Biology subjects

Dalby, M.

Publications and source records attributed to Dalby, M..

15 recordsLinked to original sources

bioRxiv2026

Mechanical evolution of 3T3 fibroblastic cells exposed to nanovibrational stimulation

Cells are mechanosensitive, responding to external mechanical stimulation. Nanovibrational stimulation has been shown to enhance cell contractility and actin stress fibre formation. These changes in morphology occur quickly, alongside associated mechanical changes. Here, the relationship between acute morphological and mechanical changes in NIH 3T3 fibroblastic cells in response to nanovibrational stimulation is presented. A 1 kHz, 30 nm vibration is applied continuously for 72 hours. Atomic force microscopy (AFM) quantifies mechanical properties of the nucleus and cytoplasm at multiple timepoints, while immunofluorescence tracks morphological changes. Within 3 hours of stimulation, both nuclear and cytoplasmic stiffness increase significantly, accompanied by a decrease in the cellular fluid exponent, suggesting a shift of the cell towards more solid-like behaviour. These changes correlate with increased nuclear area. Actin polymerisation also increases within 24 hours, although variably. To understand the role of the cytoskeleton, actin polymerisation and contraction are inhibited using cytochalasin D and blebbistatin. Results show that inhibition prevents stiffness increases and results in a higher fluid exponent, indicating a more fluid-like state. These findings demonstrate that actin-myosin dynamics mediate cell stiffening under nanovibrational stimulation. Interestingly, prolonged stimulation appears to reverse this effect, suggesting that temporal optimisation of stimulation may enhance long-term mechanotransducive responses.

cell biology↗

bioRxiv2025

Nanovibrational stimulation of osteogenesis engages non-canonical Wnt signalling and NF-κB regulator BCL3 as a mechanotransducer

Enhancing osteogenesis in mesenchymal stromal (stem) cells is essential for advancing cellular therapies that target and alleviate skeletal pathologies. By employing a bespoke bioreactor capable of delivering nano-amplitude vibration (30 nm, 1kHz) to human adipose-derived mesenchymal stromal cells, we observed osteo-specific differentiation. We related this to the mechanotransductive mechanism by showing that inhibition of intracellular tension results in the loss of cytoskeletal organisation and myosin activation driven by nanovibration. Further, we dissect the mechanism of osteogenesis using a panel of Wnt agonists and antagonists and highlight the role of non-canonical Wnt. Then, using Bcl3-/- cells and by stimulating with BCL3 peptide, we show that non-canonical Wnt, osteogenesis-related inflammation and osteogenesis itself are all regulated by BCL3. This is important as nanoscale direct cell-stimulation is gaining interest, and there is an emerging consensus that such signals can be osteogenic. While prior research has only broadly hinted at how nanovibrational signals convert to an osteogenic phenotype, this new work pinpoints critical mechanistic insights, thereby advancing our understanding of this promising avenue in musculoskeletal cell therapy.

bioengineering↗

bioRxiv2025

Synthetic peptide hydrogels as a model of the bone marrow niche demonstrate efficacy of a combined CRISPR-CAR T-cell therapy for acute myeloid leukaemia

Leukaemias, driven by mutations in hematopoietic stem cells (HSCs), rely on interactions with the bone marrow (BM) niche and other cell populations such as mesenchymal stromal cells (MSCs) for growth and survival. While chimeric antigen receptor (CAR) T-cell therapy shows promise for other hematological malignancies, its application to acute myeloid leukaemia (AML) is hindered by tumour heterogeneity and off-target toxicity. Combining CRISPR-Cas9 gene editing with CAR T-cell therapy has potential for selectively targeting AML cells while sparing healthy tissue. However, validating the efficacy of these treatments prior to clinical trial is hampered by the differences between humans and animal models typically used for pre-clinical testing. Furthermore, traditional in vitro models fail to replicate the complexity of the BM niche and often overestimate treatments efficacy. Here, we present a bioengineered human-cell containing BM niche model combining a fibronectin-presenting polymeric surface and a synthetic peptide hydrogel (PeptiGel) that mimics native BM tissues mechanical properties. This platform supports niche phenotypes in MSCs and HSCs and enables the evaluation of combined CRISPR-CAR T-cell therapy, demonstrating potential as a preclinical human model for testing novel therapies.

bioengineering↗

bioRxiv2024

Early life milk diets shape infant gut microbiota: evidence of microbial plasticity in response to breast and formula milk.

