• The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis.

      Chen, Daliang; Janganan, Thamarai K.; Chen, Gongyou; Marques, Everaldo R.; Kress, Marcia R.; Goldman, Gustavo H.; Walmsley, Adrian R.; Borges-Walmsley, Maria Ines; University of Durham; Universidade de Sao Paulo (Wiley, 2007-08-30)
      Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Galpha (Gpa1-3), Gbeta (Gpb1) and Ggamma (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch.
    • Cellular mechanisms governing glucose-dependent insulinotropic polypeptide secretion.

      Reimann, Frank; Diakogiannaki, Eleftheria; Moss, Catherine E.; Gribble, Fiona M.; Wellcome Trust; University of Cambridge (Elsevier, 2020-11-19)
      Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone secreted from the upper small intestine, which plays an important physiological role in the control of glucose metabolism through its incretin action to enhance glucose-dependent insulin secretion. GIP has also been implicated in postprandial lipid homeostasis. GIP is secreted from enteroendocrine K-cells residing in the intestinal epithelium. K-cells sense a variety of components found in the gut lumen following food consumption, resulting in an increase in plasma GIP signal dependent on the nature and quantity of ingested nutrients. We review the evidence for an important role of sodium-coupled glucose uptake through SGLT1 for carbohydrate sensing, of free-fatty acid receptors FFAR1/FFAR4 and the monoacyl-glycerol sensing receptor GPR119 for lipid detection, of the calcium-sensing receptor CASR and GPR142 for protein sensing, and additional modulation by neurotransmitters such as somatostatin and galanin. These pathways have been identified through combinations of in vivo, in vitro and molecular approaches.
    • Changes of synovial fluid protein concentrations in supra-patellar bursitis patients after the injection of different molecular weights of hyaluronic acid

      Chen, Carl P.C.; Hsu, Chin Chin; Pei, Yu-Cheng; Chen, Ruoli; Zhou, Shaobo; Shen, Hsuan-Chen; Lin, Shih-Cherng; Tsai, Wen-Chung; ; Chang Gung University; et al. (Elsevier, 2014-01-30)
      Knee pain is commonly seen in orthopedic and rehabilitation outpatient clinical settings, and in the aging population. Bursitis of the knee joint, especially when the volume of the synovial fluid is large enough, can compress and distend the nearby soft tissues, causing pain in the knee joint. Out of all the bursae surrounding the knee joint, supra-patellar bursitis is most often associated with knee pain. Treatment strategies in managing supra-patellar bursitis include the aspiration of joint synovial fluid and then followed by steroid injection into the bursa. When supra-patellar bursitis is caused by degenerative disorders, the concept of viscosupplementation treatment may be effective by injecting hyaluronic acid into the bursa. However, the rheology or the changes in the concentrations of proteins (biomarkers) that are related to the development of bursitis in the synovial fluid is virtually unexplored. Therefore, this study aimed to identify the concentration changes in the synovial fluid total protein amount and individual proteins associated with supra-patellar bursitis using the Bradford protein assay and western immunoglobulin methods. A total of 20 patients were divided into two groups with 10 patients in each group. One group received the high molecular weight hyaluronic acid product of Synvisc Hylan G-F 20 and the other group received the low molecular weight hyaluronic acid product of Hya-Joint Synovial Fluid Supplement once per week injection into the bursa for a total of 3. weeks. Significant decreases in the synovial fluid total protein concentrations were observed after the second dosage of high molecular weight hyaluronic acid injections. Apolipoprotein A-I, interleukin 1 beta, alpha 1 antitrypsin, and matrix metalloproteinase 1 proteins revealed a trend of decreasing western immunoblotting band densities after hyaluronic acid injections. The decreases in apolipoprotein A-I and interleukin 1 beta protein band densities were significant in the high molecular weight hyaluronic acid injection group. Transthyretin, complement 5, and matrilin 3 proteins revealed a trend of increasing western immunoblotting band densities after hyaluronic acid injections. Transthyretin revealed significant increases in protein band densities in both the high and low molecular weight hyaluronic acid injection groups. This study may provide the rationale for targeting several biomarkers associated with lipid transport, inflammation, and anti-aging as possible disease modifying therapies for the treatment of supra-patellar bursitis and even degenerative joint disorders. © 2014 Elsevier B.V.
    • Characterisation of hepcidin response to holotransferrin treatment in CHO TRVb-1 cells

