Biology subjects

Lyons, A. C.

Publications and source records attributed to Lyons, A. C..

4 recordsLinked to original sources

bioRxiv2026

Dynamic visualization of physiological CaMKII activity using sensitive FRET biosensors

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key mediator of complex physiological processes throughout the body, from the brain to the reproductive system, where CaMKII translates spatiotemporally dynamic calcium elevations into specific biological functions. Directly visualizing CaMKII activity dynamics in living cells using genetically encoded fluorescent biosensors can thus provide crucial insights into the molecular regulation of health and disease. Yet the ability to sensitively and specifically monitor endogenous CaMKII activity in physiologically relevant contexts is limited by the lack of sensors that can achieve robust, quantitative visualization of CaMKII responses. Here, we leveraged a recent serine/threonine kinome-wide substrate atlas to rationally engineer a powerful suite of Forster resonance energy transfer (FRET)-based CaMKII kinase activity reporters with high specificity, sensitivity, and signal-to-noise ratio. Using these biosensors, we were able to sensitively and robustly visualize endogenous CaMKII activity dynamics in both cultured cell lines and primary cells, including cardiomyocytes, oocytes, and neurons. We further utilized 2pFLIM imaging of organotypic hippocampal slices to quantitatively track LTP-induced CaMKII activity within single dendritic spines, highlighting a major advance in the study of physiological CaMKII signaling.

bioengineering↗

bioRxiv2024

An Infusible Extracellular Matrix Biomaterial Improves Survival in a Model of Severe Systemic Inflammation

Excess systemic inflammation can often be lethal in septic and trauma patients due to onset of multiple organ dysfunction syndrome (MODS). As of right now, there are no effective immunomodulatory therapeutics that can promote survival within this patient population. Pro-regenerative extracellular matrix (ECM) biomaterials have shown success for treatment of local inflammation but have not been fully explored for treating systemic inflammation. Here, we demonstrate efficacy of an intravenously delivered infusible ECM (iECM) material, which promotes increased survival in a murine model of MODS by decreasing systemic mediators of inflammation. Lung and kidney failure are associated with higher mortality in MODS compared to other organ failures, and we demonstrate that iECM localizes primarily to kidney and lung tissues during systemic inflammation induced by endotoxin. iECM successfully lowered vascular permeability within lung tissue and lowered levels of inflammatory cytokine signaling such as IL-6, verified via ELISA and gene expression analyses. We also demonstrated that immune cell infiltration into lung tissue was modulated with iECM treatment, with an increase in neutrophil retention in the lung and decreases in pro-inflammatory macrophage presence. In summation, iECM improves survival from severe systemic inflammation by decreasing the local and systemic inflammatory signaling pathways that contribute to MODS. These results provide a strong rationale for translational studies of iECM treatment in systemic inflammatory syndromes, including sepsis and trauma.

bioengineering↗

bioRxiv2024

Far-red chemigenetic biosensors for multi-dimensional and super-resolved kinase activity imaging

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

bioengineering↗

bioRxiv2020

Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that the simple inclusion of bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) in the fibrinogen solvent slows the degradation rate of fibrin by providing additional crosslinking. In this study, we aimed to further improve the longevity of fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. In this study, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.

bioengineering↗