Chemical and Biological Aspects of Secondary Metabolites from Tongan Marine Sponges

<p>This thesis describes the isolation and structural elucidation of 17 new secondary metabolites from Tongan marine sponges including examples of alkaloids, polyketides and terpenoids. In the process of this work, 19 sponge specimens were subjected to preliminary NMR-guided investigation. Nine organisms were selected for further analysis on the basis of the structural novelty perceived within the HMBC spectrum of crude fractions generated by the first chromatographic purification of their crude extracts, and the apparent rarity of the specimen. Investigation of two different demosponge specimens afforded the γ-hydroxybutenolide sesterterpenes (23 and 24), and small quantities of the potently cytotoxic alkaloid 14-bromohomofascaplysin (29). The analysis of two samples of a dictyoceratid sponge yielded the new labdane diterpenes luakuliides A–C (33–36), characterised by a bridging hemi-acetal function on the B-ring of the labdane bicycle. Luakuliide A (33) and its methyl acetal derivative 34 were found to display interesting immunomodulatory activity. Seven new α-pyrone polyketides, lehualides E–K (69–75), were isolated from a Plakortis sp. Lehualides H–K (72–75) display a range of sulfur functionalities, the natures of which were determined by spectroscopic comparison with synthesised model compounds. Another plakinid sponge specimen contained four new polyketides (95–98), all of which possess different cyclic peroxide moieties. Cyclic peroxides 95, 97 and 98 displayed potent cytoxicity against human promyelocytic leukemia cells (HL- 60). Chemical genetic and phenoytypic profiling studies of 95 were undertaken in Saccharomyces cerevisiae yeast using the homozygous diploid and heterozygous diploid deletion libraries. These studies indicate that 95 acts to disrupt Ca2+ homeostasis, leading to elevation of intracellular Ca2 levels.</p>

Chemical and Biological Aspects of Secondary Metabolites from Tongan Marine Sponges

<p>This thesis describes the isolation and structural elucidation of 17 new secondary metabolites from Tongan marine sponges including examples of alkaloids, polyketides and terpenoids. In the process of this work, 19 sponge specimens were subjected to preliminary NMR-guided investigation. Nine organisms were selected for further analysis on the basis of the structural novelty perceived within the HMBC spectrum of crude fractions generated by the first chromatographic purification of their crude extracts, and the apparent rarity of the specimen. Investigation of two different demosponge specimens afforded the γ-hydroxybutenolide sesterterpenes (23 and 24), and small quantities of the potently cytotoxic alkaloid 14-bromohomofascaplysin (29). The analysis of two samples of a dictyoceratid sponge yielded the new labdane diterpenes luakuliides A–C (33–36), characterised by a bridging hemi-acetal function on the B-ring of the labdane bicycle. Luakuliide A (33) and its methyl acetal derivative 34 were found to display interesting immunomodulatory activity. Seven new α-pyrone polyketides, lehualides E–K (69–75), were isolated from a Plakortis sp. Lehualides H–K (72–75) display a range of sulfur functionalities, the natures of which were determined by spectroscopic comparison with synthesised model compounds. Another plakinid sponge specimen contained four new polyketides (95–98), all of which possess different cyclic peroxide moieties. Cyclic peroxides 95, 97 and 98 displayed potent cytoxicity against human promyelocytic leukemia cells (HL- 60). Chemical genetic and phenoytypic profiling studies of 95 were undertaken in Saccharomyces cerevisiae yeast using the homozygous diploid and heterozygous diploid deletion libraries. These studies indicate that 95 acts to disrupt Ca2+ homeostasis, leading to elevation of intracellular Ca2 levels.</p>

