Biology subjects

Martin, C.

Publications and source records attributed to Martin, C..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

A minor impact of VGLUT1 expression level on quantal size revealed through the characterization of VGLUT1mEos2 knock-down new mouse model

Synaptic vesicles (SVs) are small organelles secreting neurotransmitters at synapses. By fusing a photoactivated fluorescent protein to VGLUT1, we generated a VGLUT1mEos2 knock-in mouse. VGLUT1mEos2 knock-in mice are viable and healthy, but exhibit a severe reduction in VGLUT1 expression levels. Using VGLUT1mEos2 expressing neurons, we established paradigms to trace individual SV mobility at the single-molecule level or via massive photoconversion. Hippocampal neurons with significantly diminished VGLUT1 expression maintain unaltered miniature glutamate release characteristics in terms of quantal size and frequency. We demonstrate that VGLUT1 expression level are not correlated in a linear fashion with the vesicular glutamate content. In conclusion, the VGLUT1mEos2 mouse line serves as a powerful tool for exploring SV mobility properties and elucidating the contributions of VGLUT1 to excitatory neurotransmission and cognitive processes.

neuroscience↗

bioRxiv2024

Temperature drives seagrass recovery across the Western North Atlantic

Climate-driven shifts in herbivores, temperature and nutrient runoff threaten coastal ecosystem resilience. However, our understanding of ecological resilience, particularly for foundation species, remains limited due to a rarity of field experiments that are conducted across appropriate spatial and temporal scales and that investigate multiple stressors. This study aimed to evaluate the resilience of a widespread tropical marine plant (turtlegrass) to disturbances across its geographic range and how this is impacted by environmental gradients in (a)biotic factors. We assessed the resilience (i.e. recovery) of turtlegrass to a simulated disturbance (complete above- and belowground biomass removal) over a year. Contrary to temperate studies, higher temperature generally enhanced seagrass recovery. While nutrients and light availability had minimal impact, combined high levels of nutrients and herbivore grazing (meso and megaherbivore) reduced aboveground recovery. Our results suggest that the resilience of some tropical species, especially in cooler subtropical waters, may initially increase with warming.

ecology↗

bioRxiv2024

Protein Set Transformer: A protein-based genome language model to power high diversity viromics

Exponential increases in microbial and viral genomic data demand transformational advances in scalable, generalizable frameworks for their interpretation. Standard homology-based functional analyses are hindered by the rapid divergence of microbial and especially viral genomes and proteins that significantly decreases the volume of usable data. Here, we present Protein Set Transformer (PST), a protein-based genome language model that models genomes as sets of proteins without considering sparsely available functional labels. Trained on >100k viruses, PST outperformed other homology- and language model-based approaches for relating viral genomes based on shared protein content. Further, PST demonstrated protein structural and functional awareness by clustering capsid-fold-containing proteins with known capsid proteins and uniquely clustering late gene proteins within related viruses. Our data establish PST as a valuable method for diverse viral genomics, ecology, and evolutionary applications. We posit that the PST framework can be a foundation model for microbial genomics when trained on suitable data.

bioinformatics↗

bioRxiv2024

Prophage-DB: A comprehensive database to explore diversity, distribution, and ecology of prophages

BackgroundViruses that infect prokaryotes (phages) constitute the most abundant group of biological agents, playing pivotal roles in microbial systems. They are known to impact microbial community dynamics, microbial ecology, and evolution. Efforts to document the diversity, host range, infection dynamics, and effects of bacteriophage infection on host cell metabolism are extremely underexplored. Phages are classified as virulent or temperate based on their life cycles. Temperate phages adopt the lysogenic mode of infection, where the genome integrates into the host cell genome forming a prophage. Prophages enable viral genome replication without host cell lysis, and often contribute novel and beneficial traits to the host genome. Current phage research predominantly focuses on lytic phages, leaving a significant gap in knowledge regarding prophages, including their biology, diversity, and ecological roles. ResultsHere we develop and describe Prophage-DB, a database of prophages, their proteins, and associated metadata that will serve as a resource for viral genomics and microbial ecology. To create the database, we identified and characterized prophages from genomes in three of the largest publicly available databases. We applied several state-of-the-art tools in our pipeline to annotate these viruses, cluster and taxonomically classify them, and detect their respective auxiliary metabolic genes. In total, we identify and characterize over 350,000 prophages and 35,000 auxiliary metabolic genes. Our prophage database is highly representative based on statistical results and contains prophages from a diverse set of archaeal and bacterial hosts which show a wide environmental distribution. ConclusionProphages are particularly overlooked in viral ecology and merit increased attention due to their vital implications for microbiomes and their hosts. Here, we created Prophage-DB to advance our comprehension of prophages in microbiomes through a comprehensive characterization of prophages in publicly available genomes. We propose that Prophage-DB will serve as a valuable resource for advancing phage research, offering insights into viral taxonomy, host relationships, auxiliary metabolic genes, and environmental distribution.