In early life, diet plays a key role in shaping the infant microbiota, yet the impact of breast milk and formula on microbial ecosystems at different stages of infant development remains poorly understood. Here, we performed static batch culture experiments using infant faecal samples at ages 1, 12 and 18 months of age, supplemented with either breast milk or formula for 48 hours. We further removed small metabolites (e.g. small carbohydrates, proteins and lipids) from breast and formula milk through dialysis and also added this to the static batch cultures with faecal samples from each infant. Our results show that the one-month-old faecal microbiota exhibited the greatest sensitivity to dietary intervention, with significant changes in microbial composition, metabolites and lipids, particularly in response to formula supplementation. In contrast, the microbiota at 12 months displayed increased stability, while the 18-month-old infant samples were the most resilient to different dietary supplementation. We also observed that triglyceride (TG46:1) was produced in the youngest infant samples but consumed by the older infant microbiota, suggesting a shift in metabolic interactions as the gut microbiota diversifies with age. Metabolite profiles linked to KEGG pathways further indicated the older infants diverse microbiotas had greater functional capacity in comparison to the one-month-old infant, particularly in response to breast milk. Fewer overall metabolic pathways were affected when the infant samples were grown in formula. Collectively, our data underscores the importance of early life diet in shaping microbiota profiles, with more stable microbial dynamics in more mature gut ecosystems, in response to dietary changes which are also associated with downstream functional (i.e. metabolite/lipid) readouts. Notably, the removal of small metabolites from breast milk by dialysis highlighted the potential role of milk lipids in promoting growth of foundational microbiota members like Bifidobacterium. Our data, along with further studies, are required to probe the mechanisms by which specific nutrients, particularly lipids, modulate microbial composition and support infant health during the first 2 years of life.

microbiology↗

bioRxiv2021

Clathrin-mediated Endocytosis Facilitates Internalization of Magnaporthe oryzae Effectors into Rice Cells

Fungi and oomycetes deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. Little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here we show that cytoplasmic effectors within BICs are packaged into vesicles that are occasionally observed in the host cytoplasm. Live cell imaging with fluorescently-labeled rice showed that effector vesicles colocalize with plant plasma membrane and with clathrin light chain-1, a marker for clathrin-mediated endocytosis (CME). Inhibition of CME using Virus-Induced Gene Silencing (VIGS) and chemical treatments results in cytoplasmic effectors in swollen BICs lacking vesicles. In contrast, fluorescent marker co-localization, VIGS and chemical inhibitor studies failed to support a major role for clathrin-independent endocytosis in effector vesicle formation. Localization studies of two novel effectors, and of known effectors after CME inhibition, indicate that cytoplasmic effector translocation occurs underneath appressoria before invasive hyphal growth. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by clathrin-mediated endocytosis in BICs and suggests a role for M. oryzae effectors in co-opting plant endocytosis.

plant biology↗

bioRxiv2020

PEA polymer-coated nanotopography delivers solid-state BMP2, enhances mesenchymal stem cell adhesion, prevents bacterial biofilm formation and protects cells from quorum sensing virulence factors

Post-operative infection is a major complication in patients recovering from orthopaedic surgery. As such, there is a clinical need to develop biomaterials for use in regenerative surgery that can promote mesenchymal stem cell (MSC) osteospecific differentiation and that can prevent infection caused by biofilm-forming pathogens. Nanotopographical approaches to pathogen control are being identified, including in orthopaedic materials such as titanium and its alloys. These topographies use high aspect ratio nanospikes or nanowires to prevent bacterial adhesion but these features puncture adhering cells, thus also reducing MSC adhesion. Here, we use a poly(ethyl acrylate) (PEA) polymer coating on titanium nanowires to spontaneously organise fibronectin (FN) and to deliver bone morphogenetic protein 2 (BMP2) to enhance MSC adhesion and osteospecific signalling. This nanotopography when combined with the PEA coating enhanced osteogenesis and reduced adhesion of Pseudomonas aeruginosa in culture. Using a novel MSC-Pseudomonas aeruginosa co-culture, we also show that the coated nanotopographies protect MSCs from cytotoxic quorum sensing and signalling molecules. We conclude that the PEA polymer-coated nanotopography can both support MSCs and prevent pathogens from adhering to a biomaterial surface, thus protecting from biofilm formation and bacterial infection and supporting osteogenic repair.

bioengineering↗

bioRxiv2020

Nanovibrational stimulation of mesenchymal stem cells induces therapeutic reactive oxygen species and inflammation for 3D bone tissue engineering

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilise these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in 2D and 3D but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz, and assess MSC metabolite, gene and protein changes. These approaches reveal that dose-dependent changes occur in MSCs responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression, and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation, as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells mechanical memory, driven by the stronger N90 stimulation. These new mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

bioengineering↗

bioRxiv2020

Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumour growth via a mast cell-dependent pathway

