Biology subjects

Newsome, T. P.

Publications and source records attributed to Newsome, T. P..

4 recordsLinked to original sources

bioRxiv2024

Large scale semi-automated quantification of viral spread reveals multi-phasic structure of plaque formation

Plaque assays are typically quantified at an endpoint and fail to capture the temporal dynamics of viral infection and spread. In this study we introduce a high-throughput method utilising live-cell imaging and a recombinant vaccinia virus (VACV) engineered to express two fluorescent transgenes linked to distinct replication phases. This novel approach allows for real-time tracking of VACV plaques from their inception at a single infected cell through to multicellular expansion, enabling detailed kinetic analysis of plaque development. Using this analysis pipeline we categorise VACV plaque formation into three distinct phases: Establishment, Expansion, and Exhaustion, and quantitatively describe their dynamics. Our findings reveal significant variability in the initiation time of plaque spread (from single-cell to multicellular stages) and the growth rate of plaques, both between individual plaques and across different cell lines. This variability underscores the influence of host cellular factors on the kinetics of viral replication. Additionally, we reveal that the viral replication cycle dramatically accelerates through the early phases of plaque formation, which we hypothesise is driven by the changing multiplicity of cellular infection at the plaque edge. Finally, we demonstrate that differences in plaque sizes between two cell lines (BS-C-1 and HaCaT) can be largely attributed to variations in viral replication rate. This research reinforces the value of live-cell fluorescence microscopy in elucidating the complex spatiotemporal dynamics of viral infections, and contributes to a deeper understanding of the mechanisms driving viral spread and replication kinetics.

microbiology↗

bioRxiv2024

Antimicrobial mechanism of in-situ plasma activated water treatment of pathogenic Escherichia coli and Staphylococcus aureus biofilms

AimsThis study investigated the efficacy and mechanisms of inactivation of against Escherichia coli UTI89 and Staphylococcus aureus NCTC8325 through an in-situ plasma-activated water (PAW) treatment. Methods and ResultsPAW was prepared by discharging atmospheric pressure cold plasma beneath the surface of sterile distilled water. The study investigated the inactivation of biofilm cells and biofilm matrix. A complete killing of biofilm cells was achieved on both of E. coli (6.76 {+/-} 0.01 log CFU/mL) and S. aureus (6.82 {+/-} 0.02 log CFU/mL). This process happened earlier in S. aureus. Simultaneously, PAW treatment disrupted the biofilm structure, inducing a significant reduction in general biofilm biomass and extracellular polymer substances (EPS) matrix. With the disruption of EPS, PAW was enabled to further interact with the bacterial membrane, causing a significant increase in membrane permeability and disrupted membrane structure. Finally, PAW treatment led to a significant accumulation of intracellular reactive oxygen and nitrogen species within the biofilm cells. ConclusionsCollectively, these findings indicate that PAW effectively inactivates biofilms by mechanically targeting the biofilm EPS matrix and biofilm cells in both gram-negative and gram-positive bacteria. Impact statementThis study contributes novel insights into plasma-activated waters mechanisms of action, particularly its impact on the biofilm extracellular polymeric substances matrix (exopolysaccharides, extracellular DNA, and protein), cell membrane permeability, depolarization, and intracellular ROS and RNS accumulation in both of Gram-positive and Gram-negative species. These findings highlight PAW-based treatments against biofilm-related challenges in antimicrobial development and water system decontamination.

microbiology↗

bioRxiv2022

Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection

Intracellular mature virions (IMV) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMV can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMV, like intracellular enveloped virions (IEV), the second form of vaccinia, undergo anterograde transport and recruit kinesin-1. In vitro reconstitution of virion transport reveals that IMV and IEV move toward microtubule plus-ends with respective velocities of 0.66 and 0.56 m/s. Quantitative imaging establishes IMV and IEV recruit an average of 65 and 115 kinesin-1 motor complexes respectively. In the absence of kinesin-1 there is a near-complete loss of in vitro motility and defects in the cellular spread of both virions. Our observations demonstrate kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to investigate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.

cell biology↗

bioRxiv2019

TNF deficiency dysregulates inflammatory cytokine production leading to lung pathology and death during respiratory poxvirus infection

Excessive tumor necrosis factor (TNF) is known to cause significant pathology. Paradoxically, deficiency in TNF (TNF-/-) also caused significant pathology during respiratory ectromelia virus (ECTV) infection, a surrogate mouse model for smallpox. TNF-/- mice succumbed to fulminant disease whereas wild-type mice, and those expressing only transmembrane TNF, recovered. TNF deficiency did not affect viral load or leukocyte recruitment but caused severe lung pathology and excessive production of the cytokines IL-6, IL-10, TGF-{beta}, and IFN-{gamma}. Blockade of these cytokines reduced lung pathology concomitant with induction of protein inhibitor of activated STAT3 (PIAS3) and/or suppressor of cytokine signaling 3 (SOCS3), factors that inhibit STAT3 activation. Short-term inhibition of STAT3 activation in ECTV-infected TNF-/- mice with an inhibitor reduced lung pathology. TNF is essential for regulating inflammation and its deficiency exacerbates ECTV infection as a consequence of significant lung pathology caused by dysregulation of inflammatory cytokine production, in part via overactivation of STAT3.

immunology↗