Marine dinoflagellates produce a wide range of bioactive metabolites, including unique pigments and proteins. In this study, ethanol (EtE) and aqueous (AqE) extracts of the marine dinoflagellate Prorocentrum cordatum, containing secondary metabolites, were investigated. For extract analysis, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Analysis with the AB Sciex 5800 TOF/TOF 5800 System operating in positive ion mode enabled the rapid and accurate characterization of pigments and proteins in the dinoflagellate extracts. The study revealed the presence of peridinin and chlorophyll a in the EtE and allowed the characterization of the most abundant protein groups in the AqE, including chaperones, cytoprotective and antioxidant proteins. The obtained extracts were tested for antitumor and migrastatic activity using adherent mouse hepatoma MH22a cells and suspension human leukemia K562 cells as target models. According to the MTS colorimetric assay, both EtE and AqE showed no toxic effects on MH22a cells, in contrast to the K562 culture. Flow cytometry analysis showed that EtE induced G2/M phase arrest in MH22a cells and had no effect on the K562 cell cycle, while AqE promoted the accumulation of both K562 and MH22a cells in the S phase. A cell adhesion assay demonstrated that EtE significantly reduced the adhesion ability of MH22a cells, decreasing the number of adhered cells by 4.8 times. In a wound healing assay, EtE suppressed MH22a cell migration by 2.5 times. AqE had no effect on either adhesion or motility. Thus, pigment-containing ethanol extracts, while exhibiting low toxicity, demonstrated pronounced migrastatic and anti-adhesive activity against MH22a cells.

This research continues a series of studies of physiology and phylogeny of the potentially toxic dinoflagellate Prorocentrum cordatum (Ostenfeld) Dodge, 1975, a eukaryotic unicellular alga capable of forming harmful algal blooms (HABs) in marine coastal areas.P. cordatum is a highly adaptable invasive species, which recently colonized brackish-water Baltic Sea.HAB dynamics of this and other dinoflagellates are influenced by various abiotic and biotic factors, such as nutrient enrichment, temperature, salinity and irradiance.Light plays an important role in regulating the growth rate and nutrient assimilation by phytoplankton.Some dinoflagellate species possess unique pigments, such as peridinin, which is associated with light-harvesting protein complex acpPC, belonging to the LHC protein family.This protein binds chlorophyll a/c and carotenoids and functions as a light-harvesting antenna.In this work, we phylogenetically characterized the LHC-like amino acid sequences of dinoflagellates found in unannotated transcriptomes of the Marine Microbial Eukaryote Transcriptome Sequencing Project database (MMETSP).The obtained LHC-like sequences have strong phylogenetic relationships with homologous proteins of algae from other taxa and share typical conservative amino acid motifs with them.Phylogenetic analysis showed that the LHC-like sequenced dinoflagellates grouped together with other fucoxanthin-chlorophyll a/c-containing algae.Within this group, acpPC sequences from the peridinin-containing dinoflagellate species formed a well-supported distinct clade.The evolutionary relationships of peridinincontaining dinoflagellates P. cordatum with other dinoflagellates and various algal taxa were analyzed.The obtained results provide a deeper insight into advanced physiological adaptation strategies of bloom-forming dinoflagellates thus contributing to HABs modeling, forecasting and management.

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a–c compounds with promising antiproliferative activity, previously synthetized and screened against human erythroleukemic cell line K562 tumor cell line. Cytotoxicity of 4a–c against murine fibroblast 3T3 and SV-40 transformed murine fibroblast 3T3-SV40 cell lines were evaluated. The 4a and 4c compounds were cytotoxic against 3T3-SV40 cells in comparison with those of 3T3. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G0/G1 phase. Using confocal microscopy, we found that with 4a and 4c treatment of 3T3 cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 82–97% of cells. The number of 3T3-SV40 cells with stress fibers increased to 7–30% against 2% in control. We discovered that transformed 3T3-SV40 cells after treatment with compounds 4a and 4c significantly reduced the number of cells with filopodium-like membrane protrusions (from 86 % in control cells to 6–18% after treatment), which indirectly suggests a decrease in cell motility. We can conclude that the studied compounds 4a and 4c have a cytostatic effect, which can lead to a decrease in the number of filopodium-like membrane protrusions.

