Biology subjects

Hart, E.

Publications and source records attributed to Hart, E..

12 recordsLinked to original sources

bioRxiv2026

CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin

Adeno-Associated Viruses (AAVs) are powerful platforms for delivering therapeutic transgenes via recombinant AAV (rAAV) vectors. However, a limited understanding of the regulation of AAV gene expression has narrowed the ability to efficiently express therapeutic transgenes from rAAV vectors. Since rAAVs retain only the wtAAV inverted terminal repeats (ITR), we hypothesized that regulatory elements outside the ITR that govern wild-type AAV (wtAAV) gene expression can be used to modify rAAV genomes to enhance vector performance. Through in silico analysis, biochemical pulldowns, and high-throughput sequencing, we have identified that the host architectural protein CCCTC-binding Factor (CTCF) associates with the wtAAV type 2 (wtAAV2) genome but is absent from rAAV vectors. Global knockdown and site-specific deletion revealed that the CTCF binding element (CBE) on the wtAAV2 genome, located upstream of the viral P5 promoter, regulates expression of the viral Rep68/78 genes. We have re-engineered new rAAV vectors expressing a GFP reporter transgene to contain the wtAAV2-CBE upstream of the vector promoter. Our results show that CTCF binding dramatically increased rAAV transduction efficiency and GFP expression by up to four-fold across multiple cell types. This enhancement was independent of the AAV capsid serotype used for packaging rAAV vectors. CUT&RUN analysis revealed that this CBE was necessary and sufficient to regulate the chromatin landscape of wtAAV2 and rAAV2. Finally, we observed that CTCF-mediated chromatin remodeling of rAAV2 led to increased production of nascent RNA transcripts from the vector genome. Based on our findings, we propose that CTCF supports wtAAV2/rAAV gene expression by shaping the local chromatin landscape. SIMPLE ABSTRACTRecombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of transgenes using current rAAV technologies require high doses, which can lead to sporadic toxic side effects. We hypothesized that uncharacterized regulatory elements in the wtAAV2 genome drive efficient viral gene expression and are absent from the current generation of rAAV vectors. Using in-silico analysis combined with biochemical pulldowns, high-throughput sequencing, and mutant viral systems, we have identified a novel cis-acting element bound by the cellular architectural protein CCCTC-binding Factor (CTCF). This CTCF binding element is necessary for wtAAV2 gene expression and is sufficient to enhance the rAAV vectors ability to express reporter transgenes. This CTCF-binding element regulates the chromatin landscape of the virus and its vectors. Our discovery that adding the 19-bp AAV2 CTCF-binding element enhances transgene expression without affecting vector production efficiency presents a promising new rAAV gene therapy platform that is likely to reduce clinical doses and minimize toxicity in therapeutic applications.

microbiology↗

bioRxiv2026

Netrin-1 drives cell-type-specific plasticity of human dopaminergic neurons during circuit integration in a Parkinsonian model

The survival of transplanted ventral midbrain (vm) dopaminergic neurons (DAn) and their innervation of host striatal tissue are crucial for ameliorating motor symptoms in Parkinsons disease (PD). However, human pluripotent stem cells (hPSC) show inferior axonal plasticity compared to fetal donor tissue. While modulation of the host environment with trophic cues, such as glial cell-derived neurotrophic factor (GDNF), can improve graft outcomes, these cues lack specificity for DAn, resulting in plasticity of other neurons within the graft. Using single nuclei RNA sequencing, we identified axonal guidance pathways (Semaphorin, Netrin and Wnt) that were preferentially activated in DAn within the graft. Overexpression of Semaphorin3A/SEMA3A, Netrin1/NTN1 or WNT5A in the striatum of Parkinsonian mice following vm DA progenitor transplantation promoted A9-DA specification and selectively increased DA innervation of the host striatum, without off-target extrastriatal DA innervation observed in response to GDNF. In Parkinsonian rats, NTN1 overexpression promoted graft-induced motor recovery, selective DA plasticity and activation of postsynaptic striatal neurons without evidence of non-DAn plasticity. Further, snRNA-sequencing of NTN1 or GDNF-treated grafts confirmed the upregulation of DA-specific plasticity by NTN1, while GDNF promoted plasticity in both DA and non-DAn. These findings highlight the capacity to improve on-target integration of hPSC-derived DAn in grafts by selectively targeting DA-specific plasticity. Taken together, these results demonstrate the utility of DA-specific cues to promote functional recovery, improve graft predictability, and limit off-target innervation.