The diverse community of commensal microbes that comprise the gut microbiota is known to play an integral role in human health, not least through its ability to regulate host immune responses and metabolic pathways. Alterations to the homeostasis of this community, including through the use of broad-spectrum antibiotics, have already been associated with the progression of several cancers, namely melanoma and liver. The aggressive nature of breast cancer (BrCa), largely due to its ability to metastasize early, has ranked the disease with the second highest mortality rate of all cancers globally. Yet the body of research into the complex relationship between the microbiota and BrCa is still limited. This study found that a depletion of the microbiota, through the administration of antibiotics, significantly increased the rate of primary tumour progression in mouse BrCa models. We show that antibiotic-induced microbiota disturbances lead to changes in behaviour of a relatively obscure tumour-immune cell population: mast cells. We observed increases in tumour stroma-associated mast cells in antibiotic treated animals. Moreover, inhibition of mast cell degranulation, via cromolyn, slowed tumour progression in antibiotic treated animals but not in control animals. Thus, it appears that a perturbed microbiota drives stroma-associated mast cell recruitment and activation, which in turn promotes primary tumour growth through an as yet unknown mechanism. One Sentence Summary: We show that breast cancer progression is accelerated through a unique/novel immune response involving mast cells as a result of an antibiotic induced perturbation of the gut microbiota in a mouse model.

cancer biology↗

bioRxiv2019

Microbiota supplementation with Bifidobacterium and Lactobacillus modifies the preterm infant gut microbiota and metabolome

Supplementation with members of the early-life microbiota or probiotics is becoming increasingly popular to attempt to beneficially manipulate the preterm gut microbiota. We performed a large longitudinal study comprising two preterm groups; 101 orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 non-supplemented (Control) matched by age, sex, birth-mode, and diet. 16S rRNA metataxonomic profiling on stool samples (n = 592) indicated a predominance of Bifidobacterium, and a reduction of pathobionts in the Bif/Lacto group. Metabolic phenotyping found a parallel increase in fecal acetate and lactate in the Bif/Lacto group compared to the Control group, which positively correlated with Bifidobacterium abundance consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides and reduced gut pH. This study demonstrates that microbiota supplementation can modify the preterm microbiome and the gastrointestinal environment to more closely resemble that of a full-term infant.

microbiology↗

bioRxiv2019

Customizable, engineered substrates for rapid screening of cellular cues

Biophysical cues robustly direct cell responses and are thus important tools for in vitro and translational biomedical applications. High throughput platforms exploring substrates with varying physical properties are therefore valuable, however, currently existing platforms are limited in throughput, the biomaterials used, the capability to segregate between different cues and the assessment of dynamic cellular responses. Here we present a multiwell array (3x8) using a substrate engineered with patterns that present topography or rigidity cues welded to a bottomless plate with a 96-well format. Both the patterns on the engineered substrate and the well plate format can be easily customized, permitting systematic and efficient screening of biophysical cues. Here, we demonstrate three multiwell arrays patterned with a variety of topographical and mechanical cues (nano-grooves, soft pillars and nano pillars) tested with three different cell types. Using the multiwell array, we were able to measure cell functionality using analytical modalities such as live microscopy, qPCR and fluorescent immunochemistry. Cardiomyocytes cultured on 5{micro}m grooves showed less variation in electrophysiology and contractile function. Nanopillars with 127 nm height, 100 nm diameter and 300 nm pitch showed improved chondrogenic maintenance from matrix deposition and chondrogenic gene expression. High aspect ratio pillars with an elastic shear modulus of 16 kPa mimicking the cortical bone altered cell adhesion, morphology, and increased expression of osteogenic genes. We have demonstrated the bespoke, controlled and high-throughput properties of the multiwell array that are currently unparalleled in the field today.

bioengineering↗

bioRxiv2019

Nanotopography reveals metabolites that maintain the immunosuppressive phenotype of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent stem cells that are immunosuppressive and thus of considerable therapeutic potential in transplant operations. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunosuppressive therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can modulate paracrine activities of MSCs. Here, we show for the first time that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension increasing oxidative glycolysis. We also use the nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture. Our findings show a novel route to support large-scale expansion of functional MSCs for therapeutic purposes

bioengineering↗

bioRxiv2019

Perturbation of the gut microbiota by antibiotics results in accelerated breast tumour growth and metabolic dysregulation

