Biology subjects

Green, R.

Publications and source records attributed to Green, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Dopaminergic Modulation of Short-Term Associative Memory in Caenorhabditis elegans

Forgetting, the inability to retrieve previously encoded memories, is an active process involving neurotransmission, second messenger signalling, and cytoskeletal modifications. Forgetting is thought to be essential to remove irrelevant memories and to increase the capacity to encode new memories. Therefore, identifying key regulators of active forgetting is crucial to advance our understanding of neuroplasticity. In this study, we utilised the compact and tractable Caenorhabditis elegans model to investigate the role of the neurotransmitter dopamine in forgetting. We conducted butanone associative learning assays based on an established protocol and used mutant strains deficient in dopamine synthesis (tyrosine hydroxylase CAT-2 and dopamine transporter DAT-1) and signalling (G protein-coupled receptors DOP-1, DOP-2, and DOP-3) to assess the impact on learning and memory retention. Learning was measured immediately post-training, and memory retention was evaluated every 0.5 hours up to 2 hours. Our results show that animals lacking dopamine display a modest enhancement in learning relative to wild-type, with the learned association persisting for at least 2 hours after training. We also found that D2-like receptors DOP-2 and DOP-3 act together to modulate the forgetting process, with D1-like receptor DOP-1 functioning redundantly. Furthermore, re-expression of CAT-2 tyrosine hydroxylase in ADE and/or CEP neurons was unable to rescue the memory retention phenotype observed in cat-2 mutants, suggesting that dopamine release from all dopaminergic neurons is required to modulate forgetting. These findings highlight the critical role of dopamine in forgetting, consistent with findings in Drosophila, and suggest potential relevance for understanding memory retention during healthy ageing and in conditions with dopamine imbalances such as Parkinsons disease.

neuroscience↗

bioRxiv2025

Evolutionary potential of the Escherichia coli antimutator ΔnudJ is reduced via altered mutational spectrum

The rate of spontaneous mutation is a key factor in determining the capacity of a population to adapt to a novel environment, for example a bacterial population exposed to antibiotics. Genetic and environmental factors controlling the mutation rate commonly also cause shifts in the relative rates of different mutational classes, i.e. the spectrum of spontaneous mutations. When the mutational spectrum is altered, the relatively enriched and depleted mutations may differ in their fitness effects. Here we aim to explore how a reduced mutation rate and altered mutational spectrum can contribute to adaptation in Escherichia coli. We measure mutation rates across a set of Nudix hydrolase deletants, finding multiple strains with an antimutator phenotype. We focus on the antimutator {Delta}nudJ which can cause a 6-fold mutation rate reduction relative to the wildtype with an altered mutational spectrum. Not only does nudJ deletion reduce the probability of antibiotic resistance arising but, in the case of rifampicin, the accessible resistance mutations under this altered mutation spectrum have greater fitness costs. While this effect is, so far, specific to this drug, it opens up the possibility of reducing the likelihood of resistance mutations establishing through simultaneous modification of mutation rate and spectrum. The identification and characterisation of antimutator alleles such as {Delta}nudJ provides potential targets for antievolution drugs which could be applied during antibiotic treatment to inhibit NudJ activity. Such an approach could suppress spontaneous resistance evolution both through fewer resistance mutations and by limiting access to the fittest mutations.

evolutionary biology↗

bioRxiv2024

The RNA helicase HrpA rescues collided ribosomes in E. coli

Although many antibiotics inhibit bacterial ribosomes, loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in E. coli, suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in E. coli. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryo-EM structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA, such that by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics.

molecular biology↗

bioRxiv2024

Bispecific GD2 x B7-H3 Antibody Improves Tumor Targeting and Reduces Toxicity while Maintaining Efficacy for Neuroblastoma

The current treatment for neuroblastoma involves an immunotherapy regimen that includes a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues but is expressed on nerves. Thus, anti-GD2 treatment causes substantial, dose-limiting, neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, with minimal normal cell expression and is absent on nerves. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight-binding of tumor cells. Our preclinical research shows that when compared to an anti-GD2 monospecific antibody, the GD2xB7-H3 bsAb has improved tumor specificity with similar efficacy and reduced toxicity. Since this bsAb does not bind to nerves, it may be possible to administer increased or additional doses beyond the tolerable dose of monospecific anti-GD2 antibodies, which could improve therapeutic efficacy and quality of life for patients with neuroblastoma.

