How to obtain an integrated picture of the molecular networks involved in adaptation to microgravity in different biological systems?

Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.

Starvation resistance in planarians: multiple strategies to get a thrifty phenotype.

Starvation resistance is a life-saving mechanism for many organisms facing food availability fluctuation in the natural environment. Different strategies have been episodically identified for some model organisms, the first of which was the ability to suppress metabolic rate. Among the identified strategies, the ability of planarians to shrink their body under fasting conditions and revert the process after feeding (the growth-degrowth process) represents a fascinating mechanism to face long periods of fasting. The growth-degrowth process is strictly related to the capability of planarians to continuously maintain tissue homeostasis and body proportions even in challenging conditions, thanks to the presence of a population of pluripotent stem cells. Here, we take advantage of several previous studies describing the growth-degrowth process and of recent progress in the understanding of planarian homeostasis mechanisms, to identify tissue-selective transcriptional downregulation as a driving strategy for the development of a thrifty phenotype, and the p53 transcription factor as a player in adjusting tissue homeostasis in accordance with food availability.

A Network of MicroRNAs and mRNAs Involved in Melanosome Maturation and Trafficking Defines the Lower Response of Pigmentable Melanoma Cells to Targeted Therapy.

BACKGROUND: The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma cells. METHODS: Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos). RESULTS: Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as GPR143, OCA2, RAB27A, RAB32 and TYRP1 mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes-due to increased maturation and/or trafficking-confers resistance to vem. CONCLUSION: We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells.

Autophagy Activation Associates with Suppression of Prion Protein and Improved Mitochondrial Status in Glioblastoma Cells.

Cells from glioblastoma multiforme (GBM) feature up-regulation of the mechanistic Target of Rapamycin (mTOR), which brings deleterious effects on malignancy and disease course. At the cellular level, up-regulation of mTOR affects a number of downstream pathways and suppresses autophagy, which is relevant for the neurobiology of GBM. In fact, autophagy acts on several targets, such as protein clearance and mitochondrial status, which are key in promoting the malignancy GBM. A defective protein clearance extends to cellular prion protein (PrPc). Recent evidence indicates that PrPc promotes stemness and alters mitochondrial turnover. Therefore, the present study measures whether in GBM cells abnormal amount of PrPc and mitochondrial alterations are concomitant in baseline conditions and whether they are reverted by mTOR inhibition. Proteins related to mitochondrial turnover were concomitantly assessed. High amounts of PrPc and altered mitochondria were both mitigated dose-dependently by the mTOR inhibitor rapamycin, which produced a persistent activation of the autophagy flux and shifted proliferating cells from S to G1 cell cycle phase. Similarly, mTOR suppression produces a long-lasting increase of proteins promoting mitochondrial turnover, including Pink1/Parkin. These findings provide novel evidence about the role of autophagy in the neurobiology of GBM.

Autopsy Study of Testicles in COVID-19: Upregulation of Immune-Related Genes and Downregulation of Testis-Specific Genes.

CONTEXT: Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility. OBJECTIVE: Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes. METHODS: Three groups were compared: (a) uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); (b) subjects dying of COVID-19 (virus-negative in testes; n = 15); (c) subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, and immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method. RESULTS: SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups. CONCLUSION: In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.

Microtubule-associated protein 1B is implicated in stem cell commitment and nervous system regeneration in planarians.

Microtubule-associated 1B (MAP1B) proteins are expressed at the nervous system level where they control cytoskeleton activity and regulate neurotransmitter release. Here, we report about the identification of a planarian MAP1B factor (DjMap1B) that is enriched in cephalic ganglia and longitudinal nerve cords but not in neoblasts, the plentiful population of adult stem cells present in planarians, thanks to which these animals can continuously cell turnover and regenerate any lost body parts. DjMap1B knockdown induces morphological anomalies in the nervous system and affects neoblast commitment. Our data put forward a correlation between a MAP1B factor and stem cells and suggest a function of the nervous system in non-cell autonomous control of planarian stem cells.

Cell quiescence in planarian stem cells, interplay between p53 and nutritional stimuli.

Cell quiescence appeared early in evolution as an adaptive response to adverse conditions (i.e. nutrient depletion). In metazoans, quiescence has been involved in additional processes like tissue homeostasis, which is made possible by the presence of adult stem cells (ASCs). Cell cycle control machinery is a common hub for quiescence entrance, and evidence indicates a role for p53 in establishing the quiescent state of undamaged cells. Mechanisms responsible for waking up quiescent cells remain elusive, and nutritional stimulus, as a legacy of its original role, still appears to be a player in quiescence exit. Planarians, rich in ASCs, represent a suitable system in which we characterized a quiescent population of ASCs, the dorsal midline cord (DMC) cells, exhibiting unique transcriptional features and maintained quiescent by p53 and awakened upon feeding. The function of DMC cells is puzzling and we speculate that DMC cells, despite retaining ancient properties, might represent a functional drift in which quiescence has been recruited to provide evolutionary advantages.

