekin-Taner, Nilufer; Logue, Mark; Baldwin, Clinton T; Green, Robert C; Barnes, Lisa L; Cantwell, Laura B; Fallin, M Daniele; Go, Rodney C P; Griffith, Patrick; Obisesan, Thomas O; Manly, Jennifer J; Lunetta, Kathryn L; Kamboh, M Ilyas; Lopez, Oscar L; Bennett, David A; Hendrie, Hugh; Hall, Kathleen S; Goate, Alison M; Byrd, Goldie S; Kukull, Walter A; Foroud, Tatiana M; Haines, Jonathan L; Farrer, Lindsay A; Pericak-Vance, Margaret A; Schellenberg, Gerard D; Mayeux, Richard

2013-04-10

Genetic variants associated with susceptibility to late-onset Alzheimer disease are known for individuals of European ancestry, but whether the same or different variants account for the genetic risk of Alzheimer disease in African American individuals is unknown. Identification of disease-associated variants helps identify targets for genetic testing, prevention, and treatment. To identify genetic loci associated with late-onset Alzheimer disease in African Americans. The Alzheimer Disease Genetics Consortium (ADGC) assembled multiple data sets representing a total of 5896 African Americans (1968 case participants, 3928 control participants) 60 years or older that were collected between 1989 and 2011 at multiple sites. The association of Alzheimer disease with genotyped and imputed single-nucleotide polymorphisms (SNPs) was assessed in case-control and in family-based data sets. Results from individual data sets were combined to perform an inverse variance-weighted meta-analysis, first with genome-wide analyses and subsequently with gene-based tests for previously reported loci. Presence of Alzheimer disease according to standardized criteria. Genome-wide significance in fully adjusted models (sex, age, APOE genotype, population stratification) was observed for a SNP in ABCA7 (rs115550680, allele = G; frequency, 0.09 cases and 0.06 controls; odds ratio [OR], 1.79 [95% CI, 1.47-2.12]; P = 2.2 × 10(-9)), which is in linkage disequilibrium with SNPs previously associated with Alzheimer disease in Europeans (0.8 < D' < 0.9). The effect size for the SNP in ABCA7 was comparable with that of the APOE ϵ4-determining SNP rs429358 (allele = C; frequency, 0.30 cases and 0.18 controls; OR, 2.31 [95% CI, 2.19-2.42]; P = 5.5 × 10(-47)). Several loci previously associated with Alzheimer disease but not reaching significance in genome-wide analyses were replicated in gene-based analyses accounting for linkage disequilibrium between markers and correcting for number of tests performed per gene (CR1, BIN1, EPHA1, CD33; 0.0005 < empirical P < .001). In this meta-analysis of data from African American participants, Alzheimer disease was significantly associated with variants in ABCA7 and with other genes that have been associated with Alzheimer disease in individuals of European ancestry. Replication and functional validation of this finding is needed before this information is used in clinical settings.

Altered surfactant homeostasis and recurrent respiratory failure secondary to TTF-1 nuclear targeting defect.

PubMed

Peca, Donatella; Petrini, Stefania; Tzialla, Chryssoula; Boldrini, Renata; Morini, Francesco; Stronati, Mauro; Carnielli, Virgilio P; Cogo, Paola E; Danhaive, Olivier

2011-08-25

Mutations of genes affecting surfactant homeostasis, such as SFTPB, SFTPC and ABCA3, lead to diffuse lung disease in neonates and children. Haploinsufficiency of NKX2.1, the gene encoding the thyroid transcription factor-1 (TTF-1)--critical for lung, thyroid and central nervous system morphogenesis and function--causes a rare form of progressive respiratory failure designated brain-lung-thyroid syndrome. Molecular mechanisms involved in this syndrome are heterogeneous and poorly explored. We report a novel TTF-1 molecular defect causing recurrent respiratory failure episodes in an infant. The subject was an infant with severe neonatal respiratory distress syndrome followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities. Lung histology and ultrastructure were assessed by surgical biopsy. Surfactant-related genes were studied by direct genomic DNA sequencing and array chromatine genomic hybridization (aCGH). Surfactant protein expression in lung tissue was analyzed by confocal immunofluorescence microscopy. For kinetics studies, surfactant protein B and disaturated phosphatidylcholine (DSPC) were isolated from serial tracheal aspirates after intravenous administration of stable isotope-labeled (2)H(2)O and (13)C-leucine; fractional synthetic rate was derived from gas chromatography/mass spectrometry (2)H and (13)C enrichment curves. Six intubated infants with no primary lung disease were used as controls. Lung biopsy showed desquamative interstitial pneumonitis and lamellar body abnormalities suggestive of genetic surfactant deficiency. Genetic studies identified a heterozygous ABCA3 mutation, L941P, previously unreported. No SFTPB, SFTPC or NKX2.1 mutations or deletions were found. However, immunofluorescence studies showed TTF-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. This pattern has not been reported in human and was not found in two healthy controls and in five ABCA3 mutation carriers. Kinetic studies demonstrated a marked reduction of SP-B synthesis (43.2 vs. 76.5 ± 24.8%/day); conversely, DSPC synthesis was higher (12.4 vs. 6.3 ± 0.5%/day) compared to controls, although there was a marked reduction of DSPC content in tracheal aspirates (29.8 vs. 56.1 ± 12.4% of total phospholipid content). Defective TTF-1 signaling may result in profound surfactant homeostasis disruption and neonatal/pediatric diffuse lung disease. Heterozygous ABCA3 missense mutations may act as disease modifiers in other genetic surfactant defects.

Altered surfactant homeostasis and recurrent respiratory failure secondary to TTF-1 nuclear targeting defect

PubMed Central

2011-01-01

Background Mutations of genes affecting surfactant homeostasis, such as SFTPB, SFTPC and ABCA3, lead to diffuse lung disease in neonates and children. Haploinsufficiency of NKX2.1, the gene encoding the thyroid transcription factor-1 (TTF-1) - critical for lung, thyroid and central nervous system morphogenesis and function - causes a rare form of progressive respiratory failure designated brain-lung-thyroid syndrome. Molecular mechanisms involved in this syndrome are heterogeneous and poorly explored. We report a novel TTF-1 molecular defect causing recurrent respiratory failure episodes in an infant. Methods The subject was an infant with severe neonatal respiratory distress syndrome followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities. Lung histology and ultrastructure were assessed by surgical biopsy. Surfactant-related genes were studied by direct genomic DNA sequencing and array chromatine genomic hybridization (aCGH). Surfactant protein expression in lung tissue was analyzed by confocal immunofluorescence microscopy. For kinetics studies, surfactant protein B and disaturated phosphatidylcholine (DSPC) were isolated from serial tracheal aspirates after intravenous administration of stable isotope-labeled 2H2O and 13C-leucine; fractional synthetic rate was derived from gas chromatography/mass spectrometry 2H and 13C enrichment curves. Six intubated infants with no primary lung disease were used as controls. Results Lung biopsy showed desquamative interstitial pneumonitis and lamellar body abnormalities suggestive of genetic surfactant deficiency. Genetic studies identified a heterozygous ABCA3 mutation, L941P, previously unreported. No SFTPB, SFTPC or NKX2.1 mutations or deletions were found. However, immunofluorescence studies showed TTF-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. This pattern has not been reported in human and was not found in two healthy controls and in five ABCA3 mutation carriers. Kinetic studies demonstrated a marked reduction of SP-B synthesis (43.2 vs. 76.5 ± 24.8%/day); conversely, DSPC synthesis was higher (12.4 vs. 6.3 ± 0.5%/day) compared to controls, although there was a marked reduction of DSPC content in tracheal aspirates (29.8 vs. 56.1 ± 12.4% of total phospholipid content). Conclusion Defective TTF-1 signaling may result in profound surfactant homeostasis disruption and neonatal/pediatric diffuse lung disease. Heterozygous ABCA3 missense mutations may act as disease modifiers in other genetic surfactant defects. PMID:21867529

Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages

PubMed Central

Mao, Min; Lei, Han; Liu, Qing; Chen, Yaxi; Zhao, Lei; Li, Qing; Luo, Suxin; Zuo, Zhong; He, Quan; Huang, Wei; Zhang, Nan; Zhou, Chao; Ruan, Xiong Z.

2014-01-01

The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages. PMID:25329888

ABCA1 agonist peptides for the treatment of disease

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

Bielicki, John K.

Purpose of review The review summarizes information pertaining to the preclinical development of new apolipoprotein (apo) E mimetic peptides that stimulate cellular cholesterol efflux. Recent findings Small α-helical peptides based on the C-terminal domain of apoE have been developed for therapeutic applications. These peptides stimulate cellular cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1) with high potency, like native apolipoproteins on a molar basis. This potent activity has been related to the unique ability of these peptides to maintain α-helix structure upon dilution. Recent structure-activity studies improving the safety features of these mimetic peptides have greatly improved their potentialmore » for clinical use. Structural features of the class A α-helix motif that induce muscle toxicity and hypertriglyceridemia have been identified. These may have implications for the design of other HDL mimetic peptides. Summary ABCA1 is an integral membrane protein that plays a central role in biology. Its principal function is to mediate the efflux of cholesterol and phospholipid from cells to extracellular apo, preventing a build-up of excess cholesterol in membranes. This process generates HDL particles that perform a variety of functions to protect against disease. A number of these functions can be viewed as directly or indirectly supporting ABCA1 activity, thus constituting a positive feedback system to optimize cellular lipid efflux responses and disease prevention. Consequently, therapeutic approaches that mimic the activities of apos may prove highly effective to combat disease. One such approach involves the use of peptides. The broad biological relevance of ABCA1 suggests these apo mimetic peptides may be useful for the treatment of a number of diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.« less

ABCA1 agonist peptides for the treatment of disease

DOE PAGES

Bielicki, John K.

2016-02-01

Purpose of review The review summarizes information pertaining to the preclinical development of new apolipoprotein (apo) E mimetic peptides that stimulate cellular cholesterol efflux. Recent findings Small α-helical peptides based on the C-terminal domain of apoE have been developed for therapeutic applications. These peptides stimulate cellular cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1) with high potency, like native apolipoproteins on a molar basis. This potent activity has been related to the unique ability of these peptides to maintain α-helix structure upon dilution. Recent structure-activity studies improving the safety features of these mimetic peptides have greatly improved their potentialmore » for clinical use. Structural features of the class A α-helix motif that induce muscle toxicity and hypertriglyceridemia have been identified. These may have implications for the design of other HDL mimetic peptides. Summary ABCA1 is an integral membrane protein that plays a central role in biology. Its principal function is to mediate the efflux of cholesterol and phospholipid from cells to extracellular apo, preventing a build-up of excess cholesterol in membranes. This process generates HDL particles that perform a variety of functions to protect against disease. A number of these functions can be viewed as directly or indirectly supporting ABCA1 activity, thus constituting a positive feedback system to optimize cellular lipid efflux responses and disease prevention. Consequently, therapeutic approaches that mimic the activities of apos may prove highly effective to combat disease. One such approach involves the use of peptides. The broad biological relevance of ABCA1 suggests these apo mimetic peptides may be useful for the treatment of a number of diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.« less

17β-estradiol suppresses the macrophage foam cell formation associated with SOCS3.

PubMed

Liang, X; He, M; Chen, T; Wu, Y; Tian, Y; Zhao, Y; Shen, Y; Liu, Y; Yuan, Z

2013-06-01

Evidence from clinical trials and animal experiments has shown that estrogen has anti-atherosclerotic effects when administered to young women or experimental animals. The mechanisms involve the modulation of vascular inflammation, growth factor expression, and oxidative stress injured arteries. However, whether estrogen modulates the foam cell formation in plaque remains unknown. Here, we investigated the effects of 17β-estradiol (E2) on cholesterol efflux in vivo and in vitro. ApoE null mice underwent an ovariectomy at 5(th) week of age and then were treated with E2 or vehicle for the following 8 weeks. Compared with the vehicle-treated mice, the serum total cholesterol level, atherosclerotic plaque size, and lipid deposits were decreased and meanwhile ATP-binding cassette transporter A1 (ABCA1) expression in the plaque was increased in mice with E2 treatment. E2 also increased suppressor of cytokine signaling 3 (SOCS3) expression in the atherosclerotic plaques and in RAW264.7 cells. In vitro, E2 treatment reversed janus kinase/signal transducers and activators of transcription (JAK/STAT)-inhibited ABCA1 expression in RAW264.7 cells but had no effect on ABCA1 expression in SOCS3 knockdown cells. SOCS3 overexpression elevated ABCA1 expression through the inhibition of JAK2/STAT3 phosphorylation. Finally, we also found that E2 enhanced the cholesterol efflux to apoA I in RAW264.7 cells. In summary, E2 reduces atherosclerosis in ApoE null mice associated with upregulating ABCA1 expression and modulating the cholesterol efflux, which are dependent on SOCS3 upregulation. These results provide new insight into the athero-protective effects of estrogen. © Georg Thieme Verlag KG Stuttgart · New York.

Genetic variants of ABCA1 modify Alzheimer disease risk and quantitative traits related to beta-amyloid metabolism.

PubMed

Katzov, Hagit; Chalmers, Katy; Palmgren, Juni; Andreasen, Niels; Johansson, Boo; Cairns, Nigel J; Gatz, Margaret; Wilcock, Gordon K; Love, Seth; Pedersen, Nancy L; Brookes, Anthony J; Blennow, Kaj; Kehoe, Patrick G; Prince, Jonathan A

2004-04-01

Linkage studies have provided evidence that one or more loci on chromosome 9q influence Alzheimer disease (AD). The gene encoding the ATP-binding cassette A1 transporter (ABCA1) resides within proximity of previously identified linkage peaks and represents a plausible biological candidate for AD due to its central role in cellular lipid homeostasis. Several single nucleotide polymorphisms (SNPs) spanning ABCA1 have been genotyped and haplotype-based association analyses performed in four independent case-control samples, consisting of over 1,750 individuals from three European populations representing both early and late-onset AD. Prominent effects were observed for a common (H2) and rarer haplotype (H5) that were enriched in AD cases across studied populations (odds ratio [OR] 1.59, 95% confidence interval [CI] 1.36-1.82; P<0.00001 and OR 2.90; 95% CI 2.54-3.27; P<0.00001, respectively). Two other common haplotypes in the studied region (H1 and H3) were significantly under-represented in AD cases, suggesting that they may harbor alleles that decrease disease risk (OR 0.79, 95% CI 0.64-0.94; P=0.0065 and OR 0.70, 95% CI 0.46-0.93; P=0.011, respectively). While findings were significant in both early and late-onset samples, haplotype effects were more distinct in early-onset materials. For late-onset samples, ancillary evidence was obtained that both single marker alleles and haplotypes of ABCA1 contribute to variable cerebrospinal fluid tau and beta amyloid (Abeta42) protein levels, and brain Abeta load. Results indicate that variants of ABCA1 may affect the risk of AD, providing further support for a genetic link between AD and cholesterol metabolism. Copyright 2004 Wiley-Liss, Inc.

Glucagon-like peptide-1 contributes to increases ABCA1 expression by downregulating miR-758 to regulate cholesterol homeostasis.

PubMed

Yao, Yue; Li, Qiang; Gao, Ping; Wang, Wei; Chen, Lili; Zhang, Jinchao; Xu, Yi

2018-03-04

Abnormal regulation of lipid metabolism is associated with type 2 diabetes mellitus (T2DM). GLP-1 as a new treatment for T2DM, has unique effects in modulating cholesterol homeostasis. However, the mechanism of this effect is largely missing. The aim of this study was to determine the effects of GLP-1 on cholesterol-induced lipotoxicity in hepatocytes and examine the underlying mechanisms. The cell viability was determined, and caspase-3 was used to detect the effects of GLP-1 on cholesterol-induced apoptosis. The alterations of miR-758 and ATP-binding cassette transporter A1 (ABCA1) resulting from cholesterol incubation or GLP-1 were detected by qRT-PCR and Western blot assays. Overexpression of miR-758 abrogated the GLP-1-mediated ABCA1 expression, and conversely, down-regulation of miR-758 aggravated GLP-1's action and revealed significant promotion effects. BODIPY-Cholesterol efflux assay revealed that treatment with miR-758 inhibitor significantly enhanced ABCA1-dependent cholesterol efflux, resulting in reduced total cholesterol. Furthermore, Oil red O staining and cholesterol measurement were used to detect lipid accumulation. As a result, cholesterol significantly attenuated cell viability, promoted cell apoptosis, and facilitated lipid accumulation, and these effects were reversed by GLP-1. This study provides evidence that, in HepG2 cells, GLP-1 may affect cholesterol homeostasis by regulating the expression of miR-758 and ABCA1. These data can inform the development of biomarkers for miR-758, and potentially other drugs, on the key pathways of lipid metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

PubMed Central

2011-01-01

Background Synthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. Methods RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux. Results Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells. Conclusion 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway. PMID:22129452

Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1 macrophages.

PubMed

Mao, Min; Lei, Han; Liu, Qing; Chen, Yaxi; Zhao, Lei; Li, Qing; Luo, Suxin; Zuo, Zhong; He, Quan; Huang, Wei; Zhang, Nan; Zhou, Chao; Ruan, Xiong Z

2014-01-01

The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.

A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.

PubMed

Díaz-Badillo, Alvaro; Muñoz, María de Lourdes; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G; Martínez-Muñoz, Jorge P; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

2014-04-25

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

PubMed Central

Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

2014-01-01

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

PGD and aneuploidy screening for 24 chromosomes: advantages and disadvantages of competing platforms.

PubMed

Bisignano, A; Wells, D; Harton, G; Munné, S

2011-12-01

Diagnosis of embryos for chromosome abnormalities, i.e. aneuploidy screening, has been invigorated by the introduction of microarray-based testing methods allowing analysis of 24 chromosomes in one test. Recent data have been suggestive of increased implantation and pregnancy rates following microarray testing. Preimplantation genetic diagnosis for infertility aims to test for gross chromosome changes with the hope that identification and transfer of normal embryos will improve IVF outcomes. Testing by some methods, specifically single-nucleotide polymorphism (SNP) microarrays, allow for more information and potential insight into parental origin of aneuploidy and uniparental disomy. The usefulness and validity of reporting this information is flawed. Numerous papers have shown that the majority of meiotic errors occur in the egg, while mitotic errors in the embryo affect parental chromosomes at random. Potential mistakes made in assigning an error as meiotic or mitotic may lead to erroneous reporting of results with medical consequences. This study's data suggest that the bioinformatic cleaning used to 'fix' the miscalls that plague single-cell whole-genome amplification provides little improvement in the quality of useful data. Based on the information available, SNP-based aneuploidy screening suffers from a number of serious issues that must be resolved. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

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

Achyuthan, Komandoor E.; Wheeler, David R.

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of themore » YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.« less

Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

DOE PAGES

Achyuthan, Komandoor E.; Wheeler, David R.

2015-08-27

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of themore » YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.« less

Contribution of 20 single nucleotide polymorphisms of 13 genes to dyslipidemia associated with antiretroviral therapy.

PubMed

Arnedo, Mireia; Taffé, Patrick; Sahli, Roland; Furrer, Hansjakob; Hirschel, Bernard; Elzi, Luigia; Weber, Rainer; Vernazza, Pietro; Bernasconi, Enos; Darioli, Roger; Bergmann, Sven; Beckmann, Jacques S; Telenti, Amalio; Tarr, Philip E

2007-09-01

HIV-1 infected individuals have an increased cardiovascular risk which is partially mediated by dyslipidemia. Single nucleotide polymorphisms in multiple genes involved in lipid transport and metabolism are presumed to modulate the risk of dyslipidemia in response to antiretroviral therapy. The contribution to dyslipidemia of 20 selected single nucleotide polymorphisms of 13 genes reported in the literature to be associated with plasma lipid levels (ABCA1, ADRB2, APOA5, APOC3, APOE, CETP, LIPC, LIPG, LPL, MDR1, MTP, SCARB1, and TNF) was assessed by longitudinally modeling more than 4400 plasma lipid determinations in 438 antiretroviral therapy-treated participants during a median period of 4.8 years. An exploratory genetic score was tested that takes into account the cumulative contribution of multiple gene variants to plasma lipids. Variants of ABCA1, APOA5, APOC3, APOE, and CETP contributed to plasma triglyceride levels, particularly in the setting of ritonavir-containing antiretroviral therapy. Variants of APOA5 and CETP contributed to high-density lipoprotein-cholesterol levels. Variants of CETP and LIPG contributed to non-high-density lipoprotein-cholesterol levels, a finding not reported previously. Sustained hypertriglyceridemia and low high-density lipoprotein-cholesterol during the study period was significantly associated with the genetic score. Single nucleotide polymorphisms of ABCA1, APOA5, APOC3, APOE, and CETP contribute to plasma triglyceride and high-density lipoprotein-cholesterol levels during antiretroviral therapy exposure. Genetic profiling may contribute to the identification of patients at risk for antiretroviral therapy-related dyslipidemia.

Facile generation of cell microarrays using vacuum degassing and coverslip sweeping.

PubMed

Wang, Min S; Luo, Zhen; Cherukuri, Sundar; Nitin, Nitin

2014-07-15

A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays. Copyright © 2014 Elsevier Inc. All rights reserved.

Screening of biomarkers for prediction of response to and prognosis after chemotherapy for breast cancers

PubMed Central

Bing, Feng; Zhao, Yu

2016-01-01

Objective To screen the biomarkers having the ability to predict prognosis after chemotherapy for breast cancers. Methods Three microarray data of breast cancer patients undergoing chemotherapy were collected from Gene Expression Omnibus database. After preprocessing, data in GSE41112 were analyzed using significance analysis of microarrays to screen the differentially expressed genes (DEGs). The DEGs were further analyzed by Differentially Coexpressed Genes and Links to construct a function module, the prognosis efficacy of which was verified by the other two datasets (GSE22226 and GSE58644) using Kaplan–Meier plots. The involved genes in function module were subjected to a univariate Cox regression analysis to confirm whether the expression of each prognostic gene was associated with survival. Results A total of 511 DEGs between breast cancer patients who received chemotherapy or not were obtained, consisting of 421 upregulated and 90 downregulated genes. Using the Differentially Coexpressed Genes and Links package, 1,244 differentially coexpressed genes (DCGs) were identified, among which 36 DCGs were regulated by the transcription factor complex NFY (NFYA, NFYB, NFYC). These 39 genes constructed a gene module to classify the samples in GSE22226 and GSE58644 into three subtypes and these subtypes exhibited significantly different survival rates. Furthermore, several genes of the 39 DCGs were shown to be significantly associated with good (such as CDC20) and poor (such as ARID4A) prognoses following chemotherapy. Conclusion Our present study provided a serial of biomarkers for predicting the prognosis of chemotherapy or targets for development of alternative treatment (ie, CDC20 and ARID4A) in breast cancer patients. PMID:27217777

Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray

PubMed Central

Bayer, D K; Martinez, C A; Sorte, H S; Forbes, L R; Demmler-Harrison, G J; Hanson, I C; Pearson, N M; Noroski, L M; Zaki, S R; Bellini, W J; Leduc, M S; Yang, Y; Eng, C M; Patel, A; Rodningen, O K; Muzny, D M; Gibbs, R A; Campbell, I M; Shaw, C A; Baker, M W; Zhang, V; Lupski, J R; Orange, J S; Seeborg, F O; Stray-Pedersen, A

2014-01-01

In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8+ T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20–30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5–10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients. PMID:25046553

Recessive Stargardt Disease Phenocopying Hydroxychloroquine Retinopathy

PubMed Central

Noupuu, Kalev; Lee, Winston; Zernant, Jana; Greenstein, Vivienne C.; Tsang, Stephen; Allikmets, Rando

2015-01-01

Purpose To describe a series of Stargardt disease (STGD1) patients exhibiting a phenotype usually associated with hydroxychloroquine (HCQ) retinopathy on spectral domain-optical coherence tomography (SD-OCT). Methods Observational case series from Columbia University Medical Center involving 8 patients with genetically-confirmed STGD1. Patients selected for the study presented no history of HCQ use. Horizontal macular SD-OCT scans and accompanying 488nm autofluorescence (AF) images, color fundus photographs, and full-field electroretinograms were analyzed. Results All study patients exhibited an abrupt thinning of the parafoveal region or disruption of the outer retinal layers on SD-OCT resembling the transient HCQ retinopathy phenotype. Funduscopy and AF imaging revealed variations of bull’s eye maculopathy (BEM). Five patients exhibited local fleck-like deposits around the lesion. Genetic screening confirmed two disease-causing ABCA4 mutations in 5 patients and one mutation in 3 patients. Conclusions A transient SD- OCT phenotype ascribed to patients with HCQ retinopathy is associated with an early subtype of STGD1. This finding may also present with HCQ retinopathy-like BEM lesions on AF imaging and funduscopy. A phenotypic overlap may not be surprising given certain shared mechanistic disease processes between the two conditions. A thorough work-up, including screening of genes that are causal in retinal dystrophies associated with foveal sparing, may prevent the misdiagnoses of more ambiguous cases. PMID:26311262

Deployable Pan-Flavivirus and Pan-alphavirus Assays for Screening Pools of Medically Relevant Arthropod

DTIC Science & Technology

2011-09-01

Venezuelan equine encephalitis virus (TC-83 strain). The flaviviruses included West Nile Virus and Dengue 3 virus. We chose these organisms because these...and Venezuelan Equine Encephalitis (VEE, TC-83 strain). Flaviviruses: Dengue 3 and West Nile Virus (WNV). We purified these lysates using the...mixed 1:1 with 6xSSC. 30ul of each of these PCR mixes were incubated for 30 minutes in separate sectors of a 4x2k microarray with detection probes

Breast Reference Set Application: Karen Anderson-ASU (2014) — EDRN Public Portal

Cancer.gov

In order to increase the predictive value of tumor-specific antibodies for use as immunodiagnostics, our EDRN BDL has developed a novel protein microarray technology, termed Nucleic Acid Protein Programmable Array (NAPPA), which circumvents many of the limitations of traditional protein microarrays. NAPPA arrays are generated by printing full-length cDNAs encoding the target proteins at each feature of the array. The proteins are then transcribed and translated by a cell-free system and immobilized in situ using epitope tags fused to the proteins. Sera are added, and bound IgG is detected by standard secondary reagents. Using a sequential screening strategy to select AAb from 4,988 candidate tumor antigens, we have identified 28 potential AAb biomarkers for the early detection of breast cancer, and here we propose to evaluate these biomarkers using the EDRN Breast Cancer Reference Set.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration.

PubMed

Chen, Yuanyuan; Chen, Yu; Jastrzebska, Beata; Golczak, Marcin; Gulati, Sahil; Tang, Hong; Seibel, William; Li, Xiaoyu; Jin, Hui; Han, Yong; Gao, Songqi; Zhang, Jianye; Liu, Xujie; Heidari-Torkabadi, Hossein; Stewart, Phoebe L; Harte, William E; Tochtrop, Gregory P; Palczewski, Krzysztof

2018-05-17

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC 50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.

High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

PubMed

Landry, James P; Fei, Yiyan; Zhu, X D

2011-12-01

Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

PubMed

Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

2015-04-03

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Construction of robust prognostic predictors by using projective adaptive resonance theory as a gene filtering method.

PubMed

Takahashi, Hiro; Kobayashi, Takeshi; Honda, Hiroyuki

2005-01-15

For establishing prognostic predictors of various diseases using DNA microarray analysis technology, it is desired to find selectively significant genes for constructing the prognostic model and it is also necessary to eliminate non-specific genes or genes with error before constructing the model. We applied projective adaptive resonance theory (PART) to gene screening for DNA microarray data. Genes selected by PART were subjected to our FNN-SWEEP modeling method for the construction of a cancer class prediction model. The model performance was evaluated through comparison with a conventional screening signal-to-noise (S2N) method or nearest shrunken centroids (NSC) method. The FNN-SWEEP predictor with PART screening could discriminate classes of acute leukemia in blinded data with 97.1% accuracy and classes of lung cancer with 90.0% accuracy, while the predictor with S2N was only 85.3 and 70.0% or the predictor with NSC was 88.2 and 90.0%, respectively. The results have proven that PART was superior for gene screening. The software is available upon request from the authors. honda@nubio.nagoya-u.ac.jp

Systematic Omics Analysis Review (SOAR) Tool to Support Risk Assessment

PubMed Central

McConnell, Emma R.; Bell, Shannon M.; Cote, Ila; Wang, Rong-Lin; Perkins, Edward J.; Garcia-Reyero, Natàlia; Gong, Ping; Burgoon, Lyle D.

