Biology subjects

Knight, M.

Publications and source records attributed to Knight, M..

26 records · Page 2Linked to original sources

bioRxiv2020

A nanocompartment containing the peroxidase DypB contributes to defense against oxidative stress in M. tuberculosis

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelles consisting of an encapsulin protein shell that encloses a protein cargo1. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes2. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogen Mycobacterium tuberculosis (Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DypB. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

microbiology↗

bioRxiv2020

Plasmablast-derived antibody response to acute SARS-CoV-2 infection in humans

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 13.0% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-RBD, three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. At last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

microbiology↗

bioRxiv2020

A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy

In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provides a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain (NTD) of the viral S glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain (RBD) of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic virus infection in a standard assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.

immunology↗

bioRxiv2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

microbiology↗

bioRxiv2020

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a fingerprint of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (g-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

biochemistry↗

bioRxiv2018

Competition and Synergy Between Prochlorococcus and Synechococcus Under Ocean Acidification Conditions

Anthropogenic CO2 emissions are projected to lower the pH of the open ocean by 0.2 to 0.3 units over the next century. Laboratory experiments show that different phytoplankton taxa exhibit a wide variety of responses, with some strains having higher fitness under projected future conditions, and others being negatively impacted. Previous studies have suggested that Prochlorococcus and Synechococcus, the numerically dominant picophytoplankton in the oceans, have very different responses to elevated CO2 that may result in a dramatic shift in their relative abundances in future oceans. Here we show that these two genera experience faster exponential growth rates under future CO2 conditions, similar to most other cyanobacteria that have been studied. However, Prochlorococcus strains have significantly lower realized growth rates due to more extreme lag periods after exposure to fresh culture media. Surprisingly, however, Synechococcus was unable to outcompete Prochlorococcus in co-culture at elevated CO2. Under these conditions, Prochlorococcus poor response to elevated CO2 disappeared, and it showed negative frequency dependence in its relative fitness compared to Synechococcus, with a significant fitness advantage when it was initially rare. Moreover, both Synechococcus and Prochlorococcus had faster growth rates in co-culture with each other than either had in unialgal culture. We speculate that this negative frequency dependence is an outgrowth of reductive Black Queen evolution operating on both taxa that has resulted in a passively mutualistic relationship analogous to that connecting Prochlorococcus with the \"helper\" heterotrophic microbes in its environment.

microbiology↗

bioRxiv2018

Low dose resveratrol promotes hypertrophy in wildtype skeletal muscle and reduces damage in skeletal muscle of exercised mdx mice.

Duchenne muscular dystrophy (DMD) is a progressive and fatal neuromuscular disorder for which there is no treatment. Therapies to restore dystrophin deficiency are not ready for clinical use and long-term efficiency is yet to be established. Therefore, there is a need to develop alternative strategies to treat DMD. Resveratrol is a nutraceutical with anti-inflammatory properties and previous studies have shown that high doses can benefit mdx mice. We treated 4-week-old mdx and wildtype mice with low-dose resveratrol (5mg/kg bodyweight/day) for 15 weeks. A voluntary exercise protocol was added to test if low dose resveratrol could reduce exercise-induced damage. We showed that resveratrol promoted skeletal muscle hypertrophy in the wildtype mice. There was no change in markers of pathology in the mdx mice; however, the low-dose resveratrol reduced exercised induced damage. Gene expression of immune cell markers such as CD86, CD163 and PCNA was reduced; however signalling targets associated with resveratrols mechanism of action of action including SIRT1 and NF-{kappa}B were unchanged. In conclusion, low-dose resveratrol was not effective in reducing disease pathology; however, its ability to promote hypertrophy in wildtype skeletal muscle could have direct applications to the livestock industry or in sports medicine.

physiology↗

bioRxiv2018

Benfotiamine reduces pathology and improves muscle function in mdx mice.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal neuromuscular disease which arises from mutations in the dystrophin gene (DMD) that result in the absence or severe reduction of the cytoskeletal protein dystrophin. In addition to the primary dystrophin defect, secondary processes such as inflammation, calcium influx, dysregulated autophagy and fibrosis exacerbate dystrophic pathology and thus increase disease progression. While therapies to restore dystrophin deficiency are being developed, strategies which target these secondary processes could be of benefit to patients. Benfotiamine is a lipid soluble precursor to thiamine that can reduce secondary processes such as inflammation and oxidative stress in diabetic patients. As such we tested it in the mdx mouse model of DMD and found that benfotiamine reduced multiple markers of dystrophic pathology and improved grip strength. In addition, members of the utrophin and dystrophin glycoprotein complexes were significantly increased at the sarcolemma which could improve cell adhesion. We also demonstrated that benfotiamine treatment lowered the expression of macrophage markers and pro-inflammatory cytokines suggesting that benfotiamine is reducing dystrophic pathology by acting on inflammatory processes.

physiology↗