Suppression of Pituitary Hormone Genes in Subjects Who Died From COVID-19 Independently of Virus Detection in the Gland.

CONTEXT: Involvement of the pituitary gland in SARS-CoV-2 infection has been clinically suggested by pituitary hormone deficiency in severe COVID-19 cases, by altered serum adrenocorticotropic hormone (ACTH) levels in hospitalized patients, and by cases of pituitary apoplexy. However, the direct viral infection of the gland has not been investigated. OBJECTIVE: To evaluate whether the SARS-CoV-2 genome and antigens could be present in pituitary glands of lethal cases of COVID-19, and to assess possible changes in the expression of immune-related and pituitary-specific genes. METHODS: SARS-CoV-2 genome and antigens were searched in the pituitary gland of 23 patients who died from COVID-19 and, as controls, in 12 subjects who died from trauma or sudden cardiac death. Real-time reverse transcription polymerase chain reaction (PCR), in situ hybridization, immunohistochemistry, and transmission electron microscopy were utilized. Levels of mRNA transcripts of immune-related and pituitary-specific genes were measured by the nCounter assay. RESULTS: The SARS-CoV-2 genome and antigens were detected in 14/23 (61%) pituitary glands of the COVID-19 group, not in controls. In SARS-CoV-2-positive pituitaries, the viral genome was consistently detected by PCR in the adeno- and the neurohypophysis. Immunohistochemistry, in situ hybridization, and transmission electron microscopy confirmed the presence of SARS-CoV-2 in the pituitary. Activation of type I interferon signaling and enhanced levels of neutrophil and cytotoxic cell scores were found in virus-positive glands. mRNA transcripts of pituitary hormones and pituitary developmental/regulatory genes were suppressed in all COVID-19 cases irrespective of virus positivity. CONCLUSION: Our study supports the tropism of SARS-CoV-2 for human pituitary and encourages exploration of pituitary dysfunction after COVID-19.

Sub-Lethal 5-Fluorouracil Dose Challenges Planarian Stem Cells Promoting Transcriptional Profile Changes in the Pluripotent Sigma-Class Neoblasts.

Under physiological conditions, the complex planarian neoblast system is a composite of hierarchically organized stem cell sub-populations with sigma-class neoblasts, including clonogenic neoblasts, endowed with larger self-renewal and differentiation capabilities, thus generating all the other sub-populations and dominating the regenerative process. This complex system responds to differentiated tissue demands, ensuring a continuous cell turnover in a way to replace aged specialized cells and maintain tissue functionality. Potency of the neoblast system can be appreciated under challenging conditions in which these stem cells are massively depleted and the few remaining repopulate the entire body, ensuring animal resilience. These challenging conditions offer the possibility to deepen the relationships among different neoblast sub-populations, allowing to expose uncanonical properties that are negligible under physiological conditions. In this paper, we employ short, sub-lethal 5-fluorouracil treatment to specifically affect proliferating cells passing through the S phase and demonstrate that S-phase slowdown triggers a shift in the transcriptional profile of sigma neoblasts, which reduces the expression of their hallmark sox-P1. Later, some cells reactivate sox-P1 expression, suggesting that some neoblasts in the earlier steps of commitment could modulate their expression profile, reacquiring a wider differentiative potential.

Stem Cells and Innate Immunity in Aquatic Invertebrates: Bridging Two Seemingly Disparate Disciplines for New Discoveries in Biology.

The scopes related to the interplay between stem cells and the immune system are broad and range from the basic understanding of organism's physiology and ecology to translational studies, further contributing to (eco)toxicology, biotechnology, and medicine as well as regulatory and ethical aspects. Stem cells originate immune cells through hematopoiesis, and the interplay between the two cell types is required in processes like regeneration. In addition, stem and immune cell anomalies directly affect the organism's functions, its ability to cope with environmental changes and, indirectly, its role in ecosystem services. However, stem cells and immune cells continue to be considered parts of two branches of biological research with few interconnections between them. This review aims to bridge these two seemingly disparate disciplines towards much more integrative and transformative approaches with examples deriving mainly from aquatic invertebrates. We discuss the current understanding of cross-disciplinary collaborative and emerging issues, raising novel hypotheses and comments. We also discuss the problems and perspectives of the two disciplines and how to integrate their conceptual frameworks to address basic equations in biology in a new, innovative way.

Morphology, clearing efficacy, and mTOR dependency of the organelle autophagoproteasome.

