Biology subjects

Flint, G.

Publications and source records attributed to Flint, G..

8 recordsLinked to original sources

bioRxiv2025

Genome-wide mapping of DNA G-quadruplexes in Trypanosoma brucei chromatin reveals enrichment in coding regions

G-quadruplexes (G4s) are non-canonical DNA structures formed in guanine-rich sequences that are proposed to act as regulatory elements in trypanosomatid parasites, including Trypanosoma brucei, the causative agent of African sleeping sickness. However, their functional roles remain poorly understood, largely due to limited knowledge of their genomic distribution. Herein, we performed computational analyses across 63 trypanosomatid species uncovering high degree of variability in G4-prevalence and species-specific patterns. We generated the first genome-wide map of G4s in T. brucei using G4 chromatin immunoprecipitation followed by sequencing (G4 ChIP-Seq), which revealed a striking enrichment of G4s within coding DNA sequences (CDSs). This pattern diverges markedly from in silico predictions and previous genome-wide G4 mapping studies in humans, suggesting that G4s may play unique roles exclusive to trypanosome biology. To investigate their functional relevance, we profiled the transcriptome of T. brucei upon treatment with the G4-stabilising ligand PhenDC3. We observed that PhenDC3 exerts targeted gene expression perturbation of genes bearing G4s, particularly those located within coding CDSs, where G4s are mostly enriched. Altogether, our findings highlight a distinctive role for G4s in the regulation of gene expression in T. brucei and support their potential as therapeutic targets in the treatment of African sleeping sickness. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/666098v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1a2e0b5org.highwire.dtl.DTLVardef@4eba13org.highwire.dtl.DTLVardef@9a46fdorg.highwire.dtl.DTLVardef@aabc90_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

G-quadruplex structures regulate long-range transcriptional reprogramming to promote drug resistance in ovarian cancer

Epigenetic evolution is a common mechanism used by cancer cells to evade the therapeutic effects of drug treatment. In ovarian cancers, epigenetically-driven resistance may be responsible for a large number of late-stage patient deaths. Here, we describe the first investigation into the role of G-quadruplex (G4) DNA secondary structures in mediating epigenetic regulation in drug-resistant ovarian cancer cells. Through genome-wide mapping of G4s in paired drug-sensitive and drug-resistant cell lines, we find that increased G4 formation is associated with significant increase in gene expression, with high enrichment in signalling pathways previously established to promote drug-resistant states. However, in contrast to previous studies, the expression-enhancing effects of G4s were not found at gene promoters, but intergenic and intronic regions, indicating that G4s promote long-range transcriptional regulation in drug-resistant cells. Furthermore, we discovered that clusters of G4s (super-G4s) are associated with particularly high levels of transcriptional enhancement that surpass the effects of super-enhancers, which act as well established regulatory sites in many cancers. Finally, we demonstrate that targeting G4s with small molecules results in significant down-regulation of pathways associated with drug-resistance, which results in resensitisation of resistant cells to chemotherapy agents. These findings indicate that G4 structures are critical for the epigenetic regulatory networks of drug-resistant cells and may represent a promising target to treat drug-tolerant ovarian cancer.

cancer biology↗

bioRxiv2023

Cell based dATP delivery as a therapy for chronic heart failure.

Withdrawal StatementThe authors have withdrawn this manuscript because the senior authors have identified inconsistencies in the data presented in this manuscript, which affect the reliability of the main conclusions. In keeping with our commitment to scientific rigor and transparency, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors.

pathology↗

bioRxiv2023

Danicamtiv increases myosin recruitment and alters the chemomechanical cross bridge cycle in cardiac muscle

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the "on" state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue. Significance StatementDirectly augmenting sarcomere function has potential to overcome limitations of currently used inotropic agents to improve cardiac contractility. Myosin modulation is a novel mechanism for increased contraction in cardiomyopathies. Danicamtiv is a myosin activator that is currently under investigation for use in cardiomyopathy patients. Our study is the first detailed mechanism of how Danicamtiv increases force and alters kinetics of cardiac activation and relaxation. This new understanding of the mechanism of action of Danicamtiv can be used to help identify patients that could benefit most from this treatment.