neuroscience↗

bioRxiv2026

Recapitulating whipworm development in vitro using caecaloids

Whipworms (Trichuris spp.) are intracellular intestinal parasites that develop within the host caecal epithelium, yet the host signals that regulate their growth and developmental progression remain poorly understood. Progress in studying these processes has been limited by the lack of physiologically relevant in vitro systems capable of supporting sustained whipworm development. Here, we established an in vitro infection system using caecal organoids (caecaloids) and evaluated their capacity to support sustained growth and morphological development of Trichuris muris larvae. To rigorously validate this system, we generated a comprehensive and up-to-date anatomical and biometrical reference dataset describing the whole-body growth and tissue-level morphogenesis of T. muris throughout its life cycle in vivo. Quantitative analysis across larval and adult stages confirmed that the trajectory of parasite growth is largely conserved across host mouse strains and provided a detailed contextualised description of the development of key anatomical structures of T. muris. Using this reference framework, we evaluated parasite growth and development in long-term T. muris-caecaloid co-cultures. Larvae invading the caecaloid epithelium remained intracellular within syncytial tunnels and exhibited sustained growth over extended culture periods. in vitro parasites developed increasing anatomical complexity, including formation of the bacillary band, stichosome, intestine, and rectum. Importantly, quantitative comparisons revealed that larvae developing within caecaloids follow growth trajectories and morphological developmental patterns closely resembling those observed in vivo. This study therefore presents the first detailed anatomical and morphometric framework for validating whipworm development in an organoid system and provides concrete evidence that the caecaloid epithelium is sufficient to trigger and sustain whipworm growth and morphogenesis, establishing caecaloids as a powerful experimental platform for investigating Trichuris infection and development.

microbiology↗

bioRxiv2025

Investigating diurnal effects and joint nest defense behavior in Herring Gulls

Nest defense in birds is vital for the protection of their young, but can prove energetically costly. Birds often show plasticity in nest defense depending on factors such as threat level, mate presence, nest stage, and body condition. Such factors can vary over temporal extents ranging from a diel cycle to a breeding season or an individuals lifetime. Understanding diurnal variation in nest defense intensity can be particularly useful when studying a breeding population to help investigators minimize nest disturbance, yet few studies have explored diurnal variation in nest defense intensity. Here we investigated how nest defense by Herring Gulls (Larus smithsonianus) varies with time of day. We simulated predatory threat at 26 Herring Gull nests during four different times of day: "Early Morning" (0530-0600), "Late Morning" (1000-1030), "Afternoon" (1400-1430), and "Evening" (1900-1930). Based on previous findings of diurnal activity patterns in a Herring Gull, we predicted that nest defense intensity--measured as aggressiveness of response and latency to calm--would be greater during the early morning and late evening than at other times. Contrary to our prediction, we found that the time of day did not affect nest defense intensity in Herring Gulls. However, independent of time of day, we found that when both mates were present at the nest, aggression scores were elevated. Our results suggest that joint nest defense in Herring Gulls permits greater aggressiveness towards predators, perhaps due to division of labor or lowered risk of complete nest failure if one parent is injured or killed. Further, our results indicate that researchers can minimize nest disturbance and accompanying stress by limiting research activities at Herring Gull nests when both parents are present.

animal behavior and cognition↗

bioRxiv2025

Persistent representation of a prior schema in the orbitofrontal cortex facilitates learning of a conflicting schema

Schemas allow efficient behavior in new situations, but reliance on them can impair flexibility when new demands conflict, culminating in psychopathology. Evidence implicates the orbitofrontal cortex (OFC) in deploying schemas in new situations congruent with previously acquired knowledge. But how does this role affect learning of a conflicting behavioral schema? Here we addressed this question by recording single-unit activity in the OFC of rats learning odor problems with identical external information but orthogonal rules governing reward. Consistent with schema formation, OFC representations adapted to track the underlying rules, and both performance and encoding was faster on subsequent than initial problems. Surprisingly however, when the rule governing reward changed, persistent representation of the prior schema was correlated with acquisition of the new. Thus, OFC was not a source of interference and instead supported new learning by accurately and independently representing the old schema as the new was acquired.

neuroscience↗

bioRxiv2024

Optimisation of single-nuclei isolation and RNA sequencing of parasitic nematodes

