Biology subjects

Powell, H.

Publications and source records attributed to Powell, H..

7 recordsLinked to original sources

bioRxiv2026

STEER-seq: a recombinase based platform for pooled multimodal single-cell perturbation screening

Pooled gain-of-function screening is a powerful method for screening for transcription factors whose overexpression leads to changes in cellular identity. However, variable transgene copy number, integration site and genomic context from random viral integration can confound quantitative comparisons between perturbations. We present STEER-seq (Single-cell Transgene Expression via Exchange Recombination sequencing), a virus-free platform for pooled ORF gain-of-function screening in human induced pluripotent stem cells (hiPSCs). STEER-seq combines irreversible Bxb1-mediated integration at a defined genomic safe-harbour locus with promoterless donor libraries and barcode-enabled perturbation tracking, enabling single-copy expression from a common promoter and genomic context and direct recovery of perturbation identity by single-cell sequencing. In a focused haematopoietic screen, SPI1 expression alone was sufficient to drive differentiation towards HSC/MPP-like progenitors in the absence of exogenous cytokines, with transient induction generating progenitors that retained multilineage haematopoietic differentiation potential. Joint RNA-chromatin accessibility profiling linked transcription factor-induced transcriptional responses to chromatin remodelling and enabled reconstruction of perturbation-induced gene regulatory networks, including a RUNX2-driven osteoblast programme. Scaling STEER-seq to around 2,000 human coding sequences enabled systematic discovery of transcription factors capable of inducing distinct cell-state programmes, including neural progenitor-, melanocyte- and vascular smooth muscle-like states, as well as maintenance of pluripotency. STEER-seq provides a scalable, quantitative framework for single-cell and multimodal gain-of-function screening, enabling functional discovery and engineering of human cell identity.

genomics↗

bioRxiv2025

A spatiotemporal cancer cell trajectory underlies glioblastoma heterogeneity

Cancer cells display highly heterogeneous and plastic states in glioblastoma, an incurable brain tumour. However, how these malignant states arise and whether they follow defined cellular trajectories across tumours is poorly understood. Here, we generated a deep single cell and spatial multi-omic atlas of human glioblastoma that pairs transcriptomic, epigenomic and genomic profiling of 12 tumours across multiple regions. We identify that glioblastoma heterogeneity is driven by spatially-patterned transitions of cancer cells from developmental-like states towards those defined by a glial injury response and hypoxia. This cellular trajectory regionalises tumours into distinct tissue niches and manifests in a molecularly conserved manner across tumours as well as genetically distinct tumour subclones. Moreover, using a new deep learning framework to map cancer cell states jointly with clones in situ, we show that tumour subclones are finely spatially intermixed through glioblastoma tissue niches. Finally, we show that this cancer cell trajectory is intimately linked to myeloid heterogeneity and unfolds across regionalised myeloid signalling environments. Our findings define a stereotyped trajectory of cancer cells in glioblastoma and unify glioblastoma tumour heterogeneity into a tractable cellular and tissue framework.

cancer biology↗

bioRxiv2024

Decoupling of GABA and Glutamine-Glutamate Dynamics and their role in tactile perception: An fMRS Study

Tactile processing is fundamental for our daily lives. In particular, adaptation, the mechanism by which neural (and behavioural) responses change due to repeated stimulation, is key in adjusting our responses to the environment and is often affected in neurodevelopmental conditions such as autism and ADHD. While GABA and glutamate--the main inhibitory and excitatory neurotransmitters-- are known to be fundamental for encoding sensory input, we know little regarding the dynamic responses of the GABA and glutamatergic systems during tactile processing. Here, we examine how GABA and glutamine+glutamate (Glx) in vivo dynamics change during repetitive tactile stimulation and how these changes relate to tactile perception in a healthy population, using functional magnetic resonance spectroscopy (fMRS). Our study showed that repetitive tactile stimulation induced a decoupling between GABA and Glx during the first stimulation blocks as suggested by a negative correlation between GABA and Glx, which changed from a positive correlation at baseline. Subsequently, a multivariate time series analysis showed a predictive temporal relationship between Glx and GABA, showing that changes in these metabolites are temporally linked with an estimated lag of 6 seconds informing on a complex metabolite response function. The absence of gross metabolite change suggests that Glx and GABA adjust in relation to each other in response to repeated tactile stimulation. Furthermore, individual differences in the changed GABA and Glx levels correlated with perceptual measures of touch. Together, our study highlights the complex relationship between GABA and glutamate in tactile processing and demonstrates that experience-dependence plasticity induces a decoupling between these key metabolites. Further study into their dynamic interplay may be key to understanding adaptation as meso-levels in the brain and how these mechanisms differ in neurodevelopmental and neurological conditions.