bioinformatics↗

bioRxiv2024

Endemism shapes viral ecology and evolution in globally distributed hydrothermal vent ecosystems

Viruses are ubiquitous in deep-sea hydrothermal environments, where they exert a major influence on microbial communities and biogeochemistry. Yet, viral ecology and evolution remain understudied in these environments. Here, we identified 49,962 viruses from 52 globally distributed hydrothermal vent samples (10 plumes, 40 deposits, and 2 diffuse flow), and reconstructed 5,708 viral metagenome-assembled genomes (vMAGs), the majority of which were bacteriophages. Hydrothermal viruses were largely endemic. However, some viruses were shared between geographically separated vents, predominantly between the Lau Basin and Brothers Volcano in the Pacific Ocean. Geographically distant viruses often shared proteins related to core functions such as structural proteins, and rarely, proteins of auxiliary functions. Common microbial hosts of viruses included members of Campylobacterota, Alpha-, and Gammaproteobacteria in deposits, and Gammaproteobacteria in plumes. Campylobacterota- and Gammaproteobacteria-infecting viruses reflected variations in hydrothermal chemistry and functional redundancy in their predicted microbial hosts, suggesting that hydrothermal geology is a driver of viral ecology and coevolution of viruses and hosts. Our study indicates that viral ecology and evolution in globally distributed hydrothermal vents is shaped by endemism, and thus may have increased susceptibility to the negative impacts of deep-sea mining and anthropogenic change in ocean ecosystems.

microbiology↗

bioRxiv2024

Two Stabiliser loci in Antirrhinum suppress the transposition of Tam3 without compromising transposase production

Antirrhinum majus has been known to exhibit occasional instabilities that are manifested as variegations and morphological chimeras. Stabiliser (St) is a historical locus that stabilizes phenotypically unstable or mutable traits in Antirrhinum. Here, we characterized two St loci, the previously described Old Stabiliser (OSt) and New Stabiliser (NSt), in Antirrhinum that specifically suppress the transposition of the Class II DNA transposable element, Tam3. Both St loci involve derivatives of Tam3 with unique structures: OSt has a pseudo-Tam3 copy whose 5-terminal region has been rearranged compared to the cognate Tam3 element, and NSt consists of two intact copies of Tam3 in a head-to-head orientation. Neither locus interferes with the production of the intact Tam3 transposase (TPase) or the nuclear import of TPase. Both OSt and NSt produce specific sRNAs from their 5 terminal regions containing multiple TPase binding motifs. These specific sRNAs could repress Tam3 transposition by interacting with the TPase binding motifs within the Tam3 element or with the TPase itself.

genetics↗

bioRxiv2024

Modeling epithelial homeostasis and perturbation in three-dimensional human esophageal organoids

BackgroundEsophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbeccos Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. MethodsWe have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-{beta} receptor-mediated signaling, both key regulators of proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. ResultsThe formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGF{beta} receptor-mediated signaling. In optimized HOME0, normal human esophageal organoid formation was improved, whereas IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

cell biology↗

bioRxiv2024

KIRA6 is an effective and versatile mast cell inhibitor of IgE-mediated activation

