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Proteomics and immunoproteomics of Anoplocephala perfoliata

ABSTRACT: Anoplocephala perfoliata is a common tapeworm in horse with an ability to cause colic and increase mortality. At present, current diagnostic methods are not sensitive enough to detect A. perfoliata and immunoreactive excretory/secretory (E/S) proteins produced by this parasite, which comprise the most promising candidates for diagnostic tests. Here, we compared the E/S proteins produced by A. perfoliata worms of two different physiological states (small- and large-sized worms) in vitro by label-free quantitative (LFQ) proteomics with protein database composed of proteins encoded by Hymenolepis diminuta, Echinococcus multilocularis/granulosus and Taenia aseatica for protein identifications. The quantitative proteomics data was complemented with one-dimensional gel electrophoresis combined with immunoblotting using antisera from horses diagnosed positive and negative for this tapeworm. Protein bands cross-reacting most efficiently with the anti-A. perfoliata antibodies were subjected to in-gel digestion with LC-MS/MS identification. Both the non-gel and gel-based proteomic results were compared to propose a list of candidate proteins for further studies. These proteins will be validated for their suitability as diagnostic markers specific to cestodes and A. perfoliata.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Anoplocephala Perfoliata

SUBMITTER: Tuula Nyman

LAB HEAD: Tuula Nyman

PROVIDER: PXD029144 | Pride | 2022-11-11

REPOSITORIES: Pride

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Project description:The principal toxicity of acute organophosphate (OP) pesticides poisoning is the disruption of neurotransmission through inhibition of acetylcholinesterase (AChE). However, other mechanisms leading to persistent effects and neurodegeneration remain controversial and difficult to detect. Because Caenorhabditis elegans is relatively resistant to OP lethalityM-bM-^@M-^Tparticularly through the inhibition of AChEM-bM-^@M-^Tstudies in this nematode provide an opportunity to observe alterations in global gene expression following OP exposure that cannot be readily observed in less resistant organisms. We exposed cultures of worms in axenic, defined medium to dichlorvos under three exposure protocols. In the first, worms were exposed continuously throughout the experiment. In the second and third, the worms were exposed for either 2 or 8 h, the dichlorvos was washed out of the culture, and the worms were allowed to recover. We then analyzed gene expression using whole genome microarrays from RNA sampled at multiple time points throughout the exposure. The worms showed a time-dependent response that progressively worsened. Early in the exposure, the predominant effect was on metabolic processes, while at later times, an immune-like response and cellular repair mechanisms dominated the expression pattern. Following removal of dichlorvos, the gene expression in the worms appeared to relatively, rapidly return to steady-state levels. The changes in gene expression observed in the worms following exposure to dichlorvos point towards two potential mechanisms of toxicity: inhibition of AChE and mitochondrial disruption. Synchronized cultures of worms at the L3/L4 molt were treated with dichlorvos (0.6 M-BM-5M or 15 M-BM-5M) or maintained as a controls according to one of three protocols. In the first protocol, the set of flasks was incubated without interruption for the duration of the experiment with flasks being harvested at 2, 8, 14, 20, and 26 h. The other two sets were incubated for 2 or 8 h, at which time the worms were centrifuged out of the exposure medium, washed 3 times with a modified CeHR medium (Washout Buffer), resuspended in fresh CeHR medium without dichlorvos, and returned to the incubator. Flasks were harvested at 6 h intervals following the washout through 26 h. Flasks of worms were also harvested prior to beginning the exposure as the 0 h controls. Total RNA was isolated from the frozen harvested worms then analyzed using Affymetrix GeneChips. A total of 148 microarrays were processed (47 per exposure protocol with 4 replicates) however only 146 were included in final analysis as 2 falied to pass QC criteria.

