Molecular Dynamics of Emiliania huxleyi and Cooccurring Viruses during Two Separate Mesocosm Studies

In this study we used denaturing gradient gel electrophoresis, sequencing analysis, and analytical flow cytometry to monitor the dynamics and genetic richness of Emiliania huxleyi isolates and cooccurring viruses during two mesocosm experiments in a Norwegian fjord in 2000 and 2003. We exploited variations in a gene encoding a protein with calcium-binding motifs (GPA) and in the major capsid protein (MCP) gene to assess allelic and genotypic richness within E. huxleyi and E. huxleyi-specific viruses (EhVs), respectively. To our knowledge, this is the first report that shows the effectiveness of the GPA gene for analysis of natural communities of E. huxleyi. Our results revealed the existence of a genetically rich, yet stable E. huxleyi and EhV community in the fjordic environment. Incredibly, the same virus and host genotypes dominated in separate studies conducted 3 years apart. Both E. huxleyi-dominated blooms contained the same six E. huxleyi alleles. In addition, despite the presence of at least six and four EhV genotypes at the start of the blooms in 2000 and 2003, respectively, the same two virus genotypes dominated the naturally occurring infections during the exponential and termination phases of the blooms in both years.     

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi–EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range.     

ASSESSING THE ROLE OF CASPASE ACTIVITY AND METACASPASE EXPRESSION ON VIRAL SUSCEPTIBILITY OF THE COCCOLITHOPHORE,EMILIANIA HUXLEYI (HAPTOPHYTA)

As part of their strategy to infect the globally important coccolithophore, Emiliania huxleyi (Lohmann) W.W. Hay & H.P. Mohler, Coccolithoviruses trigger and regulate the host's programmed cell death (PCD) machinery during lytic infection. The induction and recruitment of host metacaspases, specialized, ancestral death proteases that facilitate viral lysis, suggests they may be important subcellular determinants to infection. We examined the “basal” levels and patterns of caspase activity and metacaspase expression in exponentially growing resistant and sensitive E. huxleyi strains and linked them with susceptibility to E. huxleyi virus 1 (EhV1). Resistant E. huxleyi strains were consistently characterized by low caspase specific activity and a relatively simple metacaspase expression profile. In contrast, sensitive E. huxleyi strains had markedly elevated caspase specific activity and consistently expressed more diverse metacaspase proteins. Using pooled data sets from triplicate experiments, we observed statistically significant linear correlations between infectivity, caspase activity, and metacaspase expression, with each strain forming distinct clusters, within a gradient in viral susceptibility. At the same time, we observed positive correlations between the expression of a subset of metacaspase proteins and lower susceptibility, suggestive of potential protective roles. Our findings implicate the importance of subtle differences in the basal physiological regulation of the PCD machinery to viral resistance or sensitivity and cell fate.     

Temperature-Induced Viral Resistance in Emiliania huxleyi (Prymnesiophyceae)

Annual Emiliania huxleyi blooms (along with other coccolithophorid species) play important roles in the global carbon and sulfur cycles. E. huxleyi blooms are routinely terminated by large, host-specific dsDNA viruses, (Emiliania huxleyi Viruses; EhVs), making these host-virus interactions a driving force behind their potential impact on global biogeochemical cycles. Given projected increases in sea surface temperature due to climate change, it is imperative to understand the effects of temperature on E. huxleyi’s susceptibility to viral infection and its production of climatically active dimethylated sulfur species (DSS). Here we demonstrate that a 3°C increase in temperature induces EhV-resistant phenotypes in three E. huxleyi strains and that successful virus infection impacts DSS pool sizes. We also examined cellular polar lipids, given their documented roles in regulating host-virus interactions in this system, and propose that alterations to membrane-bound surface receptors are responsible for the observed temperature-induced resistance. Our findings have potential implications for global biogeochemical cycles in a warming climate and for deciphering the particular mechanism(s) by which some E. huxleyi strains exhibit viral resistance.     

Ecological significance of extracellular vesicles in modulating host-virus interactions during algal blooms

Extracellular vesicles are produced by organisms from all kingdoms and serve a myriad of functions, many of which involve cell-cell signaling, especially during stress conditions and host-pathogen interactions. In the marine environment, communication between microorganisms can shape trophic level interactions and population succession, yet we know very little about the involvement of vesicles in these processes. In a previous study, we showed that vesicles produced during viral infection by the ecologically important model alga Emiliania huxleyi, could act as a pro-viral signal, by expediting infection and enhancing the half-life of the virus in the extracellular milieu. Here, we expand our laboratory findings and show the effect of vesicles on natural populations of E. huxleyi in a mesocosm setting. We profile the small-RNA (sRNA) cargo of vesicles that were produced by E. huxleyi during bloom succession, and show that vesicles applied to natural assemblages expedite viral infection and prolong the half-life of this major mortality agent of E. huxleyi. We subsequently reveal that exposure of the natural assemblage to E. huxleyi-derived vesicles modulates not only host-virus dynamics, but also other components of the microbial food webs, thus emphasizing the importance of extracellular vesicles to microbial interactions in the marine environment.Subject terms: Virus-host interactions, Microbial ecology, Water microbiology     

