• Negative regulation of autophagy in activating nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes in the hippocampus of an epilepsy rat model

      Wu, Jia-Mei; Chen, Liqiang; Wang, Shuo; Li, Yingyu; Liu, Lei; Chen, Guang; Wang, Shu-Qiu; Zhou, Shaobo (American Scientific Publishers, 2019-06-25)
      Epilepsy, characterized by unpredictable and periodic seizures, is associated with chronic hippocampal inflammation and autophagy. Moreover, a molecular relationship between autophagy and inflammation in neurodegenerative disorders has been reported, highlighting the role of autophagy in the regulation of inflammation. To the best of our knowledge, there is no previous evidence of an association between nucleotide-binding oligomerization domain-like receptor family pyrin domaincontaining 3 (NLRP3) inflammasome and autophagy in epilepsy. Hence, we in this study aimed at investigating the possible association between NLRP3 inflammasome activation and autophagy in the development of epilepsy. A rat model of temporal lobe epilepsy was induced with lithiumpilocarpine. Five groups, i.e., control (n=20), status epilepticus (SE, n=30), SE+control (siRNA; n=15), SE+NLRP3 siRNA (n=30), and SE+wortmannin (n=30), were investigated. Real-time quantitative reverse transcription polymerase chain reaction, Western blotting, and quantum dotbased immunohistochemistry were used to detect the mRNA/protein expression levels of NLRP3, caspase-1, interleukin (IL)-1, IL-18, Beclin-1, and microtubule-associated protein light chain 3 (LC3) in the hippocampus. In addition, transmission electron microscopy was utilized to investigate autophagosome in the hippocampus of SE rats. We found that mRNA and protein expressions of NLRP3, caspase-1, IL-1, IL-18, LC3, and Beclin-1 were activated in the hippocampus. Gene silencing of NLRP3 suppressed caspase-1, IL-1, and IL-18 release and significantly ameliorated hippocampal damage. Furthermore, the LC3 and Beclin-1 expression levels decreased significantly after treatment with wortmannin. Importantly, NLRP3 inflammasome activation and IL-1/IL-18 releases were significantly enhanced after treatment with wortmannin, which implied a negative association between autophagy inhibition and NLRP3 inflammasome activation. Our study provides the first evidence that autophagy plays an important role in NLRP3 inflammasome activation in the development of epilepsy. These findings suggest that regulation of autophagy may be a promising potential strategy for treating patients with epilepsy.
    • A new family of periplasmic-binding proteins that sense arsenic oxyanions

      Badilla, Consuelo; Osborne, Thomas H.; Cole, Ambrose; Watson, Cameron; Djordjevic, Snezana; Santini, Joanne M. (SpringerNature, 2018-04-19)
      Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that’s role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate.
    • Non-destructive genome skimming for aquatic copepods

      Vakati, Vinod; Dodsworth, Steven; Neijiang Normal University; Hanyang University; University of Bedfordshire (Springer, 2020-01-21)
      Copepods are important ecologically and represent a large amount of aquatic biomass in both freshwater and marine systems. Despite this, the taxonomy of copepods and other meiofauna is not well understood, hampered by tiny sizes, cryptic taxa, intraspecific polymorphisms and total specimen destruction where DNA methods are employed. In this article we highlight these issues and propose a more up-to-date approach for dealing with them. Namely, we recommend non-destructive DNA extraction methods, coupled with high-throughput sequencing (HTS). Whilst DNA yields may be low, they should still be sufficient for HTS library preparation and DNA sequencing. At the same time morphological specimens can be preserved and the crucial link between morphology and DNA sequence is maintained. This is critical for an integrative taxonomy and a fuller understanding of biodiversity patterns as well as evolutionary processes in meiofauna.
    • Non-redundant role for the transcription factor Gli1 at multiple stages of thymocyte development

      Drakopoulou, Ekati; Outram, Susan V.; Rowbotham, Nicola J.; Ross, Susan; Furmanski, Anna L.; Saldana, Jose Ignacio; Hager-Theodorides, Ariadne L.; Crompton, Tessa (Taylor & Francis, 2010-10-15)
      The Hedgehog (Hh) signaling pathway influences multiple stages of murine T-cell development. Hh signaling mediates transcriptional changes by the activity of the Gli family of transcription factors, Gli1, Gli2 and Gli3. Both Gli2 and Gli3 are essential for mouse developmentand can be processed to function as transcriptional repressors or transcriptional activators, whereas Gli1, itself a transcriptional target of Hh pathway activation, can only function as a transcriptional activator and is not essential for mouse development. Gli1-deficient mice are healthy and appear normal and nonredundant functions for Gli1 have been difficult to identify. Here we show that Gli1 is non-redundant in the regulation of T-cell development in the thymus, at multiple developmental stages. Analysis of Gli1-deficient embryonic mouse thymus shows a role for Gli1 to promote the differentiation of CD4⁻CD8⁻ double negative (DN) thymocytes before pre- TCR signal transduction, and a negative regulatory function after pre-TCR signaling. In addition, introduction of a Class I-restricted transgenic TCR into the adult Gli1-deficient and embryonic Gli2-deficient thymus showed that both Gli1 and Gli2 influence its selection to the CD8 lineage.
    • A novel 3D in vitro model of glioblastoma reveals resistance to temozolomide which was potentiated by hypoxia

      Musah-Eroje, Ahmed; Watson, Sue; University of Nottingham; University of Bedfordshire (Springer, 2019-01-29)
      Glioblastoma (GBM) is the most common invasive malignant brain tumour in adults. It is traditionally investigated in vitro by culturing cells as a monolayer (2D culture) or as neurospheres (clusters enriched in cancer stem cells) but neither system accurately reflects the complexity of the three-dimensional (3D) chemoresistant microenvironment of GBM. Using three GBM cell-lines (U87, U251, and SNB19), the effect of culturing cells in a Cultrex-based basement membrane extract (BME) [3D Tumour Growth Assay (TGA)] on morphology, gene expression, metabolism, and temozolomide chemoresistance was investigated. Cells were easily harvested from the 3D model and cultured as a monolayer (2D) and neurospheres. Indeed, the SNB19 cells formed neurospheres only after they were first cultured in the 3D model. The expression of CD133 and OCT4 was upregulated in the neurosphere and 3D assays respectively. Compared with cells cultured in the 2D model, cells were more resistant to temozolomide in the 3D model and this resistance was potentiated by hypoxia. Taken together, these results suggest that micro-environmental factors influence GBM sensitivity to temozolomide. Knowledge of the mechanisms involved in temozolomide resistance in this 3D model might lead to the identification of new strategies that enable the more effective use of the current standard of care agents. PURPOSE MATERIALS AND METHODS RESULTS CONCLUSION
    • A novel approach to oral iron delivery using ferrous sulphate loaded solid lipid nanoparticles

      Zariwala, Mohammed Gulrez; Elsaid, Naba; Jackson, Timothy L.; Corral López, Francisco; Farnaud, Sébastien; Somavarapu, Satyanarayana; Renshaw, Derek; University of Westminster; University College London; King's College Hospital; et al. (Elsevier, 2013-11-18)
      Iron (Fe) loaded solid lipid nanoparticles (SLN's) were formulated using stearic acid and iron absorption was evaluated in vitro using the cell line Caco-2 with intracellular ferritin formation as a marker of iron absorption. Iron loading was optimised at 1% Fe (w/w) lipid since an inverse relation was observed between initial iron concentration and SLN iron incorporation efficiency. Chitosan (Chi) was included to prepare chitosan coated SLN's. Particle size analysis revealed a sub-micron size range (300.3±31.75 nm to 495.1±80.42 nm), with chitosan containing particles having the largest dimensions. As expected, chitosan (0.1%, 0.2% and 0.4% w/v) conferred a net positive charge on the particle surface in a concentration dependent manner. For iron absorption experiments equal doses of Fe (20 μM) from selected formulations (SLN-FeA and SLN-Fe-ChiB) were added to Caco-2 cells and intracellular ferritin protein concentrations determined. Caco-2 iron absorption from SLN-FeA (583.98±40.83 ng/mg cell protein) and chitosan containing SLN-Fe-ChiB (642.77±29.37 ng/mg cell protein) were 13.42% and 24.9% greater than that from ferrous sulphate (FeSO4) reference (514.66±20.43 ng/mg cell protein) (p≤0.05). We demonstrate for the first time preparation, characterisation and superior iron absorption in vitro from SLN's, suggesting the potential of these formulations as a novel system for oral iron delivery.
    • Novel classes of fatty acid and retinol binding protein from nematodes