The interplay between autophagy (ATG) and ubiquitin proteasome (UP) cell-clearing systems was recently evidenced at biochemical and morphological levels, where subunits belonging to both pathways co-localize within a novel organelle named autophagoproteasome (APP). We previously documented that APP occurs at baseline conditions, while it is hindered by neurotoxicant administration. This is bound to the activity of the mechanistic target of rapamycin (mTOR), since APP is stimulated by mTOR inhibition, which in turn, is correlated with cell protection. In this brief report, we provide novel, morphological and biochemical evidence on APP, suggesting the presence of active UP subunits within ATG vacuoles. Although a stream of interpretation considers such a merging as a catabolic pathway to clear inactive UP subunits, our data further indicate that UP-ATG merging may rather provide an empowered catalytic organelle.

Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded protein response.

Planarians are able to stand long periods of starvation by maintaining adult stem cell pools and regenerative capacity. The molecular pathways that are needed for the maintenance of regeneration during starvation are not known. Here, we show that down-regulation of chaperonin TRiC/CCT subunits abrogates the regeneration capacity of planarians during starvation, but TRiC/CCT subunits are dispensable for regeneration in fed planarians. Under starvation, they are required to maintain mitotic fidelity and for blastema formation. We show that TRiC subunits modulate the unfolded protein response (UPR) and are required to maintain ATP levels in starved planarians. Regenerative defects in starved CCT-depleted planarians can be rescued by either chemical induction of mild endoplasmic reticulum stress, which leads to induction of the UPR, or by the supplementation of fatty acids. Together, these results indicate that CCT-dependent UPR induction promotes regeneration of planarians under food restriction.

Artificially altered gravity elicits cell homeostasis imbalance in planarian worms, and cerium oxide nanoparticles counteract this effect.

Gravity alterations elicit complex and mostly detrimental effects on biological systems. Among these, a prominent role is occupied by oxidative stress, with consequences for tissue homeostasis and development. Studies in altered gravity are relevant for both Earth and space biomedicine, but their implementation using whole organisms is often troublesome. Here we utilize planarians, simple worm model for stem cell and regeneration biology, to characterize the pathogenic mechanisms brought by artificial gravity alterations. In particular, we provide a comprehensive evaluation of molecular responses in intact and regenerating specimens, and demonstrate a protective action from the space-apt for nanotechnological antioxidant cerium oxide nanoparticles.

Glutathione-S-transferase omega 1 and nurse cell formation during experimental Trichinella infection.

The glutathione-S-transferases omega (GSTO) are multifunctional enzymes involved in cellular defense. During the nurse cell (NC) formation in Trichinella spiralis infection, the structural and regulatory genes of the skeletal muscle cell are downregulated and a new phenotype is acquired which advances parasite growth and survival. Previous studies showed that the GSTO1 is overexpressed in the NC during T. spiralis infection. To clarify the role of GSTO1 during NC formation, we evaluated the production of this enzyme by immunohistochemistry (IHC) in the diaphragms of mice experimentally infected with T. spiralis at 15, 28 and 60 days post infection (dpi); phosphorylation of Akt (p-Akt) and JNK1 (p-JNK1) were also evaluated. Furthermore, we evaluated the in vitro effects of T. spiralis excretory/secretory (ES) products from muscle larvae on specific functions (viability, proliferative response, apoptosis) in two cell lines (HeLa and U937), as well as its ability to induce GSTO1, p-AkT, p-ERK1/2 and p-JNK1. Results showed that GSTO1 was elevated in NC present in the diaphragms of T. spiralis experimentally infected mice at 15 dpi and progressively increased up to 60 dpi. The activation pattern of Akt in NC was similar to that of GSTO1, whereas JNK1 was never phosphorylated. ES induced a dose-dependent proliferative response in U937 cells, at 24 h and 48 h of treatment, but not in HeLa cells. However, after 72 h following treatment, significant cell death was observed in both cell lines at all doses. The apoptotic index (a.i.) was significantly higher than in untreated cells in both cell lines but only at the highest concentration of ES tested. Furthermore, Western Blots revealed that cells treated with ES for 24, 48 and 72 h, exhibited time-dependent overexpression of GSTO1, whereas p-Akt appeared only after 24 h of treatment. The p-ERK-1/2 peaked at 24 h then declined at 48 h and 72 h after treatment; however, it remained significantly higher than in untreated cells. No changes were observed in p-JNK1 at 24 and 48 h after treatment but a sharp increase in p-JNK1 was observed at 72 h. Also in HeLa cells, ES induced a small but significant increase in GSTO1 expression after 24 and 48 h of treatment where p-JNK1 was present only after 72 h of treatment. In conclusion, T. spiralis ES can reproduce in vitro the modifications observed inside the NC during experimental infection in mice.

Stem cell and tissue regeneration analysis in low-dose irradiated planarians treated with cerium oxide nanoparticles.