cancer biology↗

bioRxiv2023

LARP1 senses free ribosomes to coordinate supply and demand of ribosomal proteins

Terminal oligopyrimidine motif-containing mRNAs (TOPs) encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies implicate LARP1 in 40S- or 80S-ribosome complexes that repress and stabilize TOPs. However, a mechanistic understanding of how LARP1 and TOPs interact with these complexes to coordinate TOP outcomes is lacking. Here, we show that LARP1 senses the cellular supply of ribosomes by directly binding non-translating ribosomal subunits. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the 40S mRNA channel. Free cytosolic ribosomes induce sequestration of TOPs in repressed 80S-LARP1-TOP complexes independent of alterations in mTOR signaling. Together, this work demonstrates a general ribosome-sensing function of LARP1 that allows it to tune ribosome protein synthesis to cellular demand. One-Sentence SummaryLARP1 directly binds free ribosomal subunits to repress TOP mRNAs

molecular biology↗

bioRxiv2023

BTR: A Bioinformatics Tool Recommendation System

MotivationThe rapid expansion of Bioinformatics research has resulted in a vast array of computational tools utilized in the development of scientific analysis pipelines. However, constructing these pipelines is a laborious and intricate task, one which demands extensive domain knowledge and careful consideration at all stages. As the Bioinformatics landscape continues to evolve, researchers, both novice and expert, may find themselves overwhelmed when working in unfamiliar fields. Consequently, this may result in the selection of unsuitable or suboptimal tools during workflow development. ResultsIn this paper, we propose the Bioinformatics Tool Recommendation system (BTR), an innovative deep learning model designed to recommend the most suitable tools for a given workflow-in-progress. BTR utilizes recent advances in graph neural network technology and introduces a novel approach, representing the entire workflow as a graph to capture essential context and structural information. Additionally, natural language processing techniques are integrated to enhance the quality of tool recommendations by analyzing associated tool descriptions. Experiments demonstrate that BTR outperforms the existing Galaxy tool recommendation system, highlighting its potential to greatly facilitate scientific workflow construction. Availability and implementationThe Python source code is available at https://github.com/ryangreenj/bioinformatics_tool_recommendation

bioinformatics↗

bioRxiv2023

Working together to control mutation: how collective peroxide detoxification determines microbial mutation rate plasticity

Mutagenesis is responsive to many environmental factors. Evolution therefore depends on the environment not only for selection but also in determining the variation available in a population. One such environmental dependency is the inverse relationship between mutation rates and population density in many microbial species. Here we determine the mechanism responsible for this mutation rate plasticity. Using dynamical computational modelling and in vivo mutation rate estimation we show that the negative relationship between mutation rate and population density arises from the collective ability of microbial populations to control concentrations of hydrogen peroxide. We demonstrate a loss of this density-associated mutation rate plasticity when Escherichia coli populations are deficient in the degradation of hydrogen peroxide. We further show that the reduction in mutation rate in denser populations is restored in peroxide degradation-deficient cells by the presence of wild-type cells in a mixed population. Together, these model-guided experiments provide a mechanistic explanation for density-associated mutation rate plasticity, applicable across all domains of life, and frames mutation rate as a dynamic trait shaped by microbial community composition.

evolutionary biology↗

bioRxiv2023

Comparison of concentrations of lead (Pb) in meat from wild-shot common pheasants killed using shotgun pellets principally composed of lead, iron (Fe), bismuth (Bi) and zinc (Zn)

