Biology subjects

Fernig, D. G.

Publications and source records attributed to Fernig, D. G..

10 recordsLinked to original sources

bioRxiv2026

The potent anti-influenza virus activity of FluPep is mediated by interactions with cellular anionic polysaccharides

FluPep is a 16 amino-acid peptide with potent anti-viral activity against influenza A virus, including the H1N1 subtype. To understand how FluPep exerts its effects, gold nanoparticles were functionalised with FluPep, since gold nanoparticles provide a very sensitive probe that can be detected optically. Our results demonstrate that nanoparticle-FluPep conjugates did not bind to influenza virus particles in vitro. However, the nanoparticle-FluPep conjugates bound to polyanions on the cell surface, and after washing cells they remained cell-associated and were able to inhibit flu virus infectivity. Nanoparticle-FluPep conjugates bound to heparin affinity columns and their binding to cells could be reduced by pre-treatment of the cells with heparinase and neuraminidase, but not with chondroitinase ABC. These data indicated that FluPep bound to heparan sulfate and sialic acid-containing glycans in the pericellular matrix and in doing so prevented viral infectivity. The importance of the interaction of FluPep with heparan sulfate (HS) was examined by extending FluPep at its C-terminus with amino acid sequences responsible for the interactions of growth factors with heparin. The resultant two new engineered peptides, termed superFluPep1 and superFluPep2, were more potent than parental FluPep. The results demonstrate that FluPep likely exerts its antiviral activity by interfering with virus-cell interactions and that enhancing the binding of such peptides to heparan sulfate is an effective strategy to increase their anti viral potency.

biochemistry↗

bioRxiv2026

FLEX: A heparin-binding fusion partner engineered from fibroblast growth factor 1 to enhance protein expression, solubility and purity

Expression of aggregation-prone, unstable, or cytotoxic recombinant proteins remains a major bottleneck in both academic and industrial research. Although solubility-enhancing affinity tags can improve expression, they often compromise purification stringency, increase construct size, or require additional downstream processing. Here we report FLEX, a compact 15.5-kDa dual-function fusion tag engineered from human fibroblast growth factor-1 (FGF1) that integrates intrinsic protein-stabilising properties with high-affinity heparin binding. Structure-guided computational redesign of the FGF1 scaffold reduced exposed hydrophobic residues, removed flexible protease-susceptible regions, and expanded the electropositive surface while preserving the canonical heparin-binding interface. FLEX exhibits markedly improved thermal and chemical stability relative to wild-type FGF1 together with enhanced heparin affinity, enabling high-stringency washing and improved purity in a single affinity step. We demonstrate the broad utility of FLEX by expressing and purifying a panel of challenging proteins in Escherichia coli, including cytotoxic Pseudomonas aeruginosa virulence factors that are difficult to obtain in active form. Unexpectedly, FLEX also performed robustly in mammalian expression systems, where transiently expressed FLEX-tagged proteins were recovered at higher yield and purity than with gold standard Myc and Strep tags, including difficult targets such as Tribbles 3 (TRIB3). These findings establish FLEX as a versatile affinity-and-stabilisation tag that improves expression and purification across diverse systems, providing a practical new tool for structural, biochemical, and translational studies of otherwise intractable proteins.

biochemistry↗

bioRxiv2025

Concentration- and time-dependence of phosphorylation events stimulated by fibroblast growth factor-4 (FGF-4) in Rama 27 fibroblasts

Gradients of FGFs are an essential feature of many developmental processes. Thus, FGF2 stimulates different responses at high and low concentrations, while FGF4 gradients are critical, e.g., in limb development., but it is not known if responses to FGF4 differ in a concentration-dependent manner. Employing rat mammary (Rama) 27 fibroblasts, we therefore measured the FGF4 concentration- and time-dependence of the simulation of phosphorylation of FRS2, MAPK1 and MAPK3 which are downstream of the FGF receptor (FGFR1c). At 10 pg/mL FGF4 caused a very weak phosphorylation of FRS2 at Y435 and Y196 and a stronger one of MAPK1 and MAPK3 that oscillated with maximum levels after 30 min and 240 min stimulation. The phosphorylation of these proteins at 1 ng/mL FGF4 was considerably stronger and showed a similar oscillation. At 10 ng/mL FGF4, the phosphorylation of FRS2 reached an early peak after 5 min, declined at 15 min and then rose again at 30 min before declining to the end of the time-course at 240 min, whereas the phosphorylation of MAPK1 and MAPK3 was strong after 5 minutes, reached a maximum after 30 minutes and then declined gradually. At 1 {micro}g/mL and 3 {micro}g/mL FGF4 after 15 min and 60 min the phosphorylation of FRS2, MAPK1 and MAPK3 was much lower at 60 min compared to that observed at lower concentrations of FGF4. The data indicate that at low concentrations FGF4 elicits an oscillatory response at the level of phosphorylation of FRS2 and of MAPK1 and MAPK3, whereas a bell-shaped dose response may occur at the highest concentrations of FGF4. The addition of exogenous heparin, an effective mimic of endogenous heparan sulfate, suggests that there may be an influence of the interaction of FGF4 with pericellular matrix and the dose-and time-dependence of the phosphorylation of FRS2 and MAPK1and MAPK3.

