Biology subjects

Hamilton, G.

Publications and source records attributed to Hamilton, G..

12 recordsLinked to original sources

bioRxiv2025

Gut Microbe-Derived N-Acyl Serinol Lipids Shape Host Postprandial Metabolic Homeostasis

Although strong evidence links the gut microbiome to metabolic disease, the mechanisms linking microbiota to hormonal and metabolic responses to food are not well understood. After a meal, gut bacteria produce a wide array of small molecule, protein, and lipid metabolites originating from bacterial sources. Annotating physiological function to select gut microbe-derived metabolites is critical to understanding diet-microbe-host interactions, and to developing microbiome-inspired therapies to improve human health. Here, we have investigated the role of a poorly annotated class of gut microbiome-derived lipids called N-acyl amides in postprandial metabolic physiology. Here we show both bacterial overproduction and provision of exogenous N-acyl amides reorganize host hormone-driven metabolic transition after a meal. Moreover, N-acyl amides exert broad effects on the meal- and circadian-related reorganization of gene expression, metabolic hormones, and gut microbiome composition. Collectively, these results demonstrate that microbiota-derived N-acyl amides play a physiologic role in postprandial metabolic homeostasis in the host.

physiology↗

bioRxiv2024

Differential KEAP1/NRF2 mediated signaling widens the therapeutic window of redox-targeting drugs in SCLC therapy

Small cell lung cancer (SCLC) patients frequently experience a remarkable response to first-line therapy. Follow up maintenance treatments aim to control residual tumor cells, but generally fail due to cross-resistance, inefficient targeting of tumor vulnerabilities, or dose-limiting toxicity, resulting in relapse and disease progression. Here, we show that SCLC cells, similar to their cells of origin, pulmonary neuroendocrine cells (PNECs), exhibit low activity in pathways protecting against reactive oxygen species (ROS). When exposed to a novel thioredoxin reductase 1 (TXNRD1) inhibitor, these cells quickly exhaust their ROS-scavenging capacity, regardless of their molecular subtype or resistance to first-line therapy. Importantly, unlike non-cancerous cells, SCLC cells cannot adapt to drug-induced ROS stress due to the suppression of ROS defense mechanisms by multiple layers of epigenetic and transcriptional regulation. By exploiting this difference in oxidative stress management, we safely increased the therapeutic dose of TXNRD1 inhibitors in vivo by pharmacological activation of the NRF2 stress response pathway. This resulted in improved tumor control without added toxicity to healthy tissues. These findings underscore the therapeutic potential of TXNRD1 inhibitors for maintenance therapy in SCLC. Graphical summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/621846v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@2a7336org.highwire.dtl.DTLVardef@f21de9org.highwire.dtl.DTLVardef@189d062org.highwire.dtl.DTLVardef@cff38b_HPS_FORMAT_FIGEXP M_FIG C_FIG Pharmacological induction of NRF2 leads to differential cyto-protection against TXNRD1 inhibitors in normal tissue but not in SCLC tumor cells. This results in a reduction of adverse effects, allowing to increase the therapeutic dose.

cancer biology↗

bioRxiv2024

Nanopore sequencing reveals that DNA replication compartmentalisation dictates genome stability and instability in Trypanosoma brucei

The genome of Trypanosoma brucei is structurally complex. Eleven megabase-sized chromosomes each comprise a transcribed core flanked by silent subtelomeres, housing thousands of Variant Surface Glycoprotein (VSG) genes. Additionally, silent VSGs are also found on hundreds of sub-megabase chromosomes that harbour 177 bp repeats of unknown function, and multiple VSG transcription sites localise to the telomeres of both chromosome types. DNA replication dynamics have been described in the megabase chromosome cores but not in the subtelomeres or sub-megabase chromosomes, and targeted early replication of the single active VSG transcription site is unexplained. Here, using Nanopore assembly, we mapped DNA replication across this compartmentalised genome. We show that subtelomeres display a paucity of replication initiation events relative to the core, correlating with increased instability in the silent VSG archive. In addition, early replication of the active VSG transcription site is shown to originate from the telomere, likely causing targeted VSG recombination. Lastly, we demonstrate that the 177 bp repeats act as widespread, conserved DNA replication origins, explaining mitotic stability of the abundant small chromosomes and early DNA replication of megabase chromosome centromeres. Compartmentalized DNA replication dynamics therefore explains how T. brucei balances stable genome transmission with localised instability driving immune evasion.