      McDermott, Lindsay C.; Cooper, Alan; Kennedy, Malcolm W.; University of Glasgow (Springer, 1999-02-28)
      Parasitic nematodes have recently been found to produce proteins which represent two new classes of fatty acid and retinoid binding protein. The first is the nematode polyprotein allergens/antigens (NPAs) which, as their name suggests, are synthesised as large polyproteins which are subsequently cleaved at regularly spaced sites to form multiple copies of a fatty acid binding protein of approximately 14.5 kDa. Binding studies using molecular environment-sensitive fluorescent ligands have shown that the binding site is highly unusual, producing blue-shifting in fluorescence to an unprecedented degree, suggesting a remarkably non-polar environment and isolation from solvent water. Computer-based structural predictions and biophysical observations have identified the NPAs as highly helical proteins which might form a four helix bundle, so constitute a new class of lipid binding protein from animals. The second class, like the NPAs, binds both fatty acids and retinol, but with a higher affinity for the latter. These are also highly helical but are structurally distinct from the NPAs. The biological function of these new classes of protein are discussed in the context of both the metabolic requirements of the parasites and the possible role of the proteins in control of the immune and inflammatory environment of the tissue sites parasitised.
    • A novel human neuronal cell model to study iron accumulation in Parkinson’s disease

      Mehta, Kosha; Ahmed, Bushra Y.; Farnaud, Sébastien; University of Bedfordshire (OMICS International, 2019-02-11)
      Objectives: With an estimated seven to ten million sufferers worldwide, Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder. Progress in elucidating its causes has been slow, partly due to the lack of human-relevant models. Similarly, while the contribution of iron is increasingly advocated, identifying its role in disease progression remains challenging mainly due to the lack of valid model. In this study, we created Parkinson-like conditions in a human neuron model and conducted preliminary studies on iron-related parameters to assess whether these cells replicated iron accumulation observed in Parkinsonism. Methods: ReNcell VM (human neural progenitor) were differentiated into dopaminergic neurons (dDCNs) and treated with neurotoxin 6-hydroxy dopamine (100 μM) to mimic Parkinsonism. Total intracellular, mitochondrial and cytoplasmic iron was measured by ferrozine assay. Expression of iron-related genes TFRC, SLC40A1, HAMP and SLC25A37 were assessed through real-time PCR. Results: Data showed that the treated dDCNs accumulated iron over time and exceeded levels measured in untreated dDCNs by 2.5-fold at 48 h (p<0.02). Following the treatment, the treated cells showed lower expression of TFRC (p<0.05), but substantially higher mRNA expressions of SLC40A1 (9-fold; p<0.02) and HAMP (5.7-fold; p<0.05), along with higher intracellular iron (p<0.05). Higher iron accumulation in the mitochondria than cytosol (p<0.05), was also observed with increased expression of the mitochondrial iron-importer SLC25A37 (p=0.08). Conclusion: Our Parkinsonian model demonstrates iron accumulation and elevated HAMP expression as previously described in PD phenotype. The observed mitochondrial iron shuttling, which is proposed to be one of the primary contributors of oxidative stress in PD, calls for further investigation. The differences observed in distribution of iron in our human model and with the expression of major iron-related proteins, indicate that our model reproduces the disease state successfully, and suggests that further study could help in advancing our understanding of PD.
    • A novel orvinol analog, BU08028, as a safe opioid analgesic without abuse liability in primates