Single-cell/nuclei transcriptomics has revolutionised our understanding of cell, tissue, and organismal biology during health and disease of humans and model organisms. However, for non-model species, reassessment and refinement of conventional methods to address novel technical and biological challenges are needed. Here, we evaluate three established nuclei isolation protocols and, building on experiences, optimised single nuclei isolation for parasitic nematodes (SNIP). We test the versatility of SNIP across diverse species and life stages, validating it with snRNA-seq to reveal sex, stage, and tissue-specific gene expression. Optimised protocols will advance the understanding of parasitic nematodes, from fundamental biology to infection and disease.

molecular biology↗

bioRxiv2024

Random subsamples of animal populations can reveal intrinsic differences in sociality with key implications in ecology, conservation and disease transmission

Animal populations are under mounting stress from the dual threats of climate change and rapid global human population growth, raising significant concerns about declining wildlife and the rising risk of zoonotic diseases. In many species, social interactions can be a highly plastic suite of behaviours that are responsive to these disturbances and are consequential to other processes like disease transmission and population dynamics. Studying social interactions can be challenging in that researchers often rely on wildlife population subsamples due to practical constraints and costs, which can introduce biases in the reliability of social network metrics. We investigated the extent to which subsamples can depict intrinsic characteristics of wildlife populations using data from three distinct species: peri-urban fallow deer, Alpine ibex and Angolan giraffe. We showed that random subsamples of these populations could still reveal differences in their social behaviour, indicating that, as long as researchers have a reliable estimate of population size, subsampling animal populations can be an effective and precise method to infer their sociality and offer valuable empirical data for management, conservation and zoonotic disease ecology. Furthermore, we demonstrate that non-random sampling, influenced for instance by animal personality and related trappability, can introduce significant biases in social network estimates. These findings underscore the importance of accounting for sampling biases in social network analysis and offer a robust framework for using partial networks in ecological studies and conservation management.

ecology↗

bioRxiv2024

Hatching of whipworm eggs induced by bacterial contact is serine-protease dependent

Whipworms (Trichuris spp) are ubiquitous parasites of humans and domestic and wild mammals that cause chronic disease, considerably impacting human and animal health. Egg hatching is a critical phase in the whipworm life cycle that marks the initiation of infection, with newly hatched larvae rapidly migrating to and invading host intestinal epithelial cells. Hatching is triggered by the host microbiota; however, the physical and chemical interactions between bacteria and whipworm eggs, as well as the bacterial and larval responses that result in the disintegration of the polar plug and larval eclosion, are not completely understood. Here, we examined hatching in the murine whipworm, Trichuris muris, and investigated the role of specific bacterial and larval structures and molecules in this process. Using scanning and transmission electron microscopy, we characterised the physical interactions of both fimbriated (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) and non-fimbriated (Staphylococcus aureus) bacteria with the egg polar plugs during the induction/initiation stage and visualised the process of degradation of the polar plugs that leads to larval eclosion. Further, we found that protease inhibitors blocked whipworm hatching induced by both fimbriated and non-fimbriated bacteria in a dose-dependent manner, suggesting the partial involvement of bacterial enzymes in this process. In addition, we identified the minimal egg developmental timing required for whipworm hatching, and transcriptomic analysis of T. muris eggs through embryonation revealed the specific upregulation of serine proteases (S01A family) in fully embryonated eggs containing hatch-ready L1 larvae. Finally, we demonstrated that inhibition of serine proteases with the serine-protease inhibitor Pefabloc ablated T. muris egg hatching induced by bacteria. Collectively, our findings unravel the temporal and physicochemical bacterial-egg interactions leading to whipworm hatching and indicate serine proteases of both bacterial and larval origin mediate these processes. Author summaryHuman whipworms are parasites that cause the gastrointestinal disease trichuriasis in millions of people around the world. Infections occur when whipworm eggs, ingested in contaminated food and water, hatch in the intestine in response to gut bacteria (microbiota). The egg encloses a larva within an egg-shell and has a plug at each end. Hatching liberates the larva that burrows inside the cells that line the gut. Interactions between the microbiota of the gut and whipworm eggs are needed for hatching but are poorly understood. In this study, using the natural mouse whipworm as an infection model, we show that bacteria bind the whipworm egg plugs during the initial stages of hatching, resulting in their degradation and leading to larval exit. We further show that disintegration of the egg plugs is caused by protein-degrading enzymes produced by the bacteria and the larvae. The production of those enzymes by the parasite is dependent on the full development of the larva inside the whipworm egg. These new mechanistic insights pave the way for future studies to understand human whipworm infection and develop new tools to tackle these globally important parasites.

