Biology subjects

Ratcliffe, J.

Publications and source records attributed to Ratcliffe, J..

9 recordsLinked to original sources

bioRxiv2024

Self-healing, biocompatible bioinks from self-assembled peptide and alginate hybrid hydrogels

1.There is a pressing need for new biomaterials that are printable, stiff and highly biocompatible. This is primarily due to the inverse relationship between the printability and viscosity of hydrogels. Cell-laden, printable, rigid biomaterials are needed for replicating stiffer tissues such as cartilage in regenerative medicine, modelling the fibrosis of tissue and cancer microenvironments, as well as in non-cellular research fields such as biosensors. Here, we have designed a hybrid material compromised of self-assembled Fmoc-FF peptide assemblies dispersed throughout a sodium alginate matrix. The resultant hybrid bioink has a stiffness up to 10 times greater than sodium alginate alone but remains highly printable, even when laden with high concentrations of cells. In addition, the thixotropic self-assembled peptide assemblies gave the hybrid bioinks highly desirable self-healing capabilities. The choice of solvent used to initially dissolve the peptides made significant differences to both the physical properties and the biocompatibility of the bioinks, with the best performing able to support the growth of encapsulated macrophages over 5 days. Our developed hybrid materials allow the bioprinting of materials previously considered too stiff to extrude without causing shear induced cytotoxicity with applications in tissue engineering and biosensing.

bioengineering↗

bioRxiv2024

Cell invasive amyloid assemblies from SARS-CoV-2 peptides can form multiple polymorphs with varying neurotoxicity

The neurological symptoms of COVID-19, such as memory loss, cognitive and sensory disruption (neuro-COVID) are well reported. These neurological symptoms frequently persist for months (post-acute sequalae of COVID-19 or PASC). The molecular origins of neuro-COVID and how it contributes to PASC are unknown, however a growing body of research highlights that the self-assembly of protein fragments from SARS-CoV-2 into amyloid nanofibrils may play a causative role. Previously, we identified two fragments from the proteins Open Reading Frame 6 (ORF6) and ORF10 that self-assemble into neurotoxic amyloid assemblies. Here we further our understanding of the self-assembly mechanisms and nano-architectures formed by these fragments as well as performing a more in-depth study of the biological responses of co-cultured neurons. By solubilising the peptides in a fluorinated solvent we eliminate insoluble aggregates in the starting materials (seeds) that change the polymorphic landscape of the assemblies. The resultant assemblies are dominated by structures with higher free energies (e.g. ribbons and amorphous aggregates) that are less toxic to cultured neurons. We also show the first direct evidence of cellular uptake by viral amyloids. This work highlights the importance of understanding the polymorphic behaviour of amyloids particularly in the context of neuro-COVID and PASC. Graphical Abstract for ToCThe neurological symptoms of COVID-19 are likely to be, in part, caused by the aggregation of viral proteins into neurotoxic amyloid nanofibrils. Changes in aggregation conditions alters the balance of fibril structures formed (polymorphism), influencing their toxicity to a neuronal cell line. These findings increase our understanding of viral amyloids and highlight the importance of careful choice of experimental protocol when studying these systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/594465v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@68f505org.highwire.dtl.DTLVardef@ae3624org.highwire.dtl.DTLVardef@164a1f1org.highwire.dtl.DTLVardef@1c3f9d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Structural and biochemical characterization of LIG1 during mutagenic nick sealing of oxidatively damaged ends at the final step of DNA repair

DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair intermediate with 3-oxidative damage incorporated by DNA polymerase (pol) {beta} at the downstream steps of base excision repair (BER) pathway. Here, we determined X-ray structures of LIG1/nick DNA complexes containing 3-8oxodG and 3-8oxorG opposite either a templating Cytosine or Adenine and demonstrated that the ligase active site engages with mutagenic repair intermediates during steps 2 and 3 of the ligation reaction referring to the formation of DNA-AMP intermediate and a final phosphodiester bond, respectively. Furthermore, we showed the mutagenic nick sealing of DNA substrates with 3-8oxodG:A and 3-8oxorG:A by LIG1 wild-type, immunodeficiency disease-associated variants, and DNA ligase 3 (LIG3) in vitro. Finally, we observed that LIG1 and LIG3 seal resulting nick after an incorporation of 8oxorGTP:A by pol{beta} and AP-Endonuclease 1 (APE1) can clean oxidatively damaged ends at the final steps. Overall, our findings uncover a mechanistic insight into how LIG1 discriminates DNA or DNA/RNA junctions including oxidative damage and a functional coordination between the downstream enzymes, pol{beta}, APE1, and BER ligases, to process mutagenic repair intermediates to maintain repair efficiency.

biochemistry↗

bioRxiv2023

Beclin1 is essential for intestinal homeostasis.

BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrates autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice led to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibited extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells displayed morphological abnormalities. Deletion of the autophagy regulator, ATG7, failed to elicit similar effects, suggesting novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice showed E-cadherin mislocalisation providing a mechanism linking endocytic trafficking mediated by Beclin1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has significant implications for our understanding of intestinal pathophysiology.

cell biology↗

bioRxiv2022

Incorporation of bioactive peptides into peptide nanofibrillar hydrogels affects their nanostructure, mechanical properties and biocompatibility

Self-assembling peptides are promising candidates as scaffolds for 3D cell cultures. These hydrogels offer favourable biocompatibility, nanofibrillar structures that mimic native tissues, and the convenient integration of bioactive peptide sequences, such as arginine-glycine-aspartic acid (RGD), which can enable the development of therapeutically valuable cell types. In the treatment of osteoarthritis (OA) attempts have been made to combine hydrogel scaffolds with mesenchymal stem cells (MSCs) to harness their regenerative potential. This involves the deposition of extracellular matrix (ECM) components like collagen and proteoglycans. Here, we employ the hydrogel-forming peptide Fmoc-diphenylalanine (Fmoc-FF) and incorporate stoichiometric amounts of Fmoc-RGD. We investigate the impact of RGD on nanofibrillar morphologies, hydrogel stability, MSC viability, and the deposition of collagen, proteoglycans, and glycosaminoglycans. Elevating RGD content enhances cell viability and collagen deposition. However, at higher RGD concentrations, the stability of the hydrogels is compromised. To characterise collagen deposition, we introduce a non-destructive and label-free method using a plasmon-enhanced colorimetric histology technique. This innovation provides a practical means to image collagen without resorting to intricate and destructive sample processing and complex immunohistological staining procedures. This simple approach holds broad potential for routine and label-free quantification of collagen-rich biomaterials, promising widespread applications across various research and clinical settings.

bioengineering↗

bioRxiv2021

Neurotoxic Amyloidogenic Peptides Identified in the Proteome of SARS-COV2: Potential Implications for Neurological Symptoms in COVID-19

COVID-19 is primarily known as a respiratory disease caused by the virus SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, cognitive and psychiatric issues, severe headaches, and even stroke are reported in as many as 30% of cases and can persist even after the infection is over (so-called long COVID). These neurological symptoms are thought to be caused by brain inflammation, triggered by the virus infecting the central nervous system of COVID-19 patients, however we still dont fully understand the mechanisms for these symptoms. The neurological effects of COVID-19 share many similarities to neurodegenerative diseases such as Alzheimers and Parkinsons in which the presence of cytotoxic protein-based amyloid aggregates is a common etiological feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we performed a bioinformatic scan of the SARS-CoV-2 proteome, detecting peptide fragments that were predicted to be highly amyloidogenic. We selected two of these peptides and discovered that they do rapidly self-assemble into amyloid. Furthermore, these amyloid assemblies were shown to be highly toxic to a neuronal cell line. We introduce and support the idea that cytotoxic amyloid aggregates of SARS-CoV-2 proteins are causing some of the neurological symptoms commonly found in COVID-19 and contributing to long COVID, especially those symptoms which are novel to long COVID in contrast to other post-viral syndromes.

biochemistry↗

bioRxiv2020

Directed evolution for improved total secretory protein production in Escherichia coli

Production of secretory protein in Gram-negative bacteria simplifies downstream processing in recombinant protein production, accelerates protein engineering, and advances synthetic biology. Signal peptides and secretory carrier proteins are commonly used to effect the secretion of heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have successfully applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate the directed evolution workflow. In this study, we applied directed evolution on OsmY to enhance its total secretory protein production. After just one round of directed evolution followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 {+/-} 0.3 fold and 2.9 {+/-} 0.8 fold more secretory Tfu0937 {beta}-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 {degrees}C, 30 {degrees}C and 25 {degrees}C). Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 {+/-} 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. This is the first report of applying directed evolution on a carrier protein to enhance total secretory protein production. The methodology can be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli and other bacteria. In this study, OsmY(M3) improved the production of three proteins, originating from diverse organisms and with diverse properties, in secreted form, clearly demonstrating its wide-ranging applications.

synthetic biology↗

bioRxiv2019

Subtle, seasonal and potentially important: Are phytotoxic foliage leachates overlooked in plant invasion studies?

Invasive species pose a threat to global biodiversity. Recently there has been a growth in research into the chemical aspect of invasions, with some plants reported to introduce compounds to the soil which impair the growth of competing native plants. One such species is Rhododendron ponticum, which has become a damaging invasive shrub in Britain. Here we investigate whether water-soluble compounds introduced from R. ponticum leaves in Ceredigion, Wales, suppressed seed germination, and whether this varied temporally. Leachates from leaf material collected monthly (March to July) were analysed for their chemical composition by liquid chromatography-mass spectrometry and Lactuca sotiva bioassays. In total, 19 compounds were identified, displaying both qualitative and quantitative seasonal variation. Total concentrations peaked in June, however L sativa germination inhibition was only found in leachates from April and May (15% and 17% respectively). The study provides evidence that phytotoxic chemical introduction from R. ponticum leaves is seasonal and most bioactive at a time of year when the seeds of other species germinate. We propose this is one of several mechanisms including shading, lowering of soil pH and the deposition of thick litter, that this species exploits when in competition with other species. We argue that for studies on the phytotoxicity of invasive plants, there is a need to consider temporal variation in the rate and quality of bioactive chemicals produced and released, local precipitation rates, persistence of chemicals in the soil plus synergistic interactions between individual chemicals.

ecology↗

bioRxiv2017

Fungus causing White-Nose Syndrome in bats accumulates genetic variability in North America and shows no sign of recombination

Emerging fungal diseases of wildlife are on the rise worldwide (3) and the best lens on the evolution of the fungal pathogens is population genomics. Our genome-wide analysis shows that the newly introduced North American population of Pseudogymnoascus destructans, the causal agent of White-Nose Syndrome (WNS) in bats, has expanded in size, has begun to accumulate variation through mutation, and presents no evidence as yet for genetic exchange and recombination among individuals.

evolutionary biology↗