Biology subjects

Kelly, L.

Publications and source records attributed to Kelly, L..

35 records · Page 2Linked to original sources

bioRxiv2023

Magnetogenetic cell activation using endogenous ferritin

The ability to precisely control the activity of defined cell populations enables studies of their physiological roles and may provide therapeutic applications. While prior studies have shown that magnetic activation of ferritin-tagged ion channels allows cell-specific modulation of cellular activity, the large size of the constructs made the use of adeno-associated virus, AAV, the vector of choice for gene therapy, impractical. In addition, simple means for generating magnetic fields of sufficient strength have been lacking. Toward these ends, we first generated a novel anti-ferritin nanobody that when fused to transient receptor potential cation channel subfamily V member 1, TRPV1, enables direct binding of the channel to endogenous ferritin in mouse and human cells. This smaller construct can be delivered in a single AAV and we validated that it robustly enables magnetically induced cell activation in vitro. In parallel, we developed a simple benchtop electromagnet capable of gating the nanobody-tagged channel in vivo. Finally, we showed that delivering these new constructs by AAV to pancreatic beta cells in combination with the benchtop magnetic field delivery stimulates glucose-stimulated insulin release to improve glucose tolerance in mice in vivo. Together, the novel anti-ferritin nanobody, nanobody-TRPV1 construct and new hardware advance the utility of magnetogenetics in animals and potentially humans.

bioengineering↗

bioRxiv2023

Preneoplastic cells switch to Warburg metabolism from their inception exposing multiple vulnerabilities for targeted elimination

Otto Warburg first described tumour cells as displaying enhanced aerobic glycolysis whilst maintaining defective oxidative phosphorylation (OXPHOS) for energy production almost 100 years ago 1,2. Since then, the Warburg effect has been widely accepted as a key feature of rapidly proliferating cancer cells3,4. Targeting cancer metabolism is now being considered as a promising precision oncology therapeutic approach5. What is not clear is how early "Warburg metabolism" initiates during cancer progression and whether changes in energy metabolism might influence tumour progression ab initio. We set out to investigate energy metabolism in HRASG12V driven preneoplastic cell (PNC) at inception, in a zebrafish skin PNC model; and to test how the impact of manipulating energy metabolism in the whole animal may impact PNC initiation. We find that, within 24 hours of HRASG12V induction, PNCs upregulate "Warburg metabolism", and that this is required for their expansion. We show that blocking glycolysis reduces PNC proliferation, whilst increasing available glucose both enhances PNC proliferation and also reduces apoptosis. Impaired OXPHOS of PNCs might be exploited therapeutically since a mild complex I inhibitor, metformin, selectively induces apoptosis and suppresses proliferation of PNCs. In addition, we find mitochondrial fragmentation in PNCs occur prior to metabolic alteration and this is important for their survival since exposure to Drp1/Dnml1 inhibitor, mdivi, which blocks mitochondrial fragmentation leads to enhanced PNC apoptosis. Our data indicate that altered energy metabolism is one of the earliest events upon oncogene activation in somatic cells, which provide a targeted and effective tumour prevention therapy. Key findingsO_LIGlycolysis is upregulated in HRASG12V expressing preneoplastic cells (PNCs) in zebrafish skin, and is required for PNC proliferation C_LIO_LIOXPHOS respiration is impaired in PNCs and metformin complex I inhibition specifically eliminates PNCs C_LIO_LIPNC undergo mitochondrial fragmentation and exhibit reduced membrane potential C_LIO_LIMdivi reverses mitochondrial fragmentation in PNCs and triggers apoptosis C_LI

cancer biology↗

bioRxiv2022

Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimers disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/{beta}-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we developed L6-F4-2, a non-lipidated, FZD4-specific surrogate with significantly improved sub-picomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reversed neonatal retinal angiogenesis deficits, but also restored BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduced BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with general applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

physiology↗

bioRxiv2022

Patterns of NFkB activation resulting from damage, reactive microglia, cytokines, and growth factors in the mouse retina