Incidents of IgE-mediated, mast cell (MC)-driven allergic diseases are constantly rising and there is an urgent need for the development of novel pharmacological MC stabilizers. Allergen/antigen (Ag)-triggered activation of MCs via crosslinking of the high-affinity receptor for IgE (Fc{varepsilon}RI) is regulated, amongst others, by the coordinated action of various cytosolic tyrosine kinases of the SRC family, e.g. LYN and FYN, which exert positive as well as negative functions. We report that KIRA6, an inhibitor developed for the endoplasmic reticulum (ER) stress sensor IRE1, suppresses IgE-mediated pro-inflammatory MC activation by inhibiting both LYN and FYN. KIRA6 dose-dependently and effectively attenuates Ag-stimulated early signaling (e.g. substrate tyrosine phosphorylation, Ca2+ mobilization, and activation of MAPK pathways) as well as effector functions such as degranulation and pro-inflammatory cytokine production/secretion in murine bone marrow-derived MCs (BMMCs). Moreover, Ag-triggered bronchoconstriction in an ex vivo model of precision-cut lung slices (PCLS), and IgE-mediated stimulation of human MCs were repressed by KIRA6. To get in-depth inside into KIRA6 interaction with three MC-relevant tyrosine kinases, LYN, FYN, and KIT, and to elicit the potential of KIRA6 structure to serve as pharmacophore for the development of respective single-, dual-, or triple-specificity inhibitors, we modeled and evaluated the binding of KIRA6 on the three kinases by applying homology modeling and molecular dynamics simulations, as well as MM GBSA calculations. We found that KIRA6 has a high propensity to bind the inactive state of LYN, FYN, and KIT with comparable affinities. In conclusion, our data suggest the use of novel inhibitors based on the KIRA6 pharmacophore as effective MC stabilizers to improve treatment of pro-inflammatory diseases with MC involvement in need of effective pharmacological interventions.

immunology↗

bioRxiv2024

Systematic genome-wide discovery of host factors governing bacteriophage infectivity

Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host genes impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways previously unknown to affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers valuable insights into phage-host interactions, phage evolution, and bacterial resistance.

synthetic biology↗

bioRxiv2024

Evolutionary paleoecology of European rhinocerotids across the Oligocene-Miocene transition

The Oligocene-Miocene transition witnessed great environmental and faunal changes, but its drivers and consequences on mammals remain poorly understood. Rhinocerotoids are among the most affected taxa, reflected by great taxonomical (extinction of all non-rhinocerotids) and morphological (e.g., more mediportal forms) changes observed during this interval. However, potential associated changes in ecology have not been explored. Here, we investigated the paleoecology of 10 rhinocerotid species coming from 15 localities across Western and Central Europe and ranging from Mammal Paleogene reference level 28 to Mammal Neogene zone 3. We explored evolutionary trends for their diet, physiology, and habitat via dental wear, hypoplasia, body mass estimates, and stable isotopy. Our results (isotopy, dental wear) suggest that all rhinocerotids studied were C3 feeders, whether browsing or mixed-feeding, but niche partitioning was assumed at some localities: distinct dietary preferences and/or habitats were highlighted at Gaimersheim, Ulm-Westtangente, and Rickenbach, whereas a potential competition for the resources or different niche partitioning strategies (body mass, feeding height) are hypothesized at others localities (La Milloque, Thezels, Paulhiac). We reconstructed similar warm conditions at all localities except Gaimersheim, but found greater variations in precipitation. Indeed, a clear shift in {delta}13CCO3, enamel values was noticed at the end of the Oligocene, consistent with climatic changes (Mi-1) and vegetation modifications reported at that time. Despite these great changes, the prevalence of hypoplasia was low (< 10 %) to moderate (< 20 %) except at some localities (Pappenheim, Rickenbach, Engehalde) and for some species (Brachydiceratherium aurelianense and B. lamilloquense). Teleoceratines were particularly affected, suggesting a potential phylogenetic effect. Fourth milk molars and third molars were the most affected loci, indicating stresses around birth and environmental stresses respectively. Body mass co-variated with hypoplasia prevalence, indicating that larger species might be more susceptible to stresses and environmental changes. The tracking of rhinocerotids paleoecological preferences during the Oligocene-Miocene revealed several changes in diet and habitat (temperature, precipitation, vegetation) consistent with global changes during this period.

paleontology↗

bioRxiv2024

The Sociodemographic and Lifestyle Correlates of Epigenetic Aging in a Nationally Representative U.S. Study of Younger Adults

