Biology subjects

Brown, M. E.

Publications and source records attributed to Brown, M. E..

16 recordsLinked to original sources

bioRxiv2026

Hormetic heat shock activates HLH-30/TFEB independently of canonical nutrient-sensing pathways

In Caenorhabditis elegans, a brief, sublethal heat shock (HS) induces a hormetic response that increases resistance to subsequent stress and extends lifespan. These benefits require hlh-30, the ortholog of mammalian transcription factor EB (TFEB). Although HS induces robust HLH-30 nuclear translocation, how this response is regulated remains poorly understood. Nutrient- and energy-sensing pathways, including mTORC1 and AMPK, regulate HLH-30/TFEB subcellular localization under other physiological conditions, but whether they mediate its nuclear translocation during HS is unknown. Here, we show that, although HS inhibited mTORC1 and dephosphorylated its conserved HLH-30 S201 target site, HLH-30 S201 phosphorylation was dispensable for HS-induced HLH-30 nuclear localization and hormetic protection. Moreover, HS remained protective in hlh-30 mutants when mTORC1 activity was reduced, revealing an HLH-30-independent component of the hormetic response. HS also activated AMPK and aak-2 was required for hormetic protection but dispensable for HS-induced HLH-30 nuclear localization and autophagosome formation. Together, these findings demonstrate that although HS engages canonical mTORC1 and AMPK signaling, these pathways do not account for HS-induced HLH-30 nuclear localization and instead make distinct contributions to hormetic protection. Our findings reveal stress-specific regulation of HLH-30/TFEB and point to additional mechanisms that drive its activation during heat stress.

cell biology↗

bioRxiv2026

CRISPR-Mediated Targeting of BRAF Oncogenes in Pediatric Low-Grade Glioma

SummaryA catch-all intronic guide RNA pair excises the KIAA1549--BRAF oncofusion across its major variants, with productive junction excision confirmed by gain-of-function PCR in patient-derived glioma cells. An allele-specific guide selectively disrupts BRAF V600E, in patient-derived pediatric low-grade glioma cells. Pediatric low-grade glioma (pLGG) is the most common brain tumor of childhood, accounting for 30--50% of all pediatric central nervous system malignancies1. The disease is almost universally driven by activating mutations in the BRAF serine/threonine kinase: a chromosomal tandem duplication generating the KIAA1549--BRAF oncofusion in approximately 70% of cases, or the BRAF V600E gain-of-function point mutation in approximately 15%2. Current targeted pharmacotherapies, including the RAF inhibitor tovorafenib, require continuous dosing, are not allele-specific, and carry risks of long-term toxicity in children. A one-time genomic intervention that permanently disables the oncogenic BRAF alteration while preserving wild-type BRAF signaling represents a compelling therapeutic alternative. In this study, we describe the design and experimental validation of allele-specific CRISPR guide RNAs targeting both the KIAA1549--BRAF oncofusion and the BRAF V600E point mutation. For the oncofusion, we developed a double-cut intronic excision strategy in which a guide RNA targeting KIAA1549 intron 14 is paired with a guide RNA targeting BRAF intron 11. Because the genomic breakpoints of all four major fusion variants (KB 16:9, 15:9, 16:11, and 15:11) fall within these introns, a single guide pair can address the full landscape of fusion heterogeneity in a single intervention. For BRAF V600E, we exploited a unique PAM sequence created by the pathogenic TBA transversion at codon 600, enabling allele-specific SpCas9 and AsCas12a guide designs that distinguish the mutant from the wild-type allele at single-nucleotide resolution. We screened guide RNA candidates by ribonucleoprotein (RNP) nucleofection in A375 human melanoma cells (BRAF V600E homozygous) and in patient-derived 3635 PXA glioma cells (BRAF V600E heterozygous). The top KIAA1549 intron 14 guide, K9_i14_A_Cas9, achieved 66% indel frequency in A375 cells. The top BRAF intron 11 guides, B_i11_A_Cas9 and B_i11_D_Cas9, achieved 84% and 85% indel frequency, respectively. For BRAF V600E, the best allele-specific SpCas9 guide achieved l57% editing in A375 cells and l74% editing in 3635 PXA patient-derived glioma cells. Dual-cut excision of the KIAA1549--BRAF junction was confirmed by a gain-of-function PCR assay designed to detect the excision junction amplicon ([~]191 bp) produced by NHEJ-mediated rejoining of the KIAA1549 intron 14 and BRAF intron 11 cut ends.

cancer biology↗

bioRxiv2026

Cellular senescence is associated with age-related loss of liver zonation and hepatocyte function

The liver is organized into tightly regulated zones with distinct metabolic functions but zonation erodes with age. Cellular senescence contributes to aging and liver diseases, however, its impact on aging biology is ill-defined. As part of The Cellular Senescence Network Consortium, we used multiple spatial transcriptomics approaches (GeoMx, Visium, CosMx) with snRNA-seq to profile senescence signatures, zonation markers, and metabolic pathways in livers from wild-type (WT) mice of multiple ages. We observed a loss of canonical zone signatures in aged mouse livers characterized by "expansion" of midlobular (zone 2) marker gene expression, accompanied by diminished expression of zone 3 marker genes by middle-age (18 months), indicative of loss of cell identity. Multiple analytic approaches identified distinct age-, zone- and sex-specific senescence signatures, which were significantly associated with zonation markers changes. This was recapitulated in Ercc1 mutant models of accelerated senescence, supporting a causal role of senescent cells in liver aging. A "no-zone" hepatocyte-like cluster expanded with age and with the strongest Senescence-Associated Secretory Phenotype (SASP) profile. Gene expression profiles from senescent hepatocytes implicate decreased WNT signaling and increased BMP as contributing to age-related loss of zonation. Together, these data elucidate the role of senescent cells in driving aging biology in non-diseased liver through disruption of cell:cell signaling and the loss of metabolic and cell identity gene expression necessary for hepatocyte function.

cell biology↗

bioRxiv2026

The NBSGW RIP-DTR Mouse: An Integrated Platform for Diabetes Induction, Human Immune Reconstitution and Transplantation Studies

Using toxin receptor-mediated cell ablation, a diabetes mouse model was generated that supports engraftment of human hematopoietic stem/progenitor cells (HSPCs) without the need for irradiation. The NBSGW immunodeficient strain was crossed with the NSG RIP-DTR which carries the diphtheria toxin receptor (hDTR) under the control of the rat insulin promoter to generate the NBSGW RIP-DTR mouse. This model enables controlled {beta}-cell ablation, robust human immune system reconstitution without myeloablative conditioning, and evaluation of human immune-mediated graft rejection within a single platform. NBSGW RIP-DTR mice exhibited reproducible and titratable diabetes induction, supported durable human islet engraftment and glycemic correction, and retained efficient human hematopoietic reconstitution comparable to the parental NBSGW strain. In humanized mice, diphtheria toxin-mediated diabetes induction was well tolerated and enabled assessment of human immune responses to allogeneic islet grafts. Collectively, these findings establish the NBSGW RIP-DTR mouse as an integrated and clinically relevant platform for studying {beta}-cell replacement therapies and human immune-mediated graft rejection. Article HighlightsO_LICurrent preclinical models do not simultaneously support controlled diabetes induction, human islet transplantation, and durable human immune reconstitution without irradiation. C_LIO_LIThis study asked whether the NBSGW RIP-DTR mouse could integrate diphtheria toxin-mediated {beta}-cell ablation with irradiation-free humanization in a single platform. C_LIO_LINBSGW RIP-DTR mice demonstrated reproducible diabetes induction, supported functional human islet engraftment, and retained robust human hematopoietic reconstitution comparable to parental strains. C_LIO_LIThese findings establish the NBSGW RIP-DTR model as a clinically relevant platform for studying {beta}-cell replacement and human immune response to islet allografts, xenografts and stem cell-derived islets. C_LI

immunology↗

bioRxiv2026

Generation of hypoimmunogenic gastric insulin-secreting organoids

Gastric insulin-secreting organoids (GINS) represent a promising source of {beta}-like cells for type 1 diabetes (T1D) therapy. In same-donor comparisons with induced pluripotent stem cell-derived islets (iPSC-islets), GINS displayed robust glucose responsiveness and reduced expression of key T1D autoantigens. Importantly, GINS exhibited decreased susceptibility to cytotoxicity mediated by engineered HLA-matched preproinsulin-specific effector T cells (Avatar Teffs) and a distinct transcriptional profile enriched for immune-modulatory and stress-adaptive gene programs. To enhance immune evasion, we engineered gastric stem cells to overexpress Programmed Death Ligand 1 (PD-L1) in an inducible manner. PD-L1+ GINS maintained normal functionality, while exhibiting improved survival under allogeneic Avatar Teff challenge in a MHC class I-independent fashion. We evaluated PD-L1-mediated protection against autologous Avatar Teff attack using an endothelialized microfluidic platform recapitulating physiologic immune interactions. T cells show reduced infiltration into PD-L1 GINS, resulting in significantly higher organoid viability compared to control GINS. Together, these findings identify GINS as a functional and engineerable {beta}-like cell platform with intrinsic hypoimmunogenic features, and support PD-L1 engineering as a strategy to enhance immune protection for both allogeneic and autologous transplantation in T1D.

immunology↗

bioRxiv2025

Human Stem Cell-Derived β-cells Expressing An Optimized CD155 Reduce Cytotoxic Immune Cell Function for Application in Type 1 Diabetes

Insulin-producing {beta}-cell replacement therapies offers a potential treatment for type 1 diabetes (T1D) but faces challenges from donor shortages and immune rejection. Stem cell-derived {beta}-cells (sBC) provide a renewable source but remain vulnerable to immune attack. We engineered human pluripotent stem cells to express either the wildtype (WT) or a high-affinity mutant (Mut) variant (rs1058402, G>A; Ala67Thr) of the NK and T cell checkpoint inhibitor CD155 before differentiation into sBC. Modified sBC maintained upregulated CD155 expression and showed enhanced binding to co-receptor ligands. Co-culture studies revealed that CD155 Mut-expressing sBC suppressed CD8+ T cell and NK cell activation and proliferation by preferentially engaging the co-inhibitory receptor TIGIT. Both CD155 Mut sBC lines reduced autoreactive CD8+ T cell- and NK cell-mediated sBC destruction and cytotoxic molecule secretion. This protection was lost with TIGIT blockade, confirming the role of CD155-TIGIT signaling in antagonizing immune cell-mediated killing. Our findings suggest that high-affinity CD155 expression enhances immune evasion of sBC, improving their potential for restorative therapy in T1D. TeaserEngineered {beta}-cells with a mutant CD155 help evade immune attack, offering a promising therapeutic approach for type 1 diabetes.

immunology↗

bioRxiv2025

Pathobiont-triggered induction of epithelial IDO1 drives regional susceptibility to Inflammatory Bowel Disease

The structure and function of the mammalian gut vary by region, yet why inflammatory diseases manifest in specific regions and not others remains unclear. We use a TNF-overexpressing Crohns disease (CD) model (Tnf{Delta}ARE/+), which typically presents in the terminal ileum (TI), to investigate how environmental factors interact with the hosts immune susceptibility to drive region-specific disease. We identified Chlamydia muridarum, an intracellular bacterium and murine counterpart to the human sexually transmitted C. trachomatis, as necessary and sufficient to trigger disease manifestation in the ascending colon (AC), another common site of human CD. Disease manifestation in the AC depends on indoleamine 2,3-dioxygenase (IDO1) activity induced by hypersensitive surface secretory cells in genetically susceptible hosts. Single-cell and microbial analyses of human specimens also implicates this pathobiont-epithelial IDO1 pathway in patients with a history of CD in the AC. Our findings demonstrate that genetic and microbial factors can independently drive region-specific disease and provide a unique model to study CD specific to the AC.