      Ding, Huiping; Czoty, Paul W.; Kiguchi, Norikazu; Cami-Kobeci, Gerta; Sukhtankar, Devki D.; Nader, Michael A.; Husbands, Stephen M.; Ko, Mei-Chuan; ; Wake Forest University; et al. (National Academy of Sciences, 2016-08-29)
      Despite the critical need, no previous research has substantiated safe opioid analgesics without abuse liability in primates. Recent advances in medicinal chemistry have led to the development of ligands with mixed mu opioid peptide (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor agonist activity to achieve this objective. BU08028 is a novel orvinol analog that displays a similar binding profile to buprenorphine with improved affinity and efficacy at NOP receptors. The aim of this preclinical study was to establish the functional profile of BU08028 in monkeys using clinically used MOP receptor agonists for side-by-side comparisons in various wellhoned behavioral and physiological assays. Systemic BU08028 (0.001-0.01 mg/kg) produced potent long-lasting (i.e., >24 h) antinociceptive and antiallodynic effects, which were blocked by MOP or NOP receptor antagonists. More importantly, the reinforcing strength of BU08028 was significantly lower than that of cocaine, remifentanil, or buprenorphine in monkeys responding under a progressive-ratio schedule of drug self-administration. Unlike MOP receptor agonists, BU08028 at antinociceptive doses and ?10-to 30-fold higher doses did not cause respiratory depression or cardiovascular adverse events as measured by telemetry devices. After repeated administration, the monkeys developed acute physical dependence on morphine, as manifested by precipitated withdrawal signs, such as increased respiratory rate, heart rate, and blood pressure. In contrast, monkeys did not show physical dependence on BU08028. These in vivo findings in primates not only document the efficacy and tolerability profile of bifunctional MOP/NOP receptor agonists, but also provide a means of translating such ligands into therapies as safe and potentially abusefree opioid analgesics.
    • A novel role for Hedgehog in T-cell receptor signaling: implications for development and immunity

      Rowbotham, Nicola J.; Hager-Theodorides, Ariadne L.; Furmanski, Anna L.; Crompton, Tessa (Taylor & Francis, 2007-09-07)
      The Hedgehog (Hh) signaling pathway is a key regulator of both embryonic development and homeostasis of adult tissues, including thymus and blood. In the thymus, Hh signals for differentiation, survival and proliferation in the early stages of T cell development, before TCR gene rearrangement. Our recent data has shown that Hh signaling also modulates T cell receptor (TCR) signal strength in more mature T lineage cells. We showed that constitutive activation of the Hh pathway in thymocytes (by transgenic expression of the transcriptional activator form of Gli2) decreased TCR signal strength with profound consequences for the thymus--allowing self-reactive T cells to escape deletion and altering T cell CD4/CD8 lineage decisions. In contrast, in the Sonic Hh deficient thymus, TCR signaling was increased, again influencing both TCR repertoire selection and CD4/8 lineage commitment. In peripheral T cells, the transcriptional changes induced by activation of the Hh signaling pathway lead to reduced T cell activation. Hh signaling also attenuated ERK phosphorylation and proliferation in mature T cells on TCR ligation. Modulation of TCR signal strength by Hh pathway activation has importance for immunity as the presence or absence of Hh in the environment in which a T cell is activated would shape the immune response.
    • A nuclear phylogenomic study of the angiosperm order Myrtales, exploring the potential and limitations of the universal Angiosperms353 probe set

