Correlation of CLEC1B haplotypes with plasma levels of soluble CLEC-2 in healthy individuals.

Binding of podoplanin to the C-type lectin-like receptor 2 (CLEC-2) promotes platelet activation and soluble CLEC-2 (sCLEC-2) is shed from activated platelets. The role of sCLEC-2 in the plasma is unknown. The expression level and plasma concentration of sCLEC-2 could be affected by variants of the corresponding gene, CLEC1B. Here, we genotyped SNVs in the promoter and coding region of CLEC1B and determined plasma levels of sCLEC-2 in healthy individuals. We genotyped 516 healthy blood donors for 7 SNVs (rs10505743, rs11053538, rs4764178, rs76016091, rs2273986, rs2273987, rs521040) by using PCR methods and calculated haplotypes from the SNV genotypes. For 313 of the donors we measured the sCLEC-2 concentration in EDTA plasma samples by using a commercial ELISA. SNV typing revealed allele frequencies comparable to database information. None of the SNVs showed significant correlation with sCLEC-2 plasma levels. Haplotype analysis indicated 6 haplotypes with frequencies >1% and haplotype h3 was the most frequent (33.8%). Donors homozygous for h3 (n = 37) showed significantly lower sCLEC-2 plasma levels (median 0.95 ng/mL) than donors being h3 negative or heterozygous (n = 276; 1.44 ng/mL; p = .0203). We found that the sCLEC-2 plasma concentration is variable in healthy individuals and the CLEC1B genotype contributes to the expression level.

Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis.

The ADP receptor P2Y12, the thromboxane A2 receptor (TXA2R) and the C-type lectin-like receptor 2 (CLEC-2) mediate platelet activation by different mechanisms. Only little is known about the expression of the receptors in human megakaryopoiesis. Our study aimed to establish a flow cytometry (FC) method for the measurement of P2Y12, TXA2R, and CLEC-2 on platelets of healthy donors and to monitor receptor expression in ex vivo megakaryopoiesis. We determined mean fluorescence intensity (MFI) values of FITC, PE, or APC labeled antibodies binding to the receptors on platelets of 90 healthy donors. For cord blood-derived megakaryopoiesis (CBMK) differentiation of CD34+ cells was induced by IL-3, SCF, and TPO. At 6 time points between day 0 and day 21 of cell culture the MFI values for CD34, CD41, CD61, P2Y12, TXA2R, and CLEC-2 were measured. Quantitative PCR was used for relative quantification of the corresponding mRNA. Transcription factor (TF) binding sites were predicted by in silico analysis of the genes. Platelets showed expectable high MFI values for the platelet marker CD41 (13,716 median MFI). Lower MFI was found for P2Y12 (2,847 median MFI) and CLEC-2 (1,211 median MFI), whereas, binding of the TXA2R antibody revealed even higher values (21,458 median MFI) than CD41. In CBMK the CD34+ cells were negative for P2Y12, TXA2R, and CLEC-2 at day 0. A maximum of 21-fold and 6-fold increase of P2Y12 and TXA2R MFI values, respectively, was found on day 14 to 17. MFI for CLEC-2 increased by 58-fold within the first week and reached a maximum of 1,572-fold increase within the first two weeks of CBMK. Very similar results were obtained on the RNA level. The differential regulation of receptor expression in CBMK was further supported by significant differences in the numbers and types of TF binding sites. P2Y12 and TXA2R, both upregulated only to a low extent in CBMK, probably, are dispensable for megakaryopoiesis. Furthermore, we speculate that CLEC-2 strongly upregulated in early CMBK is important for megakaryopoiesis.

DIANA-mirExTra v2.0: Uncovering microRNAs and transcription factors with crucial roles in NGS expression data.

Differential expression analysis (DEA) is one of the main instruments utilized for revealing molecular mechanisms in pathological and physiological conditions. DIANA-mirExTra v2.0 (http://www.microrna.gr/mirextrav2) performs a combined DEA of mRNAs and microRNAs (miRNAs) to uncover miRNAs and transcription factors (TFs) playing important regulatory roles between two investigated states. The web server uses as input miRNA/RNA-Seq read count data sets that can be uploaded for analysis. Users can combine their data with 350 small-RNA-Seq and 65 RNA-Seq in-house analyzed libraries which are provided by DIANA-mirExTra v2.0.The web server utilizes miRNA:mRNA, TF:mRNA and TF:miRNA interactions derived from extensive experimental data sets. More than 450 000 miRNA interactions and 2 000 000 TF binding sites from specific or high-throughput techniques have been incorporated, while accurate miRNA TSS annotation is obtained from microTSS experimental/in silico framework. These comprehensive data sets enable users to perform analyses based solely on experimentally supported information and to uncover central regulators within sequencing data: miRNAs controlling mRNAs and TFs regulating mRNA or miRNA expression. The server also supports predicted miRNA:gene interactions from DIANA-microT-CDS for 4 species (human, mouse, nematode and fruit fly). DIANA-mirExTra v2.0 has an intuitive user interface and is freely available to all users without any login requirement.