Owing to the self-renewing reactive oxygen species scavenger capability of cerium oxide nanoparticles (nanoceria), we tested in vivo radioprotective effects on stem cells and tissue regeneration using low-dose irradiated planarians as model system. We treated planarians with nanoceria or gum Arabic, as control, and we analyzed the expression of stem cell molecular markers and tissue regeneration capability, as well as cell death and DNA damage in non-irradiated and in low-dose irradiated animals. Our findings show that nanoceria increase the number of stem cells and tissue regenerative capability, and reduce cell death and DNA damage after low-dose irradiation, suggesting a protective role on stem cells.

5-Fluorouracil-treated planarians, a versatile model system for studying stem cell heterogeneity and tissue aging.

BACKGROUND INFORMATION: Planarians are a sound, well-established model system for molecular studies in the field of stem cells, cell differentiation, developmental biology and translational research. Treated stem cell-less planarians produced by X-ray treatment are commonly used to study stem cell transcriptional profile and their role in planarian biological processes. X-ray induces oxidative and DNA damage to differentiated cells, requires expensive radiation machines that are not available in most of the research centres and demand rigorous risk management and dedicated staff. RESULTS: We tested the use of the well-known antimetabolite genotoxic drug 5-fluorouracil which mainly affects proliferating cells in way to demonstrate its use in replacing X-ray treatment. We succeeded in demonstrating ability of high doses of 5-fluorouracil to deplete Dugesia japonica stem cells and in identifying a 5-fluorouracil transiently resistant population of lineage committed stem cells. CONCLUSIONS AND SIGNIFICANCE: Our results encourage the use of 5-fluorouracil-treated planarians as a model system for studying mechanisms of resistance to genotoxicants, planarian stem cell heterogeneity and molecular cascades of tissue aging.

"Candidatus Trichorickettsia mobilis", a Rickettsiales bacterium, can be transiently transferred from the unicellular eukaryote Paramecium to the planarian Dugesia japonica.

Most of the microorganisms responsible for vector-borne diseases (VBD) have hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms phylogenetically related to agents of VBD were found in a variety of aquatic eukaryotic hosts; in particular, numerous new bacterial species related to the genus Rickettsia (Alphaproteobacteria, Rickettsiales) were discovered in protist ciliates and other unicellular eukaryotes. Although their pathogenicity for humans and terrestrial animals is not known, several indirect indications exist that these bacteria might act as etiological agents of possible VBD of aquatic organisms, with protists as vectors. In the present study, a novel strain of the Rickettsia-Like Organism (RLO) endosymbiont "Candidatus (Ca.) Trichorickettsia mobilis" was identified in the macronucleus of the ciliate Paramecium multimicronucleatum. We performed transfection experiments of this RLO to planarians (Dugesia japonica) per os. Indeed, the latter is a widely used model system for studying bacteria pathogenic to humans and other Metazoa. In transfection experiments, homogenized paramecia were added to food of antibiotic-treated planarians. Treated and non-treated (i.e. control) planarians were investigated at day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be transiently transferred from ciliates to metazoans, being detected up to day 7 in treated planarians' enterocytes. Our findings might offer insights into the potential role of ciliates or other protists as putative vectors for diseases caused by Rickettsiales or other RLOs and occurring in fish farms or in the wild.

Dynamics of interaction and effects of microplastics on planarian tissue regeneration and cellular homeostasis.

Increasing microplastics pollution of marine and terrestrial water is a concerning issue for ecosystems and human health. Nevertheless, the interaction of microplastics with freshwater biota is still a poorly explored field. In order to achieve information concerning the uptake, distribution and effect of microplastics in planarians, Dugesia japonica specimens have been fed with mixtures of food and differently shaped and sized plastic particles. Feeding activity and food intake were non-altered by the presence of high concentrations of different types of plastic particles. However, the persistence of microplastic within the planarian body was a function of size/shape, being small spheres (<10 µm in diameter) and short fibers (14 µm large and 5/6 µm length) more persisting than larger spheres and longer fibers which were eliminated almost entirely by ejection in a few hours. Transmission electron microscopy analysis demonstrated that at least part of microplastics was phagocytized by the enterocytes. Chronic exposure to small plastic did not alter the regenerative ability but caused a significant reduction of the gut epithelium thickness and lipid content of enterocytes, together with the induction of apoptotic cell death, modulation of Djgata 4/5/6 expression and reduced growth rate. The ability of microplastic to perturb planarian homeostasis is concerning being them extremely resilient against mechanical and chemical insults and suggests possible harmful effects upon other more susceptible species in freshwater ecosystems.

Antitumoral effects of attenuated Listeria monocytogenes in a genetically engineered mouse model of melanoma.

Attenuated Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models.