Biology subjects

Stewart, C. M.

Publications and source records attributed to Stewart, C. M..

10 recordsLinked to original sources

bioRxiv2025

Evaluation of the safety and insecticidal efficacy of ivermectin-treated bird feed formulations in different avian species

BackgroundWest Nile virus (WNV) is maintained in an enzootic cycle between reservoir host birds and Culex (Cx.) spp. mosquitoes. This relationship presents a potential target for vector control strategies. Ivermectin (IVM), an endectocidal drug that selectively affects invertebrates while remaining safe at high concentrations in mammals and birds, can be delivered to Cx. tarsalis via blood meals from birds fed IVM-treated bird feed. In this study we evaluated the safety, efficacy, and utility of IVM-treated bird feed as a novel vector control strategy by assessing its impact on multiple bird species and mosquitoes. MethodsMosquitoes were collected during peak WNV transmission season in Northern Colorado and DNA extracted from blood meals to determine host species. Chickens, pigeons, zebra finches, and house sparrows were fed different formulations of IVM-treated bird feed and observed for clinical signs, and their sera were fed to Cx. tarsalis mosquitoes to evaluate mosquitocidal efficacy. Feeding rates and IVM serum concentrations in birds were analyzed by unpaired t-test and one-way ANOVA, and mosquito survivorship was analyzed by Kaplan-Meier curves and compared using paired log-rank tests. IVM serum concentration and mosquito survivorship were compared using Spearman correlation. ResultsSpeciation analyses conducted on blood meals from Cx. tarsalis collected during peak WNV transmission season in Northern Colorado determined that they feed primarily on songbird species that commonly visit bird feeders, with house sparrows representing the most frequent blood meal host. In laboratory experiments using multiple formulations and doses of IVM, chickens, pigeons, zebra finches, and house sparrows ate comparable amounts of IVM-treated bird feed compared to untreated feed, had similar weight gain, and exhibited no clinical signs of toxicity. Both colony-reared and locally captured Cx. tarsalis showed significant mortality after feeding on sera from IVM-treated birds compared to controls. ConclusionsThese results suggest that targeting songbirds with IVM-treated bird feed should be safe for wildlife and may elicit high rates of IVM-induced mortality by reaching a large proportion of WNV vector mosquitoes via their proclivity for feeding on passerine birds.

microbiology↗

bioRxiv2025

Sleeping Beauty mutagenesis identifies BACH2 and other regulators of CD8+T cell exhaustion, persistence in vivo, and CAR-T function under tumor-associated chronic antigen stimulation

Genes that enhance T cell function represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T cell persistence, employing Sleeping Beauty (SB) insertional mutagenesis, which induces both gain-(GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying Doxycycline (Dox)-inducible SB mutagenesis system (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T cell persistence under chronic antigen exposure. Specifically, CD8 T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 T cells using enhanced-specificity tagmentation sequencing (esTag-seq) and RNA-seq, respectively. Under chronic stimulation, SB-mutagenized CD8 T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation stress. Among these, T2/Onc2 insertions into Bach2 and Elmo1 were repeatedly found at the genomic level and were associated with altered nascent transcript expression. Bach2, known as a key regulator of T cell memory formation and resistance to chronic viral infection but less characterized in engineered T cells for cancer therapy, was found to enhance in vivo tumor persistence in the B16-Ova tumor model. We showed that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2low signature allowed differentiation into both KLRG1 and CD62L phenotypes, whereas Bach2high restricted differentiation predominantly to the CD62L subset. Finally, in human CART19-28{zeta} cells, BACH2 overexpression enhanced cytotoxicity and improved tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T cell therapeutic phenotypes. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