      Maurin, Olivier; Anest, Artemis; Bellot, Sidonie; Biffin, Edward; Brewer, Grace E.; Charles-Dominique, Tristan; Cowan, Robyn S.; Dodsworth, Steven; Epitawalage, Niroshini; Gallego, Berta; et al. (Wiley, 2021-07-31)
      To further advance the understanding of the species-rich, economically and ecologically important angiosperm order Myrtales in the rosid clade, comprising nine families, approximately 400 genera and almost 14,000 species occurring on all continents (except Antarctica), we tested the Angiosperms353 probe kit. We combined high-throughput sequencing and target enrichment with the Angiosperms353 probe kit to evaluate a sample of 485 species across 305 genera (76% of all genera in the order). Results provide the most comprehensive phylogenetic hypothesis for the order to date. Relationships at all ranks, such as the relationship of the early-diverging families, often reflect previous studies, but gene conflict is evident, and relationships previously found to be uncertain often remain so. Technical considerations for processing HTS data are also discussed. High-throughput sequencing and the Angiosperms353 probe kit are powerful tools for phylogenomic analysis, but better understanding of the genetic data available is required to identify genes and gene trees that account for likely incomplete lineage sorting and/or hybridization events.
    • The occurrence and potential health risk of microcystins in drinking water of rural areas in China.

      Zheng, Weiwei; Yang, Lan; Ma, Wuren; Huang, Yu; Crabbe, M. James C.; Qu, Weidong (Elsevier, 2018-12-26)
      Large-scale use of nitrogen and phosphorus fertilizers in agricultural production, environmental pollution and climate warming cause frequent algal blooms and the generation of algal toxins in water bodies in China. Algal pollution is increasing and microcystins (MCs) are detectable in both surface and ground water in China at sub- μg/L and μg/L levels. Toxicological studies show that microcystins have hepatic and  renal toxicity, genotoxicity, tumor-promoting effects, neurotoxicity, reproductive and developmental toxicity. Epidemiological evidence from China further reveals that chronic exposure to MCs through drinking water and liver cancer are positively correlated and demonstrate that MCs in drinking water are a main risk factor in liver cancer. Effectively controlled water pollution, reduced sewage discharge, and enhanced wastewater treatments are pivotal measures to control algal pollution and toxins in the drinking water of rural China.
    • On the origin of giant seeds: the macroevolution of the double coconut (Lodoicea maldivica) and its relatives (Borasseae, Arecaceae)

      Bellot, Sidonie; Bayton, Ross P.; Couvreur, Thomas L.P.; Dodsworth, Steven; Eiserhardt, Wolf L.; Guignard, Maite S.; Pritchard, Hugh W.; Roberts, Lucy; Toorop, Peter E.; Baker, William J. (Wiley, 2020-06-16)
      Seed size shapes plant evolution and ecosystems, and may be driven by plant size and architecture, dispersers, habitat and insularity. How these factors influence the evolution of giant seeds is unclear, as are the rate of evolution and the biogeographical consequences of giant seeds. We generated DNA and seed size data for the palm tribe Borasseae (Arecaceae) and its relatives, which show a wide diversity in seed size and include the double coconut (Lodoicea maldivica), the largest seed in the world. We inferred their phylogeny, dispersal history and rates of change in seed size, and evaluated the possible influence of plant size, inflorescence branching, habitat and insularity on these changes. Large seeds were involved in 10 oceanic dispersals. Following theoretical predictions, we found that: taller plants with fewer-branched inflorescences produced larger seeds; seed size tended to evolve faster on islands (except Madagascar); and seeds of shade-loving Borasseae tended to be larger. Plant size and inflorescence branching may constrain seed size in Borasseae and their relatives. The possible roles of insularity, habitat and dispersers are difficult to disentangle. Evolutionary contingencies better explain the gigantism of the double coconut than unusually high rates of seed size increase.
    • Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner

      Janganan, Thamarai K.; Zhang, Li; Bavro, Vassiliy N; Matak-Vinkovic, Dijana; Barrera, Nelson P.; Burton, Matthew F.; Steel, Patrick G.; Robinson, Carol V.; Borges-Walmsley, Maria Ines; Walmsley, Adrian R.; et al. (American Society for Biochemistry and Molecular Biology, 2011-02-18)
      The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.
    • Optimising the release rate of naproxen liqui-pellet: a new technology for emerging novel oral dosage form