2014-01-01

Environmental health risk assessors are challenged to understand and incorporate new data streams as the field of toxicology continues to adopt new molecular and systems biology technologies. Systematic screening reviews can help risk assessors and assessment teams determine which studies to consider for inclusion in a human health assessment. A tool for systematic reviews should be standardized and transparent in order to consistently determine which studies meet minimum quality criteria prior to performing in-depth analyses of the data. The Systematic Omics Analysis Review (SOAR) tool is focused on assisting risk assessment support teams in performing systematic reviews of transcriptomic studies. SOAR is a spreadsheet tool of 35 objective questions developed by domain experts, focused on transcriptomic microarray studies, and including four main topics: test system, test substance, experimental design, and microarray data. The tool will be used as a guide to identify studies that meet basic published quality criteria, such as those defined by the Minimum Information About a Microarray Experiment standard and the Toxicological Data Reliability Assessment Tool. Seven scientists were recruited to test the tool by using it to independently rate 15 published manuscripts that study chemical exposures with microarrays. Using their feedback, questions were weighted based on importance of the information and a suitability cutoff was set for each of the four topic sections. The final validation resulted in 100% agreement between the users on four separate manuscripts, showing that the SOAR tool may be used to facilitate the standardized and transparent screening of microarray literature for environmental human health risk assessment. PMID:25531884

Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry*

PubMed Central

Palma, Angelina S.; Liu, Yan; Zhang, Hongtao; Zhang, Yibing; McCleary, Barry V.; Yu, Guangli; Huang, Qilin; Guidolin, Leticia S.; Ciocchini, Andres E.; Torosantucci, Antonella; Wang, Denong; Carvalho, Ana Luísa; Fontes, Carlos M. G. A.; Mulloy, Barbara; Childs, Robert A.; Feizi, Ten; Chai, Wengang

2015-01-01

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides. PMID:25670804

Panel-Based Clinical Genetic Testing in 85 Children with Inherited Retinal Disease.

PubMed

Taylor, Rachel L; Parry, Neil R A; Barton, Stephanie J; Campbell, Christopher; Delaney, Claire M; Ellingford, Jamie M; Hall, Georgina; Hardcastle, Claire; Morarji, Jiten; Nichol, Elisabeth J; Williams, Lindsi C; Douzgou, Sofia; Clayton-Smith, Jill; Ramsden, Simon C; Sharma, Vinod; Biswas, Susmito; Lloyd, I Chris; Ashworth, Jane L; Black, Graeme C; Sergouniotis, Panagiotis I

2017-07-01

To assess the clinical usefulness of genetic testing in a pediatric population with inherited retinal disease (IRD). Single-center retrospective case series. Eighty-five unrelated children with a diagnosis of isolated or syndromic IRD who were referred for clinical genetic testing between January 2014 and July 2016. Participants underwent a detailed ophthalmic examination, accompanied by electrodiagnostic testing (EDT) and dysmorphologic assessment where appropriate. Ocular and extraocular features were recorded using Human Phenotype Ontology terms. Subsequently, multigene panel testing (105 or 177 IRD-associated genes) was performed in an accredited diagnostic laboratory, followed by clinical variant interpretation. Diagnostic yield and clinical usefulness of genetic testing. Overall, 78.8% of patients (n = 67) received a probable molecular diagnosis; 7.5% (n = 5) of these had autosomal dominant disease, 25.4% (n = 17) had X-linked disease, and 67.2% (n = 45) had autosomal recessive disease. In a further 5.9% of patients (n = 5), a single heterozygous ABCA4 variant was identified; all these participants had a spectrum of clinical features consistent with ABCA4 retinopathy. Most participants (84.7%; n = 72) had undergone EDT and 81.9% (n = 59) of these patients received a probable molecular diagnosis. The genes most frequently mutated in the present cohort were CACNA1F and ABCA4, accounting for 14.9% (n = 10) and 11.9% (n = 8) of diagnoses respectively. Notably, in many cases, genetic testing helped to distinguish stationary from progressive IRD subtypes and to establish a precise diagnosis in a timely fashion. Multigene panel testing pointed to a molecular diagnosis in 84.7% of children with IRD. The diagnostic yield in the study population was significantly higher compared with that in previously reported unselected IRD cohorts. Approaches similar to the one described herein are expected to become a standard component of care in pediatric ophthalmology. We propose the introduction of genetic testing early in the diagnostic pathway in children with clinical and/or electrophysiologic findings, suggestive of IRD. Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube format.

PubMed

Schüler, Susann; Wenz, Ingrid; Wiederanders, B; Slickers, P; Ehricht, R

2006-06-12

Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes. Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures. In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well. As the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.

Screening of potential genes contributing to the macrocycle drug resistance of C. albicans via microarray analysis

PubMed Central

Yang, Jing; Zhang, Wei; Sun, Jian; Xi, Zhiqin; Qiao, Zusha; Zhang, Jinyu; Wang, Yan; Ji, Ying; Feng, Wenli

2017-01-01

The aim of the present study was to investigate the potential genes involved in drug resistance of Candida albicans (C. albicans) by performing microarray analysis. The gene expression profile of GSE65396 was downloaded from the Gene Expression Omnibus, including a control, 15-min and 45-min macrocyclic compound RF59-treated group with three repeats for each. Following preprocessing using RAM, the differentially expressed genes (DEGs) were screened using the Limma package. Subsequently, the Kyoto Encyclopedia of Genes and Genomes pathways of these genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery. Based on interactions estimated by the Search Tool for Retrieval of Interacting Gene, the protein-protein interaction (PPI) network was visualized using Cytoscape. Subnetwork analysis was performed using ReactomeFI. A total of 154 upregulated and 27 downregulated DEGs were identified in the 15-min treated group, compared with the control, and 235 upregulated and 233 downregulated DEGs were identified in the 45-min treated group, compared with the control. The upregulated DEGs were significantly enriched in the ribosome pathway. Based on the PPI network, PRP5, RCL1, NOP13, NOP4 and MRT4 were the top five nodes in the 15-min treated comparison. GIS2, URA3, NOP58, ELP3 and PLP7 were the top five nodes in the 45-min treated comparison, and its subnetwork was significantly enriched in the ribosome pathway. The macrocyclic compound RF59 had a notable effect on the ribosome and its associated pathways of C. albicans. RCL1, NOP4, MRT4, GIS2 and NOP58 may be important in RF59-resistance. PMID:28944888

On the mechanism for PPAR agonists to enhance ABCA1 gene expression

PubMed Central

Ogata, Masaki; Tsujita, Maki; Hossain, Mohammad Anwar; Akita, Nobukatsu; Gonzalez, Frank J.; Staels, Bart; Suzuki, Shogo; Fukutomi, Tatsuya; Kimura, Genjiro; Yokoyama, Shinji

2009-01-01

Expression of ATP binding cassette transporter A1 (ABCA1), a major regulator of high density lipoprotein (HDL) biogenesis, is known to be up-regulated by the transcription factor liver X receptor (LXR) α, and expression is further enhanced by activation of the peroxisome proliferator activated receptors (PPARs). We investigated this complex regulatory network using specific PPAR agonists: four fibrates (fenofibrate, bezafibrate, gemfibrozil and LY518674), a PPAR δ agonist (GW501516) and a PPAR γ agonist (pioglitazone). All of these compounds increased the expression of LXRs, PPARs and ABCA1 mRNAs, and associated apoA-I-mediated lipid release in THP-1 macrophage, WI38 fibroblast and mouse fibroblast. When mouse fibroblasts lacking expression of PPAR α were examined, the effects of fenofibrate and LY518674 were markedly diminished while induction by other ligands were retained. The PPAR α promoter was activated by all of these compounds in an LXR α-dependent manner, and partially in a PPAR α-dependent manner, in mouse fibroblast. The LXR responsive element (LXRE)-luciferase activity was enhanced by all the compounds in an LXR α-dependent manner in mouse fibroblast. This activation was exclusively PPAR α-dependent by fenofibrate and LY518674, but nonexclusively by the others. We conclude that PPARs and LXRs are involved in the regulation of ABCA1 expression and HDL biogenesis in a cooperative signal transduction pathway. PMID:19201410

Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury

PubMed Central

Pedigo, Christopher E.; Ducasa, Gloria Michelle; Leclercq, Farah; Sloan, Alexis; Hashmi, Tahreem; Molina-David, Judith; Ge, Mengyuan; Lassenius, Mariann I.; Groop, Per-Henrik; Kretzler, Matthias; Martini, Sebastian; Reich, Heather; Wahl, Patricia; Ghiggeri, GianMarco; Burke, George W.; Kretz, Oliver; Huber, Tobias B.; Mendez, Armando J.; Merscher, Sandra

2016-01-01

High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol–dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1–mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol–dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol–dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels. PMID:27482889

Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages

PubMed Central

Kim, Hye Sun; Park, Woo Jung; Kim, Joo-Yun; Chung, Dae Kyun

2016-01-01

Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent. PMID:27120199

Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages.

PubMed

Hong, Yi-Fan; Kim, Hangeun; Kim, Hye Sun; Park, Woo Jung; Kim, Joo-Yun; Chung, Dae Kyun

2016-01-01

Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent.

High-density polymer microarrays: identifying synthetic polymers that control human embryonic stem cell growth.

PubMed

Hansen, Anne; Mjoseng, Heidi K; Zhang, Rong; Kalloudis, Michail; Koutsos, Vasileios; de Sousa, Paul A; Bradley, Mark

2014-06-01

The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cosegregation and functional analysis of mutant ABCR (ABCA4) alleles in families that manifest both Stargardt disease and age-related macular degeneration.

PubMed

Shroyer, N F; Lewis, R A; Yatsenko, A N; Wensel, T G; Lupski, J R

2001-11-01

Mutations in ABCR (ABCA4) have been reported to cause a spectrum of autosomal recessively inherited retinopathies, including Stargardt disease (STGD), cone-rod dystrophy and retinitis pigmentosa. Individuals heterozygous for ABCR mutations may be predisposed to develop the multifactorial disorder age-related macular degeneration (AMD). We hypothesized that some carriers of STGD alleles have an increased risk to develop AMD. We tested this hypothesis in a cohort of families that manifest both STGD and AMD. With a direct-sequencing mutation detection strategy, we found that AMD-affected relatives of STGD patients are more likely to be carriers of pathogenic STGD alleles than predicted based on chance alone. We further investigated the role of AMD-associated ABCR mutations by testing for expression and ATP-binding defects in an in vitro biochemical assay. We found that mutations associated with AMD have a range of assayable defects ranging from no detectable defect to apparent null alleles. Of the 21 missense ABCR mutations reported in patients with AMD, 16 (76%) show abnormalities in protein expression, ATP-binding or ATPase activity. We infer that carrier relatives of STGD patients are predisposed to develop AMD.

C20-D3-vitamin A Slows Lipofuscin Accumulation and Electrophysiological Retinal Degeneration in a Mouse Model of Stargardt Disease*

PubMed Central

Ma, Li; Kaufman, Yardana; Zhang, Junhua; Washington, Ilyas

2011-01-01

Stargardt disease, also known as juvenile macular degeneration, occurs in approximately one in 10,000 people and results from genetic defects in the ABCA4 gene. The disease is characterized by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE) of the eye and by vision loss. No cure or treatment is available. Although lipofuscin is considered a hallmark of Stargardt disease, its mechanism of formation and its role in disease pathogenesis are poorly understood. In this work we investigated the effects of long-term administration of deuterium-enriched vitamin A, C20-D3-vitamin A, on RPE lipofuscin deposition and eye function in a mouse model of Stargardt's disease. Results support the notion that lipofuscin forms partly as a result of the aberrant reactivity of vitamin A through the formation of vitamin A dimers, provide evidence that preventing vitamin A dimerization may slow disease related, retinal physiological changes and perhaps vision loss and suggest that administration of C20-D3-vitamin A may be a potential clinical strategy to ameliorate clinical symptoms resulting from ABCA4 genetic defects. PMID:21156790

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

Protein Microarray Analysis in Patients With Asthma*

PubMed Central

Kim, Hyo-Bin; Kim, Chang-Keun; Iijima, Koji; Kobayashi, Takao; Kita, Hirohito

2010-01-01

Background Microarray technology offers a new opportunity to gain insight into global gene and protein expression profiles in asthma. To identify novel factors produced in the asthmatic airway, we analyzed sputum samples by using a membrane-based human cytokine microarray technology in patients with bronchial asthma (BA). Methods Induced sputum was obtained from 28 BA subjects, 20 nonasthmatic atopic control (AC) subjects, and 38 nonasthmatic nonatopic normal control (NC) subjects. The microarray samples of subjects were randomly selected from nine BA subjects, three AC subjects, and six NC subjects. Sputum supernatants were analyzed using a custom human cytokine array (RayBio Custom Human Cytokine Array; RayBiotech; Norcross, GA) designed to analyze 79 specific cytokines simultaneously. The levels of growth-regulated oncogene (GRO)-α, eotaxin-2, and pulmonary and activation-regulated chemokine (PARC)/CCL18 were measured by sandwich enzyme-linked immunosorbent assays (ELISAs), and eosinophil-derived neurotoxin (EDN) was measured by radioimmunoassay. Results By microarray, the signal intensities for GRO-α, eotaxin-2, and PARC were significantly higher in BA subjects than in AC and NC subjects (p = 0.036, p = 0.042, and p = 0.033, respectively). By ELISA, the sputum PARC protein levels were significantly higher in BA subjects than in AC and NC subjects (p < 0.0001). Furthermore, PARC levels correlated significantly with sputum eosinophil percentages (r = 0.570, p < 0.0001) and the levels of EDN(r = 0.633, p < 0.0001), the regulated upon activation, normal T cell expressed and secreted cytokine (r = 0.440, p < 0.001), interleukin-4 (r = 0.415, p < 0.01), and interferon-γ (r = 0.491, p < 0.001). Conclusions By a nonbiased screening approach, a chemokine, PARC, is elevated in sputum specimens from patients with asthma. PARC may play important roles in development of airway eosinophilic inflammation in asthma. PMID:19017877

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

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminatedmore » the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.« less

Cholesterol efflux is differentially regulated in neurons and astrocytes: implications for brain cholesterol homeostasis

PubMed Central

Chen, Jing; Zhang, Xiaolu; Kusumo, Handojo; Costa, Lucio G.; Guizzetti, Marina

2012-01-01

Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood-brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS. PMID:23010475

Andrographolide Inhibits Oxidized LDL-Induced Cholesterol Accumulation and Foam Cell Formation in Macrophages.

PubMed

Lin, Hung-Chih; Lii, Chong-Kuei; Chen, Hui-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Chen, Haw-Wen

2018-01-01

oxLDL is involved in the pathogenesis of atherosclerotic lesions through cholesterol accumulation in macrophage foam cells. Andrographolide, the bioactive component of Andrographis paniculata, possesses several biological activities such as anti-inflammatory, anti-oxidant, and anticancer functions. Scavenger receptors (SRs), including class A SR (SR-A) and CD36, are responsible for the internalization of oxLDL. In contrast, receptors for reverse cholesterol transport, including ABCA1 and ABCG1, mediate the efflux of cholesterol from macrophage foam cells. Transcription factor liver X receptor [Formula: see text] (LXR[Formula: see text] plays a key role in lipid metabolism and inflammation as well as in the regulation of ABCA1 and ABCG1 expression. Because of the contribution of inflammation to macrophage foam cell formation and the potent anti-inflammatory activity of andrographolide, we hypothesized that andrographolide might inhibit oxLDL-induced macrophage foam cell formation. The results showed that andrographolide reduced oxLDL-induced lipid accumulation in macrophage foam cells. Andrographolide decreased the mRNA and protein expression of CD36 by inducing the degradation of CD36 mRNA; however, andrographolide had no effect on SR-A expression. In contrast, andrographolide increased the mRNA and protein expression of ABCA1 and ABCG1, which were dependent on LXR[Formula: see text]. Andrographolide enhanced LXR[Formula: see text] nuclear translocation and DNA binding activity. Treatment with the LXR[Formula: see text] antagonist GGPP and transfection with LXR[Formula: see text] siRNA reversed the ability of andrographolide to stimulate ABCA1 and ABCG1 protein expression. In conclusion, inhibition of CD36-mediated oxLDL uptake and induction of ABCA1- and ABCG1-dependent cholesterol efflux are two working mechanisms by which andrographolide inhibits macrophage foam cell formation, which suggests that andrographolide could be a potential candidate to prevent atherosclerosis.

ABCA7 frameshift deletion associated with Alzheimer disease in African Americans

PubMed Central

Cukier, Holly N.; Kunkle, Brian W.; Vardarajan, Badri N.; Rolati, Sophie; Hamilton-Nelson, Kara L.; Kohli, Martin A.; Whitehead, Patrice L.; Dombroski, Beth A.; Van Booven, Derek; Lang, Rosalyn; Dykxhoorn, Derek M.; Farrer, Lindsay A.; Cuccaro, Michael L.; Vance, Jeffery M.; Gilbert, John R.; Beecham, Gary W.; Martin, Eden R.; Carney, Regina M.; Mayeux, Richard; Schellenberg, Gerard D.; Byrd, Goldie S.; Haines, Jonathan L.

2016-01-01

Objective: To identify a causative variant(s) that may contribute to Alzheimer disease (AD) in African Americans (AA) in the ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene, a known risk factor for late-onset AD. Methods: Custom capture sequencing was performed on ∼150 kb encompassing ABCA7 in 40 AA cases and 37 AA controls carrying the AA risk allele (rs115550680). Association testing was performed for an ABCA7 deletion identified in large AA data sets (discovery n = 1,068; replication n = 1,749) and whole exome sequencing of Caribbean Hispanic (CH) AD families. Results: A 44-base pair deletion (rs142076058) was identified in all 77 risk genotype carriers, which shows that the deletion is in high linkage disequilibrium with the risk allele. The deletion was assessed in a large data set (531 cases and 527 controls) and, after adjustments for age, sex, and APOE status, was significantly associated with disease (p = 0.0002, odds ratio [OR] = 2.13 [95% confidence interval (CI): 1.42–3.20]). An independent data set replicated the association (447 cases and 880 controls, p = 0.0117, OR = 1.65 [95% CI: 1.12–2.44]), and joint analysis increased the significance (p = 1.414 × 10−5, OR = 1.81 [95% CI: 1.38–2.37]). The deletion is common in AA cases (15.2%) and AA controls (9.74%), but in only 0.12% of our non-Hispanic white cohort. Whole exome sequencing of multiplex, CH families identified the deletion cosegregating with disease in a large sibship. The deleted allele produces a stable, detectable RNA strand and is predicted to result in a frameshift mutation (p.Arg578Alafs) that could interfere with protein function. Conclusions: This common ABCA7 deletion could represent an ethnic-specific pathogenic alteration in AD. PMID:27231719

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

Yue, Jianmei; Department of Endocrinology, The First Hospital of Zibo, 4# E Mei Shan Dong Road, Zibo 255200; Li, Bo, E-mail: libosubmit@163.com

Objectives: Cholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages. Methods: After PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [{sup 3}H] cholesterol (1.0 μCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL{sub 2}more » or HDL{sub 3}. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism. Results: The results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα. Conclusion: These results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway. - Highlights: • Sal B promotes the expression of ABCA1. • Sal B promotes cholesterol efflux in macrophages. • Sal B promotes the expression of ABCA1 and cholesterol efflux through PPAR-γ/LXRα signaling pathway.« less

Gene-Gene Combination Effect and Interactions among ABCA1, APOA1, SR-B1, and CETP Polymorphisms for Serum High-Density Lipoprotein-Cholesterol in the Japanese Population

PubMed Central

Nakamura, Akihiko; Niimura, Hideshi; Kuwabara, Kazuyo; Takezaki, Toshiro; Morita, Emi; Wakai, Kenji; Hamajima, Nobuyuki; Nishida, Yuichiro; Turin, Tanvir Chowdhury; Suzuki, Sadao; Ohnaka, Keizo; Uemura, Hirokazu; Ozaki, Etsuko; Hosono, Satoyo; Mikami, Haruo; Kubo, Michiaki; Tanaka, Hideo

2013-01-01

Background/Objective Gene-gene interactions in the reverse cholesterol transport system for high-density lipoprotein-cholesterol (HDL-C) are poorly understood. The present study observed gene-gene combination effect and interactions between single nucleotide polymorphisms (SNPs) in ABCA1, APOA1, SR-B1, and CETP in serum HDL-C from a cross-sectional study in the Japanese population. Methods The study population comprised 1,535 men and 1,515 women aged 35–69 years who were enrolled in the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. We selected 13 SNPs in the ABCA1, APOA1, CETP, and SR-B1 genes in the reverse cholesterol transport system. The effects of genetic and environmental factors were assessed using general linear and logistic regression models after adjusting for age, sex, and region. Principal Findings Alcohol consumption and daily activity were positively associated with HDL-C levels, whereas smoking had a negative relationship. The T allele of CETP, rs3764261, was correlated with higher HDL-C levels and had the highest coefficient (2.93 mg/dL/allele) among the 13 SNPs, which was statistically significant after applying the Bonferroni correction (p<0.001). Gene-gene combination analysis revealed that CETP rs3764261 was associated with high HDL-C levels with any combination of SNPs from ABCA1, APOA1, and SR-B1, although no gene-gene interaction was apparent. An increasing trend for serum HDL-C was also observed with an increasing number of alleles (p<0.001). Conclusions The present study identified a multiplier effect from a polymorphism in CETP with ABCA1, APOA1, and SR-B1, as well as a dose-dependence according to the number of alleles present. PMID:24376512

Variants in the ATP-Binding Cassette Transporter (ABCA7), Apolipoprotein E ε4, and the Risk of Late-Onset Alzheimer Disease in African Americans

PubMed Central

Reitz, Christiane; Jun, Gyungah; Naj, Adam; Rajbhandary, Ruchita; Vardarajan, Badri Narayan; Wang, Li-San; Valladares, Otto; Lin, Chiao-Feng; Larson, Eric B.; Graff-Radford, Neill R.; Evans, Denis; De Jager, Philip L.; Crane, Paul K.; Buxbaum, Joseph D.; Murrell, Jill R.; Raj, Towfique; Ertekin-Taner, Nilufer; Logue, Mark; Baldwin, Clinton T.; Green, Robert C.; Barnes, Lisa L.; Cantwell, Laura B.; Fallin, M. Daniele; Go, Rodney C. P.; Griffith, Patrick; Obisesan, Thomas O.; Manly, Jennifer J.; Lunetta, Kathryn L.; Kamboh, M. Ilyas; Lopez, Oscar L.; Bennett, David A.; Hendrie, Hugh; Hall, Kathleen S.; Goate, Alison M.; Byrd, Goldie S.; Kukull, Walter A.; Foroud, Tatiana M.; Haines, Jonathan L.; Farrer, Lindsay A.; Pericak-Vance, Margaret A.; Schellenberg, Gerard D.; Mayeux, Richard

2013-01-01

Importance Genetic variants associated with susceptibility to late-onset Alzheimer disease are known for individuals of European ancestry, but whether the same or different variants account for the genetic risk of Alzheimer disease in African American individuals is unknown. Identification of disease-associated variants helps identify targets for genetic testing, prevention, and treatment. Objective To identify genetic loci associated with late-onset Alzheimer disease in African Americans. Design, Setting, and Participants The Alzheimer Disease Genetics Consortium (ADGC) assembled multiple data sets representing a total of 5896 African Americans (1968 case participants, 3928 control participants) 60 years or older that were collected between 1989 and 2011 at multiple sites. The association of Alzheimer disease with genotyped and imputed single-nucleotide polymorphisms (SNPs) was assessed in case-control and in family-based data sets. Results from individual data sets were combined to perform an inverse variance–weighted meta-analysis, first with genome-wide analyses and subsequently with gene-based tests for previously reported loci. Main Outcomes and Measures Presence of Alzheimer disease according to standardized criteria. Results Genome-wide significance in fully adjusted models (sex, age, APOE genotype, population stratification) was observed for a SNP in ABCA7 (rs115550680, allele = G; frequency, 0.09 cases and 0.06 controls; odds ratio [OR], 1.79 [95% CI, 1.47-2.12]; P = 2.2 × 10–9), which is in linkage disequilibrium with SNPs previously associated with Alzheimer disease in Europeans (0.8

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples.

PubMed

Sanchis, Ana; Salvador, J-Pablo; Campbell, Katrina; Elliott, Christopher T; Shelver, Weilin L; Li, Qing X; Marco, M-Pilar

2018-07-01

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal or polyclonal antibodies raised against important families of chemical pollutants such as triazine biocide (i.e. Irgarol 1051®), sulfonamide and chloramphenicol antibiotics, polybrominated diphenyl ether flame-retardant (PBDE, i.e. BDE-47), hormone (17β-estradiol), and algae toxin (domoic acid). These contaminants were selected as model analytes, however, the platform developed has the potential to detect a broader group of compounds based on the cross-reactivity of the immunoreagents used. The microarray chip is able to simultaneously determine these families of contaminants directly in seawater samples reaching limits of detection close to the levels found in contaminated areas (Irgarol 1051®, 0.19 ± 0,06 µg L -1 ; sulfapyridine, 0.17 ± 0.07 µg L -1 ; chloramphenicol, 0.11 ± 0.03 µg L -1 ; BDE-47, 2.71 ± 1.13 µg L -1 ; 17β-estradiol, 0.94 ± 0.30 µg L -1 and domoic acid, 1.71 ± 0.30 µg L -1 ). Performance of the multiplexed microarray chip was assessed by measuring 38 blind spiked seawater samples containing either one of these contaminants or mixtures of them. The accuracy found was very good and the coefficient of variation was < 20% in all the cases. No sample pre-treatment was necessary, and the results could be obtained in just 1 h 30 min. The microarray shows high sample throughput capabilities, being able to measure simultaneously more than 68 samples and screen them for a significant number of chemical contaminants of interest in environmental screening programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Preventive effects of heregulin-beta1 on macrophage foam cell formation and atherosclerosis.

PubMed

Xu, Gang; Watanabe, Takuya; Iso, Yoshitaka; Koba, Shinji; Sakai, Tetsuo; Nagashima, Masaharu; Arita, Shigeko; Hongo, Shigeki; Ota, Hidekazu; Kobayashi, Youichi; Miyazaki, Akira; Hirano, Tsutomu

2009-08-28

Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1. The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis. Plasma heregulin-beta(1) levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3+/-0.3, 2.0+/-0.4 versus 7.6+/-1.4, 8.2+/-1.2 ng/mL; P<0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-beta(1) levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-beta(1) was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-beta(1), but not heregulin-alpha, significantly reduced acetylated low-density lipoprotein-induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-beta(1) significantly decreased endocytic uptake of [(125)I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-beta(1) into ApoE(-/-) mice significantly suppressed the development of atherosclerotic lesions. This study provided the first evidence that heregulin-beta(1) inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

Improved microarray methods for profiling the yeast knockout strain collection

PubMed Central

Yuan, Daniel S.; Pan, Xuewen; Ooi, Siew Loon; Peyser, Brian D.; Spencer, Forrest A.; Irizarry, Rafael A.; Boeke, Jef D.

2005-01-01

A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens. PMID:15994458

An efficient method to identify differentially expressed genes in microarray experiments

PubMed Central

Qin, Huaizhen; Feng, Tao; Harding, Scott A.; Tsai, Chung-Jui; Zhang, Shuanglin

2013-01-01

Motivation Microarray experiments typically analyze thousands to tens of thousands of genes from small numbers of biological replicates. The fact that genes are normally expressed in functionally relevant patterns suggests that gene-expression data can be stratified and clustered into relatively homogenous groups. Cluster-wise dimensionality reduction should make it feasible to improve screening power while minimizing information loss. Results We propose a powerful and computationally simple method for finding differentially expressed genes in small microarray experiments. The method incorporates a novel stratification-based tight clustering algorithm, principal component analysis and information pooling. Comprehensive simulations show that our method is substantially more powerful than the popular SAM and eBayes approaches. We applied the method to three real microarray datasets: one from a Populus nitrogen stress experiment with 3 biological replicates; and two from public microarray datasets of human cancers with 10 to 40 biological replicates. In all three analyses, our method proved more robust than the popular alternatives for identification of differentially expressed genes. Availability The C++ code to implement the proposed method is available upon request for academic use. PMID:18453554

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

PubMed

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Genetic basis for childhood interstitial lung disease among Japanese infants and children.

PubMed

Hayasaka, Itaru; Cho, Kazutoshi; Akimoto, Takuma; Ikeda, Masahiko; Uzuki, Yutaka; Yamada, Masafumi; Nakata, Koh; Furuta, Itsuko; Ariga, Tadashi; Minakami, Hisanori

2018-02-01

BackgroundGenetic variants responsible for childhood interstitial lung disease (chILD) have not been studied extensively in Japanese patients.MethodsThe study population consisted of 62 Japanese chILD patients. Twenty-one and four patients had pulmonary hypertension resistant to treatment (PH) and hypothyroidism, respectively. Analyses of genetic variants were performed in all 62 patients for SFTPC and ABCA3, in all 21 PH patients for FOXF1, and in a limited number of patients for NKX2.1.ResultsCausative genetic variants for chILD were identified in 11 (18%) patients: SFTPC variants in six, NKX2.1 variants in three, and FOXF1 variants in two patients. No patients had ABCA3 variants. All three and two patients with NKX2.1 variants had hypothyroidism and developmental delay, respectively. We found six novel variants in this study.ConclusionMutations in SFTPC, NKX2.1, and FOXF1 were identified among Japanese infants and children with chILD, whereas ABCA3 mutations were rare.