      Mehta, Kosha; Greenwell, Pamela; Renshaw, Derek; Busbridge, Mark; Garcia, Mitla; Farnaud, Sébastien; Patel, Vinood B.; University of Westminster; Coventry University; Imperial College Healthcare NHS Trust; et al. (Elsevier, 2015-08-28)
      Iron overload coupled with low hepcidin levels are characteristics of hereditary haemochromatosis. To understand the role of transferrin receptor (TFR) and intracellular iron in hepcidin secretion, Chinese hamster ovary transferrin receptor variant (CHO TRVb-1) cells were used that express iron-response-element-depleted human TFRC mRNA (TFRC∆IRE). Results showed that CHO TRVb-1 cells expressed higher basal levels of cell-surface TFR1 than HepG2 cells (2.2-fold; p < 0.01) and following 5 g/L holotransferrin treatment maintained constitutive over-expression at 24h and 48 h, contrasting the HepG2 cells where the receptor levels significantly declined. Despite this, the intracellular iron content was neither higher than HepG2 cells nor increased over time under basal or holotransferrin-treated conditions. Interestingly, hepcidin secretion in CHO TRVb-1 cells exceeded basal levels at all time-points (p < 0.02) and matched levels in HepG2 cells following treatment. While TFRC mRNA expression showed expected elevation (2h, p < 0.03; 4h; p < 0.05), slc40a1 mRNA expression was also elevated (2 h, p < 0.05; 4 h, p < 0.03), unlike the HepG2 cells. In conclusion, the CHO TRVb-1 cells prevented cellular iron-overload by elevating slc40a1 expression, thereby highlighting its significance in the absence of iron-regulated TFRC mRNA. Furthermore, hepcidin response to holotransferrin treatment was similar to HepG2 cells and resembled the human physiological response.
    • Characterization of a two-component signal transduction system that controls arsenite oxidation in the chemolithoautotroph NT-26

      Sardiwal, Sunita; Santini, Joanne M.; Osborne, Thomas H.; Djordjevic, Snezana; University College London (Wiley, 2010-10-07)
      NT-26 is a chemolithoautotrophic arsenite oxidizer. Understanding the mechanisms of arsenite signalling, tolerance and oxidation by NT-26 will have significant implications for its use in bioremediation and arsenite sensing. We have identified the histidine kinase (AroS) and the cognate response regulator (AroR) involved in the arsenite-dependent transcriptional regulation of the arsenite oxidase aroBA operon. AroS contains a single periplasmic sensory domain that is linked through transmembrane helices to the HAMP domain that transmits the signal to the kinase core of the protein. AroR belongs to a family of AAA+ transcription regulators that interact with DNA through a helix-turn-helix domain. The presence of the AAA+ domain as well as the RNA polymerase σ(54) -interaction sequence motif suggests that this protein regulates transcription through interaction with RNA polymerase in a σ(54) -dependent fashion. The kinase core of AroS and the receiver domain of AroR were heterologously expressed and purified and their autophosphorylation and transphosphorylation activities were confirmed. Using site-directed mutagenesis, we have identified the phosphorylation sites on both proteins. Mutational analysis in NT-26 confirmed that both proteins are essential for arsenite oxidation and the AroS mutant affected growth with arsenite, also implicating it in the regulation of arsenite tolerance. Lastly, arsenite sensing does not appear to involve thiol chemistry.
    • Characterization of Colletotrichum gloeosporioides, as the main causal agent of citrus anthracnose, and C. karstii as species preferentially associated with lemon twig dieback in Portugal

      Ramos, Ana Paula; Talhinhas, Pedro; Sreenivasaprasad, Surapareddy; Oliveira, Helena (Springer Netherlands, 2016-09-13)
      In the last two decades significant losses in citrus production in Portugal related to anthracnose symptoms have been recorded. These symptoms were attributed to Colletotrichum gloeosporioides, but preliminary population diversity evidence suggested that other Colletotrichum species could be involved in the disease. In this work, a field survey of the main citrus growing areas in Portugal was conducted and the pathogenicity of a group of Colletotrichum spp. isolates was studied along with morphological and genetic variability characterization [including Inter-Simple Sequence Repeats (ISSR) profiles and DNA sequence data of rDNA-ITS and β-tubulin 2 gene regions]. Colletotrichum karstii (from the C. boninense species complex) and C. gloeosporioides were identified from symptoms on leaves, branches, flowers and fruits of several citrus cultivars. However, C. acutatum, the species most commonly associated with citrus anthracnose in the Americas, was never detected. While C. gloeosporioides was isolated at higher frequency overall (87 %), C. karstii was more frequent in branches and leaves of lemon in specific geographic locations. Only C. gloeosporioides was detected in flowers. Colletotrichum karstii and C. gloeosporioides were pathogenic to sweet orange flowers and fruits and to leaves of sweet orange, mandarin and lemon, while reference C. acutatum citrus isolates were pathogenic to Key lime flowers and leaves.
    • Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells

      Mehta, Kosha; Busbridge, Mark; Renshaw, Derek; Evans, Robert W.; Farnaud, Sébastien; Patel, Vinood B.; University of Westminster; Imperial College Healthcare NHS Trust; Coventry University; Brunel University; et al. (Elsevier, 2016-06-30)
      Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.
    • Characterization of Linaria KNOX genes suggests a role in petal-spur development

      Box, Mathew S.; Dodsworth, Steven; Rudall, Paula J.; Bateman, Richard M.; Glover, Beverley J. (Wiley, 2011-07-25)
      Spurs are tubular outgrowths of perianth organs that have evolved iteratively among angiosperms. They typically contain nectar and often strongly influence pollinator specificity, potentially mediating reproductive isolation. The identification of Antirrhinum majus mutants with ectopic petal spurs suggested that petal-spur development is dependent on the expression of KNOTTED 1-like homeobox (KNOX) genes, which are better known for their role in maintaining the shoot apical meristem. Here, we tested the role of KNOX genes in petal spur development by isolating orthologs of the A. majus KNOX genes Hirzina (AmHirz) and Invaginata (AmIna) from Linaria vulgaris, a related species that differs from A. majus in possessing long, narrow petal spurs. We name these genes LvHirz and LvIna, respectively. Using quantitative reverse-transcription PCR, we show that LvHirz is expressed at high levels in the developing petals and demonstrate that the expression of petal associated KNOX genes is sufficient to induce sac-like outgrowths on petals in a heterologous host. We propose a model in which KNOX gene expression during early petal-spur development promotes and maintains further morphogenetic potential of the petal, as previously described for KNOX gene function in compound leaf development. These data indicate that petal spurs could have evolved by changes in regulatory gene expression that cause rapid and potentially saltational phenotypic modifications. Given the morphological similarity of spur ontogeny in distantly related taxa, changes in KNOX gene expression patterns could be a shared feature of spur development in angiosperms.
    • Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

      Janganan, Thamarai K.; Mullin, Nic; Tzokov, Svetomir B.; Stringer, Sandra; Fagan, Robert P.; Hobbs, Jamie K.; Moir, Anne; Bullough, Per A.; Institute of Food Research; University of Sheffield (Elsevier, 2016-06-06)
      Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.
    • cis-(Benzenethiolato)bis(tert-butyl isocyanide)carbonyl(cyclopentadienyl)molybdenum(II), [Mo(C6H5S)(C5H9N)(2)-(C5H5)(CO)]

      McDermott, Lindsay C.; Muir, K.W.; Petillon, F.Y.; PoderGuillou, S.; Schollhammer, P. (Wiley, 1996-01-15)
    • Cleavage of mer tyrosine kinase (MerTK) from the cell surface contributes to the regulation of retinal phagocytosis

      Law, Ah-Lai; Parinot, Celia; Chatagnon, Jonathan; Gravez, Basile; Sahel, Jose-Alain; Bhattacharya, Shomi S.; Nandrot, Emeline F.; ; Sorbonne Universite; University College London; et al. (American Society for Biochemistry and Molecular Biology Inc., 2014-12-23)
      Background: The MerTK receptor is necessary for retinal phagocytosis and its daily rhythm. Results: MerTK is cleaved from the apical cell surface in vitro and in vivo, reducing the phagocytic capacity of RPE cells. Conclusion: MerTK cleavage might help control the duration of the daily phagocytic peak. Significance: Our data show that extracellular cleavage of MerTK partly regulates retinal phagocytosis.
    • The climatic challenge: which plants will people use in the next century?

      Borrell, J.S.; Dodsworth, Steven; Forest, Felix; Pérez-Escobar, Oscar Alejandro; Lee, M.A.; Mattana, E.; Stevenson, P.C.; Howes, M.-J.R.; Pritchard, Hugh W.; Ballesteros, D.; et al. (Elsevier, 2019-08-30)
      More than 31,000 useful plant species have been documented to fulfil needs and services for humans or the animals and environment we depend on. Despite this diversity, humans currently satisfy most requirements with surprisingly few plant species; for example, just three crops – rice, wheat and maize – comprise more than 50% of plant derived calories. Here, we synthesize the projected impact of global climatic change on useful plants across the spectrum of plant domestication. We illustrate the demographic, spatial, ecophysiological, chemical, functional, evolutionary and cultural traits that are likely to characterise useful plants and their resilience in the next century. Using this framework, we consider a range of possible pathways for future human use of plants. These are centred on two trade-offs: i) diversification versus specialization in the range of species we utilize, and ii) substitutionof the species towards those better suited to future climate versus facilitating adaptation in our existing suite of dominant useful plants. In the coming century, major challenges to agriculture and biodiversity will be dominated by increased climatic variation, shifting species ranges, disruption to biotic interactions, nutrient limitation and emerging pests and pathogens. These challenges must be mitigated, whilst enhancing sustainable production to meet the needs of a growing population and a more resource intensive standard of living. With the continued erosion of biodiversity, our future ability to choose among these pathways and trade-offs is likely to be diminished.
    • Cold-adapted arsenite oxidase from a psychrotolerant Polaromonas species

      Osborne, Thomas H.; Heath, Matthew D.; Martin, Andrew C.R.; Pankowski, Jaroslaw A.; Hudson-Edwards, Karen A.; Santini, Joanne M.; University College London; Birkbeck, University of London (Royal Society of Chemistry, 2012-10-31)
      Polaromonas sp. str. GM1 is an aerobic, psychrotolerant, heterotrophic member of the Betaproteobacteria and is the only isolate capable of oxidising arsenite at temperatures below 10 °C. Sequencing of the aio gene cluster in GM1 revealed the presence of the aioB and aioA genes, which encode the arsenite oxidase but the regulatory genes typically found upstream of aioB in other members of the Proteobacteria were absent. The GM1 Aio was purified to homogeneity and was found to be a heterodimer. The enzyme contained Mo and Fe as cofactors and had, using the artificial electron acceptor 2,6-dichlorophenolindophenol, a Km for arsenite of 111.70 ± 0.88 μM and a Vmax of 12.16 ± 0.30 U mg(-1), which is the highest reported specific activity for any known Aio. The temperature-activity profiles of the arsenite oxidases from GM1 and the mesophilic betaproteobacterium Alcaligenes faecalis were compared and showed that the GM1 Aio was more active at low temperatures than that of A. faecalis. A homology model of the GM1 Aio was made using the X-ray crystal structure of the Aio from A. faecalis as the template. Structural changes that account for cold adaptation were identified and it was found that these resulted in increased enzyme flexibility and a reduction in the hydrophobicity of the core.
    • Combination of Sanger and target-enrichment markers supports revised generic delimitation in the problematic ‘Urera clade’ of the nettle family (Urticaceae)

      Wells, Tom; Maurin, Olivier; Dodsworth, Steven; Friis, Ib; Cowan, Robyn S.; Epitawalage, Niroshini; Brewer, Grace E.; Forest, Felix; Baker, William; Monro, Alexandre; et al. (Elsevier, 2020-11-05)
      Urera Gaudich, s.l. is a pantropical genus comprising c. 35 species of trees, shrubs, and vines. It has a long history of taxonomic uncertainty, and is repeatedly recovered as polyphyletic within a poorly resolved complex of genera in the Urticeae tribe of the nettle family (Urticaceae). To provide generic delimitations concordant with evolutionary history, we use increased taxonomic and genomic sampling to investigate phylogenetic relationships among Urera and associated genera. A cost-effective two-tier genome-sampling approach provides good phylogenetic resolution by using (i) a taxon-dense sample of Sanger sequence data from two barcoding regions to recover clades of putative generic rank, and (ii) a genome-dense sample of target-enrichment data for a subset of representative species from each well-supported clade to resolve relationships among them. The results confirm the polyphyly of Urera s.l. with respect to the morphologically distinct genera Obetia, Poikilospermum and Touchardia. Afrotropic members of Urera s.l. are recovered in a clade sister to the xerophytic African shrubs Obetia; and Hawaiian ones with Touchardia, also from Hawaii. Combined with distinctive morphological differences between Neotropical and African members of Urera s.l., these results lead us to resurrect the previously synonymised name Scepocarpus Wedd. for the latter. The new species epiphet Touchardia oahuensis T.Wells &amp; A.K. Monro is offered as a replacement name for Touchardia glabra non H.St.John, and subgenera are created within Urera s.s. to account for the two morphologically distinct Neotropical clades. This new classification minimises taxonomic and nomenclatural disruption, while more accurately reflecting evolutionary relationships within the group.
    • Comparison of two reef sites on the north coast of Jamaica over a 15-year period

