Biology subjects

Shoemaker, R.

Publications and source records attributed to Shoemaker, R..

8 recordsLinked to original sources

bioRxiv2026

Physiological cerebrospinal fluid like medium reveals autophagy dependency of leukaemia in the central nervous system

Nutrient availability is a critical environmental factor that influences the metabolism and adaptability of cancer cells, including acute lymphoblastic leukaemia (ALL) cells, prone to relapse in the central nervous system (CNS). Currently available cell culture media contain supraphysiological nutrient levels and do not represent the restricted metabolic environment of CNS-ALL which resides in the leptomeninges surrounded by cerebrospinal fluid (CSF). Therefore, we formulated a novel physiological CSF-like cell culture medium (CSFmax) that recapitulates the unique metabolite composition of the CSF. Through in vitro and in vivo metabolic and functional studies, we demonstrate that ALL cells cultured in CSFmax rewire their metabolism, closely mimicking the metabolic phenotype of CNS-ALL, including their metabolic activity and redox state. Utilising CSFmax, in comparison to conventional nutrient-rich culture media, we identified an essential role for autophagy in ALL adaptation to the CNS niche. This was evident by increased autophagic activity and selective sensitisation of ALL cells to pharmacological inhibition of autophagy and genetic knockout of Unc-51 Like Autophagy Activating Kinase 1 (ULK1) or autophagy related 7 (ATG7). Importantly, using a robust preclinical in vivo model, mice xenografted with ULK1 and ATG7 deficient ALL cells exhibited reduced CNS disease burden when compared to mice xenografted with control cells. Overall, our findings provide strong evidence that physiological CSFmax is superior to current in vitro culture systems in recapitulating the metabolic signature of CNS resident ALL cells. By exploiting this system, we revealed for the first time autophagy as a targetable therapeutic vulnerability in CNS-ALL. Key PointsO_LICulturing ALL cells in bespoke CSF-like medium (CSFmax) recapitulates the metabolic adaptation of ALL cells in the CNS niche C_LIO_LIAutophagy is critical for metabolic adaptation and survival of CNS resident ALL cells C_LI

cancer biology↗

bioRxiv2025

Combining a rhesus cytomegalovirus/SIV vaccine with neutralizing antibody to protect against SIV challenge in rhesus macaques

A vaccine is widely regarded as necessary for the control of the HIV pandemic and eventual eradication of AIDS. Neutralizing antibodies and MHC-E-restricted CD8+ T cells have both been shown capable of vaccine protection against the simian counterpart of HIV, SIV, in rhesus macaques. Here we provide preliminary evidence that combining these orthogonal antiviral mechanisms can provide increased protection against SIV challenge such that replication arrest observed following vaccination with a rhesus cytomegalovirus (RhCMV/SIV)-based vaccine was enhanced in the presence of passively administered incompletely protective levels of neutralizing antibody. The report invites studies involving larger cohorts of macaques and alternate routes of providing neutralizing antibody.

immunology↗

bioRxiv2024

Detection and decontamination of chronic wasting disease prions during venison processing

Prion diseases, including chronic wasting disease (CWD), are caused by prions, which are misfolded aggregates of normal cellular prion protein. Prions possess many characteristics that distinguish them from conventional pathogens, in particular, an extraordinary recalcitrance to inactivation and a propensity to avidly bind to surfaces. In mid to late stages of CWD, prions begin accumulating in cervid muscle tissues. These features collectively create scenarios where occupational hazards arise for workers processing venison and pose risks to consumers through direct prion exposure via ingestion and cross-contamination of food products. In this work, we show that steel and plastic surfaces used in venison processing can be directly contaminated with CWD prions and that cross-contamination of CWD-negative venison can occur from equipment that had previously been used with CWD-positive venison. We also show that several decontaminant solutions (commercial bleach and potassium peroxymonosulfate) are efficacious for prion inactivation on these same surfaces.

microbiology↗

bioRxiv2022

Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends

EB1 is a key cellular protein that delivers critical regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a [~]70-fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on tubulin hydrolysis state, recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with the chemotherapy drug Eribulin. We found that Eribulin blocked EB1 protofilament-edge binding, which led to a dramatic decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites increases the efficiency of EB1 tip tracking at the growing microtubule plus end.