Muller glia are a cellular source for neuronal regeneration in vertebrate retinas. However, the capacity for retinal regeneration varies widely across species. Understanding the mechanisms that regulate the reprogramming of Muller glia into progenitor cells is key to reversing the loss of vision that occurs with retinal diseases. In the mammalian retina, NF{kappa}B signaling promotes glial reactivity and represses the reprogramming of Muller glia into progenitor cells. Here we investigate different cytokines, growth factors, cell signaling pathways, and damage paradigms that influence NF{kappa}B-signaling in the mouse retina. We find that exogenous TNF and IL1{beta} potently activate NF{kappa}B-signaling in Muller glia in undamaged retinas, and this activation is independent of microglia. By comparison, TLR1/2 agonist indirectly activates NF{kappa}B-signaling in Muller glia, and this activation depends on the presence of microglia as Tlr2 is predominantly expressed by microglia, but not other types of retinal cells. Exogenous FGF2 did not activate NF{kappa}B-signaling, whereas CNTF, Osteopontin, WNT4, or inhibition of GSK3{beta} activated NF{kappa}B in Muller glia in the absence of neuronal damage. By comparison, dexamethasone, a glucocorticoid agonist, suppressed NF{kappa}B-signaling in Muller glia in damaged retinas, in addition to reducing numbers of dying cells and the accumulation of reactive microglia. Although NMDA-induced retinal damage activated NF{kappa}B in Muller glia, optic nerve crush had no effect on NF{kappa}B activation within the retina, whereas glial cells within the optic nerve were responsive. We conclude that the NF{kappa}B pathway is activated in retinal Muller glia in response to many different cell signaling pathways, and activation often depends on signals produced by reactive microglia.

neuroscience↗

bioRxiv2022

Genomic evidence of natural selection for ash dieback resistance

Rapid evolution via small shifts in allele frequencies at thousands of loci are a long- standing neo-Darwinian prediction but are hard to characterize in the wild. European ash tree (Fraxinus excelsior) populations have recently come under strong selection by the invasive fungal pathogen Hymenoscyphus fraxineus. Using genomic prediction models based on field trial phenotypes and 7,985 loci, we show a shift in genomically estimated breeding values in an ancient woodland, between adult trees established before the epidemic started and juvenile trees established since. Using simulations, we estimate that natural selection has eliminated 31% of the juvenile population. Thus, we document a highly polygenic heritable micro-evolutionary adaptive change over a single generation in the wild. One-Sentence SummarySubtle changes at thousands of genomic locations in one generation allow woodland trees to respond to a new selective pressure

evolutionary biology↗

bioRxiv2022

The polycomb repressor complex 2 and Enhancer of Zeste 2 regulate glial reactivity and are required for the formation of Muller glia-derived progenitor cells in the retina

Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein we investigate the expression and functions of S-Adenosylhomocysteine Hydrolase (SAHH; AHCY) and Histone Methyltransferases (HMTs) during the formation of Muller glia-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1, AHCYL2 and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to covey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the formation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick, but not the mouse, the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.

neuroscience↗

bioRxiv2022

Prenatal development of neonatal vocalizations

Human and non-human primates produce rhythmical sounds as soon as they are born. These early vocalizations are important for soliciting the attention of caregivers. How they develop, remains a mystery. The orofacial movements necessary for producing these vocalizations have distinct spatiotemporal signatures. Therefore, their development could potentially be tracked over the course of prenatal life. We densely and longitudinally sampled fetal head and orofacial movements in marmoset monkeys using ultrasound imaging. We show that orofacial movements necessary for producing rhythmical vocalizations differentiate from a larger movement pattern that includes the entire head. We also show that signature features of marmoset infant contact calls emerge prenatally as a distinct pattern of orofacial movements. Our results establish that aspects of the sensorimotor development necessary for vocalizing occur prenatally, even before the production of sound.

neuroscience↗

bioRxiv2022

The HH-GLI2-CKS1B network regulates the proliferation-to-maturation transition of human cardiomyocytes

Cardiomyocyte (CM) proliferation and maturation are highly linked processes, however, the extent to which these processes are controlled by a single signaling axis is unclear. Here, we find the Hedgehog (HH)-GLI2-CKS1B cascade regulates the transition between proliferation and maturation in hiPSC-CMs. Initially, we found a significant enrichment of GLI2-signaling in CMs from patients with ischemic heart failure (HF) or dilated-cardiomyopathy (DCM), indicating initiation of fetal programs in the stressed heart. Developmentally, we showed downregulation of GLI-signaling in adult human CM, adult murine CM, and in late-stage hiPSC-CM. In early-stage, proliferative hiPSC-CM, inhibition of Hh- or GLI-proteins enhanced CM maturation. Mechanistically, we identified CKS1B, a new effector of GLI2 and showed that GLI2 binds the CKS1B promoter to regulate its expression. CKS1B overexpression in late-stage hiPSC-CMs led to increased proliferation with loss of maturation. Thus, the Hh-GLI2-CKS1B axis regulates the proliferation-maturation transition and provides targets to enhance cardiac tissue engineering and regenerative therapies.

