Genome-wide transcriptome analysis of Echinococcus multilocularis larvae and germinative cell cultures reveals genes involved in parasite stem cell function.

The lethal zoonosis alveolar echinococcosis is caused by tumour-like growth of the metacestode stage of the tapeworm Echinococcus multilocularis within host organs. We previously demonstrated that metacestode proliferation is exclusively driven by somatic stem cells (germinative cells), which are the only mitotically active parasite cells that give rise to all differentiated cell types. The Echinococcus gene repertoire required for germinative cell maintenance and differentiation has not been characterised so far. We herein carried out Illumina sequencing on cDNA from Echinococcus metacestode vesicles, from metacestode tissue depleted of germinative cells, and from Echinococcus primary cell cultures. We identified a set of ~1,180 genes associated with germinative cells, which contained numerous known stem cell markers alongside genes involved in replication, cell cycle regulation, mitosis, meiosis, epigenetic modification, and nucleotide metabolism. Interestingly, we also identified 44 stem cell associated transcription factors that are likely involved in regulating germinative cell differentiation and/or pluripotency. By in situ hybridization and pulse-chase experiments, we also found a new general Echinococcus stem cell marker, EmCIP2Ah, and we provide evidence implying the presence of a slow cycling stem cell sub-population expressing the extracellular matrix factor Emkal1. RNA-Seq analyses on primary cell cultures revealed that metacestode-derived Echinococcus stem cells display an expanded differentiation capability and do not only form differentiated cell types of the metacestode, but also cells expressing genes specific for protoscoleces, adult worms, and oncospheres, including an ortholog of the schistosome praziquantel target, EmTRPMPZQ. Finally, we show that primary cell cultures contain a cell population expressing an ortholog of the tumour necrosis factor α receptor family and that mammalian TNFα accelerates the development of metacestode vesicles from germinative cells. Taken together, our analyses provide a robust and comprehensive characterization of the Echinococcus germinative cell transcriptome, demonstrate expanded differentiation capability of metacestode derived stem cells, and underscore the potential of primary germinative cell cultures to investigate developmental processes of the parasite. These data are relevant for studies into the role of Echinococcus stem cells in parasite development and will facilitate the design of anti-parasitic drugs that specifically act on the parasite germinative cell compartment.

Anticancer effects of NSC­631570 (Ukrain) in head and neck cancer cells: In vitro analysis of growth, invasion, angiogenesis and gene expression.

NSC­631570 (Ukrain) is an aqueous extract of Chelidonium majus, a herbaceous perennial plant, one of two species in the genus Chelidonium, which has been demonstrated to selectively kill tumor cells without affecting non­malignant cells. In the present study, the components of NSC­631570 were examined by combined liquid chromatography/mass spectroscopy (LC­MS) and the effects of NSC­631570 on HNSCC cell lines, as well as primary cells, were analyzed with respect to growth, apoptosis, invasion, angiogenesis and gene expression. LC­MS identified chelerythrine and allocryptopine as the major alkaloids of the extract. Moreover, NSC­631570 suppressed the growth of all tested HNSCC cell lines, including a paclitaxel­resistant and P­glycoprotein (MDR1)­overexpressing cell line. Mucosal keratinocytes were also affected by the extract, while fibroblasts proved to be much more resistant. In contrast to allocryptopine, chelerythrine had toxic effects on HNSCC cell lines at low doses. NSC­631570 significantly induced apoptosis in the FaDu and HLaC78 cell lines. As analyzed by a spheroid­based invasion assay, cell migration was significantly suppressed by NSC­631570 in FaDu cells on gelatine, fibronectin, collagen, laminin and Matrigel®. Migration of the highly invasive cell line HLaC78 was also inhibited, albeit to a lesser extent (not significant on laminin). Microarray analysis revealed the downregulation of genes encoding key regulators, including EGFR, AKT2, JAK1, STAT3 and ß­catenin (CTNNB1), all of which are involved in cell proliferation, migration, angiogenesis, apoptosis as well as the radiation­ and chemo­resistance of HNSCC. The strongest upregulation occurred for cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), involved in the metabolism of xenobiotics. Upregulation of CYP1A1 was at least partially caused by chelerythrine and allocryptopine, as shown by RT­qPCR in two HNSCC cell lines. In addition, NSC­631570 showed a high anti­angiogenic action on the tube formation ability of human umbilical vein endothelial cells (HUVECs). In conclusion, this study highlights NSC­631570 as a promising therapeutic approach for HNSCC.

Effect of chelidonine on growth, invasion, angiogenesis and gene expression in head and neck cancer cell lines.

The greater celandine 'Chelidonium majus' and its main alkaloid chelidonine have previously been shown to exert high cytotoxicity against cancer cells. Furthermore, chelidonine is proposed to possess pro-apoptotic and anti-metastatic properties. Within the present study, the effects chelidonine on several HNSCC cell lines, as well as primary cells, were analyzed with respect to growth, migration, angiogenesis and apoptosis. Chelidonine suppressed the growth of all tested HNSCC cell lines, including a paclitaxel-resistant and P-glycoprotein (MDR1) overexpressing cell line, but not in a clear dose-dependent manner. Mucosal keratinocytes were also strongly affected by chelidonine, while fibroblasts proved to be much more resistant. Chelidonine failed to trigger apoptosis at physiological concentrations in HNSCC cell lines. Based on a spheroid invasion model chelidonine suppressed invasion of FaDu cells effectively on gelatin, fibronectin, collagen I, laminin and Matrigel®. However, invasion inhibition of the more aggressively invading cell line HLaC78 largely failed. Using the tube formation assay, chelidonine effectively inhibited angiogenesis. Expression analysis revealed an upregulation of the xenobiotic metabolism genes CYP1A1 and MDR1 by chelidonine. In summary, chelidonine appeared to exert only minor impact on head and neck cancer cells. Chelidonine did not produce clear dose-dependent and cell-type specific cytotoxicity nor did it trigger apoptosis strongly.

Comparative genomics analysis of Streptococcus isolates from the human small intestine reveals their adaptation to a highly dynamic ecosystem.

The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine.