Publications by authors named "Andrew J Thompson"

151 Publications

A synthetic peptide rescues rat cortical neurons from anesthetic-induced cell death, perturbation of growth and synaptic assembly.

Sci Rep 2021 Feb 25;11(1):4567. Epub 2021 Feb 25.

University of Calgary, Calgary, T2N 4N1, Canada.

Anesthetics are deemed necessary for all major surgical procedures. However, they have also been found to exert neurotoxic effects when tested on various experimental models, but the underlying mechanisms remain unknown. Earlier studies have implicated mitochondrial fragmentation as a potential target of anesthetic-induced toxicity, although clinical strategies to protect their structure and function remain sparse. Here, we sought to determine if preserving mitochondrial networks with a non-toxic, short-life synthetic peptide-P110, would protect cortical neurons against both inhalational and intravenous anesthetic-induced neurotoxicity. This study provides the first direct and comparative account of three key anesthetics (desflurane, propofol, and ketamine) when used under identical conditions, and demonstrates their impact on neonatal, rat cortical neuronal viability, neurite outgrowth and synaptic assembly. Furthermore, we discovered that inhibiting Fis1-mediated mitochondrial fission reverses anesthetic-induced aberrations in an agent-specific manner. This study underscores the importance of designing mitigation strategies invoking mitochondria-mediated protection from anesthetic-induced toxicity in both animals and humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-84168-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907385PMC
February 2021

Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

bioRxiv 2021 Feb 4. Epub 2021 Feb 4.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S - insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complimentary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.02.03.429627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872350PMC
February 2021

De Novo Genome Assembly Of Solanum Sitiens Reveals Structural Variation Associated With Drought And Salinity Tolerance.

Bioinformatics 2021 Jan 30. Epub 2021 Jan 30.

The Bioinformatics Group, School of Water, Energy and Environment, Cranfield University, College Road, Bedford, MK43 0AL, UK T.

Motivation: Solanum sitiens is a self-incompatible wild relative of tomato, characterised by salt and drought resistance traits, with the potential to contribute through breeding programmes to crop improvement in cultivated tomato. This species has a distinct morphology, classification and ecotype compared to other stress resistant wild tomato relatives such as S. pennellii and S. chilense. Therefore, the availability of a reference genome for S. sitiens will facilitate the genetic and molecular understanding of salt and drought resistance.

Results: A high-quality de novo genome and transcriptome assembly for S. sitiens (Accession LA1974) has been developed. A hybrid assembly strategy was followed using Illumina short reads (∼159X coverage) and PacBio long reads (∼44X coverage), generating a total of ∼262 Gbp of DNA sequence. A reference genome of 1,245 Mbp, arranged in 1,483 scaffolds with a N50 of 1.826 Mbp was generated. Genome completeness was estimated at 95% using the Benchmarking Universal Single-Copy Orthologs (BUSCO) and the K-mer Analysis Tool (KAT). In addition, ∼63 Gbp of RNA-Seq were generated to support the prediction of 31,164 genes from the assembly, and to perform a de novo transcriptome. Lastly, we identified three large inversions compared to S. lycopersicum, containing several drought resistance related genes, such as beta-amylase 1 and YUCCA7.

Availability: S. sitiens (LA1974) raw sequencing, transcriptome and genome assembly have been deposited at the NCBI's Sequence Read Archive, under the BioProject number "PRJNA633104".All the commands and scripts necessary to generate the assembly are available at the following github repository: https://github.com/MCorentin/Solanum_sitiens_assembly.

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btab048DOI Listing
January 2021

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.

Proc Natl Acad Sci U S A 2020 11 5;117(47):29595-29601. Epub 2020 Nov 5.

Department of Chemistry, University of York, York YO10 5DD, United Kingdom;

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from GlcManGlcNAc precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole -acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2013620117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703563PMC
November 2020

Adaptation of Influenza Viruses to Human Airway Receptors.

J Biol Chem 2020 11 3. Epub 2020 Nov 3.

Molecular Medicine, The Scripps Research Institute, United States.

Through annual epidemics and global pandemics, influenza A viruses (IAVs) remain a significant threat to human health as the leading cause of severe respiratory disease. Within the last century, four global pandemics have resulted from the introduction of novel IAVs into humans, with components of each originating from avian viruses. IAVs infect many avian species wherein they maintain a diverse natural reservoir, posing a risk to humans through the occasional emergence of novel strains with enhanced zoonotic potential. One natural barrier for transmission of avian IAVs into humans is the specificity of the receptor-binding protein, hemagglutinin (HA), that recognizes sialic acid-containing glycans on host cells. HAs from human IAVs exhibit "human-type" receptor specificity, binding exclusively to glycans on cells lining the human airway where terminal sialic acids are attached in the α2-6 configuration (NeuAcα2-6Gal). In contrast, HAs from avian viruses exhibit specificity for "avian-type" α2-3-linked (NeuAcα2-3Gal) receptors, and thus require adaptive mutations to bind human-type receptors. Since all human IAV pandemics can be traced to avian origins, there remains ever-present concern over emerging IAVs with human-adaptive potential that might lead to the next pandemic. This concern has been brought into focus through emergence of SARS-CoV-2, aligning both scientific and public attention to the threat of novel respiratory viruses from animal sources. In this review we summarize receptor-binding adaptations underlying the emergence of all prior IAV pandemics in humans, maintenance and evolution of human-type receptor specificity in subsequent seasonal IAVs, and potential for future human-type receptor adaptation in novel avian HAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.REV120.013309DOI Listing
November 2020

Transcriptome and phytohormone changes associated with ethylene-induced onion bulb dormancy.

Postharvest Biol Technol 2020 Oct;168:111267

Cranfield University, Bedfordshire, MK43 0AL, UK.

Control of dormancy and sprouting in onion bulbs is commercially important for postharvest management. Although ethylene application is sometimes used to extend dormancy, the underlying mechanisms regulating dormancy transition remain unclear. Since the sprout leaves emerge from the bulb baseplate, we used this tissue to assess the impact of ethylene treatment and storage time on the hormone profile and the transcriptome. Reads from 30 libraries were assembled and annotated, with 94,840 unigenes retained after filtering. The transcriptome assembly was of high quality and continuity (N50: 1809 bp, GC content: 36.21 %), and was used to analyse differential expression and Gene Onotologies. Across two years, applied ethylene resulted in delayed dormancy break and reduced post-dormancy sprout vigour. Ethylene supplementation enhanced endogenous ethylene production and caused a transient climacteric-like increase in respiration. Significant changes in hormone and associated transcript profiles occurred through storage and in response to ethylene. In particular, abscisic acid (ABA) and its metabolite phaseic acid (PA) increased under ethylene during the longer dormancy period; however, cytokinin increases observed during storage appeared largely independent of ethylene treatment. Several hormone-related transcripts showed differential expression over time and/or in response to ethylene. Expression of ethylene biosynthesis (), receptor () and transcription factor () genes were modified by ethylene, as were ABA biosynthesis genes such and cytokinin biosynthesis genes such as and . We conclude that ethylene substantially modifies expression of genes in several phytohormone pathways, and some of these changes may underlie the dormancy-extending effects of exogenous ethylene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.postharvbio.2020.111267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398043PMC
October 2020

Memory CD8 T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Cell Metab 2020 Sep 31;32(3):457-467.e5. Epub 2020 Jul 31.

Department of Biomedicine, Immunobiology, University of Basel, 4031 Basel, Switzerland; Department of Medicine, CITIID, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK. Electronic address:

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8 T cells to produce IFN-γ. Whether acetate modulates memory CD8 T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8 T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2020.07.004DOI Listing
September 2020

Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses.

Science 2020 06;368(6497):1335-1340

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaz5143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412937PMC
June 2020

Impact of overexpression of 9-cis-epoxycarotenoid dioxygenase on growth and gene expression under salinity stress.

Plant Sci 2020 Jun 12;295:110268. Epub 2019 Sep 12.

Department of Plant Nutrition, CEBAS-CSIC, Murcia, Spain.

