Biology subjects

Hull, J.

Publications and source records attributed to Hull, J..

5 recordsLinked to original sources

bioRxiv2025

An In Vitro BRAF Activation Assay Elucidates Molecular Mechanisms Driving the Disassembly of the Autoinhibited BRAF State

The RAF kinases (ARAF, BRAF and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-32:MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential. Significance StatementBRAF is a central intermediate in RAS pathway signaling, and its activity is often elevated in human cancers and RASopathy disorders. Due to the complexity of BRAF activation, identifying compounds that sustainably inhibit BRAF function has proven difficult, emphasizing the need for a more comprehensive understanding of BRAF regulation. Here, we have developed an in vitro BRAF activation assay that elucidates key steps in this process. Our findings demonstrate that RAS binding not only recruits BRAF to the plasma membrane but initiates the disassembly of the autoinhibited monomer, which in the context of the membrane, facilitates BRAF dimerization and activation. This assay advances our understanding of BRAF regulation and provides a novel platform for drug discovery efforts targeting BRAF.

biochemistry↗

bioRxiv2025

Spatial and longitudinal tracking of enhancer-AAV vectors that target transgene expression to injured mouse myocardium

Tissue regeneration enhancer elements (TREEs) direct expression of target genes in injured and regenerating tissues. Additionally, TREEs of zebrafish origin were shown to direct the expression of transgenes in border zone regions after cardiac injury when packaged into recombinant AAV vectors and introduced into mice. Future implementation of TREEs into AAV-based vectors as research tools and potential gene therapy modalities will require a more detailed understanding of expression dynamics and potential off-target effects. Here, we applied in vivo bioluminescent imaging to mice systemically injected with AAV vectors containing different combinations of capsids, enhancers, and timing of delivery. Longitudinal tracking of TREE-based expression over time revealed distinct amplitudes and durations of reporter gene expression in the injured heart directed by different TREEs. The liver-de-targeted AAV capsid, AAV.cc84, was able to deliver TREEs either pre- or post-cardiac injury to negate off-target expression in the liver while maintaining transduction in the heart. By screening AAV9-based capsid libraries dosed systemically in mice post-cardiac injury, we discovered a new capsid variant, AAV.IR41, with enhanced transduction at cardiac border zone regions, and with elevated transduction of TREE driven transgenes versus conventional AAV9 vectors. In vivo bioluminescence imaging offers insights into how enhancers and engineered capsids can be implemented to modulate spatiotemporal transgene expression for future targeted therapies.

developmental biology↗

bioRxiv2022

A CMOS-based highly scalable flexible neural electrode interface

Perception, thoughts, and actions are encoded by the coordinated activity of large neuronal populations spread over large areas. Using thin film electrocorticography (ECoG) arrays, this cortical activity has been used to decode speech and individual finger movements, enabling neuroprosthetics, and to localize epileptic foci. However, the connectorization of these multi-thousand channel thin-film arrays to external circuitry is challenging; current state-of-the-art methods are complex, bulky, and unscalable. We address this shortcoming by developing an electrode connector based on an ultra-conformable thin film electrode array that self-assembles onto hard silicon chip sensors, such as microelectrode arrays (MEAs) or camera sensors enabling large channel counts at high density. The interconnects are formed using microfabricated electrode pads suspended by thin support arms, termed flex2chip. Capillary-assisted assembly drives the pads to deform towards the chip surface, and van der Waals forces maintain this deformation, establishing mechanical and Ohmic contact onto individual pixels. We demonstrate a 2200-channel array with a channel density of 272 channels / mm2 connected to the MEA through the flex2chip interconnection method. Thin film electrode arrays connected through the flex2chip successfully measured extracellular action potentials ex vivo. Furthermore, in a transgenic mouse model for absence epilepsy, Scn8a+/-, we observed highly variable propagation trajectories at micrometer scales, even across the duration of a single spike- and-wave discharge (SWD).

bioengineering↗

bioRxiv2022

A spatiotemporal map of the aging mouse brain reveals white matter tracts as vulnerable foci

Aging is the key risk factor for cognitive decline, yet the molecular changes underlying brain aging remain poorly understood. Here, we conducted spatiotemporal RNA-seq of the mouse brain, profiling 1,076 samples from 15 regions across 7 ages and 2 rejuvenation interventions. Our analysis identified a brain-wide gene signature of aging in glial cells, which exhibited spatially defined changes in magnitude. By integrating spatial and single-nucleus transcriptomics, we found that glia aging was particularly accelerated in white matter compared to cortical regions, while specialized neuronal populations showed region-specific expression changes. Rejuvenation interventions, including young plasma injection and dietary restriction, exhibited distinct effects on gene expression in specific brain regions. Furthermore, we discovered differential gene expression patterns associated with three human neurodegenerative diseases, highlighting the importance of regional aging as a potential modulator of disease. Our findings identify molecular foci of brain aging, providing a foundation to target age-related cognitive decline.

neuroscience↗

bioRxiv2019

Stable isotope composition of feathers help track the migratory patterns of Sharp-shinned Hawks (Accipiter striatus) along Western Flyways of North America

The large-scale patterns of movement for the Sharp-shinned Hawk (Accipiter striatus), a small forest hawk found throughout western North America, are largely unknown. However, based on field observations we set out to test the hypothesis that juvenile migratory A. striatus caught along two distinct migration routes on opposite sides of the Sierra Nevada Mountains of North America (Pacific Coast and Intermountain Migratory Flyways) come from geographically different natal populations. We applied stable isotope analysis of hydrogen (H) and oxygen (O) of feathers, and large scale models of spatial isotopic variation (isoscapes) to formulate spatially explicit predictions of the origin of the migrant birds. Novel relationships were assessed between the measured hydrogen and oxygen isotope values of feathers from A. striatus museum specimens of known origin and the isoscape modeled hydrogen and oxygen isotope values of precipitation at those known locations. We used these relationships to predict the origin regions for birds migrating along the two flyways from the measured isotope values of migrants feathers and the associated hydrogen and oxygen isotopic composition of precipitation where these feathers were formed. The birds from the two migration routes had overlap in their natal/breeding origins and did not differentiate into fully separate migratory populations, with birds from the Pacific Coast Migratory Flyway showing broader natal geographic origins then those from the Intermountain Flyway. The methodology based on oxygen isotopes had, in general, less predictive power than the one based on hydrogen. There was broad agreement between the two isotope approaches in the geographic assignment of the origins of birds migrating along the Pacific Coast Flyway, but not for those migrating along the Intermountain Migratory Flyway. These results are discussed in terms of their implications for conservation efforts of A. striatus in western North America, and the use of combined hydrogen and oxygen stable isotope analysis to track the movement of birds of prey on continental scales.

zoology↗