Project description:Hookworms are parasitic nematodes that chronically infect 170-440 million people, despite healthy human immune systems ending most infections quickly. How hookworms successfully overcome host resistance is unclear, but is thought to involve hookworm proteins that either digest host tissues or dampen the host's immune system. To find such proteins in the model hookworm Ancylostoma ceylanicum, we identified hookworm genes encoding proteins excreted into the environment, genes expressed in the intestine, and genes transcriptionally upregulated in response to a normal host immune system. To maximize our accuracy, we repredicted 33,190 protein-coding genes in A. ceylanicum with a BUSCO completeness of 95.3%. We collected excreted-secreted (ES) proteins from two sets of adult hookworms in vitro, and used mass spectrometry to identify 565 A. ceylanicum genes encoding ES proteins; we also used RNA-seq to identify A. ceylanicum genes expressed both in young adults (12 days post-infection) and in intestines and non-intestinal tissues dissected from mature adults (19 days post-infection), with hamster hosts that either had normal immune systems or were immunosuppressed by dexamethasone. In adult A. ceylanicum, we observed 2,145 and 1,694 genes with intestine- and non-intestine-biased expression, respectively. Comparing hookworm gene activity in normal versus immunosuppressed hosts, we observed essentially no changes of gene activity in non-blood-feeding 12-day young adults or non-intestinal 19-day adult tissues. However, in intestinal 19-day adult tissues, we observed 671 genes upregulated at least two-fold in normal hosts versus immunosuppressed hosts (positively immunoregulated), and 75 genes that were negatively immunoregulated; thus, immunoregulation was observed only in mature adult hookworm intestine, directly exposed to host blood. Of the positively immunoregulated intestinal genes, 590 (87.9%) showed male-biased gene expression (9.8-fold greater overlap than chance), suggesting that male and female hookworms have different responses to host immune systems; of the other 81 non-male-biased genes, 53 encoded ES proteins, a 38-fold greater overlap than expected by chance. Of these 53 immunoregulated ES genes, 26 had ES gene homologs in the closely related hookworm Ancylostoma caninum, five in the human hookworm Necator americanus, and four in the more distantly related strongylid parasite Haemonchus contortus. Such a mixture of rapidly evolving and conserved genes could serve as virulence factors enabling infection.

Project description:Metastasis and drug-resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil (5-FU), methotrexate (MTX), doxorubicin (DOX) or oxaliplatin (OXA). Drug-resistant invasive cells were compared to non invasive cells using cDNA microarray, qRT-PCR, flow cytometry, immunoblots and ELISA. Functional and cellular signaling analyses were undertaken using pharmacological inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-FU- and MTX-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behaviour in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, mAb 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 is associated with up-regulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2a and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from colon cancer patients treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis can potentially counteract the emergence of the invasive metastatic behaviour in clonal derivatives of drug-resistant colon cancer cells. Samples: HT-29 cell clones were obtained by limiting dilution from HT-29 subpopulations resistant to methothrexate (HT-29 5M21 cell clone) and or to 5-Fluoro-uracil (HT-29 5F7 and HT-29 5F31). Samples were provided by Dr. T Lesuffleur. Xenografts: HT-29 5M21, HT-29 5F7, and HT-29 5F31 xenografts were obtained by a first subcutaneous inoculation of cells in Nude mice and then fragments of subcutaneous tumors were reimplantated in SCID mice. First experiment of microarrays: HT-29 5M21 and HT-29 5F7 cells (that are invasive in in vitro assays on type I collagen) were compared to HT-29 5F31 cells (that are not invasive in vitro on type I collagen). Samples were co-hybridized on Agilent Human 44K GEP arrays (respectively 5F31 vs 5M21, 5F31 vs 5F7, 5M21 vs 5F31 and 5F7 vs 5F31). Second experiment of microarrays: HT-29 5M21 and HT-29 5F7 xenografts (that develop metastases in immuno-deficient mice lungs) were compared to HT-29 5F31 xenografts (that do not develop metastases in immuno-deficient mice lungs). Samples were consequently co-hybridized on human and mouse 4x44K GEP arrays (respectively AG41_5F31-5M21-138869, AG39_5F31-5F7-138867, AG42_5M21-5F31-138870 and AG40_5F7-5F31-138868 for human microarrays; 5M21_5F31_27033_4, 5F31_5M21_27033_3, 5F7_5F31_27033_2 and 5F31_5F7_27033_1_1 for mouse microarrays).

			Action	DRS
	SRR5176611.fastq.gz	Fastqsanger.gz
	SRR5176612.fastq.gz	Fastqsanger.gz
	SRR5176613.fastq.gz	Fastqsanger.gz
	SRR5176614.fastq.gz	Fastqsanger.gz

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Homo sapiens

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