      Lam, Matthew; Ghafourian, Taravat; Nokhodchi, Ali; (Springer, 2019-07-08)
      Liqui-pellet is a new dosage form stemming from pelletisation technology and concept from liquisolid technology. In spite of liqui-pellet overcoming a major hurdle in liquisolid technology through achieving excellent flow property with high liquid load factor, the formulation requires to be optimised in order to improve drug release rate. Liqui-pellets of naproxen containing Tween 80, Primojel, Avicel and Aerosil were extruded and spheronised. Flowability test confirmed that all liqui-pellet formulations have excellent-good flow property (Carr’s index between 3.9–11.17%), including liqui-pellets with a high liquid load factor of 1.52, where 38% of the total mass is co-solvent. This shows a relatively high liquid load factor can be achieved in liqui-pellet without compromising the flowability, which is one of the key novelty of this work. It was found that the improved drug release rate was due to the remarkably improved disintegration of the supposedly non-disintegrating microcrystalline-based pellet; the optimised liqui-pellet seems to explode into fragments in the dissolution medium. At pH 1.2, the optimised formulation had ~ 10% more drug release than non-optimised formulation after 2 h, and at pH 7.4, the drug release of the optimised pellet was nearing 100% at ~ 15 min, whereas the none-optimised pellet only achieved ~ 79% drug release after 2 h. DSC and XRPD indicated an increase in the dissolution rate could be due to molecularly dispersion of naproxen in the pellets. Overall results showed that liqui-pellet exhibited an enhanced drug release and the capacity for high liquid load factor whilst maintaining excellent flowability, rendering it a potentially commercially feasible drug delivery system.
    • Oral ingestion of bacterially expressed dsrna can silence genes and cause mortality in a highly invasive, tree-killing pest, the emerald ash borer

      Leelesh, Ramya Shanivarsanthe; Rieske, Lynne K.; University of Kentucky; University of Bedfordshire (MDPI, 2020-07-14)
      RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since its discovery in 2002, prompting the need for innovative management strategies. In our study, bacterial expression and synthesis of dsRNA were performed with E. coli strain HT115 using the L4440 expression vector. EAB-specific dsRNAs (shi and hsp) over-expressed in E. coli were toxic to neonate EAB after oral administration, successfully triggering gene silencing and subsequent mortality; however, a non-specific dsRNA control was not included. Our results suggest that ingestion of transformed E. coli expressing dsRNAs can induce an RNAi response in EAB. To our knowledge, this is the first example of an effective RNAi response induced by feeding dsRNA-expressing bacteria in a forest pest.
    • The origin and diversification of the hyperdiverse flora in the Chocó biogeographic region

      Pérez-Escobar, Oscar Alejandro; Lucas, Eve; Jaramillo, Carlos; Monro, Alexandre; Morris, Sarah K.; Bogarín, Diego; Greer, Deborah; Dodsworth, Steven; Aguilar-Cano, José; Sanchez Meseguer, Andrea; et al. (Frontiers, 2019-12-06)
      Extremely high levels of plant diversity in the American tropics are derived from multiple interactions between biotic and abiotic factors. Previous studies have focused on macro-evolutionary dynamics of the Tropical Andes, Amazonia, and Brazil’s Cerrado and Atlantic forests during the last decade. Yet, other equally important Neotropical biodiversity hotspots have been severely neglected. This is particularly true for the Chocó region on the north-western coast of South and Central America. This geologically complex region is Earth’s ninth most biodiverse hotspot, hosting approximately 3% of all known plant species. Here, we test Gentry’s [1982a,b] hypothesis of a northern Andean-Central American Pleistocene origin of the Chocoan flora using phylogenetic reconstructions of representative plant lineages in the American tropics. We show that plant diversity in the Chocó is derived mostly from Andean immigrants. Contributions from more distant biogeographical areas also exist but are fewer. We also identify a strong floristic connection between the Chocó and Central America, revealed by multiple migrations into the Chocó during the last 5 Ma. The dated phylogenetic reconstructions suggest a Plio-Pleistocene onset of the extant Chocó flora. Taken together, these results support to a limited extend Gentry’s hypothesis of a Pleistocene origin and of a compound assembly of the Chocoan biodiversity hotspot. Strong Central American–Chocoan floristic affinity may be partly explained by the accretion of a land mass derived from the Caribbean plate to north-western South America. Additional densely sampled phylogenies of Chocoan lineages also well represented across the Neotropics could enlighten the role of land mass movements through time in the assembly of floras in Neotropical biodiversity hotspots.
    • The Ov20 protein of the parasitic nematode Onchocerca volvulus - A structurally novel class of small helix-rich retinol-binding proteins