neuroscience↗

bioRxiv2023

The developmental trajectory of 1H-MRS brain metabolites from childhood to adulthood.

Human brain development is ongoing throughout childhood, with for example myelination of nerve fibres and refinement of synaptic connections continuing until early adulthood. 1H-Magnetic Resonance Spectroscopy (1H-MRS) can be used to quantify the concentrations of endogenous metabolites (e.g., glutamate and {gamma}-aminobutyric acid (GABA)) in the human brain in vivo and so can provide valuable, tractable insight into the biochemical processes that support postnatal neurodevelopment. This can feasibly provide new insight into and aid management of neurodevelopmental disorders by providing chemical markers of atypical development. This study aims to characterize the normative developmental trajectory of various brain metabolites, as measured by 1H-MRS from a midline posterior parietal voxel. We find significant non-linear trajectories for GABA+, Glx, tNAA and tCr concentrations. Glx and GABA+ concentrations steeply decrease across childhood. tNAA concentrations are relatively stable in childhood but gradually decrease from early adulthood, while tCr concentrations increase from childhood to early adulthood. tCho was the only metabolite to have a strictly linear association with age. Trajectories likely reflect fundamental neurodevelopmental processes (including local circuit refinement) which occur from childhood to early adulthood and can be associated with cognitive development; we find GABA+ concentrations significantly positively correlate with recognition memory scores across post-natal development.

neuroscience↗

bioRxiv2021

HA and M2 sequences alter the replication of 2013-16 H1 Live Attenuated Influenza Vaccine Infection in Human Nasal Epithelial Cell cultures

From 2013-2016, the H1N1 component of live, attenuated influenza vaccine (LAIV) performed very poorly in contrast to the inactivated influenza vaccine. We utilized a primary, differentiated human nasal epithelial (hNEC) culture system to assess the replication differences between isogenic LAIVs containing the HA segment from either A/Bolivia/559/2013 (rBol), which showed poor vaccine efficacy, and A/Slovenia/2903/2015 (rSlov), which had restored reasonable vaccine efficacy. While there were minimal differences in infectious virus production in Madin-Darby Canine Kidney (MDCK) cells, the rSlov LAIV showed markedly improved replication in hNEC cultures at both 32{degrees}C and 37{degrees}C, demonstrating that the HA segment alone could impact LAIV replication. The rSlov-infected hNEC cultures showed stronger production of interferon and proinflammatory chemokines which might also be contributing to the increased overall vaccine effectiveness of the rSlov LAIV through enhanced recruitment and activation of immune cells. The introduction of an M2-S86A mutation had no positive effects on H1 LAIV replication in hNEC cultures, in contrast to the increased infectious virus production seen with that mutation in an H3 LAIV. No obvious defects in viral RNA packaging were detected, suggesting the HA function may be driving the differential infectious virus production in hNEC cultures. The use of physiologically relevant temperatures and primary cell cultures demonstrated that candidate LAIVs can replicate efficiently, which is a necessary property for effective vaccines.

microbiology↗

bioRxiv2020

Identification of H3N2 NA and PB1-F2 genetic variants and their association with disease symptoms in the 2014-15 influenza season