A genome-wide portrait of pervasive drug contaminants

Using a validated yeast chemogenomic platform, we characterized the genome-wide effects of several pharmaceutical contaminants, including three N-nitrosamines (NDMA, NDEA and NMBA), two related compounds (DMF and 4NQO) and several of their metabolites. A collection of 4800 non-essential homozygous diploid yeast deletion strains were screened in parallel and the strain abundance was quantified by barcode sequencing. These data were used to rank deletion strains representing genes required for resistance to the compounds to delineate affected cellular pathways and to visualize the global cellular effects of these toxins in an easy-to-use searchable database. Our analysis of the N-nitrosamine screens uncovered genes (via their corresponding homozygous deletion mutants) involved in several evolutionarily conserved pathways, including: arginine biosynthesis, mitochondrial genome integrity, vacuolar protein sorting and DNA damage repair. To investigate why NDMA, NDEA and DMF caused fitness defects in strains lacking genes of the arginine pathway, we tested several N-nitrosamine metabolites (methylamine, ethylamine and formamide), and found they also affected arginine pathway mutants. Notably, each of these metabolites has the potential to produce ammonium ions during their biotransformation. We directly tested the role of ammonium ions in N-nitrosamine toxicity by treatment with ammonium sulfate and we found that ammonium sulfate also caused a growth defect in arginine pathway deletion strains. Formaldehyde, a metabolite produced from NDMA, methylamine and formamide, and which is known to cross-link free amines, perturbed deletion strains involved in chromatin remodeling and DNA repair pathways. Finally, co-administration of N-nitrosamines with ascorbic or ferulic acid did not relieve N-nitrosamine toxicity. In conclusion, we used parallel deletion mutant analysis to characterize the genes and pathways affected by exposure to N-nitrosamines and related compounds, and provide the data in an accessible, queryable database.

Identification of Genetic Modifiers of TDP-43: Inflammatory Activation of Astrocytes for Neuroinflammation

Transactive response DNA-binding protein 43 (TDP-43) is a ubiquitously expressed DNA/RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 has been implicated in numerous aspects of the mRNA life cycle, as well as in cell toxicity and neuroinflammation. In this study, we used the toxicity of the TDP-43 expression in Saccharomyces cerevisiae as an assay to identify TDP-43 genetic interactions. Specifically, we transformed human TDP-43 cDNAs of wild-type or disease-associated mutants (M337V and Q331K) en masse into 4,653 homozygous diploid yeast deletion mutants and then used next-generation sequencing readouts of growth to identify yeast toxicity modifiers. Genetic interaction analysis provided a global view of TDP-43 pathways, some of which are known to be involved in cellular metabolic processes. Selected putative loci with the potential of genetic interactions with TDP-43 were assessed for associations with neurotoxicity and inflammatory activation of astrocytes. The pharmacological inhibition of succinate dehydrogenase flavoprotein subunit A (SDHA) and voltage-dependent anion-selective channel 3 (VDAC3) suppressed TDP-43-induced expression of proinflammatory cytokines in astrocytes, indicating the critical roles played by SDHA and VDAC3 in TDP-43 pathways during inflammatory activation of astrocytes and neuroinflammation. Thus, the findings of our TDP-43 genetic interaction screen provide a global landscape of TDP-43 pathways and may help improve our understanding of the roles of glia and neuroinflammation in ALS and FTD pathogenesis.

Comparative Analysis of Morphology, Photosynthetic Physiology, and Transcriptome Between Diploid and Tetraploid Barley Derived From Microspore Culture

Polyploids play an important role in the breeding of plant for superior characteristics, and many reports have focused on the effects upon photosynthesis from polyploidization in some plant species recently, yet surprisingly little of this is known for barley. In this study, homozygous diploid and tetraploid plants, derived from microspore culturing of the barley cultivar “H30,” were used to assess differences between them in their cellular, photosynthetic, and transcriptomic characteristics. Our results showed that tetraploid barley has the distinct characteristics of polyploids, namely thicker and heavier leaves, enlarged stomata size or stomatal guard cell size, and more photosynthetic pigments and improved photosynthesis (especially under high light intensity). This enhanced photosynthesis of tetraploid barley was confirmed by several photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), maximum net photosynthetic rate (Pmax), light saturation point (LSP), maximum RuBP saturated rate carboxylation (Vcmax), and maximum rate of electron transport (Jmax). Transcriptomic analyses revealed that just ~2.3% of all detected genes exhibited differential expression patterns [i.e., differentially expressed genes (DEGs)], and that most of these – 580 of 793 DEGs in total – were upregulated in the tetraploid barley. The follow-up KEGG analysis indicated that the most enriched pathway was related to photosynthesis-antenna proteins, while the downregulation of DEGs was related mainly to the light-harvesting cholorophyII a/b-binding protein (Lhcb1) component, both validated by quantitative PCR (qPCR). Taken together, our integrated analysis of morphology, photosynthetic physiology, and transcriptome provides evidences for understanding of how polyploidization enhances the photosynthetic capacity in tetraploids of barley.