Project description:Purpose: The goal of this study was to compare the genome-wide promoter methylation alterations in macrophages and endothelial cells during hindlimb ischemia among normal, hyperlipidemic and type-2 diabetic mice. Methods: Unilateral hindlimb ischemia was induced by ligating femoral artery proximal to the bifurcation of superficial and deep femoral artery in mice deficient of LDL receptor and expressing only apolipoprotein B100 (LDLR-/-ApoB100/100, C57BL/6J background) (The Jackson Laboratory, Bar Harbor,USA) and mice with Î²-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/LDLR-/-ApoB100/100, C57BL/6J background) with type 2 diabetic features on high-fat diet (TD 88173, Harlan Teklad: 42% of calories from fat and 0.15% from cholesterol, no sodium cholate) 8 weeks prior to surgery and continued throughout the study 1. C57BL/6J (WT) mice fed with regular chow-diet (R36, Lactamin) served as controls. All animals were aged between 20 to 24 weeks at the time of hindlimb operations. For sorting macrophages from ischemic muscles, ischemic gastrocnemius muscles were minced and enzymatically dissociated using a cocktail containing 450 U/mL Collagenase I, 125 U/mL Collagenase XI, 60 U/mL DNAseI, and 60 U/mL hyaluronidase (Sigma Aldrich) for 1 h at 37Â°C. The cells were then counted and divided into CD31+ve and CD31-vefractions using CD31 magnetic bead enrichment (Miltenyi Biotec). For macrophage sorting CD31-ve fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with FITC conjugated rat anti-mouse F4/80 antibody (Serotec) for 20 minutes at 4ËC. For endothelial sorting CD31+ fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with APC conjugated rat anti-mouse CD31 antibody (BD-pharmingen) and FITC conjugated rat anti-mouse CD45 ((BD-pharmingen) for 20 minutes at 4ËC. FACS sorting was performed on FACS AriaIII (BD Biosciences). Genomic DNA was isolated from FACS sorted macrophages and endothelial cells using AllPrep DNA/RNA/Protein Mini Kit (Qiagen Finland, Helsinki, Finland) according to manufacturer's instructions. Results: The sample similarity as assessed by Pearsonâs correlation matrix and Hierarchial clustering showed high correalation among macrophages, as well as endothelial cells. There was a clear clustering of macrophages and endothelial cells as evidence by their CpG methylation clustering, furthermore macrophages from HL and T2DM mice showed clear clustering compared to control macrophages. Differential methylation analysis of RRBS methylation data from macrophages and endothelial cells was performed using Methylkit. Using a threshold of adjusted p value (Q) <0.05 and percentage methylation difference of >5%, we identified 198 and 272 genes whose promoters were hypomethylated in HL and T2DM macrophages. Similarly, there were 102 and 136 gene promoters were hypermethylated in HL and T2DM macrophages, respectively compare to control macrophages. Thus, proximal promoter methylation suggested that HL and T2DM have convergent influences on the proximal promoter methylation of numerous macrophage specific genes. In order to find out whether these genes with differential methylated promoters were differentially expressed at mRNA expression level in purified macrophages, we further compared our data with the GEO datasets as above. Of the 198 genes with promoter hypomethylation in HL macrophages 72 genes were suggested to be upregulated in M1- MÏs; whereas, of the 102 genes with promoter hypermethylation, 51 genes were suggested to be upregulated in M2- MÏs. Similarly, out of 272 genes with differentially methylated promoters in T2DM macrophages 88 genes were suggested to be upregulated in M1-MÏs; whereas, out of 136 genes with promoter hypermethylation 60 genes were suggested to be upregulated in M2- MÏs. Thus a significant promoter hypomethylation of M1-MÏ and hypermethylation of M2-MÏ genes suggested the predominance of proinflammatory M1-MÏs in ischemic muscles of HL and T2DM compared to M2-MÏs in control mice. Conclusions: We found significant promoter hypomethylation of genes typical for proinflammatory M1-MÏs and hypermethylation of anti-inflammatory, proangiogenic M2-MÏ associated genes in HL and T2DM ischemic muscles. Epigenetic alterations skewing MÏ phenotype towards proinflammatory as opposed to anti-inflammatory, proangiogenic and tissue repair phenotype may contribute to impaired adaptive vascular growth in these pathological conditions. Macrophages and endothelial whole genome DNA methylation was performed in triplicates (Each sample was pooled from 3-4 mice) by RRBS Sequencing approach using Illumina HiSeq 2500. qRTâPCR validation was performed using TaqMan assays.

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Proteomics and immunoproteomics of Anoplocephala perfoliata

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