The source of almost all of the lead (Pb) found in meat from carcasses of wild-shot small game animals is often thought to be small embedded fragments of the lead shotgun pellets usually used by hunters to kill them. Available circumstantial evidence supports this conjecture, but an unknown proportion of the lead in game meat might be biologically-incorporated and absorbed by the game animal from ingested shotgun pellets and other environmental sources, such as soil and residues from mining. A critical test comparing lead concentrations in meat from animals known to have been killed using different types of shotgun ammunition has not been performed until now. We compared lead concentrations in samples of edible meat from carcasses of 27 wild-shot common pheasants (Phasianus colchicus) from which only lead shotgun pellets were recovered post mortem with concentrations for 20 birds from which only iron pellets were recovered. Shotgun pellets were removed from the meat samples before analysis. The mean concentration of lead was about 30 times greater in the meat of pheasants shot with lead than those shot with iron and was similar to mean concentrations of lead reported previously from Europe-wide samples of meat from wild-shot small game animals, including pheasants, killed using unknown types of ammunition. These results support the hypothesis that changing the type of shotgun ammunition in use for hunting from lead to iron would greatly reduce the concentration of lead in meat from wild-shot small game.

pharmacology and toxicology↗

bioRxiv2023

B. subtilis MutS2 splits stalled ribosomesinto subunits without mRNA cleavage

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains and reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation by the ribosome quality control pathway. Notably, we see no evidence of mRNA cleavage by MutS2, nor does it promote ribosome rescue by tmRNA as SmrB cleavage does in E. coli. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in various bacteria.

molecular biology↗

bioRxiv2022

Bursting Translation on Single mRNAs in Live Cells

Stochasticity has emerged as a mechanism to control gene expression. Much of this so-called "noise" has been attributed to bursting transcription. However, the stochasticity of translation has not similarly been investigated due to a lack of enabling imaging technologies. We developed techniques to track single mRNAs and their translation in live cells for hours, allowing measurement of previously uncharacterized translation dynamics. We applied genetic and pharmacological perturbations to control translation kinetics. Like transcription, translation is not a constitutive process but instead cycles between inactive and active states or "bursts". But unlike transcription, which is largely frequency modulated, complex structure in the 5-untranslated region alters burst amplitude. Bursting frequency can be controlled through cap-proximal sequences and trans-acting factors such as eIF4F. We coupled single molecule imaging with stochastic modeling to deduce the fundamental kinetic parameters of translational bursting, a new dimension of translational control. HighlightsO_LILong-term tracking of single mRNAs reveals multi-state, bursting translation C_LIO_LIStructure in the 5-untranslated region modulates translational burst amplitude C_LIO_LI5-cap proximal sequences modulate translational burst frequency C_LIO_LImTOR signaling adjusts translation bursting to respond to environmental cues C_LI

molecular biology↗

bioRxiv2022

A small molecule RIG-I agonist serves to adjuvant broad multifaceted influenza virus vaccine immunity

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulates the immune response against many RNA viruses. We identified a small molecule compound, KIN1148, that directly binds RIG-I to drive IRF3activation to impart the expression of IRF3-target genes, including specific immunomodulatory cytokines and chemokines. KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I interferon (IFN) treatment. When administered in combination with a vaccine against influenza A virus (IAV), KIN1148 induces both neutralizing antibody and broadly cross-protective IAV-specific T cell responses compared to vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal H1N1 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing non-canonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination. SummaryHemann, et. al. identify a small-molecule RIG-I agonist (KIN1148) that directly binds RIG-I for non-canonical activation and adjuvants pandemic and avian influenza virus vaccination. KIN1148 augments broadly neutralizing antibody and T cell responses in mice and enhances human DC maturation and CD8+ T cell activation.

immunology↗

bioRxiv2022

Implications for food safety of the size and location of fragments of lead shotgun pellets embedded in hunted carcasses of small game animals intended for human consumption

Carcasses of common pheasants (Phasianus colchicus) killed by hunters using shotguns are widely used or sold in the United Kingdom and elsewhere for human consumption. Almost all of the birds are shot using shotgun pellets composed principally of lead (Pb). Lead shotgun pellets often fragment on impact within the bodies of gamebirds, leaving small lead particles in the meat that are difficult for consumers to detect and remove and from which a greater proportion of lead is likely to be absorbed. Chronic exposure to even low levels of lead is associated with negative health effects in humans and especially in groups particularly vulnerable to the effects of lead, which include young children and pregnant women. Our study used a high-resolution computerised tomography X-ray scanner to locate, in three dimensions, metal fragments embedded within carcasses of eight pheasants sold for human consumption in the UK. Small radio-dense fragments (<2 mm diameter), assumed to be metallic lead, were present in all of the pheasant carcasses examined (mean number: 39 per carcass) and many were too small (<0.1 mm diameter) and too distant from the nearest large shotgun pellet for it to be practical for consumers to detect and remove them without discarding a large proportion of otherwise usable meat. Consumers of carcasses of pheasants killed using lead shotgun ammunition are likely to be exposed to elevated levels of dietary lead, even if careful food preparation is practiced to remove shotgun pellets and the most damaged tissue.