The 2014-15 influenza season saw the emergence of an H3N2 antigenic drift variant that formed the 3C.2a HA clade. Whole viral genomes were sequenced from nasopharyngeal swabs of 94 patients with confirmed influenza A virus infection and primary human nasal epithelial cell cultures used to efficiently isolate H3N2 viruses. The isolates were classified by HA clade and the presence of a new set of co-selected mutations in NA (a glycosylation site, NAg+) and PB1-F2 (H75P). The NA and PB1-F2 mutations were present in a subset of clade 3C.2a viruses (NAg+F2P) which dominated during the subsequent influenza seasons. In human nasal epithelial cell cultures, a virus with the novel NAg+F2P genotype replicated less well compared to a virus with the parental genotype. Retrospective analyses of clinical data showed that NAg+F2P genotype viruses were associated with increased cough and shortness of breath in infected patients.

microbiology↗

bioRxiv2020

Neuraminidase antigenic drift of influenza A virus H3N2 clade 3c.2a viruses alters virus replication, enzymatic activity and inhibitory antibody binding

In the 2014-2015 influenza season a novel neuraminidase (NA) genotype emerged in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance (JH CEIRS) surveillance network as well as globally. This novel genotype encoded a glycosylation site at position 245-247 in the NA protein from clade 3c.2a H3N2 viruses. In the years following the 2014-2015 season, this novel NA glycosylation genotype quickly dominated the human H3N2 population of viruses. To assess the effect this novel glycosylation has on virus fitness and antibody binding, recombinant viruses with (NA Gly+) or without (NA Gly-) the novel NA glycosylation were created. Viruses with the 245 NA Gly+ genotype grew to a significantly lower infectious virus titer on primary, differentiated human nasal epithelial cells (hNEC) compared to viruses with the 245 NA Gly-genotype, but growth was similar on immortalized cells. The 245 NA Gly+ blocked human and rabbit monoclonal antibodies that target the enzymatic site from binding to their epitope. Additionally, viruses with the 245 NA Gly+ genotype had significantly lower enzymatic activity compared to viruses with the 245 NA Gly-genotype. Human monoclonal antibodies that target residues near the 245 NA glycosylation were less effective at inhibiting NA enzymatic activity and virus replication of viruses encoding an NA Gly+ protein compared to ones encoding NA Gly-protein. Additionally, a recombinant H6N2 virus with the 245 NA Gly+ protein was more resistant to enzymatic inhibition from convalescent serum from H3N2-infected humans compared to viruses with the 245 NA Gly-genotype. Finally, the 245 NA Gly+ protected from NA antibody mediated virus neutralization. These results suggest that while the 245 NA Gly+ decreases virus replication in hNECs and decreases enzymatic activity, the glycosylation blocks the binding of monoclonal and human serum NA specific antibodies that would otherwise inhibit enzymatic activity and virus replication. Author SummaryInfluenza virus infects millions of people worldwide and leads to thousands of deaths and millions in economic loss each year. During the 2014/2015 season circulating human H3N2 viruses acquired a novel mutation in the neuraminidase (NA) protein. This mutation has since fixed in human H3N2 viruses. This mutation at position 245 through 247 in the amino acid sequence of NA encoded an N-linked glycosylation. Here, we studied how this N-linked glycosylation impacts virus fitness and protein function. We found that this N-linked glycosylation on the NA protein decreased viral replication fitness on human nasal epithelial cells (hNEC) but not immortalized Madin-Darby Canine Kidney (MDCK) cells. We also determined this glycosylation decreases NA enzymatic activity, enzyme kinetics and affinity for substrate. Furthermore, we show that this N-linked glycosylation at position 245 blocks some NA specific inhibitory antibodies from binding to the protein, inhibiting enzymatic activity, and inhibiting viral replication. Finally, we showed that viruses with the novel 245 N-linked glycosylation are more resistant to convalescent human serum antibody mediated enzymatic inhibition. While this 245 N-linked Glycan decreases viral replication and enzymatic activity, the 245 N-linked glycosylation protects the virus from certain NA specific inhibitory antibodies. Our study provides new insight into the function of this dominant H3N2 NA mutation and how it impacts antigenicity and fitness of circulating H3N2 viruses.

microbiology↗