Biology subjects

Phillips, W.

Publications and source records attributed to Phillips, W..

6 recordsLinked to original sources

bioRxiv2024

Cell invasive amyloid assemblies from SARS-CoV-2 peptides can form multiple polymorphs with varying neurotoxicity

The neurological symptoms of COVID-19, such as memory loss, cognitive and sensory disruption (neuro-COVID) are well reported. These neurological symptoms frequently persist for months (post-acute sequalae of COVID-19 or PASC). The molecular origins of neuro-COVID and how it contributes to PASC are unknown, however a growing body of research highlights that the self-assembly of protein fragments from SARS-CoV-2 into amyloid nanofibrils may play a causative role. Previously, we identified two fragments from the proteins Open Reading Frame 6 (ORF6) and ORF10 that self-assemble into neurotoxic amyloid assemblies. Here we further our understanding of the self-assembly mechanisms and nano-architectures formed by these fragments as well as performing a more in-depth study of the biological responses of co-cultured neurons. By solubilising the peptides in a fluorinated solvent we eliminate insoluble aggregates in the starting materials (seeds) that change the polymorphic landscape of the assemblies. The resultant assemblies are dominated by structures with higher free energies (e.g. ribbons and amorphous aggregates) that are less toxic to cultured neurons. We also show the first direct evidence of cellular uptake by viral amyloids. This work highlights the importance of understanding the polymorphic behaviour of amyloids particularly in the context of neuro-COVID and PASC. Graphical Abstract for ToCThe neurological symptoms of COVID-19 are likely to be, in part, caused by the aggregation of viral proteins into neurotoxic amyloid nanofibrils. Changes in aggregation conditions alters the balance of fibril structures formed (polymorphism), influencing their toxicity to a neuronal cell line. These findings increase our understanding of viral amyloids and highlight the importance of careful choice of experimental protocol when studying these systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/594465v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@68f505org.highwire.dtl.DTLVardef@ae3624org.highwire.dtl.DTLVardef@164a1f1org.highwire.dtl.DTLVardef@1c3f9d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

CRISPR/Cas9 gene editing to generate Drosophila LexA lines in secondary school classes

Genome editing in vivo with CRISPR/Cas9 generates powerful tools to study gene regulation and function. We developed CRISPR-based methods that permitted secondary school student scientists to convert Drosophila GAL4 lines to LexA lines. Our novel curricula implement a new donor strain optimizing Homology-assisted CRISPR knock-in (HACK) that simplifies screening using light microscopy. Successful curricula adoption by a consortium of schools led to the generation and characterization of 16 novel LexA lines. This includes extensive comparative tissue expression analysis between the parental Gal4 and derived LexA lines. From this collaboration, we established a workflow to systematically generate LexA lines from frequently-used GAL4 lines. Modular courses developed from this effort can be tailored to specific secondary school scheduling needs, and serve as a template for science educators to innovate courses and instructional goals. Our unique collaborations highlight that resources and expertise harnessed by university-based research laboratories can transform experiential science instruction in secondary schools while addressing research needs for the community of science.

genetics↗

bioRxiv2022

Comprehensive multiomic profiling of somatic mutations in malformations of cortical development

Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, and in promoting neuronal hyperexcitability after birth.

genetics↗

bioRxiv2020

Characterization of gene repression by designed transcription activator-like effector dimer proteins

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the E. coli lac operon switch, where DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed Transcription Activator-Like Effector Dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system. Statement of SignificanceWe are designing and testing in living bacteria artificial DNA looping proteins engineered based on principles learned from studies of the E. coli lac repressor. The engineered proteins are based on artificial dimers of Transcription Activator-Like Effector (TALE) proteins that have programmable DNA binding specificities. The current work is the first to create unique DNA repression loops using this approach. Systematic study of repression as a function of loop size, with data fitting to a thermodynamic model, now allows this system to be compared in detail with lac repressor loops, and relevant biophysical parameters to be estimated. This approach has implications for the artificial regulation of gene expression.

biophysics↗

bioRxiv2019

A Bayesian decomposition of BAC firing as a mechanism for apical amplification in neocortical pyramidal neurons

Pyramidal cells in layer 5 of the neocortex have two distinct integration sites. These cells integrate inputs to basal dendrites in the soma while integrating inputs to the tuft in a site at the top of the apical trunk. The two sites communicate by action potentials that backpropagate to the apical site and by backpropagation-activated calcium spikes (BAC firing) that travel from the apical to the somatic site. Six key messages arise from the probabilistic information-theoretic analyses of BAC firing presented here. First, we suggest that pyramidal neurons with BAC firing could convert the odds in favour of the presence of a feature given the basal data into the odds in favour of the presence of a feature given the basal data and the apical input, by a simple Bayesian calculation. Second, the strength of the cells response to basal input can be amplified when relevant to the current context, as specified by the apical input, without corrupting the message that it sends. Third, these analyses show rigorously how this apical amplification depends upon communication between the sites. Fourth, we use data on action potentials from a very detailed multi-compartmental biophysical model to study our general model in a more realistic setting, and demonstrate that it describes the data well. Fifth, this form of BAC firing meets criteria for distinguishing modulatory from driving interactions that have been specified using recent definitions of multivariate mutual information. Sixth, our general decomposition can be extended to cases where, instead of being purely driving or purely amplifying, apical and basal inputs can be partly driving and partly amplifying to various extents. These conclusions imply that an advance beyond the assumption of a single site of integration within pyramidal cells is needed, and suggest that the evolutionary success of neocortex may depend upon the cellular mechanisms of context-sensitive selective amplification hypothesized here.\n\nAuthor summaryThe cerebral cortex has a key role in conscious perception, thought, and action, and is predominantly composed of a particular kind of neuron: the pyramidal cells. The distinct shape of the pyramidal neuron with a long dendritic shaft separating two regions of profuse dendrites allows them to integrate inputs to the two regions separately and combine the results non-linearly to produce output. Here we show how inputs to this more distant site strengthen the cells output when it is relevant to the current task and environment. By showing that such neurons have capabilities that transcend those of neurons with the single site of integration assumed by many neuroscientists, this splitting of the neuronal atom offers a radically new viewpoint from which to understand the evolution of the cortex and some of its many pathologies. This also suggests that approaches to artificial intelligence using neural networks might come closer to something analogous to real intelligence, if, instead of basing them on processing elements with a single site of integration, they were based on elements with two sites, as in cortex.

neuroscience↗

bioRxiv2017

PI3K Activation In Neural Stem Cells Drives Tumorigenesis Which Can Be Suppressed By Targeting CREB

Hyperactivation of the PI3K signaling is common in human cancers, including gliomas, but the precise role of the pathway in glioma biology remains to be determined. Some limited understanding of PI3K signaling in brain cancer come from studies on neural stem/progenitor cells (NSPCs) where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate NSPC proliferation. To investigate the role for the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a Pik3caH1047A oncogenic mutation and simultaneous deletion of the PI3K negative regulator, Pten, in NSPCs. We show that the expression of a Pik3caH1047A was sufficient to initiate tumorigenesis but that simultaneous loss of Pten, was required for the development of invasive, high-grade glioma. Mutant NSPCs exhibited enhanced neurosphere forming capacity which correlated with increased Wnt signaling. We also show that loss of CREB in Pik3ca-PTEN tumors led to a longer symptom-free survival in mice. Taken together, our findings present a novel mouse model for high-grade glioma with which we demonstrate that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.

cancer biology↗