Reduced Plasma HDL Cholesterol in Hyperthyroid Mice Coincides with Decreased Hepatic ABCA1 Expression

PubMed Central

TANCEVSKI, IVAN; WEHINGER, ANDREAS; DEMETZ, EGON; ELLER, PHILIPP; DUWENSEE, KRISTINA; HUBER, JULIA; HOCHEGGER, KATHRIN; SCHGOER, WILFRIED; FIEVET, CATHERINE; STELLAARD, FRANS; RUDLING, MATS; PATSCH, JOSEF R.; RITSCH, ANDREAS

2010-01-01

The aim of the study was to investigate the influence of severe hyperthyroidism on plasma high-density lipoprotein cholesterol (HDL-C). Recently, it was shown in mice that increasing doses of triiodothyronine (T3) upregulate hepatic expression of scavenger receptor-BI (SR-BI), resulting in increased clearance of plasma HDL-C. Here we show that severe hyperthyroidism in mice did not affect hepatic expression of SR-BI, but reduced hepatic expression of ATP-binding cassette transporter 1 (ABCA1), accompanied by a 40%-reduction of HDL-C. Sterol content of bile, liver and feces was markedly increased, accompanied by upregulation of hepatic CYP7A1, and ATP-binding cassette half-transporter ABCG5, which is known to promote biliary sterol secretion upon dimerization with ABCG8. Both control and hyperthyroid mice exerted identical plasma clearance of intravenously injected [3H] HDL-C, supporting the view that severe hyperthyroidism does not affect HDL-C clearance, but rather its formation via hepatic ABCA1. PMID:18388200

Multilayer-omics analysis of renal cell carcinoma, including the whole exome, methylome and transcriptome.

PubMed

Arai, Eri; Sakamoto, Hiromi; Ichikawa, Hitoshi; Totsuka, Hirohiko; Chiku, Suenori; Gotoh, Masahiro; Mori, Taisuke; Nakatani, Tamao; Ohnami, Sumiko; Nakagawa, Tohru; Fujimoto, Hiroyuki; Wang, Linghua; Aburatani, Hiroyuki; Yoshida, Teruhiko; Kanai, Yae

2014-09-15

The aim of this study was to identify pathways that have a significant impact during renal carcinogenesis. Sixty-seven paired samples of both noncancerous renal cortex tissue and cancerous tissue from patients with clear cell renal cell carcinomas (RCCs) were subjected to whole-exome, methylome and transcriptome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Illumina Infinium HumanMethylation27 BeadArray and Agilent SurePrint Human Gene Expression microarray, respectively. Sanger sequencing and quantitative reverse transcription-PCR were performed for technical verification. MetaCore software was used for pathway analysis. Somatic nonsynonymous single-nucleotide mutations, insertions/deletions and intragenic breaks of 2,153, 359 and 8 genes were detected, respectively. Mutations of GCN1L1, MED12 and CCNC, which are members of CDK8 mediator complex directly regulating β-catenin-driven transcription, were identified in 16% of the RCCs. Mutations of MACF1, which functions in the Wnt/β-catenin signaling pathway, were identified in 4% of the RCCs. A combination of methylome and transcriptome analyses further highlighted the significant role of the Wnt/β-catenin signaling pathway in renal carcinogenesis. Genetic aberrations and reduced expression of ERC2 and ABCA13 were frequent in RCCs, and MTOR mutations were identified as one of the major disrupters of cell signaling during renal carcinogenesis. Our results confirm that multilayer-omics analysis can be a powerful tool for revealing pathways that play a significant role in carcinogenesis. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Multilayer-omics analysis of renal cell carcinoma, including the whole exome, methylome and transcriptome

PubMed Central

Arai, Eri; Sakamoto, Hiromi; Ichikawa, Hitoshi; Totsuka, Hirohiko; Chiku, Suenori; Gotoh, Masahiro; Mori, Taisuke; Nakatani, Tamao; Ohnami, Sumiko; Nakagawa, Tohru; Fujimoto, Hiroyuki; Wang, Linghua; Aburatani, Hiroyuki; Yoshida, Teruhiko; Kanai, Yae

2014-01-01

The aim of this study was to identify pathways that have a significant impact during renal carcinogenesis. Sixty-seven paired samples of both noncancerous renal cortex tissue and cancerous tissue from patients with clear cell renal cell carcinomas (RCCs) were subjected to whole-exome, methylome and transcriptome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Illumina Infinium HumanMethylation27 BeadArray and Agilent SurePrint Human Gene Expression microarray, respectively. Sanger sequencing and quantitative reverse transcription-PCR were performed for technical verification. MetaCore software was used for pathway analysis. Somatic nonsynonymous single-nucleotide mutations, insertions/deletions and intragenic breaks of 2,153, 359 and 8 genes were detected, respectively. Mutations of GCN1L1, MED12 and CCNC, which are members of CDK8 mediator complex directly regulating β-catenin-driven transcription, were identified in 16% of the RCCs. Mutations of MACF1, which functions in the Wnt/β-catenin signaling pathway, were identified in 4% of the RCCs. A combination of methylome and transcriptome analyses further highlighted the significant role of the Wnt/β-catenin signaling pathway in renal carcinogenesis. Genetic aberrations and reduced expression of ERC2 and ABCA13 were frequent in RCCs, and MTOR mutations were identified as one of the major disrupters of cell signaling during renal carcinogenesis. Our results confirm that multilayer-omics analysis can be a powerful tool for revealing pathways that play a significant role in carcinogenesis. PMID:24504440

Regulation of gene expression in the mammalian eye and its relevance to eye disease.

PubMed

Scheetz, Todd E; Kim, Kwang-Youn A; Swiderski, Ruth E; Philp, Alisdair R; Braun, Terry A; Knudtson, Kevin L; Dorrance, Anne M; DiBona, Gerald F; Huang, Jian; Casavant, Thomas L; Sheffield, Val C; Stone, Edwin M

2006-09-26

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

Printing Proteins as Microarrays for High-Throughput Function Determination

NASA Astrophysics Data System (ADS)

MacBeath, Gavin; Schreiber, Stuart L.

2000-09-01

Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

Mapping the affinity landscape of Thrombin-binding aptamers on 2΄F-ANA/DNA chimeric G-Quadruplex microarrays

PubMed Central

Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos

2017-01-01

Abstract In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2΄-Fluoroarabinonucleic acid (2΄F-ANA) is a prime candidate for such use in microarrays. Indeed, 2΄F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2΄F-ANA and 2΄F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2΄F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2΄F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2΄F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2΄F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays. PMID:28100695

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.

PubMed

Drobnik, Wolfgang; Borsukova, Hana; Böttcher, Alfred; Pfeiffer, Alexandra; Liebisch, Gerhard; Schütz, Gerhard J; Schindler, Hansgeorg; Schmitz, Gerd

2002-04-01

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL3-mediated lipid efflux.

A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens

PubMed Central

Dotsey, Emmanuel Y.; Gorlani, Andrea; Ingale, Sampat; Achenbach, Chad J.; Forthal, Donald N.; Felgner, Philip L.; Gach, Johannes S.

2015-01-01

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV. PMID:25938510

Association studies of several cholesterol-related genes (ABCA1, CETP and LIPC) with serum lipids and risk of Alzheimer’s disease

PubMed Central

2012-01-01

Objectives Accumulating evidence suggested that dysregulation of cholesterol homeostasis might be a major etiologic factor in initiating and promoting neurodegeneration in Alzheimer’s disease (AD). ATP-binding cassette transporter A1 (ABCA1), hepatic lipase (HL, coding genes named LIPC) and cholesteryl ester transfer protein (CETP) are important components of high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT) implicated in atherosclerosis and neurodegenerative diseases. In the present study, we will investigate the possible association of several common polymorphisms (ABCA1R219K, CETPTaqIB and LIPC-250 G/A) with susceptibility to AD and plasma lipid levels. Methods Case–control study of 208 Han Chinese (104 AD patients and 104 non-demented controls) from Changsha area in Hunan Province was performed using the PCR-RFLP analysis. Cognitive decline was assessed using Mini Mental State Examination (MMSE) as a standardized method. Additionally, fasting lipid profile and the cognitive testing scores including Wechsler Memory Scale (WMS) and Wisconsin Card Sorting Test (WCST) were recorded. Results and conclusions We found significant differences among the genotype distributions of these three genes in AD patients when compared with controls. But after adjusting other factors, multivariate logistic regression analysis showed only ABCA1R219K (B = −0.903, P = 0.005, OR = 0.405, 95%CI:0.217-0.758) and LIPC-250 G/A variants(B = −0.905, P = 0.018, OR = 0.405, 95%CI:0.191-0.858) were associated with decreased AD risk. There were significantly higher levels of high-density lipoprotein cholesterol (HDL-C) and apolipoproteinA-I in the carriers of KK genotype and K allele (P < 0.05), and B2B2 genotype of CETP Taq1B showed significant association with higher HDL-C levels than other genotypes (F = 5.598, P = 0.004), while -250 G/A polymorphisms had no significant effect on HDL-C. In total population, subjects carrying ABCA1219K allele or LIPC-250A allele obtained higher MMSE or WMS scores than non-carriers, however, no significant association was observed in AD group or controls. Therefore, this preliminary study showed that the gene variants of ABCA1R219K and LIPC-250 G/A might influence AD susceptibility in South Chinese Han population, but the polymorphism of CETPTaq1B didn't show any association in despite of being a significant determinant of HDL-C. PMID:23181436

Microarray expression profiling identifies genes with altered expression in HDL-deficient mice

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

Callow, Matthew J.; Dudoit, Sandrine; Gong, Elaine L.

2000-05-05

Based on the assumption that severe alterations in the expression of genes known to be involved in HDL metabolism may affect the expression of other genes we screened an array of over 5000 mouse expressed sequence tags (ESTs) for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apo AI) knockout mice, Scavenger Receptor BI (SR-BI) transgenic mice and control mice were co-hybridized to microarrays. Two-sample t-statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared withmore » the control mice. In the SR-BI group we found 9 array elements representing at least 5 genes to be significantly altered on the basis of an adjusted p value of less than 0.05. In the apo AI knockout group 8 array elements representing 4 genes were altered compared with the control group (p < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments of apo AI knockout and SR-BI transgenic mice.« less

[DNA microarray reveals changes in gene expression of endothelial cells under shear stress].

PubMed

Cheng, Min; Zhang, Wensheng; Chen, Huaiqing; Wu, Wenchao; Huang, Hua

2004-04-01

cDNA microarray technology is used as a powerful tool for rapid, comprehensive, and quantitative analysis of gene profiles of cultured human umbilical vein endothelial cells(HUVECs) in the normal static group and the shear stressed (4.20 dyne/cm2, 2 h) group. The total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP, and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP. The expression ratios reported are the average from the two separate experiments. After bioinformatics analysis, we identified a total of 108 genes (approximately 0.026%) revealing differential expression. Of these 53 genes expressions were up-regulated, the most enhanced ones being human homolog of yeast IPP isomerase, human low density lipoprotein receptor gene, Squalene epoxidase gene, 7-dehydrocholesterol reductase, and 55 were down-regulated, the most decreased ones being heat shock 70 kD protein 1, TCB gene encoding cytosolic thyroid hormone-binding protein in HUVECs exposed to low shear stress. These results indicate that the cDNA microarray technique is effective in screening the differentially expressed genes in endothelial cells induced by various experimental conditions and the data may serve as stimuli to further researches.

Diazonium-protein adducts for graphite electrode microarrays modification: direct and addressed electrochemical immobilization.

PubMed

Corgier, Benjamin P; Marquette, Christophe A; Blum, Loïc J

2005-12-28

Diazonium cation electrodeposition was investigated for the direct and electro-addressed immobilization of proteins. For the first time, this reaction was triggered directly onto diazonium-modified proteins. Screen-printed (SP) graphite electrode microarrays were studied as active support for this immobilization. A 10-microelectrode (eight working electrodes, 0.2 mm2 each; one reference; and one auxiliary) setup was used to study the addressing possibilities of the method. These electrode microarrays were shown to be able to covalently graft diazonium cations through electrochemical reduction. Cyclic voltammetry and X-ray photoelectron spectroscopy were used to characterize the electrochemical grafting onto our SP graphite surface and suggested that a diazonium monolayer was deposited. Rabbit and human immunoglobulins (IgGs) were then chemically coupled to an aniline derivative (4-carboxymethylaniline), followed by diazotation to form an aryl diazonium function available for the electrodeposition. These modified proteins were both successfully electro-addressed at the surface of the graphite electrodes without cross-talk or interference. The immuno-biochip obtained using this novel approach enabled the specific detection of anti-rabbit IgG antibodies with a detection limit of 50 fmol of protein. A promising strategy to immobilize markedly different biological entities was then presented, providing an excellent spatial specificity of the electro-addressing.

A Rational Approach for Discovering and Validating Cancer Markers in Very Small Samples Using Mass Spectrometry and ELISA Microarrays

DOE PAGES

Zangar, Richard C.; Varnum, Susan M.; Covington, Chandice Y.; ...

2004-01-01

Identifying useful markers of cancer can be problematic due to limited amounts of sample. Some samples such as nipple aspirate fluid (NAF) or early-stage tumors are inherently small. Other samples such as serum are collected in larger volumes but archives of these samples are very valuable and only small amounts of each sample may be available for a single study. Also, given the diverse nature of cancer and the inherent variability in individual protein levels, it seems likely that the best approach to screen for cancer will be to determine the profile of a battery of proteins. As a result,more » a major challenge in identifying protein markers of disease is the ability to screen many proteins using very small amounts of sample. In this review, we outline some technological advances in proteomics that greatly advance this capability. Specifically, we propose a strategy for identifying markers of breast cancer in NAF that utilizes mass spectrometry (MS) to simultaneously screen hundreds or thousands of proteins in each sample. The best potential markers identified by the MS analysis can then be extensively characterized using an ELISA microarray assay. Because the microarray analysis is quantitative and large numbers of samples can be efficiently analyzed, this approach offers the ability to rapidly assess a battery of selected proteins in a manner that is directly relevant to traditional clinical assays.« less

The FLIGHT Drosophila RNAi database

PubMed Central

Bursteinas, Borisas; Jain, Ekta; Gao, Qiong; Baum, Buzz; Zvelebil, Marketa

2010-01-01

FLIGHT (http://flight.icr.ac.uk/) is an online resource compiling data from high-throughput Drosophila in vivo and in vitro RNAi screens. FLIGHT includes details of RNAi reagents and their predicted off-target effects, alongside RNAi screen hits, scores and phenotypes, including images from high-content screens. The latest release of FLIGHT is designed to enable users to upload, analyze, integrate and share their own RNAi screens. Users can perform multiple normalizations, view quality control plots, detect and assign screen hits and compare hits from multiple screens using a variety of methods including hierarchical clustering. FLIGHT integrates RNAi screen data with microarray gene expression as well as genomic annotations and genetic/physical interaction datasets to provide a single interface for RNAi screen analysis and datamining in Drosophila. PMID:20855970

Nanotechnology: moving from microarrays toward nanoarrays.

PubMed

Chen, Hua; Li, Jun

2007-01-01

Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

AFM 4.0: a toolbox for DNA microarray analysis

PubMed Central

Breitkreutz, Bobby-Joe; Jorgensen, Paul; Breitkreutz, Ashton; Tyers, Mike

2001-01-01

We have developed a series of programs, collectively packaged as Array File Maker 4.0 (AFM), that manipulate and manage DNA microarray data. AFM 4.0 is simple to use, applicable to any organism or microarray, and operates within the familiar confines of Microsoft Excel. Given a database of expression ratios, AFM 4.0 generates input files for clustering, helps prepare colored figures and Venn diagrams, and can uncover aneuploidy in yeast microarray data. AFM 4.0 should be especially useful to laboratories that do not have access to specialized commercial or in-house software. PMID:11532221

Fundus autofluorescence findings in a mouse model of retinal detachment.

PubMed

Secondi, Roberta; Kong, Jian; Blonska, Anna M; Staurenghi, Giovanni; Sparrow, Janet R

2012-08-07

Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD). RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4-5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis. Results. In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence. In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.

Cholesterol uptake in the mouse aorta increases during Chlamydia pneumoniae infection.

PubMed

Edvinsson, Marie; Tallkvist, Jonas; Nyström-Rosander, Christina; Ilbäck, Nils-Gunnar

2017-01-01

Chlamydia pneumoniae has been suggested as a stimulator of the atherosclerotic process. Mice fed a normal diet were infected intranasally with C. pneumoniae and given one intraperitoneal injection of 14C-cholesterol tracer per day for 12 days. Bacteria were demonstrated in the aorta in the early phase of infection and in lungs and liver throughout the study period of 20 days. 14C-cholesterol was not affected in the heart but increased in the blood, liver and aorta on day 4 when the infection was clinically most severe. Furthermore, on day 20 14C-cholesterol tended to be increased in the aorta. Accordingly, copper- and zinc levels and expressions of the infection biomarkers Cxcl2 and Ifng increased in the liver on day 4 with a tendency of increased of copper, zinc and Ifng on day 20. In mice where bacteria could be cultivated from the lungs, expressions of cholesterol transporters Abca1 and Idol were both increased in the liver on day 4. The increased levels of 14C-cholesterol in blood and aorta together with increased Abca1 and Idol in the liver during C. pneumoniae infection in mice fed a normal diet suggest that this pathogen may have a role in the initiation of the atherosclerotic process. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

PubMed Central

van Huet, Ramon A. C.; Pierrache, Laurence H.M.; Meester-Smoor, Magda A.; Klaver, Caroline C.W.; van den Born, L. Ingeborgh; Hoyng, Carel B.; de Wijs, Ilse J.; Collin, Rob W. J.; Hoefsloot, Lies H.

2015-01-01

Purpose To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon–intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis. PMID:25999674

Association of GWAS Top Genes With Late-Onset Alzheimer's Disease in Colombian Population.

PubMed

Moreno, Diana Jennifer; Ruiz, Susana; Ríos, Ángela; Lopera, Francisco; Ostos, Henry; Via, Marc; Bedoya, Gabriel

2017-02-01

The association of variants in CLU, CR1, PICALM, BIN1, ABCA7, and CD33 genes with late-onset Alzheimer's disease (LOAD) was evaluated and confirmed through genome-wide association study. However, it is unknown whether these associations can be replicated in admixed populations. The association of 14 single-nucleotide polymorphisms in those genes was evaluated in 280 LOAD cases and 357 controls from the Colombian population. In a multivariate analysis using age, gender, APOE∊4 status, and admixture covariates, significant associations were obtained ( P < .05) for variants in BIN1 (rs744373, odds ratio [OR]: 1.42), CLU (rs11136000, OR: 0.66), PICALM (rs541458, OR: 0.69), ABCA7 (rs3764650, OR: 1.7), and CD33 (rs3865444, OR: 1.12). Likewise, a significant interaction effect was observed between CLU and CR1 variants with APOE. This study replicated the associations previously reported in populations of European ancestry and shows that APOE variants have a regulatory role on the effect that variants in other loci have on LOAD, reflecting the importance of gene-gene interactions in the etiology of neurodegenerative diseases.

There is no silver bullet--a guide to low-level data transforms and normalisation methods for microarray data.

PubMed

Kreil, David P; Russell, Roslin R

2005-03-01

To overcome random experimental variation, even for simple screens, data from multiple microarrays have to be combined. There are, however, systematic differences between arrays, and any bias remaining after experimental measures to ensure consistency needs to be controlled for. It is often difficult to make the right choice of data transformation and normalisation methods to achieve this end. In this tutorial paper we review the problem and a selection of solutions, explaining the basic principles behind normalisation procedures and providing guidance for their application.

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community.

PubMed

Yokoi, Takahide; Kaku, Yoshiko; Suzuki, Hiroyuki; Ohta, Masayuki; Ikuta, Hajime; Isaka, Kazuichi; Sumino, Tatsuo; Wagatsuma, Masako

2007-08-01

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.

Visual function in patients with cone-rod dystrophy (CRD) associated with mutations in the ABCA4(ABCR) gene.

PubMed

Birch, D G; Peters, A Y; Locke, K L; Spencer, R; Megarity, C F; Travis, G H

2001-12-01

Mutations in the ABCA4(ABCR) gene cause autosomal recessive Stargardt disease (STGD). ABCR mutations were identified in patients with cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) by direct sequencing of all 50 exons in 40 patients. Of 10 patients with RP, one contained two ABCR mutations suggesting a compound heterozygote. This patient had a characteristic fundus appearance with attenuated vessels, pale disks and bone-spicule pigmentation. Rod electroretinograms (ERGs) were non-detectable, cone ERGs were greatly reduced in amplitude and delayed in implicit time, and visual fields were constricted to 10 degrees diameter. Eleven of 30 (37%) patients with CRD had mutations in ABCR. In general, these patients showed reduced but detectable rod ERG responses, reduced and delayed cone responses, and poor visual acuity. Rod photoresponses to high intensity flashes were of reduced maximum amplitude but showed normal values for the gain of phototransduction. Most CRD patients with mutations in ABCR showed delayed recovery of sensitivity (dark adaptation) following exposure to bright light. Pupils were also significantly smaller in these patients compared to controls at 30 min following light exposure, consistent with a persistent 'equivalent light' background due to the accumulation of a tentatively identified 'noisy' photoproduct. Copyright 2001 Academic Press.

Bimodal imprint chips for peptide screening: integration of high-throughput sequencing by MS and affinity analyses by surface plasmon resonance imaging.

PubMed

Wang, Weizhi; Li, Menglin; Wei, Zewen; Wang, Zihua; Bu, Xiangli; Lai, Wenjia; Yang, Shu; Gong, He; Zheng, Hui; Wang, Yuqiao; Liu, Ying; Li, Qin; Fang, Qiaojun; Hu, Zhiyuan

2014-04-15

Peptide probes and drugs have widespread applications in disease diagnostics and therapy. The demand for peptides ligands with high affinity and high specificity toward various targets has surged in the biomedical field in recent years. The traditional peptide screening procedure involves selection, sequencing, and characterization steps, and each step is manual and tedious. Herein, we developed a bimodal imprint microarray system to embrace the whole peptide screening process. Silver-sputtered silicon chip fabricated with microwell array can trap and pattern the candidate peptide beads in a one-well-one-bead manner. Peptides on beads were photocleaved in situ. A portion of the peptide in each well was transferred to a gold-coated chip to print the peptide array for high-throughput affinity analyses by surface plasmon resonance imaging (SPRi), and the peptide left in the silver-sputtered chip was ready for in situ single bead sequencing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the bimodal imprint chip system, affinity peptides toward AHA were efficiently screened out from the 7 × 10(4) peptide library. The method provides a solution for high efficiency peptide screening.

miR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice

PubMed Central

Goedeke, Leigh; Rotllan, Noemi; Ramírez, Cristina M.; Aranda, Juan F.; Canfrán-Duque, Alberto; Araldi, Elisa; Fernández-Hernando, Ana; Langhi, Cedric; de Cabo, Rafael; Baldán, Ángel; Suárez, Yajaira; Fernández-Hernando, Carlos

2015-01-01

Rationale Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and Results Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50–fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10–20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. Conclusions The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels. PMID:26520906

The impact of APOA5, APOB, APOC3 and ABCA1 gene polymorphisms on ischemic stroke: Evidence from a meta-analysis.

PubMed

Au, Anthony; Griffiths, Lyn R; Irene, Looi; Kooi, Cheah Wee; Wei, Loo Keat

2017-10-01

Genetic studies have been reported on the association between APOA5, APOB, APOC3 and ABCA1 gene polymorphisms and ischemic stroke, but results remain controversial. Hence, this meta-analysis aimed to infer the causal relationships of APOA5 (rs662799, rs3135506), APOB (rs693, rs1042031, rs1801701), APOC3 (rs4520, rs5128, rs2854116, rs2854117) and ABCA1 rs2230806 with ischemic stroke risk. A systematic review was performed for all the articles retrieved from multiple databases, up until March 2017. Data were extracted from all eligible studies, and meta-analysis was carried out using RevMan 5.3 and R package 3.2.1. The strength of association between each studied polymorphism and ischemic stroke risk was measured as odds ratios (ORs) and 95% confidence intervals (CIs), under fixed- and random-effect models. A total of 79 studies reporting on the association between the studied polymorphisms and ischemic stroke risk were identified. The pooled data indicated that all genetic models of APOA5 rs662799 (ORs = 1.23-1.43), allelic and over-dominant models of APOA5 rs3135506 (ORs = 1.77-1.97), APOB rs1801701 (ORs = 1.72-2.13) and APOB rs1042031 (ORs = 1.66-1.88) as well as dominant model of ABCA1 rs2230806 (OR = 1.31) were significantly associated with higher risk of ischemic stroke. However, no significant associations were observed between ischemic stroke and the other five polymorphisms, namely ApoB (rs693) and APOC3 (rs4520, rs5128, rs2854116 and rs2854117), under any genetic model. The present meta-analysis confirmed a significant association of APOA5 rs662799 CC, APOA5 rs3135506 CG, APOB rs1801701 GA, APOB rs1042031 GA and ABCA1 rs2230806 GG with increased risk of ischemic stroke. Copyright © 2017 Elsevier B.V. All rights reserved.

The Effect of Gestational Age on Angiogenic Gene Expression in the Rat Placenta

PubMed Central

Vaswani, Kanchan; Hum, Melissa Wen-Ching; Chan, Hsiu-Wen; Ryan, Jennifer; Wood-Bradley, Ryan J.; Nitert, Marloes Dekker; Mitchell, Murray D.; Armitage, James A.; Rice, Gregory E.

2013-01-01

The placenta plays a central role in determining the outcome of pregnancy. It undergoes changes during gestation as the fetus develops and as demands for energy substrate transfer and gas exchange increase. The molecular mechanisms that coordinate these changes have yet to be fully elucidated. The study performed a large scale screen of the transcriptome of the rat placenta throughout mid-late gestation (E14.25–E20) with emphasis on characterizing gestational age associated changes in the expression of genes invoved in angiogenic pathways. Sprague Dawley dams were sacrificed at E14.25, E15.25, E17.25 and E20 (n = 6 per group) and RNA was isolated from one placenta per dam. Changes in placental gene expression were identifed using Illumina Rat Ref-12 Expression BeadChip Microarrays. Differentially expressed genes (>2-fold change, <1% false discovery rate, FDR) were functionally categorised by gene ontology pathway analysis. A subset of differentially expressed genes identified by microarrays were confirmed using Real-Time qPCR. The expression of thirty one genes involved in the angiogenic pathway was shown to change over time, using microarray analysis (22 genes displayed increased and 9 gene decreased expression). Five genes (4 up regulated: Cd36, Mmp14, Rhob and Angpt4 and 1 down regulated: Foxm1) involved in angiogenesis and blood vessel morphogenesis were subjected to further validation. qPCR confirmed late gestational increased expression of Cd36, Mmp14, Rhob and Angpt4 and a decrease in expression of Foxm1 before labour onset (P<0.0001). The observed acute, pre-labour changes in the expression of the 31 genes during gestation warrant further investigation to elucidate their role in pregnancy. PMID:24391823

Deficiency of ABCA1 and ABCG1 in Macrophages Increases Inflammation and Accelerates Atherosclerosis in Mice

PubMed Central

Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Pagler, Tamara A.; Vengrenyuk, Yuliya; Kappus, Mojdeh S.; Gorman, Darren J.; Nagareddy, Prabhakara R.; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S.; Welch, Carrie; Fisher, Edward A.; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R.

2013-01-01

Rationale Plasma HDL levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is due to the ability of HDL to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results We developed MAC-ABCDKO mice with efficient deletion of the ATP Binding Cassette Transporters A1 and G1 (ABCA1 and ABCG1) in macrophages but not in hematopoietic stem or progenitor populations. MAC-ABCDKO bone marrow (BM) was transplanted into Ldlr-/- recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared to controls. On the Western type diet (WTD), MAC-ABCDKO BM transplanted Ldlr-/- mice had disproportionate atherosclerosis, considering they also had lower VLDL/LDL cholesterol levels than controls. ABCA1/G1 deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, WTD-fed MAC-ABCDKO BM transplanted Ldlr-/- mice displayed monocytosis and neutrophilia in the absence of HSPC proliferation. Mechanistic studies revealed increased expression of M-CSF and G-CSF in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusion These studies 1) show that macrophage deficiency of ABCA1/G1 is pro-atherogenic likely by promoting plaque inflammation and 2) uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways. PMID:23572498

ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines

PubMed Central

Sporstøl, Marita; Mousavi, Seyed Ali; Eskild, Winnie; Roos, Norbert; Berg, Trond

2007-01-01

Background Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. Results By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. Conclusion Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR. PMID:17241464

ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines.

PubMed

Sporstøl, Marita; Mousavi, Seyed Ali; Eskild, Winnie; Roos, Norbert; Berg, Trond

2007-01-22

Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR.

Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice.

PubMed

van der Sluis, Ronald J; Nahon, Joya E; Reuwer, Anne Q; Van Eck, Miranda; Hoekstra, Menno

2015-05-01

Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (-31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (-70%) and secretion (-28%) by peritoneal macrophages. Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis. © 2015 The British Pharmacological Society.

Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.

