Let-7f: A New Potential Circulating Biomarker Identified by miRNA Profiling of Cells Isolated from Human Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a progressive vascular disease responsible for 1-4% of the deaths in elderly men. This study aimed to characterize specific microRNA (miRNA) expression in aneurysmal smooth muscle cells (SMCs) and macrophages in order to identify circulating miRNAs associated with AAA. We screened 850 miRNAs in aneurysmal SMCs, M1 and M2 macrophages, and in control SMCs isolated by micro-dissection from aortic biopsies using microarray analysis. In all, 92 miRNAs were detected and 10 miRNAs were selected for validation by qRT-PCR in isolated cells (n = 5), whole control and aneurysmal aorta biopsies (n = 13), and plasma from patients (n = 24) undergoing AAA (over 50 mm) repair matched to patients (n = 18) with peripheral arterial disease (PAD) with atherosclerosis but not AAA. Seven miRNAs were modulated similarly in all aneurysmal cells. The Let-7f was downregulated in aneurysmal cells compared to control SMCs with a significant lower expression in M1 compared to M2 macrophages (0.1 fold, p = 0.03), correlated with a significant downregulation in whole aneurysmal aorta compared to control aorta (0.2 fold, p = 0.03). Significant levels of circulating let-7f (p = 0.048) were found in AAA patients compared to PAD patients with no significant correlation with aortic diameter (R2 = 0.03). Our study underlines the utility of profiling isolated aneurysmal cells to identify other miRNAs for which the modulation of expression might be masked when the whole aorta is used. The results highlight let-7f as a new potential biomarker for AAA.

LKB1 specifies neural crest cell fates through pyruvate-alanine cycling.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

Adventitial Tertiary Lymphoid Organs as Potential Source of MicroRNA Biomarkers for Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology.

Cryptosporidium parvum-induced ileo-caecal adenocarcinoma and Wnt signaling in a mouse model.

Cryptosporidium species are apicomplexan protozoans that are found worldwide. These parasites constitute a large risk to human and animal health. They cause self-limited diarrhea in immunocompetent hosts and a life-threatening disease in immunocompromised hosts. Interestingly, Cryptosporidium parvum has been related to digestive carcinogenesis in humans. Consistent with a potential tumorigenic role of this parasite, in an original reproducible animal model of chronic cryptosporidiosis based on dexamethasone-treated or untreated adult SCID mice, we formerly reported that C. parvum (strains of animal and human origin) is able to induce digestive adenocarcinoma even in infections induced with very low inoculum. The aim of this study was to further characterize this animal model and to explore metabolic pathways potentially involved in the development of C. parvum-induced ileo-caecal oncogenesis. We searched for alterations in genes or proteins commonly involved in cell cycle, differentiation or cell migration, such as ß-catenin, Apc, E-cadherin, Kras and p53. After infection of animals with C. parvum we demonstrated immunohistochemical abnormal localization of Wnt signaling pathway components and p53. Mutations in the selected loci of studied genes were not found after high-throughput sequencing. Furthermore, alterations in the ultrastructure of adherens junctions of the ileo-caecal neoplastic epithelia of C. parvum-infected mice were recorded using transmission electron microscopy. In conclusion, we found for the first time that the Wnt signaling pathway, and particularly the cytoskeleton network, seems to be pivotal for the development of the C. parvum-induced neoplastic process and cell migration of transformed cells. Furthermore, this model is a valuable tool in understanding the host-pathogen interactions associated with the intricate infection process of this parasite, which is able to modulate host cytoskeleton activities and several host-cell biological processes and remains a significant cause of infection worldwide.

Involvement of the immune system, endocytosis and EIF2 signaling in both genetically determined and sporadic forms of Parkinson's disease.

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is a common genetic cause of Parkinson's disease (PD). Although patients with sporadic PD and individuals with LRRK2-linked PD display the classical PD phenotype, it is not known whether or not the same biological pathways are deregulated in each context. By using transcriptome profiling, we investigated the deregulation of various biological pathways in a total of 47 peripheral blood mononuclear cell (PBMC) samples from patients with sporadic PD, patients heterozygous for the LRRK2 G2019S mutation compared to healthy controls. We found that the deregulation patterns were indeed similar in PBMCs obtained from patients with sporadic PD and from LRRK2 G2019S carriers, with dysfunctions in mitochondrial pathways, cell survival signaling, cancerization, endocytosis signaling and iron metabolism. Analysis of our PBMC data and other publicly available transcriptome datasets (for whole blood samples) showed that deregulation of the immune system, endocytosis and eukaryotic initiation factor 2 (EIF2) signaling are the main features of transcriptome profiles in PD (since they are also present in the transcriptome of dopaminergic neurons from patients). Transcriptome analysis of PBMCs is thus valuable for (i) characterizing the pathophysiological pathways shared by genetic and sporadic forms of PD and (ii) identifying potential biomarkers and therapeutic targets. This minimally invasive approach opens up tremendous perspectives for better diagnosis and therapy of neurodegenerative diseases because it can be applied from the earliest stages of the disease onwards.

Structural determination and Toll-like receptor 2-dependent proinflammatory activity of dimycolyl-diarabino-glycerol from Mycobacterium marinum.

Although it was identified in the cell wall of several pathogenic mycobacteria, the biological properties of dimycolyl-diarabino-glycerol have not been documented yet. In this study an apolar glycolipid, presumably corresponding to dimycolyl-diarabino-glycerol, was purified from Mycobacterium marinum and subsequently identified as a 5-O-mycolyl-ß-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1')-glycerol (designated Mma_DMAG) using a combination of nuclear magnetic resonance spectroscopy and mass spectrometry analyses. Lipid composition analysis revealed that mycolic acids were dominated by oxygenated mycolates over α-mycolates and devoid of trans-cyclopropane functions. Highly purified Mma_DMAG was used to demonstrate its immunomodulatory activity. Mma_DMAG was found to induce the secretion of proinflammatory cytokines (TNF-α, IL-8, IL-1ß) in human macrophage THP-1 cells and to trigger the expression of ICAM-1 and CD40 cell surface antigens. This activation mechanism was dependent on TLR2, but not on TLR4, as demonstrated by (i) the use of neutralizing anti-TLR2 and -TLR4 antibodies and by (ii) the detection of secreted alkaline phosphatase in HEK293 cells co-transfected with the human TLR2 and secreted embryonic alkaline phosphatase reporter genes. In addition, transcriptomic analyses indicated that various genes encoding proinflammatory factors were up-regulated after exposure of THP-1 cells to Mma_DMAG. Importantly, a wealth of other regulated genes related to immune and inflammatory responses, including chemokines/cytokines and their respective receptors, adhesion molecules, and metalloproteinases, were found to be modulated by Mma_DMAG. Overall, this study suggests that DMAG may be an active cell wall glycoconjugate driving host-pathogen interactions and participating in the immunopathogenesis of mycobacterial infections.

AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis, is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis.

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both beta-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.