Miranda Judd

Dinoflagellates are a diverse group of eukaryotes in the Alveolate lineage known for a wide array of ecological adaptations that depend on a combination of photoautotrophy and mixotrophy. Current methods for measuring both photosynthesis and respiration in algae have limitations, but Resipher (Lucid Scientific), a long-term, non-invasive oxygen monitoring system that uses platinum (Pl) fluorescent technology and patented signal-processing methods, may overcome these constraints. The purpose of this study was to assess whether Resipher can be used to measure photosynthesis and respiration in the dinoflagellates Amphidinium carterae and Symbiodinium natans. Oxygen concentration measurements were first collected from wells of media under various light intensities and wavelengths provided by LED sources below the culture plate to determine the effect light had on the device’s sensors. Oxygen measurements were then obtained from each dinoflagellate species at various cell densities to estimate the optimal concentration needed for each species during an experiment. Finally, oxygen concentration was measured for each species exposed to the metabolic inhibitors DCMU, rotenone, and propyl gallate to determine whether the device was sensitive to changes in algal physiology. Results suggest that any variances introduced by green or blue light are minor, and that the device can distinguish between oxygen production and consumption in a cell density-dependent manner. This study ultimately confirms that Resipher can measure both photosynthesis and respiration in dinoflagellate cultures.

Dinoflagellates are ecologically vital marine microalgae with notoriously large and complex genomes, marked by extensive repetition and unconventional gene structures. Here, we present a near-complete genome assembly for Amphidinium carterae, assembled exclusively using Oxford Nanopore long-read sequencing and comprehensively annotated using a transcriptome-guided approach. The 1.25 Gb nuclear genome spans 2,478 contigs with a coverage of 60X, N50 of 1.1 Mb, and contains 55% repetitive elements. Annotation was challenging due to the prevalence of short exons, long introns, tandem gene duplication, and frequent nesting of genes. Standard BUSCO analysis using AUGUSTUS underestimated completeness; however, super-transcriptome-guided correction revealed over 89.6% of conserved genes, underscoring the importance of tailored annotation approaches for dinoflagellates. For the 68 densely packed tandemly repeated gene sets, with copy numbers ranging from 3 to 52, identification and annotation required a combination of methods including manual curation. Comparison to a recent independent assembly of an alternate A. carterae strain corroborated the results with slight variation in gene copy and nucleotide sequence but overall showed strong conservation of tandem gene sets. Many tandem repeat gene regions displayed high conservation across both coding and non-coding segments, implying DNA-based duplication. Gene expression analyses revealed that tandem gene arrays contributed significantly to transcriptional output, with expression levels positively correlated with copy number (p = 1.7e-15), suggesting a dose-dependent regulatory mechanism. The most highly duplicated gene, Major Basic Nuclear Protein (MBNP), was also one of the most highly expressed gene families and existed in distinct long and short isoforms arranged in alternating tandem patterns. This genome represents a comprehensive assembly with exhaustive annotation of tandem gene sets within a free-living dinoflagellate genome and offers new insights into gene organization, expression regulation, and repeat-driven genome architecture. Our findings challenge the long-held view of dinoflagellates as genomic “hoarders”, instead revealing a strategy of selective gene retention, curation, and amplification related to selection pressures. This work provides a valuable reference for future genomic studies and paves the way for improved annotation of complex, repeat-rich eukaryotic genomes.

Dinoflagellates are one of the largest groups of marine microalgae and exhibit diverse trophic strategies. Some dinoflagellates can produce secondary metabolites that are known to be toxic, which can lead to ecologically harmful blooms. Amphidinium carterae is one species of dinoflagellate that produces toxic compounds and is used as a model for dinoflagellate studies. The impact of the microbiome on A. carterae growth and metabolite synthesis is not yet fully understood, nor is the impact of bacterial data on sequencing and assembly. An antibiotic cocktail was previously shown to eliminate 16S amplification from the dinoflagellate culture. Even with drastically reduced bacterial numbers during antibiotic treatment, bacterial sequences were still present. In this experiment, we used novel Nanopore long-read sequencing techniques on A. carterae cultures to assemble 15 full bacterial genomes ranging from 2.9 to 6.0 Mb and found that the use of antibiotics decreased the percentage of reads mapping back to bacteria. We also identified shifts in the microbiome composition and identified a potentially deleterious bacterial species arising in the absence of the antibiotic treatment. Multiple antibiotic resistance genes were identified, as well as evidence that the bacterial population does not contribute to toxic secondary metabolite synthesis.

Dinoflagellates are one of the largest groups of marine microalgae and exhibit diverse trophic strategies. Within these niches, dinoflagellates can produce secondary metabolites that are known to be toxic, which can lead to ecologically harmful blooms. Amphidinium carterae is one species of dinoflagellate that produces toxic compounds and is used as a model for dinoflagellate studies. The extent to which the microbiome surrounding A. carterae affects its growth and secondary metabolite synthesis is still being assessed, as well as the impact of bacterial data on sequencing and assembly. An antibiotic cocktail was previously shown to eliminate 16S amplification from the dinoflagellate culture. Even with drastically reduced bacterial numbers, bacterial populations are likely to be present. In this experiment we used novel Nanopore long-read sequencing techniques on A. carterae cultures to assemble 15 full bacterial genomes ranging from 2.9-6.0 Mb and found that the use of antibiotics decreased the percentage of reads mapping back to bacteria. We also identified shifts in the microbiome composition and identified a potentially deleterious bacterial species arising in the absence of the antibiotic-treatment. Multiple antibiotic-resistance genes were identified, as well as evidence that the bacterial population do not contribute to toxic secondary metabolite synthesis.

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5′-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis.

(Br J Anaesth. 2019;122(6):751–759) Gestational alterations to coagulation factor concentrations include elevated levels of factor (F) VII, FVIII, and von Willebrand factor (vWF) in comparison to the blood of nonpregnant (NP) reference patients. These elevated factors serve as an advantage in regards to prevention of postpartum hemorrhage. Standard treatment of obstetrical hemorrhage includes fluid replacement, transfusion, vasoactive therapy, and surgical intervention. This treatment can lead to hemodilution after hemorrhage, and consequently progressive coagulopathy, but unfortunately optimal hemostatic intervention(s) and coagulation monitoring are not yet standardized.