Subcompartmentalisation of Proteins in the Rhoptries Correlates with Ordered Events of Erythrocyte Invasion by the Blood Stage Malaria Parasite

Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction - the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion. © 2012 Zuccala et al

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

International audienceABSTRACT: BACKGROUND: Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. METHODS: Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. RESULTS: Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of DR3 that has no trans-membrane domain and no death domain. CONCLUSION: Colon cancer cells acquire an increased capacity to survive via the activation of the PI3K/NFκB pathway following the stimulation of DR3 by E-selectin. Generation of a DR3 splice variant devoid of death domain can further contribute to protect against apoptosis

High Resolution Melt analysis for mutation screening in PKD1 and PKD2

<p>Abstract</p> <p>Background</p> <p>Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. <it>PKD1 </it>and <it>PKD2 </it>have been implicated in ADPKD pathogenesis but genetic features and the size of <it>PKD1 </it>make genetic diagnosis tedious.</p> <p>Methods</p> <p>We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in <it>PKD1 </it>and <it>PKD2 </it>with HRM in 37 unrelated patients with ADPKD.</p> <p>Results</p> <p>We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in <it>PKD1 </it>and 3 in <it>PKD2 </it>) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in <it>PKD1 </it>and two in <it>PKD2</it>.</p> <p>Conclusion</p> <p>HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.</p

Influence of folate status on genomic DNA methylation in colonic mucosa of subjects without colorectal adenoma or cancer

DNA hypomethylation may increase the risk of colorectal cancer. The main aim of this study was to assess the influence of folate status (serum and erythrocyte folate and plasma homocysteine concentrations) on DNA methylation. Methylenetetrahydrofolate reductase (MTHFR 677C → T and 1298A → C), methionine synthase (MS 2756A → G) and cystathionine synthase (CBS 844ins68) polymorphisms were measured to account for potential confounding effects on folate status and DNA methylation. A total of 68 subjects (33 men and 35 women, 36–78 years) free from colorectal polyps or cancer were recruited in a cross-sectional study. Tissue biopsies were obtained at colonoscopy for the determination of DNA methylation in colonic mucosa using an in vitro radiolabelled methyl acceptance assay. Serum and erythrocyte folate were inversely correlated with plasma homocysteine (r=−0.573, P<0.001 and r=−0.307, P=0.01 respectively) and DNA hypomethylation in colonic mucosa (r=−0.311, P=0.01 and r=−0.356, P=0.03). After adjusting for gender, age, body mass index, smoking and genotype, there were weak negative associations between serum and erythrocyte folate and colonic DNA hypomethylation (P=0.07 and P=0.08, respectively)

Human Pentraxin 3 Binds to the Complement Regulator C4b-Binding Protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1–3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage

Analyses of genome architecture and gene expression reveal novel candidate virulence factors in the secretome of Phytophthora infestans

<p>Abstract</p> <p>Background</p> <p><it>Phytophthora infestans </it>is the most devastating pathogen of potato and a model organism for the oomycetes. It exhibits high evolutionary potential and rapidly adapts to host plants. The <it>P. infestans </it>genome experienced a repeat-driven expansion relative to the genomes of <it>Phytophthora sojae </it>and <it>Phytophthora ramorum </it>and shows a discontinuous distribution of gene density. Effector genes, such as members of the RXLR and Crinkler (CRN) families, localize to expanded, repeat-rich and gene-sparse regions of the genome. This distinct genomic environment is thought to contribute to genome plasticity and host adaptation.</p> <p>Results</p> <p>We used <it>in silico </it>approaches to predict and describe the repertoire of <it>P. infestans </it>secreted proteins (the secretome). We defined the "plastic secretome" as a subset of the genome that (i) encodes predicted secreted proteins, (ii) is excluded from genome segments orthologous to the <it>P. sojae </it>and <it>P. ramorum </it>genomes and (iii) is encoded by genes residing in gene sparse regions of <it>P. infestans </it>genome. Although including only ~3% <it>of P. infestans </it>genes, the plastic secretome contains ~62% of known effector genes and shows >2 fold enrichment in genes induced <it>in planta</it>. We highlight 19 plastic secretome genes induced <it>in planta </it>but distinct from previously described effectors. This list includes a trypsin-like serine protease, secreted oxidoreductases, small cysteine-rich proteins and repeat containing proteins that we propose to be novel candidate virulence factors.</p> <p>Conclusions</p> <p>This work revealed a remarkably diverse plastic secretome. It illustrates the value of combining genome architecture with comparative genomics to identify novel candidate virulence factors from pathogen genomes.</p