microbiology↗

bioRxiv2022

Assessment of Human Renal Transporter Based Drug-Drug Interactions Using Proximal Tubule Kidney-Chip

Study of renal transporters is crucial for understanding drug disposition and toxicity, and more importantly, predicting potential drug-drug interactions (DDIs). However, conventional in vitro models often fail to predict renal transporter activity and are not scalable to a predictive clinical outcome due to in vitro-in vivo discrepancy. Here, we successfully developed a human Proximal Tubule Kidney-Chip model that emulated in vivo renal physiology and function to assess renal transporter-based DDIs. Active and improved functionality of key renal transporters including p-glycoprotein (P-gp), multidrug and toxin extrusion (MATE) 1 and 2-K, organic anion transporter (OAT) 1 and 3, and organic cation transporter (OCT) 2 were demonstrated using appropriate probe substrates in Kidney-Chips compared to transwell controls. Moreover, comparative transcriptomic analysis revealed that key efflux and uptake transporters were expressed significantly higher in the Kidney-Chip compared to the transwell. Additionally, key parameters obtained from substrate-inhibitor interactions in the model were used to predict clinical DDIs as well as clearance values, which were closer to in vivo clearances. Overall, these results support that the human Proximal Tubule Kidney-Chip can reliably assess the role of human renal transporters in drug disposition and drug interactions, providing a critical tool to assess renal transport in vitro.

cell biology↗

bioRxiv2022

Cell-type specific transcriptional networks in root xylem adjacent cell layers

Transport of water, ions and signals from roots to leaves via the xylem vessels is essential for plant life and needs to be tightly regulated. The final composition of the transpiration stream before passage into the shoots is controlled by the xylem-adjacent cell layers, namely xylem parenchyma and pericycle, in the upper part of the root. To unravel regulatory networks in this strategically important location, we generated Arabidopsis lines expressing a nuclear tag under the control of the HKT1 promoter. HKT1 retrieves sodium from the xylem to prevent toxic levels in the shoot, and this function depends on its specific expression in upper root xylem-adjacent tissues. Based on FACS RNA-sequencing and INTACT ChIP-sequencing, we identified the gene repertoire that is preferentially expressed in the tagged cell types and discovered transcription factors experiencing cell-type specific loss of H3K27me3 demethylation. For one of these, ZAT6, we show that H3K27me3-demethylase REF6 is required for de-repression. Analysis of zat6 mutants revealed that ZAT6 activates a suite of cell-type specific downstream genes and restricts Na+ accumulation in the shoots. The combined Files open novel opportunities for bottom-up causal dissection of cell-type specific regulatory networks that control root-to-shoot communication under environmental challenge.

plant biology↗

bioRxiv2021

Effective Computational Framework for Pre-Interventional Planning of Peripheral Arteriovenous Malformations with In Vivo and In Vitro Validation

PurposePeripheral arteriovenous malformations (pAVMs) are congenital lesions characterised by abnormal high-flow, low-resistance vascular connections - constituting the so-called nidus - between arteries and veins. The mainstay treatment typically involves the embolisation of the nidus with embolic and sclerosant agents, however the complexity of AVMs often leads to uncertain outcomes. This study aims at developing a simple, yet effective computational framework to aid the clinical decision making around the treatment of pAVMs. MethodsA computational model was developed to simulate the pre-, intra-, and post-intervention haemodynamics of an AVM. A porous medium of varying permeability was used to simulate the effect that the sclerosant has on the blood flow through the nidus. The computational model was informed by computed tomography (CT) scans and digital subtraction angiography (DSA) images, and the results were compared against clinical data and experimental results. ResultsThe computational model was able to simulate the blood flow through the AVM throughout the intervention and predict (direct and indirect) haemodynamic changes due to the embolisation. The simulated transport of the dye in the AVM was compared against DSA time-series obtained at different intervention stages, providing confidence in the results. Moreover, experimental data obtained via a mock circulatory system involving a patient specific 3D printed phantom of the same AVM provided further validation of the simulation results. ConclusionWe developed a simple computational framework to simulate AVM haemodynamics and predict the effects of the embolisation procedure. The developed model lays the foundation of a new, computationally driven treatment planning tool for AVM embolisation procedures.

