S. mediterranea ETS-1 regulates the function of cathepsin-positive cells and the epidermal lineage landscape via basement membrane remodeling.

Extracellular matrix (ECM) is an important component of stem cell niche. Remodeling of ECM mediated by ECM regulators, such as matrix metalloproteinases (MMPs) plays a vital role in stem cell function. However, the mechanisms that modulate the function of ECM regulators in the stem cell niche are understudied. Here, we explored the role of the transcription factor (TF) ETS-1, which is expressed in the cathepsin-positive cell population, in regulating the expression of the ECM regulator, mt-mmpA, thereby modulating basement membrane thickness. In planarians, the basement membrane around the gut/inner parenchyma is thought to act as a niche for pluripotent stem cells. It has been shown that the early epidermal progenitors migrate outwards from this region and progressively differentiate to maintain the terminal epidermis. Our data shows that thickening of the basement membrane in the absence of ets-1 results in defective migration of stem cell progeny. Furthermore, the absence of ets-1 leads to a defective epidermal progenitor landscape, despite its lack of expression in those cell types. Together, our results demonstrate the active role of ECM remodeling in regulating tissue homeostasis and regeneration in the planarian Schmidtea mediterranea. This article has an associated First Person interview with one of the co-first authors of the paper.

Maritalea mediterranea sp. nov., isolated from marine plastic residues.

A novel Gram-reaction-negative, aerobic, motile, rod-shaped, grey bacterium, strain P4.10XT, was isolated from plastic debris sampled from shallow waters in the Mediterranean Sea (Valencia, Spain). P4.10XT was catalase- and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA gene sequences revealed that P4.10XT was closely related to Maritalea myrionectae DSM 19524T and Maritalea mobilis E6T (98.25 and 98.03 % sequence similarity, respectively). The DNA G+C content of the genome sequence of P4.10XT was 53.66 %. The genomic indexes average nucleotide identity by blast (ANIb) and digital DNA-DNA hybridization (dDDH) confirmed its classification as representing a novel species of the genus Maritalea. The predominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c) and C18 : 1 ω7c 11-methyl. The results of this polyphasic study confirm that P4.10XT represents a novel species of the genus Maritalea, for which the name Maritalea mediterranea sp. nov. is proposed (type strain P4.10XT=CECT 30306T = DSM 112386T).

Schmidtea mediterranea as a Model Organism to Study the Molecular Background of Human Motile Ciliopathies.

Cilia and flagella are evolutionarily conserved organelles that form protrusions on the surface of many growth-arrested or differentiated eukaryotic cells. Due to the structural and functional differences, cilia can be roughly classified as motile and non-motile (primary). Genetically determined dysfunction of motile cilia is the basis of primary ciliary dyskinesia (PCD), a heterogeneous ciliopathy affecting respiratory airways, fertility, and laterality. In the face of the still incomplete knowledge of PCD genetics and phenotype-genotype relations in PCD and the spectrum of PCD-like diseases, a continuous search for new causative genes is required. The use of model organisms has been a great part of the advances in understanding molecular mechanisms and the genetic basis of human diseases; the PCD spectrum is not different in this respect. The planarian model (Schmidtea mediterranea) has been intensely used to study regeneration processes, and-in the context of cilia-their evolution, assembly, and role in cell signaling. However, relatively little attention has been paid to the use of this simple and accessible model for studying the genetics of PCD and related diseases. The recent rapid development of the available planarian databases with detailed genomic and functional annotations prompted us to review the potential of the S. mediterranea model for studying human motile ciliopathies.

Mitogen-like Cerium-Based Nanoparticles Protect Schmidtea mediterranea against Severe Doses of X-rays.

