Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms

Background Early developmental patterns of flatworms are extremely diverse and difficult to compare between distant groups. In parasitic flatworms, such as tapeworms, this is confounded by highly derived life cycles involving indirect development, and even the true orientation of the tapeworm antero-posterior (AP) axis has been a matter of controversy. In planarians, and metazoans generally, the AP axis is specified by the canonical Wnt pathway, and we hypothesized that it could also underpin axial formation during larval metamorphosis in tapeworms. Results By comparative gene expression analysis of Wnt components and conserved AP markers in the tapeworms Echinococcus multilocularis and Hymenolepis microstoma, we found remarkable similarities between the early stages of larval metamorphosis in tapeworms and late embryonic and adult development in planarians. We demonstrate posterior expression of specific Wnt factors during larval metamorphosis and show that scolex formation is preceded by localized expression of Wnt inhibitors. In the highly derived larval form of E. multilocularis, which proliferates asexually within the mammalian host, we found ubiquitous expression of posterior Wnt factors combined with localized expression of Wnt inhibitors that correlates with the asexual budding of scoleces. As in planarians, muscle cells are shown to be a source of secreted Wnt ligands, providing an explanation for the retention of a muscle layer in the immotile E. multilocularis larva. Conclusions The strong conservation of gene expression between larval metamorphosis in tapeworms and late embryonic development in planarians suggests, for the first time, a homologous developmental period across this diverse phylum. We postulate these to represent the phylotypic stages of these flatworm groups. Our results support the classical notion that the scolex is the true anterior end of tapeworms. Furthermore, the up-regulation of Wnt inhibitors during the specification of multiple anterior poles suggests a mechanism for the unique asexual reproduction of E. multilocularis larvae

Editorial: Novel Frontiers in Helminth Genomics

No abstract available

Metformin suppresses development of the Echinococcus multilocularis larval stage by targeting the TOR pathway

Alveolar echinococcosis (AE) is a severe disease caused by the larval stage of the tapeworm Echinococcus multilocularis. Current chemotherapeutic treatment options based on benzimidazoles are of limited effectiveness, which underlines the need to find new antiechinococcosis drugs. Metformin is an antihyperglycemic and antiproliferative agent that shows activity against the related parasite Echinococcus granulosus. Hence, we assessed the in vitro and in vivo effects of the drug on E. multilocularis. Metformin exerted significant dose-dependent killing effects on in vitro cultured parasite stem cells and protoscoleces and significantly reduced the dedifferentiation of protoscoleces into metacestodes. Likewise, oral administration of metformin (50 mg/kg of body weight/day for 8 weeks) was effective in achieving a significant reduction of parasite weight in a secondary murine AE model. Our results revealed mitochondrial membrane depolarization, activation of Em-AMPK, suppression of Em-TOR, and overexpression of Em-Atg8 in the germinal layer of metformin-treated metacestode vesicles. The opposite effects on the level of active Em-TOR in response to exogenous insulin and rapamycin suggest that Em-TOR is part of the parasite’s insulin signaling pathway. Finally, the presence of the key lysosomal pathway components, through which metformin reportedly acts, was confirmed in the parasite by in silico assays. Taken together, these results introduce metformin as a promising candidate for AE treatment. Although our study highlights the importance of those direct mechanisms by which metformin reduces parasite viability, it does not necessarily preclude any additional systemic effects of the drug that might reduce parasite growth in vivo.Fil: Loos, Julia Alexandra. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología. Laboratorio de Zoonosis Parasitarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Dávila, Valeria Analia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología. Laboratorio de Zoonosis Parasitarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Brehm, Klaus. Universität Würzburg; AlemaniaFil: Cumino, Andrea Carina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología. Laboratorio de Zoonosis Parasitarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentin

Fit and Healthy in Middle Adulthood – Do Fitness Levels Make a Difference

Strong evidence exists that fitness is a physical health resource, which serves to protect one’s health. There is still uncertainty about which fitness level provides the best health outcome and which measurements can be used for analyzing this question. This cross-sectional study analyzed 462 (64.07% female) German middle-aged adults regarding their fitness status, physical activity (Non-Exercise test), body composition (Body Mass Index) and heart-related health status. Motor tests were used to measure the health-related fitness status. The heart-related health status was surveyed by questionnaire and diagnosis was done in part by a physician. Relationships between risk factors and fitness factors are visible during the correlative analysis. They are substantially more visible in the differentiation of people with and without risks. People with low fitness show noticeable risks in activity, Body Mass Index and heart-related health. People with high fitness show health resources for activity, Body Mass Index and heart-related health. This study points out that all fitness dimensions influence one’s heart-related health in a positive way. Fitness is measured objectively and includes all health-related fitness dimensions such as endurance, strength, coordination and flexibility. Apart from this standardization, we ask for more longitudinal studies and more objective health measurements

Loss of Cx43 in Murine Sertoli Cells Leads to Altered Prepubertal Sertoli Cell Maturation and Impairment of the Mitosis-Meiosis Switch.