PubMed

Cuchel, Marina; Lund-Katz, Sissel; de la Llera-Moya, Margarita; Millar, John S; Chang, David; Fuki, Ilia; Rothblat, George H; Phillips, Michael C; Rader, Daniel J

2010-03-01

Reconstituted high-density lipoprotein (rHDL) is of interest as a potential novel therapy for atherosclerosis because of its ability to promote free cholesterol (FC) mobilization after intravenous administration. We performed studies to identify the underlying molecular mechanisms by which rHDL promote FC mobilization from whole body in vivo and macrophages in vitro. Wild-type (WT), SR-BI knockout (KO), ABCA1 KO, and ABCG1 KO mice received either rHDL or phosphate-buffered saline intravenously. Blood was drawn before and at several time points after injection for apolipoprotein A-I, phosphatidylcholine, and FC measurement. In WT mice, serum FC peaked at 20 minutes and rapidly returned toward baseline levels by 24 hours. Unexpectedly, ABCA1 KO and ABCG1 KO mice did not differ from WT mice regarding the kinetics of FC mobilization. In contrast, in SR-BI KO mice the increase in FC level at 20 minutes was only 10% of that in control mice (P<0.01). Bone marrow-derived macrophages from WT, SR-BI O, ABCA1 KO, and ABCG1 KO mice were incubated in vitro with rHDL and cholesterol efflux was determined. Efflux from SR-BI KO and ABCA1 KO macrophages was not different from WT macrophages. In contrast, efflux from ABCG1 KO macrophages was approximately 50% lower as compared with WT macrophages (P<0.001). The bulk mobilization of FC observed in circulation after rHDL administration is primarily mediated by SR-BI. However, cholesterol mobilization from macrophages to rHDL is primarily mediated by ABCG1.

Genetic variant of V825I in the ATP-binding cassette transporter A1 gene and serum lipid levels in the Guangxi Bai Ku Yao and Han populations

PubMed Central

2011-01-01

Background Several genetic variants in the ATP-binding cassette transporter A1 (ABCA1) gene have associated with modifications of serum high-density lipoprotein cholesterol (HDL-C) levels and the susceptibility for coronary heart disease, but the findings are still controversial in diverse racial/ethnic groups. Bai Ku Yao is an isolated subgroup of the Yao minority in southern China. The present study was undertaken to detect the possible association of V825I (rs2066715) polymorphism in the ABCA1 gene and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations. Methods A total of 677 subjects of Bai Ku Yao and 646 participants of Han Chinese were randomly selected from our previous stratified randomized cluster samples. Polymerase chain reaction and restriction fragment length polymorphism assay combined with gel electrophoresis were performed for the genotyping of V825I variant, and then confirmed by direct sequencing. Results The levels of serum total cholesterol (TC), HDL-C, apolipoprotein (Apo) AI and ApoB were lower in Bai Ku Yao than in Han (P < 0.01 for all). The frequency of G and A alleles was 57.4% and 42.6% in Bai Ku Yao, and 57.7% and 42.3% in Han (P > 0.05); respectively. The frequency of GG, GA and AA genotypes was 33.7%, 47.4% and 18.9% in Bai Ku Yao, and 33.4%, 48.6% and 18.0% in Han (P > 0.05); respectively. There was no difference in the genotypic and allelic frequencies between males and females in the both ethnic groups. The subjects with AA genotype in Bai Ku Yao had higher serum TC levels than the subjects with GG and GA genotypes (P < 0.05). The participants with AA genotype in Han had lower serum HDL-C and ApoAI levels than the participants with GG and GA genotypes (P < 0.05 for each), but these results were found in males but not in females. Multivariate linear regression analysis showed that the levels of TC in Bai Ku Yao and HDL-C and ApoAI in male Han were correlated with genotypes (P < 0.05 for all). Serum lipid parameters were also correlated with sex, age, body mass index, alcohol consumption, and blood pressure in both ethnic groups (P < 0.05-0.001). Conclusion The present study suggests that the V825I polymorphism in the ABCA1 gene is associated with male serum HDL-C and ApoAI levels in the Han, and serum TC levels in the Bai Ku Yao populations. The difference in the association of V825I polymorphism and serum lipid levels between the two ethnic groups might partly result from different ABCA1 gene-enviromental interactions. PMID:21247457

Assembly of high-density lipoprotein.

PubMed

Yokoyama, Shinji

2006-01-01

Mammalian somatic cells do not catabolize cholesterol and need to export it for its homeostasis at the levels of cells and whole bodies. This reaction may reduce intracellularly accumulated cholesterol in excess and would contribute to prevention or regression of the initial stage of atherosclerosis. High-density lipoprotein (HDL) is thought to play a main role in this reaction, and 2 independent mechanisms are proposed for this reaction. First, cholesterol is exchanged in a nonspecific physicochemical manner between cell surface and extracellular lipoproteins, and cholesterol esterification on HDL provides a driving force for net removal of cell cholesterol. Second, apolipoproteins directly interact with cells and generate HDL by removing cellular phospholipid and cholesterol. This reaction is a major source of plasma HDL and is mediated by a membrane protein, ABCA1. Lipid-free or lipid-poor helical apolipoproteins primarily recruit cellular phospholipid to assemble HDL particles, and cholesterol enrichment in these particles is regulated independently. ABCA1 is a rate-limiting factor of the HDL assembly and is regulated by transcriptional factors and posttranscriptional factors. Posttranscriptional regulation of ABCA1 includes modulation of its calpain-mediated degradation.

Common variants near ABCA1, AFAP1 and GMDS confer risk of primary open-angle glaucoma

PubMed Central

Fogarty, Rhys; Sharma, Shiwani; Hewitt, Alex W.; Martin, Sarah; Law, Matthew H.; Cremin, Katie; Bailey, Jessica N. Cooke; Loomis, Stephanie J.; Pasquale, Louis R.; Haines, Jonathan L.; Hauser, Michael A.; Viswanathan, Ananth C.; McGuffin, Peter; Topouzis, Fotis; Foster, Paul J.; Graham, Stuart L; Casson, Robert J; Chehade, Mark; White, Andrew J; Zhou, Tiger; Souzeau, Emmanuelle; Landers, John; Fitzgerald, Jude T; Klebe, Sonja; Ruddle, Jonathan B; Goldberg, Ivan; Healey, Paul R; Mills, Richard A.; Wang, Jie Jin; Montgomery, Grant W.; Martin, Nicholas G.; Radford-Smith, Graham; Whiteman, David C.; Brown, Matthew A.; Wiggs, Janey L.; Mackey, David A; Mitchell, Paul; MacGregor, Stuart; Craig, Jamie E.

2014-01-01

Primary open-angle glaucoma (POAG) is a major cause of irreversible blindness worldwide. We performed a genome-wide association study in an Australian discovery cohort comprising 1,155 advanced POAG cases and 1,992 controls. Association of the top SNPs from the discovery stage was investigated in two Australian replication cohorts (total 932 cases, 6,862 controls) and two US replication cohorts (total 2,616 cases, 2,634 controls). Meta-analysis of all cohorts revealed three novel loci associated with development of POAG. These loci are located upstream of ABCA1 (rs2472493 [G] OR=1.31, P= 2.1 × 10−19), within AFAP1 (rs4619890 [G] OR=1.20, P= 7.0 × 10−10) and within GMDS (rs11969985 [G] OR=1.31, and P= 7.7 × 10−10). Using RT-PCR and immunolabelling, we also showed that these genes are expressed within human retina, optic nerve and trabecular meshwork and that ABCA1 and AFAP1 are also expressed in retinal ganglion cells. PMID:25173105

A non-retinoid antagonist of Retinol-Binding Protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

PubMed

Racz, Boglarka; Varadi, Andras; Kong, Jian; Allikmets, Rando; Pearson, Paul G; Johnson, Graham; Cioffi, Christopher L; Petrukhin, Konstantin

2018-06-05

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Development of a genotyping microarray for Usher syndrome.

PubMed

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner-Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva-Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-02-01

Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

Development of a genotyping microarray for Usher syndrome

PubMed Central

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner‐Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva‐Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-01-01

Background Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein‐coding exons. Methods: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele‐specific oligonucleotides corresponding to all 298 Usher syndrome‐associated sequence variants known to date, 76 of which are novel, were arrayed. Results Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. Conclusion The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first‐pass screening tool. PMID:16963483

Use of whole genome expression analysis in the toxicity screening of nanoparticles

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

Fröhlich, Eleonore, E-mail: eleonore.froehlich@medunigraz.at; Meindl, Claudia; Wagner, Karin

2014-10-15

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays formore » NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay.« less

Screening for Intellectual Disability Using High-Resolution CMA Technology in a Retrospective Cohort from Central Brazil

PubMed Central

Pereira, Rodrigo Roncato; Pinto, Irene Plaza; Minasi, Lysa Bernardes; de Melo, Aldaires Vieira; da Cruz e Cunha, Damiana Mirian; Cruz, Alex Silva; Ribeiro, Cristiano Luiz; da Silva, Cláudio Carlos; de Melo e Silva, Daniela; da Cruz, Aparecido Divino

2014-01-01

Intellectual disability is a complex, variable, and heterogeneous disorder, representing a disabling condition diagnosed worldwide, and the etiologies are multiple and highly heterogeneous. Microscopic chromosomal abnormalities and well-characterized genetic conditions are the most common causes of intellectual disability. Chromosomal Microarray Analysis analyses have made it possible to identify putatively pathogenic copy number variation that could explain the molecular etiology of intellectual disability. The aim of the current study was to identify possible submicroscopic genomic alterations using a high-density chromosomal microarray in a retrospective cohort of patients with otherwise undiagnosable intellectual disabilities referred by doctors from the public health system in Central Brazil. The CytoScan HD technology was used to detect changes in the genome copy number variation of patients who had intellectual disability and a normal karyotype. The analysis detected 18 CNVs in 60% of patients. Pathogenic CNVs represented about 22%, so it was possible to propose the etiology of intellectual disability for these patients. Likely pathogenic and unknown clinical significance CNVs represented 28% and 50%, respectively. Inherited and de novo CNVs were equally distributed. We report the nature of CNVs in patients from Central Brazil, representing a population not yet screened by microarray technologies. PMID:25061755

MIPHENO: Data normalization for high throughput metabolic analysis.

EPA Science Inventory

High throughput methodologies such as microarrays, mass spectrometry and plate-based small molecule screens are increasingly used to facilitate discoveries from gene function to drug candidate identification. These large-scale experiments are typically carried out over the course...

Temperature-controlled microintaglio printing for high-resolution micropatterning of RNA molecules.

PubMed

Kobayashi, Ryo; Biyani, Manish; Ueno, Shingo; Kumal, Subhashini Raj; Kuramochi, Hiromi; Ichiki, Takanori

2015-05-15

We have developed an advanced microintaglio printing method for fabricating fine and high-density micropatterns and applied it to the microarraying of RNA molecules. The microintaglio printing of RNA reported here is based on the hybridization of RNA with immobilized complementary DNA probes. The hybridization was controlled by switching the RNA conformation via the temperature, and an RNA microarray with a diameter of 1.5 µm and a density of 40,000 spots/mm(2) with high contrast was successfully fabricated. Specifically, no size effects were observed in the uniformity of patterned signals over a range of microarray feature sizes spanning one order of magnitude. Additionally, we have developed a microintaglio printing method for transcribed RNA microarrays on demand using DNA-immobilized magnetic beads. The beads were arrayed on wells fabricated on a printing mold and the wells were filled with in vitro transcription reagent and sealed with a DNA-immobilized glass substrate. Subsequently, RNA was in situ synthesized using the bead-immobilized DNA as a template and printed onto the substrate via hybridization. Since the microintaglio printing of RNA using DNA-immobilized beads enables the fabrication of a microarray of spots composed of multiple RNA sequences, it will be possible to screen or analyze RNA functions using an RNA microarray fabricated by temperature-controlled microintaglio printing (TC-µIP). Copyright © 2014 Elsevier B.V. All rights reserved.

A fisheye viewer for microarray-based gene expression data

PubMed Central

Wu, Min; Thao, Cheng; Mu, Xiangming; Munson, Ethan V

2006-01-01

Background Microarray has been widely used to measure the relative amounts of every mRNA transcript from the genome in a single scan. Biologists have been accustomed to reading their experimental data directly from tables. However, microarray data are quite large and are stored in a series of files in a machine-readable format, so direct reading of the full data set is not feasible. The challenge is to design a user interface that allows biologists to usefully view large tables of raw microarray-based gene expression data. This paper presents one such interface – an electronic table (E-table) that uses fisheye distortion technology. Results The Fisheye Viewer for microarray-based gene expression data has been successfully developed to view MIAME data stored in the MAGE-ML format. The viewer can be downloaded from the project web site . The fisheye viewer was implemented in Java so that it could run on multiple platforms. We implemented the E-table by adapting JTable, a default table implementation in the Java Swing user interface library. Fisheye views use variable magnification to balance magnification for easy viewing and compression for maximizing the amount of data on the screen. Conclusion This Fisheye Viewer is a lightweight but useful tool for biologists to quickly overview the raw microarray-based gene expression data in an E-table. PMID:17038193

Microarray analysis of potential genes in the pathogenesis of recurrent oral ulcer.

PubMed

Han, Jingying; He, Zhiwei; Li, Kun; Hou, Lu

2015-01-01

Recurrent oral ulcer seriously threatens patients' daily life and health. This study investigated potential genes and pathways that participate in the pathogenesis of recurrent oral ulcer by high throughput bioinformatic analysis. RT-PCR and Western blot were applied to further verify screened interleukins effect. Recurrent oral ulcer related genes were collected from websites and papers, and further found out from Human Genome 280 6.0 microarray data. Each pathway of recurrent oral ulcer related genes were got through chip hybridization. RT-PCR was applied to test four recurrent oral ulcer related genes to verify the microarray data. Data transformation, scatter plot, clustering analysis, and expression pattern analysis were used to analyze recurrent oral ulcer related gene expression changes. Recurrent oral ulcer gene microarray was successfully established. Microarray showed that 551 genes involved in recurrent oral ulcer activity and 196 genes were recurrent oral ulcer related genes. Of them, 76 genes up-regulated, 62 genes down-regulated, and 58 genes up-/down-regulated. Total expression level up-regulated 752 times (60%) and down-regulated 485 times (40%). IL-2 plays an important role in the occurrence, development and recurrence of recurrent oral ulcer on the mRNA and protein levels. Gene microarray can be used to analyze potential genes and pathways in recurrent oral ulcer. IL-2 may be involved in the pathogenesis of recurrent oral ulcer.

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

PubMed Central

Morgan, Hugh P.; Estibeiro, Peter; Wear, Martin A.; Max, Klaas E.A.; Heinemann, Udo; Cubeddu, Liza; Gallagher, Maurice P.; Sadler, Peter J.; Walkinshaw, Malcolm D.

2007-01-01

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function. PMID:17488853

Combined analysis of transcriptome and proteome data as a tool for the identification of candidate biomarkers in renal cell carcinoma

PubMed Central

Seliger, Barbara; Dressler, Sven P.; Wang, Ena; Kellner, Roland; Recktenwald, Christian V.; Lottspeich, Friedrich; Marincola, Francesco M.; Baumgärtner, Maja; Atkins, Derek; Lichtenfels, Rudolf

2012-01-01

Results obtained from expression profilings of renal cell carcinoma using different “ome”-based approaches and comprehensive data analysis demonstrated that proteome-based technologies and cDNA microarray analyses complement each other during the discovery phase for disease-related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to upregulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX-defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%) and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome-based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely 3 candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin alpha-1A chain and ubiquitin carboxyl-terminal hydrolase L1 the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors. PMID:19235166

Regulation of gene expression in the mammalian eye and its relevance to eye disease

PubMed Central

Scheetz, Todd E.; Kim, Kwang-Youn A.; Swiderski, Ruth E.; Philp, Alisdair R.; Braun, Terry A.; Knudtson, Kevin L.; Dorrance, Anne M.; DiBona, Gerald F.; Huang, Jian; Casavant, Thomas L.; Sheffield, Val C.; Stone, Edwin M.

2006-01-01

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR/JrHsd × SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (α = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5′ flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor β2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet–Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease. PMID:16983098

Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice

PubMed Central

van der Sluis, Ronald J; Nahon, Joya E; Reuwer, Anne Q; Van Eck, Miranda; Hoekstra, Menno

2015-01-01

Background and Purpose Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Experimental Approach Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Key Results Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (−31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (−70%) and secretion (−28%) by peritoneal macrophages. Conclusions and Implications Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis. PMID:25572138

Prenatal Ethanol Exposure Up-Regulates the Cholesterol Transporters ATP-Binding Cassette A1 and G1 and Reduces Cholesterol Levels in the Developing Rat Brain.

PubMed

Zhou, Chunyan; Chen, Jing; Zhang, Xiaolu; Costa, Lucio G; Guizzetti, Marina

2014-11-01

Cholesterol plays a pivotal role in many aspects of brain development; reduced cholesterol levels during brain development, as a consequence of genetic defects in cholesterol biosynthesis, leads to severe brain damage, including microcephaly and mental retardation, both of which are also hallmarks of the fetal alcohol syndrome. We had previously shown that ethanol up-regulates the levels of two cholesterol transporters, ABCA1 (ATP binding cassette-A1) and ABCG1, leading to increased cholesterol efflux and decreased cholesterol content in astrocytes in vitro. In the present study we investigated whether similar effects could be seen in vivo. Pregnant Sprague-Dawley rats were fed liquid diets containing 36% of the calories from ethanol from gestational day (GD) 6 to GD 21. A pair-fed control groups and an ad libitum control group were included in the study. ABCA1 and ABCG1 protein expression and cholesterol and phospholipid levels were measured in the neocortex of female and male fetuses at GD 21. Body weights were decreased in female fetuses as a consequence of ethanol treatments. ABCA1 and ABCG1 protein levels were increased, and cholesterol levels were decreased, in the neocortex of ethanol-exposed female, but not male, fetuses. Levels of phospholipids were unchanged. Control female fetuses fed ad libitum displayed an up-regulation of ABCA1 and a decrease in cholesterol content compared with pair-fed controls, suggesting that a compensatory up-regulation of cholesterol levels may occur during food restriction. Maternal ethanol consumption may affect fetal brain development by increasing cholesterol transporters' expression and reducing brain cholesterol levels. © The Author 2014. Medical Council on Alcohol and Oxford University Press. All rights reserved.

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.

4p terminal deletion and 11p subtelomeric duplication detected by genomic microarray in a patient with Wolf-Hirschhorn syndrome and an atypical phenotype.

PubMed

Stevenson, David A; Carey, John C; Cowley, Brett C; Bayrak-Toydemir, Pinar; Mao, Rong; Brothman, Arthur R

2004-12-01

We report a de novo cryptic 11p duplication found by genomic microarray with a cytogenetically detected 4p deletion. Terminal 4p deletions cause Wolf-Hirschhorn syndrome, but the phenotype probably was modified by the paternally derived 11p duplication. This emphasizes the clinical utility of genomic microarray.

Fundus Autofluorescence Findings in a Mouse Model of Retinal Detachment

PubMed Central

Secondi, Roberta; Kong, Jian; Blonska, Anna M.; Staurenghi, Giovanni; Sparrow, Janet R.

2012-01-01

Purpose. Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD). Methods. RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4–5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis. Results. In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence. Conclusions. In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells. PMID:22786896

Genes that characterize T3-predominant Graves' thyroid tissues.

PubMed

Matsumoto, Chisa; Ito, Mitsuru; Yamada, Hiroya; Yamakawa, Noriko; Yoshida, Hiroshi; Date, Arisa; Watanabe, Mikio; Hidaka, Yoh; Iwatani, Yoshinori; Miyauchi, Akira; Takano, Toru

2013-02-01

3,5,3'-Triiodothyronine (T(3))-predominant Graves' disease is characterized by the increasing volume of thyroid goiter resulting in poor prognosis. Although type 1 and type 2 iodothyronine deiodinases (DIO1 and DIO2 respectively) are known to be overexpressed in the thyroid tissues of T(3)-predominant Graves' disease, the pathogenesis of this disease is still unclear. The aim of our study is to identify genes that characterize T(3)-predominant Graves' disease tissue in order to clarify the molecular mechanism of this disease. mRNAs from two thyroid tissues of both typical T(3)-predominant and common-type Graves' disease were analyzed with DNA microarrays with probes for 28 869 genes. Genes identified to be differentially expressed between the two groups were further analyzed in the second and third screenings using 70 Graves' thyroid tissues by real-time quantitative RT-PCR. Twenty-three candidate genes were selected as being differentially expressed in the first screening with microarrays. Among these, seven genes, leucine-rich repeat neuronal 1 (LRRN1), bone morphogenetic protein 8a (BMP8A), N-cadherin (CDH2), phosphodiesterase 1A (PDE1A), creatine kinase mitochondrial 2 (CKMT2), integrin beta-3 (ITGB3), and protein tyrosine phosphatase non-receptor type 4 (PTPN4), were confirmed to be differentially expressed in DIO1 or DIO2 over- and underexpressing Graves' tissues. These genes are related to the characteristics of T(3)-predominant Graves' disease, such as high titer level of serum anti-TSH receptor antibody, high free T(3) to free thyroxine ratio, and a large goiter size. They might play a role in the pathogenesis of T(3)-predominant Graves' disease.

Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays.

PubMed

Hu, Chao-Jun; Song, Guang; Huang, Wei; Liu, Guo-Zhen; Deng, Chui-Wen; Zeng, Hai-Pan; Wang, Li; Zhang, Feng-Chun; Zhang, Xuan; Jeong, Jun Seop; Blackshaw, Seth; Jiang, Li-Zhi; Zhu, Heng; Wu, Lin; Li, Yong-Zhe

2012-09-01

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.

[Study of generational risk in deafness inflicted couples using deafness gene microarray technique].

PubMed

Wang, Ping; Zhao, Jia; Yu, Shu-yuan; Jin, Peng; Zhu, Wei; DU, Bo

2011-06-01

To explored the significance of screening the gene mutations of deafness related in deaf-mute (deaf & dumb) family using DNA microarray. Total of 52 couples of deaf-mute were recruited from Changchun deaf-mute community. With an average age of (58.3 ± 6.7) years old (x(-) ± s). Blood samples were obtained with informed consent. Their genomic DNA was extracted from peripheral blood and PCR was performed. Nine of hot spot mutations in four most common deafness pathologic gene were examined with the DNA microarray, including GJB2, GJB3, PDS and mtDNA 12S rRNA genes. At the same time, the results were verified with the traditional methods of sequencing. Fifty of normal people served as a control group. All patients were diagnosed non-syndromic sensorineural hearing loss by subjective pure tone audiometry. Thirty-two of 104 cases appeared GJB2 gene mutation (30.7%), the mutation sites included 35delG, 176del16, 235delC and 299delAT. Eighteen of 32 cases of GJB2 mutations were 235delC (59.1%). Seven of 104 cases appeared SLC26A4 gene IVS7-2 A > G mutation. Questionnaire survey and gene diagnosis revealed that four of 52 families have deaf offspring (7.6%). When a couple carries the same gene mutation, the risk of their children deafness was 100%. The results were confirmed with the traditional methods of sequencing. There is a high risk of deafness if a deaf-mute family is planning to have a new baby. It is very important and helpful to avoid deaf newborns again in deaf-mute family by DNA microarray.

EMAAS: An extensible grid-based Rich Internet Application for microarray data analysis and management

PubMed Central

Barton, G; Abbott, J; Chiba, N; Huang, DW; Huang, Y; Krznaric, M; Mack-Smith, J; Saleem, A; Sherman, BT; Tiwari, B; Tomlinson, C; Aitman, T; Darlington, J; Game, L; Sternberg, MJE; Butcher, SA

2008-01-01

Background Microarray experimentation requires the application of complex analysis methods as well as the use of non-trivial computer technologies to manage the resultant large data sets. This, together with the proliferation of tools and techniques for microarray data analysis, makes it very challenging for a laboratory scientist to keep up-to-date with the latest developments in this field. Our aim was to develop a distributed e-support system for microarray data analysis and management. Results EMAAS (Extensible MicroArray Analysis System) is a multi-user rich internet application (RIA) providing simple, robust access to up-to-date resources for microarray data storage and analysis, combined with integrated tools to optimise real time user support and training. The system leverages the power of distributed computing to perform microarray analyses, and provides seamless access to resources located at various remote facilities. The EMAAS framework allows users to import microarray data from several sources to an underlying database, to pre-process, quality assess and analyse the data, to perform functional analyses, and to track data analysis steps, all through a single easy to use web portal. This interface offers distance support to users both in the form of video tutorials and via live screen feeds using the web conferencing tool EVO. A number of analysis packages, including R-Bioconductor and Affymetrix Power Tools have been integrated on the server side and are available programmatically through the Postgres-PLR library or on grid compute clusters. Integrated distributed resources include the functional annotation tool DAVID, GeneCards and the microarray data repositories GEO, CELSIUS and MiMiR. EMAAS currently supports analysis of Affymetrix 3' and Exon expression arrays, and the system is extensible to cater for other microarray and transcriptomic platforms. Conclusion EMAAS enables users to track and perform microarray data management and analysis tasks through a single easy-to-use web application. The system architecture is flexible and scalable to allow new array types, analysis algorithms and tools to be added with relative ease and to cope with large increases in data volume. PMID:19032776

High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

PubMed

Inoue, Daisuke; Hinoura, Takuji; Suzuki, Noriko; Pang, Junqin; Malla, Rabin; Shrestha, Sadhana; Chapagain, Saroj Kumar; Matsuzawa, Hiroaki; Nakamura, Takashi; Tanaka, Yasuhiro; Ike, Michihiko; Nishida, Kei; Sei, Kazunari

2015-01-01

Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1-37 pathogen species/groups, including 1-27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

Identification of placental nutrient transporters associated with intrauterine growth restriction and pre-eclampsia.

PubMed

Huang, Xiao; Anderle, Pascale; Hostettler, Lu; Baumann, Marc U; Surbek, Daniel V; Ontsouka, Edgar C; Albrecht, Christiane

2018-03-02

Gestational disorders such as intrauterine growth restriction (IUGR) and pre-eclampsia (PE) are main causes of poor perinatal outcomes worldwide. Both diseases are related with impaired materno-fetal nutrient transfer, but the crucial transport mechanisms underlying IUGR and PE are not fully elucidated. In this study, we aimed to identify membrane transporters highly associated with transplacental nutrient deficiencies in IUGR/PE. In silico analyses on the identification of differentially expressed nutrient transporters were conducted using seven eligible microarray datasets (from Gene Expression Omnibus), encompassing control and IUGR/PE placental samples. Thereby 46 out of 434 genes were identified as potentially interesting targets. They are involved in the fetal provision with amino acids, carbohydrates, lipids, vitamins and microelements. Targets of interest were clustered into a substrate-specific interaction network by using Search Tool for the Retrieval of Interacting Genes. The subsequent wet-lab validation was performed using quantitative RT-PCR on placentas from clinically well-characterized IUGR/PE patients (IUGR, n = 8; PE, n = 5; PE+IUGR, n = 10) and controls (term, n = 13; preterm, n = 7), followed by 2D-hierarchical heatmap generation. Statistical evaluation using Kruskal-Wallis tests was then applied to detect significantly different expression patterns, while scatter plot analysis indicated which transporters were predominantly influenced by IUGR or PE, or equally affected by both diseases. Identified by both methods, three overlapping targets, SLC7A7, SLC38A5 (amino acid transporters), and ABCA1 (cholesterol transporter), were further investigated at the protein level by western blotting. Protein analyses in total placental tissue lysates and membrane fractions isolated from disease and control placentas indicated an altered functional activity of those three nutrient transporters in IUGR/PE. Combining bioinformatic analysis, molecular biological experiments and mathematical diagramming, this study has demonstrated systematic alterations of nutrient transporter expressions in IUGR/PE. Among 46 initially targeted transporters, three significantly regulated genes were further investigated based on the severity and the disease specificity for IUGR and PE. Confirmed by mRNA and protein expression, the amino acid transporters SLC7A7 and SLC38A5 showed marked differences between controls and IUGR/PE and were regulated by both diseases. In contrast, ABCA1 may play an exclusive role in the development of PE.

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

PubMed Central

Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

2005-01-01

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

Bayesian hierarchical modeling for subject-level response classification in peptide microarray immunoassays

PubMed Central

Imholte, Gregory; Gottardo, Raphael

2017-01-01

Summary The peptide microarray immunoassay simultaneously screens sample serum against thousands of peptides, determining the presence of antibodies bound to array probes. Peptide microarrays tiling immunogenic regions of pathogens (e.g. envelope proteins of a virus) are an important high throughput tool for querying and mapping antibody binding. Because of the assay’s many steps, from probe synthesis to incubation, peptide microarray data can be noisy with extreme outliers. In addition, subjects may produce different antibody profiles in response to an identical vaccine stimulus or infection, due to variability among subjects’ immune systems. We present a robust Bayesian hierarchical model for peptide microarray experiments, pepBayes, to estimate the probability of antibody response for each subject/peptide combination. Heavy-tailed error distributions accommodate outliers and extreme responses, and tailored random effect terms automatically incorporate technical effects prevalent in the assay. We apply our model to two vaccine trial datasets to demonstrate model performance. Our approach enjoys high sensitivity and specificity when detecting vaccine induced antibody responses. A simulation study shows an adaptive thresholding classification method has appropriate false discovery rate control with high sensitivity, and receiver operating characteristics generated on vaccine trial data suggest that pepBayes clearly separates responses from non-responses. PMID:27061097

Gene expression analysis using a highly sensitive DNA microarray for colorectal cancer screening.

