Sponge Cells Research Articles

Novel use of a - 20°C cryoprotectant yields high viability and improved aggregation of marine sponge cells.

Novel use of a - 20°C cryoprotectant yields high viability and improved aggregation of marine sponge cells.

Transient Interphase Microtubules Appear in Differentiating Sponge Cells.

Microtubules are an indispensable component of all eukaryotic cells due to their role in mitotic spindle formation, yet their organization and number can vary greatly in the interphase. The last common ancestor of all eukaryotes already had microtubules and microtubule motor proteins moving along them. Sponges are traditionally regarded as the oldest animal phylum. Their body does not have a clear differentiation into tissues, but it contains several distinguishable cell types. The choanocytes stand out among them and are responsible for creating a flow of water with their flagella and increasing the filtering and feeding efficiency of the sponge. Choanocyte flagella contain microtubules, but thus far, observing a developed system of cytoplasmic microtubules in non-flagellated interphase sponge cells has been mostly unsuccessful. In this work, we combine transcriptomic analysis, immunofluorescence, and electron microscopy with time-lapse recording to demonstrate that microtubules appear in the cytoplasm of sponge cells only when transdifferentiation processes are activated. We conclude that dynamic cytoplasmic microtubules in the cells of sponges are not a persistent but rather a transient structure, associated with cellular plasticity.

Algorithm Training and Testing for a Nonendoscopic Barrett’s Esophagus Detection Test in Prospective Multicenter Cohorts

Endoscopic Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) detection is invasive and expensive. Nonendoscopic BE/EAC detection tools are guideline-endorsed alternatives. We previously described a 5-methylated DNA marker (MDM) panel assayed on encapsulated sponge cell collection device (CCD) specimens. We aimed to train a new algorithm using a 3-MDM panel and test its performance in an independent cohort. Algorithm training and test samples were from 2 prospective multicenter cohorts. All BE cases had esophageal intestinal metaplasia (with or without dysplasia/EAC); control subjects had no endoscopic evidence of BE. The CCD procedure was followed by endoscopy. From CCD cell lysates, DNA was extracted, bisulfite treated, and MDMs were blindly assayed. The algorithm was set and locked using cross-validated logistic regression (training set) and its performance was assessed in an independent test set. Training (N= 352) and test (N= 125) set clinical characteristics were comparable. The final panel included 3 MDMs (NDRG4, VAV3, ZNF682). Overall sensitivity was 82% (95% CI, 68%-94%) at 90% (79%-98%) specificity and 88% (78%-94%) sensitivity at 84% (70%-93%) specificity in training and test sets, respectively. Sensitivity was 90% and 68% for all long- and short-segment BE, respectively. Sensitivity for BE with high-grade dysplasia and EAC was 100% in training and test sets. Overall sensitivity for nondysplastic BE was 82%. Areas under the receiver operating characteristic curves for BE detection were 0.92 and 0.94 in the training and test sets, respectively. A locked 3-MDM panel algorithm for BE/EAC detection using a nonendoscopic CCD demonstrated excellent sensitivity for high-risk BE cases in independent validation samples. (Clinical trials.gov: NCT02560623, NCT03060642.).

Cell cycle dynamics of food-entrapping cells of sponges: an experimental approach.

Sponges (phylum Porifera) exhibit surprisingly complex tissue dynamics, maintaining constant cell turnover and migration, rearranging internal structures, and regenerating after severe injuries. Such tissue plasticity relies on the activity of proliferating cells represented primarily by the food-entrapping cells, choanocytes. Although there are plenty of studies regarding the dynamics of regeneration and tissue rearrangement in sponges, cell cycle kinetics of choanocytes in intact tissues remains a controversial issue. This study is devoted to the comparative description of choanocyte cell cycle dynamics in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia corallorrhiza (class Calcarea). We have identified populations of proliferating cells and synchronized them in the S-phase to estimate the growth fraction of cycling cells. Using continuous exposure to labeled thymidine analog ethynyl deoxyuridine (EdU), we calculated choanocyte cell cycle duration and the length of the S phase. We also applied double labeling with EdU and antibodies against phosphorylated histone 3 to estimate the lengths of choanocyte M and G2 phases. Finally, flow-cytometry-based quantitative analysis of DNA content provided us with the lengths of G2 and G1 phases. We found that tissue growth and renewal in the studied sponges are generally maintained by a relatively large population of slowly cycling choanocytes with a total cell cycle duration of 40 h in H. dujardinii and 60 h in L. corallorrhiza. In both species, choanocytes are characterized by an extremely short M-phase and heterogeneity in the duration of the G2 phase.

