Biology subjects

Scott, S.

Publications and source records attributed to Scott, S..

25 records · Page 2Linked to original sources

bioRxiv2019

Single-molecule visualization of the effects of ionic strength and crowding on structure-mediated interactions in supercoiled DNA molecules

DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe-plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe-plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNAs proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe-plasmid interactions once the site is unwound.

biophysics↗

bioRxiv2018

The bat influenza H17N10 can be neutralized by broadly-neutralizing monoclonal antibodies and its neuraminidase can facilitate viral egress.

The diversity of subtypes within the Influenza A virus genus has recently expanded with the identification of H17N10 and H18N11 from bats. In order to further study the tropism and zoonotic potential of these viruses, we have successfully produced lentiviral pseudotypes bearing both haemagglutinin H17 and neuraminidase N10. These pseudotypes were shown to be efficiently neutralized by the broadly-neutralizing monoclonal antibodies CR9114 and FI6. Our studies also confirm previous reports that H17 does not use sialic acid as its cellular receptor, as pseudotypes bearing the H17 envelope glycoprotein are released into the cell supernatant in the absence of NA. However, we demonstrate that N10 facilitates heterosubtypic (H5 and H7) influenza HA-bearing pseudotype release in the absence of another source of NA, significantly increasing luciferase pseudotype production titres. Despite this, N10 shows no activity in the enzyme-linked lectin assay used for traditional sialidases. These findings suggest that this protein plays an important role in viral egress, but is perhaps involved in further accessory roles in the bat influenza lifecycle that are yet to be discovered. Thus we show the lentiviral pseudotype system is a useful research tool, and amenable for investigation of bat influenza tropism, restriction and sero-epidemiology, without the constraints or safety issues with producing a replication-competent virus, to which the human population is naive.\n\nSignificance statementInfluenza virus is responsible for mortality and morbidity across the globe; human populations are constantly at risk of newly emerging strains from the aquatic bird reservoir which harbors most of the subtypes of influenza A (H1-H16). Recently identified subtypes (H17N10, H18N11) from bats have broadened the reservoir from which potential pandemic strains of influenza can emerge. To evaluate the potential for these novel subtypes to cross over into human populations, their ability to establish an infection, in addition to the extent of cross-reactive immunity established by human seasonal strains needs to be investigated. This study highlights a novel platform for the study of the bat H17 and N10 envelope glycoproteins, using a lentiviral pseudotype system. Following the generation of this pseudotype it was employed in cell entry and microneutralization assays. These showed that two well-characterised monoclonal antibodies (mAb) which target avian and human influenza subtypes will also neutralize H17. Furthermore the data presented in this study show a novel aspect of the N10 glycoprotein in its ability to facilitate the budding of pseudotypes bearing different influenza HAs.

microbiology↗

bioRxiv2018

Development and use of Lentiviral Vectors Pseudotyped with Influenza B Haemagglutinins: application to vaccine immunogenicity, mAb potency and sero-surveillance studies

Influenza B viruses cause respiratory disease epidemics in human populations and are included in seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, the haemagglutination inhibition assay, which represents the gold standard for assessing the immunogenicity of influenza vaccines, has been shown to be relatively insensitive for the detection of antibodies against influenza B viruses. Furthermore, this assay, and the serial radial haemolysis assay are not able to detect stalk-directed cross-reactive antibodies. For these reasons there is a need to develop new assays that can overcome these limitations. The use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A haemagglutinins, in microneutralization assays is a safe and sensitive alternative to study antibody responses elicited by natural infection or vaccination. We have produced Influenza B haemagglutinin-pseudotypes using plasmid-directed transfection. To activate influenza B haemagglutinin, we have explored the use of proteases by adding relevant encoding plasmids to the transfection mixture. When tested for their ability to transduce target cells, the newly produced influenza B pseudotypes exhibit tropism for different cell lines. Subsequently the pseudotypes were evaluated as surrogate antigens in microneutralization assays using reference sera, monoclonal antibodies, human sera collected during a vaccine immunogenicity study and surveillance sera from seals. The influenza B pseudotype virus neutralization assay was found to effectively detect neutralizing and cross-reactive responses despite lack of significant correlation with the haemagglutinin inhibition assay.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC=\"FIGDIR/small/492785_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (19K):\norg.highwire.dtl.DTLVardef@d5d8e7org.highwire.dtl.DTLVardef@1934a06org.highwire.dtl.DTLVardef@e0d0b8org.highwire.dtl.DTLVardef@1a717e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2018

Heteromeric GABAA receptor structures in positively-modulated active states

Type-A {gamma}-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of /{beta}/{gamma} subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human 1{beta}3{gamma}2 GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered 5 and 5{gamma}2 GABAAR constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABAAR activation.

biophysics↗

bioRxiv2018

Visualizing structure-mediated interactions in supercoiled DNA molecules

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmids structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

biophysics↗

bioRxiv2017

The use of hyperimmune chicken reference sera is not appropriate for the validation of influenza pseudotype neutralization assays

Pseudotype particle neutralization (pp-NT) is a next-generation serological assay employed for the sensitive study of influenza antibody responses, especially haemagglutinin stalk-directed antibodies. However, to date a validation of this assay has not been performed, and this limits its use to primarily research laboratories. To identify possible serological standards to be used in optimization and validation of the pp-NT, we have evaluated the cross-reactivity of hyperimmune chicken reference antisera in this assay. Our findings show that the cross-reactivity detected by the pp-NT assay is only in part explained by phylogenetic relationships and protein homology between the HA subtypes analysed; further studies are necessary to understand the origin of the cross-reactivity detected, and reference standards with higher specificity should be evaluated or generated de novo for future use in pp-NT.

microbiology↗

bioRxiv2017

Conditionally Redundant Bacteriocin Targeting by Pseudomonas syringae

The widespread use of antimicrobials under clinical and agricultural settings has resulted in the evolution of resistance to these compounds. To combat the emergence of resistance, current research efforts are focusing on designing treatments to exploit combinations of antimicrobials, where the evolution of resistance confers sensitivity to alternative compounds. In this work we demonstrate that strains of Pseudomonas syringae possess a natural analogue to this strategy. Specifically, we demonstrate that a single strain produces multiple bacteriocins that can target another strain, but antimicrobial activity of the second bacteriocin is manifest only after resistance to the first emerges. The evolution of resistance also sensitizes the target strain to bacteriocins from a variety of other strains. Strains of P. syringae therefore encode multiple bacteriocins that can act in a conditionally redundant manner. It is possible that combinations of bacteriocins could be applied as a cocktail or sequentially, to potentially achieve durable pathogen control.

microbiology↗