Taxonomic difference in marine bloom-forming phytoplanktonic species affects the dynamics of both bloom-responding prokaryotes and prokaryotic viruses.

The production of dissolved organic matter during phytoplankton blooms and consumption by heterotrophic prokaryotes promote marine carbon biogeochemical cycling. Although prokaryotic viruses presumably affect this process, their dynamics during blooms are not fully understood. Here, we investigated the effects of taxonomic difference in bloom-forming phytoplankton on prokaryotes and their viruses. We analyzed the dynamics of coastal prokaryotic communities and viruses under the addition of dissolved intracellular fractions from taxonomically distinct phytoplankton, the diatom Chaetoceros sp. (CIF) and the raphidophycean alga Heterosigma akashiwo (HIF), using microcosm experiments. Ribosomal RNA gene amplicon and viral metagenomic analyses revealed that particular prokaryotes and prokaryotic viruses specifically increased in either CIF or HIF, indicating that taxonomic difference in bloom-forming phytoplankton promotes distinct dynamics of not only the prokaryotic community but also prokaryotic viruses. Furthermore, combining our microcosm experiments with publicly available environmental data mining, we identified both known and novel possible host-virus pairs. In particular, the growth of prokaryotes associating with phytoplanktonic organic matter, such as Bacteroidetes (Polaribacter and NS9 marine group), Vibrio spp., and Rhodobacteriales (Nereida and Planktomarina), was accompanied by an increase in viruses predicted to infect Bacteroidetes, Vibrio, and Rhodobacteriales, respectively. Collectively, our findings suggest that changes in bloom-forming species can be followed by an increase in a specific group of prokaryotes and their viruses and that elucidating these tripartite relationships among specific phytoplankton, prokaryotes, and prokaryotic viruses improves our understanding of coastal biogeochemical cycling in blooms.IMPORTANCEThe primary production during marine phytoplankton bloom and the consumption of the produced organic matter by heterotrophic prokaryotes significantly contribute to coastal biogeochemical cycles. While the activities of those heterotrophic prokaryotes are presumably affected by viral infection, the dynamics of their viruses during blooms are not fully understood. In this study, we experimentally demonstrated that intracellular fractions of taxonomically distinct bloom-forming phytoplankton species, the diatom Chaetoceros sp. and the raphidophycean alga Heterosigma akashiwo, promoted the growth of taxonomically different prokaryotes and prokaryotic viruses. Based on their dynamics and predicted hosts of those viruses, we succeeded in detecting already-known and novel possible host-virus pairs associating with either phytoplankton species. Altogether, we propose that the succession of bloom-forming phytoplankton would change the composition of the abundant prokaryotes, resulting in an increase in their viruses. These changes in viral composition, depending on bloom-forming species, would alter the dynamics and metabolism of prokaryotes, affecting biogeochemical cycling in blooms.

Prevalence of Viral Frequency-Dependent Infection in Coastal Marine Prokaryotes Revealed Using Monthly Time Series Virome Analysis.

Viruses infecting marine prokaryotes have a large impact on the diversity and dynamics of their hosts. Model systems suggest that viral infection is frequency dependent and constrained by the virus-host encounter rate. However, it is unclear whether frequency-dependent infection is pervasive among the abundant prokaryotic populations with different temporal dynamics. To address this question, we performed a comparison of prokaryotic and viral communities using 16S rRNA amplicon and virome sequencing based on samples collected monthly for 2 years at a Japanese coastal site, Osaka Bay. Concurrent seasonal shifts observed in prokaryotic and viral community dynamics indicated that the abundance of viruses correlated with that of their predicted host phyla (or classes). Cooccurrence network analysis between abundant prokaryotes and viruses revealed 6,423 cooccurring pairs, suggesting a tight coupling of host and viral abundances and their "one-to-many" correspondence. Although stable dominant species, such as SAR11, showed few cooccurring viruses, a fast succession of their viruses suggests that viruses infecting these populations changed continuously. Our results suggest that frequency-dependent viral infection prevails in coastal marine prokaryotes regardless of host taxa and temporal dynamics. IMPORTANCE There is little room for doubt that viral infection is prevalent among abundant marine prokaryotes regardless of their taxa or growth strategy. However, comprehensive evaluations of viral infections in natural prokaryotic communities are still technically difficult. In this study, we examined viral infection in abundant prokaryotes by monitoring the monthly dynamics of prokaryotic and viral communities at a eutrophic coastal site, Osaka Bay. We compared the community dynamics of viruses with those of their putative hosts based on genome-based in silico host prediction. We observed frequent cooccurrence among the predicted virus-host pairs, suggesting that viral infection is prevalent in abundant prokaryotes regardless of their taxa or temporal dynamics. This likely indicates that frequent lysis of the abundant prokaryotes via viral infection has a considerable contribution to the biogeochemical cycling and maintenance of prokaryotic community diversity.

