Catalytic Olefin Transpositions Facilitated by Ruthenium N,N,N-Pincer Complexes.

In this report, we demonstrate olefin transposition/isomerization reactions catalyzed by a series of N,N,N-pincer (1,3-bis(2-pyridylimino)isoindoline) Ru-hydride complexes. The protocol proceeds at room temperature for most substrates, achieving excellent yields, regioselectivity, and diastereoselectivity in short reaction times. The air-stable Ru-chloride derivatives of these complexes exhibit comparable reactivity enabling benchtop setup and synthetic versatility. Furthermore, we demonstrate the potential for one-pot cascade sequences of the products derived from the transposition reactions.

MEM-based pangenome indexing for k-mer queries.

Pangenomes are growing in number and size, thanks to the prevalence of high-quality long-read assemblies. However, current methods for studying sequence composition and conservation within pangenomes have limitations. Methods based on graph pangenomes require a computationally expensive multiple-alignment step, which can leave out some variation. Indexes based on k-mers and de Bruijn graphs are limited to answering questions at a specific substring length k. We present Maximal Exact Match Ordered (MEMO), a pangenome indexing method based on maximal exact matches (MEMs) between sequences. A single MEMO index can handle arbitrary-length queries over pangenomic windows. MEMO enables both queries that test k-mer presence/absence (membership queries) and that count the number of genomes containing k-mers in a window (conservation queries). MEMO's index for a pangenome of 89 human autosomal haplotypes fits in 2.04 GB, 8.8× smaller than a comparable KMC3 index and 11.4× smaller than a PanKmer index. MEMO indexes can be made smaller by sacrificing some counting resolution, with our decile-resolution HPRC index reaching 0.67 GB. MEMO can conduct a conservation query for 31-mers over the human leukocyte antigen locus in 13.89 seconds, 2.5x faster than other approaches. MEMO's small index size, lack of k-mer length dependence, and efficient queries make it a flexible tool for studying and visualizing substring conservation in pangenomes.

Architectural Editing of Polyesters and Polyurethanes via Palladium(II)-Catalyzed [3,3]-Sigmatropic Oxo-Rearrangements.

Architecture underlies the thermomechanical properties of polymers. Yet, few strategies are available to tune a polymer's architecture after it is prepared without altering its chemical composition. The ability to edit the architecture of a polymer would dramatically expand the accessible architecture-property space of polymeric materials. Herein, we disclose a backbone rearrangement approach to tune the short-chain branching of polymers. Specifically, we demonstrate that palladium(II)-catalyzed [3,3]-sigmatropic oxo-rearrangements can transform branched polyesters and polyurethanes to their linear counterparts. While the effects on materials properties are generally subtle in the case of polyesters, more dramatic changes are observed in the case of polyurethanes: two polyurethanes undergo a soluble-to-insoluble transition, and one exhibits a dramatic increase in both strain at break and toughness after rearrangement. Additionally, the incorporation of alkenes in the polymer backbone through the rearrangement enables facile deconstruction via ethenolysis. In all, we disclose a powerful and broad-scope strategy to edit the architecture of polymer backbones and thereby tune their physical and chemical properties.

Appended Lewis Acids Enable Dioxygen Reactivity and Catalytic Oxidations with Ni(II).

We disclose a suite of Ni(II) complexes featuring secondary sphere Lewis acids of varied Lewis acidity and tether lengths. Several of these complexes feature atypical behavior of Ni(II): reactivity with O2 that occurs only in the presence of a tethered Lewis acid. In situ UV-vis spectroscopy revealed that, although adducts are stable at -40 °C, complexes containing 9-borabicyclo[3.3.1]nonane (9-BBN) Lewis acids underwent irreversible oxidative deborylation when warmed to room temperature. We computationally and experimentally identified that oxidative instability of appended 9-BBN moieties can be mitigated using weaker Lewis acids such as pinacolborane (BPin). These insights enabled the realization of catalytic reactions: hydrogen atom abstraction from phenols and room temperature oxygen atom transfer to PPh3.

NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development.

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generated retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing (scRNA-Seq) across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNA-Seq data, we reconstruct the rod photoreceptor developmental lineage and identify a branch point unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different from those observed in Nr2e3 rodent models. Together, these data provide a road map of human photoreceptor development and leverage patient induced pluripotent stem cells to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

Iron-Catalyzed C-H Oxygenation Using Perchlorate Enabled by Secondary Sphere Hydrogen Bonds.

