Biology subjects

Egan, C.

Publications and source records attributed to Egan, C..

6 recordsLinked to original sources

bioRxiv2026

Streamlined Production of Recombinant Adeno-Associated Viruses Using Gateway Cloning Technology for Neural Circuit All-Optical Interrogation

The advent of optogenetics and the rapid development of genetically encoded actuators and reporters for calcium, voltage, neurotransmitters, and other molecules have revolutionized neuroscience research by enabling precise, non-invasive optical interrogation of neural circuits. Successful implementation, however, critically depends on efficient reporter expression in defined neuronal populations. We present a Gateway(R)-based cloning platform providing a robust pipeline for rapid, efficient construction of recombinant adeno-associated virus (rAAV) vectors optimized for neuroscience applications. This platform addresses promoter selection, indicator engineering, and capsid-type optimization, and enables systematic optimization of vector components across a range of optical tools. The benefit of this modular approach is illustrated in the case of ULoVE, a two-photon microscopy method based on acousto-optic deflectors (AODs). This method provides serial light-targeting with kHz sampling rates and high signal-to-noise ratio in vivo, which imposes stringent requirements on indicator expression -- including cell-type specificity, sparse labelling, and precise control of expression levels-- efficiently met through the combinatorial flexibility of the Gateway pipeline. As specific examples, Gateway-constructed Cre-driver viruses combined with Cre-dependent reporters enabled cell-type-specific labelling for two complementary applications. In cerebellar Purkinje cells, an L7::Cre driver virus paired with a Gateway-constructed voltage indicator (JEDI2P-Kv) and ULoVE two-photon excitation at ~5 kHz enables resolution of sub-millisecond dendritic voltage dynamics in awake, behaving mice, including optical detection of dendritic spikelets previously accessible only via intracellular electrophysiology. The same driver virus paired with calcium indicators (GCaMP6f, jRGECO1a) resolves climbing fiber-evoked calcium kinetics under the same conditions. In acute cerebellar slices, a kit::Cre driver virus enabled cell-type-specific expression of the optogenetic actuator ChR2(H134R) in molecular layer interneurons, where ULoVE doughnut-pattern photostimulation achieved single-cell, sub-millisecond optogenetic activation with micron-scale spatial resolution. Beyond ULoVE applications, the same sparse, strong labelling also proved suitable for anatomical tracing of axonal projections in cleared cerebellar tissue, resolving individual DCN axon terminals at the granule cell layer --molecular layer boundary. Taken together, our dual-front approach --combining a modular AAV expression pipeline with AOD-based optical acquisition --provides an integrated platform for developing sophisticated optical tools, including but not limited to AOD-based microscopy, for neural circuit interrogation.

neuroscience↗

bioRxiv2024

Disconnection Between Microvascular Damage and Neurodegeneration in Early Diabetic Retinopathy

AimDiabetic retinopathy (DR) is a common complication of diabetes mellitus and can result in vision loss. Early clinically diagnosed signs of DR are linked to vascular damage, impacting on the neural retina typically at later stages. However, vascular changes and potential effects on neural cells before clinical diagnosis of DR are less understood. MethodsTo learn more about the earliest stages of DR we studied postmortem retina from diabetic donors who did not have clinical DR. Histological phenotyping and quantitative analysis were carried out on retina from 14 donors (3 controls, 10 diabetics, and 1 DR case) to examine capillary loss in the deeper vascular plexus (DVP) and the superficial vascular plexus (SVP) of the retinal vasculature and to study effects on the neural retina. ResultThe advanced DR case exhibited profound vascular and neural damage as expected, whereas none of the ten randomly selected diabetic donors had any DR signs that would have been diagnosed clinically. Within that group, two showed very minor capillary dropout in the SVP, whilst in the remaining diabetic cases the SVP was indistinguishable from the controls. In contrast, over half of the diabetic retinas showed capillary dropout in the DVP and increased capillary diameter. Furthermore, a pan retinal loss of inner nuclear layer cells was observed in those diabetics with capillary dropout compared to the controls (p<0.05), but there was no local spatial correlation between neural cell loss and capillary dropout. ConclusionsOur study has established a novel histological biomarker for diabetes related tissue damage at the earliest stages of DR in human postmortem retina, which appears to be common in people with diabetes before DR can be clinically diagnosed. Furthermore, the spatial mismatch between local capillary dropout and the diffuse neural loss in the INL suggests that at this very early stage of DR, microvascular loss may not causally be directly connected to neurodegeneration and that diabetes may affect the two readouts independently. RESEARCH IN CONTEXTWhat is already known about this subject? O_LIEarly DR is currently clinically defined by visible microvascular changes. C_LIO_LITests of retinal function in people with diabetes imply neuroretinal impairments in early DR. C_LIO_LIClinical imaging in people with diabetes suggests reduced perfusion in the deep retinal vasculature plexus. C_LI What is the key question? (one bullet point only; formatted as a question) O_LIHow does retinal microvascular damage relate to neuronal damage? C_LI What are the new findings? O_LIWe have developed a sensitive histological biomarker for microvascular damage in postmortem retinal tissue. C_LIO_LIEarly vascular changes in the deeper plexus occur in diabetes even in the absence of manifest DR. C_LIO_LINeurodegenerative changes are distributed throughout the retina in diabetes and do not spatially correlate with microvascular nonperfusion. C_LI How might this impact on clinical practice in the foreseeable future? O_LIDeeper plexus perfusion can be a useful early biomarker to assess DR. C_LI