      Crabbe, M. James C.; University of Oxford; University of Bedfordshire (Scientific Research Publishing, 2016-03-12)
      This paper compares two reef sites near Discovery Bay, Jamaica, Dairy Bull and Dancing Lady, from 2000 to 2015. At Dairy Bull reef, with low macroalgal cover (8% in 2002 falling to 1% in 2015) and significant number of Diadema antillarum urchins (c. 5 m−2), live coral cover increased from 13% ± 5% in 2006 after the bleaching event in 2005, to 31% ± 7% in 2008, while live Acropora cervicornis increased from 2% ± 2% in 2006 to 28% ± 5% in 2015. Coral cover levels were at least maintained until 2015, owing mostly to a slight increase in A. cervicornis. Dancing Lady reef however was dominated by macroalgae throughout this period (cover of c. 76% ± 7%), with no D. antillarum and showed little decrease in the already low (6% ± 1%) coral cover in 2005. Growth rates for Siderastrea siderea were similar for both sites (7 mm∙yr−1), while growth rates of A. cervicornis and A. palmata were 120.0 ± 30 mm∙yr−1 and 71.0 ± 29 mm∙yr−1 respectively at Dairy Bull in 2015. At Dancing Lady reef, A. cervicornis colonies which were present from 2003 to 2005 had disappeared in 2006, possibly as a result of the mass bleaching event. It appears that A. cervicornis was the most impacted species during the 2005 bleaching event, but was also the species that recovered fastest after its decline at Dairy Bull.
    • Comparison study of oral iron preparations using a human intestinal model

      Zariwala, Mohammed Gulrez; Somavarapu, Satyanarayana; Farnaud, Sébastien; Renshaw, Derek; University of Westminster; University College London; University of Bedfordshire (Österreichischer Apothekerverlag, 2013-06-21)
      Iron deficiency and related iron deficiency anaemia (IDA) are the most prevalent nutritional disorders worldwide. The standard treatment involves supplementation with solid or liquid iron supplement preparations, usually based on a ferrous salt such as ferrous sulphate, ferrous fumarate, or ferrous gluconate. In the present study, we compared iron uptake and absorption from various solid and liquid iron supplement preparations currently available in the United Kingdom using the well-characterised human epithelial adenocarcinoma cell line Caco-2. Intracellular ferritin protein formation by the Caco-2 cell was considered an indicator of cellular iron uptake and absorption. We investigated the effects of formulation ingredients at a defined pH on iron uptake and absorption, and designed a novel two-stage dissolution-absorption protocol that mimicked physiological conditions. Our experiments revealed wide variations in the rate of dissolution between the various solid iron preparations. Conventional-release ferrous iron tablets dissolved rapidly (48 ± 4 mins to 64 ± 4 mins), whereas modified-released tablets and capsules took significantly longer to undergo complete dissolution (274 ± 8 to 256 ± 8 mins). Among the solid iron preparations, ferrous sulphate conventional-release tablets demonstrated the highest iron absorption, whereas modified-release ferrous preparations demonstrated uniformly low iron absorption, as compared to the control (P < 0.05). Taken together, our results demonstrate that there are wide-ranging variations in dissolution times and iron uptake from oral iron preparations, with the physical characteristics of the preparation as well as the form of iron playing a key role.
    • A comprehensive phylogenomic platform for exploring the angiosperm tree of life