Investigation of Tychonema sp. tropism to the sponge body in the experiment of co-cultivation of cyanobacteria with primmorphs

Mass death of endemic sponges accompanied by the growth of cyanobacterial biofilms on their bodies has been registered in recent years in Lake Baikal. These biofilms are formed by the cyanobacterium Tychonema sp., which grows on various types of substrates in the littoral zone and is often found on sponges. The aim of the study was to conduct an experiment on co-cultivation of Tychonema sp. with Baikalospongia sp. primomorphs to test the hypothesis about the tropism of Baikal cyanobacterium to the body of sponges. In the experiment, gradual fouling of the primmorphs with cyanobacterium filaments, destruction of their structure and death of sponge cells were registered. In the control without addition of cyanobacteria, the primmorphs remained alive during the whole time of the experiment.

Characteristics of δ-Aminolevulenic Acid Dehydratase of the Cold-Water Sponge <i>Halisarca dujardinii</i>

Delta-aminolevulinic acid dehydratase (ALAD) is a key enzyme in the cytoplasmic pathway of heme biosynthesis. Here, a primary structure of the ALAD gene of the marine cold-water sponge Halisarca dujardinii, a multimeric structure of the ALAD/hemB protein, and the ALAD gene expression during the sponge annual reproductive cycle were analyzed. On the base of results obtained one can suppose that the sponge ALAD gene expression is regulated by the transcription factor GATA-1 and DNA methylation. Re-aggregation of the sponge cells was accompanied by a decrease in ALAD expression and a change in the cellular content of the active ALAD/hemB form. Further study of heme biosynthesis and the role of ALAD/hemB in morphogenesis of basal animals may provide new opportunities for correcting pathologies in higher animals.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Candidatus Nemesobacterales is a sponge-specific clade of the candidate phylum Desulfobacterota adapted to a symbiotic lifestyle.

Members of the candidate phylum Dadabacteria, recently reassigned to the phylum Candidatus Desulfobacterota, are cosmopolitan in the marine environment found both free-living and associated with hosts that are mainly marine sponges. Yet, these microorganisms are poorly characterized, with no cultured representatives and an ambiguous phylogenetic position in the tree of life. Here, we performed genome-centric metagenomics to elucidate their phylogenomic placement and predict the metabolism of the sponge-associated members of this lineage. Rank-based phylogenomics revealed several new species and a novel family (Candidatus Spongomicrobiaceae) within a sponge-specific order, named here Candidatus Nemesobacterales. Metabolic reconstruction suggests that Ca. Nemesobacterales are aerobic heterotrophs, capable of synthesizing most amino acids, vitamins and cofactors and degrading complex carbohydrates. We also report functional divergence between sponge- and seawater-associated metagenome-assembled genomes. Niche-specific adaptations to the sponge holobiont were evident from significantly enriched genes involved in defense mechanisms against foreign DNA and environmental stressors, host-symbiont interactions and secondary metabolite production. Fluorescence in situ hybridization gave a first glimpse of the morphology and lifestyle of a member of Ca. Desulfobacterota. Candidatus Nemesobacterales spp. were found both inside sponge cells centred around sponge nuclei and in the mesohyl of the sponge Geodia barretti. This study sheds light on the enigmatic group Ca. Nemesobacterales and their functional characteristics that reflect a symbiotic lifestyle.

A novel in-vivo phagocytosis assay to gain cellular insights on sponge-microbe interactions

IntroductionSponges harbor diverse, specific, and stable microbial communities, but at the same time, they efficiently feed on microbes from the surrounding water column. This filter-feeding lifestyle poses the need to distinguish between three categories of bacteria: food to digest, symbionts to incorporate, and pathogens to eliminate. How sponges discriminate between these categories is still largely unknown. Phagocytosis is conceivable as the cellular mechanism taking part in such discrimination, but experimental evidence is missing. We developed a quantitative in-vivo phagocytosis assay using an emerging experimental model, the sponge Halichondria panicea.MethodsWe incubated whole sponge individuals with different particles, recovered the sponge (host) cells, and tracked the incorporation of these particles into the sponge cells. Fluorescence-activated cell sorting (FACS) and fluorescent microscopy were used to quantify and verify phagocytic activity, defined here as the population of sponge cells with incorporated particles. Sponges were incubated with a green microalgae to test if particle concentration in the seawater affects the percentage of phagocytic activity, and to determine the timing where the maximum of phagocytic cells are captured in a pulse-chase experiment. Lastly, we investigated the application of our phagocytic assay with other particle types (i.e., fluorescently-labelled bacteria and fluorescent beads).Results and discussionThe percentage of sponge cells that had incorporated algae, bacteria, and beads ranged between 5 to 24%. These phagocytic sponge cells exhibited different morphologies and sizes depending on the type of particle presented to the sponge. Particle incorporation into sponge cells was positively related to algal concentration in the seawater, suggesting that sponge cells adjust their phagocytic activity depending on the number of particles they encounter. Our results further revealed that sponge phagocytosis initiates within minutes after exposure to the particles. Fluorescent and TEM microscopy rectified algal internalization and potential digestion in sponge cells. To our knowledge, this is the first quantitative in-vivo phagocytosis assay established in sponges that could be used to further explore phagocytosis as a cellular mechanism for sponges to differentiate between different microorganisms.