BackgroundBreast cancer is the second most prevalent cancer worldwide with around 1.7 million new cases diagnosed every year. Whilst prognosis is generally favourable in early stages, this worsens significantly in advanced disease. Therefore, it is pertinent to focus on mitigating factors that may slow growth or progression. Recently, the gut microbiome has been implicated in a wide-range of roles in tumour biology. Through modulation of immunity, the gut microbiota can improve the efficacy of several immunotherapies. However, despite the prevalence of breast cancer, there is still a lack of microbiota studies in this field, including exploring the influence of external microbiome-modulating factors such as antibiotics. We describe herein how disruption of the gut microbiota via antibiotics may be detrimental to patient outcomes through acceleration of tumour growth. ResultsSupplementing animals with a cocktail of antibiotics leads to gut microbiota alterations and is accompanied by significant acceleration of tumour growth. Surprisingly, and distinct from previous microbiome-tumour studies, the mechanism driving these effects do not appear to be due to gross immunological changes. Analysis of intratumoural immune cell populations and cytokine production are not affected by antibiotic administration. Through global tumour transcriptomics, we have uncovered dysregulated gene expression networks relating to protein and lipid metabolism that are correlated with accelerated tumour growth. Fecal metabolomics revealed a reduction of the microbial-derived short-chain fatty acid butyrate that may contribute to accelerated tumour growth. Finally, through use of a routinely administered antibiotic in breast cancer patients, Cephalexin, we have shown that tumour growth is also significantly affected. Metataxanomic sequencing and analysis highlighted significant antibiotic-associated reductions in the butyrate producing genera Odoribacter and Anaeotruncus, and increased abundance of Bacteroides. ConclusionsOur data indicate that perturbation of the microbiota by antibiotics may have negative impacts on breast cancer patient outcomes. This is of importance as antibiotics are regularly prescribed to breast cancer patients undergoing mastectomy or breast reconstruction. We have also shown that the metabolic impact of disruption to the microbiome should be considered alongside the potent immunological effects. We believe our work lays the foundation for improving the use of antibiotics in patients, and with further investigation could potentially inform clinical practice.

cancer biology↗

bioRxiv2018

Effector Gene Reshuffling Involves Dispensable Mini-chromosomes in the Wheat Blast Fungus

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. To understand genetic diversity in wheat-infecting strains, we report a near-finished reference genome of a recent field isolate generated using long read sequencing and a novel scaffolding approach with long-distance paired genomic sequences. The genome assemblage includes seven core chromosomes and sequences from a dispensable mini-chromosome that harbors effector genes normally found on the ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and two mini-chromosomes from another field isolate each contain different effector homologous genes and core chromosome end sequences. The mini-chromosome is highly repetitive and is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and non-dispensable core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.\n\nSignificance statementThe emerging blast disease on wheat is proving even harder to control than the ancient, still-problematic rice blast disease. Potential wheat resistance identified using strains isolated soon after disease emergence are no longer effective in controlling recent aggressive field isolates from wheat in South America and South Asia. We report that recent wheat pathogens can contain one or two highly-variable conditionally-dispensable mini-chromosomes, each with an amalgamation of effector sequences that are duplicated or absent from pathogen core chromosome ends. Well-studied effectors found on different core chromosomes in rice pathogens appear side-by-side in wheat pathogen mini-chromosomes. The rice pathogen often overcomes deployed resistance genes by deleting triggering effector genes. Localization of effectors on mini-chromosomes, which are unstably transmitted during growth, would accelerate pathogen adaptation in the field.

genomics↗

bioRxiv2017

Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter encoded enhancer activities

Mammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. Here we investigate the transcription initiation landscape and its associated RNA decay in D. melanogaster. Surprisingly, we find that the majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. We observe quantitative relationships between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The different layers of regulation mediated by gene-distal enhancers and gene promoters are also reflected in chromatin interaction data. Our results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

genomics↗

bioRxiv2017

Transcriptional decomposition reveals active chromatin architectures and cell specific regulatory interactions

Transcriptional regulation is tightly coupled with chromosomal positioning and three-dimensional chromatin architecture. However, it is unclear what proportion of transcriptional activity is reflecting such organisation, how much can be informed by RNA expression alone, and how this impacts disease. Here, we develop a transcriptional decomposition approach separating the proportion of expression associated with genome organisation from independent effects not directly related to genomic positioning.\n\nWe show that positionally attributable expression accounts for a considerable proportion of total levels and is highly informative of topological associating domain activities and organisation, revealing boundaries and chromatin compartments. Furthermore, expression data alone accurately predicts individual enhancer-promoter interactions, drawing features from expression strength, stabilities, insulation and distance. We further characterise commonalities and differences across predictions in 76 human cell types, observing extensive sharing of domains, yet highly cell-type specific enhancer-promoter interactions and strong enrichments in relevant trait-associated variants. Our work demonstrates a close relationship between transcription and chromatin architecture, presenting a novel strategy and an unprecedented resource for investigating regulatory organisations and interpretations of disease associated genetic variants across cell types.

genomics↗