To better understand abscisic acid (ABA)'s role in the salinity response of tomato (Solanum lycopersicum L.), two independent transgenic lines, sp5 and sp12, constitutively overexpressing the LeNCED1 gene (encoding 9-cis-epoxycarotenoid dioxygenase, a key enzyme in ABA biosynthesis) and the wild type (WT) cv. Ailsa Craig, were cultivated hydroponically with or without the addition of 100 mM NaCl. Independent of salinity, LeNCED1 overexpression (OE) increased ABA concentration in leaves and xylem sap, and salinity interacted with the LeNCED1 transgene to enhance ABA accumulation in xylem sap and roots. Under control conditions, LeNCED1 OE limited root and shoot biomass accumulation, which was correlated with decreased leaf gas exchange. In salinized plants, LeNCED1 OE reduced the percentage loss in shoot and root biomass accumulation, leading to a greater total root length than WT. Root qPCR analysis of the sp12 line under control conditions revealed upregulated genes related to ABA, jasmonic acid and ethylene synthesis and signalling, gibberellin and auxin homeostasis and osmoregulation processes. Under salinity, LeNCED1 OE prevented the induction of genes involved in ABA metabolism and GA and auxin deactivation that occurred in WT, but the induction of ABA signalling and stress-adaptive genes was maintained. Thus, complex changes in phytohormone and stress-related gene expression are associated with constitutive upregulation of a single ABA biosynthesis gene, alleviating salinity-dependent growth limitation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.plantsci.2019.110268DOI Listing
June 2020

An Epoxide Intermediate in Glycosidase Catalysis.

ACS Cent Sci 2020 May 16;6(5):760-770. Epub 2020 Apr 16.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 -α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope () conformation. Kinetic isotope effects (/) for anomeric-H and anomeric-C support an oxocarbenium ion-like transition state, and that for C2-O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscentsci.0c00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256955PMC
May 2020

Salmonella Typhoid Toxin PltB Subunit and Its Non-typhoidal Salmonella Ortholog Confer Differential Host Adaptation and Virulence.

Cell Host Microbe 2020 06 11;27(6):937-949.e6. Epub 2020 May 11.

Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA. Electronic address:

Typhoidal and non-typhoidal Salmonelleae (NTS) cause typhoid fever and gastroenteritis, respectively, in humans. Salmonella typhoid toxin contributes to typhoid disease progression and chronic infection, but little is known about the role of its NTS ortholog. We found that typhoid toxin and its NTS ortholog induce different clinical presentations. The PltB subunit of each toxin exhibits different glycan-binding preferences that correlate with glycan expression profiles of host cells targeted by each bacterium at the primary infection or intoxication sites. Through co-crystal structures of PltB subunits bound to specific glycan receptor moieties, we show that they induce markedly different glycan-binding preferences and virulence outcomes. Furthermore, immunization with the NTS S. Javiana or its toxin offers cross-reactive protection against lethal-dose typhoid toxin challenge. Cumulatively, these results offer insights into the evolution of host adaptations in Salmonella AB toxins, their cell and tissue tropisms, and the design for improved typhoid vaccines and therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2020.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292776PMC
June 2020

The effects of knock-down resistance mutations and alternative splicing on voltage-gated sodium channels in Musca domestica and Drosophila melanogaster.

Insect Biochem Mol Biol 2020 07 4;122:103388. Epub 2020 May 4.

School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom. Electronic address:

Voltage-gated sodium channels (VGSCs) are a major target site for the action of pyrethroid insecticides and resistance to pyrethroids has been ascribed to mutations in the VGSC gene. VGSCs in insects are encoded by only one gene and their structural and functional diversity results from posttranscriptional modification, particularly, alternative splicing. Using whole cell patch clamping of neurons from pyrethroid susceptible (wild-type) and resistant strains (s-kdr) of housefly, Musca domestica, we have shown that the V for activation and steady state inactivation of sodium currents (I) is significantly depolarised in s-kdr neurons compared with wild-type and that 10 nM deltamethrin significantly hyperpolarised both of these parameters in the neurons from susceptible but not s-kdr houseflies. Similarly, tail currents were more sensitive to deltamethrin in wild-type neurons (EC 14.5 nM) than s-kdr (EC 133 nM). We also found that in both strains, I are of two types: a strongly inactivating (to 6.8% of peak) current, and a more persistent (to 17.1% of peak) current. Analysis of tail currents showed that the persistent current in both strains (wild-type EC 5.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-type EC 35.1 nM). It has been shown previously, that the presence of exon l in the Drosophila melanogaster VGSC gives rise to a more persistent I than does the alternative splice variant containing exon k and we used PCR with housefly head cDNA to confirm the presence of the housefly orthologues of splice variants k and l. Their effect on deltamethrin sensitivity was determined by examining I in Xenopus oocytes expressing either the k or l variants of the Drosophila para VGSC. Analysis of tail currents, in the presence of various concentrations of deltamethrin, showed that the l splice variant was significantly more sensitive (EC 42 nM) than the k splice variant (EC 866 nM). We conclude that in addition to the presence of point mutations, target site resistance to pyrethroids may involve the differential expression of splice variants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ibmb.2020.103388DOI Listing
July 2020

Human Influenza Virus Hemagglutinins Contain Conserved Oligomannose N-Linked Glycans Allowing Potent Neutralization by Lectins.