Algal symbionts of the freshwater sponge Ephydatia muelleri

The freshwater sponge, Ephydatia muelleri, is an emerging model system for studying animal:microbe symbioses. Intracellular green microalgae are one of the more common symbionts that live in a facultative mutualism with E. muelleri. While these symbioses have long been known, the identity of the algal symbionts in E. muelleri cells has not been studied in detail. Here, we isolate and characterize endosymbiotic algae from E. muelleri collected from different geographic locations. We find that the algae can be transmitted through asexually produced gemmules and importantly that they can form symbioses with different, differentiated sponge cell types in the adult sponge. Our findings indicate that at least two algal lineages form endosymbioses with E. muelleri. One of the lineages includes species commonly found in samples from two locations in Canada and one in the United States (clade 1: closely related to Auxenochlorella pyrenoidosa). The other clade includes algae found in sponges from one site in Maine, USA, and Lewiniosphaera symbiontica, which is a strain isolated in 1956 from the freshwater sponge Spongilla. We compared microbiomes found in cultures of microalgae as well as the original sponge hosts, and found that very similar bacterial microbiomes associate with both clades (91 orders of Bacteria are shared among the samples we compared). The microbiomes found in the cultures resemble, with a high degree of overlap, the microbiome associated with the sponge host.

Cross-phyla protein annotation by structural prediction and alignment

BackgroundProtein annotation is a major goal in molecular biology, yet experimentally determined knowledge is typically limited to a few model organisms. In non-model species, the sequence-based prediction of gene orthology can be used to infer protein identity; however, this approach loses predictive power at longer evolutionary distances. Here we propose a workflow for protein annotation using structural similarity, exploiting the fact that similar protein structures often reflect homology and are more conserved than protein sequences.ResultsWe propose a workflow of openly available tools for the functional annotation of proteins via structural similarity (MorF: MorphologFinder) and use it to annotate the complete proteome of a sponge. Sponges are highly relevant for inferring the early history of animals, yet their proteomes remain sparsely annotated. MorF accurately predicts the functions of proteins with known homology in >90%\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${>}90\\%$$\\end{document} cases and annotates an additional 50%\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$50\\%$$\\end{document} of the proteome beyond standard sequence-based methods. We uncover new functions for sponge cell types, including extensive FGF, TGF, and Ephrin signaling in sponge epithelia, and redox metabolism and control in myopeptidocytes. Notably, we also annotate genes specific to the enigmatic sponge mesocytes, proposing they function to digest cell walls.ConclusionsOur work demonstrates that structural similarity is a powerful approach that complements and extends sequence similarity searches to identify homologous proteins over long evolutionary distances. We anticipate this will be a powerful approach that boosts discovery in numerous -omics datasets, especially for non-model organisms.

Calcareous sponges can synthesize their skeleton under short-term ocean acidification

Calcifying organisms are considered as threatened by ocean acidification, because of their calcium carbonate skeleton. This study investigated if a calcareous sponge could synthesize its skeleton (i.e. spicules) under ocean-acidification conditions. Sponge cell aggregates that have the potential to develop into a functional sponge, called primmorphs, were submitted to a 5-day experiment, with two treatments: control (pH 8.1) and acidified conditions (pH 7.6). Primmorphs of the calcareous sponge Paraleucilla magna were able to synthesize a skeleton, even under low pH, and to develop into functional sponges. The spicules had the same shape in both conditions, although the spicules synthesized in low pH were slightly thinner than those in the control. These results suggest that P. magna may be able to survive near-future ocean-acidification conditions.