ImportanceEpigenetic clocks represent molecular evidence of disease risk and aging processes and have been used to identify how social and lifestyle characteristics are associated with accelerated biological aging. However, most of this research is based on older adult samples who already have measurable chronic disease. ObjectiveTo investigate whether and how sociodemographic and lifestyle characteristics are related to biological aging in a younger adult sample across a wide array of epigenetic clock measures. DesignNationally representative prospective cohort study. SettingUnited States (U.S.). ParticipantsData come from the National Longitudinal Study of Adolescent to Adult Health, a national cohort of adolescents in grades 7-12 in U.S. in 1994 followed for 25 years over five interview waves. Our analytic sample includes participants followed-up through Wave V in 2016-18 who provided blood samples for DNA methylation (DNAm) testing (n=4237) at Wave V. ExposureSociodemographic (sex, race/ethnicity, immigrant status, socioeconomic status, geographic location) and lifestyle (obesity status, exercise, tobacco, and alcohol use) characteristics. Main OutcomeBiological aging assessed from blood DNAm using 16 epigenetic clocks when the cohort was aged 33-44 in Wave V. ResultsWhile there is considerable variation in the mean and distribution of epigenetic clock estimates and in the correlations among the clocks, we found sociodemographic and lifestyle factors are more often associated with biological aging in clocks trained to predict current or dynamic phenotypes (e.g., PhenoAge, GrimAge and DunedinPACE) as opposed to clocks trained to predict chronological age alone (e.g., Horvath). Consistent and strong associations of faster biological aging were found for those with lower levels of education and income, and those with severe obesity, no weekly exercise, and tobacco use. Conclusions and RelevanceOur study found important social and lifestyle factors associated with biological aging in a nationally representative cohort of younger-aged adults. These findings indicate that molecular processes underlying disease risk can be identified in adults entering midlife before disease is manifest and represent useful targets for interventions to reduce social inequalities in heathy aging and longevity. Key PointsO_ST_ABSQuestionC_ST_ABSAre epigenetic clocks, measures of biological aging developed mainly on older-adult samples, meaningful for younger adults and associated with sociodemographic and lifestyle characteristics in expected patterns found in prior aging research? FindingsSociodemographic and lifestyle factors were associated with biological aging in clocks trained to predict morbidity and mortality showing accelerated aging among those with lower levels of education and income, and those with severe obesity, no weekly exercise, and tobacco use. MeaningAge-related molecular processes can be identified in younger-aged adults before disease manifests and represent potential interventions to reduce social inequalities in heathy aging and longevity.

genomics↗

bioRxiv2024

Genome-Wide Association Study in Outbred Heterogeneous Stock Rats Identifies Multiple Loci for the Incentive Salience of Reward Cues.

Addiction vulnerability is associated with the tendency to attribute incentive salience to reward predictive cues; both addiction and the attribution of incentive salience are influenced by environmental and genetic factors. To characterize the genetic contributions to incentive salience attribution, we performed a genome-wide association study (GWAS) in a cohort of 1,645 genetically diverse heterogeneous stock (HS) rats. We tested HS rats in a Pavlovian conditioned approach task, in which we characterized the individual responses to food-associated stimuli ("cues"). Rats exhibited either cue-directed "sign-tracking" behavior or food-cup directed "goal-tracking" behavior. We then used the conditioned reinforcement procedure to determine whether rats would perform a novel operant response for unrewarded presentations of the cue. We found that these measures were moderately heritable (SNP heritability, h2 = .189-.215). GWAS identified 14 quantitative trait loci (QTLs) for 11 of the 12 traits we examined. Interval sizes of these QTLs varied widely. 7 traits shared a QTL on chromosome 1 that contained a few genes (e.g. Tenm4, Mir708) that have been associated with substance use disorders and other mental health traits in humans. Other candidate genes (e.g. Wnt11, Pak1) in this region had coding variants and expression-QTLs in mesocorticolimbic regions of the brain. We also conducted a Phenome-Wide Association Study (PheWAS) on other behavioral measures in HS rats and found that regions containing QTLs on chromosome 1 were also associated with nicotine self-administration in a separate cohort of HS rats. These results provide a starting point for the molecular genetic dissection of incentive salience and provide further support for a relationship between attribution of incentive salience and drug abuse-related traits.

