Biology subjects

Burgess, A.

Publications and source records attributed to Burgess, A..

11 recordsLinked to original sources

bioRxiv2026

CD19 CAR Engineered T Regulatory Cells Promote Durable Immune Tolerance in Experimental Systemic Lupus Erythematosus

While the immune reset induced by CD19 CAR T therapies has shown striking promise in systemic lupus erythematosus (SLE), the nature and duration of benefit mediated by B cell depletion remains unclear. Loss of tolerance in SLE is driven by extrafollicular and germinal center interactions between dendritic cells and self-reactive T and B cells. We hypothesized that CD19 CAR-engineered regulatory T cells (CAR19 EngTreg), capable of suppressing multiple autoimmune effector cell types, might promote long-term tolerance without producing high-levels of inflammatory mediators. To generate CAR19 EngTreg, we combined CD19 CAR delivery with homology directed repair at the FOXP3 locus to drive constitutive, stable FOXP3 expression in CD4 T cells. CAR19 EngTreg expressed canonical Treg markers; exhibited minimal inflammatory cytokine production; and blocked human B cell differentiation into antibody secreting plasma cells. In humanized mouse models, transfer of CAR19 EngTreg led to a reduction in B cell numbers with low inflammatory cytokine production relative to CD19 CAR Teff, and reduced tissue inflammation. In a short-term, in vivo imiquimod-induced inflammatory model, murine CAR19 EngTreg reduced skin pathology, spleen size, and inflammatory cytokines. Finally, in two distinct mouse models of lupus, CAR19 EngTreg persisted for >4 months leading to reduced autoimmune T and B effector populations, sustained reduction in multiple autoantibodies and improved renal function and histopathology. Thus, CAR19 EngTreg may offer a promising and durable therapeutic avenue for B cell-mediated autoimmune disorders capable of long-term regulation of both autoreactive B and T cells.

immunology↗

bioRxiv2026

Direct visualization of lysosome-mediated intracellular transport and membrane anchoring of TNF-α in human T cells

Tumor necrosis factor-alpha (TNF-) represents a major pro-inflammatory cytokine associated with chronic inflammation. Enhanced understanding of intracellular trafficking of TNF- enables the identification of drug targets that modulate TNF- secretion at the cellular source. However, conventional cytokine assays provide only bulk measurements of secreted TNF- or static measurements in fixed cells and therefore cannot provide mechanistic information. Here, we report a fluorescence imaging platform to visualize and quantify intracellular TNF- trafficking in human T cells. Using a Jurkat T cell line stably expressing TNF- fused to mStayGold as a model system, we revealed through single-particle tracking (SPT) that T cell activation increased the motility of TNF- vesicles and that microtubule perturbation disrupted this increased motility. 3D structured illumination microscopy uncovered spatial associations between TNF- vesicles and lysosomes, and SPT further demonstrated their coordinated transport. Using total internal reflection fluorescence microscopy, we captured the sub-second splitting of single TNF- vesicles into multiple puncta that subsequently separated, suggesting delivery of TNF- from the vesicle to the plasma membrane. Confocal-based super-resolution microscopy further resolved TNF- vesicles fusing with the plasma membrane and transmembrane TNF-. Together, these imaging capabilities for tracking and super-resolving the intracellular transport and membrane delivery of TNF- in live T cells enable mechanistic studies of TNF- trafficking and release.

biophysics↗

bioRxiv2026

Neonatal Muscle Tone Predicts Cerebellar Morphology Later in Development Without Mediating Autistic Traits