A series of heterocyclic compounds containing a spiro-fused pyrrolo[3,4-a]pyrrolizine and tryptanthrin framework have been synthesized and studied as potential antitumor agents. Cytotoxicity of products was screened against human erythroleukemia (K562) and human cervical carcinoma (HeLa) cell lines. Among the screened compounds. 4a, 4b and 5a were active against human erythroleukemia (K562) cell line, while 4a and 5a were active against cervical carcinoma (HeLa) cell line. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G2/M phase and induced apoptosis. Using confocal microscopy, we found that with 4a and 5a treatment of HeLa cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 76–91% of cells. We discovered that HeLa cells after treatment with compounds 4a and 5a significantly reduced the number of cells with filopodium-like membrane protrusions (from 63 % in control cells to 29% after treatment) and a decrease in cell motility.

The marine planktonic dinoflagellate Prorocentrum cordatum (syn.Prorocentrum minimum) is a thecate species capable of ecdysis, i.e. rearrangement of the cell covering in response to different stressors.During the process of ecdysis, P. cordatum cells shed the plasma membrane and outer amphiesmal vesicle membrane, and become immotile, covered only by thecal plates and the new plasma membrane.At this stage, the cells can be considered as thecal cysts.Then, they complete ecdysis by leaving the old thecal plates and restoring motility, i.e. excyst.To elucidate the role of vesicular transport in the cell covering rearrangement, we tested the effect of brefeldin A, the inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi complex widely used in cell biology, on the cellular structure of P. cordatum and its ability to ecdyse.We found out that brefeldin A blocked vesicular trafficking from the endoplasmic reticulum to the Golgi complex, since we observed disassembly of the Golgi complex in cells treated by brefeldin A at 0.1 µg/ml for 1 h.Moreover, such a treatment did not increase mortality in P. cordatum culture at the time scale of 6 h.Nevertheless, our experiments demonstrated that brefeldin A affected neither shedding of outer membranes nor discarding of thecal plates during the stressorinduced ecdysis.Based on our findings, we conclude that vesicle trafficking from the endoplasmic reticulum to the Golgi complex and further to the plasma membrane is not essential to the process of membrane and theca shedding.However, this route still can be involved in the process of new amphiesma' maturation, which is discussed in this work.

Potentially toxic dinoflagellate species Prorocentrum minimum (Pavillard) Schiller, 1933 colonized the brackish-water Baltic Sea in the early 1980s, where thereafter it has been forming regular harmful blooms. Heat shock proteins (HSP) play an important role in their ability to adapt to environmental stress. In this study, we evaluated how the synthesis of heme oxygenase-1 (HO-1/HSP32), an important part of cellular machinery for cell protection, changes in P. minimum under the influence of salinity stress during the day-night cycle. We identified sequences of the HO-1 homologs in the unannotated P. minimum transcriptomes, represented in the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database. Phylogenetic analysis showed that this protein clustered in a distinct clade and demonstrated an evolutionary relationship with the HO-1 homologs of other taxonomic groups. To identify P. minimum cells that express the HO-1 protein, we used fluorescent flow cytometry. In control conditions (17 parts per thousand), the number of cells expressing this protein was 2.2 times higher during the day than at night, with the same average fluorescence intensity (1.7 units). During the daytime, the critical salinity conditions of 8 parts per thousand did not lead to significant changes in the number of cells expressing HO-1, while in the night culture this salinity shift caused a 2.7-fold increase in the number of cells expressing HO-1, and led to an increase in the intensity of fluorescence up to 2.0 units. The overall DNA and RNA synthesis rates were estimated by the incorporation of H-3-thymidine and H-3-uridine, respectively. Transcription and replication levels were significantly higher under critical salinity conditions (8 parts per thousand) compared to the control (17 parts per thousand) and typical sea salinity conditions (35 parts per thousand). The greatest increase in the DNA synthesis rates (up to 2.7 times) was observed during the daytime in cells exposed to salinity stress at 8 parts per thousand. In contrast, the greatest increase in the RNA synthesis rates (2.7 times) was registered in cells exposed to salinity of 8 parts per thousand at night. Thus, the replication activity of cells in response to salinity stress increased significantly during the day, while transcription levels reached their maximum values at night. These results indicate that a significant increase in the synthesis of both nucleic acids and the HO-1 stress protein can serve as a biomarker of the impact of environmental stress factors, such as salinity. This allowed concluding that the studied parameters demonstrate a high potential for future modeling of harmful algae blooms, particularly those formed by the invasive potentially toxic species under horohaline conditions of the Baltic Sea.