biochemistry↗

bioRxiv2024

Stable topical application of antimicrobials using plumbing rings in an ex vivo porcine corneal infection model

Microbial keratitis (MK) is a substantial cause of clinical blindness worldwide. Pseudomonas aeruginosa is an opportunistic gram-negative bacterium and is the leading cause of MK. Infection models are vital tools in understanding host-pathogen interactions and the development of novel therapies. As well as ethical and practical advantages, ex vivo infection models can allow researchers to better investigate host-pathogen interactions more accurately than traditional cell culture systems. The versatility of porcine corneal ex vivo models have been employed to study various pathogens (for example Staphylococcus aureus and Acanthamoeba) and has enabled innovation of novel MK therapies. Here, we describe an improved porcine corneal ex vivo protocol, which uses plumbing rings and medical adhesive to circumvent several distinct limitations and challenges. The application of a 10 mm plumbing ring to the center of the cornea allows localized inoculation of pathogens of interest, maintaining them at the site of infection, rather than running the risk of "run off" of topically added aqueous solutions. The second important advantage is that topically applied therapeutic agents can be properly maintained on the cornea within the plumbing ring reservoir, allowing more accurate study of antimicrobial effects. In this contextualized protocol, we infected porcine corneas with the P. aeruginosa strain PA103 with topical treatments of moxifloxacin. PA103 colony forming unit (CFU) deduction, quantification of corneal opacity and histology analysis (hematoxylin and eosin staining), was used to assess infection over 48h, which was alleviated by moxifloxacin in a dose-dependent manner.

pathology↗

bioRxiv2024

Macromolecular crowding by hyaluronan: implications for binding reactions in the pericellular matrix and at the cell surface

In common with many glycosaminoglycan binding growth factors, the interactions of fibroblast growth factor-2 (FGF-2) with heparin/heparan sulfate regulate its biological activity. Consequently, much effort has been expended in characterising growth factor-glycoaminoglycan interactions. Many experiments are performed in vitro in dilute solution. However, the cell surface is an extremely crowded environment. We have, therefore, examined the parameters of FGF-2 binding to a heparin-derived tetradecasaccharide in the presence of two different crowding agents, dextran, a commonly used crowding agent, as well as hyaluronan (HA), which is normally present at the cell surface. The heparin tetradecassaccaride was immobilised through its reducing end onto a biosensor surface and the crowding agents were used in solution. The results show that the observed on-rate rate of association (kon) decreased over two-fold with increasing concentrations of both crowding agents. The slope of initial rate of association similarly decreased, indicating that as the solution became more crowded the diffusion of FGF-2 becomes increasingly limited. One thousand kDa HA was shown to be the most effective crowding agent, as diffusion became limited at concentrations above 0.5 mg/ml, compared to 1 mg/ml and 10 mg/ml for 100kDa HA and dextran, respectively. Such concentrations of hyaluronan are readily found extracellularly. The results, therefore, suggest that the effect of crowding at the cell surface and in the extracellular matrix may play an important role in governing the interactions of at least FGF-2 with its heparan sulfate co-receptor.

biochemistry↗

bioRxiv2020

Pixatimod (PG545), a clinical-stage heparan sulfate mimetic, is a potent inhibitor of the SARS-CoV-2 virus

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a co-receptor with the ACE2 protein for recognition of the S1 spike protein on SARS-CoV-2 virus, providing a tractable new target for therapeutic intervention. Clinically-used heparins demonstrate inhibitory activity, but world supplies are limited, necessitating alternative solutions. Synthetic HS mimetic pixatimod is a drug candidate for cancer with immunomodulatory and heparanase-inhibiting properties. Here we show that pixatimod binds to and destabilizes the SARS-CoV-2 spike protein receptor binding domain (S1-RBD), and directly inhibits its binding to human ACE2, consistent with molecular modelling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of live SARS-CoV-2 virus show that pixatimod potently inhibits infection of monkey Vero E6 and human bronchial epithelial cells at concentrations within its safe therapeutic dose range. Furthermore, in a K18-hACE2 mouse model pixatimod demonstrates that pixatimod markedly attenuates SARS-CoV-2 viral titer and COVID-19-like symptoms. This demonstration of potent anti-SARS-CoV-2 activity establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics. Together with other known activities of pixatimod our data provides a strong rationale for its clinical investigation as a potential multimodal therapeutic to address the COVID-19 pandemic.

microbiology↗

bioRxiv2020

Raman mapping glucose metabolites during human mesenchymal stem cell adipogenesis