pharmacology and toxicology↗

bioRxiv2022

Fgf8 dosage regulates jaw shape and symmetry through pharyngeal-cardiac tissue relationships

BackgroundAsymmetries in craniofacial anomalies are commonly observed. With respect to the facial skeleton, the left side is more commonly and/or severely affected than the right. Such asymmetries complicate treatment options. Mechanisms underlying variation in disease severity between individuals as well as within individuals (asymmetries) are still relatively unknown. ResultsDevelopmental reductions in Fibroblast growth factor 8 (Fgf8) have a dosage dependent effect on jaw size, shape, and symmetry. Further, Fgf8 mutants have directionally asymmetric jaws with the left side being more affected than the right. Defects in lower jaw development begin with an early disruption to Meckels cartilage, which is discontinuous and appears as two separate condensations in Fgf8 mutants. All skeletal elements associated with the proximal condensation are dysmorphic in the mutants, which is exemplified by a malformed and mis-oriented malleus. At later stages, Fgf8 mutants exhibit syngnathia, which falls into 2 broad categories: bony fusion of the maxillary and mandibular alveolar ridges and zygomatico-mandibular fusion. All of these morphological defects exhibit both inter- and intra-individual variation. ConclusionsWe hypothesize that these asymmetries are linked to asymmetries in heart development resulting in higher levels of Fgf8 on the right side of the face during development, which may buffer the right side to mild developmental perturbations. This mutant mouse is a good model for future investigations of mechanisms underlying human syngnathia and facial asymmetry.

developmental biology↗

bioRxiv2022

The inositol-requiring enzyme 1α/X-box protein 1 (IRE1α/XBP1) pathway of the unfolded protein response is impaired in pediatric cholestatic liver disease explants

Background/AimsCholestatic liver diseases (CLD) are the leading indication for pediatric liver transplantation. Increased intrahepatic bile acid concentrations cause endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is activated to maintain homeostasis. UPR dysregulation, including the inositol-requiring enzyme 1/X-box protein 1 (IRE1/XBP1) pathway, is associated with several adult liver diseases. We evaluated hepatic UPR expression in pediatric patients with end-stage CLD and hypothesize that an inability to appropriately activate the hepatic IRE1/XBP1 pathway is associated with the pathogenesis of CLD. MethodsWe evaluated 34 human liver explants. Cohorts included: pediatric CLD (Alagille, ALGS, and progressive familial intrahepatic cholestasis, PFIC), pediatric non-cholestatic liver disease controls (autoimmune hepatitis, AIH), adult CLD, and normal controls. We performed RNA-seq, quantitative PCR, and western blotting to measure expression differences of the hepatic UPR and other signaling pathways. ResultsMetascape pathway analysis demonstrated that the KEGG protein processing in ER pathway was downregulated in pediatric CLD compared to normal controls. Pediatric CLD had decreased hepatic IRE1/XBP1 pathway gene expression and decreased protein expression of p-IRE1 compared to normal controls. These CLD changes were not disease-specific to ALGS or PFIC. IRE1/XBP1 pathway gene expression was decreased in pediatric CLD compared to AIH disease controls. ConclusionPediatric CLD explants have decreased gene and protein expression of the protective IRE1/XBP1 pathway and down-regulated KEGG protein processing in the ER pathways. IRE1/XBP1 pathway expression differences occur when compared to both normal and non-cholestatic disease controls. Attenuated expression of the IRE1/XBP1 pathway is associated with cholestatic diseases and could be targeted to treat pediatric CLD.

cell biology↗

bioRxiv2022

Hepatic Deletion of X-box Binding Protein 1 in Farnesoid X Receptor Null Mice Leads to Enhanced Liver Injury