The potentially toxic marine dinoflagellates Prorocentrum cordatum (Ostenfeld) Dodge, 1975 are responsible for harmful algal blooms in many coastal ecosystems and have recently colonized the brackish-water Baltic Sea.Their ability to adapt to changing environmental conditions is partly mediated by cytoprotective proteins that provide the effective physiological stress response.One such protein, heme oxygenase (HO/HSP32), not only catalyzes the degradation of heme but also protects cells from oxidative stress caused by a number of environmental factors.In this study, we phylogenetically characterized the HO-like protein sequences of dinoflagellates found in the unannotated transcriptomes represented in the Marine Microbial Eukaryote Transcriptome Sequencing Project database.The homologues sequences identified in the database shared amino acid identity with HO family proteins of other taxa and contained typical conserved motifs.Phylogenetic analysis showed that HO-like homologs are widely represented in the dinoflagellate transcriptomes.Overall, sequences of dinoflagellates can be classified into two distinct groups.The first group is closely related to other unicellular protists and cyanobacteria.The second group clusters separately from all other taxa.We made a comparative analysis of the HO-1-like and HO-2-like protein trees to evaluate topological and branch length differences between the trees.We found that both trees possessed different topologies, thus indicating that these two proteins evolved at different rates.

Many dinoflagellate species are sensitive to mechanical disturbances, for instance, to mixing, which hampers effective continuous culturing of these organisms.Such sensitivity may be linked to the induction of ecdysis, a process of the cell covering rearrangement, followed by temporary quiescence of the dinoflagellate cells.Ecdysis can be induced in a significant fraction of a dinoflagellate population by unfavorable environmental conditions and is often involved in the formation of their cysts.Previously it was demonstrated that the bacteriostatic antibiotic tetracycline, which is widely used against bacteria in aquaculture, decreased the natural ecdysis rate in the culture of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller, 1933.In this study, we tested the ability of this compound to lower the rate of ecdysis induced by mechanical stimuli.We showed that mechanical disturbances associated with centrifugation, stirring, and shaking equally induced ecdysis in the tetracyclinetreated and control cultures.Thus, tetracycline did not make dinoflagellates less susceptible to the mechanical stressors and cannot be used to surpass their sensitivity to mixing during continuous culturing.

Ecdysis, the process of extensive cell covering rearrangement, represents a remarkable physiological trait of dinoflagellates. It is involved in the regulation of the population and bloom dynamics of these microorganisms, since it is required for the formation of their thin-walled cysts. This study presents laboratory data on ecdysis in Prorocentrum cordatum, a harmful dinoflagellate species of high environmental significance. We studied external stressors triggering this process and changes in the cell ultrastructure accompanying it. Our experiments showed that mass ecdysis and formation of cysts in P. cordatum could be induced by centrifugation, temperature decrease, changes in salinity, and treatment by 2,6-dichlorobenzonitrile, whereas temperature increase, changes in pH and treatment by tetracycline did not have this effect. Obtained cysts of P. cordatum did not contain the pellicular layer and were formed in the end of the first stage of this process, i.e. removal of the plasma membrane and the outer amphiesmal vesicle membrane, whereas its second stage, removal of theca, represented excystment. Based on our findings, we conclude that such cysts can be attributed to thecate cysts and suggest P. cordatum as a promising model organism for the investigation of cellular and molecular aspects of ecdysis in dinoflagellates.