PubMed

Koga, Yoshikatsu; Yamazaki, Nobuyoshi; Takizawa, Satoko; Kawauchi, Junpei; Nomura, Osamu; Yamamoto, Seiichiro; Saito, Norio; Kakugawa, Yasuo; Otake, Yosuke; Matsumoto, Minori; Matsumura, Yasuhiro

2014-01-01

Half of all patients with small, right-sided, non-metastatic colorectal cancer (CRC) have negative results for the fecal occult blood test (FOBT). In the present study, the usefulness of CRC screening with a highly sensitive DNA microarray was evaluated in comparison with that by FOBT using fecal samples. A total of 53 patients with CRC and 61 healthy controls were divided into "training" and "validation sets". For the gene profiling, total RNA extracted from 0.5 g of feces was hybridized to a highly sensitive DNA chip. The expressions of 43 genes were significantly higher in the patients with CRC than in healthy controls (p<0.05). In the training set, the sensitivity and specificity of the DNA chip assay using six genes were 85.4% and 85.2%, respectively. On the other hand, in the validation set, the sensitivity and specificity of the DNA chip assay were 85.2% and 85.7%, respectively. The sensitivities of the DNA chip assay were higher than those of FOBT in cases of the small, right-sided, early-CRC, tumor invading up to the muscularis propria (i.e. surface tumor) subgroups. In particular, the sensitivities of the DNA chip assay in the surface tumor and early-CRC subgroups were significantly higher than those of FOBT (p=0.023 and 0.019, respectively.). Gene profiling assay using a highly sensitive DNA chip was more effective than FOBT at detecting patients with small, right-sided, surface tumor, and early-stage CRC.

Sorting out co-occurrence of rare monogenic retinopathies: Stargardt disease co-existing with congenital stationary night blindness.

PubMed

Huynh, Nancy; Jeffrey, Brett G; Turriff, Amy; Sieving, Paul A; Cukras, Catherine A

2014-03-01

Inherited retinal diseases are uncommon, and the likelihood of having more than one hereditary disorder is rare. Here, we report a case of Stargardt disease and congenital stationary night blindness (CSNB) in the same patient, and the identification of two novel in-frame deletions in the GRM6 gene. The patient underwent an ophthalmic exam and visual function testing including: visual acuity, color vision, Goldmann visual field, and electroretinography (ERG). Imaging of the retina included fundus photography, spectral-domain optical coherence tomography (OCT), and fundus autofluorescence. Genomic DNA was PCR-amplified for analysis of all coding exons and flanking splice sites of both the ABCA4 and GRM6 genes. A 46-year-old woman presented with recently reduced central vision and clinical findings of characteristic yellow flecks consistent with Stargardt disease. However, ERG testing revealed an ERG phenotype unusual for Stargardt disease but consistent with CSNB1. Genetic testing revealed two previously reported mutations in the ABCA4 gene and two novel deletions in the GRM6 gene. Diagnosis of concurrent Stargardt disease and CSNB was made on the ophthalmic history, clinical examination, ERG, and genetic testing. This case highlights that clinical tests need to be taken in context, and that co-existing retinal dystrophies and degenerations should be considered when clinical impressions and objective data do not correlate.

Two-color Dye-swap DNA Microarray approach toward confident gene expression profiling in PMCAO mouse model for ischemia-related and PACAP38-influenced genes

PubMed Central

Hori, Motohide; Shibato, Junko; Nakamachi, Tomoya; Rakwal, Randeep; Ogawa, Tetsuo; Shioda, Seiji; Numazawa, Satoshi

2015-01-01

Toward twin goals of identifying molecular factors in brain injured by ischemic stroke, and the effects of neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain, we have established the permanent middle cerebral artery occlusion (PMCAO) mouse model and utilized the Agilent mouse whole genome 4 × 44 K DNA chip. PACAP38 (1 pmol) injection was given intracerebroventrically in comparison to a control saline (0.9% NaCl) injection, to screen genes responsive to PACAP38. Two sets of tissues were prepared, whole hemispheres (ischemic and non-ischemic) and infract core and penumbra regions at 6 and 24 h. In this study, we have detailed the experimental design and protocol used therein and explained the quality controls for the use of total RNA in the downstream DNA microarray experiment utilizing a two-color dye-swap approach for stringent and confident gene identification published in a series of papers by Hori and coworkers (Hori et al., 2012–2015). PMID:26484166

Construction of diagnosis system and gene regulatory networks based on microarray analysis.

PubMed

Hong, Chun-Fu; Chen, Ying-Chen; Chen, Wei-Chun; Tu, Keng-Chang; Tsai, Meng-Hsiun; Chan, Yung-Kuan; Yu, Shyr Shen

2018-05-01

A microarray analysis generally contains expression data of thousands of genes, but most of them are irrelevant to the disease of interest, making analyzing the genes concerning specific diseases complicated. Therefore, filtering out a few essential genes as well as their regulatory networks is critical, and a disease can be easily diagnosed just depending on the expression profiles of a few critical genes. In this study, a target gene screening (TGS) system, which is a microarray-based information system that integrates F-statistics, pattern recognition matching, a two-layer K-means classifier, a Parameter Detection Genetic Algorithm (PDGA), a genetic-based gene selector (GBG selector) and the association rule, was developed to screen out a small subset of genes that can discriminate malignant stages of cancers. During the first stage, F-statistic, pattern recognition matching, and a two-layer K-means classifier were applied in the system to filter out the 20 critical genes most relevant to ovarian cancer from 9600 genes, and the PDGA was used to decide the fittest values of the parameters for these critical genes. Among the 20 critical genes, 15 are associated with cancer progression. In the second stage, we further employed a GBG selector and the association rule to screen out seven target gene sets, each with only four to six genes, and each of which can precisely identify the malignancy stage of ovarian cancer based on their expression profiles. We further deduced the gene regulatory networks of the 20 critical genes by applying the Pearson correlation coefficient to evaluate the correlationship between the expression of each gene at the same stages and at different stages. Correlationships between gene pairs were calculated, and then, three regulatory networks were deduced. Their correlationships were further confirmed by the Ingenuity pathway analysis. The prognostic significances of the genes identified via regulatory networks were examined using online tools, and most represented biomarker candidates. In summary, our proposed system provides a new strategy to identify critical genes or biomarkers, as well as their regulatory networks, from microarray data. Copyright © 2018. Published by Elsevier Inc.

Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization.

PubMed

Forreryd, Andy; Johansson, Henrik; Albrekt, Ann-Sofie; Lindstedt, Malin

2014-05-16

Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection (GARD) assay is an in vitro alternative to animal testing for identification of skin sensitizers, classifying chemicals by evaluating transcriptional levels of a genomic biomarker signature. During assay development and biomarker identification, genome-wide expression analysis was applied using microarrays covering approximately 30,000 transcripts. However, the microarray platform suffers from drawbacks in terms of low sample throughput, high cost per sample and time consuming protocols and is a limiting factor for adaption of GARD into a routine assay for screening of potential sensitizers. With the purpose to simplify assay procedures, improve technical parameters and increase sample throughput, we assessed the performance of three high throughput gene expression platforms--nCounter®, BioMark HD™ and OpenArray®--and correlated their performance metrics against our previously generated microarray data. We measured the levels of 30 transcripts from the GARD biomarker signature across 48 samples. Detection sensitivity, reproducibility, correlations and overall structure of gene expression measurements were compared across platforms. Gene expression data from all of the evaluated platforms could be used to classify most of the sensitizers from non-sensitizers in the GARD assay. Results also showed high data quality and acceptable reproducibility for all platforms but only medium to poor correlations of expression measurements across platforms. In addition, evaluated platforms were superior to the microarray platform in terms of cost efficiency, simplicity of protocols and sample throughput. We evaluated the performance of three non-array based platforms using a limited set of transcripts from the GARD biomarker signature. We demonstrated that it was possible to achieve acceptable discriminatory power in terms of separation between sensitizers and non-sensitizers in the GARD assay while reducing assay costs, simplify assay procedures and increase sample throughput by using an alternative platform, providing a first step towards the goal to prepare GARD for formal validation and adaption of the assay for industrial screening of potential sensitizers.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants*

PubMed Central

Carmona, Santiago J.; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C.; Campetella, Oscar; Buscaglia, Carlos A.; Agüero, Fernán

2015-01-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. PMID:25922409

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

PubMed

Carmona, Santiago J; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C; Campetella, Oscar; Buscaglia, Carlos A; Agüero, Fernán

2015-07-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

PubMed

Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

2017-12-15

A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarrays. 22-24 January 2001, Zurich, Switzerland.

PubMed

Jain, K K

2001-02-01

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarray technology covered the latest advances in this technology and applications in life sciences. Highlights of the meetings are reported briefly with emphasis on applications in genomics, drug discovery and molecular diagnostics. There was an emphasis on microfluidics because of the wide applications in laboratory and drug discovery. The lab-on-a-chip provides the facilities of a complete laboratory in a hand-held miniature device. Several microarray systems have been used for hybridisation and detection techniques. Oligonucleotide scanning arrays provide a versatile tool for the analysis of nucleic acid interactions and provide a platform for improving the array-based methods for investigation of antisense therapeutics. A method for analysing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional scanner has considerable potential in molecular diagnostics. Various applications of microarray technology for high-throughput screening in drug discovery and single nucleotide polymorphisms (SNP) analysis were discussed. Protein chips have important applications in proteomics. With the considerable amount of data generated by the different technologies using microarrays, it is obvious that the reading of the information and its interpretation and management through the use of bioinformatics is essential. Various techniques for data analysis were presented. Biochip and microarray technology has an essential role to play in the evolving trends in healthcare, which integrate diagnosis with prevention/treatment and emphasise personalised medicines.

Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray

PubMed Central

von Schalburg, Kristian R; Rise, Matthew L; Cooper, Glenn A; Brown, Gordon D; Gibbs, A Ross; Nelson, Colleen C; Davidson, William S; Koop, Ben F

2005-01-01

Background We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested. Results We have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA. Conclusion We validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content. PMID:16164747

Cancer-associated autoantibodies to MUC1 and MUC4--a blinded case–control study of colorectal cancer in UK collaborative trial of ovarian cancer screening.

PubMed

Pedersen, Johannes W; Gentry-Maharaj, Aleksandra; Nøstdal, Alexander; Fourkala, Evangelia-Ourania; Dawnay, Anne; Burnell, Matthew; Zaikin, Alexey; Burchell, Joy; Papadimitriou, Joyce Taylor; Clausen, Henrik; Jacobs, Ian; Menon, Usha; Wandall, Hans H

2014-05-01

Recent reports suggest that autoantibodies directed to aberrantly glycosylated mucins, in particular MUC1 and MUC4, are found in patients with colorectal cancer. There is, however, limited information on the autoantibody levels before clinical diagnosis, and their utility in cancer screening in the general population. In our study, we have generated O-glycosylated synthetic MUC1 and MUC4 peptides in vitro, to mimic cancer-associated glycoforms, and displayed these on microarrays. The assay's performance was tested through an initial screening of serum samples taken from patients at the time of colorectal cancer diagnosis and healthy controls. Subsequently, the selected biomarkers were evaluated in a blinded nested case–control study using stored serum samples from among the 50,640 women randomized to the multimodal arm of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS), where women gave annual blood samples for several years. Cases were 97 postmenopausal women who developed colorectal cancer after recruitment and were age-matched to 97 women without any history of cancer. MUC1-STn and MUC1-Core3 IgG autoantibodies identified cases with 8.2 and 13.4% sensitivity, respectively, at 95% specificity. IgA to MUC4 glycoforms were unable to discriminate between cases and controls in the UKCTOCS sera. Additional analysis was undertaken by combining the data of MUC1-STn and MUC1-Core3 with previously generated data on autoantibodies to p53 peptides, which increased the sensitivity to 32.0% at 95% specificity. These findings suggest that a combination of antibody signatures may have a role as part of a biomarker panel for the early detection of colorectal cancer.

Mutation spectrum and differential gene expression in cystic and solid vestibular schwannoma.

PubMed

Zhang, Zhihua; Wang, Zhaoyan; Sun, Lianhua; Li, Xiaohua; Huang, Qi; Yang, Tao; Wu, Hao

2014-03-01

We sought to characterize the mutation spectrum of NF2 and the differential gene expression in cystic and solid vestibular schwannomas. We collected tumor tissue and blood samples of 31 cystic vestibular schwannomas and 114 solid vestibular schwannomas. Mutation screening of NF2 was performed in both tumor and blood DNA samples of all patients. cDNA microarray was used to analyze the differential gene expression between 11 cystic vestibular schwannomas and 6 solid vestibular schwannomas. Expression levels of top candidate genes were verified by quantitative reverse transcription PCR. NF2 mutations were identified in 34.5% of sporadic vestibular schwannomas, with all mutations being exclusively somatic. No significant difference was found between the mutation detection rates of cystic vestibular schwannoma (35.5%) and solid vestibular schwannoma (34.2%). cDNA microarray analysis detected a total of 46 differentially expressed genes between the cystic vestibular schwannoma and solid vestibular schwannoma samples. The significantly decreased expression of four top candidate genes, C1orf130, CNTF, COL4A3, and COL4A4, was verified by quantitative reverse transcription PCR. NF2 mutations are not directly involved in the cystic formation of vestibular schwannoma. In addition, the differential gene expression of cystic vestibular schwannoma reported in our study may provide useful insights into the molecular mechanism underlying this process.

Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4.

PubMed

Thiel, Cora S; Huge, Andreas; Hauschild, Swantje; Tauber, Svantje; Lauber, Beatrice A; Polzer, Jennifer; Paulsen, Katrin; Lier, Hartwin; Engelmann, Frank; Schmitz, Burkhard; Schütte, Andreas; Layer, Liliana E; Ullrich, Oliver

2017-01-01

In the last decades, a plethora of in vitro studies with living human cells contributed a vast amount of knowledge about cellular and molecular effects of microgravity. Previous studies focused mostly on the identification of gravity-responsive genes, whereas a multi-platform analysis at an integrative level, which specifically evaluates the extent and robustness of transcriptional response to an altered gravity environment was not performed so far. Therefore, we investigated the stability of gene expression response in non-activated human Jurkat T lymphocytic cells in different gravity environments through the combination of parabolic flights with a suborbital ballistic rocket and 2D clinostat and centrifuge experiments, using strict controls for excluding all possible other factors of influence. We revealed an overall high stability of gene expression in microgravity and identified olfactory gene expression in the chromosomal region 11p15.4 as particularly robust to altered gravity. We identified that classical reference genes ABCA5 , GAPDH , HPRT1 , PLA2G4A , and RPL13A were stably expressed in all tested gravity conditions and platforms, while ABCA5 and GAPDH were also known to be stably expressed in U937 cells in all gravity conditions. In summary, 10-20% of all transcripts remained totally unchanged in any gravitational environment tested (between 10 -4 and 9 g), 20-40% remained unchanged in microgravity (between 10 -4 and 10 -2  g) and 97-99% were not significantly altered in microgravity if strict exclusion criteria were applied. Therefore, we suppose a high stability of gene expression in microgravity. Comparison with other stressors suggests that microgravity alters gene expression homeostasis not stronger than other environmental factors.

Cost-Effectiveness of Old and New Technologies for Aneuploidy Screening.

PubMed

Sinkey, Rachel G; Odibo, Anthony O

2016-06-01

Cost-effectiveness analyses allow assessment of whether marginal gains from new technology are worth increased costs. Several studies have examined cost-effectiveness of Down syndrome (DS) screening and found it to be cost-effective. Noninvasive prenatal screening also appears to be cost-effective among high-risk women with respect to DS screening, but not for the general population. Chromosomal microarray (CMA) is a genetic sequencing method superior to but more expensive than karyotype. In light of CMAs greater ability to detect genetic abnormalities, it is cost-effective when used for prenatal diagnosis of an anomalous fetus. This article covers methodology and salient issues of cost-effectiveness. Copyright © 2016 Elsevier Inc. All rights reserved.

Segmental Duplications and Copy-Number Variation in the Human Genome

PubMed Central

Sharp, Andrew J. ; Locke, Devin P. ; McGrath, Sean D. ; Cheng, Ze ; Bailey, Jeffrey A. ; Vallente, Rhea U. ; Pertz, Lisa M. ; Clark, Royden A. ; Schwartz, Stuart ; Segraves, Rick ; Oseroff, Vanessa V. ; Albertson, Donna G. ; Pinkel, Daniel ; Eichler, Evan E. 

2005-01-01

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders. PMID:15918152

Brain penetrant liver X receptor (LXR) modulators based on a 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole core.

PubMed

Tice, Colin M; Noto, Paul B; Fan, Kristi Yi; Zhao, Wei; Lotesta, Stephen D; Dong, Chengguo; Marcus, Andrew P; Zheng, Ya-Jun; Chen, Guozhou; Wu, Zhongren; Van Orden, Rebecca; Zhou, Jing; Bukhtiyarov, Yuri; Zhao, Yi; Lipinski, Kerri; Howard, Lamont; Guo, Joan; Kandpal, Geeta; Meng, Shi; Hardy, Andrew; Krosky, Paula; Gregg, Richard E; Leftheris, Katerina; McKeever, Brian M; Singh, Suresh B; Lala, Deepak; McGeehan, Gerard M; Zhuang, Linghang; Claremon, David A

2016-10-15

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aβ) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aβ levels was detected. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efficacy of a therapeutic vaccine using mutated β-amyloid sensitized dendritic cells in Alzheimer's mice.

PubMed

Luo, Zhongqiu; Li, Jialin; Nabar, Neel R; Lin, Xiaoyang; Bai, Ge; Cai, Jianfeng; Zhou, Shu-Feng; Cao, Chuanhai; Wang, Jinhuan

2012-09-01

Despite FDA suspension of Elan's AN-1792 amyloid beta (Aβ) vaccine in phase IIb clinical trials, the implications of this study are the guiding principles for contemporary anti-Aβ immunotherapy against Alzheimer's disease (AD). AN-1792 showed promising results with regards to Aβ clearance and cognitive function improvement, but also exhibited an increased risk of Th1 mediated meningoencephalitis. As such, vaccine development has continued with an emphasis on eliciting a notable anti-Aβ antibody titer, while avoiding the unwanted Th1 pro-inflammatory response. Previously, we published the first report of an Aβ sensitized dendritic cell vaccine as a therapeutic treatment for AD in BALB/c mice. Our vaccine elicited an anti-Aβ titer, with indications that a Th1 response was not present. This study is the first to investigate the efficacy and safety of our dendritic cell vaccine for the prevention of AD in transgenic mouse models (PDAPP) for AD. We also used Immunohistochemistry to characterize the involvement of LXR, ABCA1, and CD45 in order to gain insight into the potential mechanisms through which this vaccine may provide benefit. The results indicate that (1) the use of mutant Aβ1-42 sensitized dendritic cell vaccine results in durable antibody production, (2) the vaccine provides significant benefits with regards to cognitive function without the global (Th1) inflammation seen in prior Aβ vaccines, (3) histological studies showed an overall decrease in Aβ burden, with an increase in LXR, ABCA1, and CD45, and (4) the beneficial results of our DC vaccine may be due to the LXR/ABCA1 pathway. In the future, mutant Aβ sensitized dendritic cell vaccines could be an efficacious and safe method for the prevention or treatment of AD that circumvents problems associated with traditional anti-Aβ vaccines.

Treatment Failure and Miltefosine Susceptibility in Dermal Leishmaniasis Caused by Leishmania Subgenus Viannia Species

PubMed Central

Obonaga, Ricardo; Fernández, Olga Lucía; Valderrama, Liliana; Rubiano, Luisa Consuelo; Castro, Maria del Mar; Barrera, Maria Claudia; Gomez, Maria Adelaida

2014-01-01

Treatment failure and parasite drug susceptibility in dermal leishmaniasis caused by Leishmania (Viannia) species are poorly understood. Prospective evaluation of drug susceptibility of strains isolated from individual patients before drug exposure and at clinical failure allows intrinsic and acquired differences in susceptibility to be discerned and analyzed. To determine whether intrinsic susceptibility or loss of susceptibility to miltefosine contributed to treatment failure, we evaluated the miltefosine susceptibility of intracellular amastigotes and promastigotes of six Leishmania (Viannia) braziliensis and six Leishmania (Viannia) panamensis strains isolated sequentially, at diagnosis and treatment failure, from two children and four adults ≥55 years old with concurrent conditions. Four patients presented only cutaneous lesions, one had mucosal disease, and one had disseminated mucocutaneous disease. Expression of the Leishmania drug transporter genes abca2, abca3, abcc2, abcc3, abcg4, abcg6, and LbMT was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Intracellular amastigotes (median 50% effective concentration [EC50], 10.7 μmol/liter) were more susceptible to miltefosine than promastigotes (median EC50, 55.3 μmol/liter) (P < 0.0001). Loss of susceptibility at failure, demonstrated by a miltefosine EC50 of >32 μmol/liter (the upper limit of intracellular amastigote assay), occurred in L. panamensis infection in a child and in L. braziliensis infection in an adult and was accompanied by decreased expression of the miltefosine transporter LbMT (LbMT/β-tubulin, 0.42- to 0.26-fold [P = 0.039] and 0.70- to 0.57-fold [P = 0.009], respectively). LbMT gene polymorphisms were not associated with susceptibility phenotype. Leishmania ABCA3 transporter expression was inversely correlated with miltefosine susceptibility (r = −0.605; P = 0.037). Loss of susceptibility is one of multiple factors involved in failure of miltefosine treatment in dermal leishmaniasis. PMID:24145529

Atheroprotective Effects of Methotrexate on Reverse Cholesterol Transport Proteins and Foam Cell Transformation in THP-1 Human Monocytes/Macrophages

PubMed Central

Reiss, Allison B.; Carsons, Steven E.; Anwar, Kamran; Rao, Soumya; Edelman, Sari D.; Zhang, Hongwei; Fernandez, Patricia; Cronstein, Bruce N.; Chan, Edwin S.L.

2008-01-01

OBJECTIVE: To determine whether MTX can overcome the atherogenic effect of COX-2 inhibitors and IFN-γ, both of which suppress cholesterol efflux protein levels and promote foam cell transformation in THP-1 human monocytes/macrophages. METHODS: Message and protein level of the reverse cholesterol transport (RCT) proteins cholesterol 27-hydroxylase and ABCA1 in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFN-γ with/without MTX. Foam cell transformation of lipid-loaded THP-1 macrophages was detected with oil red O staining and light microscopy. RESULTS: MTX increased 27-hydroxylase message and completely blocked NS398-induced downregulation of 27-hydroxylase (112.8±13.1% for NS398+MTX versus 71.1±4.3% for NS398 alone, with untreated as 100%, n=3, p<0.01). MTX also negated COX-2 inhibitor-mediated downregulation of ABCA1. Reversal of inhibitory effects on 27-hydroxylase and ABCA1 in the presence of MTX were blocked by the adenosine A2A receptor-specific antagonist ZM-241385. MTX also prevented NS398 and IFN-γ from increasing transformation of lipid-loaded THP-1 macrophages into foam cells. CONCLUSIONS: This study provides evidence supporting the atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes RCT and limits foam cell formation in THP-1 macrophages. This is the first evidence that any commonly used medication can increase expression of anti-atherogenic RCT proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular mortality in RA patients is through facilitation of cholesterol outflow from cells of the artery wall. PMID:19035488

Atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human THP-1 monocyte/macrophages.

PubMed

Reiss, Allison B; Carsons, Steven E; Anwar, Kamran; Rao, Soumya; Edelman, Sari D; Zhang, Hongwei; Fernandez, Patricia; Cronstein, Bruce N; Chan, Edwin S L

2008-12-01

To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages. Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy. MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells. This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.

Living-Cell Microarrays

PubMed Central

Yarmush, Martin L.; King, Kevin R.

2011-01-01

Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

Identification and validation of vesicant therapeutic targets using a high, throughput siRNA screening approach

DTIC Science & Technology

2014-12-24

toxlet.2011.04.007 Rogers JV, Choi YW, Kiser RC et al (2004) Microarray analysis of gene expression in murine skin exposed to sulfur mustard. J Bio...Chemotactic factors released in culture by intact developing and healing skin lesions produced in rabbits by the irritant sulfur mustard. Inflam- mation 21(2...Project ID Number CBM.CUTOC.04.10. RC 00114. ABSTRACT See reprint. 15. SUBJECT TERMS sulfur mustard, cutaneous injury, siRNA, high-throughput screening

Whole CMV Proteome Pattern Recognition Analysis after HSCT Identifies Unique Epitope Targets Associated with the CMV Status

PubMed Central

Pérez-Bercoff, Lena; Valentini, Davide; Gaseitsiwe, Simani; Mahdavifar, Shahnaz; Schutkowski, Mike; Poiret, Thomas; Pérez-Bercoff, Åsa; Ljungman, Per; Maeurer, Markus J.

2014-01-01

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/− for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the ‘exclusive recognition analysis (ERA)’ to identify unique CMV epitope responses for each patient group. The ‘exclusive recognition analysis’ of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D−/R−). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution. PMID:24740411

Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma

PubMed Central

Konishi, H; Ichikawa, D; Komatsu, S; Shiozaki, A; Tsujiura, M; Takeshita, H; Morimura, R; Nagata, H; Arita, T; Kawaguchi, T; Hirashima, S; Fujiwara, H; Okamoto, K; Otsuji, E

2012-01-01

Background: Recently, it was reported that plasma microRNAs (miRNAs) are low-invasive useful biomarkers for cancer. We attempted to isolate gastric cancer (GC)-associated miRNAs comparing pre- and post-operative paired plasma, thereby excluding the possible effects of individual variability. Methods: This study was divided into four steps: (1) microarray analysis comparing pre- and post-operative plasma; (2) validation of candidate miRNAs by quantitative RT–PCR; (3) validation study of selected miRNAs using paired plasma; and (4) comparison of the levels of selected miRNAs in plasma between healthy controls and patients. Results: From the results of microarray analysis, nine candidate miRNAs the levels of which were markedly decreased in post-operative plasma were selected for further studies. After confirmation of their post-operative marked reduction, two candidate miRNAs, miR-451 and miR-486, were selected as plasma biomarkers, considering the abundance in plasma, and marked decrease in post-operative samples. In validation, the two miRNAs were found to decrease in post-operative plasma in 90 and 93% of patients (both P<0.01). In comparison with healthy controls, the levels of both miRNAs were found to be significantly higher in patients, and the area under the curve values were high at 0.96 and 0.92. Conclusion: Plasma miR-451 and miR-486 could be useful blood-based biomarkers for screening GC. PMID:22262318

The Role of Cytokine PF4 in the Antiviral Immune Response of Shrimp

PubMed Central

Chen, Yulei; Cao, Jiao; Zhang, Xiaobo

2016-01-01

During viral infection in vertebrates, cytokines play important roles in the host defense against the virus. However, the function of cytokines in invertebrates has not been well characterized. In this study, shrimp cytokines involved in viral infection were screened using a cytokine antibody microarray. The results showed that three cytokines, the Fas receptor (Fas), platelet factor 4 (PF4) and interleukin-22 (IL-22), were significantly upregulated in the white spot syndrome virus (WSSV)-challenged shrimp, suggesting that these cytokines played positive regulatory roles in the immune response of shrimp against the virus. Further experiments revealed that PF4 had positive effects on the antiviral immunity of shrimp by enhancing the shrimp phagocytic activity and inhibiting the apoptotic activity of virus-infected hemocytes. Therefore, our study presented a novel mechanism of cytokines in the innate immunity of invertebrates. PMID:27631372

Late-onset Stargardt disease is associated with missense mutations that map outside known functional regions of ABCR (ABCA4).

PubMed

Yatsenko, A N; Shroyer, N F; Lewis, R A; Lupski, J R

2001-04-01

Based on recent studies of the photoreceptor-specific ABC transporter gene ABCR (ABCA4) in Stargardt disease (STGD1) and other retinal dystrophies, we and others have developed a model in which the severity of retinal disease correlates inversely with residual ABCR activity. This model predicts that patients with late-onset STGDI may retain partial ABCR activity attributable to mild missense alleles. To test this hypothesis, we used late-onset STGDI patients (onset: > or =35 years) to provide an in vivo functional analysis of various combinations of mutant alleles. We sequenced directly the entire coding region of ABCR and detected mutations in 33/50 (66%) disease chromosomes, but surprisingly, 11/33 (33%) were truncating alleles. Importantly, all 22 missense mutations were located outside the known functional domains of ABCR (ATP-binding or transmembrane), whereas in our general cohort of STGDI subjects, alterations occurred with equal frequency across the entire protein. We suggest that these missense mutations in regions of unknown function are milder alleles and more susceptible to modifier effects. Thus, we have corroborated a prediction from the model of ABCR pathogenicity that (1) one mutant ABCR allele is always missense in late-onset STGD1 patients, and (2) the age-of-onset is correlated with the amount of ABCR activity of this allele. In addition, we report three new pseudodominant families that now comprise eight of 178 outbred STGD1 families and suggest a carrier frequency of STGD1-associated ABCR mutations of about 4.5% (approximately 1/22).

