Computational protocol: Involvement of β-defensin 130 (DEFB130) in the macrophage microbicidal mechanisms for killing Plasmodium falciparum

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Protocol publication

[…] Microarray analysis was conducted as previously described. Briefly, cy3-labeled complementary RNA probe synthesis was initiated with 50 ng of total RNA by using the Agilent Low Input Quick Amp Labeling kit, one color (Agilent Technologies). Agilent SurePrint G3 Human GE 8 × 60 K microarrays (G4851) were used according to the manufacturer’s instructions. Slides were scanned with an Agilent’s High-Resolution Microarray Scanner, and image data were processed by using Agilent Feature Extraction software, version All data were subsequently uploaded into GeneSpring GX, version 12.6 for data analysis. Each gene expression array dataset was normalized to the in silico pool for the macrophages cultured with RBCs. Statistically significant differences in gene expression between macrophages cultured with RBCs and those cultured with iRBCs were determined by using a moderated T-test (P < 0.05). Genes that showed at least a 2.0-fold change in their expression with a statistical significant difference (P < 0.05) were assigned to a Gene Ontology group in the DAVID Bioinformatics Database ( GO analysis was used to classify the genes based on their known functions. For all analyses, P values were calculated by using the Fisher’s exact test and considered to be significant at P < 0.05. The microarray data are publically available at the Gene Expression Omnibus (GEO) database (http: under the accession number GSE77122. [...] Total RNA was reverse transcribed to first-strand cDNA (Invitrogen) using oligo-dT primer, according to manufacturer’s instructions. The expression of targeted genes was analyzed with qRT–PCR using an ABI Prism Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) with SYBR Green (Applied Biosystems) and the specific primers listed in the (). The primers for qRT–PCR were designed with the Primer Express software (Applied Biosystems). Specific gene expression was normalized to the expression of ubiquitin, by using the β-actin housekeeping gene. Relative gene expression was calculated with the 2−ΔΔCt method and the fold-increase was determined based on the gene expression of macrophages cultured with RBCs. [...] Recombinant DEFB130 was produced in Escherichia coli as a 6xHistidine-tag fusion protein. Briefly, the coding region was amplified from cDNA prepared from human macrophages with the following specific primer set: 5′-AGGATCCGGCGTTATTCCAGGACAAAA-3′, which includes a BamHI restriction enzyme site (underlined), and 5′-ACTCGAGTTAAGGAGATTTTCCTTTG-3′, which includes a XhoI restriction enzyme site (underlined). PCR products were digested with the appropriate restriction enzymes and then ligated into a pET-28a expression vector (Novagen), which had been digested with the same restriction enzymes. Recombinant protein was expressed in large-scale E. coli DH5α strain cultures (Takara Bio Inc., Osaka, Japan) and purified by means of Ni-NTA affinity chromatography, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The purified recombinant protein was dialyzed in PBS, subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie Brilliant Blue. The recombinant DEFB130 was stored at 4 °C until use. To measure the Circular Dichroism (CD) spectra, 150 μg/ml of recombinant DEFB130 and synthetic peptide were prepared in appropriate buffer (pH 7.4), and the CD spectra were measured with a Jasco J-820 Circular dichroism dispersion meter in a 0.1-cm quartz cell at room temperature. The spectra were the averages of eight consecutive scans from 260 to 195 nm, recorded with a bandwidth of 1 nm, a time constant of 4 sec, and a scan rate of 20 nm/min. The 3D structure modeling of DEFB130 was performed by using SWISS-MODEL and modeling structures of DEFB3 (1KJ6). The electrostatic surface potentials were calculated by using the program APBS and are represented by PyMOL ( […]

Pipeline specifications

Software tools Agilent Feature Extraction, GeneSpring GX, DAVID, Primer Express
Databases GEO
Applications Gene expression microarray analysis, qPCR
Organisms Plasmodium falciparum, Plasmodium yoelii
Diseases Parasitic Diseases