developmental biology↗

bioRxiv2021

Nuclear Factor I in neurons, glia and during the formation of Muller glia-derived progenitor cells in avian, porcine and primate retinas

The regenerative potential of Muller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Muller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina (Clark et al., 2019) and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina (Hoang et al., 2020). Accordingly, we sought to characterize the patterns of expression NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single cell RNA-sequencing (scRNA-seq) and immunolabeling we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), up-regulated in E8 RPCs, further up-regulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further down-regulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.

neuroscience↗

bioRxiv2021

Resolving the structure of phage-bacteria interactions in the context of natural diversity

Microbial communities are shaped by viral predators1. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity2. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in "the wild" remain poorly resolved3, 4. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain quantitative estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that killing in environmental interaction networks is sparse - with phage predator loads low for most bacterial strains and phages host-strain-specific in their killing. Paradoxically, we also find that although overlap in killing is generally rare between phages, recombination is common. Together, these results indicate that the number of hosts that phages infect is often larger than the number that they kill and suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it will be important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on killing host range.

microbiology↗

bioRxiv2020

Resolving phylogeny and polyploid parentage using genus-wide genome-wide sequence data from birch trees

Numerous plant genera have a history including frequent hybridisation and polyploidisation, which often means that their phylogenies are not yet fully resolved. The genus Betula, which contains many ecologically important allopolyploid tree species, is a case in point. We generated genome-wide sequence data for 27 diploid and 31 polyploid Betula species or subspecies using restriction site associated DNA (RAD) sequences assembled into contigs with a mean length of 675 bp. We reconstructed the evolutionary relationships among diploid Betula species using both supermatrix and species tree methods. We identified progenitors of the polyploids according to the relative rates at which their reads mapped to contigs from different diploid species. We sorted the polyploid reads into different putative sub-genomes and used the extracted contigs, along with the diploid sequences, to build new phylogenies that included the polyploid sub-genomes. This approach yielded a highly evidenced phylogenetic hypothesis for the genus Betula, including the complex reticulate origins of the majority of its polyploid taxa. The genus was split into two well supported clades, which differ in their seed-wing morphology. We propose a new taxonomy for Betula, splitting it into two subgenera. We have resolved the parentage of many widespread and economically important polyploid tree species, opening the way for their population genomic study.

evolutionary biology↗

bioRxiv2020

Isolation and chacterisation of Klebsiella phages for phage therapy

Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection and sepsis. Bacteriophage (phage) therapy is being considered as a primary option for the treatment of drugresistant infections of these types. We report the successful isolation and characterisation of 30 novel, genetically diverse Klebsiella phages. The isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages. Of the Klebsiella-infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Importantly, when applied alone, none of the characterised phages were able to suppress the growth of Klebsiella for more than 12 hours, with some phages only able to suppress growth for 3 hours, likely due to inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.

microbiology↗

bioRxiv2020

Identification of amyloid beta oligomers in locus coeruleus (LC) neurons of Alzheimer's patients and their impact on LC oxidative stress, inhibitory neurotransmitter receptors and neuronal excitability

Amyloid {beta} oligomers (A{beta}O) are potent modulators of two key Alzheimers pathological processes, namely synaptic dysfunction and tau tangle formation in various brain regions. Remarkably, the impact of A{beta}O in one of the earliest brain regions to exhibit Alzheimers pathology, the locus coeruleus (LC), remains to be determined. Of particular importance is the effect of A{beta}O on the excitability of individual LC neurons. This parameter determines brain-wide noradrenaline (NA) release, and thus NA-mediated brain functions, including cognition, emotion and immune function, which are all severely compromised in Alzheimers. Using a mouse model of increased A{beta} production (APP-PSEN1), together with correlative histopathological analyses in post mortem Alzheimers patient samples, we determined the impact of A{beta} pathology on various correlates of LC neuronal integrity. A{beta}O immunoreactivity in the LC of APP-PSEN1 mice was replicated in patient samples, presenting as individual clusters located both intraneuronally, in mitochondrial compartments, as well as extracellularly in association with inhibitory synapses. No specific signal was detected in either patient control or wild type mouse samples. Accompanying this A{beta}O expression profile was LC neuronal hyperexcitability and indicators of oxidative stress in APP-PSEN1 mice. LC hyperexcitability arose from a diminished inhibitory effect of GABA, due to impaired expression and function of the GABA-A receptor (GABAAR) 3 subunit. Importantly, this altered LC 3-GABAAR expression profile overlapped with A{beta}O expression in both APP-PSEN1 mice and Alzheimers patient samples. Finally, strychnine-sensitive glycine receptors (GlyRs) remained resilient to A{beta}O-induced changes and their activation reversed LC hyperexcitability. Alongside this first demonstration of A{beta}O expression in the LC of Alzheimers patients, the study is also first to reveal a direct association between A{beta}O and LC neuronal excitability. GlyR-3-GABAAR modulation of A{beta}O-dependent LC hyperexcitability could delay the onset of cognitive and psychiatric symptoms arising from LC-NA deficits, thereby significantly diminishing the disease burden for Alzheimers patients.