Perchlorate (ClO4-) is a groundwater pollutant that is challenging to remediate. We report a strategy to use Fe(II) tris(2-pyridylmethyl)amine (TPA) complexes featuring appended aniline hydrogen bonds (H-bonds) to promote ClO4- reduction. These complexes facilitate oxygen atom transfer from ClO4- to PPh3 and C-H oxygenation reactions of organic substrates. Catalytic reactions using 15 mol % afforded excellent yields for oxygenation of anthracene and cyclic alkyl aromatics, and this methodology tolerates aryl halides as well as heterocycles containing either O, S, or N.

A metal-free strategy to construct fluoroalkyl-olefin linkages using fluoroalkanes.

We present a metal-free strategy to access fluoroalkyl-olefin linkages from fluoroalkane precursors and vinyl-pinacol boronic ester (BPin) reagents. This reaction sequence is templated by the boron reagent, which induces C-C bond formation upon oxidation. We developed this strategy into a one-pot synthetic protocol using RCF2H precursors directly with vinyl-BPin reagents in the presence of a Brønsted base, which tolerated oxygen- and nitrogen-containing heterocycles, and aryl halogens. We also found that HCF3 (HCF-23; a byproduct of the Teflon industry) and CH2F2 (HCF-32; a low-cost refrigerant) are amenable to this protocol, representing distinct strategies to generate RCF2H and RCF3 molecules. Finally, we demonstrate that the vinyldifluoromethylene products can be readily derivatized, representing an avenue for late-stage modification after installing the fluoroalkyl unit.

Movi: a fast and cache-efficient full-text pangenome index.

Efficient pangenome indexes are promising tools for many applications, including rapid classification of nanopore sequencing reads. Recently, a compressed-index data structure called the "move structure" was proposed as an alternative to other BWT-based indexes like the FM index and r-index. The move structure uniquely achieves both O(r) space and O(1)-time queries, where r is the number of runs in the pangenome BWT. We implemented Movi, an efficient tool for building and querying move-structure pangenome indexes. While the size of the Movi's index is larger than the r-index, it scales at a smaller rate for pangenome references, as its size is exactly proportional to r, the number of runs in the BWT of the reference. Movi can compute sophisticated matching queries needed for classification - such as pseudo-matching lengths and backward search - at least ten times faster than the fastest available methods, and in some cases more than 30-fold faster. Movi achieves this speed by leveraging the move structure's strong locality of reference, incurring close to the minimum possible number of cache misses for queries against large pangenomes. We achieve still further speed improvements by using memory prefetching to attain a degree of latency hiding that would be difficult with other index structures like the r-index. Movi's fast constant-time query loop makes it well suited to real-time applications like adaptive sampling for nanopore sequencing, where decisions must be made in a small and predictable time interval.

CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.

Rhodopsin (RHO) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOpCRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

Elucidating the impact of bacterial lipases, human serum albumin, and FASII inhibition on the utilization of exogenous fatty acids by Staphylococcus aureus.

IMPORTANCE: Incorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that glycerol ester hydrolase (Geh) is the primary lipase hydrolyzing cholesteryl esters and, to a lesser extent, triglycerides and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs but high levels of HSA inhibit it. The fact that the type II fatty acid synthesis (FASII) inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane properties, respectively.

Gene Expression Within a Human Choroidal Neovascular Membrane Using Spatial Transcriptomics.

Purpose: Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process. Methods: To study how gene expression is altered in focal areas of pathology, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We performed differential expression to identify genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes. Results: Within the area of neovascularization, endothelial cells demonstrated increased expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we investigated regional gene expression patterns within the macular neural retina and between the macular and peripheral choroid. Conclusions: Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

Differences in Life Space Activity Patterns Between Older Adults With Mild Cognitive Impairment Living Alone or as a Couple: Cohort Study Using Passive Activity Sensing.