Cell Host Microbe 2020 05 15;27(5):725-735.e5. Epub 2020 Apr 15.

Department of Molecular Medicine, Scripps Research, La Jolla, CA 92037, USA; Department of Immunology & Microbiology, Scripps Research, La Jolla, CA 92037, USA. Electronic address:

Hemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2020.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158820PMC
May 2020

Major antigenic site B of human influenza H3N2 viruses has an evolving local fitness landscape.

Nat Commun 2020 03 6;11(1):1233. Epub 2020 Mar 6.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

Antigenic drift of influenza virus hemagglutinin (HA) is enabled by facile evolvability. However, HA antigenic site B, which has become immunodominant in recent human H3N2 influenza viruses, is also evolutionarily constrained by its involvement in receptor binding. Here, we employ deep mutational scanning to probe the local fitness landscape of HA antigenic site B in six different human H3N2 strains spanning from 1968 to 2016. We observe that the fitness landscape of HA antigenic site B can be very different between strains. Sequence variants that exhibit high fitness in one strain can be deleterious in another, indicating that the evolutionary constraints of antigenic site B have changed over time. Structural analysis suggests that the local fitness landscape of antigenic site B can be reshaped by natural mutations via modulation of the receptor-binding mode. Overall, these findings elucidate how influenza virus continues to explore new antigenic space despite strong functional constraints.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-15102-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060233PMC
March 2020

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3-subunit.

FASEB J 2020 03 16;34(3):3537-3553. Epub 2020 Jan 16.

Deparment of Biochemistry, University of Cambridge, Cambridge, UK.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated β-subunits. The β3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length β3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the β3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the β3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the β3-subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the β3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the β3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the β3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201701473RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079131PMC
March 2020

Over-accumulation of abscisic acid in transgenic tomato plants increases the risk of hydraulic failure.

Plant Cell Environ 2020 03 9;43(3):548-562. Epub 2020 Jan 9.

EGFV, Bordeaux-Sciences Agro, INRA, Univ. Bordeaux, ISVV, Villenave d'Ornon, France.

Climate change threatens food security, and plant science researchers have investigated methods of sustaining crop yield under drought. One approach has been to overproduce abscisic acid (ABA) to enhance water use efficiency. However, the concomitant effects of ABA overproduction on plant vascular system functioning are critical as it influences vulnerability to xylem hydraulic failure. We investigated these effects by comparing physiological and hydraulic responses to water deficit between a tomato (Solanum lycopersicum) wild type control (WT) and a transgenic line overproducing ABA (sp12). Under well-watered conditions, the sp12 line displayed similar growth rate and greater water use efficiency by operating at lower maximum stomatal conductance. X-ray microtomography revealed that sp12 was significantly more vulnerable to xylem embolism, resulting in a reduced hydraulic safety margin. We also observed a significant ontogenic effect on vulnerability to xylem embolism for both WT and sp12. This study demonstrates that the greater water use efficiency in the tomato ABA overproducing line is associated with higher vulnerability of the vascular system to embolism and a higher risk of hydraulic failure. Integrating hydraulic traits into breeding programmes represents a critical step for effectively managing a crop's ability to maintain hydraulic conductivity and productivity under water deficit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/pce.13703DOI Listing
March 2020

Evidence for an effect of receptor density on ligand occupancy and agonist EC.

Sci Rep 2019 12 13;9(1):19111. Epub 2019 Dec 13.

Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, UK.

Drug-receptor interaction theory predicts that proportional receptor occupancy is a function of ligand concentration as defined by a ligand-receptor affinity constant, and is independent of receptor density. However, we previously observed that the EC of 5-HT reduced as the density of 5-HT receptors increased, suggesting an effect of receptor density on occupancy. The current study was designed to maximise variability in experimentally observed currents and confirm this apparent contradiction prospectively. Xenopus oocytes were injected with RNA encoding 5-HTA receptors under conditions designed to achieve varying receptor expression levels and 5-HT-evoked currents measured using two electrode voltage clamp. Results from 99 oocytes showed that as the maximal peak current increased from 0.05 µA to 12.1 µA there was a 3.7-fold reduction in EC. Since occupancy and conductance are directly related in this system, this indicates that for a given concentration of 5-HT, proportional occupancy increases with increased receptor density. We conclude that normalising data masks this correlation, and can result in reduced accuracy of pharmacological measurements. We propose a mechanistic explanation for our observations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-55361-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910986PMC
December 2019

The mechanism of root growth inhibition by the endocrine disruptor bisphenol A (BPA).