Male factor infertility is a problem in today's society but many underlying causes are still unknown. The generation of a conditional Sertoli cell (SC)-specific connexin 43 (Cx43) knockout mouse line (SCCx43KO) has provided a translational model. Expression of the gap junction protein Cx43 between adjacent SCs as well as between SCs and germ cells (GCs) is known to be essential for the initiation and maintenance of spermatogenesis in different species and men. Adult SCCx43KO males show altered spermatogenesis and are infertile. Thus, the present study aims to identify molecular mechanisms leading to testicular alterations in prepubertal SCCx43KO mice. Transcriptome analysis of 8-, 10- and 12-day-old mice was performed by next-generation sequencing (NGS). Additionally, candidate genes were examined by qRT-PCR and immunohistochemistry. NGS revealed many significantly differentially expressed genes in the SCCx43KO mice. For example, GCspecific genes were mostly downregulated and found to be involved in meiosis and spermatogonial differentiation (e.g., Dmrtb1, Sohlh1). In contrast, SC-specific genes implicated in SC maturation and proliferation were mostly upregulated (e.g., Amh, Fshr). In conclusion, Cx43 in SCs appears to be required for normal progression of the first wave of spermatogenesis, especially for the mitosis-meiosis switch, and also for the regulation of prepubertal SC maturation

Health-Related Fitness in Adults From Eight European Countries—An Analysis Based on Data From the European Fitness Badge

Background: There are conflicting reports about the fitness status of European adults, partly due to the lack of a standardized fitness test battery used across Europe. The European Fitness Badge (EFB) was developed in 2017 as an online-based tool to assess the health-related fitness of persons aged ≥ 18 years residing in European countries. We examined the demographic characteristics and fitness status of persons who completed the EFB between June 2017 and May 2019. Methods: We conducted a multinational study in eight European countries. Participants completed the EFB which includes eleven validated motor tests to measure endurance, strength, coordination and flexibility performance, under the supervision of an EFB instructor in different settings (e.g. sports club sessions, public events). Two different test batteries (test profiles, TP) are available to distinguish between less active (TP1) and active individuals (TP2). We calculated descriptive statistics and conducted analyses of variance to examine sample characteristics and a potential impact of sex, age, body mass index (BMI), physical activity and posture on fitness as assessed by the EFB. Results: The sample included 6,019 adults (68.7% females; mean age 52.7 years; age range 18-89 years). Participants who completed TP1 were older (TP1: 61.4 years; TP2: 44.2 years; p = 0.00), reported a lower level of physical activity (TP1: 3.8; TP2: 4.0; p = 0.00), had a higher BMI (TP1: 25.7; TP2: 24.3; p = 0.00) and a higher frequency of postural abnormalities (TP1: 43%; TP2: 33%; p = 0.00) than TP2 participants. Among 3,034 participants who completed TP2, males had higher performance in endurance, strength and overall fitness, whereas females performed better in coordination and flexibility tests. In addition, younger age, lower BMI and higher level of physical activity engagement was associated with better EFB test performance. Conclusions: The EFB can be used to assess the health-related fitness status of individuals aged ≥ 18 years. Our results show that TP1 and TP2 were completed by persons from the respective target groups (i.e. less active vs. active), and also confirm findings from previous studies on potential determinants of fitness such as sex or age

Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto.

Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s

Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro.

Echinococcosis represents a major public health problem worldwide and is considered a neglected disease by the World Health Organization. The etiological agents are Echinococcus tapeworms, which display elaborate developmental traits that imply a complex control of gene expression. MicroRNAs (miRNAs), a class of small regulatory RNAs, are involved in the regulation of many biological processes such as development and metabolism. They act through the repression of messenger RNAs (mRNAs) usually by binding to the 3' untranslated region (3'UTR). Previously, we described the miRNome of several Echinococcus species and found that miRNAs are highly expressed in all life cycle stages, suggesting an important role in gene expression regulation. However, studying the role of miRNAs in helminth biology remains a challenge. To develop methodology for functional analysis of miRNAs in tapeworms, we performed miRNA knockdown experiments in primary cell cultures of Echinococcus multilocularis, which mimic the development of metacestode vesicles from parasite stem cells in vitro. First, we analysed the miRNA repertoire of E. multilocularis primary cells by small RNA-seq and found that miR-71, a bilaterian miRNA absent in vertebrate hosts, is one of the top five most expressed miRNAs. Using genomic information and bioinformatic algorithms for miRNA binding prediction, we found a high number of potential miR-71 targets in E. multilocularis. Inhibition of miRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) that block miRNA function. To this end, we evaluated a variety of chemically modified anti-miRs for miR-71 knockdown. Electroporation of primary cells with 2'-O-methyl modified anti-miR-71 led to significantly reduced miR-71 levels. Transcriptomic analyses showed that several predicted miR-71 targets were up-regulated in anti-miR-treated primary cells, including genes potentially involved in parasite development, host parasite interaction, and several genes of as yet unknown function. Notably, miR-71-silenced primary cell cultures showed a strikingly different phenotype from control cells and did not develop into fully mature metacestodes. These findings indicate an important function of miR-71 in Echinococcus development and provide, for the first time, methodology to functionally study miRNAs in a tapeworm