Biology subjects

Walls, A.

Publications and source records attributed to Walls, A..

4 recordsLinked to original sources

bioRxiv2026

Medical Grade Manuka Honey Inhibits A23187 Induced Mast Cell Degranulation and Cytokine Release via Downregulation of the ERK Signalling Pathways

RationaleMast degranulation is a driver of several pathologies. Several endogenous peptides have been established as stimulators of mast cell degranulation. Compounds that stabilise mast cells have in part demonstrated inhibitory effect on the inflammatory cascade. Manuka honey has been widely used for the treatments of various inflammatory diseases. However, its effect on A23187 (Calcium ionophore) stimulated mast cell degranulation, cytokine release and cellular signalling pathways has not yet been investigated. Aim of the studyWe aim to investigate the effect of Medical Grade Manuka Honey (MGMH) on A23187 induced mast cell degranulation, cytokine release and downstream signalling pathways in a human mast cell lines LAD2 model. Materials and methodsThe cytotoxic effect of MGMH on LAD2 cells was assessed using LDH assay. LAD2 cells were pre-incubated with MGMH and challenged with A23187.Mast cell degranulation was measured by {beta}-hexosaminidase release whilst histamine and cytokines released were measured by ELISA. The effect of MGMH on downstream signalling of Mitogen Activated Protein Kinase (MAPK) pathways was determined and quantified by SDS-PAGE western blotting. ResultsMGMH at 2% and 4% was well tolerated by LAD2 cells. MGMH at all concentrations tested significantly inhibited the A23187 triggered release of {beta}-hexosaminidase but failed to inhibit the release of histamine. MGMH 4% significantly inhibited the release of GM-CSF and IL-8. MGMH (2% and 4%) significantly down regulated the expression of both ERK I and ERK II. However, MGMH at all the doses tested had no effect on the expressions of JNK and p38. On the contrary, an increase expression of these p38 and JNK were noted with MGMH pre-incubation. ConclusionOur present study provides evidence that MGMH inhibition of A23187 stimulated degranulation of mast cells and cytokine release through down regulation of ERK I and II signalling. The results suggest potential use as a mast cell stabiliser and effective treatments of mast cell mediated inflammatory diseases. ImpactThis study provides the first evidence that Medical Grade Manuka Honey (MGMH) can attenuate A23187-induced mast cell activation and pro-inflammatory cytokine release in human LAD2 mast cells through modulation of ERK1/2 signalling pathways. The findings advance our understanding of the cellular and molecular mechanisms underlying the anti-inflammatory properties of Manuka honey. The demonstration that MGMH inhibits {beta}-hexosaminidase release suggests a mast cell-stabilising effect, highlighting its potential as a novel natural therapeutic agent for mast cell-mediated disorders, including allergic diseases, interstitial cystitis, asthma, chronic inflammatory skin conditions, and mast cell activation syndromes. The observed reduction in GM-CSF and IL-8 production further indicates that MGMH may help limit the amplification and persistence of inflammatory responses. Mechanistically, the selective downregulation of ERK1/2 signalling provides new insight into how MGMH exerts its biological effects and identifies a potential molecular target through which its anti-inflammatory activity is mediated. These findings contribute to the growing evidence base supporting the therapeutic value of Manuka honey beyond its established antimicrobial and wound-healing properties. Overall, this work lays the foundation for future preclinical and clinical studies investigating MGMH as a safe, naturally derived mast cell stabiliser and anti-inflammatory intervention, with potential applications across a broad spectrum of allergic and inflammatory diseases.

immunology↗

bioRxiv2023

Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

Engineering bacterial metabolism to efficiently produce chemicals and materials from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important metabolic engineering tools for programming multi-gene expression regulation. However, poor predictability of guide RNA folding can disrupt enzyme balance through unreliable expression control. We devised a set of computational parameters that can describe guide RNA folding, and we expect them to be broadly applicable across CRISPR-Cas9 systems. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a kinetic parameter describing folding rate into the active structure. This parameter also enables forward design of new scRNAs, with no observed failures in our screen. We use CRISPRa target sequences from this set to design a system of three synthetic promoters that can orthogonally activate and tune expression of chosen outputs over a >35-fold dynamic range. Independent activation tuning allows experimental exploration of a three-dimensional expression design space via a 64-member combinatorial triple-scRNA library. We apply these CRISPRa programs to two biosynthetic pathways, demonstrating production of valuable pteridine and human milk oligosaccharide products in E. coli. Profiling these design spaces indicated expression combinations producing up to 2.3-fold higher titer than that produced by maximal expression. Mapping production can also identify bottlenecks as targets for pathway redesign, improving titer of the oligosaccharide lacto-N-tetraose by 6-fold. Aided by computational scRNA efficacy prediction, the combinatorial CRISPRa strategy enables effective optimization of multi-step metabolic pathways. More broadly, the guide RNA design rules uncovered here may enable the routine design of effective multi-guide programs for a wide range of model- and data-driven applications of CRISPR gene regulation in bacterial hosts.

synthetic biology↗

bioRxiv2021

QSMxT: Robust Masking and Artefact Reduction for Quantitative Susceptibility Mapping

PurposeQuantitative Susceptibility Mapping (QSM) is a post-processing technique applied to gradient-echo phase data. QSM algorithms require a signal mask to delineate regions with reliable phase signal for subsequent susceptibility estimation. Existing masking techniques used in QSM have limitations that introduce artefacts, exclude anatomical detail, and rely on parameter tuning and anatomical priors that narrow their application. Here, a robust masking and reconstruction procedure is presented to overcome these limitations and enable automated QSM processing for a wide range of use-cases implemented in an open-source software framework: QSMxT. MethodsA robust masking technique that automatically separates reliable from less reliable phase regions was developed and combined with a two-pass reconstruction procedure that operates on the separated sources before combination, extracting more information while reducing the influence of artefacts. ResultCompared with standard masking and reconstruction procedures, the two-pass inversion reduces streaking artefacts caused by unreliable phase and high dynamic ranges of susceptibility sources. QSMxT is robust across a range of datasets at 3 T in healthy volunteers and phantoms, at 7 T in tumour patients, and in the QSM challenge 2.0 simulated brain dataset, with significant artefact and error reductions, greater anatomical detail, and minimal parameter tuning. ConclusionQSMxT generates masks for QSM that separate reliable from less reliable phase regions, enables a more accurate QSM reconstruction that mitigates artefacts, operates without anatomical priors, and requires minimal parameter tuning. QSMxT makes QSM processing more accessible, reliable and reproducible.

neuroscience↗

bioRxiv2020

De novo design of picomolar SARS-CoV-2 miniprotein inhibitors

We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC50 values ranging from 35 pM to 35 nM; the most potent of these -- 56 and 64 residue hyperstable proteins made using the second approach -- are roughly six times more potent on a per mass basis (IC50 ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.

synthetic biology↗