Biology subjects

Simmons, T.

Publications and source records attributed to Simmons, T..

5 recordsLinked to original sources

bioRxiv2026

Tiled base editing screens uncover rare second-site mutations that rescue a misfolded disease protein

Therapeutic base editing can directly revert ~30% of disease-causing missense mutations to the wild-type sequence. One strategy to broaden its reach is to install second-site corrector mutations that improve protein stability or function across multiple pathogenic alleles. Here, we use tiled adenine and cytosine base editing screens to identify such corrector mutations in NPC1, a lysosomal cholesterol transporter whose misfolding causes Niemann-Pick type C disease. Tiling mutagenesis of the endogenous NPC1 locus in haploid human cells carrying the prevalent NPC1I1061T disease allele, combined with phenotypic reporters for cholesterol transport and protein abundance, uncovered rare intragenic suppressor mutations distributed across multiple domains. Unexpectedly, we identified a promiscuous second-site variant that can correct many different pathogenic NPC1 mutations. These correctors improve protein stability, promote ER exit and lysosomal trafficking, and restore cholesterol clearance in patient-derived fibroblasts and induced neurons. Our results establish in situ base editing screens for the discovery of second-site corrector mutations that are actionable for therapeutic base editing when the original mutation cannot be corrected.

cell biology↗

bioRxiv2026

Antibiotic Persistence Emerges from Cell-State-Driven Transcriptional Reprogramming

Antibiotic persistence allows a subpopulation of bacterial cells to survive antibiotic treatment without acquiring resistance mutations, often contributing to treatment failure. Although many genes have been linked to persistence, their deletion rarely abolishes the phenotype, highlighting redundancy in persistence mechanisms. Moreover, the transient and heterogeneous nature of persister formation has made it difficult to resolve its molecular basis using bulk analyses. Here, we use bacterial single-cell RNA sequencing and functional assays in Klebsiella pneumoniae to demonstrate how transcriptional heterogeneity and redundancy in stress responses shape persistence outcomes. Using growth phase as a biologically meaningful axis of transcriptional variation, we reveal that even within an isogenic population, distinct transcriptional responses can be induced and co-contribute to survival. These responses are shaped by the cells pre-treatment transcriptional state and the mechanism of antibiotic action. Genetic and environmental perturbations, such as rpoS deletion and nutrient supplementation, shift pre-treatment cell states and alter persistence frequencies. Our findings establish the biological significance of transcriptional heterogeneity shaped by pre-treatment cell states, providing a systems-level framework for understanding persistence and suggesting strategies to enhance antibiotic efficacy by modulating cell states.

microbiology↗

bioRxiv2026

Understanding heterogeneous responses to T cell engagers: Binding characteristics and dosing thresholds determine cytotoxic efficacy

Therapeutic efficacy of multivalent T cell engagers varies widely across individuals, but the basis for this heterogeneity remains poorly understood. Here, we integrate in vitro experiments of antitumor immune responses with a mechanistic modeling framework to investigate sources of response variability across T cell donors and TE constructs, focusing on a novel multivalent bispecific T cell engager currently in development. We identify parameter regimes that accurately recapitulate dose-response behaviors across T cell donors and doses, and perform cross-validation studies that demonstrate the models predictive accuracy. We find that variability in therapy efficacy is governed by the relationship between binding affinity and dose. When dose exceeds the binding affinity, responses are relatively robust across donors; when dose is below the binding affinity, responses are more donor dependent. At smaller doses, the TE-specific shape characteristics of the tumor-binding dose response, its steepness in particular, is a key marker of therapy efficacy. More generally, our integrated modeling and experimental framework offers insights and tools that are applicable to other bispecific T-cell engagers, and provides a quantitative foundation for the systematic, in silico optimization of TE design and dosing strategies.

immunology↗

bioRxiv2024

Cholesterol inhibits oncogenic EphA2 activation

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

biochemistry↗

bioRxiv2022

Parallel evolution and cryptic diversification in the common and widespread Amazonian tree, Protium subserratum

The lowland Amazon rainforest houses some of the greatest tree diversity on earth. While the vast majority of these species are rare, a small number are common and widespread and thus considered to play a disproportionate role in many of the global ecosystem services provided by the Amazon. However, the extent to which dominant Amazonian tree species actually include multiple clades, each on their own unique evolutionary trajectory, is unknown. Here we investigate the extent to which lineage divergence may be occurring within Protium subserratum (Burseraceae), a common and widespread tree species that is monophyletic with populations exhibiting genotypic and phenotypic differences associated with soil and geography. Utilizing a combination of phylogenomic and population genomic methods with sampling from across the range, we found that P. subserratum contains at least eight distinct clades. Specialization onto white-sand soils has evolved independently at least two times within the species; however, phenotype is not correlated with soil type. Finally, cryptic diversity at the base of the Andes is associated with elevational shifts. Together these results lend support to the hypothesis that common and widespread Amazon tree species may not represent evolutionary cohesive units. Instead, these dominant species may more commonly represent species complexes, undergoing evolutionary transitions on a trajectory to become multiple range restricted, specialist species.

evolutionary biology↗