First continuous marine sponge cell line established

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Bacterial aerobic methane cycling by the marine sponge-associated microbiome

BackgroundMethanotrophy by the sponge-hosted microbiome has been mainly reported in the ecological context of deep-sea hydrocarbon seep niches where methane is either produced geothermically or via anaerobic methanogenic archaea inhabiting the sulfate-depleted sediments. However, methane-oxidizing bacteria from the candidate phylum Binatota have recently been described and shown to be present in oxic shallow-water marine sponges, where sources of methane remain undescribed.ResultsHere, using an integrative -omics approach, we provide evidence for sponge-hosted bacterial methane synthesis occurring in fully oxygenated shallow-water habitats. Specifically, we suggest methane generation occurs via at least two independent pathways involving methylamine and methylphosphonate transformations that, concomitantly to aerobic methane production, generate bioavailable nitrogen and phosphate, respectively. Methylphosphonate may be sourced from seawater continuously filtered by the sponge host. Methylamines may also be externally sourced or, alternatively, generated by a multi-step metabolic process where carnitine, derived from sponge cell debris, is transformed to methylamine by different sponge-hosted microbial lineages. Finally, methanotrophs specialized in pigment production, affiliated to the phylum Binatota, may provide a photoprotective function, closing a previously undescribed C1-metabolic loop that involves both the sponge host and specific members of the associated microbial community.ConclusionGiven the global distribution of this ancient animal lineage and their remarkable water filtration activity, sponge-hosted methane cycling may affect methane supersaturation in oxic coastal environments. Depending on the net balance between methane production and consumption, sponges may serve as marine sources or sinks of this potent greenhouse gas.3rpQ8ar2d6vfELht75ymrgVideo

Transfection of Sponge Cells and Intracellular Localization of Cancer-Related MYC, RRAS2, and DRG1 Proteins

The determination of the protein's intracellular localization is essential for understanding its biological function. Protein localization studies are mainly performed on primary and secondary vertebrate cell lines for which most protocols have been optimized. In spite of experimental difficulties, studies on invertebrate cells, including basal Metazoa, have greatly advanced. In recent years, the interest in studying human diseases from an evolutionary perspective has significantly increased. Sponges, placed at the base of the animal tree, are simple animals without true tissues and organs but with a complex genome containing many genes whose human homologs have been implicated in human diseases, including cancer. Therefore, sponges are an innovative model for elucidating the fundamental role of the proteins involved in cancer. In this study, we overexpressed human cancer-related proteins and their sponge homologs in human cancer cells, human fibroblasts, and sponge cells. We demonstrated that human and sponge MYC proteins localize in the nucleus, the RRAS2 in the plasma membrane, the membranes of the endolysosomal vesicles, and the DRG1 in the cell's cytosol. Despite the very low transfection efficiency of sponge cells, we observed an identical localization of human proteins and their sponge homologs, indicating their similar cellular functions.

Characteristics of δ-Aminolevulinic Acid Dehydratase of the Cold-Water Sponge Halisarca dujardinii

δ-Aminolevulinic acid dehydratase (ALAD) is a key enzyme of the cytoplasmic heme biosynthesis pathway. The primary structure of the ALAD gene, the multimeric structure of the ALAD/hemB protein, and ALAD expression during the annual reproductive cycle were studied in the cold-water marine sponge Halisarca dujardinii. The results implicated the GATA-1 transcription factor and DNA methylation in regulating ALAD expression. Re-aggregation of sponge cells was accompanied by a decrease in ALAD expression and a change in the cell content of an active ALAD/hemB form. Further study of heme biosynthesis and the role of ALAD/hemB in morphogenesis of basal animals may provide new opportunities for treating pathologies in higher animals.

Toxic Effect of Anionic Surfactants on Freshwater Sponge Lubomirskia baikalensis and Its Endosymbiotic Microalgae Chlorella sp.