      Kennedy, Malcolm W.; Garside, Lisa H.; Goodrick, Lucy E.; McDermott, Lindsay C.; Brass, Andrew; Price, Nicholas C.; Kelly, Sharon M.; Cooper, Alan; Bradley, Jannette E.; University of Glasgow; et al. (American Society for Biochemistry and Molecular Biology, 1997-11-21)
      Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-DL-alpha-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized beta-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a alpha-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of beta structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 degrees C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.
    • Peptide-specific, TCR-alpha-driven, coreceptor-independent negative selection in TCR alpha-chain transgenic mice

      Furmanski, Anna L.; Bartok, Istvan; Chai, Jian-Guo; Singh, Yogesh; Ferreira, Cristina; Scott, Diane; Holland, Stephen J.; Bourdeaux, Christophe; Crompton, Tessa; Dyson, Julian (American Association of Immunologists, 2010-01-06)
      As thymocytes differentiate, Ag sensitivity declines, with immature CD4-CD8- double-negative (DN) cells being most susceptible to TCRsignaling events. We show that expression of alphabetaTCR from the DN3 stage lowers the threshold for activation, allowing recognition of MHC peptides independently of the TCR beta-chain and without either T cell coreceptor. The MHC class I-restricted C6 TCR recognizes the Y-chromosome-derived Ag HYK(k)Smcy. Positive selection in C6 alphabetaTCR females is skewed to the CD8 compartment, whereas transgenic male mice exhibit early clonal deletion of thymocytes. We investigated the effect of the HYK(k)Smcy complex on developing thymocytes expressing the C6 TCR alpha-chain on a TCR-alpha(-/-) background. On the original selecting haplotype, the skew to the CD8 lineage is preserved. This is MHC dependent, as the normal bias to the CD4 subset is seen on an H2b background. In male H2k C6 alpha-only mice, the presence of the HYK(k)Smcy complex leads to a substantial deletion of thymocytes from the DN subset. This phenotype is replicated in H2k C6 alpha-only female mice expressing an Smcy transgene. Deletion is not dependent on the beta variable segment of the C6 TCR or on a restricted TCR-beta repertoire. In contrast, binding of HYK(k)Smcy and Ag-specific activation of mature CD8+ T cells is strictly dependent on the original C6 beta-chain. These data demonstrate that, in comparison with mature T cells, alphabetaTCR+ immature thymocytes can recognize and transduce signals in response to specific MHC-peptide complexes with relaxed binding requirements.
    • Petal, sepal, or tepal? B-genes and monocot flowers

      Dodsworth, Steven; Royal Botanic Gardens, Kew (Elsevier Ltd, 2016-11-25)
      In petaloid monocots expansion of B-gene expression into whorl 1 of the flower results in two whorls of petaloid organs (tepals), as opposed to sepals in whorl 1 of typical eudicot flowers. Recently, new gene-silencing technologies have provided the first functional data to support this, in the genus Tricyrtis (Liliaceae).