Background & AimsFarnesoid X receptor (FXR) regulates bile acid metabolism and FXR null (Fxr-/-) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway is a protective pathway of the unfolded protein response (UPR) that is activated in response to ER stress. In this study we sought to determine the role of the UPR in Fxr-/- mice. Approach & ResultsWe examined hepatic UPR gene and protein expression in 10- and 24-week-old wild type (WT) and Fxr-/- mice. Hepatic XBP1 and other UPR pathways were activated in 24-week-old Fxr-/- mice, but not WT mice. To further determine the role of the liver UPR activation in Fxr-/- mice, we generated mice with FXR and liver-specific XBP1 double knockout (DKO, Fxr-/-Xbp1LKO) and Fxr-/- Xbp1fl/fl single knockout (SKO) mice and characterized their phenotypes at different ages. DKO mice demonstrated enhanced liver injury, apoptosis and fibrosis compared with SKO mice. RNA-seq revealed increased gene expression in apoptosis, inflammation and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein (CHOP) pathway was activated in DKO mice. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. ConclusionsThe hepatic XBP1 pathway is activated in older Fxr-/- mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.

molecular biology↗

bioRxiv2022

Expression of Unfolded Protein Response Genes in Post-transplantation Liver Biopsies

Cholestatic liver diseases are a major source of morbidity and mortality that can progress to end-stage liver disease. There are few effective medical therapies for primary biliary cholangitis, primary sclerosing cholangitis and other cholestatic liver diseases, in part, due to our incomplete understanding of the pathogenesis of cholestatic liver injury. The hepatic unfolded protein response (UPR) is an adaptive cellular response to endoplasmic reticulum stress that is important in the pathogenesis of many liver diseases and recent animal studies have demonstrated the importance of the UPR in the pathogenesis of cholestatic liver injury. However, the role of the UPR in human cholestatic liver diseases is largely unknown. In this study, we utilized liver biopsies from patients after liver transplantation as a disease model to determine the transcriptional profile and hepatic UPR gene expression that is associated with liver injury and cholestasis. RNA-seq analysis revealed that patients with hyperbilirubinemia had enhanced expression of hepatic UPR pathways. Alternatively, liver biopsy samples from patients with acute rejection had enhanced gene expression of LAG3 and CDK1. Pearson correlation analysis of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels demonstrated significant correlations with the hepatic expression of several UPR genes, as well as genes involved in hepatic bile acid metabolism and inflammation. In contrast, serum alkaline phosphatase levels were correlated with the level of hepatic bile acid metabolism gene expression but not liver UPR gene expression. ConclusionOverall, these data indicate that hepatic UPR pathways are increased in cholestatic human liver biopsy samples and supports an important role of the UPR in the mechanism of human cholestatic liver injury.

molecular biology↗

bioRxiv2021

Distinct ribosome states trigger diverse mRNA quality control pathways

Key protein adapters couple translation to mRNA decay on specific classes of problematic mRNAs in eukaryotes. Slow decoding on non-optimal codons leads to codon-optimality-mediated decay (COMD) and prolonged arrest at stall sites leads to no-go decay (NGD). The identities of the decay factors underlying these processes and the mechanisms by which they respond to translational distress remain open areas of investigation. We use carefully-designed reporter mRNAs to perform genetic screens and functional assays in S. cerevisiae. We characterize the roles of Hel2 and Syh1 in coordinating translational repression and mRNA decay on NGD reporter mRNAs, finding that Syh1 acts as the primary link to mRNA decay in NGD. Importantly, we observe that these NGD factors are not involved in the degradation of mRNAs enriched in non-optimal codons. Further, we establish that a key factor previously implicated in COMD, Not5, contributes modestly to the degradation of an NGD-targeted mRNA. Finally, we use ribosome profiling to reveal distinct ribosomal states associated with each reporter mRNA that readily rationalize the contributions of NGD and COMD factors to degradation of these reporters. Taken together, these results provide new mechanistic insight into the role of Syh1 in NGD and define the molecular triggers that determine how distinct pathways target mRNAs for degradation in yeast.

molecular biology↗

bioRxiv2021

Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB

Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions; cryo-EM structures of collided disomes from E. coli and B. subtilis reveal a distinct and conserved arrangement of the individual ribosomes and the composite SmrB binding site. These findings reveal the underlying mechanism by which ribosome collisions trigger ribosome rescue in bacteria.

molecular biology↗