Role of Cancer Microenvironment in Metastasis: Focus on Colon Cancer

One person on three will receive a diagnostic of cancer during his life. About one third of them will die of the disease. In most cases, death will result from the formation of distal secondary sites called metastases. Several events that lead to cancer are under genetic control. In particular, cancer initiation is tightly associated with specific mutations that affect proto-oncogenes and tumour suppressor genes. These mutations lead to unrestrained growth of the primary neoplasm and a propensity to detach and to progress through the subsequent steps of metastatic dissemination. This process depends tightly on the surrounding microenvironment. In fact, several studies support the point that tumour development relies on a continuous cross-talk between cancer cells and their cellular and extracellular microenvironments. This signaling cross-talk is mediated by transmembrane receptors expressed on cancer cells and stromal cells. The aim of this manuscript is to review how the cancer microenvironment influences the journey of a metastatic cell taking liver invasion by colorectal cancer cells as a model

A “Candidate-Interactome” Aggregate Analysis of Genome-Wide Association Data in Multiple Sclerosis

Though difficult, the study of gene-environment interactions in multifactorial diseases is crucial for interpreting the relevance of non-heritable factors and prevents from overlooking genetic associations with small but measurable effects. We propose a "candidate interactome" (i.e. a group of genes whose products are known to physically interact with environmental factors that may be relevant for disease pathogenesis) analysis of genome-wide association data in multiple sclerosis. We looked for statistical enrichment of associations among interactomes that, at the current state of knowledge, may be representative of gene-environment interactions of potential, uncertain or unlikely relevance for multiple sclerosis pathogenesis: Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, HHV8-Kaposi sarcoma, H1N1-influenza, JC virus, human innate immunity interactome for type I interferon, autoimmune regulator, vitamin D receptor, aryl hydrocarbon receptor and a panel of proteins targeted by 70 innate immune-modulating viral open reading frames from 30 viral species. Interactomes were either obtained from the literature or were manually curated. The P values of all single nucleotide polymorphism mapping to a given interactome were obtained from the last genome-wide association study of the International Multiple Sclerosis Genetics Consortium & the Wellcome Trust Case Control Consortium, 2. The interaction between genotype and Epstein Barr virus emerges as relevant for multiple sclerosis etiology. However, in line with recent data on the coexistence of common and unique strategies used by viruses to perturb the human molecular system, also other viruses have a similar potential, though probably less relevant in epidemiological terms

V392 Persei: a γ-ray bright nova eruption from a known dwarf nova

V392 Persei is a known dwarf nova (DN) that underwent a classical nova eruption in 2018. Here we report ground-based optical, Swift UV and X-ray, and Fermi-LAT γ-ray observations following the eruption for almost three years. V392 Per is one of the fastest evolving novae yet observed, with a t2 decline time of 2 days. Early spectra present evidence for multiple and interacting mass ejections, with the associated shocks driving both the γ-ray and early optical luminosity. V392 Per entered Sun-constraint within days of eruption. Upon exit, the nova had evolved to the nebular phase, and we saw the tail of the super-soft X-ray phase. Subsequent optical emission captured the fading ejecta alongside a persistent narrow line emission spectrum from the accretion disk. Ongoing hard X-ray emission is characteristic of a standing accretion shock in an intermediate polar. Analysis of the optical data reveals an orbital period of 3.230 ± 0.003 days, but we see no evidence for a white dwarf (WD) spin period. The optical and X-ray data suggest a high mass WD, the pre-nova spectral energy distribution (SED) indicates an evolved donor, and the post-nova SED points to a high mass accretion rate. Following eruption, the system has remained in a nova-like high mass transfer state, rather than returning to the pre-nova DN low mass transfer configuration. We suggest that this high state is driven by irradiation of the donor by the nova eruption. In many ways, V392 Per shows similarity to the well-studied nova and DN GK Persei