bioengineering↗

bioRxiv2020

Modeling alcoholic liver disease in a human Liver-Chip

Fatty liver disease (FLD), is a major public health burden that affects up to 30% of people in Western countries and leads to progressive liver injury, comorbidities, and increased mortality. Key risk factors for developing FLD are obesity and alcohol consumption, both of which are growing in prevalence worldwide. There is an urgent need for human-relevant preclinical models to improve our understanding of FLD progression to steatohepatitis and for the development of sensitive noninvasive diagnostics and therapies. Alcohol-induced liver disease (ALD) represents an ideal case for modeling FDL as ethanol exposure is a comparatively simpler trigger for experimental induction of the pathology, as opposed to the complexity of modeling the diet- and life-style induced FLD. Further, despite their different root causes, the two pathologies share several common characteristics in disease progression and deterioration of liver function, highlighting the potential of an ALD microphysiological model for broad application in translational research. Here, we leverage our recently reported human Liver-Chip for toxicity applications, to expand the capabilities of the platform for broad application in translational research. We report the first in vitro modeling of ALD that uses human relevant blood alcohol concentrations (BAC) and affords multimodal profiling of clinically relevant endpoints. Our ALD Liver-Chip recapitulates established FLD markers in response to ethanol in a concentration-dependent manner, including lipid accumulation and oxidative stress. Importantly, we show that the ALD Liver-Chip supports the study of secondary insults common in patients with advanced ALD, such as high blood endotoxin levels due to alcohol-associated increased intestinal permeability and barrier dysfunction. Moreover, owing to new developments in the design, the ALD Liver-Chip enables the measurement of structural changes of the bile canaliculi (BC) network as a novel in vitro quantitative readout of alcoholic liver toxicity. In summary, we report the development of a human ALD Liver-Chip as a new platform for modeling the progression of alcohol-induced liver injury with direct translation to clinical research.

bioengineering↗

bioRxiv2020

Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform.

Accurate decoding of nucleic acid variation is important to understand the complexity and regulation of genome function. Here we introduce a single-molecule platform based on magnetic tweezer (MT) technology that can identify and map the positions of sequence variation and multiple base modifications together in the same single molecules of DNA or RNA at single base resolution. Using synthetic templates, we demonstrate that our method can distinguish the most common epigenetic marks on DNA and RNA with high sensitivity, specificity and precision. We also developed a highly specific CRISPR-Cas enrichment strategy to target genomic regions in native DNA without amplification. We then used this method to enrich native DNA from E. coli and characterized the differential levels of adenine and cytosine base modifications together in molecules of up to 5 kb in length. Finally, we enriched the 5 UTR of FMR1 from cells derived from a Fragile X carrier and precisely measured the repeat expansion length and methylation status of each molecule. These results demonstrate that our platform can detect a variety of genetic, epigenetic and base modification changes concomitantly within the same single molecules.

genomics↗

bioRxiv2020

A Micro-Engineered Airway Lung-Chip Models Key Features of Viral-Induced Exacerbation of Asthma