A number of reports on sponge diseases, including from Lake Baikal, have increased dramatically all over the world in recent years. Herewith, there are various hypotheses for sponge mortality. Lubomirskia baikalensis (phylum Porifera, order Spongillida) is a unique endemic freshwater sponge of Lake Baikal that contains a complex community of eukaryotic and prokaryotic endosymbiotic microorganisms. In this work, we present the first results for the effect of anionic surfactants viz. linear alkylbenzene sulphonates (LAS) at low 10 and 20 µg L−1 concentrations on Baikal sponge species and their symbiotic community as an experimental model. A new toxicity test protocol under conditions close to natural is proposed. It uses the sponge amoebocytes called as SA1-cells, which contain eukaryotic green microalgae Chlorella sp. These SA1-cells are shown to be representative indicator in assessing the impact of anionic surfactants. The acute toxic effect resulted in 97–100% sponge cell death in less than 48 h, as well as 100% symbiotic microalgae Chlorella sp. death over 72 h was noted under LAS solution (20 μg L−1) exposure during in vivo experiments. This includes the cell membrane fatty acid changes, change in the cell sizes, cell swelling, and lysis. Long term exposure to LAS solution (10 μg L−1) reflected in cellular stress (oxidative stress) and accompanied by malondialdehyde formation (0.16–2.0 μg g−1 of dry weight) during 14-day exposure was noted. Oxidative stress and mortality of L. baikalensis are associated with their low antioxidant activity. Trolox-equivalent antioxidant capacity (TEAC) found in the range from 0.00031 to 0.00077 Trolox equivalents for these freshwater sponges.

A 3D Bioengineered Neural Tissue Model Generated from Human iPSC-Derived Neural Precursor Cells.

Available models to study neuropathological diseases include cell cultures and animal models. Brain pathologies, however, are often poorly recapitulated in animal models. 2D cell culture systems are well established and have beenused since the early 1900s to grow cells on flat dishes. However, conventional 2D neural culture systems, which lack key features of the brain's 3D microenvironment, often inaccurately represent the diversity and maturation of multiple cell types and their interaction under physiological and pathological conditions.To improve CNS modeling, we have designed a 3D bioengineered neural tissue model generated from human iPSC-derived neural precursor cells (NPCs). This NPC-derived biomaterial scaffold, composed of silk fibroin with an intercalated hydrogel, matches the mechanical properties of native brain tissue and supports the long-term differentiation of neural cells in a donut-shaped sponge within an optically clear central window. This chapter describes integrating iPSC-derived NPCs in these silk-collagen scaffolds and differentiating them into neural cells over time.

Economical alternatives for the production of fungal β-1,3-glucanase using easily obtainable industrial substrates

The β-1,3-glucanases synthesized by filamentous fungi have wide applicability in the food, chemical, and pharmaceutical industries. However, its obtainment can be costly, especially due to substrates used to induce its synthesis. Therefore, the objective of this work was to produce β-1,3-glucanase by T. harzianum Rifai using free and immobilized cells in synthetic and plant sponges, using different inducing substrates that could provide better cost-effectiveness for the industrial production of the enzyme. The Petri dish zymogram technique proved to be efficient for screening substrates inducing β -1,3-glucanases against species of filamentous fungi. It was possible to perform the immobilization of T. harzianum in a synthetic sponge allowing the realization of repetitive batches for enzymatic production. All tested substrates resulted in the synthesis of β-1,3-glucanase, including succinoglycan, proposed innovatively in this study. Fungal biomass resulted in the best inducing substrate under conditions of free and immobilized cells, with a production of β-1,3-glucanases of 0.73 U and 0.80 U of β -1,3-glucanases. The substrates corn starch and cassava showed promise in the production of β-1,3-glucanase and maintained production until the fourth batch was evaluated, with values of 0.51 U and 0.46 U of β-1.3-glucanases, respectively. The results obtained in this study showed that the zymogram is a practical method for screening substrates induced by the fungus T. harzianum. Corn starch and cassava are accessible and low-cost sources for β-1,3-glucanase synthesis in repetitive batches, including the use of immobilized and free cells.

Rheology of marine sponges reveals anisotropic mechanics and tuned dynamics.

Sponges are animals that inhabit many aquatic environments while filtering small particles and ejecting metabolic wastes. They are composed of cells in a bulk extracellular matrix, often with an embedded scaffolding of stiff, siliceous spicules. We hypothesize that the mechanical response of this heterogeneous tissue to hydrodynamic flow influences cell proliferation in a manner that generates the body of a sponge. Towards a more complete picture of the emergence of sponge morphology, we dissected a set of species and subjected discs of living tissue to physiological shear and uniaxial deformations on a rheometer. Various species exhibited rheological properties such as anisotropic elasticity, shear softening and compression stiffening, negative normal stress, and non-monotonic dissipation as a function of both shear strain and frequency. Erect sponges possessed aligned, spicule-reinforced fibres which endowed three times greater stiffness axially compared with orthogonally. By contrast, tissue taken from shorter sponges was more isotropic but time-dependent, suggesting higher flow sensitivity in these compared with erect forms. We explore ecological and physiological implications of our results and speculate about flow-induced mechanical signalling in sponge cells.