Novel radioprotectors are strongly demanded due to their numerous applications in radiobiology and biomedicine, e.g., for facilitating the remedy after cancer radiotherapy. Currently, cerium-containing nanomaterials are regarded as promising inorganic radioprotectors due to their unrivaled antioxidant activity based on their ability to mimic the action of natural redox enzymes like catalase and superoxide dismutase and to neutralize reactive oxygen species (ROS), which are by far the main damaging factors of ionizing radiation. The freshwater planarian flatworms are considered a promising system for testing new radioprotectors, due to the high regenerative potential of these species and an excessive amount of proliferating stem cells (neoblasts) in their bodies. Using planarian Schmidtea mediterranea, we tested CeO2 nanoparticles, well known for their antioxidant activity, along with much less studied CeF3 nanoparticles, for their radioprotective potential. In addition, both CeO2 and CeF3 nanoparticles improve planarian head blastema regeneration after ionizing irradiation by enhancing blastema growth, increasing the number of mitoses and neoblasts' survival, and modulating the expression of genes responsible for the proliferation and differentiation of neoblasts. The CeO2 nanoparticles' action stems directly from their redox activity as ROS scavengers, while the CeF3 nanoparticles' action is mediated by overexpression of "wound-induced genes" and neoblast- and stem cell-regulating genes.

Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea.

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and disubstituted O-N-acetylgalactosamine structures. Extensions made of hexoses and 3-O-methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, 60 genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. Although a majority of the genes involved in mucin-type O-glycan biosynthesis were highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

Regenerative responses following DNA damage - ß-catenin mediates head regrowth in the planarian Schmidtea mediterranea.

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component ß-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Metabolic cost of development, regeneration, and reproduction in the planarian Schmidtea mediterranea.

Planaria are known for their ability to completely regenerate upon fissioning or experimental amputation. Yet, metabolic costs of regeneration have not been directly measured in planaria. Our goal was to establish the relationships between oxygen consumption (V̇O2), regeneration, and reproductive mode for asexual and sexual strains of Schmidtea mediterranea. We hypothesized that V̇O2 would vary by regeneration day for both sexual and asexual S. mediterranea, reflecting different costs of tissue reconstruction, but with an additional cost for regenerating sexual organs. Testes regeneration and body mass, as indicators of regeneration progress, and routine mass-specific V̇O2 as a function of maturity, regeneration, and reproductive mode, were measured over a 22-day regeneration period. Testes growth was highest in sexually mature adults, ~1/2 that in 14-day post-amputation sexual adults, and not detectable in juveniles and hatchlings. Mass-specific routine V̇O2 in sexuals was highest in mature controls at ~23 µl O2/g/h, but only half that in juveniles, hatchlings, and 14 day post-amputation adults. Both intact and 14-day post-amputation asexuals had a mass-specific routine V̇O2 of ~10-12 µl O2/g/h. The sum of V̇O2 of all amputated sections was ~100% higher than pre-amputation levels in the first 6 days of regeneration in asexuals, but not sexuals. There was no significant difference in V̇O2 of head, middle, and tail sections during regeneration. Overall, the highest metabolic costs associated with regeneration occurred during the initial 1-6 days of regeneration in both strains, but regeneration costs for sexual structures were not reflected in major V̇O2 differences between sexual and asexual strains.

Stepwise endo-/epicardial catheter ablation for atrial fibrillation: The Mediterranea approach.

BACKGROUND: Outcomes of catheter ablation (CA) among patients with nonparoxysmal atrial fibrillation (AF) are largely disappointing. OBJECTIVE: We sought to evaluate the feasibility, effectiveness, and safety of a single-stage stepwise endo-/epicardial approach in patients with persistent/longstanding-persistent AF. METHODS: We enrolled 25 consecutive patients with symptomatic persistent (n = 4) or longstanding-persistent (n = 21) AF and at least one prior endocardial procedure, who underwent CA using an endo-/epicardial approach. Our anatomical stepwise protocol included multiple endocardial as well as epicardial (Bachmann's bundle [BB] and ligament of Marshall ablations) components, and entailed ablation of atrial tachycardias emerging during the procedure. The primary outcome was freedom from any AF/atrial tachycardia episode after a 3-month blanking period. The secondary outcome was patients' symptom status during follow-up. RESULTS: The stepwise endo-/epicardial approach allowed sinus rhythm restoration in 72% of patients, either directly (n = 6, 24%) or after AF organization into atrial tachycardia (n = 12, 48%). BB's ablation was commonly implicated in arrhythmia termination. After a median follow-up of 266 days (interquartile range, 96 days), survival free from AF/atrial tachycardia was 88%. Antiarrhythmic drugs could be discontinued in 22 patients (88%). As compared to baseline, more patients were asymptomatic at 9-month follow-up (0% vs. 56%, p = .02). Five patients (20%) developed mild medical complications, whereas one subject (4%) had severe kidney injury requiring dialysis. CONCLUSION: A single-stage endo-/epicardial CA resulted in favorable rhythm and symptom outcomes in a cohort of patients with symptomatic persistent/longstanding-persistent AF and one or more prior endocardial procedures. Epicardial ablation of BB was commonly implicated in procedural success.