cell biology↗

bioRxiv2024

Altered cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation

Functional loss of the motor protein, Myosin Vb (MYO5B), induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). Utilizing the MVID model mice, Vil1-CreERT2;Myo5bflox/flox (MYO5B{Delta}IEC) and Vil1-CreERT2;Myo5bflox/G519R(MYO5B(G519R)), we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5B{Delta}IEC small intestine. Consistent with these observations in vivo, RNA-sequencing of enteroids generated from two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, lysophosphatidic acid (LPA) signaling ameliorates epithelial cell defects in MYO5B{Delta}IEC tissues and enteroids. The present study demonstrates that the highly soluble LPAR5-preferred agonist, Compound-1, improved sodium transporter localization and absorptive function, and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on intestine with MYO5B functional loss through epithelial cell-autonomous pathways that may improve the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for treatment of MVID symptoms induced by different point mutations in MYO5B. NEW & NOTEWOTHYThis study demonstrates the importance of MYO5B for cellular lipid metabolism and mitochondria in intestinal epithelial cells, a previously unexplored function of MYO5B. Alterations in cellular metabolism may underlie the progenitor cell malfunction observed in microvillus inclusion disease (MVID). To examine the therapeutic potential of progenitor-targeted treatments, the effects of LPAR5-preferred agonist, Compound-1, was investigated utilizing several MVID model mice and enteroids. Our observations suggests that Compound-1 may provide a therapeutic approach for treating MVID. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/610579v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@54b5borg.highwire.dtl.DTLVardef@1966a5aorg.highwire.dtl.DTLVardef@2073f1org.highwire.dtl.DTLVardef@9c12e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Diminished Immune Cell Adhesion in Hypoimmune ICAM-1 Knockout Pluripotent Stem Cells

Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g., T cell) immune responses, but have largely not addressed the innate immune cells (e.g., monocytes, neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies, we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responses in vitro and in longer in vivo retention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding, thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases. HighlightsO_LIAntibody blocking of ICAM-1 on human pluripotent stem cell-derived cells inhibits immune cell adhesion C_LIO_LICRISPR/Cas9 knock-out of ICAM-1 ablates surface and secreted ICAM-1 protein and inhibits immune adhesion C_LIO_LIICAM-1 knock-out results in decreased T cell proliferative responses to human pluripotent stem cell-derived grafts in vitro, and resistance to immune-mediated graft loss in vivo C_LIO_LIAddition of ICAM-1 knock-out to first generation MHC knock-out human pluripotent stem cells confers protection against immune adhesion C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/597791v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@890b56org.highwire.dtl.DTLVardef@147449borg.highwire.dtl.DTLVardef@1e94f08org.highwire.dtl.DTLVardef@43c55c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO ICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse. Abbreviations: PSC-EC - pluripotent stem cell-derived endothelial cells; KO - knock-out; dSMAC - distal supramolecular activation complex; pSMAC - peripheral supramolecular activation complex; cSMAC - central supramolecular activation complex. C_FIG

immunology↗

bioRxiv2023

Mitochondrial citrate carrier SLC25A1 is a dosage-dependent regulator of metabolic reprogramming and morphogenesis in the developing heart

The developing mammalian heart undergoes an important metabolic shift from glycolysis toward mitochondrial oxidation, such that oxidative phosphorylation defects may present with cardiac abnormalities. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mice with systemic loss of the mitochondrial citrate carrier SLC25A1. Slc25a1 null embryos displayed impaired growth, cardiac malformations, and aberrant mitochondrial function. Importantly, Slc25a1 heterozygous embryos, which are overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggesting Slc25a1 haploinsuffiency and dose-dependent effects. Supporting clinical relevance, we found a near-significant association between ultrarare human pathogenic SLC25A1 variants and pediatric congenital heart disease. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of gene expression to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis and cardiac metabolic maturation and suggests a role in congenital heart disease.

developmental biology↗

bioRxiv2023

Enthesis Maturation in Engineered Ligaments is Differentially Driven by Loads that Mimic Slow Growth Elongation and Rapid Cyclic Muscle Movement

Entheses are complex attachments that translate load between elastic-ligaments and stiff-bone via organizational and compositional gradients. Neither natural healing, repair, nor engineered replacements restore these gradients, contributing to high re-tear rates. Previously, we developed a novel culture system which guides ligament fibroblasts in high-density collagen gels to develop early postnatal-like entheses, however further maturation is needed. Mechanical cues, including slow growth elongation and cyclic muscle activity, are critical to enthesis development in vivo but these cues have not been widely explored in engineered entheses and their individual contribution to maturation is largely unknown. Our objective here was to investigate how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, individually drive enthesis maturation in our system so to shed light on the cues governing enthesis development, while further developing our engineered replacements. Interestingly, we found these loads differentially drive organizational maturation, with slow stretch driving improvements in the interface/enthesis region, and cyclic load improving the ligament region. However, despite differentially affecting organization, both loads produced improvements to interface mechanics and zonal composition. This study provides new insight into how mechanical cues differentially affect enthesis development, while producing some of the most organized engineered enthesis to date.

bioengineering↗

bioRxiv2022

Temporal Application of Lysyl Oxidase during Hierarchical Collagen Fiber Formation Differentially Effects Mechanics in Engineered Tissues

The primary source of strength in musculoskeletal menisci, tendons, and ligaments are hierarchical collagen fibers; however, these fibers are not regenerated after injury nor in engineered replacements, resulting in limited repair options. Collagen strength is reliant on fiber alignment, density, diameter, and crosslinking. Recently, we developed a culture system which guides cells in high-density collagen gels to develop native-like hierarchically organized collagen fibers, which match native alignment and fiber diameters by 6 weeks. However, tissue mechanics plateau at 1 MPa, suggesting crosslinking is lacking. Collagen crosslinking is regulated by lysyl oxidase (LOX) which forms immature crosslinks that condense into mature trivalent crosslinks. Trivalent crosslinks are thought to be the primarily source of strength in fibers, but its not well understood how they form. The objective of this study was to evaluate the effect of exogenous LOX treatment at different stages of hierarchical fiber formation in our culture system to produce functional engineered replacements and to better understand factors effecting collagen crosslink maturation. We found LOXL2 treatment did not restrict hierarchical fiber formation, with constructs still forming aligned collagen fibrils by 2 weeks, larger fibers by 4 weeks, and early fascicles by 6 weeks. However, LOXL2 treatment did significantly increase mature pyridinium crosslink accumulation and tissue mechanics, with timing of LOXL2 supplementation during fiber formation having a significant effect. Overall, we found one week of LOXL2 supplementation at 4 weeks produced constructs with native fiber organization, increased PYD accumulation, and increased mechanics, ultimately matching the tensile modulus of immature bovine menisci. Statement of SignificanceCollagen fibers are the primarily source of strength and function in connective tissues throughout the body, however it remains a challenge to develop these fibers in engineered replacements, greatly reducing treatment options. Here we demonstrate lysyl oxidase like 2 (LOXL2) can be used to significantly improve the mechanics of tissue engineered constructs, but timing of application is important and will most likely depend on degree of collagen organization or maturation. Currently there is limited understanding of how collagen crosslinking is regulated, and this system is a promising platform to further investigate cellular regulation of LOX crosslinking. Understanding the mechanism that regulates LOX production and activity is needed to ultimately regenerate functional repair or replacements for connective tissues throughout the body.

bioengineering↗

bioRxiv2022

Insulin-like Growth Factor-1 Synergizes with IL-2 to Induce Homeostatic Proliferation of Regulatory T cells