BACKGROUND: Measuring function with passive in-home sensors has the advantages of real-world, objective, continuous, and unobtrusive measurement. However, previous studies have focused on 1-person homes only, which limits their generalizability. OBJECTIVE: This study aimed to compare the life space activity patterns of participants living alone with those of participants living as a couple and to compare people with mild cognitive impairment (MCI) with cognitively normal participants in both 1- and 2-person homes. METHODS: Passive infrared motion sensors and door contact sensors were installed in 1- and 2-person homes with cognitively normal residents or residents with MCI. A home was classified as an MCI home if at least 1 person in the home had MCI. Time out of home (TOOH), independent life space activity (ILSA), and use of the living room, kitchen, bathroom, and bedroom were calculated. Data were analyzed using the following methods: (1) daily averages over 4 weeks, (2) hourly averages (time of day) over 4 weeks, or (3) longitudinal day-to-day changes. RESULTS: In total, 129 homes with people living alone (n=27, 20.9%, MCI and n=102, 79.1%, no-MCI homes) and 52 homes with people living as a couple (n=24, 46.2%, MCI and n=28, 53.8%, no-MCI homes) were included with a mean follow-up of 719 (SD 308) days. Using all 3 analysis methods, we found that 2-person homes showed a shorter TOOH, a longer ILSA, and shorter living room and kitchen use. In MCI homes, ILSA was higher in 2-person homes but lower in 1-person homes. The effects of MCI status on other outcomes were only found when using the hourly averages or longitudinal day-to-day changes over time, and they depended on the household type (alone vs residing as a couple). CONCLUSIONS: This study shows that in-home behavior is different when a participant is living alone compared to when they are living as a couple, meaning that the household type should be considered when studying in-home behavior. The effects of MCI status can be detected with in-home sensors, even in 2-person homes, but data should be analyzed on an hour-to-hour basis or longitudinally.

Serratia marcescens Outbreak at a Correctional Facility: Environmental Sampling, Laboratory Analyses and Genomic Characterization to Assess Sources and Persistence.

Serratia marcescens is an environmental bacterium and clinical pathogen that can cause an array of infections. We describe an environmental sampling and comparative genomics approach used to investigate a multi-year outbreak of S. marcescens at a correctional facility. Whole genome sequencing analysis revealed a predominant cluster of clonally related S. marcescens from nine patient cases and items associated with illicit drug use. Closely related strains found among items associated with case-patient cells and diluted Cell Block 64 (CB64), a quaternary ammonium disinfectant, and Break Out (BO), a multipurpose cleaner, highlighted their role as environmental reservoirs for S. marcescens in this outbreak. Comparative genomic analysis suggested outbreak strains were both persistent (identical strains found over long periods and in multiple locations of the correctional facility) and diverse (strains clustered with multiple global samples from NCBI database). No correlation was found between antimicrobial resistance (AMR) genes of outbreak strains; NCBI strains have more AMR genes. Principal component analysis (PCA) of virulence factors associated with persistence and infectivity indicated variation based on phylogroups, including the predominant cluster; identifiable variations among environmental versus clinical strains were not observed. Identification of multiple distinct genetic groups highlights the importance of putting epidemiological genomic studies in a proper genetic context.

Elucidating the Impact of Bacterial Lipases, Human Serum Albumin, and FASII Inhibition on the Utilization of Exogenous Fatty Acids by Staphylococcus aureus.

Staphylococcus aureus only synthesizes straight-chain or branched-chain saturated fatty acids (SCFAs or BCFAs) via the type II fatty acid synthesis (FASII) pathway, but as a highly adaptive pathogen, S. aureus can also utilize host-derived exogenous fatty acids (eFAs), including SCFAs and unsaturated fatty acids (UFAs). S. aureus secretes three lipases, Geh, sal1, and SAUSA300_0641, which could perform the function of releasing fatty acids from host lipids. Once released, the FAs are phosphorylated by the fatty acid kinase, FakA, and incorporated into the bacterial lipids. In this study, we determined the substrate specificity of S. aureus secreted lipases, the effect of human serum albumin (HSA) on eFA incorporation, and the effect of FASII inhibitor, AFN-1252, on eFA incorporation using comprehensive lipidomics. When grown with major donors of fatty acids, cholesteryl esters (CEs) and triglycerides (TGs), Geh was found to be the primary lipase responsible for hydrolyzing CEs, but other lipases could compensate for the function of Geh in hydrolyzing TGs. Lipidomics showed that eFAs were incorporated into all major S. aureus lipid classes and that fatty acid-containing HSA can serve as a source of eFAs. Furthermore, S. aureus grown with UFAs displayed decreased membrane fluidity and increased production of reactive oxygen species (ROS). Exposure to AFN-1252 enhanced UFAs in the bacterial membrane, even without a source of eFAs, indicating a FASII pathway modification. Thus, the incorporation of eFAs alters the S. aureus lipidome, membrane fluidity, and ROS formation, which could affect host-pathogen interactions and susceptibility to membrane-targeting antimicrobials.