physiology↗

bioRxiv2023

Correcting dilated cardiomyopathy with fibroblast-targeted p38 deficiency

Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension generation, are principal drivers of dilated cardiomyopathy (DCM)- the leading cause of heart failure1,2. Progress towards developing precision therapeutics for and defining the underlying determinants of DCM has been cardiomyocyte centric with negligible attention directed towards fibroblasts despite their role in regulating the best predictor of DCM severity, cardiac fibrosis3,4. Given that failure to reverse fibrosis is a major limitation of both standard of care and first in class precision therapeutics for DCM, this study examined whether cardiac fibroblast-mediated regulation of the hearts material properties is essential for the DCM phenotype. Here we report in a mouse model of inherited DCM that prior to the onset of fibrosis and dilated myocardial remodeling both the myocardium and extracellular matrix (ECM) stiffen from switches in titin isoform expression, enhanced collagen fiber alignment, and expansion of the cardiac fibroblast population, which we blocked by genetically suppressing p38 in cardiac fibroblasts. This fibroblast-targeted intervention unexpectedly improved the primary cardiomyocyte defect in contractile function and reversed ECM and dilated myocardial remodeling. Together these findings challenge the long-standing paradigm that ECM remodeling is a secondary complication to inherited defects in cardiomyocyte contractile function and instead demonstrate cardiac fibroblasts are essential contributors to the DCM phenotype, thus suggesting DCM-specific therapeutics will require fibroblast-specific strategies.

cell biology↗

bioRxiv2022

dATP Elevation Induces Myocardial Metabolic Remodeling to Support Improved Cardiac Function

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ~1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs. HighlightsO_LIUbiquitylation-resistant variant RRB in a transgenic mice model (TgRRB) elevates dATP level up to 1% (of the total ATP pool) in the heart and improves function. C_LIO_LITgRRB-CMs show greater basal oxygen consumption due to changes in myosin state by dATP. C_LIO_LITgRRB-CMs respond to elevated function with a metabolic shift, such that there are higher pools of oxidative metabolites, with elevated OXPHOS, FAO, and energy reserve. C_LIO_LILong-term mitochondrial remodeling may occur to accommodate for the higher energy demands of the high functioning TgRRB-CMs. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/515235v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@11fa00borg.highwire.dtl.DTLVardef@14460e9org.highwire.dtl.DTLVardef@369f31org.highwire.dtl.DTLVardef@d879f_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2021

The Food Additive Xanthan Gum Drives Adaptation of the Human Gut Microbiota

The diets of industrialized countries reflect the increasing use of processed foods, often with the introduction of novel food additives. Xanthan gum is a complex polysaccharide with unique rheological properties that have established its use as a widespread stabilizer and thickening agent1. However, little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other, chemically-distinct dietary fiber polysaccharides. Here, we show that the ability to digest xanthan gum is surprisingly common in industrialized human gut microbiomes and appears to be contingent on the activity of a single bacterium that is a member of an uncultured bacterial genus in the family Ruminococcaceae. We used a combination of enrichment culture, multi-omics, and recombinant enzyme studies to identify and characterize a complete pathway in this uncultured bacterium for the degradation of xanthan gum. Our data reveal that this keystone degrader cleaves the xanthan gum backbone with a novel glycoside hydrolase family 5 (GH5) enzyme before processing the released oligosaccharides using additional enzymes. Surprisingly, some individuals harbor a Bacteroides species that is capable of consuming oligosaccharide products generated by the keystone Ruminococcaceae or a purified form of the GH5 enzyme. This Bacteroides symbiont is equipped with its own distinct enzymatic pathway to cross-feed on xanthan gum breakdown products, which still harbor the native linkage complexity in xanthan gum, but it cannot directly degrade the high molecular weight polymer. Thus, the introduction of a common food additive into the human diet in the past 50 years has promoted the establishment of a food chain involving at least two members of different phyla of gut bacteria.

microbiology↗

bioRxiv2021

Fibroblast State Reversal By MBNL1-Dependent Transcriptome Modification Regulates Cardiac Repair

Dynamic fibroblast state transitions are responsible for the hearts fibrotic response to injury, raising the possibility that tactical control of these transitions could alter maladaptive fibrotic outcomes. Transcriptome maturation by the RNA binding protein Muscleblind Like 1 (MBNL1) has emerged as a potential driver of differentiated cell states. Here genetic lineage tracing of myofibroblasts in the injured heart demonstrated that gains in MBNL1 function corresponded to profibrotic fibroblast states. Similarly, in mice cardiac fibroblast specific MBNL1 overexpression induced a transcriptional myofibroblast profile in healthy cardiac fibroblasts that prevented the fibroproliferative phase of cardiac wound healing. By contrast loss of MBNL1 reverted cardiac fibroblasts to a pro-proliferative epicardial progenitor state that limited cardiac fibrosis following myocardial infarction. This progenitor state transition was associated with an MBNL1-dependent destabilization of the mesenchymal transition gene, Sox9. These findings suggest that MBNL1 regulation of the fibroblast transcriptome drives state transitions underlying cardiac fibrosis and repair.

cell biology↗