neuroscience↗

bioRxiv2024

A 20-year time-series of a freshwater lake reveals seasonal dynamics and environmental drivers of viral diversity, ecology, and evolution

Long-term ecological studies are powerful tools to investigate microbiomes and ecosystem change but have mostly ignored viruses. Here, we leveraged a 20-year time-series of a freshwater lake to characterize 1.3 million viral genomes over time, seasonality, and environmental factors. We identified 578 auxiliary metabolic gene (AMG) clusters representing over 150,000 AMGs, the most abundant of which, including psbA for photosynthesis, pmoC for methane oxidation, and katG for hydrogen peroxide decomposition, were consistently represented in viruses across decades and seasons. We observed positive associations and niche differentiation between virus-host pairs during seasonal change including in keystone taxa, Cyanobacteria, methanotrophs, and Nanopelagicales. Environmental constraints, specifically inorganic carbon and ammonium influenced viral abundances over time, and highlighted roles of viruses in both "top-down" and "bottom-up" interactions. Key evolutionary processes shaping gene and genome-wide selection included favored fitness genes, reduced genomic heterogeneity, and dominant sub-populations carrying specific genes. Overall, our study advances understanding of diversity, ecological dynamics, and evolutionary trajectories of viruses in Earths microbiomes and ecosystems.

microbiology↗

bioRxiv2024

SAR324 and related lineages are associated with the evolutionary history and origins of dsr-mediated sulfur oxidation

Microorganisms play vital roles in sulfur cycling through the oxidation of elemental sulfur and reduction of sulfite. These metabolisms are catalyzed by dissimilatory sulfite reductases (dsr) which function in the reductive (dsr) or reverse, oxidative direction (rdsr). Dsr-based sulfite reduction is an ancient metabolism and has been proposed to fuel energy metabolism in some of Earths earliest microorganisms. Conversely, sulfur oxidation is believed to have evolved later in association with the widespread availability of oxygen on Earth. Organisms are generally believed to carry out either the reductive or oxidative pathway, yet a subset of bacterial phyla have recently been discovered with gene combinations that implicate them in both pathways. A comprehensive global investigation into the metabolisms of these phyla regarding dsr can shed light on the evolutionary underpinnings of sulfur metabolism but is currently lacking. In this study, we selected one of these phyla, the abundant and metabolically versatile candidate phylum SAR324, to study the ecology and evolution of dsr and rdsr. We confirmed that phylogenetically, environmentally, and geographically diverse SAR324 contained dsr, rdsr, or both. Comprehensive phylogenetic analyses with other dsr-encoding bacterial and archaeal phyla revealed that organisms encoding both dsr and rdsr genes are constrained to a few phyla, which we term "transitionary clades for sulfur oxidation", and these phyla are phylogenetically positioned at the interface between well-defined oxidative and reductive bacterial clades. Together, this research suggests that SAR324 and other transitionary clades are associated with the evolutionary history and origins of the reverse dsr pathway in bacteria.

microbiology↗

bioRxiv2023

Combining transcutaneous spinal stimulation and functional electrical stimulation increases force generated by lower limbs: When more is more

BackgroundTranscutaneous Spinal Stimulation (TSS) has been shown to promote activation of the lower limb and trunk muscles and is being actively explored for improving the motor outcomes of people with neurological conditions. However, individual responses to TSS vary, and often the muscle responses are insufficient to produce enough force for self-supported standing. Functional electrical stimulation (FES) can activate individual muscles and assist in closing this functional gap, but it introduces questions regarding timing between modalities. MethodsTo assess the effects of TSS and FES on force generation, ten neurologically intact participants underwent (1) TSS only, (2) FES only, and (3) TSS + FES. TSS was delivered using four electrodes placed at T10-T11 through the L1-L2 intervertebral spaces simultaneously, while FES was delivered to the skin over the right knee extensors and plantarflexors. For all conditions, TSS and FES were delivered using three 0.5 ms biphasic square-wave pulses at 15 Hz. During the TSS + FES condition, timing between the two modalities was adjusted in increments of [1/4] time between pulses (16.5 ms). ResultsWhen TSS preceded FES, a larger force production was observed. We also determined several changes in muscle activation amplitude at different relative stimulus intervals, which help characterize our finding and indicate the facilitating and inhibitory effects of the modalities. ConclusionsUtilizing a delay ranging from 15 to 30 ms between stimuli resulted in higher mean force generation in both the knee and ankle joints, regardless of the selected FES location (Average; knee: 112.0%, ankle: 103.1%).