Automated microfluidic assay system for autoantibodies found in autoimmune diseases using a photoimmobilized autoantigen microarray.

PubMed

Matsudaira, Takahiro; Tsuzuki, Saki; Wada, Akira; Suwa, Akira; Kohsaka, Hitoshi; Tomida, Maiko; Ito, Yoshihiro

2008-01-01

Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and autoimmune diabetes are characterized by the production of autoantibodies that serve as useful diagnostic markers, surrogate markers, and prognostic factors. We devised an in vitro system to detect these clinically pivotal autoantibodies using a photoimmobilized autoantigen microarray. Photoimmobilization was useful for preparing the autoantigen microarray, where autoantigens are covalently immobilized on a plate, because it does not require specific functional groups of the autoantigens and any organic material can be immobilized by a radical reaction induced by photoirradiation. Here, we prepared the microarray using a very convenient method. Aqueous solutions of each autoantigen were mixed with a polymer of poly(ethylene glycol) methacrylate and a photoreactive crosslinker, and the mixtures were microspotted on a plate and dried in air. Finally, the plate was irradiated with an ultraviolet lamp to obtain immobilization. In the assay, patient serum was added to the microarray plate. Antigen-specific IgG adsorbed on the microspotted autoantigen was detected by peroxidase-conjugated anti-IgG antibody. The chemical luminescence intensities of the substrate decomposed by the peroxidase were detected with a sensitive CCD camera. All autoantigens were immobilized stably by this method and used to screen antigen-specific IgG. In addition, the plate was covered with a polydimethylsiloxane sheet containing microchannels and automated measurement was carried out.

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway.

PubMed

Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

2013-11-15

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. Copyright © 2013 Elsevier Inc. All rights reserved.

Comparison of L1000 and Affymetrix Microarray for In Vitro Concentration-Response Gene Expression Profiling (SOT)

EPA Science Inventory

Advances in high-throughput screening technologies and in vitro systems have opened doors for cost-efficient evaluation of chemical effects on a diversity of biological endpoints. However, toxicogenomics platforms remain too costly to evaluate large libraries of chemicals in conc...

EVALUATION OF DNA CHIPS (MICROARRAYS) FOR DETERMINING VIRULENCE FACTOR ACTIVITY RELATIONSHIPS (VFARS)

EPA Science Inventory

Computational toxicology is a rapid approach to screening for toxic effects and looking for common outcomes that can result in predictive models. The long term project will result in the development of a database of mRNA responses to known water-borne pathogens. An understanding...

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

PubMed Central

Ren, Juan; Zhang, Yuelang; Cai, Hui; Ma, Hongbing; Zhao, Dongli; Zhang, Xiaozhi; Li, Zongfang; Wang, Shufeng; Wang, Jiangsheng; Liu, Rui; Li, Yi; Qian, Jiansheng; Wei, Hongxia; Niu, Liying; Liu, Yan; Xiao, Lisha; Ding, Muyang; Jiang, Shiwen

2014-01-01

G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism. PMID:24753754

ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma.

PubMed

Dréan, Antonin; Rosenberg, Shai; Lejeune, François-Xavier; Goli, Larissa; Nadaradjane, Aravindan Arun; Guehennec, Jérémy; Schmitt, Charlotte; Verreault, Maïté; Bielle, Franck; Mokhtari, Karima; Sanson, Marc; Carpentier, Alexandre; Delattre, Jean-Yves; Idbaih, Ahmed

2018-03-08

ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p < 0.01) and multivariate analyses including MGMT promoter methylation (p = 0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM-PDCL. ABCA13 expression is an independent prognostic factor in newly diagnosed GBM patients. Further prospective studies are warranted to investigate whether ABCA13 expression can be used to further personalize treatments for GBM.

Diosgenin inhibits atherosclerosis via suppressing the MiR-19b-induced downregulation of ATP-binding cassette transporter A1.

PubMed

Lv, Yun-cheng; Yang, Jing; Yao, Feng; Xie, Wei; Tang, Yan-yan; Ouyang, Xin-ping; He, Ping-ping; Tan, Yu-lin; Li, Liang; Zhang, Min; Liu, Dan; Cayabyab, Francisco S; Zheng, Xi-Long; Tang, Chao-ke

2015-05-01

Diosgenin (Dgn), a structural analogue of cholesterol, has been reported to have the hypolipidemic and antiatherogenic properties, but the underlying mechanisms are not fully understood. Given the key roles of macrophages in cholesterol metabolism and atherogenesis, it is critical to investigate macrophage cholesterol efflux and development of atherosclerotic lesion after Dgn treatment. This study was designed to evaluate the potential effects of Dgn on macrophage cholesterol metabolism and the development of aortic atherosclerosis, and to explore its underlying mechanisms. Dgn significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) protein, but didn't affect liver X receptor α levels in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by western blotting. The miR-19b levels were markedly down-regulated in Dgn-treated THP-1 macrophages/MPM-derived foam cells. Cholesterol transport assays revealed that treatment with Dgn alone or together with miR-19b inhibitor notably enhanced ABCA1-dependent cholesterol efflux, resulting in the reduced levels of total cholesterol, free cholesterol and cholesterol ester as determined by high-performance liquid chromatography. The fecal 3H-sterol originating from cholesterol-laden MPMs was increased in apolipoprotein E knockout mice treated with Dgn or both Dgn and antagomiR-19b. Treatment with Dgn alone or together with antagomiR-19b elevated plasma high-density lipoprotein levels, but reduced plasma low-density lipoprotein levels. Accordingly, aortic lipid deposition and plaque area were reduced, and collagen content and ABCA1 expression were increased in mice treated with Dgn alone or together with antagomiR-19b. However, miR-19b overexpression abrogated the lipid-lowering and atheroprotective effects induced by Dgn. The present study demonstrates that Dgn enhances ABCA1-dependent cholesterol efflux and inhibits aortic atherosclerosis progression by suppressing macrophage miR-19b expression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Novel association of the R230C variant of the ABCA1 gene with high triglyceride levels and low high-density lipoprotein cholesterol levels in Mexican school-age children with high prevalence of obesity.

PubMed

Gamboa-Meléndez, Marco Alberto; Galindo-Gómez, Carlos; Juárez-Martínez, Liliana; Gómez, F Enrique; Diaz-Diaz, Eulises; Ávila-Arcos, Marco Antonio; Ávila-Curiel, Abelardo

2015-08-01

Metabolic syndrome (MetS) is a disorder that includes a cluster of several risk factors for the development of type 2 diabetes and cardiovascular disease. The R230C variant of the ABCA1 gene has been associated with low HDL-cholesterol in several studies, but its association with MetS in children remains to be determined. The aim of this study was to analyze the association of the R230C variant with MetS and other metabolic traits in school-aged Mexican children. The study was performed in seven urban primary schools in the State of Mexico. Four hundred thirty-two Mexican school-age children 6-13 years old were recruited. MetS was identified using the International Diabetes Federation definition. The R230C variant of the ABCA1 gene was genotyped to seek associations with MetS and other metabolic traits. The prevalence of MetS was 29% in children aged 10-13 years. The R230C variant was not associated with MetS (OR = 1.65; p = 0.139). Furthermore, in the whole population, the R230C variant was associated with low HDL-cholesterol levels (β coefficient = -3.28, p <0.001). Interestingly, in the total population we found a novel association of this variant with high triglyceride levels (β coefficient = 14.34; p = 0.027). We found a new association of the R230C variant of the ABCA1 gene with high triglyceride levels. Our findings also replicate the association of this variant with low HDL-cholesterol levels in Mexican school-age children. Copyright © 2015 IMSS. Published by Elsevier Inc. All rights reserved.

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity.

PubMed

Cukier, Alexandre M O; Therond, Patrice; Didichenko, Svetlana A; Guillas, Isabelle; Chapman, M John; Wright, Samuel D; Kontush, Anatol

2017-09-01

High-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised. Reconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL+LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages. rHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux. Increasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics. Non-standard abbreviations and acronyms: AAPH, 2,2'-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide. Copyright © 2017 Elsevier B.V. All rights reserved.

Hepatic Overexpression of Endothelial Lipase Lowers HDL (High-Density Lipoprotein) but Maintains Reverse Cholesterol Transport in Mice: Role of SR-BI (Scavenger Receptor Class B Type I)/ABCA1 (ATP-Binding Cassette Transporter A1)-Dependent Pathways.

PubMed

Takiguchi, Shunichi; Ayaori, Makoto; Yakushiji, Emi; Nishida, Takafumi; Nakaya, Kazuhiro; Sasaki, Makoto; Iizuka, Maki; Uto-Kondo, Harumi; Terao, Yoshio; Yogo, Makiko; Komatsu, Tomohiro; Ogura, Masatsune; Ikewaki, Katsunori

2018-05-10

Reverse cholesterol transport (RCT) is a major mechanism by which HDL (high-density lipoprotein) protects against atherosclerosis. Endothelial lipase (EL) reportedly reduces HDL levels, which, in theory, would increase atherosclerosis. However, it remains unclear whether EL affects RCT in vivo. Adenoviral vectors expressing EL or luciferase were intravenously injected into mice, and a macrophage RCT assay was performed. As expected, hepatic EL overexpression markedly reduced HDL levels. In parallel, plasma 3 H-cholesterol counts from the EL-expressing mice decreased by 85% compared with control. Surprisingly, there was no difference in fecal 3 H-cholesterol excretion between the groups. Kinetic studies revealed increased catabolism/hepatic uptake of 3 HDL-cholesteryl ether, resulting in no change in fecal HDL-cholesteryl ester excretion in the mice. To explore underlying mechanisms for the preservation of RCT despite low HDL levels in the EL-expressing mice, we investigated the effects of hepatic SR-BI (scavenger receptor class B type I) knockdown. RCT assay revealed that knockdown of SR-BI alone reduced fecal excretion of macrophage-derived 3 H-cholesterol. Interestingly, hepatic EL overexpression under SR-BI inhibition further attenuated fecal tracer counts as compared with control. Finally, we observed that EL overexpression enhanced in vivo RCT under pharmacological inhibition of hepatic ABCA1 (ATP-binding cassette transporter A1) by probucol. Hepatic EL expression compensates for reduced macrophage-derived cholesterol efflux to plasma because of low HDL levels by promoting cholesterol excretion to bile/feces via an SR-BI pathway, maintaining overall RCT in vivo. In contrast, EL-modified HDL might negatively regulate RCT via hepatic ABCA1. Despite extreme hypoalphalipoproteinemia, RCT is maintained in EL-expressing mice via SR-BI/ABCA1-dependent pathways. © 2018 American Heart Association, Inc.

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.

PubMed

Laassri, Majid; Bidzhieva, Bella; Speicher, James; Pletnev, Alexander G; Chumakov, Konstantin

2011-05-01

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G(2337) C (Met(457) Ile) in the E gene and A(6751) G (Lys(125) Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains. Copyright © 2011 Wiley-Liss, Inc.

Microarray Hybridization for Assessment of the Genetic Stability of Chimeric West Nile/Dengue 4 Virus

PubMed Central

Laassri, Majid; Bidzhieva, Bella; Speicher, James; Pletnev, Alexander G.; Chumakov, Konstantin

2012-01-01

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G2337C (Met457Ile) in the E gene and A6751G (Lys125Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this paper could be useful for monitoring the molecular consistency and quality control of vaccine strains. PMID:21360544

Cone and Rod Loss in Stargardt Disease Revealed by Adaptive Optics Scanning Light Ophthalmoscopy

PubMed Central

Song, Hongxin; Rossi, Ethan A.; Latchney, Lisa; Bessette, Angela; Stone, Edwin; Hunter, Jennifer J.; Williams, David R.; Chung, Mina

2015-01-01

Importance Stargardt disease (STGD1) is characterized by macular atrophy and flecks in the retinal pigment epithelium. The causative ABCA4 gene encodes a protein localizing to photoreceptor outer segments. The pathologic steps by which ABCA4 mutations lead to clinically detectable retinal pigment epithelium changes remain unclear. We investigated early STGD1 using adaptive optics scanning light ophthalmoscopy. Observations Adaptive optics scanning light ophthalmoscopy imaging of 2 brothers with early STGD1 and their unaffected parents was compared with conventional imaging. Cone and rod spacing were increased in both patients (P <.001) with a dark cone appearance. No foveal cones were detected in the older brother. In the younger brother, foveal cones were enlarged with low density (peak cone density, 48.3 × 103 cones/mm2). The ratio of cone to rod spacing was increased in both patients, with greater divergence from normal approaching the foveal center, indicating that cone loss predominates centrally and rod loss increases peripherally. Both parents had normal photoreceptor mosaics. Genetic testing revealed 3 disease-causing mutations. Conclusions and Relevance This study provides in vivo images of rods and cones in STGD1. Although the primary clinical features of STGD1 are retinal pigment epithelial lesions, adaptive optics scanning light ophthalmoscopy reveals increased cone and rod spacing in areas that appear normal in conventional images, suggesting that photoreceptor loss precedes clinically detectable retinal pigment epithelial disease in STGD1. PMID:26247787

Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.

PubMed

Charbel Issa, Peter; De Silva, Samantha R; Lipinski, Daniel M; Singh, Mandeep S; Mouravlev, Alexandre; You, Qisheng; Barnard, Alun R; Hankins, Mark W; During, Matthew J; Maclaren, Robert E

2013-01-01

Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4(-/-) mouse which is a model for Stargardt disease and in the Pde6b(rd1/rd1) mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4(-/-) mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.

Design of 240,000 orthogonal 25mer DNA barcode probes.

PubMed

Xu, Qikai; Schlabach, Michael R; Hannon, Gregory J; Elledge, Stephen J

2009-02-17

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications.

Design of 240,000 orthogonal 25mer DNA barcode probes

PubMed Central

Xu, Qikai; Schlabach, Michael R.; Hannon, Gregory J.; Elledge, Stephen J.

2009-01-01

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications. PMID:19171886

Complementary techniques: validation of gene expression data by quantitative real time PCR.

PubMed

Provenzano, Maurizio; Mocellin, Simone

2007-01-01

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

From High-Throughput Microarray-Based Screening to Clinical Application: The Development of a Second Generation Multigene Test for Breast Cancer Prognosis

PubMed Central

Brase, Jan C.; Kronenwett, Ralf; Petry, Christoph; Denkert, Carsten; Schmidt, Marcus

2013-01-01

Several multigene tests have been developed for breast cancer patients to predict the individual risk of recurrence. Most of the first generation tests rely on proliferation-associated genes and are commonly carried out in central reference laboratories. Here, we describe the development of a second generation multigene assay, the EndoPredict test, a prognostic multigene expression test for estrogen receptor (ER) positive, human epidermal growth factor receptor (HER2) negative (ER+/HER2−) breast cancer patients. The EndoPredict gene signature was initially established in a large high-throughput microarray-based screening study. The key steps for biomarker identification are discussed in detail, in comparison to the establishment of other multigene signatures. After biomarker selection, genes and algorithms were transferred to a diagnostic platform (reverse transcription quantitative PCR (RT-qPCR)) to allow for assaying formalin-fixed, paraffin-embedded (FFPE) samples. A comprehensive analytical validation was performed and a prospective proficiency testing study with seven pathological laboratories finally proved that EndoPredict can be reliably used in the decentralized setting. Three independent large clinical validation studies (n = 2,257) demonstrated that EndoPredict offers independent prognostic information beyond current clinicopathological parameters and clinical guidelines. The review article summarizes several important steps that should be considered for the development process of a second generation multigene test and offers a means for transferring a microarray signature from the research laboratory to clinical practice. PMID:27605191

Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology.

PubMed

Cirelli, C; Tononi, G

1999-06-01

The consequences of sleep and sleep deprivation at the molecular level are largely unexplored. Knowledge of such molecular events is essential to understand the restorative processes occurring during sleep as well as the cellular mechanisms of sleep regulation. Here we review the available data about changes in neural gene expression across different behavioural states using candidate gene approaches such as in situ hybridization and immunocytochemistry. We then describe new techniques for systematic screening of gene expression in the brain, such as subtractive hybridization, mRNA differential display, and cDNA microarray technology, outlining advantages and disadvantages of these methods. Finally, we summarize our initial results of a systematic screening of gene expression in the rat brain across behavioural states using mRNA differential display and cDNA microarray technology. The expression pattern of approximately 7000 genes was analysed in the cerebral cortex of rats after 3 h of spontaneous sleep, 3 h of spontaneous waking, or 3 h of sleep deprivation. While the majority of transcripts were expressed at the same level among these three conditions, 14 mRNAs were modulated by sleep and waking. Six transcripts, four more expressed in waking and two more expressed in sleep, corresponded to novel genes. The eight known transcripts were all expressed at higher levels in waking than in sleep and included transcription factors and mitochondrial genes. A possible role for these known transcripts in mediating neural plasticity during waking is discussed.

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin-3 Inhibitor.

PubMed

Dion, Johann; Advedissian, Tamara; Storozhylova, Nataliya; Dahbi, Samir; Lambert, Annie; Deshayes, Frédérique; Viguier, Mireille; Tellier, Charles; Poirier, Françoise; Téletchéa, Stéphane; Dussouy, Christophe; Tateno, Hiroaki; Hirabayashi, Jun; Grandjean, Cyrille

2017-12-14

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3'-mono- and 2,3'-diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2'-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface plasmon resonance imaging system with Mach-Zehnder phase-shift interferometry for DNA micro-array hybridization

NASA Astrophysics Data System (ADS)

Hsiu, Feng-Ming; Chen, Shean-Jen; Tsai, Chien-Hung; Tsou, Chia-Yuan; Su, Y.-D.; Lin, G.-Y.; Huang, K.-T.; Chyou, Jin-Jung; Ku, Wei-Chih; Chiu, S.-K.; Tzeng, C.-M.

2002-09-01

Surface plasmon resonance (SPR) imaging system is presented as a novel technique based on modified Mach-Zehnder phase-shifting interferometry (PSI) for biomolecular interaction analysis (BIA), which measures the spatial phase variation of a resonantly reflected light in biomolecular interaction. In this technique, the micro-array SPR biosensors with over a thousand probe NDA spots can be detected simultaneously. Owing to the feasible and swift measurements, the micro-array SPR biosensors can be extensively applied to the nonspecific adsorption of protein, the membrane/protein interactions, and DNA hybridization. The detection sensitivity of the SPR PSI imaging system is improved to about 1 pg/mm2 for each spot over the conventional SPR imaging systems. The SPR PSI imaging system and its SPR sensors have been successfully used to observe slightly index change in consequence of argon gas flow through the nitrogen in real time, with high sensitivity, and at high-throughout screening rates.

Carbohydrate Microarrays Identify Blood Group Precursor Cryptic Epitopes as Potential Immunological Targets of Breast Cancer

PubMed Central

Wang, Denong; Tang, Jin; Liu, Shaoyi

2015-01-01

Using carbohydrate microarrays, we explored potential natural ligands of antitumor monoclonal antibody HAE3. This antibody was raised against a murine mammary tumor antigen but was found to cross-react with a number of human epithelial tumors in tissues. Our carbohydrate microarray analysis reveals that HAE3 is specific for an O-glycan cryptic epitope that is normally hidden in the cores of blood group substances. Using HAE3 to screen tumor cell surface markers by flow cytometry, we found that the HAE3 glycoepitope, gpHAE3, was highly expressed by a number of human breast cancer cell lines, including some triple-negative cancers that lack the estrogen, progesterone, and Her2/neu receptors. Taken together, we demonstrate that HAE3 recognizes a conserved cryptic glycoepitope of blood group precursors, which is nevertheless selectively expressed and surface-exposed in certain breast tumor cells. The potential of this class of O-glycan cryptic antigens in breast cancer subtyping and targeted immunotherapy warrants further investigation. PMID:26539555

Suppression subtractive hybridization as a tool to identify anthocyanin metabolism-related genes in apple skin.

PubMed

Ban, Yusuke; Moriguchi, Takaya

2010-01-01

The pigmentation of anthocyanins is one of the important determinants for consumer preference and marketability in horticultural crops such as fruits and flowers. To elucidate the mechanisms underlying the physiological process leading to the pigmentation of anthocyanins, identification of the genes differentially expressed in response to anthocyanin accumulation is a useful strategy. Currently, microarrays have been widely used to isolate differentially expressed genes. However, the use of microarrays is limited by its high cost of special apparatus and materials. Therefore, availability of microarrays is limited and does not come into common use at present. Suppression subtractive hybridization (SSH) is an alternative tool that has been widely used to identify differentially expressed genes due to its easy handling and relatively low cost. This chapter describes the procedures for SSH, including RNA extraction from polysaccharides and polyphenol-rich samples, poly(A)+ RNA purification, evaluation of subtraction efficiency, and differential screening using reverse northern in apple skin.

Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

NASA Astrophysics Data System (ADS)

Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

2007-12-01

Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray spots carrying different DNA vectors was also investigated. By application of a voltage of 286 V/cm for 10 ms, transfection efficiency was doubled compared to using only transfection reagent, whilst maintaining a cell viability of 60-70% of the positive control.

A biomarker-based screen of a gene expression compendium reveals regulation of Nrf2 by CAR and STAT5b

EPA Science Inventory

Computational approaches were developed to identify factors that regulate Nrf2 in a large gene expression compendium of microarray profiles including >2000 comparisons which queried the effects of chemicals, genes, diets, and infectious agents on gene expression in the mouse l...

Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands.

PubMed

Rabinowitz, Joshua A; Lainson, John C; Johnston, Stephen Albert; Diehnelt, Chris W

2018-02-06

We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

DNA Microarray-Based Screening and Characterization of Traditional Chinese Medicine

PubMed Central

Kiyama, Ryoiti

2017-01-01

The application of DNA microarray assay (DMA) has entered a new era owing to recent innovations in omics technologies. This review summarizes recent applications of DMA-based gene expression profiling by focusing on the screening and characterization of traditional Chinese medicine. First, herbs, mushrooms, and dietary plants analyzed by DMA along with their effective components and their biological/physiological effects are summarized and discussed by examining their comprehensive list and a list of representative effective chemicals. Second, the mechanisms of action of traditional Chinese medicine are summarized by examining the genes and pathways responsible for the action, the cell functions involved in the action, and the activities found by DMA (silent estrogens). Third, applications of DMA for traditional Chinese medicine are discussed by examining reported examples and new protocols for its use in quality control. Further innovations in the signaling pathway-based evaluation of beneficial effects and the assessment of potential risks of traditional Chinese medicine are expected, just as are observed in other closely related fields, such as the therapeutic, environmental, nutritional, and pharmacological fields. PMID:28146102

Transcriptomic profiling as a screening tool to detect trenbolone treatment in beef cattle.

PubMed

Pegolo, S; Cannizzo, F T; Biolatti, B; Castagnaro, M; Bargelloni, L

2014-06-01

The effects of steroid hormone implants containing trenbolone alone (Finaplix-H), combined with 17β-oestradiol (17β-E; Revalor-H), or with 17β-E and dexamethasone (Revalor-H plus dexamethasone per os) on the bovine muscle transcriptome were examined by DNA-microarray. Overall, large sets of genes were shown to be modulated by the different growth promoters (GPs) and the regulated pathways and biological processes were mostly shared among the treatment groups. Using the Prediction Analysis of Microarray program, GP-treated animals were accurately identified by a small number of predictive genes. A meta-analysis approach was also carried out for the Revalor group to potentially increase the robustness of class prediction analysis. After data pre-processing, a high level of accuracy (90%) was obtained in the classification of samples, using 105 predictive gene markers. Transcriptomics could thus help in the identification of indirect biomarkers for anabolic treatment in beef cattle to be applied for the screening of muscle samples collected after slaughtering. Copyright © 2014 Elsevier Ltd. All rights reserved.

Practice guideline: joint CCMG-SOGC recommendations for the use of chromosomal microarray analysis for prenatal diagnosis and assessment of fetal loss in Canada

PubMed Central

Armour, Christine M; Dougan, Shelley Danielle; Brock, Jo-Ann; Chari, Radha; Chodirker, Bernie N; DeBie, Isabelle; Evans, Jane A; Gibson, William T; Kolomietz, Elena; Nelson, Tanya N; Tihy, Frédérique; Thomas, Mary Ann; Stavropoulos, Dimitri J

2018-01-01

Background The aim of this guideline is to provide updated recommendations for Canadian genetic counsellors, medical geneticists, maternal fetal medicine specialists, clinical laboratory geneticists and other practitioners regarding the use of chromosomal microarray analysis (CMA) for prenatal diagnosis. This guideline replaces the 2011 Society of Obstetricians and Gynaecologists of Canada (SOGC)-Canadian College of Medical Geneticists (CCMG) Joint Technical Update. Methods A multidisciplinary group consisting of medical geneticists, genetic counsellors, maternal fetal medicine specialists and clinical laboratory geneticists was assembled to review existing literature and guidelines for use of CMA in prenatal care and to make recommendations relevant to the Canadian context. The statement was circulated for comment to the CCMG membership-at-large for feedback and, following incorporation of feedback, was approved by the CCMG Board of Directors on 5 June 2017 and the SOGC Board of Directors on 19 June 2017. Results and conclusions Recommendations include but are not limited to: (1) CMA should be offered following a normal rapid aneuploidy screen when multiple fetal malformations are detected (II-1A) or for nuchal translucency (NT) ≥3.5 mm (II-2B) (recommendation 1); (2) a professional with expertise in prenatal chromosomal microarray analysis should provide genetic counselling to obtain informed consent, discuss the limitations of the methodology, obtain the parental decisions for return of incidental findings (II-2A) (recommendation 4) and provide post-test counselling for reporting of test results (III-A) (recommendation 9); (3) the resolution of chromosomal microarray analysis should be similar to postnatal microarray platforms to ensure small pathogenic variants are detected. To minimise the reporting of uncertain findings, it is recommended that variants of unknown significance (VOUS) smaller than 500 Kb deletion or 1 Mb duplication not be routinely reported in the prenatal context. Additionally, VOUS above these cut-offs should only be reported if there is significant supporting evidence that deletion or duplication of the region may be pathogenic (III-B) (recommendation 5); (4) secondary findings associated with a medically actionable disorder with childhood onset should be reported, whereas variants associated with adult-onset conditions should not be reported unless requested by the parents or disclosure can prevent serious harm to family members (III-A) (recommendation 8). The working group recognises that there is variability across Canada in delivery of prenatal testing, and these recommendations were developed to promote consistency and provide a minimum standard for all provinces and territories across the country (recommendation 9). PMID:29496978

Plasma long noncoding RNA expression profile identified by microarray in patients with Crohn's disease.

PubMed

Chen, Dong; Liu, Jiang; Zhao, Hui-Ying; Chen, Yi-Peng; Xiang, Zun; Jin, Xi

2016-05-21

To investigate the expression pattern of plasma long noncoding RNAs (lncRNAs) in Chrohn's disease (CD) patients. Microarray screening and qRT-PCR verification of lncRNAs and mRNAs were performed in CD and control subjects, followed by hierarchy clustering, GO and KEGG pathway analyses. Significantly dysregulated lncRNAs were categorized into subgroups of antisense lncRNAs, enhancer lncRNAs and lincRNAs. To predict the regulatory effect of lncRNAs on mRNAs, a CNC network analysis was performed and cross linked with significantly changed lncRNAs. The overlapping lncRNAs were randomly selected and verified by qRT-PCR in a larger cohort. Initially, there were 1211 up-regulated and 777 down-regulated lncRNAs as well as 1020 up-regulated and 953 down-regulated mRNAs after microarray analysis; a heat map based on these results showed good categorization into the CD and control groups. GUSBP2 and AF113016 had the highest fold change of the up- and down-regulated lncRNAs, whereas TBC1D17 and CCL3L3 had the highest fold change of the up- and down-regulated mRNAs. Six (SNX1, CYFIP2, CD6, CMTM8, STAT4 and IGFBP7) of 10 mRNAs and 8 (NR_033913, NR_038218, NR_036512, NR_049759, NR_033951, NR_045408, NR_038377 and NR_039976) of 14 lncRNAs showed the same change trends on the microarray and qRT-PCR results with statistical significance. Based on the qRT-PCR verified mRNAs, 1358 potential lncRNAs with 2697 positive correlations and 2287 negative correlations were predicted by the CNC network. The plasma lncRNAs profiles provide preliminary data for the non-invasive diagnosis of CD and a resource for further specific lncRNA-mRNA pathway exploration.

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia

PubMed Central

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-01-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre-processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)-gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein-DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid-repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF-pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF-gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA-box binding protein associated factor 1 and CCCTC-binding factor, which may be potential therapeutic targets of AML. PMID:28498449

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia.

PubMed

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-07-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre‑processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)‑gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein‑DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid‑repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF‑pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF‑gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA‑box binding protein associated factor 1 and CCCTC‑binding factor, which may be potential therapeutic targets of AML.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.