immunology↗

bioRxiv2024

Optimized CART Cell Therapy for Metastatic Aggressive Thyroid Cancer

Most thyroid cancer deaths are attributed to a subset of poorly differentiated, metastatic tumors. To improve treatment options for aggressive thyroid cancers, we developed a novel thyroid-stimulating hormone receptor (TSHR)-targeted chimeric antigen receptor T (CART) cell therapy, which demonstrated antigen-specific activation and antitumor efficacy against TSHR+ cell lines in vitro and in vivo. However, de-differentiated thyroid cancers downregulate TSHR. We therefore developed a potent treatment strategy by combining our novel TSHR-CART cells with mitogen-activated protein kinase (MAPK) inhibitors, which redifferentiate thyroid tumors and upregulate TSHR expression. In patient-derived anaplastic thyroid cancer xenografts, combination therapy of TSHR-CART cells and MAPK inhibitors led to increased TSHR expression on the tumor tissue and significantly enhanced antitumor efficacy and prolonged survival compared to TSHR-CART monotherapy. Based on our data, we are launching a phase I clinical trial for TSHR-CART cell therapy alone or in combination with MAPK inhibitors in patients with metastatic thyroid cancers. STATEMENT OF SIGNIFICANCEPoor target selection and antigen escape limit CART cell efficacy in solid tumors. We developed TSHR-CART cells to treat differentiated thyroid cancers but observed TSHR downregulation in dedifferentiated thyroid cancers. We found that MAPK inhibitors restored TSHR expression and sensitized these cancers to TSHR-CART cell therapy.

immunology↗

bioRxiv2024

A comparison between low-cost library preparation kits for low coverage sequencing

In the fields of human health and agricultural research, low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples. However, a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature. In this study, we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits--the KAPA HyperPlus kit (Roche), the DNA Prep kit (Illumina), and an IDT kit--using 96 human DNA samples. Metrics evaluated included imputation concordance with high-depth genotypes, coverage, duplication rates, time for library preparation, and additional optimization requirements. Despite slightly elevated duplication rates in IDT kits, we find that all four kits perform well in terms of imputation accuracy, with IDT kits being only marginally less performant than Illumina and Roche kits. Laboratory handling of the kits was similar: thus, the choice of a kit will largely depend on (1) existing or planned infrastructure, such as liquid handling capabilities, (2) whether a specific characteristic is desired, such as the use of full-length adapters, shorter processing times, or (3) use case, for instance, long vs short read sequencing. Our findings offer a comprehensive resource for both commercial and research workflows of low-cost library preparation methods suitable for high-throughput low coverage whole genome sequencing.

genomics↗

bioRxiv2023

Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization

Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80highSca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy.

cancer biology↗

bioRxiv2023

The Identification of IL-4 as a Regulator of Chimeric Antigen Receptor T Cell Exhaustion

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. We investigated the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using a validated in vitro model for exhaustion, RNA and ATAC sequencing on baseline and chronically stimulated CART cells, and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identified IL-4 as a key regulator of CART cell dysfunction. Further, when CART cells were treated with IL-4, they developed signs of exhaustion, but when CART cells were treated with an IL-4 monoclonal antibody, they showed improved antitumor efficacy and reduced signs of exhaustion in preclinical models. Therefore, our study identified both a novel role for IL-4 on CART cells and the improvement of CART cell therapy through IL-4 neutralization. Statement of SignificanceIdentifying regulators of CART cell exhaustion will not only enhance the fields understanding of therapeutic failure, but it will also provide avenues to enhance CART cell efficacy. This study reveals both a novel role for IL-4 in exhaustion and a strategy to improve CART cell activity through IL-4 neutralization.

immunology↗

bioRxiv2022

Deep mutational scans for ACE2 binding, RBD expression, and antibody escape in the SARS-CoV-2 Omicron BA.1 and BA.2 receptor-binding domains