neuroscience↗

bioRxiv2023

Multiple performance peaks for scale-biting in an adaptive radiation of pupfishes

The physical interactions between organisms and their environment ultimately shape their rate of speciation and adaptive radiation, but the contributions of biomechanics to evolutionary divergence are frequently overlooked. Here we investigated an adaptive radiation of Cyprinodon pupfishes to measure the relationship between feeding kinematics and performance during adaptation to a novel trophic niche, lepidophagy, in which a predator removes only the scales, mucus, and sometimes tissue from their prey using scraping and biting attacks. We used high-speed video to film scale-biting strikes on gelatin cubes by scale-eater, molluscivore, generalist, and hybrid pupfishes and subsequently measured the dimensions of each bite. We then trained the SLEAP machine-learning animal tracking model to measure kinematic landmarks and automatically scored over 100,000 frames from 227 recorded strikes. Scale-eaters exhibited increased peak gape and greater bite length; however, substantial within-individual kinematic variation resulted in poor discrimination of strikes by species or strike type. Nonetheless, a complex performance landscape with two distinct peaks best predicted gel-biting performance, corresponding to a significant nonlinear interaction between peak gape and peak jaw protrusion in which scale-eaters and their hybrids occupied a second performance peak requiring larger peak gape and greater jaw protrusion. A bite performance valley separating scale-eaters from other species may have contributed to their rapid evolution and is consistent with multiple estimates of a multi-peak fitness landscape in the wild. We thus present an efficient deep-learning automated pipeline for kinematic analyses of feeding strikes and a new biomechanical model for understanding the performance and rapid evolution of a rare trophic niche.

evolutionary biology↗

bioRxiv2023

Structural Basis of μ-Opioid Receptor-Targeting by a Nanobody Antagonist

The -opioid receptor (OR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the limitations and severe side effects of currently available opioid drugs, there is considerable interest in developing novel modulators of OR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, are emerging as alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the OR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded {micro}OR ligand and uncover the molecular basis for {micro}OR antagonism by solving the cryo-EM structure of the NbE-{micro}OR complex. NbE displays a unique ligand binding mode and achieves {micro}OR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a {beta}-hairpin loop formed by NbE that deeply inserts into the {micro}OR and centers most binding contacts, we design short peptide analogues that retain {micro}OR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel OR ligands that can serve as a basis for therapeutic developments.

molecular biology↗

bioRxiv2023

Cholinergic Modulation of Neurovascular Coupling and Neuroimaging Signals

The use of non-invasive functional neuroimaging methods to discriminate levels or patterns of brain activity between different groups, across different time points, or in response to drug treatment, depends on an understanding of neurovascular coupling relationships and how they themselves are affected by the factors of interest. For instance, Alzheimers disease (AD) features cerebral blood flow (CBF) and neurovascular unit changes as well as being characterised by a cholinergic dysfunction which impacts upon CBF regulation. The present study investigated the acute neurovascular effects of cholinergic agonist and antagonist treatment in a rat model utilising a multimodal measurement approach. Drugs were administered to rats during concurrent imaging of cortical CBF and recording of local field potential responses to somatosensory stimulation. Analysis of concurrent neuronal and vascular measures revealed a pronounced loss of neurovascular coupling following treatment with the cholinergic antagonist scopolamine. Separate analysis of CBF and neuronal responses reveal an interaction effect of stimulus input and drug treatment for both cholinergic agonist and antagonist treatment, as well as an augmentation of both neuronal and haemodynamic response magnitudes after treatment with the clinically prescribed cholinergic agonist donepezil. These findings have implications for the use and interpretation of functional neuroimaging data acquired in individuals with disease-related or pharmacological manipulations of cholinergic function.

neuroscience↗