PubMed

Chruscinski, Andrzej; Huang, Flora Y Y; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C; Kozuszko, Stella; Tinckam, Kathryn J; Rao, Vivek; Dunn, Shannon E; Persinger, Michael A; Levy, Gary A; Ross, Heather J

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation

PubMed Central

Chruscinski, Andrzej; Huang, Flora Y. Y.; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C.; Kozuszko, Stella; Tinckam, Kathryn J.; Rao, Vivek; Dunn, Shannon E.; Persinger, Michael A.; Levy, Gary A.; Ross, Heather J.

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR. PMID:26967734

Genome-wide association study of Alzheimer's disease.

PubMed

Kamboh, M I; Demirci, F Y; Wang, X; Minster, R L; Carrasquillo, M M; Pankratz, V S; Younkin, S G; Saykin, A J; Jun, G; Baldwin, C; Logue, M W; Buros, J; Farrer, L; Pericak-Vance, M A; Haines, J L; Sweet, R A; Ganguli, M; Feingold, E; Dekosky, S T; Lopez, O L; Barmada, M M

2012-05-15

In addition to apolipoprotein E (APOE), recent large genome-wide association studies (GWASs) have identified nine other genes/loci (CR1, BIN1, CLU, PICALM, MS4A4/MS4A6E, CD2AP, CD33, EPHA1 and ABCA7) for late-onset Alzheimer's disease (LOAD). However, the genetic effect attributable to known loci is about 50%, indicating that additional risk genes for LOAD remain to be identified. In this study, we have used a new GWAS data set from the University of Pittsburgh (1291 cases and 938 controls) to examine in detail the recently implicated nine new regions with Alzheimer's disease (AD) risk, and also performed a meta-analysis utilizing the top 1% GWAS single-nucleotide polymorphisms (SNPs) with P<0.01 along with four independent data sets (2727 cases and 3336 controls) for these SNPs in an effort to identify new AD loci. The new GWAS data were generated on the Illumina Omni1-Quad chip and imputed at ~2.5 million markers. As expected, several markers in the APOE regions showed genome-wide significant associations in the Pittsburg sample. While we observed nominal significant associations (P<0.05) either within or adjacent to five genes (PICALM, BIN1, ABCA7, MS4A4/MS4A6E and EPHA1), significant signals were observed 69-180 kb outside of the remaining four genes (CD33, CLU, CD2AP and CR1). Meta-analysis on the top 1% SNPs revealed a suggestive novel association in the PPP1R3B gene (top SNP rs3848140 with P = 3.05E-07). The association of this SNP with AD risk was consistent in all five samples with a meta-analysis odds ratio of 2.43. This is a potential candidate gene for AD as this is expressed in the brain and is involved in lipid metabolism. These findings need to be confirmed in additional samples.

Genome-wide association study of Alzheimer's disease

PubMed Central

Kamboh, M I; Demirci, F Y; Wang, X; Minster, R L; Carrasquillo, M M; Pankratz, V S; Younkin, S G; Saykin, A J; Jun, G; Baldwin, C; Logue, M W; Buros, J; Farrer, L; Pericak-Vance, M A; Haines, J L; Sweet, R A; Ganguli, M; Feingold, E; DeKosky, S T; Lopez, O L; Barmada, M M

2012-01-01

In addition to apolipoprotein E (APOE), recent large genome-wide association studies (GWASs) have identified nine other genes/loci (CR1, BIN1, CLU, PICALM, MS4A4/MS4A6E, CD2AP, CD33, EPHA1 and ABCA7) for late-onset Alzheimer's disease (LOAD). However, the genetic effect attributable to known loci is about 50%, indicating that additional risk genes for LOAD remain to be identified. In this study, we have used a new GWAS data set from the University of Pittsburgh (1291 cases and 938 controls) to examine in detail the recently implicated nine new regions with Alzheimer's disease (AD) risk, and also performed a meta-analysis utilizing the top 1% GWAS single-nucleotide polymorphisms (SNPs) with P<0.01 along with four independent data sets (2727 cases and 3336 controls) for these SNPs in an effort to identify new AD loci. The new GWAS data were generated on the Illumina Omni1-Quad chip and imputed at ∼2.5 million markers. As expected, several markers in the APOE regions showed genome-wide significant associations in the Pittsburg sample. While we observed nominal significant associations (P<0.05) either within or adjacent to five genes (PICALM, BIN1, ABCA7, MS4A4/MS4A6E and EPHA1), significant signals were observed 69–180 kb outside of the remaining four genes (CD33, CLU, CD2AP and CR1). Meta-analysis on the top 1% SNPs revealed a suggestive novel association in the PPP1R3B gene (top SNP rs3848140 with P=3.05E–07). The association of this SNP with AD risk was consistent in all five samples with a meta-analysis odds ratio of 2.43. This is a potential candidate gene for AD as this is expressed in the brain and is involved in lipid metabolism. These findings need to be confirmed in additional samples. PMID:22832961

Applicability of a gene expression based prediction method to SD and Wistar rats: an example of CARCINOscreen®.

PubMed

Matsumoto, Hiroshi; Saito, Fumiyo; Takeyoshi, Masahiro

2015-12-01

Recently, the development of several gene expression-based prediction methods has been attempted in the fields of toxicology. CARCINOscreen® is a gene expression-based screening method to predict carcinogenicity of chemicals which target the liver with high accuracy. In this study, we investigated the applicability of the gene expression-based screening method to SD and Wistar rats by using CARCINOscreen®, originally developed with F344 rats, with two carcinogens, 2,4-diaminotoluen and thioacetamide, and two non-carcinogens, 2,6-diaminotoluen and sodium benzoate. After the 28-day repeated dose test was conducted with each chemical in SD and Wistar rats, microarray analysis was performed using total RNA extracted from each liver. Obtained gene expression data were applied to CARCINOscreen®. Predictive scores obtained by the CARCINOscreen® for known carcinogens were > 2 in all strains of rats, while non-carcinogens gave prediction scores below 0.5. These results suggested that the gene expression based screening method, CARCINOscreen®, can be applied to SD and Wistar rats, widely used strains in toxicological studies, by setting of an appropriate boundary line of prediction score to classify the chemicals into carcinogens and non-carcinogens.

Development of Biomarkers for Screening Hepatocellular Carcinoma Using Global Data Mining and Multiple Reaction Monitoring

PubMed Central

Yu, Su Jong; Jang, Eun Sun; Yu, Jiyoung; Cho, Geunhee; Yoon, Jung-Hwan; Kim, Youngsoo

2013-01-01

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases. PMID:23717429

Development of biomarkers for screening hepatocellular carcinoma using global data mining and multiple reaction monitoring.

PubMed

Kim, Hyunsoo; Kim, Kyunggon; Yu, Su Jong; Jang, Eun Sun; Yu, Jiyoung; Cho, Geunhee; Yoon, Jung-Hwan; Kim, Youngsoo

2013-01-01

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases.

Detection of novel and potentially actionable anaplastic lymphoma kinase (ALK) rearrangement in colorectal adenocarcinoma by immunohistochemistry screening

PubMed Central

Wang, Kai; Kim, Sun Young; Jang, Jiryeon; Kim, Seung Tae; Park, Joon Oh; Lim, Ho Yeong; Kang, Won Ki; Park, Young Suk; Lee, Jiyun; Lee, Woo Yong; Park, Yoon Ah; Huh, Jung Wook; Yun, Seong Hyeon; Do, In-Gu; Kim, Seok Hyung; Balasubramanian, Sohail; Stephens, Philip J.; Ross, Jeffrey S.; Li, Gang Gary; Hornby, Zachary; Ali, Siraj M.; Miller, Vincent A.; Kim, Kyoung-Mee; Ou, Sai-Hong Ignatius

2015-01-01

Purpose Anaplastic lymphoma kinase (ALK) rearrangement has been detected in colorectal carcinoma (CRC) using advanced molecular diagnostics tests including exon scanning, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). We investigated if immunohistochemistry (IHC) can be used to detect ALK rearrangement in gastrointestinal malignancies. Experimental designs Tissue microarrays (TMAs) from consecutive gastric carcinoma (GC) and CRC patients who underwent surgical resection at Samsung Medical Center, Seoul, Korea were screened by IHC using ALK monoclonal antibody 5A4. IHC positive cases were confirmed by FISH, nCounter assays, and NGS-based comprehensive genomic profiling (CGP). ALK IHC was further applied to CRC patients enrolled in a pathway-directed therapeutic trial. Results Four hundred thirty-two GC and 172 CRC cases were screened by IHC. No GC sample was ALK IHC positive. One CRC (0.6%) was ALK IHC positive (3+) that was confirmed by ALK FISH and a novel CAD-ALK (C35; A20) fusion variant that resulted from a paracentric inversion event inv(2)(p22–21p23) was identified by CGP. One out of 50 CRC patients enrolled in a pathway-directed therapeutic trial was ALK IHC positive (3+) confirmed by ALK FISH and found to harbor the EML4-ALK (E21, A20) fusion variant by CGP. Growth of a tumor cell line derived from this EML4-ALK CRC patient was inhibited by ALK inhibitors crizotinib and entrectinib. Conclusions ALK IHC is a viable screening strategy for identifying ALK rearrangement in CRC. ALK rearrangement is a potential actionable driver mutation in CRC based on survival inhibition of patient tumor-derived cell line by potent ALK inhibitors. PMID:26172300

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

PubMed Central

List, Claudia; Qi, Weihong; Maag, Eva; Gottstein, Bruno; Müller, Norbert; Felger, Ingrid

2010-01-01

Background Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. Methodology/Principal Findings Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. Conclusions/Significance This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens. PMID:20689813

Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARα, β/δ, and γ

PubMed Central

Li, Andrew C.; Binder, Christoph J.; Gutierrez, Alejandra; Brown, Kathleen K.; Plotkin, Christine R.; Pattison, Jennifer W.; Valledor, Annabel F.; Davis, Roger A.; Willson, Timothy M.; Witztum, Joseph L.; Palinski, Wulf; Glass, Christopher K.

2004-01-01

PPARα, β/δ, and γ regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor–ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARα, β, and γ agonists on foam-cell formation and atherosclerosis in male LDL receptor–deficient (LDLR–/–) mice. Like the PPARγ agonist, a PPARα-specific agonist strongly inhibited atherosclerosis, whereas a PPARβ-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARα and PPARγ agonists, but not the PPARβ agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARα and PPARγ agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARα required LXRs, activation of PPARγ reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis. PMID:15578089

[Effect of ferulic acid on cholesterol efflux in macrophage foam cell formation and potential mechanism].

PubMed

Chen, Fu-xin; Wang, Lian-kai

2015-02-01

The formation of macrophage-derived foam cells is a typical feature of atherosclerosis (AS). Reverse cholesterol efflux (RCT) is one of important factors for the formation of macrophage foam cells. In this study, macrophage form cells were induced by oxidized low density lipoprotein (ox-LDL) and then treated with different concentrations of ferulic acid, so as to observe the effect of ferulic acid on the intracellular lipid metabolism in the ox-LDL-induced macrophage foam cell formation, the cholesterol efflux and the mRNA expression and protein levels of ATP binding cassette transporter A1 (ABCA1) and ATP binding cassette transporter G1 (ABCG1) that mediate cholesterol efflux, and discuss the potential mechanism of ferulic acid in resisting AS. According to the findings, compared with the control group, the ox-LDL-treated group showed significant increase in intracellular lipid content, especially for the cholesterol content; whereas the intracellular lipid accumulation markedly decreased, after the treatment with ferulic acid. The data also demonstrated that the mRNA and protein expressions of ABCA1 and ABCG1 significantly increased after macrophage foam cells were treated with different concentrations of ferulic acid. In summary, ferulic acid may show the anti-atherosclerosis effect by increasing the surface ABCA1 and ABCG1 expressions of macrophage form cells and promoting cholesterol efflux.

MECHANISMS OF COIX SEED COMPOSITIONS IN THE TREATMENT OF SPLEEN DEFICIENCY AND WET DAMPNESS ZHENG.

PubMed

Han, Xiaochun; Ji, Xuming; Zhao, Haijun; Zhang, Yanan; Liu, Guowei; Wang, Yanfang; Zhao, Wenxiao; Wang, Shijun

2017-01-01

Coix seed has the functions of fortifying the spleen and inhibiting the dampness. However, it remains unclear which Coix seed compositions is responsible for these functions. Previous investigations have revealed that the main compositions of Coix seed are proteins, polysaccharides, oils and starches. The objectives of this study are to explore which is the most effective compositions in fortifying the spleen and examine how Coix seed works in regulating the water transport on the spleen deficiency and wet dampness (SDWD) rat model. The rats used were divided into (i) control group, (ii) model group, (iii) decoction group, (iv) protein group, (v) polysaccharide group, (vi) oil group and (vii) starch group. The urine volume, the drinking volume and the water loading index in each group were calculated. Agilent 8*60K array was used for microarray-based gene expression analysis. The differential mRNAs related to the transport activity were screened. qRT-PCR was used to validate the mRNA microarray. The results demonstrated that all treatment groups could decrease the dampness of SDWD rats. mRNA microarray had significant effect on the protein group and the polysaccharide group in regulating the water transport, among which the most significant mRNA was Fabp6, Slc51a, Slc51b, Slc11a2, Slc4a10 and AQP3 respectively. The compositions of proteins and polysaccharides had the most significant effect in regulating the water transport of SDWD rat model. The contributing mRNA focused on Fabp, Slc and AQP family.

In vitro study of the effects of ELF electric fields on gene expression in human epidermal cells.

PubMed

Collard, Jean-Francois; Mertens, Benjamin; Hinsenkamp, Maurice

2011-01-01

An acceleration of differentiation, at the expense of proliferation, is observed after exposure of various biological models to low frequency and low amplitude electric and electromagnetic fields. Following these results showing significant modifications, we try to identify the biological mechanism involved at the cell level through microarray screening. For this study, we use epidermis cultures harvested from human abdominoplasty. Two platinum electrodes are used to apply the electric signal. The gene expressions of 38,500 well-characterized human genes are analyzed using Affymetrix(®) microarray U133 Plus 2.0 chips. The protocol is repeated on three different patients. After three periods of exposure, a total of 24 chips have been processed. After the application of ELF electric fields, the microarray analysis confirms a modification of the gene expression of epidermis cells. Particularly, four up-regulated genes (DKK1, TXNRD1, ATF3, and MME) and one down-regulated gene (MACF1) are involved in the regulation of proliferation and differentiation. Expression of these five genes was also confirmed by real-time rtPCR in all samples used for microarray analysis. These results corroborate an acceleration of cell differentiation at the expense of cell proliferation. © 2010 Wiley-Liss, Inc.

Development and Validation of Sandwich ELISA Microarrays with Minimal Assay Interference

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

Gonzalez, Rachel M.; Servoss, Shannon; Crowley, Sheila A.

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA’s ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of the multiplexed 24-assay system. We findmore » that non-specific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a “purified antigen”. We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals then within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.« less

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Microfluidics for cell-based high throughput screening platforms - A review.

PubMed

Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

2016-01-15

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

Ossification of the posterior longitudinal ligament related genes identification using microarray gene expression profiling and bioinformatics analysis.

PubMed

He, Hailong; Mao, Lingzhou; Xu, Peng; Xi, Yanhai; Xu, Ning; Xue, Mingtao; Yu, Jiangming; Ye, Xiaojian

2014-01-10

Ossification of the posterior longitudinal ligament (OPLL) is a kind of disease with physical barriers and neurological disorders. The objective of this study was to explore the differentially expressed genes (DEGs) in OPLL patient ligament cells and identify the target sites for the prevention and treatment of OPLL in clinic. Gene expression data GSE5464 was downloaded from Gene Expression Omnibus; then DEGs were screened by limma package in R language, and changed functions and pathways of OPLL cells compared to normal cells were identified by DAVID (The Database for Annotation, Visualization and Integrated Discovery); finally, an interaction network of DEGs was constructed by string. A total of 1536 DEGs were screened, with 31 down-regulated and 1505 up-regulated genes. Response to wounding function and Toll-like receptor signaling pathway may involve in the development of OPLL. Genes, such as PDGFB, PRDX2 may involve in OPLL through response to wounding function. Toll-like receptor signaling pathway enriched genes such as TLR1, TLR5, and TLR7 may involve in spine cord injury in OPLL. PIK3R1 was the hub gene in the network of DEGs with the highest degree; INSR was one of the most closely related genes of it. OPLL related genes screened by microarray gene expression profiling and bioinformatics analysis may be helpful for elucidating the mechanism of OPLL. © 2013.

A gene expression biomarker accurately predicts estrogen ...

EPA Pesticide Factsheets

The EPA’s vision for the Endocrine Disruptor Screening Program (EDSP) in the 21st Century (EDSP21) includes utilization of high-throughput screening (HTS) assays coupled with computational modeling to prioritize chemicals with the goal of eventually replacing current Tier 1 screening tests. The ToxCast program currently includes 18 HTS in vitro assays that evaluate the ability of chemicals to modulate estrogen receptor α (ERα), an important endocrine target. We propose microarray-based gene expression profiling as a complementary approach to predict ERα modulation and have developed computational methods to identify ERα modulators in an existing database of whole-genome microarray data. The ERα biomarker consisted of 46 ERα-regulated genes with consistent expression patterns across 7 known ER agonists and 3 known ER antagonists. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression data sets from experiments in MCF-7 cells. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% or 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) OECD ER reference chemicals including “very weak” agonists and replicated predictions based on 18 in vitro ER-associated HTS assays. For 114 chemicals present in both the HTS data and the MCF-7 c

Fitting new technologies into the safety paradigm: use of microarrays in transfusion.

PubMed

Fournier-Wirth, C; Coste, J

2007-01-01

Until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. The recent emergence of Nucleic acid Amplification Technologies (NAT) has revolutionised viral diagnosis, not only by increasing the level of sensitivity but also by facilitating the detection of several viruses in parallel by multiplexing specific primers. In more complex biological situations, when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. High throughput systems, such as DNA Arrays, permit a conceptually new approach. These miniaturised micro systems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, a reduction in the number of confirmation tests and a simplification of data interpretation. However, the systems currently available require additional instrumentation and reagents for sample preparation and target amplification prior to detection on the DNA array. A major challenge in the area of DNA detection is the development of methods that do not rely on target amplification systems. Likewise, the advances of protein microarrays have lagged because of poor stability of proteins, complex coupling chemistry and weak detection signals. Emerging technologies like Biosensors and nano-particle based DNA or Protein Bio-Barcode Amplification Assays are promising diagnostic tools for a wide range of clinical applications, including blood donation screening.

[Geographical characteristics of single nucleotide polymorphism of candidate genes associated with coronary artery disease in Chinese Han population].

PubMed

Wang, Sheng-Huang; Cui, Han-Bin; Wang, Dong-Qi; Chen, Xiao-Min; Zhang, Hong-Kao; Cui, Chang-Cong; Chen, Xin-Yi; Liu, Xin-Hong; Zhang, Zheng; Bai, Feng; Jb, Muhlestein

2008-01-01

To investigate the geographical characteristics of single nucleotide polymorphism (SNP) of candidate genes associated with coronary artery disease in Chinese Han population. Study population were Chinese Han nationality recruited from Xi'an, Shiyan and Ningbo districts. Patients with coronary artery disease were defined by coronary angiography with stenosis >or= 50% and control subjects with stenosis < 10%, respectively. The DNA was extracted from peripheral white blood cell by approach comprised proteinase K digestion, phenol and chloroform extraction as well as isopropanol precipitation. The SNP of ATP-binding cassette transporter (ABCA1)-G596A, cholesteryl ester transfer protein (CETP)-Taq1B, Lipoprotein lipase (LPL)-Hind III and LPL-Pvu II were genotyped by PCR-RFLPs, and verified by gene sequencing. A Total of 615 patients undertaken coronary angiography were recruited from cardiac center in Xi'an (220), Ningbo (209) and Shiyan district (186), China (mean age 60 +/- 10 years, 75.9% males). Diabetes mellitus was more prevalent in Xi'an Cohort population than Shiyan and Ningbo cohort (P < 0.01). Plasma total cholesterol, LDL cholesterol and triglyceride levels in Xi'an Cohort population were significantly higher, and HDL-C siginificantly lower than in Shiyan and Ningbo cohort population [HDL-C: (1.17 +/- 0.48) mmol/L vs. (1.25 +/- 0.33) mmol/L and (1.29 +/- 0.44) mmol/L, P < 0.05]. Distribution differences for ABCA1-G596A and CETP-Taq1B genotypes were found in Xi'an Cohort population compared to Ningbo and Shiyan cohorts (for ABCA1, Xi'an: 0.24, 0.53, 0.23 and Shiyan: 0.17, 0.62, 0.21 and Ningbo: 0.34, 0.37, 0.29, for GG, AG, AA, respectively, P < 0.01; and for CETP, Xi'an: 0.29, 0.54, 0.17 and Shiyan: 0.38, 0.40, 0.22 and Ningbo: 0.39, 0.49, 0.12 for B1B1, B1B2, B2B2, respectively, P < 0.01), but not for LPL variants. ABCA1-G596A variant predicted HDL-C [Xi'an: (1.2 +/- 0.3) mmol/L, (1.3 +/- 0.2) mmol/L and (1.4 +/- 0.4) mmol/L, P = 0.01; Shiyan: (1.1 +/- 0.4) mmol/L: (1.2 +/- 0.3) mmol/L and (1.3 +/- 0.4) mmol/L, P = 0.03; Ningbo, (1.2 +/- 0.3) mmol/L, (1.3 +/- 0.4) mmol/L and (1.4 +/- 0.3) mmol/L, across GG, GA to AA genotype, respectively, P = 0.01] and TG levels [Xi'an: (2.4 +/- 1.3) mmol/L, (1.9 +/- 0.9) mmol/L and (1.6 +/- 0.8) mmol/L, P < 0.01; Shiyan: (2.1 +/- 1.0) mmol/L, (1.9 +/- 0.8) mmol/L and (1.8 +/- 0.7) mmol/L, P = 0.03; Ningbo: (1.9 +/- 1.1) mmol/L, (1.8 +/- 0.9) mmol/L and (1.6 +/- 0.7) mmol/L, across GG, GA to AA genotype, P = 0.05] with dose-dependent relationship. LPL-Hind III (+) carriers had higher triglycerides in three cohort population [Xi'an: (2.2 +/- 1.0) mmol/L, (1.8 +/- 0.9) mmol/L, (1.6 +/- 0.7) mmol/L, P = 0.01; Shiyan: (2.1 +/- 0.7) mmol/L, (1.9 +/- 1.0) mmol/L, (1.7 +/- 0.6) mmol/L, P = 0.01; Ningbo: (1.8 +/- 1.0) mmol/L, (1.6 +/- 0.6) mmol/L and (1.4 +/- 0.5) mmol/L, for +/+, +/- and -/- genotypes, respectively, P = 0.001]. SNP of CETP-Taq1B, LPL-Hind III and LPL-Pvu II predicted HDL-C and/or TG levels in different cohort population with different manners. All these SNP were not significantly associated with the development of coronary artery disease (all P > 0.05). A geographical heterogeneity of environmental and genetic risk factors related to the development of coronary artery disease exists in Chinese Han population. Irrespective of the different geographical cohort of Chinese Han population, the SNP of candidate genes can partly predict the differences in risk-related plasma HDL-C and/or TG levels rather than angiographic coronary artery disease.

Cell-specific vacuolar calcium storage mediated by "CAX1" regulates apoplastic calcium concentration, gas exchange, and plant productivity in "Arabidopsis"

USDA-ARS?s Scientific Manuscript database

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from "Arabidopsis thaliana" leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen...

The civRT operon is important for Campylobacter jejuni strain 81-176 host cell interactions through regulation of the formate dehydrogenase operon

USDA-ARS?s Scientific Manuscript database

C. jejuni colonizes the intestinal mucosa, and the severity of disease in different strains is correlated with host cell interaction and invasion. A microarray screen to identify genes differentially regulated during C. jejuni interaction with tissue culture cells revealed the up-regulation of a two...

Tailoring Nutritional Advice for Mexicans Based on Prevalence Profiles of Diet-Related Adaptive Gene Polymorphisms

PubMed Central

Ojeda-Granados, Claudia; Panduro, Arturo; Gonzalez-Aldaco, Karina; Sepulveda-Villegas, Maricruz; Rivera-Iñiguez, Ingrid

2017-01-01

Diet-related adaptive gene (DRAG) polymorphisms identified in specific populations are associated with chronic disorders in carriers of the adaptive alleles due to changes in dietary and lifestyle patterns in recent times. Mexico’s population is comprised of Amerindians (AM) and Mestizos who have variable AM, European (EUR) and African genetic ancestry and an increased risk of nutrition-related chronic diseases. Nutritional advice based on the Mexican genome and the traditional food culture is needed to develop preventive and therapeutic strategies. Therefore, we aimed to provide a prevalence profile of several DRAG polymorphisms in the Mexican population, including Central West (CW) Mexico subpopulations. Geographic heat maps were built using ArcGIS10 (Esri, Redlands, CA, USA) software, based on the published data of the MTHFR C677T (rs1801133), ABCA1 Arg230Cys (rs9282541), APOE T388C (rs429358)/C526T (rs7412), LCT C-13910T (rs4988235) polymorphisms and AMY1 copy number variation (CNV). Also, new data obtained by allelic discrimination-real-time polymerase chain reaction (RT-PCR) assays for the MTHFR, ABCA1, and APOE polymorphisms as well as the AMY1 CNV in the CW Mexico subpopulations with different proportions of AM and EUR ancestry were included. In the CW region, the highest frequency of the MTHFR 677T, ABCA1 230C and APOE ε4 adaptive alleles was observed in the AM groups, followed by Mestizos with intermediate AM ancestry. The LCT-13910T allele frequency was highest in Mestizos-EUR but extremely low in AM, while the AMY1 diploid copy number was 6.82 ± 3.3 copies. Overall, the heat maps showed a heterogeneous distribution of the DRAG polymorphisms, in which the AM groups revealed the highest frequencies of the adaptive alleles followed by Mestizos. Given these genetic differences, genome-based nutritional advice should be tailored in a regionalized and individualized manner according to the available foods and Mexican traditional food culture that may lead to a healthier dietary pattern. PMID:29125573

Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.).

PubMed

Qi, Weiping; Ma, Xiaoli; He, Weiyi; Chen, Wei; Zou, Mingmin; Gurr, Geoff M; Vasseur, Liette; You, Minsheng

2016-09-27

ATP-binding cassette (ABC) transporters are one of the major transmembrane protein families found in all organisms and play important roles in transporting a variety of compounds across intra and extra cellular membranes. In some species, ABC transporters may be involved in the detoxification of substances such as insecticides. The diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops worldwide, is an important species to study as it is resistant to many types of insecticides as well as biological control Bacillus thuringiensis toxins. A total of 82 ABC genes were identified from our published P. xylostella genome, and grouped into eight subfamilies (ABCA-H) based on phylogenetic analysis. Genes of subfamilies ABCA, ABCC and ABCH were found to be expanded in P. xylostella compared with those in Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens. Phylogenetic analysis indicated that many of the ABC transporters in P. xylostella are orthologous to the well-studied ABC transporter genes in the seven other species. Transcriptome- and qRT-PCR-based analysis elucidated physiological effects of ABC gene expressions of P. xylostella which were developmental stage- and tissue-specific as well as being affected by whether or not the insects were from an insecticide-resistant strain. Two ABCC and one ABCA genes were preferentially expressed in midgut of the 4th-instar larvae of a susceptible strain (Fuzhou-S) suggesting their potential roles in metabolizing plant defensive chemicals. Most of the highly expressed genes in insecticide-resistant strains were also predominantly expressed in the tissues of Malpighian tubules and midgut. This is the most comprehensive study on identification, characterization and expression profiling of ABC transporter genes in P. xylostella to date. The diversified features and expression patterns of this gene family may be associated with the evolutionary capacity of this species to develop resistance to a wide range of insecticides and biological toxins. Our findings provide a solid foundation for future functional studies on specific ABC transporter genes in P. xylostella, and for further understanding of their physiological roles and regulatory pathways in insecticide resistance.

High-resolution optical coherence tomography, autofluorescence, and infrared reflectance imaging in Sjögren reticular dystrophy.

PubMed

Schauwvlieghe, Pieter-Paul; Torre, Kara Della; Coppieters, Frauke; Van Hoey, Anneleen; De Baere, Elfride; De Zaeytijd, Julie; Leroy, Bart P; Brodie, Scott E

2013-01-01

To describe the phenotype of three cases of Sjögren reticular dystrophy in detail, including high-resolution optical coherence tomography, autofluorescence imaging, and near-infrared reflectance imaging. Two unrelated teenagers were independently referred for ophthalmologic evaluation. Both underwent a full ophthalmologic workup, including electrophysiologic and extensive imaging with spectral-domain optical coherence tomography, autofluorescence imaging, and near-infrared reflectance imaging. In addition, mutation screening of ABCA4, PRPH2, and the mitochondrial tRNA gene was performed in Patient 1. Subsequently, the teenage sister of Patient 2 was examined. Strikingly similar phenotypes were present in these three patients. Fundoscopy showed bilateral foveal pigment alterations, and a lobular network of deep retinal, pigmented deposits throughout the posterior pole, tapering toward the midperiphery, with relative sparing of the immediate perifoveal macula and peripapillary area. This network is mildly to moderately hyperautofluorescent on autofluorescence and bright on near-infrared reflectance imaging. Optical coherence tomography showed abnormalities of the retinal pigment epithelium-Bruch membrane complex, photoreceptor outer segments, and photoreceptor inner/outer segment interface. The results of retinal function test were entirely normal. No molecular cause was detected in Patient 1. Imaging suggested that the lobular network of deep retinal deposits in Sjögren reticular dystrophy is the result of accumulation of both pigment and lipofuscin between photoreceptors and retinal pigment epithelium, as well as within the retinal pigment epithelium.