Solyntus, the New Highly Contiguous Reference Genome for Potato (Solanum tuberosum)

With the rapid expansion of the application of genomics and sequencing in plant breeding, there is a constant drive for better reference genomes. In potato (Solanum tuberosum), the third largest food crop in the world, the related species S. phureja, designated “DM”, has been used as the most popular reference genome for the last 10 years. Here, we introduce the de novo sequenced genome of Solyntus as the next standard reference in potato genome studies. A true Solanum tuberosum made up of 116 contigs that is also highly homozygous, diploid, vigorous and self-compatible, Solyntus provides a more direct and contiguous reference then ever before available. It was constructed by sequencing with state-of-the-art long and short read technology and assembled with Canu. The 116 contigs were assembled into scaffolds to form each pseudochromosome, with three contigs to 17 contigs per chromosome. This assembly contains 93.7% of the single-copy gene orthologs from the Solanaceae set and has an N50 of 63.7 Mbp. The genome and related files can be found at https://www.plantbreeding.wur.nl/Solyntus/. With the release of this research line and its draft genome we anticipate many exciting developments in (diploid) potato research.

Regeneration of double haploid plants from unpollinated ovary cultures of watermelon

Background: Watermelon (Citrullus lanatus), a major fresh fruit, is planted worldwide. Because double haploid plants may be used as parents to shorten watermelon breeding cycle, the present study optimized conditions for regenerating haploid plants from ovaries without pollination.Results: The results revealed that, the 10% sodium hypochlorite sterilized for 10 min is best for ovary enlargement. In addition, a dark culture period of 14 days promoted the ovary enlargement. The MS medium containing 0.5 mg/L NAA, 1.0 mg/L 6-BA and 0.5mg/L KT promoted the embryoid differentiation. The M2 medium containing 0.02 mg/L TDZ, 0.5 mg/L NAA, 0.5 mg/L 6-BA could be used for producing complete plants. The different genotypes affected the embryoid induction. The present study obtained regenerated plants that were established in field. Flow cytometry analyses revealed that the regenerated plants were haploid, diploid or tetraploid plant. The seedlings which were treated with culture medium can increase the chance of chromosome doubling. The SSR marker analyses showed that the diploid and tetraploid plants were homozygous at all six loci tested, indicating that these regenerated plants were double- or tetra-haploid plants.Conclusions: Haploid and homozygous diploid can be obtained through the culture of unpollinated ovary of watermelon, which is an effective way to innovate watermelon germplasm. The present study provides homozygous plants for future watermelon breeding.

Dissection of yeast pleiotropic drug resistance regulation reveals links between cell cycle regulation and control of drug pump expression

SummaryEukaryotes utilize a highly-conserved set of drug efflux transporters to confer pleiotropic drug resistance (PDR). Despite decades of effort interrogating this process, multiple aspects of the PDR process, in particular PDR regulation, remain mysterious. In order to interrogate the regulation of this critical process, we have developed a small-molecule responsive biosensor that couples PDR transcriptional induction to growth rate in Saccharomyces cerevisiae. We applied this system to genome-wide screens for potential PDR regulators using the homozygous diploid deletion collection. These screens identified and characterized a series of genes with significant but previously uncharacterized roles in the modulation of the yeast PDR in addition to recapitulating previously-known factors involved in PDR regulation. Furthermore, we demonstrate that disruptions of the mitotic spindle checkpoint assembly lead to elevated PDR response in response to exposure to certain compounds. These results not only establish our biosensor system as a viable tool to investigate PDR in high-throughput, but also uncovers novel control mechanisms governing PDR response and a previously uncharacterized link between this process and cell cycle regulation.SignificancePleiotropic drug resistance (PDR) is a conserved mechanism by which cells utilize membrane bound pumps to transport chemicals out of the cell. Here, we develop a growth-based biosensor system in yeast that enables high-throughput identification of factors that transcriptionally regulate PDR. Among the novel PDR regulators identified here, we show that spindle assembly checkpoint (SAC) proteins, which are important for cell cycle regulation, inhibit hyperactivation of PDR upon drug treatment. This result provides insights into PDR regulation, as well as potential targets for therapeutic intervention, particularly in chemoresistant cancers where the cell cycle regulation is often disrupted.