The increasing inflow of nitrogen (N) substrates into marine nearshore ecosystems induces proliferation of harmful algal blooms (HABs) of dinoflagellates, such as potentially toxic invasive species Prorocentrum minimum. In this study, we estimated the influence of NO3-, NH4+ and urea on transcription levels and urea transporter dur3 and nitrate transporter nrt2 genes expression in these dinoflagellates. We identified dur3 and nrt2 genes sequences in unannotated transcriptomes of P. minimum and other dinoflagellates presented in MMETSP database. Phylogenetic analysis showed that these genes of dinoflagellates clustered to the distinct Glade demonstrating evolutionary relationship with the other known dur3 and nrt2 genes of microalgae. The evaluation of expression levels of dur3 and nrt2 genes by RT-qPCR revealed their sensitivity to input of the studied N sources. Dur3 expression levels were downregulated after the supplementation of additional N sources and were 1.7-2.6-fold lower than in the nitrate-grown culture. Nrt2 expression levels decreased 1.9-fold in the presence of NH4+. We estimated total RNA and DNA synthesis rates by the analysis of incorporation of 3H-thymidine and H-3-uridine in batch and continuous cultures. Addition of N compounds did not affect the DNA synthesis rates. Transcription levels increased up to 12.5-fold after the N supplementation in urea-limited treatments. Investigation of various nitrogen sources as biomarkers of dinoflagellate proliferation due to their differentiated impact on expression of dur3 and nrt2 genes and transcription rates in P. minimum cells allowed concluding about high potential of the studied parameters for future modeling of HABs under global N pollution. (C) 2019 Elsevier Ltd. All rights reserved.

Biomarkers of temperature stress were studied as major characteristics crucial for the understanding complex processes that underlie the response of marine planktonic microorganisms to environmental factors and their sublethal effects. Using the potentially toxic dinoflagellates Prorocentrum minimum as a model object, the impact of temperature stress on viability, cell cycle, RNA synthesis and DNA replication in these protists was evaluated. It was shown by flow cytometry that stress evoked by a temperature increase from 25°C (control) to 37 or 42°C during 15 to 60 min did not cause any considerable alterations in the cell cycle, while cell death rate increased from ≤ 1% (control) to 2–12% at 37°C and 4–22% at 42°C. Along with a relatively low cell death rate, following a temperature increase to 37 and/or 42°C, P. minimum displayed the ability to boost the synthesis of DNA (1.7–1.9 and 1.2–1.6 times, respectively) and especially RNA (2.5–3.1 and 1.7–2.8 times, respectively) during the first 15–30 min after stress. At certain stages of the life cycle, this effect can be critical for maintaining the viability and normal development of the P. minimum population. The obtained results demonstrate that a significantly elevated synthesis of nucleic acids can serve as an indicator (biomarker) of sublethal environmental stress.

Dinoflagellates are an important group of unicellular eukaryotes widespread in aquatic ecosystems. Many dinoflagellates are mixotrophic, toxic or potentially toxic, highly competitive and invasive, while molecular mechanisms that underpin their success in natural communities remain enigmatic. Due to peculiar features of dinoflagellate genome, little is known about the structure and expression of genes in these organisms. We analyzed the transcriptome databases of the dinoflagellate Prorocentrum minimum in order to identify the sequences of urea transporter (dur3) and nitrate transporter (nrt2.1) genes. Taking into account prospective exon-intron organization of dinoflagellate genome we suggested two variants of choosing the primer positions. We designed six primer pairs for amplification of the urea transporter gene fragments and three – for amplification of the nitrate transporter gene fragments. As a result of PCR, fragments of target genes were obtained. Alignment of amplicons with database transcriptome sequences showed that those sequences were identical. Primers developed in this study can be further used for examination of P. minimum gene expression by RT-qPCR. This approach would provide a better understanding of the influence of various nitrogen sources on physiological characteristics of these protists responsible for their effective adaptations to fluctuating environment.