neuroscience↗

bioRxiv2020

MicroRNA composition delineates cardiosphere-derived cell and mesenchymal stem cell extracellular vesicles

Cell therapy limits ischemic injury following myocardial infarction (MI) by limiting cell death, modulating the immune response, and promoting tissue regeneration. The therapeutic efficacy of cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs) is associated with extracellular vesicle (EV) release. Despite differences in cell origin, it is unclear why EVs elicit differences in therapeutic potency between cell types. Here, we compare EVs derived from multiple MSC and CDC donors. We reveal that EV membrane protein and microRNA (miR) composition are reflective of their parent cell. Small RNA-sequencing revealed enrichment of miR-10b in MSC EVs. Our data support the hypothesis that CDC EVs are distinct from MSC-EVs and is reflected by their miR composition.

cell biology↗

bioRxiv2019

Bacterial Hyperswarming as a Protective Response to Intestinal Stress

Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. Here, we report for the first time that bacterial swarmers are associated with protection against intestinal inflammation. We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers contrast to their respective swarming-deficient, but swimming-competent isogenic strains abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family, Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal metatranscriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants conjecturing that bacterial swarming in vivo might influence SM3s access to S24-7 in the intestines. Overall, our work identifies a new paradigm in which intestinal stress allows for the emergence of swarming bacteria, which can counterintuitively heal intestinal inflammation.

microbiology↗

bioRxiv2019

Multiclass Disease Classification from Microbial Whole-Community Metagenomes using Graph Convolutional Neural Networks.

There is a wealth of information contained within ones microbiome regarding their physiology and environment, and this is a promising avenue for developing non-invasive diagnostic tools. Here, we utilize 5643 aggregated, annotated whole-community metagenomes from 19 different diseases to implement the first multiclass microbiome disease classifier of this scale. We compared three different machine learning models: random forests, deep neural nets, and a novel graph convolutional architecture which exploits the graph structure of phylogenetic trees as its input. We show that the graph convolutional model outperforms deep neural nets in terms of accuracy (achieving 75% average test-set accuracy), receiver-operator-characteristics (92.1% average AUC), and precision-recall (50% average AUPR). Additionally, the convolutional nets performance complements that of the random forest, achieving similar accuracy but better receiver-operator-characteristics and lower area under precision-recall. Lastly, we are able to achieve over 90% average top-3 accuracy across all of our models. Together, these results indicate that there are predictive, disease specific signatures across microbiomes which could potentially be used for diagnostic purposes.

bioinformatics↗

bioRxiv2019

Inferring the quasipotential landscape of microbial ecosystems with topological data analysis

Microbiome dynamics influence the health and functioning of human physiology and the environment and are driven in part by interactions between large numbers of microbial taxa, making large-scale prediction and modeling a challenge. Here, using topological data analysis, we identify states and dynamical features relevant to macroscopic processes.We show that gut disease processes and marine geochemical events are associated with transitions between community states, defined as topological features of the data density. We find a reproducible two-state succession during recovery from cholera in the gut microbiomes of multiple patients, evidence of dynamic stability in the gut microbiome of a healthy human after experiencing diarrhea during travel, and periodic state transitions in a marine Prochlorococcus community driven by water column cycling. Our approach bridges small-scale fluctuations in microbiome composition and large-scale changes in phenotype without details of underlying mechanisms, and provides a novel assessment of microbiome stability and its relation to human and environmental health.

systems biology↗