A novel parasite strain of Amoebophrya sp. infecting the toxic dinoflagellate Alexandrium catenella (Group I) and its effect on the host bloom in Osaka Bay, Japan.

The endoparasitic dinoflagellates belonging to the genus Amoebophrya can infect a broad range of free-living marine dinoflagellates, including harmful/toxic species. The parasite kills its host; the high prevalence of the parasite has been suggested to be a significant factor for the termination of dinoflagellate blooms in marine systems. The issues involved in culturing host-parasite systems have greatly restricted further research on Amoebophrya biology. Here, we established the culture of a novel strain of Amoebophrya sp. ex Alexandrium catenella (Group I) from Osaka Bay, Japan, and studied its genetic diversity, host specificity, and prevalence in the field. Genetic analysis established that the strain we isolated was a novel culture strain infecting A. catenella. Among the host species tested, the Amoebophrya sp. could infect the genera Alexandrium and Prorocentrum in culture, and the infection was also confirmed in the genus Tripos in a field sample. A maximum prevalence of 73% was recorded during the Alexandrium bloom period in Osaka Bay, after which the host cell density rapidly declined. Our results indicated that the existence of the parasite had a significant effect on the dynamics of A. catenella, especially on the termination of the blooms.

The utility of extracorporeal membrane oxygenation in patients with circulatory collapse by electrical storm.

In patients experiencing electrical storm, intensive care using extracorporeal membrane oxygenation (ECMO) is an efficient treatment to overcome a hemodynamically unstable condition. The aim of this study was to examine the utility of ECMO in patients with circulatory collapse by electrical storm. We retrospectively examined 17 consecutive patients receiving veno-arterial ECMO for electrical storm between January 2016 and December 2018 in our institution. We compared survivors (n = 11) and non-survivors (n = 6). Thirteen were weaned from ECMO, of whom 11 patients (64.7%) survived and were discharged from hospital, while 6 patients died (35.3%). In comparisons between survivors and non-survivors, blood pH before starting ECMO was significantly higher in survivors (pH 7.32) than in non-survivors (pH 6.89, p = 0.027). Blood lactate level was significantly lower in survivors (6.2 mmol/L) than in non-survivors (12.2 mmol/L, p = 0.044). Complications of hypoxic ischemic encephalopathy were found in 4 non-survivors (66.7%), compared to survivors (0%, p = 0.006). Durations of intensive care unit stay and hospital stay were significantly longer in survivors (271 h, 62 days) than in non-survivors (50 h, 3 days, respectively). Outcomes of treatment using ECMO in patients with circulatory collapse due to electrical storm proved satisfactory. Increases in blood lactate level and decreases in blood pH before starting ECMO were thought to be related to mortality due to suspected irreversible organ damage by hypoxia before ECMO.

Clinical evaluation of a new dispersive aortic cannula.

INTRODUCTION: Cerebral injury is a serious complication in open-heart surgery. Once it occurs, it causes significant disability and death. We developed a novel dispersive aortic cannula named the Stealth Flow cannula and used it as a standard aortic cannula in cardiopulmonary bypass. The aim of this study was to evaluate the efficiency of this aortic cannula. METHODS: A total of 182 consecutive patients undergoing cardiac surgery using cardiopulmonary bypass were studied. The patients were divided into two groups: the Soft-Flow cannula group (n = 89) and the Stealth Flow cannula group (n = 93). Patients with a shaggy aortic arch were excluded from this study because the cannulae were inserted at the ascending aorta with a cannula tip directed toward the aortic root in these cases. Patients with multiple arterial perfusion sites were also excluded. Complications including early mortality, perioperative stroke, and intraoperative aortic injury were compared between the two groups. RESULTS: Age, operative procedure, cardiopulmonary bypass time, and the Japan SCORE were not significantly different between the groups. In comparisons between the Stealth Flow and Soft-Flow groups, the incidences of early mortality, perioperative stroke, intraoperative aortic dissection, and all complications were 1.08% versus 1.12% (p = 0.98), 1.1% versus 2.2% (p = 0.53), 0% versus 1.1% (p = 0.33), and 1.1% versus 3.4% (p = 0.29), respectively. The incidence of major cardiovascular events, including early death, perioperative stroke, and aortic dissection, was not different. CONCLUSIONS: The Stealth Flow cannula, which was designed based on our previous experimental study, contributed to reducing cerebral and aortic events as much as the Soft-Flow cannula in the present clinical study.

