Molecular, morphological, and phytochemical evidence for a broad species concept of Plagiochila bifaria (Hepaticae)

Debate over the synonymy of the European Plagiochila killarniensis and the Neotropical P bifaria of R sect. Arrectae has focused on differences in secondary metabolite composition. The broad morphological species concept of R bifaria proposed in recent papers has now been tested by comparing nrDNA ITS1 and ITS2 sequences of R bifaria populations encompassing several different morpho- and chemotypes from the British Isles, Tenerife, Costa Rica, Brazil, Ecuador, and Bolivia, with sequences of other species of R sects. Arrectae, Rutilantes, and Fuscoluteae. Phylogenetic analyses demonstrate that specimens of P. bifaria form a well supported clade within Plagiochila sect. Arrectae. Sequences of R bifaria from the British Isles, Tenerife, and Ecuador, representing the "methyl everninate" chemotype, form a well supported subclade within the P bifaria clade. Sequences of specimens from Costa Rica, Brazil, and Bolivia are placed in the basal part of the R bifaria clade. The data support a broad species concept of P bifaria. The different chemotypes do not warrant distinct taxonomic ranks. Plagiochila centrifuga and P. compressula are treated as new synonyms of R bifaria

A mass spectrometry-guided genome mining approach for natural product peptidogenomics.

Peptide natural products show broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce here natural product peptidogenomics (NPP), a new MS-guided genome-mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo tandem MS (MS(n)) structures to genomics-based structures following biosynthetic logic. In this study, we show that NPP enabled the rapid characterization of over ten chemically diverse ribosomal and nonribosomal peptide natural products of previously unidentified composition from Streptomycete bacteria as a proof of concept to begin automating the genome-mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which are from well-characterized model Streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms

Influential factors in nectar composition and yield in Leptospermum scoparium : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science, Institute of Agriculture and the Environment, College of Sciences, Massey University, Palmerston North, New Zealand

Material omitted from digital version of thesis: Nickless, E. M., Anderson, C. W. N., Hamilton, G., Stephens, J. M., & Wargent, J. (2016). Soil influences on plant growth, floral density and nectar yield in three cultivars of manuka (Leptospermum scoparium). New Zealand Journal of Botany, 55(2), 100-117. doi:10.1080/0028825X.2016.1247732 ; Nickless, E. M., Holroyd, S. E., Stephens, J. M., Gordon, K. C., & Wargent, J. J. (2014). Analytical FT-Raman spectroscopy to chemotype Leptospermum scoparium and generate predictive models for screening for dihydroxyacetone levels in floral nectar. Journal of Raman Spectroscopy, 45(10), 890-894. doi:10.1002/jrs.4576 ; Nickless, E. M., Holroyd, S. E., Hamilton, G., Gordon, K. C., & Wargent, J. J. (2016). Analytical method development using FTIR-ATR and FT-Raman spectroscopy to assay fructose, sucrose, glucose and dihydroxyacetone, in Leptospermum scoparium nectar. Vibrational Spectroscopy, 84 (2016), 38-43. doi:10.1016/j.vibspec.2016.02.011Leptospermum scoparium (Mānuka) is the plant nectar source for medically bioactive honey, commercially marketed in New Zealand as Unique Mānuka Factor honey (UMF-honey). Methylglyoxal (MGO) is the unique bioactive component of UMF honey with Mānuka nectar containing significant amounts of the carbohydrate dihydroxyacetone (DHA), the chemical precursor for MGO. Anecdotal evidence and recently published data from nectar samples collected from various cultivars in natural sites or botanical gardens has indicated that the DHA and overall composition of L. scoparium nectar varies according to cultivar. The source of this variation is not clearly understood and although there is considerable literature on climatic and genetic influences on nectar composition and yield within various other plant species, there is little published work available on the influence of genetic and environmental factors on the composition and yield of nectar in L. scoparium. Of value to the commercial UMF honey industry in New Zealand is the ability to assess cultivars from breeding programs for the best potential to increase overall UMF honey yield. Predictive modelling of yields is invaluable to the developing honey industry to allow assessment of environmental influences that may affect overall yield along with seasonal influences on nectar production in Mānuka. The research in this thesis establishes the effect of various parameters on overall DHA yield from Mānuka and the beginnings of modelling influencing environmental factors. To determine influences on dihydroxyacetone (DHA) concentration and yield in the nectar of L. scoparium a number of studies were carried out. Methodologies for the collection and analysis of nectar were established. Ten different cultivars of L. scoparium with a range of genetic parentage were studied in controlled glasshouse conditions to assess phenotypic variability in terms of nectar composition and yield as well as plant growth and flowering amongst these cultivars. Significant differences in plant growth and flowering habits were observed amongst the ten cultivars, significant differences in nectar yield and nectar composition with regard to DHA yield were also observed. DHA yields ranged from 2714-7459 mg of DHA/kilogram normalised to 80 oBRIX, with total nectar sugar yields ranging between 0.7 and 4.8 mg amongst the ten cultivars studied. Preliminary research into the effect of temperature, radiation and humidity on nectar composition and yield were also undertaken. Effects of soil composition on these same parameters were researched with a subset of three of the ten cultivars grown on ten different soil types. Plant relative growth rates, dry weights and total plant height were measured throughout a 15 month glasshouse trial. Plant growth, flowering phenology, floral density, nectar yield and DHA composition data was gathered. Soils were analysed for various macronutrient and micronutrient levels and these parameters were modelled against plant data to determine which soil components were influencing plant parameters of interest. Soil type was shown to have no significant effect on DHA concentrations in nectar but results did show that soil type had a significant effect on flowering density amongst the three L. scoparium cultivars studied in the trial. Results from regression analysis of soil chemistry against measured plant parameters indicate that a fertiliser regime has the potential to increase nectar yields due to increased flower numbers. Multivariate analysis using partial least squares regression of soil composition data against plant parameters of value showed that soil components; phosphorus, sulphate, ferric and chloride were commonly shown to influence plant parameters measured. Analytical spectroscopy was investigated as a method to chemotype L. scoparium cultivars and also as a method for quantifying nectar components sucrose, glucose, fructose and DHA. Nectar composition was analysed using high pressure liquid chromatography (HPLC) and compared with fourier transform Raman spectroscopy (FT-Raman) and attenuated total reflectance infrared spectroscopy (ATR-FTIR) analytical spectroscopy methods. FT-Raman spectroscopy was shown to be useful in chemotyping cultivars and in addition proved to be a useful analytical method to predict DHA yield using leaf material from L. scoparium plants from the ten cultivars. FT-Raman and ATR-FTIR proved to be relatively accurate techniques to quantify L. scoparium nectar components DHA, fructose, glucose and sucrose, compared with HPLC methods which use extensive preparation techniques. R-squared values were very good for all nectar components measured excepting the sucrose model at R2 = 0.77. The R2 for the FT-Raman predictions of DHA against HPLC data are very good at 0.85. FTIR prediction data against HPLC data was also good at 0.86 R2. Overall an accurate model is possible for quantifying DHA concentrations in nectar using both FTIR-ATR and FT-Raman spectroscopy. Overall results show that various factors need to be considered when assessing plants for commercial use in the (UMF) Mānuka honey industry within New Zealand. Due to their large impact on overall nectar yield; floral density and plant growth rate parameters are the two key factors of value for commercial assessment of Mānuka cultivars. This research also highlights the importance of assessing not just DHA concentration in deducing cultivar value, but overall nectar yield. These key features must be explored when assessing L. scoparium plants within breeding programs, prior to selection for large-scale field production of high UMF Mānuka honey