Environ Pollut 2020 Feb 4;257:113516. Epub 2019 Nov 4.

Department of Molecular Biology, Sejong University, Seoul, 143-747, South Korea; Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul, 143-747, South Korea; The Plant Engineering Research Institute, Sejong University, Seoul, 143-747, South Korea. Electronic address:

Bisphenol A (BPA) is a harmful environmental contaminant acting as an endocrine disruptor in animals, but it also affects growth and development in plants. Here, we have elucidated the functional mechanism of root growth inhibition by BPA in Arabidopsis thaliana using mutants, reporter lines and a pharmacological approach. In response to 10 ppm BPA, fresh weight and main root length were reduced, while auxin levels increased. BPA inhibited root growth by reducing root cell length in the elongation zone by suppressing expansin expression and by decreasing the length of the meristem zone by repressing cell division. The inhibition of cell elongation and cell division was attributed to the enhanced accumulation/redistribution of auxin in the elongation zone and meristem zone in response to BPA. Correspondingly, the expressions of most auxin biosynthesis and transporter genes were enhanced in roots by BPA. Taken together, it is assumed that the endocrine disruptor BPA inhibits primary root growth by inhibiting cell elongation and division through auxin accumulation/redistribution in Arabidopsis. This study will contribute to understanding how BPA affects growth and development in plants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.envpol.2019.113516DOI Listing
February 2020

Control of water-use efficiency by florigen.

Plant Cell Environ 2020 01 5;43(1):76-86. Epub 2019 Nov 5.

Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Brazil.

A major issue in modern agriculture is water loss through stomata during photosynthetic carbon assimilation. In water-limited ecosystems, annual plants have strategies to synchronize their growth and reproduction to the availability of water. Some species or ecotypes of flowers are early to ensure that their life cycles are completed before the onset of late season terminal drought ("drought escape"). This accelerated flowering correlates with low water-use efficiency (WUE). The molecular players and physiological mechanisms involved in this coordination are not fully understood. We analyzed WUE using gravimetry, gas exchange, and carbon isotope discrimination in florigen deficient (sft mutant), wild-type (Micro-Tom), and florigen over-expressing (SFT-ox) tomato lines. Increased florigen expression led to accelerated flowering time and reduced WUE. The low WUE of SFT-ox was driven by higher stomatal conductance and thinner leaf blades. This florigen-driven effect on WUE appears be independent of abscisic acid (ABA). Our results open a new avenue to increase WUE in crops in an ABA-independent manner. Manipulation of florigen levels could allow us to produce crops with a life cycle synchronized to water availability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/pce.13664DOI Listing
January 2020

Anesthetics: from modes of action to unconsciousness and neurotoxicity.

J Neurophysiol 2019 08 26;122(2):760-787. Epub 2019 Jun 26.

Vi Riddell Pain Program, Alberta Children's Hospital Research Institute, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

Modern anesthetic compounds and advanced monitoring tools have revolutionized the field of medicine, allowing for complex surgical procedures to occur safely and effectively. Faster induction times and quicker recovery periods of current anesthetic agents have also helped reduce health care costs significantly. Moreover, extensive research has allowed for a better understanding of anesthetic modes of action, thus facilitating the development of more effective and safer compounds. Notwithstanding the realization that anesthetics are a prerequisite to all surgical procedures, evidence is emerging to support the notion that exposure of the developing brain to certain anesthetics may impact future brain development and function. Whereas the data in support of this postulate from human studies is equivocal, the vast majority of animal research strongly suggests that anesthetics are indeed cytotoxic at multiple brain structure and function levels. In this review, we first highlight various modes of anesthetic action and then debate the evidence of harm from both basic science and clinical studies perspectives. We present evidence from animal and human studies vis-à-vis the possible detrimental effects of anesthetic agents on both the young developing and the elderly aging brain while discussing potential ways to mitigate these effects. We hope that this review will, on the one hand, invoke debate vis-à-vis the evidence of anesthetic harm in young children and the elderly, and on the other hand, incentivize the search for better and less toxic anesthetic compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/jn.00210.2019DOI Listing
August 2019

A challenge finding P2X1 and P2X4 ligands.