Differential Responses of a Coastal Prokaryotic Community to Phytoplanktonic Organic Matter Derived from Cellular Components and Exudates.

The phytoplanktonic production and prokaryotic consumption of organic matter significantly contribute to marine carbon cycling. Organic matter released from phytoplankton via three processes (exudation of living cells, cell disruption through grazing, and viral lysis) shows distinct chemical properties. We herein investigated the effects of phytoplanktonic whole-cell fractions (WF) (representing cell disruption by grazing) and extracellular fractions (EF) (representing exudates) prepared from Heterosigma akashiwo, a bloom-forming Raphidophyceae, on prokaryotic communities using culture-based experiments. We analyzed prokaryotic community changes for two weeks. The shift in cell abundance by both treatments showed similar dynamics, reaching the first peak (~4.1×106| |cells| |mL-1) on day 3 and second peak (~1.1×106| |cells| |mL-1) on day 13. We classified the sequences obtained into operational taxonomic units (OTUs). A Bray-Curtis dissimilarity analysis revealed that the OTU-level community structure changed distinctively with the two treatments. Ten and 13 OTUs were specifically abundant in the WF and EF treatments, respectively. These OTUs were assigned as heterotrophic bacteria mainly belonging to the Alteromonadales (Gammaproteobacteria) and Bacteroidetes clades and showed successive dynamics following the addition of organic matter. We also analyzed the dynamics of these OTUs in the ocean using publicly available metagenomic data from a natural coastal bloom in Monterey Bay, USA. At least two WF treatment OTUs showed co-occurrence with H. akashiwo, indicating that the blooms of H. akashiwo also affect these OTUs in the ocean. The present results strongly suggest that the thriving and dead cells of uninfected phytoplankton differentially influence the marine prokaryotic community.

Monitoring of the toxic dinoflagellate Alexandrium catenella in Osaka Bay, Japan using a massively parallel sequencing (MPS)-based technique.

Since 2002, blooms of Alexandrium catenella sensu Fraga et al. (2015) and paralytic shellfish toxicity events have occurred almost yearly in Osaka Bay, Japan. To better understand the triggers for reoccurring A. catenella blooms in Osaka Bay, phytoplankton community was monitored during the spring seasons of 2012-2015. Monitoring was performed using massively parallel sequencing (MPS)-based technique on amplicon sequences of the 18S rRNA gene. Dense blooms of A. catenella occurred every year except in 2012, however, there was no significant correlation with the environmental parameters investigated. Plankton community diversity decreased before and middle of the A. catenella blooms, suggesting that the decline in diversity could be an indicator for the bloom occurrence. The yearly abundance pattern of A. catenella cells obtained by morphology-based counting coincided with the relative sequence abundances, which supports the effectiveness of MPS-based phytoplankton monitoring.

Correction: Locality and diel cycling of viral production revealed by a 24 h time course cross-omics analysis in a coastal region of Japan.

The original version of this Article contained an error in the main text citations and reference list. These errors have now been corrected in both the PDF and HTML versions of the Article.

Locality and diel cycling of viral production revealed by a 24 h time course cross-omics analysis in a coastal region of Japan.

Viruses infecting microorganisms are ubiquitous and abundant in the ocean. However, it is unclear when and where the numerous viral particles we observe in the sea are produced and whether they are active. To address these questions, we performed time-series analyses of viral metagenomes and microbial metatranscriptomes collected over a period of 24 h at a Japanese coastal site. Through mapping the metatranscriptomic reads on three sets of viral genomes ((i) 878 contigs of Osaka Bay viromes (OBV), (ii) 1766 environmental viral genomes from marine viromes, and (iii) 2429 reference viral genomes), we revealed that all the local OBV contigs were transcribed in the host fraction. This indicates that the majority of viral populations detected in viromes are active, and suggests that virions are rapidly diluted as a result of diffusion, currents, and mixing. Our data further revealed a peak of cyanophage gene expression in the afternoon/dusk followed by an increase of genomes from their virions at night and less-coherent infectious patterns for viruses putatively infecting various groups of heterotrophs. This suggests that cyanophages drive the diel release of cyanobacteria-derived organic matter into the environment and viruses of heterotrophic bacteria might have adapted to the population-specific life cycles of hosts.