cell biology↗

bioRxiv2021

SARS-CoV-2 Spike Protein Regulation of Angiotensin Converting Enzyme 2 and Tissue Renin-Angiotensin Systems: Influence of Biologic Sex

Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.

physiology↗

bioRxiv2021

Sustained IL-15 response signature predicts RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work suggests that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including non-canonical T cell receptor (TCR), toll-lie receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly predicting protection. The IL-15 gene expression signature was further evaluated in an independent RM IL-15 treatment cohort, revealing that in whole blood the response to IL-15 is inclusive of innate and adaptive immune gene expression networks that link with RhCMV/SIV vaccine efficacy. We also show that this IL-15 response signature similarly tracks with vaccine protection in an independent RhCMV/SIV vaccination/SIV challenge RM validation cohort. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that enable vaccine-elicited CD8+ T cells to mediate protection against highly pathogenic SIV challenge. Author SummarySIV insert-expressing vaccine vectors based on strain 68-1 RhCMV elicit robust, highly effector-memory-biased T cell responses that are associated with an unprecedented level of SIV control after challenge (replication arrest leading to clearance) in slightly over half of vaccinated monkeys. Since efficacy is not predicted by standard measures of immunogenicity, we used functional genomics analysis of RhCMV/SIV vaccinated monkeys with known challenge outcomes to identify immune correlates of protection. We found that arrest of viral replication after challenge significantly correlates with a vaccine-induced response to IL-15 that includes modulation of T cell, inflammation, TLR signaling, and cell death programming. These data suggest that RhCMV/SIV efficacy is not based on chance, but rather, results from a coordinated and sustained vaccine-mediated induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ effector-memory T cell function, that enable vaccine-elicited CD8+ T cells to mediate efficacy.

genomics↗

bioRxiv2020

Modulation of MHC-E transport by viral decoy ligands is required for RhCMV/SIV vaccine efficacy

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8+ T cells that recognize peptide epitopes presented by major histocompatibility complex (MHC)-II and MHC-E molecules, instead of MHC-Ia, and are uniquely able to mediate stringent control and subsequent clearance of highly pathogenic SIV in [~]50% of vaccinated rhesus macaques (RMs). We show that the MHC-E ligand VMAPRTLLL (VL9), encoded by the Rh67 gene (or its HCMV UL40 counterpart) is required for recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8+ T cells via its ability to promote intracellular MHC-E transport. Moreover, deletion of Rh67 from 68-1 RhCMV/SIV vectors, or mutation of its embedded VL9 ligand, abrogated induction of MHC-E-restricted CD8+ T cell responses, leaving responses that exclusively target MHC-II-restricted epitopes. These MHC-II-presented CD8+ T cell responses, though comparable in response magnitude and functional differentiation to responses arising from the efficacious 68-1 vector, did not protect RMs against SIV challenge, indicating that Rh67/UL40-enabled direct priming of MHC-E-targeted CD8+ T cells is a crucial element of RhCMV/SIV vaccine efficacy. One Sentence SummaryA cytomegalovirus protein (Rh67/UL40) that upregulates MHC-E expression on RhCMV/SIV-vector infected cells is required for induction of MHC-E-restricted CD8+ T cells and for protection against SIV.

immunology↗

bioRxiv2020

Cytomegaloviral determinants of CD8+ T cell programming and RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) insert-expressing, 68-1 Rhesus Cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex (MHC)-E- and -II-restricted, SIV-specific CD8+ T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) has not been characterized. We demonstrate that these unconventional responses resulted from a chance genetic rearrangement in 68-1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions (Rh157.5/.4 and Rh158-161). Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences (UL128/130; UL146/147) leads to either of two distinct CD8+ T cell response types - MHC-Ia-restricted-only, or a mix of MHC-II- and MHC-Ia-restricted CD8+ T cells. Despite response magnitude and functional differentiation being similar to RhCMV 68-1, neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8+ T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all the genes that inhibit these responses from the HCMV/HIV vector. One-sentence summaryEight genes in two spatially distinct RhCMV gene regions control induction of unconventionally restricted CD8+ T cell responses and the efficacy of RhCMV/SIV vaccine vectors against SIV challenge.

immunology↗