Comparing antiviral strategies against COVID-19 via multiscale within-host modelling.

Within-host models of COVID-19 infection dynamics enable the merits of different forms of antiviral therapy to be assessed in individual patients. A stochastic agent-based model of COVID-19 intracellular dynamics is introduced here, that incorporates essential steps of the viral life cycle targeted by treatment options. Integration of model predictions with an intercellular ODE model of within-host infection dynamics, fitted to patient data, generates a generic profile of disease progression in patients that have recovered in the absence of treatment. This is contrasted with the profiles obtained after variation of model parameters pertinent to the immune response, such as effector cell and antibody proliferation rates, mimicking disease progression in immunocompromised patients. These profiles are then compared with disease progression in the presence of antiviral and convalescent plasma therapy against COVID-19 infections. The model reveals that using both therapies in combination can be very effective in reducing the length of infection, but these synergistic effects decline with a delayed treatment start. Conversely, early treatment with either therapy alone can actually increase the duration of infection, with infectious virions still present after the decline of other markers of infection. This suggests that usage of these treatments should remain carefully controlled in a clinical environment.

Expression, purification and metal utilization of recombinant SodA from Borrelia burgdorferi.

Borrelia are microaerophilic spirochetes capable of causing multisystemic diseases such as Lyme disease and Relapsing Fever. The ubiquitous Fe/Mn-dependent superoxide dismutase (SOD) provides essential protection from oxidative damage by the superoxide anion. Borrelia possess a single SOD enzyme - SodA that is essential for virulence, providing protection against host-derived reactive oxygen species (ROS). Here we present a method for recombinant expression and purification of Borrelia burgdorferi SodA in E. coli. Metal exchange or insertion into the Fe/Mn-SOD is inhibited in the folded state. We therefore present a method whereby the recombinant Borrelia SodA binds to Mn under denaturing conditions and is subsequently refolded by a reduction in denaturant. SodA purified by metal affinity chromatography and size exclusion chromatography reveals a single band on SDS-PAGE. Protein folding is confirmed by circular dichroism. A coupled enzyme assay demonstrates SOD activity in the presence of Mn, but not Fe. The apparent molecular weight determined by size exclusion corresponds to a dimer of SodA; a homology model of dimeric SodA is presented revealing a surface Cys distal to the dimer interface. The method presented of acquiring a target metal under denaturing conditions may be applicable to the refolding of other metal-binding proteins.

Pectin Conformation in Solution.

The interplay of degree of methylesterification (DM), pH, temperature, and concentration on the macromolecular interactions of pectin in solution has been explored. Small-angle X-ray scattering complemented by atomic force microscopy and molecular dynamics was employed to probe chain dimensions and solution structure. Two length scales have been observed with the first level of structure characterising chain clusters with sizes ranging between 100-200 nm. The second level of structure arises from single biopolymer chains with a radius of gyration between ∼6 and 42 nm. The development of a range of macromolecular dimensions in vitro and in silico shows that the chain flexibility increases with DM and at acidic pH, whereas hydrogen bonding is the responsible thermodynamic driving force for cluster formation. High methyl pectins create structures of lower fractal dimension with less efficient packing. This work unveils pectin conformations covering most of its industrially and biologically relevant environments, enabling rational design of advanced biomaterials based on pectin.

Mesoscopic structure of pectin in solution.

Mesoscopic structure of pectin with different molecular characteristics was investigated by means of small angle X-ray scattering (SAXS), electrokinetic measurements and data modelling. The influence of a broad range of pH (2-7) on chain conformation in the dilute and semi-diluted regime was investigated. Scattering data and concomitant analysis revealed two length scales at all environmental conditions studied. pH showed greater influence at acidic values (pH 2.0) enhancing the globular component of the structure due to association of galacturonic acid residues. Double logarithmic scattering intensity plots revealed fractal dimensions of 1.9 ± 0.2 in the low-q regime and 1.5 ± 0.2 in the high q-region, irrespectively of the specific environment. Increase in branching of RG-I regions of the polysaccharide chains enhanced the compact conformation irrespectively of the pH or concentration. The present work shows that radical changes in pectin conformation can be induced only under strongly acidic conditions a finding that has important consequences in tailoring the technological performance of these biopolymers.

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging.

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly.

Communication: Non-monotonic supersaturation dependence of the nucleus size of crystals with anisotropically interacting molecules.

We study the nucleation of model two-dimensional crystals formed from anisotropically interacting molecules using kinetic Monte Carlo simulations and the forward flux sampling algorithm. The growth probability P(n) of a cluster of n molecules is measured while the supersaturation s and interaction anisotropy of the molecules are varied, in order to gain insight into the nucleation mechanism. It is found that with increasing degree of interaction anisotropy the nucleus size (defined as the cluster size at which P(n) = 0.5) can increase with increasing s, with sharp jumps at certain s values. Analysis of the cluster shape reveals that nucleation in the system studied is of a non-standard form, in that it embodies elements of both the classical nucleation theory and the density functional theory frameworks.

Alkaline induction of a novel gene locus, alx, in Escherichia coli.

A novel pH-regulated locus inducible over 100-fold in alkaline media was identified in Escherichia coli through screening of 93,000 Mu dI1734 (lacZ Kmr) operon fusions at pH 6.5 and pH 8.5. Four lacZ fusions that showed expression only at the higher pH were mapped at 67.5 min by P1 transduction crosses. The locus was designated alx.