      Baker, William J.; Bailey, Paul; Barber, Vanessa; Barker, Abigail; Bellot, Sidonie; Bishop, David; Botigué, Laura R.; Brewer, Grace E.; Carruthers, Tom; Clarkson, James J.; et al. (Oxford University Press, 2021-05-13)
      The tree of life is the fundamental biological roadmap for navigating the evolution and properties of life on Earth, and yet remains largely unknown. Even angiosperms (flowering plants) are fraught with data gaps, despite their critical role in sustaining terrestrial life. Today, high-throughput sequencing promises to significantly deepen our understanding of evolutionary relationships. Here, we describe a comprehensive phylogenomic platform for exploring the angiosperm tree of life, comprising a set of open tools and data based on the 353 nuclear genes targeted by the universal Angiosperms353 sequence capture probes. The primary goals of this paper are to (i) document our methods, (ii) describe our first data release and (iii) present a novel open data portal, the Kew Tree of Life Explorer (https://treeoflife.kew.org ). We aim to generate novel target sequence capture data for all genera of flowering plants, exploiting natural history collections such as herbarium specimens, and augment it with mined public data. Our first data release, described here, is the most extensive nuclear phylogenomic dataset for angiosperms to date, comprising 3,099 samples validated by DNA barcode and phylogenetic tests, representing all 64 orders, 404 families (96%) and 2,333 genera (17%). A "first pass" angiosperm tree of life was inferred from the data, which totalled 824,878 sequences, 489,086,049 base pairs, and 532,260 alignment columns, for interactive presentation in the Kew Tree of Life Explorer. This species tree was generated using methods that were rigorous, yet tractable at our scale of operation. Despite limitations pertaining to taxon and gene sampling, gene recovery, models of sequence evolution and paralogy, the tree strongly supports existing taxonomy, while challenging numerous hypothesized relationships among orders and placing many genera for the first time. The validated dataset, species tree and all intermediates are openly accessible via the Kew Tree of Life Explorer and will be updated as further data become available. This major milestone towards a complete tree of life for all flowering plant species opens doors to a highly integrated future for angiosperm phylogenomics through the systematic sequencing of standardised nuclear markers. Our approach has the potential to serve as a much-needed bridge between the growing movement to sequence the genomes of all life on Earth and the vast phylogenomic potential of the world's natural history collections.
    • Confocal microscopy provides visual evidence and confirms the feasibility of dsRNA delivery to emerald ash borer through plant tissues

      Pampolini, Flavia; Rodrigues, Thais B.; Leelesh, Ramya Shanivarsanthe; Kawashima, Tomokazu; Rieske, Lynne K. (Springer, 2020-05-15)
      Double-stranded RNA (dsRNA)-mediated gene silencing, or RNA interference (RNAi), is an emerging biotechnology that has been a breakthrough tool for crop protection. Exogenous dsRNA triggers the RNAi pathway, silences genes, disrupts protein function, and can cause insect mortality. However, effective delivery of the dsRNA is problematic, particularly in systems with long-lived, endophagous insects such as the emerald ash borer (EAB), Agrilus planipennis, a tree-killing nonnative invader that attacks ash, Fraxinus spp. Larvae feed on cambial tissue causing rapid tree death. EAB is susceptible to RNAi, but we lack a practical means of delivery. Here we evaluated delivery of dsRNA to green, F. pennsylvanica, and tropical ash, F. uhdei, through root and/or petiole absorption, and also demonstrated dsRNA absorption through the EAB egg chorion. We labeled exogenous dsRNA using a fluorescing label and then used confocal microscopy and RT-qPCR to evaluate its distribution in plant and insect tissues. Labeled dsRNAs are detectable in root, stem, and leaf tissues 48-h postapplication. In excised ash branches, labeled dsRNA is detectable in the inner bark and in recovered EAB neonates 8-day postapplication. Eggs and larvae emerging from treated eggs also presented fluorescing dsRNA under confocal imaging. Adult EAB-fed tropical ash leaves treated with in vitro synthesized EAB-specific dsSHI through petiole absorption experience a significant knockdown of the shi gene and a significant mortality. Our findings provide a proof of concept that delivery of dsRNAs through topical or systemic application methods is a feasible means of suppressing EAB, providing hope for future tree protection.
    • Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

      Kralt, Annemarie; Basheer, Noorjahan Jagalur; Van Den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.; ; University of Groningen; et al. (American Society for Cell Biology, 2015-07-15)
      Endoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins.
    • Corals at the EDGE of existence

      Crabbe, M. James C.; University of Oxford (OMICS International, 2016-09-16)
      There are numerous human and environmental challenges to coral reefs that leave many species of scleractinian corals globally threatened. The EDGE of Existence (Evolutionarily Distinct and Globally Endangered) programme commenced in 2007 at the Zoological Society of London (ZSL). The purpose of the programme was to focus resources for science and conservation on species where little research had been done, or where there were no conservation plans. The programme was developed to support EDGE Fellows who came from, and were based, in the countries where the EDGE species existed. So far, 58 Fellows have been supported, in 33 countries. The Fellowship lasts 2 years, and provides training to develop science and conservation techniques, including outreach and policy development. A grant enables initial implementation of the project. The EDGE programme aims to train future leaders in conservation science. In 2011 the programme was expanded from mammal, amphibian, reptile and bird species to include coral reefs. Specific training in coral reef biology, taxonomy and ecology is provided, together with SCUBA training if necessary. Marine conservationists are able to target conservation projects directed to specific species in Africa, Asia and the Americas.