1,2,6-thiadiazinones as novel narrow spectrum calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) inhibitors

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors

Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases.

The clinical success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chemical synthesis, X-ray crystallography and kinome-level biochemical profiling, we identified compounds that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these molecules is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that one compound, PP121, blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These molecules demonstrate the feasibility of accessing a chemical space that intersects two families of oncogenes

Diversity of Lecidea (Lecideaceae, Ascomycota) species revealed by molecular data and morphological characters

The diversity of lichens, especially crustose species, in continental Antarctica is still poorly known. To overcome difficulties with the morphology based species delimitations in these groups, we employed molecular data (nuclear ITS and mitochondrial SSU rDNA sequences) to test species boundaries within the genus Lecidea. Sampling was done along a north–south transect at five different areas in the Ross Sea region (Cape Hallett, Botany Bay to Mount Suess, Taylor Valley, Darwin Area and Mount Kyffin). A total of 153 specimens were collected from 13 localities. Phylogenetic analyses also include specimens from other regions in Antarctica and non-Antarctic areas. Maximum parsimony, maximum likelihood and Bayesian analyses agreed in placing the samples from continental Antarctica into four major groups. Based on this phylogenetic estimate, we restudied the micromorphology and secondary chemistry of these four clades to evaluate the use of these characters as phylogenetic discriminators. These clades are identified as the following species Lecidea cancriformis, L. andersonii as well as the new species L. polypycnidophora Ruprecht & Türk sp. nov. and another previously unnamed clade of uncertain status, referred to as Lecidea sp. (L. UCR1)

Impact of environmental factors on growth and satratoxin G production by strains of Stachybotrys chartarum

The black mould Stachybotrys chartarum and its mycotoxins have been linked to damp building-associated illnesses. The objective of this study was to determine the effects of water availability (water activity, aw) and temperature on growth and production of satratoxin G (SG) by a macrocyclic trichothecene-producing strain (IBT 7711) and non-producing strain (IBT 1495) of S. chartarum. Growth studies were carried out on potato dextrose agar modified with glycerol to 0.995-0.92 aw at 10-37 °C. Growth extension was measured and the cultures were extracted after 10 days and a competitive enzyme-linked immunosorbent assay (ELISA) method used to quantify the SG content. Growth was optimal at 25 to 30 °C at 0.995 aw, but this was modified to 0.98 aw at 30 °C for both strains (1.4- 1.6 mm/day, respectively). The ELISA method revealed that, in contrast to growth, SG production was maximal at 20 °C with highest production at 0.98 aw (approximately 250 μg/g mycelia). When water was freely available (0.995 aw), SG was maximally produced at 15 °C and decreased as temperature was increased. Interestingly, the strain classified as a non-toxigenic produced very low amounts of SG (<1.6 μg/g mycelia) that were maximal at 25 °C and 0.98 aw. Contour maps for growth and SG production were developed from these data sets. These data have shown, for the first time, that growth and SG production profiles are very different in relation to key environmental conditions in the indoor environment. This will be very useful in practically determining the risk from exposure to S. chartarum and its toxins in the built env