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

PubMed Central

Baarda, Benjamin I.; Emerson, Sarah; Proteau, Philip J.

2017-01-01

ABSTRACT The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae. A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates—NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344—in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae. IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains. PMID:28630127

Protective responses to sublytic complement in the retinal pigment epithelium

PubMed Central

Tan, Li Xuan; Toops, Kimberly A.; Lakkaraju, Aparna

2016-01-01

The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4−/− Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4−/− mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

Mechanistic and signaling analysis of Muc4-ErbB2 signaling module: new insights into the mechanism of ligand-independent ErbB2 activity.

PubMed

Kozloski, Goldi A; Carraway, Coralie A Carothers; Carraway, Kermit L

2010-09-01

The membrane mucin Muc4 is aberrantly expressed in numerous epithelial carcinomas and is currently used as a cancer diagnostic and prognostic tool. Muc4 can also potentiate signal transduction by modulating differential ErbB2 phosphorylation in the absence and in the presence of the ErbB3 soluble ligand heregulin (HRG-beta1). These features of Muc4 suggest that Muc4 is not merely a cancer marker, but an oncogenic factor with a unique-binding/activation relationship with the receptor ErbB2. In the present study, we examined the signaling mechanisms that are associated with the Muc4-ErbB2 module by analyzing ErbB2 differential signaling in response to Muc4 expression. Our study was carried out in the A375 human melanoma and BT-474 breast cancer cell lines as our model systems. Quantitative and comparative signaling modulations were evaluated by immunoblot using phospho-specific antibodies, and densitometry analysis. Signaling complex components were identified by chemical cross-linking, fractionation by gel filtration, immunoprecipitation, and immunoblotting. Activated downstream signaling pathways were analyzed by an antibody microarray screen and immunoblot analyses. Our results indicate that Muc4 modulates ErbB2 signaling potential significantly by stabilizing and directly interacting with the ErbB2-ErbB3 heterodimer. Further analyses indicate that Muc4 promotes ErbB2 autocatalysis, but it has no effect on ErbB3 phosphorylation, although the chemical cross-linking data indicated that the signaling module is composed of Muc4, ErbB2, and ErbB3. Our microarray analysis indicates that Muc4 expression promotes cell migration by increasing the phosphorylation of the focal adhesion kinase and also through an increase in the levels of beta-catenin. (c) 2010 Wiley-Liss, Inc.

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls

PubMed Central

2013-01-01

Background Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Results Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n = 4) or suffering from DCM (n = 4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p < 0.05 and fold change (FC) > 1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p > 0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p > 0.05). Conclusions Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases. PMID:23327631

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls.

PubMed

Steudemann, Carola; Bauersachs, Stefan; Weber, Karin; Wess, Gerhard

2013-01-17

Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n=4) or suffering from DCM (n=4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p<0.05 and fold change (FC)>1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p>0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p>0.05). Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases.

Screening for Pancreatic Cancer

PubMed Central

Brand, Randall E.

2007-01-01

Despite improvements in the clinical and surgical management of pancreatic cancer, limited strides have been made in the early detection of this highly lethal malignancy. The majority of localized pancreatic tumors are asymptomatic, and the recognized presenting symptoms of pancreatic adenocarcinoma are often vague and heterogeneous in nature. These factors, coupled with the lack of a sensitive and noninvasive screening method, have made population-based screening for pancreatic cancer impossible. Nevertheless, at least two large institutions have performed multimodality-screening protocols for individuals with high risk of pancreatic cancer based on genetic predisposition and strong family history. Abnormalities noted during these screening protocols prompted further investigation or surgery that resulted in the discovery of benign, potentially malignant, and malignant pancreatic lesions. In addition to ductal epithelial pancreatic intraepithelial neoplasia, greater sensitivity has recently been achieved in the identification and characterization of precancerous mucinous pancreatic tumors. Advancements in proteomics and DNA microarray technology may confirm serum-based biomarkers that could be incorporated into future screening algorithms for pancreatic cancer. PMID:21960811

Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

NASA Astrophysics Data System (ADS)

Leski, T. A.; Ansumana, R.; Jimmy, D. H.; Bangura, U.; Malanoski, A. P.; Lin, B.; Stenger, D. A.

2011-06-01

Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such assays can play an important role in outbreak investigations and environmental screening for intentional or accidental release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens. The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of hundreds of infectious agents simultaneously. The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and Mercy Hospital Research Laboratory located in Bo. We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a possible pathogen. The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases when different agents are suspected. This strategy enables quick and cost effective detection of low probability events such as outbreak of a rare disease or intentional release of a biothreat agent.

Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.

PubMed

El-Ashker, Maged; Hotzel, Helmut; Gwida, Mayada; El-Beskawy, Mohamed; Silaghi, Cornelia; Tomaso, Herbert

2015-01-30

In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

PubMed Central

Loy, Alexander; Lehner, Angelika; Lee, Natuschka; Adamczyk, Justyna; Meier, Harald; Ernst, Jens; Schleifer, Karl-Heinz; Wagner, Michael

2002-01-01

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB). PMID:12324358

Characterization of Antimicrobial Resistance Patterns and Detection of Virulence Genes in Campylobacter Isolates in Italy

PubMed Central

Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

2014-01-01

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations. PMID:24556669

Characterization of antimicrobial resistance patterns and detection of virulence genes in Campylobacter isolates in Italy.

PubMed

Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

2014-02-19

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations.

Bacterial Surface Glycans: Microarray and QCM Strategies for Glycophenotyping and Exploration of Recognition by Host Receptors.

PubMed

Kalograiaki, Ioanna; Campanero-Rhodes, María A; Proverbio, Davide; Euba, Begoña; Garmendia, Junkal; Aastrup, Teodor; Solís, Dolores

2018-01-01

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria-host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment. © 2018 Elsevier Inc. All rights reserved.

A high-density transcript linkage map with 1,845 expressed genes positioned by microarray-based Single Feature Polymorphisms (SFP) in Eucalyptus

PubMed Central

2011-01-01

Background Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers. Results SFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome. Conclusions The Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species. PMID:21492453

Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development

PubMed Central

Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia ur; Liu, Liping; Qin, Yuan

2017-01-01

Pineapple (Ananas comosus L.) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs, 20 ABCBs, 16 ABCCs, 2 ABCDs, one ABCEs, 5 ABCFs, 42 ABCGs and 9 ABCIs). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4, AcABCC7, AcABCC9, AcABCG26, AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production. PMID:29312399

Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development.

PubMed

Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia Ur; Liu, Liping; Qin, Yuan

2017-01-01

Pineapple ( Ananas comosus L .) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs , 20 ABCB s, 16 ABCCs , 2 ABCDs , one ABCEs , 5 ABCFs , 42 ABCGs and 9 ABCIs ). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4 , AcABCC7 , AcABCC9 , AcABCG26 , AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production.

Helical synthetic peptides that stimulate cellular cholesterol efflux

DOEpatents

Bielicki, John K.; Natarajan, Pradeep

2010-04-06

The present invention provides peptides comprising at least one amphipathic alpha helix and having an cholesterol mediating activity and a ABCA stabilization activity. The invention further provides methods of using such peptides.

The “Beta-Clasp” model of apolipoprotein A-I - a lipid-free solution structure determined by electron paramagnetic resonance spectroscopy

PubMed Central

Lagerstedt, Jens O.; Budamagunta, Madhu S.; Liu, Grace S.; DeValle, Nicole C.; Voss, John C.; Oda, Michael N.

2012-01-01

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free / lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. PMID:22245143

Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production*

PubMed Central

Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jörg

2012-01-01

The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

High glucose upregulates BACE1-mediated Aβ production through ROS-dependent HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization in SK-N-MC cells

PubMed Central

Lee, Hyun Jik; Ryu, Jung Min; Jung, Young Hyun; Lee, Sei-Jung; Kim, Jeong Yeon; Lee, Sang Hun; Hwang, In Koo; Seong, Je Kyung; Han, Ho Jae

2016-01-01

There is an accumulation of evidence indicating that the risk of Alzheimer’s disease is associated with diabetes mellitus, an indicator of high glucose concentrations in blood plasma. This study investigated the effect of high glucose on BACE1 expression and amyloidogenesis in vivo, and we present details of the mechanism associated with those effects. Our results, using ZLC and ZDF rat models, showed that ZDF rats have high levels of amyloid-beta (Aβ), phosphorylated tau, BACE1, and APP-C99. In vitro result with mouse hippocampal neuron and SK-N-MC, high glucose stimulated Aβ secretion and apoptosis in a dose-dependent manner. In addition, high glucose increased BACE1 and APP-C99 expressions, which were reversed by a reactive oxygen species (ROS) scavenger. Indeed, high glucose increased intracellular ROS levels and HIF-1α expression, associated with regulation of BACE1 and Liver X Receptor α (LXRα). In addition, high glucose induced ATP-binding cassette transporter A1 (ABCA1) down-regulation, was associated with LXR-induced lipid raft reorganization and BACE1 localization on the lipid raft. Furthermore, silencing of BACE1 expression was shown to regulate Aβ secretion and apoptosis of SK-N-MC. In conclusion, high glucose upregulates BACE1 expression and activity through HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization, leading to Aβ production and apoptosis of SK-N-MC. PMID:27829662

New Web-Based Tools Make Systems Pharmacology More Accessible Using Data from the NCI-60 | Center for Cancer Research

Cancer.gov

High-throughput biological techniques, like microarrays and drug screens, generate an enormous amount of data that may be critically important for cancer researchers and clinicians. Being able to manipulate the data to extract those pieces of interest, however, can require computational or bioinformatics skills beyond those of the average scientist.

A Novel Knock-Out Animal Model to Analyze Transcriptional Signaling by p53 Tumor Suppressor Protein in Breast Cancer

DTIC Science & Technology

2002-05-01

homozygous for the pcna and p21 mutant genes will be accomplised with the help of Gene Targeting and Transgenic Facility at the Rosewel Park Cancer Institute...screening of BAC library was performed with the help of the DNA Microarray Facility Facility at the Rosewel Park Cancer Institute. Sequence of mouse

Sex-Dependent Up-Regulation of Two Splicing Factors, Psf and Srp20, during Hippocampal Memory Formation

ERIC Educational Resources Information Center

Antunes-Martins, Ana; Mizuno, Keiko; Irvine, Elaine E.; Lepicard, Eve M.; Giese, K. Peter

2007-01-01

Gene transcription is required for long-term memory (LTM) formation. LTM formation is impaired in a male-specific manner in mice lacking either of the two Ca[superscript 2+] / calmodulin-dependent kinase kinase ("Camkk") genes. Since altered transcription was suggested to cause these impairments in LTM formation, we used microarrays to screen for…

A method adapting microarray technology for signature tagged mutagenesis of Dusulfovibrio dusulfuricans G20 and Shewanella oneidensis MR-1 in anaerobic sediment survival experiments

USGS Publications Warehouse

Groh, Jennifer L.; Luo, Qingwei; Ballard , Jimmy D.; Krumholz, Lee R.

2005-01-01

Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.

Characterization of Bovine Serum Albumin Blocking Efficiency on Epoxy-Functionalized Substrates for Microarray Applications.

PubMed

Sun, Yung-Shin; Zhu, Xiangdong

2016-10-01

Microarrays provide a platform for high-throughput characterization of biomolecular interactions. To increase the sensitivity and specificity of microarrays, surface blocking is required to minimize the nonspecific interactions between analytes and unprinted yet functionalized surfaces. To block amine- or epoxy-functionalized substrates, bovine serum albumin (BSA) is one of the most commonly used blocking reagents because it is cheap and easy to use. Based on standard protocols from microarray manufactories, a BSA concentration of 1% (10 mg/mL or 200 μM) and reaction time of at least 30 min are required to efficiently block epoxy-coated slides. In this paper, we used both fluorescent and label-free methods to characterize the BSA blocking efficiency on epoxy-functionalized substrates. The blocking efficiency of BSA was characterized using a fluorescent scanner and a label-free oblique-incidence reflectivity difference (OI-RD) microscope. We found that (1) a BSA concentration of 0.05% (0.5 mg/mL or 10 μM) could give a blocking efficiency of 98%, and (2) the BSA blocking step took only about 5 min to be complete. Also, from real-time and in situ measurements, we were able to calculate the conformational properties (thickness, mass density, and number density) of BSA molecules deposited on the epoxy surface. © 2015 Society for Laboratory Automation and Screening.

Direct Detection of Drug-Resistant Hepatitis B Virus in Serum Using a Dendron-Modified Microarray

PubMed Central

Kim, Doo Hyun; Kang, Hong Seok; Hur, Seong-Suk; Sim, Seobo; Ahn, Sung Hyun; Park, Yong Kwang; Park, Eun-Sook; Lee, Ah Ram; Park, Soree; Kwon, So Young; Lee, Jeong-Hoon

2018-01-01

Background/Aims Direct sequencing is the gold standard for the detection of drug-resistance mutations in hepatitis B virus (HBV); however, this procedure is time-consuming, labor-intensive, and difficult to adapt to high-throughput screening. In this study, we aimed to develop a dendron-modified DNA microarray for the detection of genotypic resistance mutations and evaluate its efficiency. Methods The specificity, sensitivity, and selectivity of dendron-modified slides for the detection of representative drug-resistance mutations were evaluated and compared to those of conventional slides. The diagnostic accuracy was validated using sera obtained from 13 patients who developed viral breakthrough during lamivudine, adefovir, or entecavir therapy and compared with the accuracy of restriction fragment mass polymorphism and direct sequencing data. Results The dendron-modified slides significantly outperformed the conventional microarray slides and were able to detect HBV DNA at a very low level (1 copy/μL). Notably, HBV mutants could be detected in the chronic hepatitis B patient sera without virus purification. The validation of our data revealed that this technique is fully compatible with sequencing data of drug-resistant HBV. Conclusions We developed a novel diagnostic technique for the simultaneous detection of several drug-resistance mutations using a dendron-modified DNA microarray. This technique can be directly applied to sera from chronic hepatitis B patients who show resistance to several nucleos(t)ide analogues. PMID:29271185

The contribution of the DNA microarray technology to gene expression profiling in Leishmania spp.: a retrospective.

PubMed

Alonso, Ana; Larraga, Vicente; Alcolea, Pedro J

2018-05-07

The first genome project of any living organism excluding viruses, the gammaproteobacteria Haemophilus influenzae, was completed in 1995. Until the last decade, genome sequencing was very tedious because genome survey sequences (GSS) and/or expressed sequence tags (ESTs) belonging to plasmid, cosmid and artificial chromosome genome libraries had to be sequenced and assembled in silico. Nowadays, no genome is completely assembled actually, because gaps and unassembled contigs are always remaining. However, most represent the whole genome of the organism of origin from a practical point of view. The first genome sequencing projects of trypanosomatid parasites were completed in 2005 following those strategies, and belong to Leishmania major, Trypanosoma cruzi and T. brucei. The functional genomics era rapidly developed on the basis of the microarray technology and has been evolving. In the case of the genus Leishmania, substantial biological information about differentiation in the digenetic life cycle of the parasite has been obtained. Later on, next generation sequencing has revolutionized genome sequencing and functional genomics, leading to more sensitive, accurate results by using much less resources. This new technology is more advantageous, but does not invalidate microarray results. In fact, promising vaccine candidates and drug targets have been found on the basis of microarray-based screening and preliminary proof-of-concept tests. Copyright © 2018. Published by Elsevier B.V.

Differential co-expression analysis of rheumatoid arthritis with microarray data.

PubMed

Wang, Kunpeng; Zhao, Liqiang; Liu, Xuefeng; Hao, Zhenyong; Zhou, Yong; Yang, Chuandong; Li, Hongqiang

2014-11-01

The aim of the present study was to investigate the underlying molecular mechanisms of rheumatoid arthritis (RA) using microarray expression profiles from osteoarthritis and RA patients, to improve diagnosis and treatment strategies for the condition. The gene expression profile of GSE27390 was downloaded from Gene Expression Omnibus, including 19 samples from patients with RA (n=9) or osteoarthritis (n=10). Firstly, the differentially expressed genes (DEGs) were obtained with the thresholds of |logFC|>1.0 and P<0.05, using the t‑test method in LIMMA package. Then, differentially co-expressed genes (DCGs) and differentially co-expressed links (DCLs) were screened with q<0.25 by the differential coexpression analysis and differential regulation analysis of gene expression microarray data package. Secondly, pathway enrichment analysis for DCGs was performed by the Database for Annotation, Visualization and Integrated Discovery and the DCLs associated with RA were selected by comparing the obtained DCLs with known transcription factor (TF)-targets in the TRANSFAC database. Finally, the obtained TFs were mapped to the known TF-targets to construct the network using cytoscape software. A total of 1755 DEGs, 457 DCGs and 101988 DCLs were achieved and there were 20 TFs in the obtained six TF-target relations (STAT3-TNF, PBX1‑PLAU, SOCS3-STAT3, GATA1-ETS2, ETS1-ICAM4 and CEBPE‑GATA1) and 457 DCGs. A number of TF-target relations in the constructed network were not within DCLs when the TF and target gene were DCGs. The identified TFs may have an important role in the pathogenesis of RA and have the potential to be used as biomarkers for the development of novel diagnostic and therapeutic strategies for RA.

Prominent Optic Disc Featured in Inherited Retinopathy.

PubMed

Todorova, M G; Bojinova, R I; Valmaggia, C; Schorderet, D F

2017-04-01

Background We investigated the relationship between prominent optic disc (POD) and inherited retinal dystrophy (IRD). Patients and Methods A cross-sectional consecutive study was performed in 10 children and 11 adults of 7 non-related families. We performed clinical phenotyping, including a detailed examination, fundus autofluorescence, and colour fundus and OCT imaging. Genetic testing was subsequently performed for all family members presenting retinal pathology. Results In 4 members of a 3-generation family, hyperfluorescent deposits on the surface of POD were related to a p.(L224M) heterozygous mutation in BEST1 . In the second family, one member presented deposits located on the surface on hyperaemic OD and a compound p.(R141H);(A195V) mutation in BEST1 . In the third family, POD was observed in father and child with early onset cone-rod dystrophy and a novel autosomal recessive p.(W31*) homozygous mutation in ABCA4 . In the fourth family, POD with "mulberry-like" deposits and attenuated vessels were observed in a 7-year old girl, with a mutation in USH1A , and with early onset rod-cone dystrophy, associated with hearing loss. In the fifth family, blurry OD with tortuous vessels was observed in 4 consanguineous female carriers and a hemizygous boy with a p.(R200H) mutation in the X-linked retinoschisis RS1 . In the sixth family, a mother and her son were both affected with POD and attenuated peripapillary vessels, and presented with a p.(Y836C) heterozygous mutation in TOPORS , thus confirming autosomal dominant RP. In the seventh family, in 3 family members with POD, compound p.(L541P;A1038 V);(G1961E) mutations in ABCA4 confirmed the diagnosis of Stargardt disease. Conclusions A variety of OD findings are found in a genetically heterogeneous group of IRDs. In the presence of POD, an inherited progressive photoreceptor disease should be ruled out. Georg Thieme Verlag KG Stuttgart · New York.

MICROARRAY DATA ANALYSIS USING MULTIPLE STATISTICAL MODELS

EPA Science Inventory

Microarray Data Analysis Using Multiple Statistical Models

Wenjun Bao1, Judith E. Schmid1, Amber K. Goetz1, Ming Ouyang2, William J. Welsh2,Andrew I. Brooks3,4, ChiYi Chu3,Mitsunori Ogihara3,4, Yinhe Cheng5, David J. Dix1. 1National Health and Environmental Effects Researc...

Genome-wide microarray analysis leads to identification of genes in response to herbicide, metribuzin in wheat leaves.

PubMed

Pilcher, Whitney; Zandkamiri, Hana; Arceneaux, Kelly; Harrison, Stephen; Baisakh, Niranjan

2017-01-01

Herbicides are an important component of weed management in wheat, particularly in the southeastern US where weeds actively compete with wheat throughout the winter for nutrients and reduce tillering and ultimately the yield of the crop. Some wheat varieties are sensitive to metribuzin, a low-cost non-selective herbicide, leading to leaf chlorosis, stand loss, and decreased yield. Knowledge of the genetics of herbicide tolerance in wheat is very limited and most new varieties have not been screened for metribuzin tolerance. The identification of genes associated with metribuzin tolerance will lead to the development of molecular markers for use in screening breeding lines for metribuzin tolerance. AGS 2035 and AGS 2060 were identified as resistant and sensitive to metribuzin in several previous field screening experiments as well as controlled condition screening of nine varieties in the present study. Genome-wide transcriptome profiling of the genes in AGS 2035 and AGS 2060 through microarray analysis identified 169 and 127 genes to be significantly (2-fold, P>0.01) up- and down-regulated, respectively in response to metribuzin. Functional annotation revealed that genes involved in cell wall biosynthesis, photosynthesis and sucrose metabolism were highly responsive to metribuzin application. (Semi)quantitative RT-PCR of seven selected differentially expressed genes (DEGs) indicated that a gene coding for alkaline alpha-galactosidase 2 (AAG2) was specifically expressed in resistant varieties only after one and two weeks of metribuzin application. Integration of the DEGs into our ongoing mapping effort and identification of the genes within the QTL region showing significant association with resistance in future will aid in development of functional markers for metribuzin resistance.

On the classification techniques in data mining for microarray data classification

NASA Astrophysics Data System (ADS)

Aydadenta, Husna; Adiwijaya

2018-03-01

Cancer is one of the deadly diseases, according to data from WHO by 2015 there are 8.8 million more deaths caused by cancer, and this will increase every year if not resolved earlier. Microarray data has become one of the most popular cancer-identification studies in the field of health, since microarray data can be used to look at levels of gene expression in certain cell samples that serve to analyze thousands of genes simultaneously. By using data mining technique, we can classify the sample of microarray data thus it can be identified with cancer or not. In this paper we will discuss some research using some data mining techniques using microarray data, such as Support Vector Machine (SVM), Artificial Neural Network (ANN), Naive Bayes, k-Nearest Neighbor (kNN), and C4.5, and simulation of Random Forest algorithm with technique of reduction dimension using Relief. The result of this paper show performance measure (accuracy) from classification algorithm (SVM, ANN, Naive Bayes, kNN, C4.5, and Random Forets).The results in this paper show the accuracy of Random Forest algorithm higher than other classification algorithms (Support Vector Machine (SVM), Artificial Neural Network (ANN), Naive Bayes, k-Nearest Neighbor (kNN), and C4.5). It is hoped that this paper can provide some information about the speed, accuracy, performance and computational cost generated from each Data Mining Classification Technique based on microarray data.

Practice guideline: joint CCMG-SOGC recommendations for the use of chromosomal microarray analysis for prenatal diagnosis and assessment of fetal loss in Canada.

PubMed

Armour, Christine M; Dougan, Shelley Danielle; Brock, Jo-Ann; Chari, Radha; Chodirker, Bernie N; DeBie, Isabelle; Evans, Jane A; Gibson, William T; Kolomietz, Elena; Nelson, Tanya N; Tihy, Frédérique; Thomas, Mary Ann; Stavropoulos, Dimitri J

2018-04-01

The aim of this guideline is to provide updated recommendations for Canadian genetic counsellors, medical geneticists, maternal fetal medicine specialists, clinical laboratory geneticists and other practitioners regarding the use of chromosomal microarray analysis (CMA) for prenatal diagnosis. This guideline replaces the 2011 Society of Obstetricians and Gynaecologists of Canada (SOGC)-Canadian College of Medical Geneticists (CCMG) Joint Technical Update. A multidisciplinary group consisting of medical geneticists, genetic counsellors, maternal fetal medicine specialists and clinical laboratory geneticists was assembled to review existing literature and guidelines for use of CMA in prenatal care and to make recommendations relevant to the Canadian context. The statement was circulated for comment to the CCMG membership-at-large for feedback and, following incorporation of feedback, was approved by the CCMG Board of Directors on 5 June 2017 and the SOGC Board of Directors on 19 June 2017. Recommendations include but are not limited to: (1) CMA should be offered following a normal rapid aneuploidy screen when multiple fetal malformations are detected (II-1A) or for nuchal translucency (NT) ≥3.5 mm (II-2B) (recommendation 1); (2) a professional with expertise in prenatal chromosomal microarray analysis should provide genetic counselling to obtain informed consent, discuss the limitations of the methodology, obtain the parental decisions for return of incidental findings (II-2A) (recommendation 4) and provide post-test counselling for reporting of test results (III-A) (recommendation 9); (3) the resolution of chromosomal microarray analysis should be similar to postnatal microarray platforms to ensure small pathogenic variants are detected. To minimise the reporting of uncertain findings, it is recommended that variants of unknown significance (VOUS) smaller than 500 Kb deletion or 1 Mb duplication not be routinely reported in the prenatal context. Additionally, VOUS above these cut-offs should only be reported if there is significant supporting evidence that deletion or duplication of the region may be pathogenic (III-B) (recommendation 5); (4) secondary findings associated with a medically actionable disorder with childhood onset should be reported, whereas variants associated with adult-onset conditions should not be reported unless requested by the parents or disclosure can prevent serious harm to family members (III-A) (recommendation 8).The working group recognises that there is variability across Canada in delivery of prenatal testing, and these recommendations were developed to promote consistency and provide a minimum standard for all provinces and territories across the country (recommendation 9). © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts

PubMed Central

Hagos, Engda G; Ghaleb, Amr M; Kumar, Amrita; Neish, Andrew S; Yang, Vincent W

2011-01-01

Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions. PMID:21892412

Identification of candidate genes in osteoporosis by integrated microarray analysis.

PubMed

Li, J J; Wang, B Q; Fei, Q; Yang, Y; Li, D

2016-12-01

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation.Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594-601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1. © 2016 Fei et al.

Clinical significance of rare copy number variations in epilepsy: a case-control survey using microarray-based comparative genomic hybridization.

PubMed

Striano, Pasquale; Coppola, Antonietta; Paravidino, Roberta; Malacarne, Michela; Gimelli, Stefania; Robbiano, Angela; Traverso, Monica; Pezzella, Marianna; Belcastro, Vincenzo; Bianchi, Amedeo; Elia, Maurizio; Falace, Antonio; Gazzerro, Elisabetta; Ferlazzo, Edoardo; Freri, Elena; Galasso, Roberta; Gobbi, Giuseppe; Molinatto, Cristina; Cavani, Simona; Zuffardi, Orsetta; Striano, Salvatore; Ferrero, Giovanni Battista; Silengo, Margherita; Cavaliere, Maria Luigia; Benelli, Matteo; Magi, Alberto; Piccione, Maria; Dagna Bricarelli, Franca; Coviello, Domenico A; Fichera, Marco; Minetti, Carlo; Zara, Federico

2012-03-01

To perform an extensive search for genomic rearrangements by microarray-based comparative genomic hybridization in patients with epilepsy. Prospective cohort study. Epilepsy centers in Italy. Two hundred seventy-nine patients with unexplained epilepsy, 265 individuals with nonsyndromic mental retardation but no epilepsy, and 246 healthy control subjects were screened by microarray-based comparative genomic hybridization. Identification of copy number variations (CNVs) and gene enrichment. Rare CNVs occurred in 26 patients (9.3%) and 16 healthy control subjects (6.5%) (P = .26). The CNVs identified in patients were larger (P = .03) and showed higher gene content (P = .02) than those in control subjects. The CNVs larger than 1 megabase (P = .002) and including more than 10 genes (P = .005) occurred more frequently in patients than in control subjects. Nine patients (34.6%) among those harboring rare CNVs showed rearrangements associated with emerging microdeletion or microduplication syndromes. Mental retardation and neuropsychiatric features were associated with rare CNVs (P = .004), whereas epilepsy type was not. The CNV rate in patients with epilepsy and mental retardation or neuropsychiatric features is not different from that observed in patients with mental retardation only. Moreover, significant enrichment of genes involved in ion transport was observed within CNVs identified in patients with epilepsy. Patients with epilepsy show a significantly increased burden of large, rare, gene-rich CNVs, particularly when associated with mental retardation and neuropsychiatric features. The limited overlap between CNVs observed in the epilepsy group and those observed in the group with mental retardation only as well as the involvement of specific (ion channel) genes indicate a specific association between the identified CNVs and epilepsy. Screening for CNVs should be performed for diagnostic purposes preferentially in patients with epilepsy and mental retardation or neuropsychiatric features.