ISOLATION AND CHARACTERIZATION OF A VIRUS THAT INFECTS EMILIANIA HUXLEYI (HAPTOPHYTA)1

The isolation and characterization of a virus (designated EhV) that infects the marine coccolithophorid Emiliania huxleyi (Lohmann) Hay & Mohler are described. Three independent clones of EhV were isolated from Norwegian coastal waters in years 1999 and 2000. EhV is a double‐stranded DNA‐containing virus with a genome size of ～415 kilo‐base pairs. The viral particle is an icosahedron with a diameter of 160–180 nm. The virus particle contains at least nine proteins ranging from 10 to 140 kDa; the major capsid protein weighs ～54 kDa. EhV has a latent period of 12–14 h and a burst size of 400–1000 (mean, 620) viral particles per cell. A phylogenetic tree based on DNA polymerase amino acid sequences indicates EhV should be assigned to the Phycodnaviridae virus family and that the virus is most closely related to viruses that infect Micromonas pusilla and certain Chlorella species.     

Black Sea algal viruses

Monitoring of the Black Sea algal viruses in Sevastopol bays and Crimean water areas has been carried out since 2002. Based on the methods that were developed and patented by the author, more than 200 strains of algal viruses of five species of microalgae that are new to science were isolated: TvV (Tetraselmis viridis virus), DvV (Dunaliella viridis virus), PtV (Phaeodactylum tricornutum virus), PpV (Prorocentrum pusillum virus) and IgV (Isochrysis galbana virus). For the first time in the Black Sea, the Emiliania huxleyi virus (EhV) of microalgae was isolated. Using the method of electron microscopy, the Black Sea algal viruses were identified as icosahedral virions with respective sizes of 56–60, 45–48, 50–53, 88–92, and 128–132 nm, for the TvV, PtV, DvV, PpV and IgV viruses. The EhV size, as determined by the method of filtration, was within the range of 50–200 nm. In the IgV and EhV viruses we revealed a viral envelope. Based on their characters the isolated algal viruses were attributed to the Phycodnaeviridae. The maximum number of algal viruses was observed in the spring and autumn seasons, which is typical for their host phytoplankton species. The Black Sea algal viruses, TvV, PpV, IgV, and EhV, displayed no strict species specificity and have a wide range of available hosts.     

The mutual interplay between calcification and coccolithovirus infection

Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non-calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV-free, ‘induced media’. Our findings show that calcification is a key component of the E. huxleyi-EhV arms race and an aspect that is critical both to the modelling of these host–virus interactions in the ocean and interpreting their impact on the global carbon cycle.     

Expression and localization of microRNAs in perinatal rat pancreas: role of miR-21 in regulation of cholesterol metabolism

Objective

To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas.

Research Design and Methods

RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells.

Results

Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA. The localization studies showed endocrine localization of six of these miRNAs (miR-21, -23a, -29a, -125b-5p, -376b-3p and -451), and all were expressed in exocrine cells at one time point at least. Pathways involving metabolic processes, terpenoid and sterol metabolism were selectively affected by concomitant regulation by miRNAs and mRNAs, and Srebf1 was validated as a target of miR-21.

Conclusions

The findings suggest that miRNAs are involved in the functional maturation of pancreatic exocrine and endocrine tissue following birth. Pathway analysis of target genes identify changes in sterol metabolism around birth as being selectively affected by differential miRNA expression during this period.     

Construction of PARPi Resistance-related Competing Endogenous RNA Network

Objective: Ovarian cancer is a kind of common gynecological malignancy in women. PARP inhibitors (PARPi) have been approved for ovarian cancer treatment. However, the primary and acquired resistance have limited the application of PARPi. The mechanisms remain to be elucidated.Methods: In this study, we characterized the expression profiles of mRNA and nonconding RNAs (ncRNAs) and constructed the regulatory networks based on RNA sequencing in PARPi Olaparib-induced ovarian cancer cells.Results: We found that the functions of the differentially expressed genes were enriched in “PI3K/AKT signaling pathway,” “MAPK signaling pathway” and “metabolic process”. The functions of DELs (cis) were enriched in “Human papillomavirus infection”“tight junction” “MAPK signaling pathway”. As the central regulator of ceRNAs, the differentially expressed miRNAs were enriched in “Human papillomavirus infection” “MAPK signaling pathway” “Ras signaling pathway”. According to the degree of interaction, we identified 3 lncRNAs, 2 circRNAs, 7 miRNAs, and 12 mRNA as the key regulatory ceRNA axis, in which miR-320b was the important mediator.Conclusion: Here, we revealed the key regulatory lncRNA (circRNA)-miRNA-mRNA axis and their involved pathways in the PARPi resistant ovarian cancer cells. These findings provide new insights into exploring the ceRNA regulatory networks and developing new targets for PARPi resistance.     