Phytochemical Investigation of New Algerian Lichen Species: Physcia Mediterranea Nimis.

The present study provides new data concerning the chemical characterisation of Physcia mediterranea Nimis, a rare Mediterranean species belonging to the family Physciaceae. The phytochemical screening was carried out using GC-MS, HPLC-ESI-MS-MS, and NMR techniques. Hot extraction of n-hexane was carried out, followed by separation of the part insoluble in methanol: wax (WA-hex), from the part soluble in methanol (ME-hex). GC-MS analysis of the ME-hex part revealed the presence of methylbenzoic acids such as sparassol and atraric acid and a diterpene with a kaurene skeleton which has never been detected before in lichen species. Out of all the compounds identified by HPLC-ESI-MS-MS, sixteen compounds are common between WA-hex and ME-hex. Most are aliphatic fatty acids, phenolic compounds and depsides. The wax part is characterised by the presence of atranorin, a depside of high biological value. Proton 1H and carbon 13C NMR have confirmed its identification. Atranol, chloroatranol (depsides compound), Ffukinanolide (sesquiterpene lactones), leprolomin (diphenyl ether), muronic acid (triterpenes), and ursolic acid (triterpenes) have also been identified in ME-hex. The results suggested that Physcia mediterranea Nimis is a valuable source of bioactive compounds that could be useful for several applications as functional foods, cosmetics, and pharmaceuticals.

Papain-Based Dissociation of Schmidtea mediterranea Cells.

The need of highly viable cells dissociated from Schmidtea mediterranea is constantly growing. In this chapter, we describe a cell dissociation method based on papain (papaya peptidase I). This enzyme, often used to dissociate cells with complex morphology, is a cysteine protease with a broad specificity and increases both the yield and the viability of the dissociated cell suspension. The papain dissociation is preceded by a pretreatment for mucus removal, as this was shown to greatly improve the yield of cell dissociation, regardless of the method used. Papain-dissociated cells are suitable for a variety of downstream applications, like live immunostaining, flow cytometry, cell sorting, transcriptomics, and cell transplantation, also at the single-cell level.

Serial block-face scanning electron microscopy of Schmidtea mediterranea.

The flatworm planarian, Schmidtea mediterranea (Smed) is a master at regenerating and rebuilding whole animals from fragments. A full understanding of Smed's regenerative capabilities requires a high-resolution characterization of organs, tissues, and the adult stem cells necessary for regeneration in their native environment. Here, we describe a serial block face scanning electron microscopy (SBF-SEM) protocol, optimized for Smed specifically, for visualizing the ultrastructure of membranes and condensed chromosomes in this model organism.

Tools for photomotor response assay standardization in ecotoxicological studies: Example of exposure to gentamicin in the freshwater planaria Schmidtea mediterranea.

Photomotor response assay (PMR) is very useful in an ecotoxicological context because it allows evaluation of behavioral response to potential toxic compounds. However, a lack of procedure standardization makes results comparison difficult between labs and organisms. Here, we aimed to propose five different tools to standardize the PMR procedure so that it may be applied to all model species, regarding: (1) the minimum total sample size, (2) the acclimation period, (3) the number and duration of light and dark phases alternation, (4) the measured behavior, and (5) the statistical analysis. As an example of procedure application, we analyzed the effect of an exposure to the antibiotic gentamicin on the locomotion behavior during PMR in an invertebrate species: the asexual freshwater planaria Schmidtea mediterranea. We encourage future studies using PMR to follow these five tools to improve data analysis and results comparability.

Conditioned place avoidance in the planaria Schmidtea mediterranea: A pre-clinical invertebrate model of anxiety-related disorders.