microbiology↗

bioRxiv2024

The conserved σD envelope stress response monitors multiple aspects of envelope integrity in corynebacteria

The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved {sigma}D envelope stress response in porin export and identified MarPas the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the {sigma}D pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the {sigma}D regulon in protein export to the MM. AUTHOR SUMMARYBacteria within the Mycobacteriales order, which includes the pathogen Mycobacterium tuberculosis, have a unique multilayered cell surface architecture. How they sense and respond to defects in the construction of this distinct envelope to maintain homeostasis remains poorly understood. Here, we used the model organism Corynebacterium glutamicum to reveal that the conserved {sigma}D envelope stress response pathway responds to two distinct signals resulting from defects in different envelope layers. The results provide new insight into how members of the Mycobacteriales monitor the integrity of their surface and pave the way for the identification of novel vulnerabilities in envelope biogenesis that may be useful for targeting with antibiotics.

microbiology↗

bioRxiv2020

Devaluation-sensitive responding to preconditioned cues requires orbitofrontal cortex during initial cue-cue learning

The orbitofrontal cortex (OFC) is necessary for value inference in tests of model-based reasoning. This ability could be accounted for by either representation of value or by representation of broader associative structure. Our lab recently reported correlates of both value and of valueless associative structure in OFC using single-unit recording (Sadacca et al., 2018). This incidental stimulus-stimulus representation was surprising since OFC was thought to be involved only when items of biological significance were driving responses. However, we did not assess whether this activity was necessary for encoding the associative information that would contribute to value inference during probe testing. Here, we used optogenetic OFC inhibition during sensory preconditioning to test this. We found that OFC inhibition during preconditioning impaired value inference during the probe test, demonstrating that the correlates we previously observed are not simply downstream readouts of sensory processing and instead contribute to encoding valueless sensory associative information.

neuroscience↗

bioRxiv2020

Varenicline rescues nicotine-induced decrease in motivation for sucrose reinforcement

Varenicline is one of the top medications used for smoking cessation and is often prescribed before termination of nicotine use. The effect of this combined nicotine and varenicline use on the reward system and motivation for primary reinforcement is underexplored. The goal of this study was to assess the effects of nicotine and varenicline on the consumption of sucrose. In Experiment 1, we first assessed the responding for sucrose after pretreatment with nicotine (0, 0.1, or 0.4 mg/kg) and varenicline (0.0, 0.1, 1.0 mg/kg) using a behavioral economics approach. The responding for sucrose was then assessed using a progressive ratio schedule of reinforcement after pretreatment with all possible combinations of nicotine and varenicline doses. In Experiment 2, rats were assessed for the consumption of sucrose in home cages after pretreatment with nicotine and varenicline. We found that a) nicotine decreased economic demand for sucrose, b) varenicline rescued nicotine-induced reduction in economic demand for sucrose, and c) history of varenicline treatment predicted responding for sucrose on a progressive ratio schedule of reinforcement where rats with a history of varenicline treatment responded significantly lower for sucrose across nicotine doses than rats that have not being exposed to varenicline. The results of Experiment 2 largely confirmed that nicotine decreases motivation for sucrose using a passive consumption protocol and that varenicline rescues this effect. Overall, these findings suggest that varenicline interacts with the effects of nicotine by restoring nicotine-induced reduction in motivation for appetitive rewards.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2017

The Healthy Brain Network Biobank: An open resource for transdiagnostic research in pediatric mental health and learning disorders

Technological and methodological innovations are equipping researchers with unprecedented capabilities for detecting and characterizing pathologic processes in the developing human brain. As a result, ambitions to achieve clinically useful tools to assist in the diagnosis and management of mental health and learning disorders are gaining momentum. To this end, it is critical to accrue large-scale multimodal datasets that capture a broad range of commonly encountered clinical psychopathology. The Child Mind Institute has launched the Healthy Brain Network (HBN), an ongoing initiative focused on creating and sharing a biobank of data from 10,000 New York area participants (ages 5-21). The HBN Biobank houses data about psychiatric, behavioral, cognitive, and lifestyle phenotypes, as well as multimodal brain imaging (resting and naturalistic viewing fMRI, diffusion MRI, morphometric MRI), electroencephalography, eye-tracking, voice and video recordings, genetics, and actigraphy. Here, we present the rationale, design and implementation of HBN protocols. We describe the first data release (n = 664) and the potential of the biobank to advance related areas (e.g., biophysical modeling, voice analysis).

neuroscience↗