GENE EXPRESSION WITHIN A HUMAN CHOROIDAL NEOVASCULAR MEMBRANE USING SPATIAL TRANSCRIPTOMICS.

Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process. In this study, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We identified genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes. Within the area of neovascularization, endothelial cells were predicted to increase expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we also investigated spatial gene expression patterns within the macular neural retina and between the macular and peripheral choroid. We recapitulated previously described regional-specific gene expression patterns across both tissues. Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

Multimodal single-cell analysis of nonrandom heteroplasmy distribution in human retinal mitochondrial disease.

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multisystem disease. The wide range of manifestations observed in patients with mitochondrial disease results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify nonrandom distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA-Seq, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profiled the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and from healthy control donors. Utilizing the retina as a model for complex multilineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the WT allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the nonrandom nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3-8.3-fold and 19-22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%-55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.

Comparison of vector elements and process conditions in transient and stable suspension HEK293 platforms using SARS-CoV-2 receptor binding domain as a model protein.

BACKGROUND: Mammalian cell lines are frequently used as protein expression hosts because of their ability to correctly fold and assemble complex proteins, produce them at high titers, and confer post-translational modifications (PTMs) critical to proper function. Increasing demand for proteins with human-like PTMs, particularly viral proteins and vectors, have made human embryonic kidney 293 (HEK293) cells an increasingly popular host. The need to engineer more productive HEK293 platforms and the ongoing nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presented an opportunity to study strategies to improve viral protein expression in transient and stable HEK293 platforms. RESULTS: Initial process development was done at 24 deep well plate (DWP) -scale to screen transient processes and stable clonal cell lines for recombinant SARS-CoV-2 receptor binding domain (rRBD) titer. Nine DNA vectors that drove rRBD production under different promoters and optionally contained Epstein-Barr virus (EBV) elements to promote episomal expression were screened for transient rRBD production at 37 °C or 32 °C. Use of the cytomegalovirus (CMV) promoter to drive expression at 32 °C led to the highest transient protein titers, but inclusion of episomal expression elements did not augment titer. In parallel, four clonal cell lines with titers higher than that of the selected stable pool were identified in a batch screen. Flask-scale transient transfection and stable fed-batch processes were then established that produced rRBD up to 100 mg/L and 140 mg/L, respectively. While a bio-layer interferometry (BLI) assay was crucial for efficiently screening DWP batch titers, an enzyme-linked immunosorbent assay (ELISA) was used to compare titers from the flask-scale batches due to varying matrix effects from different cell culture media compositions. CONCLUSION: Comparing yields from the flask-scale batches revealed that stable fed-batch cultures produced up to 2.1x more rRBD than transient processes. The stable cell lines developed in this work are the first reported clonal, HEK293-derived rRBD producers and have titers up to 140 mg/L. As stable production platforms are more economically favorable for long-term protein production at large scales, investigation of strategies to increase the efficiency of high-titer stable cell line generation in Expi293F or other HEK293 hosts is warranted.

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy.

BACKGROUND: Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation. METHODS: In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed. RESULTS: Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory. CONCLUSION: We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

Trophic ontogeny of a generalist predator is conserved across space.

Consumers can influence ecological patterns and processes through their trophic roles and contributions to the flow of energy through ecosystems. However, the diet and associated trophic roles of consumers commonly change during ontogeny. Despite the prevalence of ontogenetic variation in trophic roles of most animals, we lack an understanding of whether they change consistently across local populations and broad geographic gradients. We examined how the diet and trophic position of a generalist marine predator varied with ontogeny across seven broadly separated locations (~ 750 km). We observed a high degree of heterogeneity in prey consumed without evidence of spatial structuring in this variability. However, compound-specific isotope analysis of amino acids revealed remarkably consistent patterns of increasing trophic position through ontogeny across local populations, suggesting that the roles of this generalist predator scaled with its body size across space. Given the high degree of diet heterogeneity we observed, this finding suggests that even though the dietary patterns differed, the underlying food web architecture transcended variation in prey species across locations for this generalist consumer. Our research addresses a gap in empirical field work regarding the interplay between stage-structured populations and food webs, and suggests ontogenetic changes in trophic position can be consistent in generalist consumers.