IL-2 has been proposed to restore tolerance via regulatory T cell (Treg) expansion in autoimmunity, yet off-target effects necessitate identification of a combinatorial approach. We recently reported reduced levels of immunoregulatory insulin-like growth factor-1 (IGF1) during type 1 diabetes (T1D) progression. Thus, we hypothesized that IGF1 would synergize with IL-2 to expand Tregs. We observed IGF1R was elevated on murine memory and human naive Treg subsets. IL-2 and IGF1 promoted murine PI3K/Akt and human STAT5 signaling in Tregs. IL-2 and IGF1 treatment expanded Tregs beyond either agent alone in NOD mice. Incubation of naive human CD4+ T cells with IL-2 and IGF1 enhanced Treg proliferation in vitro, without the need for T cell receptor ligation. This synergism was attributed to increased high-affinity IL-2R expression on naive Tregs, in contrast to intermediate-affinity IL-2R{beta} and IL-2R{gamma} subunit enhancement on naive conventional T cells (Tconv). We then demonstrated that IGF1 and IL-2 or the IL2R{gamma}-chain-dependent cytokine, IL-7, can be used to induce proliferation of genetically-engineered naive Treg or Tconv cells, respectively. These data support the potential use of IGF1 in combination with common {gamma}-chain cytokines to drive T cell expansions both in vitro and in vivo for cellular therapeutics and genetic modifications.

immunology↗

bioRxiv2021

Driving Native-like Zonal Enthesis Formation in Engineered Ligaments Using Mechanical Boundary Conditions and β-Tricalcium Phosphate

Fibrocartilaginous entheses are structurally complex tissues that translate load from elastic ligaments to stiff bone via complex zonal organization with gradients in organization, mineralization, and cell phenotype. Currently, these gradients, necessary for long-term mechanical function, are not recreated in soft tissue-to-bone healing or engineered replacements, leading to high failure rates. Previously, we developed a culture system which guides ligament fibroblasts to develop aligned native-sized collagen fibers using high density collagen gels and mechanical boundary conditions. These constructs hold great promise as ligament replacements, however functional ligament-to-bone attachments, or entheses, are required for long-term function in vivo. The objective of this study was to investigate the effect of compressive mechanical boundary conditions and the addition of beta tricalcium phosphate ({beta}TCP), a known osteoconductive agent, on the development of zonal ligament-to-bone entheses. We found that compressive boundary clamps, that restrict cellular contraction and produce a zonal tensile-compressive environment, guide ligament fibroblasts to produce 3 unique zones of collagen organization, and zonal accumulation of glycosaminoglycans (GAGs), type II and type X collagen by 6 weeks of culture, ultimately resulting in similar organization and composition as immature bovine entheses. Further, {beta}TCP under the clamp enhanced the maturation of these entheses, leading to increased GAG accumulation, sheet-like mineralization, and significantly improved tensile moduli, suggesting the initiation of endochondral ossification. This culture system produced some of the most organized entheses to date, closely mirroring early postnatal enthesis development, and provides an in vitro platform to better understand the cues that drive enthesis maturation in vivo.

bioengineering↗

bioRxiv2020

An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.

immunology↗

bioRxiv2019

A dormant sub-population expressing interleukin-1 receptor characterises anti-estrogen resistant ALDH+ breast cancer stem cells

Estrogen receptor-positive (ER+) breast tumours are often treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer Stem Cells (CSCs) with high aldehyde dehydrogenase (ALDH) activity (ALDH+ cells) are reported to be enriched following AE treatment. Here we perform in vitro and in vivo functional CSC assays and gene expression analysis to characterise the ALDH+ population in AE resistant metastatic patient samples and an ER+ cell line. We show that the IL1{beta} signalling pathway is activated in ALDH+ cells and data from single cells reveals that AE treatment selects for IL1R1-expressing ALDH+ cells. Importantly, we demonstrate that increased expression of IL1R1 is observed in the tumours of patients treated with AE therapy and predicts for treatment failure. Single-cell gene expression analysis revealed that at least 2 sub-populations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy, the quiescent ALDH+IL1R1+ population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Supporting this, analysis of AE resistant dormant tumours reveals significantly increased expression of ALDH1A1, ALDH1A3 and IL1R1 genes. Thus, we propose that targeting of ALDH+IL1R1+ cells will reverse AE resistance, including in patients with minimal residual disease.

cancer biology↗