Environmental Viral Genomes Shed New Light on Virus-Host Interactions in the Ocean.

Metagenomics has revealed the existence of numerous uncharacterized viral lineages, which are referred to as viral "dark matter." However, our knowledge regarding viral genomes is biased toward culturable viruses. In this study, we analyzed 1,600 (1,352 nonredundant) complete double-stranded DNA viral genomes (10 to 211 kb) assembled from 52 marine viromes. Together with 244 previously reported uncultured viral genomes, a genome-wide comparison delineated 617 genus-level operational taxonomic units (OTUs) for these environmental viral genomes (EVGs). Of these, 600 OTUs contained no representatives from known viruses, thus putatively corresponding to novel viral genera. Predicted hosts of the EVGs included major groups of marine prokaryotes, such as marine group II Euryarchaeota and SAR86, from which no viruses have been isolated to date, as well as Flavobacteriaceae and SAR116. Our analysis indicates that marine cyanophages are already well represented in genome databases and that one of the EVGs likely represents a new cyanophage lineage. Several EVGs encode many enzymes that appear to function for an efficient utilization of iron-sulfur clusters or to enhance host survival. This suggests that there is a selection pressure on these marine viruses to accumulate genes for specific viral propagation strategies. Finally, we revealed that EVGs contribute to a 4-fold increase in the recruitment of photic-zone viromes compared with the use of current reference viral genomes. IMPORTANCE Viruses are diverse and play significant ecological roles in marine ecosystems. However, our knowledge of genome-level diversity in viruses is biased toward those isolated from few culturable hosts. Here, we determined 1,352 nonredundant complete viral genomes from marine environments. Lifting the uncertainty that clouds short incomplete sequences, whole-genome-wide analysis suggests that these environmental genomes represent hundreds of putative novel viral genera. Predicted hosts include dominant groups of marine bacteria and archaea with no isolated viruses to date. Some of the viral genomes encode many functionally related enzymes, suggesting a strong selection pressure on these marine viruses to control cellular metabolisms by accumulating genes.

Contrasting effects of stanniocalcin-related polypeptides on macrophage foam cell formation and vascular smooth muscle cell migration.

Stanniocalcin (STC) is a calcium- and phosphate-regulating hormone secreted by the corpuscles of Stannius, an endocrine gland of bony fish. Its human homologues, STC1 and STC2 showing 34% amino acid identity each other, are expressed in a variety of human tissues. To clarify their roles in atherosclerosis, we investigated the effects of their full-length proteins, STC1(18-247) and STC2(25-302), and STC2-derived fragment peptides, STC2(80-100) and STC2(85-99), on inflammatory responses in human umbilical vein endothelial cells (HUVECs), human macrophage foam cell formation, the migration and proliferation of human aortic smooth muscle cells (HASMCs) and the extracellular matrix expression. All these polypeptides suppressed lipopolysaccharide-induced expressions of interleukin-6, monocyte chemotactic protein-1, and intercellular adhesion molecule-1 in HUVECs. Oxidized low-density lipoprotein-induced foam cell formation was significantly decreased by STC1(18-247) and increased by STC2(80-100) and STC2(85-99), but not STC2(25-302), in human macrophages. Expression of acyl-CoA:cholesterol acyltransferase-1 (ACAT1) was significantly suppressed by STC1(18-247) but stimulated by STC2(80-100) and STC2(85-99). Expression of ATP-binding cassette transporter A1 was significantly stimulated by STC1(18-247). Neither STC1(18-247) nor STC2-derived peptides significantly affected CD36 expression in human macrophages or HASMC proliferation. STC2(80-100) and STC2(85-99) significantly increased HASMC migration, whereas STC1(18-247) significantly suppressed the angiotensin II-induced HASMC migration. Expressions of collagen-1, fibronectin, matrix metalloproteinase-2, and elastin were mostly unchanged with the exception of fibronectin up-regulation by STC2(80-100). Our results demonstrated the contrasting effects of STC1 and STC2-derived peptides on human macrophage foam cell formation associated with ACAT1 expression and on HASMC migration. Thus, STC-related polypeptides could serve as a novel therapeutic target for atherosclerosis.

Vasoprotective effects of urocortin 1 against atherosclerosis in vitro and in vivo.