Raman mapping was used to determine the lipid distribution inside human mesenchymal stem cells during induced adipogenesis by monitoring C-H stretching bands of the fats inside the lipid droplets. By incorporating deuterated glucose into the cell culture medium during induction it was possible to distinguish whether or not downstream metabolites, either in lipid droplets or in the cytoplasm, had been formed before or after the adipogenic cascade, because C-D stretching bands are 1/{surd}2 shifted compared to the C-H bands. Thus, metabolites formed after the initiation of the process displayed both C-H and C-D stretching bands and so were forming during induced adipogenesis rather than prior to it. With the ability to distinguish small putative lipid drops formed by the induction of adipogenesis from those pre-formed in the cell, it was possible to analyze spectral changes occurring in the droplets at the earliest stages of adipogenesis. There were two key findings. Firstly, Raman spectra of lipid droplets evolved over time, suggesting that their composition at the early stages was not the same as at the later stages. Secondly, it was apparent that the proportion of unsaturated fats in droplets was higher at early stages than it was at later stages, suggesting that unsaturated fats arrive in the droplets faster than saturated ones.

cell biology↗

bioRxiv2020

The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding.

Many pathogens take advantage of the dependence of the host on the interaction of hundreds of extracellular proteins with the glycosaminoglycans heparan sulphate to regulate homeostasis and use heparan sulphate as a means to adhere and gain access to cells. Moreover, mucosal epithelia such as that of the respiratory tract are protected by a layer of mucin polysaccharides, which are usually sulphated. Consequently, the polydisperse, natural products of heparan sulphate and the allied polysaccharide, heparin have been found to be involved and prevent infection by a range of viruses including S-associated coronavirus strain HSR1. Here we use surface plasmon resonance and circular dichroism to measure the interaction between the SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin. The data demonstrate an interaction between the recombinant surface receptor binding domain and the polysaccharide. This has implications for the rapid development of a first-line therapeutic by repurposing heparin and for next-generation, tailor-made, GAG-based antivirals.

biochemistry↗

bioRxiv2018

The heparin-binding proteome in normal pancreas and murine experimental acute pancreatitis

Acute pancreatitis (AP) is acute inflammation of the pancreas, mainly caused by gallstones and alcohol, driven by changes in communication between cells. Heparin-binding proteins (HBPs) play a central role in health and diseases. Therefore, we used heparin affinity proteomics to identify extracellular HBPs in pancreas and plasma of normal mice and in a caerulein mouse model of AP. Many new extracellular HBPs (360) were discovered in the pancreas, taking the total number of HBPs known to 786. Extracellular pancreas HBPs form highly interconnected protein-protein interaction networks in both normal pancreas (NP) and AP. Thus, HBPs represent an important set of extracellular proteins with significant regulatory potential in the pancreas. HBPs in NP are associated with biological functions such as molecular transport and cellular movement that underlie pancreatic homeostasis. However, in AP HBPs are associated with additional inflammatory processes such as acute phase response signalling, complement activation and mitochondrial dysfunction, which has a central role in the development of AP. Plasma HBPs in AP included known AP biomarkers such as serum amyloid A, as well as emerging targets such as histone H2A. Other HBPs such as alpha 2-HS glycoprotein (AHSG) and histidine-rich glycoprotein (HRG) need further investigation for potential applications in the management of AP. Pancreas HBPs are extracellular and so easily accessible and are potential drug targets in AP, whereas plasma HBPs represent potential biomarkers for AP. Thus, their identification paves the way to determine which HBPs may have potential applications in the management of AP.

biochemistry↗

bioRxiv2018

Enhanced inhibition of influenza virus infection by peptide-noble metal nanoparticle conjugates

Influenza virus is a major medical and veterinary health concern and causes global pandemics. The peptide FluPep is an established inhibitor of influenza virus infectivity in model systems. We have explored the potential for FluPep functionalised noble metal nanoparticle to enhance the antiviral activity of the peptide Flupep and determined their potential for the delivery of FluPep. The FluPep ligand designed here is FluPep extended at its N-terminus with the sequence CVVVTAAA-, to allow its incorporation into a mix matrix ligand shell of a peptidol and an alkanethiol ethyleneglycol comprising 70% H-CVVVTol and 30% HS(CH2)11(ethyleneglycol)4 (both mole/mole). Gold and silver nanoparticles (~10 nm diameter) prepared with up to 5% (mole/mole) FluPep ligand contained in the mixture of mix-matrix peptide ligands remained as stable as the control mix-matrix coated nanoparticles against ligand exchange with dithiothreitol. FluPep ligand was found to inhibit viral plaque formation in canine MDCK cells (IC50 2.1 nM), but was less potent than FluPep itself (IC50 140 pM). FluPep ligand functonalised nanoparticles retained antiviral activity in the plaque assay. Moreover, at low grafting densities (where nanoparticles incorporate ~1 FluPep ligand, the antiviral potency in terms of FluPep ligand concentration was enhanced significantly for gold and silver nanoparticles (IC50 ~8-fold and ~3-fold lower, respectively). At higher grafting density the potency relative to free FluPep ligand concentration decreased. The data demonstrate that conjugation of FluPep to gold and silver nanoparticles enhances its antiviral potency; the antimicrobial activity of silver ions may enable the design of even more potent anti-microbial inhibitors.

biochemistry↗