Ancient animal microRNAs and the evolution of tissue identity.

The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome-deuterostome ancestor.

C-Type Lectin-like Receptor 2 Expression Is Decreased upon Platelet Activation and Is Lower in Most Tumor Entities Compared to Healthy Controls.

The C-type lectin-like receptor 2 (CLEC-2) is expressed on platelets and mediates binding to podoplanin (PDPN) on various cell types. The binding to circulating tumor cells (CTCs) leads to platelet activation and promotes metastatic spread. An increased level of soluble CLEC-2 (sCLEC-2), presumably released from activated platelets, was shown in patients with thromboinflammatory and malignant disease. However, the functional role of sCLEC-2 and the mechanism of sCLEC-2 release are not known. In this study, we focused on the effect of platelet activation on CLEC-2 expression and the sCLEC-2 plasma level in patients with cancer. First, citrated blood from healthy volunteer donors (n = 20) was used to measure the effect of platelet stimulation by classical agonists and PDPN on aggregation, CLEC-2 expression on platelets with flow cytometry, sCLEC-2 release to the plasma with ELISA and total CLEC-2 expression with Western blot analysis. Second, sCLEC-2 was determined in plasma samples from healthy donors (285) and patients with colorectal carcinoma (CRC; 194), melanoma (160), breast cancer (BC; 99) or glioblastoma (49). PDPN caused a significant increase in the aggregation response induced by classical agonists. ADP or PDPN stimulation of platelets caused a significant decrease in CLEC-2 on platelets and sCLEC-2 in the plasma, whereas total CLEC-2 in platelet lysates remained the same. Thus, the increased plasma level of sCLEC-2 is not a suitable biomarker of platelet activation. In patients with CRC (median 0.9 ng/mL), melanoma (0.9 ng/mL) or BC (0.7 ng/mL), we found significantly lower sCLEC-2 levels (p < 0.0001), whereas patients with glioblastoma displayed higher levels (2.6 ng/mL; p = 0.0233) compared to healthy controls (2.1 ng/mL). The low sCLEC-2 plasma level observed in most of the tumor entities of our study presumably results from the internalization of sCLEC-2 by activated platelets or binding of sCLEC-2 to CTCs.

Hedgehog signaling regulates segment formation in the annelid Platynereis.

Annelids and arthropods share a similar segmented organization of the body whose evolutionary origin remains unclear. The Hedgehog signaling pathway, prominent in arthropod embryonic segment patterning, has not been shown to have a similar function outside arthropods. We show that the ligand Hedgehog, the receptor Patched, and the transcription factor Gli are all expressed in striped patterns before the morphological appearance of segments in the annelid Platynereis dumerilii. Treatments with small molecules antagonistic to Hedgehog signaling disrupt segment formation. Platynereis Hedgehog is not necessary to establish early segment patterns but is required to maintain them. The molecular similarity of segment patterning functions of the Hedgehog pathway in an annelid and in arthropods supports a common origin of segmentation in protostomes.

Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution.

Neurosecretory control centers form part of the forebrain in many animal phyla, including vertebrates, insects, and annelids. The evolutionary origin of these centers is largely unknown. To identify conserved, and thus phylogenetically ancient, components of neurosecretory brain centers, we characterize and compare neurons that express the prohormone vasotocin (vasopressin/oxytocin)-neurophysin in the developing forebrain of the annelid Platynereis dumerilii and of the zebrafish. These neurons express the same tissue-restricted microRNA, miR-7, and conserved, cell-type-specific combinations of transcription factors (nk2.1, rx, and otp) that specify their identity, as evidenced by the specific requirement of zebrafish rx3 for vasotocin-neurophysin expression. MiR-7 also labels another shared population of neurons containing RFamides. Since the vasotocinergic and RFamidergic neurons appear to be directly sensory in annelid and fish, we propose that cell types with dual sensory-neurosecretory properties were the starting point for the evolution of neurosecretory brain centers in Bilateria.