In vitro binding assay and co-immunoprecipitation experimentWe prepared purified S-tagged recombinant LTA and T7-tagged galectin-2 derived from E. coli using the pET system (Novagen), and combined them. The co-immunoprecipitation experiments were performed using a monoclonal antibody against LTA (R&D Systems) coupled to HiTrapTM NHS-activated Sepharose HP (Amersham). We visualized the immune complex using T7 tag antibody (Stratagene) and horseradish peroxidase (HRP) conjugated with anti-mouse IgG antibody. For coimmunoprecipitation in mammalian cells, we transfected expression plasmids of Flag or S-tagged LTA, galectin-2 and LacZ (as a negative control) into COS7 cells (HSRRB; JCRB9127) or HeLa cells using Fugene. Immunoprecipitations were done in lysis buffer (20 mM Tris pH 7.5, with 150 mM NaCl, 0.1 % Nonident P-40). Twenty-four hours after transfection, cells were lysed, and immunoprecipitations were performed using anti-Flag tag M2 agarose (Sigma). We visualized the immune complex using HRP-conjugated S-protein (Novagen), anti-Flag M2 peroxidase conjugate (Sigma) or mouse monoclonal antibody against human a-tubulin (Molecular Probes) and HRP-conjugated anti-mouse IgG antibody. Confocal microscopyPolyclonal anti-human galectin-2 antisera were raised in rabbits using recombinant protein synthesized in E. coli. The antisera showed no cross-reactivity to structurally related molecules galectin-1 and galectin-3, analysed by western blot. Polyclonal antigalectin-2 antisera and either goat anti-human LTA IgG (R&D Systems) or mouse antihuman a-tubulin monoclonal IgM antibodies were used with Alexa secondary antibodies (Molecular Probes). U937 cells (HSRRB; JCRB9021) were stimulated for 30 min with phorbol myristate acetate (PMA) (20 ng ml 21 ) and fixed. They were subsequently incubated with the corresponding primary antibodies in phosphatebuffered saline containing 3% bovine serum albumin, and the corresponding Alexa secondary antibodies. siRNA and over-expression experimentsThe target sequences for galectin-2 (5 0 -AATCCACCATTGTCTGCAACT-3 0 ) were cloned into pSilencer 2.0-U6 siRNA vector (Ambion). For the over-expression experiment, the galectin-2 was cloned into pFlag-CMV5a vector. After transfection, Jurkat cells were stimulated with PMA (20 ng ml 21 ) for 24 h, and cells and supernatants were collected separately. LTA concentration was measured using an LTA-specific ELISA system (R&D Systems), and normalized by comparison with total protein concentration. The mRNA quantification procedure has been described previously 2 . Luciferase assayA DNA fragment, corresponding to nucleotides 3,188 to 3,404 of intron-1 of LGALS2, was cloned into pGL3-enhancer vector (Promega) in the downstream of SV40 enhancer in the 5 0 to 3 0 orientation. After 24 h transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). ImmunohistochemistryTissue samples were obtained from 16 patients with MI by elective directional coronary atherectomy. Immunohistochemical protocols were carried out a...

Paraoxonase is an arylesterase enzyme that is expressed in the liver and found in the circulation in association with apoA1 and the high-density lipoprotein, and prevents the accumulation of oxidized lipids in low-density lipoproteins in vitro. Common polymorphisms in genes encoding paraoxonase are established risk factors in a variety of vascular disorders including coronary artery disease and carotid artery stenosis, but their association with Alzheimer disease (AD) is controversial. We tested the association of 29 SNPs in PON1, PON2 and PON3 with AD in 730 Caucasian and 467 African American participants of the MIRAGE Study, an ongoing multi-center family-based genetic epidemiology study of AD. Eight SNPs were associated with AD in the African American families (0.0001< or =P< or =0.04) and two SNPs were associated with AD in Caucasian families (0.01< or =P< or =0.04). Of note, the pattern of association for the PON1 promoter SNP -161[C/T] was the same in both ethnic groups (P=0.006). Haplotype analysis using sliding windows revealed 11 contiguous SNP combinations spanning the three PON genes with significant global test scores (0.006< or =P< or =0.04) in the two ethnic groups combined. The most significantly associated haplotype comprised SNPs in the region spanning the -161[C/T] SNP (P=0.00009). Our results demonstrate association between AD and variants in the PON gene cluster in Caucasians and African Americans.

BackgroundNonsteroidal anti-inflammatory drugs (NSAID) use may protect against Alzheimer's disease (AD) risk. We sought examine the association between NSAID use and risk of AD, and potential effect modification by APOE-ε4 carrier status and ethnicity.MethodsThe MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD. Subjects comprised 691 AD patients (probands) and 973 family members enrolled at 15 research centers between 1996 and 2002. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. Ethnicity was similarly examined as an effect modifier.ResultsNSAID use was less frequent in cases compared to controls in the overall sample (adjusted OR = 0.64; 95% CI = 0.38–1.05). The benefit of NSAID use appeared more pronounced among APOE-ε4 carriers (adjusted OR = 0.49; 95% CI = 0.24–0.98) compared to non-carriers, although this association was not statistically significant. The pattern of association was similar in Caucasian and African Americans.ConclusionsNSAID use is inversely associated with AD and may be modified by APOE genotype. Prospective studies and clinical trials of sufficient power to detect effect modification by APOE-ε4 carrier status are needed.

BackgroundGene set enrichment analysis and overrepresentation analyses are commonly used methods to determine the biological processes affected by a differential expression experiment. This approach requires biologically relevant gene sets, which are currently curated manually, limiting their availability and accuracy in many organisms without extensively curated resources. New feature learning approaches can now be paired with existing data collections to directly extract functional gene sets from big data.ResultsHere we introduce a method to identify perturbed processes. In contrast with methods that use curated gene sets, this approach uses signatures extracted from public expression data. We first extract expression signatures from public data using ADAGE, a neural network-based feature extraction approach. We next identify signatures that are differentially active under a given treatment. Our results demonstrate that these signatures represent biological processes that are perturbed by the experiment. Because these signatures are directly learned from data without supervision, they can identify uncurated or novel biological processes. We implemented ADAGE signature analysis for the bacterial pathogen Pseudomonas aeruginosa. For the convenience of different user groups, we implemented both an R package (ADAGEpath) and a web server (http://adage.greenelab.com) to run these analyses. Both are open-source to allow easy expansion to other organisms or signature generation methods. We applied ADAGE signature analysis to an example dataset in which wild-type and ∆anr mutant cells were grown as biofilms on the Cystic Fibrosis genotype bronchial epithelial cells. We mapped active signatures in the dataset to KEGG pathways and compared with pathways identified using GSEA. The two approaches generally return consistent results; however, ADAGE signature analysis also identified a signature that revealed the molecularly supported link between the MexT regulon and Anr.ConclusionsWe designed ADAGE signature analysis to perform gene set analysis using data-defined functional gene signatures. This approach addresses an important gap for biologists studying non-traditional model organisms and those without extensive curated resources available. We built both an R package and web server to provide ADAGE signature analysis to the community.Electronic supplementary materialThe online version of this article (10.1186/s12859-017-1905-4) contains supplementary material, which is available to authorized users.