RationaleViral-induced exacerbation of asthma remain a major cause of hospitalization and mortality. New human relevant models of the airways are urgently needed to understand how respiratory infections may trigger asthma attacks, and to advance treatment development. ObjectivesTo develop a new human relevant model of rhinovirus-induced asthma exacerbation that recapitulates viral infection of asthmatic airway epithelium, neutrophil transepithelial migration, and enables evaluation of immunomodulatory therapy. MethodsA micro-engineered model of fully differentiated human mucociliary airway epithelium was stimulated with IL-13 to induce a Th2-type asthmatic phenotype and infected with live human rhinovirus 16 (HRV16) to reproduce key features of viral-induced asthma exacerbation. Measurements and Main ResultsInfection with HRV16 replicated key hallmarks of the cytopathology and inflammatory responses observed in human airways. Generation of a Th2 microenvironment through exogenous IL-13 stimulation induced features of asthmatics airways, including goblet cell hyperplasia, reduction of cilia beating frequency, and endothelial activation, but did not alter rhinovirus infectivity or replication. High resolution kinetic analysis of secreted inflammatory markers revealed that IL-13 treatment altered the IL-6, IFN-{lambda}1, and CXCL10 secretion in response to HRV16. Neutrophil transepithelial migration was greatest when viral infection was combined with IL-13 treatment, while treatment with MK-7123, a CXCR2 antagonist, reduced neutrophil diapedesis in all conditions. ConclusionsThis micro-engineered Airway Lung-Chip provides a novel human-relevant platform for exploring the complex mechanisms underlying viral-induced asthma exacerbation. Our data suggest that IL-13 may impair the hosts ability to mount an appropriate and coordinated immune response to rhinovirus infection. We also show that the Airway Lung-Chip can be used to assess the efficacy of modulators of the immune response. NoteEmulate(R), Human Emulation System(R), S-1, ER-1, and ER-2 are trademarks of Emulate, Inc., and any other trademarks used herein remain with their respective holders. The technology disclosed in this document may be covered by one or more patents or patent applications, and no license to these is granted herein. You are solely responsible for determining whether you have all intellectual property rights that are necessary for your intended use of any of the disclosed materials, and whether you are required to obtain any additional intellectual property rights from a third party. Further information is available by contacting the authors. At a Glance CommentaryO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSNew therapies for asthma exacerbations remain a significant unmet medical need. Development of human relevant preclinical models are needed to further elucidate the complex mechanisms underlying asthma exacerbation and investigate new therapeutic strategies. What This Study Adds to the FieldUsing a human Airway Lung-Chip model, we show here for the first time a live human rhinovirus (HRV) infection of the asthmatic epithelium that recapitulates complex features of viral-induced asthma exacerbation. The dynamic microenvironment of the chip enables the real-time study of virus infection, epithelial response, and immune cell recruitment under healthy and asthmatic conditions. The model reproduces key endpoints that have been observed in asthmatics and individuals infected with rhinovirus including the ciliated cell sloughing, altered cilia beating frequency, goblet cell hyperplasia, increased expression of adhesion molecules in microvascular endothelial cells, and inflammatory mediator release. High-resolution temporal analysis of secreted inflammatory markers enabled by dynamic sampling revealed alteration of IL-6, IFN-{lambda}1 and CXCL10 secretory phases after rhinovirus infection in an IL-13 high environment. Leveraging high-content imaging and analysis of circulating inflammatory cells, we demonstrated the efficacy of a CXCR2 antagonist to reduce adhesion, motility, and transmigration of perfused human neutrophils. Thus, this micro-engineered chip may offer a powerful addition to preclinical models for understanding mechanisms underlying asthma exacerbation pathology and developing new therapeutic strategies.

bioengineering↗

bioRxiv2019

Liver-Chip: Reproducing Human and Cross-Species Toxicities

Nonclinical rodent and non-rodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans contributing to drug failures in the clinic. Here we applied microengineered Organ-on-Chip (Organ-Chip) technology to design rat, dog, and human Liver-Chips containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chips detected diverse phenotypes of liver toxicity including hepatocellular injury, steatosis, cholestasis, and fibrosis as well as species-specific toxicities when treated with tool compounds. Multi-species Liver-Chips may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.\n\nOne Sentence SummaryMicroengineered Organ-Chip technology has been used to design rat, dog and human Liver-Chips that recapitulate species-specific liver toxicities.

pharmacology and toxicology↗

bioRxiv2019

Trypanosoma brucei ribonuclease H2A is an essential enzyme that resolves R-loops associated with transcription initiation and antigenic variation

In every cell ribonucleotides represent a threat to the stability and transmission of the DNA genome. Two types of Ribonuclease H (RNase H) tackle such ribonucleotides, either by excision when they form part of the DNA strand, or by hydrolysing RNA when it base-pairs with DNA, in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast can prosper in the absence of both enzymes. Removal of RNase H1 is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of the catalytic subunit of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. In addition, differential gene expression of both RNA Pol I and II transcribed genes occurs after TbRH2A loss, including patterns that may relate to cytosolic DNA accumulation in humans with autoimmune disease. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, leading to altered variant surface glycoprotein expression. Thus, we demonstrate a separation of function between the two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

microbiology↗

bioRxiv2018

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

R-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere homeostasis, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked trans-splicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localisation was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the intergenic regions of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination, most T. brucei R-loops act in a novel role, promoting RNA Pol II movement or mRNA processing. Finally, we show there is little evidence for correlation between R-loop localisation and mapped sites of DNA replication initiation.

genomics↗