O-GlcNAc and the Epigenetic Regulation of Gene Expression

O-GlcNAcylation is an abundant nutrient-driven modification linked to cellular signaling and regulation of gene expression. Utilizing precursors derived from metabolic flux, O-GlcNAc functions as a homeostatic regulator. The enzymes of O-GlcNAc cycling, OGT and O-GlcNAcase, act in mitochondria, the cytoplasm, and the nucleus in association with epigenetic “writers” and “erasers” of the histone code. Both O-GlcNAc and O-phosphate modify repeats within the RNA polymerase II C-terminal domain (CTD). By communicating with the histone and CTD codes, O-GlcNAc cycling provides a link between cellular metabolic status and the epigenetic machinery. Thus, O-GlcNAcylation is poised to influence trans-generational epigenetic inheritance.     

Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.     

A Quest for miRNA Bio-Marker: A Track Back Approach from Gingivo Buccal Cancer to Two Different Types of Precancers

Deregulation of miRNA expression may contribute to tumorigenesis and other patho-physiology associated with cancer. Using TLDA, expression of 762 miRNAs was checked in 18 pairs of gingivo buccal cancer-adjacent control tissues. Expression of significantly deregulated miRNAs was further validated in cancer and examined in two types of precancer (leukoplakia and lichen planus) tissues by primer-specific TaqMan assays. Biological implications of these miRNAs were assessed bioinformatically. Expression of hsa-miR-1293, hsa-miR-31, hsa-miR-31* and hsa-miR-7 were significantly up-regulated and those of hsa-miR-206, hsa-miR-204 and hsa-miR-133a were significantly down-regulated in all cancer samples. Expression of only hsa-miR-31 was significantly up-regulated in leukoplakia but none in lichen planus samples. Analysis of expression heterogeneity divided 18 cancer samples into clusters of 13 and 5 samples and revealed that expression of 30 miRNAs (including the above-mentioned 7 miRNAs), was significantly deregulated in the cluster of 13 samples. From database mining and pathway analysis it was observed that these miRNAs can significantly target many of the genes present in different cancer related pathways such as “proteoglycans in cancer”, PI3K-AKT etc. which play important roles in expression of different molecular features of cancer. Expression of hsa-miR-31 was significantly up-regulated in both cancer and leukoplakia tissues and, thus, may be one of the molecular markers of leukoplakia which may progress to gingivo-buccal cancer.     

Mitochondrial sirtuins,metabolism, and aging

Maintaining metabolic homeostasis is essential for cellular and organismal health throughout life. Multiple signaling pathways that regulate metabolism also play critical roles in aging, such as PI3K/AKT, mTOR, AMPK, and sirtuins (SIRTs). Among them, sirtuins are known as a protein family with versatile functions, such as metabolic control, epigenetic modification and lifespan extension. Therefore, by understanding how sirtuins regulate metabolic processes, we can start to understand how they slow down or accelerate biological aging from the perspectives of metabolic regulation. Here, we review the biology of SIRT3, SIRT4, and SIRT5, known as the mitochondrial sirtuins due to their localization in the mitochondrial matrix. First, we will discuss canonical pathways that regulate metabolism more broadly and how these are integrated with aging regulation. Then, we will summarize the current knowledge about functional differences between SIRT3, SIRT4, and SIRT5 in metabolic control and integration in signaling networks. Finally, we will discuss how mitochondrial sirtuins regulate processes associated with aging and aging-related diseases.     

Effect of increased pCO2 on phytoplankton–virus interactions

Atmospheric carbon dioxide (CO₂) has increased since the pre-industrial period and is predicted to continue to increase throughout the twenty-first century. The ocean is a sink for atmospheric CO₂ and increased CO₂ concentration will change the carbonate equilibrium of seawater and result in lower carbonate ion concentration and lower pH. This may affect the entire marine biota but in particular calcifying organisms. In this study we investigated the effect of increased CO₂ on the virus host interaction of Emiliania huxleyi as a calcifying organism and of Phaeocystis poucheti as a non- calcifying organism. Both algae were grown in laboratory controlled conditions under past (280 ppmv), present (350 ppmv) and future (700 ppmv) CO₂ concentrations with and without added virus. Increased CO₂ had a negative effect on the growth rate of P. pouchetii, but not of E. huxleyi. No impact was found on viral lysis of P. pouchetii while increased burst size and slightly delayed lysis was observed for E. huxleyi with increased CO₂. We conclude that this short time study could not confirm earlier reports and our hypothesis of a negative effect of high CO₂ on E. huxleyi growth and E. huxleyi virus production.