AIM: Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis. METHODS: We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. RESULTS: Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe-/- mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions. CONCLUSIONS: This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.

Stimulatory effects of cardiotrophin 1 on atherosclerosis.

Cardiotrophin 1 (CT-1), an interleukin-6 family cytokine, was recently shown to be expressed in the intima of early atherosclerotic lesions in the human carotid artery. CT-1 stimulates proatherogenic molecule expression in human vascular endothelial cells and monocyte migration. However, it has not been reported whether CT-1 accelerates atherosclerosis. This study was performed to examine the stimulatory effects of CT-1 on human macrophage foam cell formation and vascular smooth muscle cell migration and proliferation in vitro, and on the development of atherosclerotic lesions in apolipoprotein E-deficient (ApoE(-/-)) mice in vivo. CT-1 was expressed at high levels in endothelial cells and macrophages in both humans and ApoE(-/-) mice. CT-1 significantly enhanced oxidized low-density lipoprotein-induced foam cell formation associated with increased levels of CD36 and acyl-CoA:cholesterol acyltransferase-1 expression in human monocyte-derived macrophages. CT-1 significantly stimulated the migration, proliferation, and collagen-1 expression in human aortic vascular smooth muscle cells. Four-week infusion of CT-1 into ApoE(-/-) mice significantly accelerated the development of aortic atherosclerotic lesions with increased monocyte/macrophage infiltration, vascular smooth muscle cell proliferation, and collagen-1 content in the aortic wall. Activation of inflammasome, such as apoptosis-associated speck-like protein containing a caspase recruitment domain, nuclear factor κB, and cyclooxygenase-2, was observed in exudate peritoneal macrophages from ApoE(-/-) mice infused with CT-1. Infusion of anti-CT-1-neutralizing antibody alone into ApoE(-/-) mice significantly suppressed monocyte/macrophage infiltration in atherosclerotic lesions. These results indicate that CT-1 accelerates the development of atherosclerotic lesions by stimulating the inflammasome, foam cell formation associated with CD36 and acyl-CoA:cholesterol acyltransferase-1 upregulation in macrophages, and migration, proliferation, and collagen-1 production in vascular smooth muscle cells.

Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella.

In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris-EDTA (TE) buffer, (2) heating at 65 °C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP.

[Validation study on a method for multiresidue analysis of pesticides in vegetables and fruits with supercritical fluid extraction].

Supercritical fluid extraction (SFE) was applied to extraction of pesticides from vegetables and fruits. Residues were extracted from homogenized samples mixed with water-absorbent polymer with supercritical carbon dioxide in a stainless steel tube, followed by elution with acetone. Co-extractives were removed by means of mini-column clean-up. Measurement was performed by GC-MS/MS. Calibration was achieved by preparing matrix-matched calibration standards to counteract matrix effects. With the Japanese method validation guideline as a reference, the method was assessed in 5 agricultural products spiked with 334 pesticides at 0.01 and 0.1 µg/g. Compounds at each level were extracted from 2 samples on 5 separate days. The trueness of the method for 189 pesticides in all samples was 70-120%, and the repeatability and within-run reproducibility were also consistent with the guideline. The trueness of the method for the other 71 pesticides was in the range of 50-70%, though the repeatability and within-run reproducibility were satisfactory. This method is available as a multiresidue analysis method for vegetables and fruits.

[Validation study of a method for multiresidue analysis of pesticides in cereals and pulses using supercritical fluid extraction].

Supercritical fluid extraction (SFE) was applied to extraction of pesticides from cereals and pulses. Residues were extracted from homogenized samples mixed with water-absorbent polymer and supercritical carbon dioxide in a stainless steel tube, followed by elution with acetonitrile. Co-extractives were removed by means of mini-column clean-up. Measurement was performed by GC-MS/MS. Calibration was achieved by preparing matrix-matched calibration standards to counteract matrix effects. With the Japanese method validation guideline for pesticide residues as a reference, the method was assessed in 5 agricultural products spiked with 334 pesticides at 0.01 and 0.1 µg/g. Compounds at each level were extracted from 2 samples on 5 separate days. The trueness of the method for 137 pesticides in all samples was 70-120%, and the repeatability and within-run reproducibility were also consistent with the guideline. The trueness of the method for the other 101 pesticides was in the range of 50-70%, though the repeatability and within-run reproducibility were satisfactory. This method is available as a multiresidue analysis method for cereals and pulses.