Neuropharmacology 2019 10 24;157:107674. Epub 2019 Jun 24.

Department of Pharmacology, University of Cambridge, Cambridge, UK. Electronic address:

Identifying novel small-molecule P2X1 and P2X4 ligands with sub-type specificity and high-affinity remains a pharmacological challenge. Here we use computational methods, electrophysiology and fluorescent microplate assays to screen for ligand candidates acting at these receptors. Modelling and docking identified 80 compounds for testing at P2X4 receptors, and 20 of these showed >50% inhibition in fluorescence-based assays, making them appealing for further SAR studies. Confirmation of activity by two-electrode voltage clamp, followed by their elaboration resulted in only minor improvements in potency, with the highest IC being 295 μM. Testing on P2X1 receptors, resulted in a series of biguanide compounds that yielded a maximum IC of 100 μM, but no consistent SAR could be found. Potencies of established antagonists gave expected results, although the measured potencies varied between techniques and no antagonism could be found for compounds such as paroxetine, carbamazepine, 9(10H)-acridanone, acridinol and phenoxazine-type heterocycles. This study highlights the challenge of identifying P2X4 and P2X1 ligands and suggests that a combination of complimentary approaches is needed if we are to be confident of ligand activities at these receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuropharm.2019.107674DOI Listing
October 2019

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

Mol Cell 2019 07 18;75(2):394-407.e5. Epub 2019 Jun 18.

Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen, Denmark. Electronic address:

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660356PMC
July 2019

Author Correction: In vivo tropism of Salmonella Typhi toxin to cells expressing a multiantennal glycan receptor.

Nat Microbiol 2019 07;4(7):1242

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0505-7DOI Listing
July 2019

Preventing an Antigenically Disruptive Mutation in Egg-Based H3N2 Seasonal Influenza Vaccines by Mutational Incompatibility.

Cell Host Microbe 2019 Jun 28;25(6):836-844.e5. Epub 2019 May 28.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Egg-based seasonal influenza vaccines are the major preventive countermeasure against influenza virus. However, their effectiveness can be compromised when antigenic changes arise from egg-adaptive mutations on influenza hemagglutinin (HA). The L194P mutation is commonly observed in egg-based H3N2 vaccine seed strains and significantly alters HA antigenicity. An approach to prevent L194P would therefore be beneficial. We show that emergence of L194P during egg passaging can be impeded by preexistence of a G186V mutation, revealing strong incompatibility between these mutations. X-ray structures illustrate that individual G186V and L194P mutations have opposing effects on the HA receptor-binding site (RBS), and when both G186V and L194P are present, the RBS is severely disrupted. Importantly, wild-type HA antigenicity is maintained with G186V, but not L194P. Our results demonstrate that these epistatic interactions can be used to prevent the emergence of mutations that adversely alter antigenicity during egg adaptation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2019.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579542PMC
June 2019

Virus recognition of glycan receptors.

Curr Opin Virol 2019 02 5;34:117-129. Epub 2019 Mar 5.

Departments of Molecular Medicine, Immunology & Microbiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Attachment of viruses to cell-surface receptors is the initial step in infection. Many mammalian viruses have evolved to recognize receptors that are glycans on cell-surface glycoproteins or glycolipids. Although glycans are a ubiquitous component of mammalian cells, the types of terminal structures expressed vary among different cell-types and tissues, and even between comparable cells and tissues from different species, frequently leading to specific tissue and species tropisms as a direct consequence of glycan receptor recognition. Covering the majority of known virus families, this review provides an overview of mammalian viruses that use glycans as receptors, and their roles in determining in host recognition and tropism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.coviro.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476673PMC
February 2019

Learning from a failure in threat assessment: 11 questions and not enough answers.

Behav Sci Law 2019 Jul 14;37(4):353-371. Epub 2019 Feb 14.

Center for the Study and Prevention of Violence, University of Colorado Boulder, Boulder, CO, USA.