The objective of the present study was to develop a model of avoidance learning and its extinction in planarians (Schmidtea mediterranea). Based on previous experiments showing conditioned place preference, we developed a procedure to investigate conditioned place avoidance (CPA) using shock as an unconditioned stimulus (US) and an automated tracking system to record the animals' behaviour. In Experiment 1, we assessed the unconditioned properties of different shock intensities by measuring post shock activity. In two subsequent experiments we investigated CPA using different designs, surfaces as conditioned stimuli (CSs; rough and smooth), and different US intensities (5 V and 10 V). In general, we observed the successful development of CPA. However, CPA was stronger with higher shock intensities, and we found that, in our preparation, a rough surface is best at entering into an association with the shock than a smooth surface. Finally, we also observed extinction of CPA. The evidence of CPA and its extinction in flatworms validates the planaria as a pre-clinical model for the study of avoidance learning, a hallmark of anxiety disorders.

Whole-Mount In Situ Hybridization in Large Sexual Schmidtea mediterranea.

Whole-mount in situ hybridization (WISH), colorimetric or fluorescent (FISH), allows for the visualization of endogenous RNA. For planarians, robust WISH protocols exist for small-sized animals (>5 mm) of the model species Schmidtea mediterranea and Dugesia japonica. However, the sexual strain of Schmidtea mediterranea studied for germline development and function reaches much larger body sizes in excess of 2 cm. The existing whole-mount WISH protocols are not optimal for such large specimens, owing to insufficient tissue permeabilization. Here, we describe a robust WISH protocol for 12-16 mm long sexually mature Schmidtea mediterranea individuals that could serve as a starting point for adapting WISH to other large planarian species.

mRNA Transfection of S. mediterranea for Luminescence Analysis.

Planarians have become a powerful model system for stem cell research and regeneration. While the tool kit for mechanistic investigations has been steadily expanding over the last decade, robust genetic tools for transgene expression are still lacking. We describe here methods for in vivo and in vitro mRNA transfection of the planarian species Schmidtea mediterranea. These methods utilize the commercially available TransIT-mRNA transfection reagent to efficiently deliver mRNA encoding a synthetic nanoluciferase reporter. Using a luminescent reporter overcomes the bright autofluorescent background of planarian tissues and allows quantitative measurements of protein expression levels. Collectively, our methods provide the means for heterologous reporter expression in planarian cells and the basis for future development of transgenic techniques.

Live Immunostaining and Flow Cytometry of Schmidtea Mediterranea Cells.

The use of flow cytometry and fluorescence-activated cell sorting to roughly separate subpopulations of cells in Schmidtea mediterranea is long established. In this chapter, we describe a method for the immunostaining-either single or double-of live planarian cells, using mouse monoclonal antibodies reactive against S. mediterranea plasma membrane antigens. This protocol allows to sort live cells according to their membrane signature, offering the possibility to further characterize the cell populations in S. mediterranea in a variety of downstream applications, like transcriptomics and cell transplantation, also at the single-cell level.

Comprehensive RNA-Seq Analysis Pipeline for Non-Model Organisms and Its Application in Schmidtea mediterranea.

RNA sequencing (RNA-seq) is a high-throughput technology that provides in-depth information on transcriptome. The advancement and dropping costs of RNA sequencing, accompanied by more available reference genomes for different species, make transcriptome analysis in non-model organisms possible. Current obstacles in analyzing RNA-seq data include a lack of functional annotation, which may complicate the process of linking genes to corresponding functions. Here, we provide a one-stop RNA-seq analysis pipeline, PipeOne-NM, for transcriptome functional annotation, non-coding RNA identification, and transcripts alternative splicing analysis of non-model organisms, intended for use with Illumina platform-based RNA-seq data. We performed PipeOne-NM on 237 Schmidtea mediterranea RNA-seq runs and assembled a transcriptome with 84,827 sequences from 49,320 genes, identifying 64,582 mRNA from 35,485 genes, 20,217 lncRNA from 17,084 genes, and 3481 circRNAs from 1103 genes. In addition, we performed a co-expression analysis of lncRNA and mRNA and identified that 1319 lncRNA co-express with at least one mRNA. Further analysis of samples from S. mediterranea sexual and asexual strains revealed the role of sexual reproduction in gene expression profiles. Samples from different parts of asexual S. mediterranea revealed that differential expression profiles of different body parts correlated with the function of conduction of nerve impulses. In conclusion, PipeOne-NM has the potential to provide comprehensive transcriptome information for non-model organisms on a single platform.

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection.

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.