SARS-CoV-2 continues to acquire mutations in the spike receptor-binding domain (RBD) that impact ACE2 receptor binding, folding stability, and antibody recognition. Deep mutational scanning prospectively characterizes the impacts of mutations on these biochemical properties, enabling rapid assessment of new mutations seen during viral surveillance. However, the effects of mutations can change as the virus evolves, requiring updated deep mutational scans. We determined the impacts of all amino acid mutations in the Omicron BA.1 and BA.2 RBDs on ACE2-binding affinity, RBD folding, and escape from binding by the LY-CoV1404 (bebtelovimab) monoclonal antibody. The effects of some mutations in Omicron RBDs differ from those measured in the ancestral Wuhan-Hu-1 background. These epistatic shifts largely resemble those previously seen in the Beta variant due to the convergent epistatically modifying N501Y substitution. However, Omicron variants show additional lineage-specific shifts, including examples of the epistatic phenomenon of entrenchment that causes the Q498R and N501Y substitutions present in Omicron to be more favorable in that background than in earlier viral strains. In contrast, the Omicron substitution Q493R exhibits no sign of entrenchment, with the derived state, R493, being as unfavorable for ACE2 binding in Omicron RBDs as in Wuhan-Hu-1. Likely for this reason, the R493Q reversion has occurred in Omicron sub-variants including BA.4/BA.5 and BA.2.75, where the affinity buffer from R493Q reversion may potentiate concurrent antigenic change. Consistent with prior studies, we find that Omicron RBDs have reduced expression, and identify candidate stabilizing mutations that ameliorate this deficit. Last, our maps highlight a broadening of the sites of escape from LY-CoV1404 antibody binding in BA.1 and BA.2 compared to the ancestral Wuhan-Hu-1 background. These BA.1 and BA.2 deep mutational scanning datasets identify shifts in the RBD mutational landscape and inform ongoing efforts in viral surveillance. Author SummarySARS-CoV-2 evolves in part through mutations in its spike receptor-binding domain. As these mutations accumulate in evolved variants, they shape the future evolutionary potential of the virus through the phenomenon of epistasis. We characterized the functional impacts of mutations in the Omicron BA.1 and BA.2 receptor-binding domains on ACE2 receptor binding, protein folding, and recognition by the clinical LY-CoV1404 antibody. We then compared the measurements to prior data for earlier variants. These comparisons identify patterns of epistasis that may alter future patterns of Omicron evolution, such as turnover in the availability of specific affinity-enhancing mutations and an expansion in the number of paths of antibody escape from a key monoclonal antibody used for therapeutic treatment of COVID-19. This work informs continued efforts in viral surveillance and forecasting.

microbiology↗

bioRxiv2022

Sphingosine kinases promote Ebola virus infection and can be targeted to inhibit filoviruses, coronaviruses, and arenaviruses using late endocytic trafficking to enter cells

Entry of enveloped viruses in host cells requires the fusion of the viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. For fusion, viral fusion proteins need to be triggered by host factors and for some viruses, such as Ebola virus (EBOV) and Lassa fever virus, this event occurs inside endosomes and/or lysosomes. Consequently, these late-penetrating viruses must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late penetrating viruses also depend on specific host factors, such as signaling molecules, for efficient viral delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1 or SK2, inhibited entry of EBOV into host cells. Mechanistically, inhibition of SK1 and/or SK2 prevented EBOV from reaching late-endosomes and lysosomes that are positive for the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests the trafficking defect caused by SK1/2 inhibition occurs independently of S1P signaling through cell-surface S1PRs. Lastly, we found that chemical inhibition of SKs prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, in addition to inhibiting infection by replication competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SKs in endocytic trafficking which can be targeted to inhibit entry of late-penetrating viruses. SK inhibitors could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

microbiology↗

bioRxiv2022

Female investment in terminal reproduction or somatic maintenance depends on infection dose

Immune responses are energetically costly to produce and often trade off with investment in reproduction and offspring provisioning. We predicted that hosts can interpret the magnitude of the infection threat from cues like initial bacterial load to adjust reproductive investment into offspring quantity and quality. To test this prediction, we exposed female flour beetles (Tribolium castaneum) to naive or sterile media controls or one of three increasing doses of heat-killed Bacillus thuringiensis (Bt). To estimate offspring quantity, we measured the number of eggs and hatched larvae from each maternal treatment group. To estimate offspring quality, we measured egg protein content, development to pupation, pupal weight, and offspring survival against Bt infection. Compared to naive controls, low and intermediate bacterial doses resulted in lower female fecundity, suggesting a shift to a somatic maintenance strategy. Meanwhile, in the highest-dose group, fecundity was negatively correlated with egg protein content, suggesting a trade-off between offspring quality and quantity that could mask terminal investment based on metrics of quantity alone. Our results underscore the need to account for the magnitude of environmental cue when quantifying plasticity and trade-offs among life history traits, and provide new insight into the trans-generational effects of immune responses.

evolutionary biology↗

bioRxiv2022

Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

microbiology↗