pathology↗

bioRxiv2022

International spread of emerging multidrug-resistant Rhodococcus equi

The recently characterized multidrug-resistant clone of the animal and human zoonotic pathogen Rhodococcus equi, MDR-RE 2287, has been circulating among horse farms in the United States (US) since the early 2000s. Here, we report the first documented detection of the MDR-RE 2287 clone outside the US. The finding highlights the risk of MDR-RE spreading internationally with the movement of horses.

microbiology↗

bioRxiv2020

Automated detection of glaucoma with interpretable machine learning using clinical data and multi-modal retinal images

Glaucoma, the leading cause of irreversible blindness worldwide, is a disease that damages the optic nerve. Current machine learning (ML) approaches for glaucoma detection rely on features such as retinal thickness maps; however, the high rate of segmentation errors when creating these maps increase the likelihood of faulty diagnoses. This paper proposes a new, comprehensive, and more accurate ML-based approach for population-level glaucoma screening. Our contributions include: (1) a multi-modal model built upon a large data set that includes demographic, systemic and ocular data as well as raw image data taken from color fundus photos (CFPs) and macular Optical Coherence Tomography (OCT) scans, (2) model interpretation to identify and explain data features that lead to accurate model performance, and (3) model validation via comparison of model output with clinician interpretation of CFPs. We also validated the model on a cohort that was not diagnosed with glaucoma at the time of imaging but eventually received a glaucoma diagnosis. Results show that our model is highly accurate (AUC 0.97) and interpretable. It validated biological features known to be related to the disease, such as age, intraocular pressure and optic disc morphology. Our model also points to previously unknown or disputed features, such as pulmonary capacity and retinal outer layers.

bioengineering↗

bioRxiv2020

Trait-based aerial dispersal of arbuscular mycorrhizal fungi

O_LIDispersal is a key process driving local-scale community assembly and global-scale biogeography of plant symbiotic arbuscular mycorrhizal (AM) fungal communities. A trait-based approach could improve predictions regarding how AM fungal aerial dispersal varies by species. C_LIO_LIWe conducted month-long collections of aerial AM fungi for 12 consecutive months in an urban mesic environment at heights of 20 m. We measured functional traits of all collected spores and assessed aerial AM fungal community structure both morphologically and with high-throughput sequencing. C_LIO_LILarge numbers of AM fungal spores were present in the air over the course of one year and these spores were more likely to exhibit traits that facilitate dispersal. Aerial spores were smaller than average for Glomeromycotinan fungi. Trait-based predictions indicate that nearly 1/3 of described species from diverse genera demonstrate the potential for aerial dispersal. Diversity of aerial AM fungi was relatively high (20 spore species and 17 virtual taxa) and both spore abundance and community structure shifted temporally. C_LIO_LIThe prevalence of aerial dispersal in arbuscular mycorrhizas is perhaps greater than previously indicated and a hypothesized model of AM fungal dispersal mechanisms is presented. Anthropogenic soil impacts may initiate the dispersal of disturbance-tolerating AM fungal species and facilitate community homogenization. C_LI

ecology↗

bioRxiv2019

Plant-microbe specificity varies as a function of elevation

Specialized associations between interacting species are a fundamental determinant of the diversity and distribution of both partners. How specialization of guilds of organisms varies along environmental gradients underpins popular theories of biogeography and macroecology, whereas the degree of specialization of a species is typically considered fixed. However, the extent to which environmental context impacts specialization dynamics is seldom examined empirically. In this study, we examine how specialization within a bipartite network consisting of three co-occurring plant species and their foliar fungal endophyte symbionts changes along a 1 000-meter elevation gradient where host species were held constant. The gradient, along the slope of Mauna Loa shield volcano, represents the entire elevational range of two of the three plants. Network and plant specialization values displayed a parabolic relationship with elevation, and were highest at middle elevations, whereas bipartite associations were least specific at low and high elevations. Shannons diversity of fungal endophytes negatively correlated with specificity, and was highest at the ends of the transects. Although plant host was a strong determinant of fungal community composition within sites, fungal species turnover was high among sites and plant host predicted a weak, though significant proportion of compositional variance. There was no evidence of spatial or elevational patterning in fungal community compositon. Our work demonstrates that specificity can be a plastic trait which is influenced by the environment and centrality of the host within its natural range.

ecology↗