Threat assessment theory and practice have evolved significantly since Columbine. The US Secret Service's guidelines for threat assessment include 11 questions that school officials should ask to identify, investigate, and manage students of concern. Yet, no research examines how school officials implement these questions. This qualitative case study examines the way that school officials used the 11 questions with a student of concern, who underwent a threat assessment and 3 months later shot and killed a classmate and himself on school grounds. The data include deposition testimony from 12 school and district officials and more than 8,000 pages of records in the case. For each of the 11 questions, the findings reveal what the threat assessment team knew and might have learned; the findings also demonstrate the importance of multiple sources of information, a multidisciplinary team, and an investigative mindset. The questions may prove difficult to answer in "loosely coupled" systems, like schools, where information is unintentionally lost due to the organization's structural hierarchy, specialization of tasks, and heavy workloads. The findings provide critical lessons learned for threat assessment, information gathering, and violence prevention in schools.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bsl.2399DOI Listing
July 2019

Flexibility of Amino Acid 226 in the Receptor-Binding Site of an H9 Subtype Influenza A Virus and Its Effect on Virus Replication, Tropism, and Transmission.

J Virol 2019 03 5;93(6). Epub 2019 Mar 5.

Poultry Diagnostic and Research Center, Department of Population Health, University of Georgia, Athens, Georgia, USA

Influenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 2015-2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from avian-like to human-like terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred an replication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variants and A single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02011-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401463PMC
March 2019

A member of the TERMINAL FLOWER 1/CENTRORADIALIS gene family controls sprout growth in potato tubers.

J Exp Bot 2019 02;70(3):835-843

The James Hutton Institute, Invergowrie, Dundee, UK.

Potato tuber bud dormancy break followed by premature sprouting is a major commercial problem which results in quality losses and decreased tuber marketability. An approach to controlling premature tuber sprouting is to develop potato cultivars with a longer dormancy period and/or reduced rate of sprout growth. Our recent studies using a potato diploid population have identified several quantitative trait loci (QTLs) that are associated with tuber sprout growth. In the current study, we aim to characterize a candidate gene associated with one of the largest effect QTLs for rapid tuber sprout growth on potato chromosome 3. Underlying this QTL is a gene encoding a TERMINAL FLOWER 1/CENTRORADIALIS homologue (PGSC0003DMG400014322). Here, we use a transgenic approach to manipulate the expression level of the CEN family member in a potato tetraploid genotype (cv. Désirée). We demonstrate a clear effect of manipulation of StCEN expression, with decreased expression levels associated with an increased rate of sprout growth, and overexpressing lines showing a lower rate of sprout growth than controls. Associated with different levels of StCEN expression were different levels of abscisic acid and cytokinins, implying a role in controlling the levels of plant growth regulators in the apical meristem.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jxb/ery387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363080PMC
February 2019

Plasticity of Amino Acid Residue 145 Near the Receptor Binding Site of H3 Swine Influenza A Viruses and Its Impact on Receptor Binding and Antibody Recognition.

J Virol 2019 01 4;93(2). Epub 2019 Jan 4.

Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens, Georgia, USA

The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of amino acid residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single-amino-acid level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without having a major impact on virus replication All substitution mutants retained receptor binding specificity, but the substitutions frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of amino acid substitutions near the RBS and the interplay between receptor binding and antigenic drift. The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the hemagglutinin (HA) of the H3N2 IAVs, the amino acid substitution N 145 K causes significant antigenic changes. We show that amino acid 145 displays remarkable amino acid plasticity , tolerating multiple amino acid substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment in virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145 K substitution in antibody immunodominance. We provide a better understanding of the functional effects of amino acid substitutions implicated in antigenic drift and its consequences for receptor binding and antigenicity. The mutation analyses presented in this report represent a significant data set to aid and test the ability of computational approaches to predict binding of glycans and in antigenic cartography analyses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01413-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321904PMC
January 2019

Avenues to Characterize the Interactions of Extended N-Glycans with Proteins by NMR Spectroscopy: The Influenza Hemagglutinin Case.

Angew Chem Int Ed Engl 2018 11 17;57(46):15051-15055. Epub 2018 Oct 17.

Dpto Química Orgánica I, Fac. Ciencias Químicas, Universidad Complutense de Madrid, Avd. Complutense s/n, 28040, Madrid, Spain.

Long-chain multiantenna N-glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X-ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N-glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N-glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N-glycan in the presence of HK/68 hemagglutinin is described.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201807162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282704PMC
November 2018