Background: Infant muscle tone reflects early central nervous system integrity and has been associated with later motor and cognitive development, including autism traits. The cerebellum regulates both motor control and higher-order socio-cognitive functions and has been repeatedly implicated in autism, but its role in linking infant muscle tone to adolescent autistic traits has not previously been studied in a large, prospective population cohort. Methods: We used data from the prospective Generation R Study. Infant muscle tone (hypotonia and hypertonia) was assessed via Prechtl examination, and third-trimester fetal transcerebellar diameter was measured using ultrasound n=6,842). Cerebellar morphology at ages 6, 10, and 14 years (n=4,861) was measured using structural MRI. Linear mixed-effects models tested associations between infant muscle tone and 35 anatomical and 10 functional cerebellar regions. Causal mediation models tested whether cerebellar volume mediated associations between infant muscle tone and adolescent autistic traits at age 14 (Social Responsiveness Scale). Results: Hypotonia predicted larger vermis IX volumes across childhood (beta=0.037, pFDR =0.043). Hypertonia showed an age-dependent association with left lateral lobule IX (beta=-0.0027, pFDR =0.041), with differences diminishing with age. Third-trimester transcerebellar diameter did not predict postnatal muscle tone. Given its significant main effect, vermis IX volume was tested as a mediator, but did not mediate the pathway to adolescent autistic traits. However, infant hypotonia showed a small direct association with elevated autistic traits at age 14, specific to girls (beta=0.0255, p=0.020). Conclusions: Infant muscle tone is associated with localized differences in cerebellar volumes. These associations are specific to vermal and left hemispheric lobule IX, a region commonly implicated in spinocerebellar postural control, axial stability, and higher-order sensorimotor integration. Furthermore, infant muscle tone was not predicted by prenatal cerebellar diameter, and cerebellar volumes did not mediate the association between infant hypotonia and adolescent autistic traits in our study. Future research should further investigate these findings in clinical populations, integrating longitudinal whole-brain, multi-modal imaging to clarify the association between infant muscle tone, the cerebellar functioning, and autistic traits.

neuroscience↗

bioRxiv2023

CTCF is essential for proper mitotic spindle structure and anaphase segregation

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus.

cell biology↗

bioRxiv2022

The in-tissue molecular architecture of β-amyloid pathology in the mammalian brain.

Amyloid plaques composed of extracellular focal deposition of A{beta} fibrils are a hallmark of Alzheimers disease (AD). Cryo-EM structures of A{beta} fibrils purified from human AD brain tissue post mortem have recently been determined. However, the molecular architecture of amyloid plaques in the context of fresh, unfixed mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the native, in situ molecular architecture of A{beta} fibrils in the brain of a mouse model containing the Arctic familial AD mutation (AppNL-G-F) and an atomic model of Arctic A{beta} fibril purified from the brains of these animals. We show that in-tissue A{beta} fibrils are arranged in a lattice or in parallel bundles within a plaque, and are interdigitated by subcellular compartments, exosomes, extracellular droplets and extracellular multilamellar bodies. At the atomic level, the Arctic A{beta} fibril differs significantly from earlier structures of A{beta} amyloid extracted from AppNL-F mice models and human AD brain tissue, showing a striking effect of the Arctic mutation (E22G) on fibril structure. Cryo-electron tomography of ex vivo purified and in-tissue amyloid revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises {beta}-amyloid plaque pathology.

neuroscience↗

bioRxiv2021

A Fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis.

The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine, appears to be attractive target for anti-Tuberculosis (TB) drug discovery. Starvation of M. tuberculosis deleted for either argB or argF genes led to rapid sterilization of these strains in mice while Chemical inhibition of ArgJ with Pranlukast was also found to clear chronic M. tuberculosis infection in animal models. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is explored using a fragment-based approach. We reveal several hits for these enzymes validated with biochemical and biophysical assays, and X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential of more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

biochemistry↗

bioRxiv2021

Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis

Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a critical regulator of cytokinesis predominantly in transformed cells and its deregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals have not been clearly defined. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated perinatal lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3{beta}/{beta}-Catenin/Myc axis in an Akt-dependent manner. The phenotype was recapitulated using human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3{beta}. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting its novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.

cell biology↗

bioRxiv2020

Resistance to platinum chemotherapy in lung adenocarcinoma is driven by a non-genetic, cell-cycle dependent mechanism

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here we advanced this model system and identified a non- genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

cell biology↗

bioRxiv2019

Cep55 overexpression promotes genomic instability and tumorigenesis in mice

High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies and association of aberrantly overexpressed CEP55 with worse prognosis, its causal role in vivo tumorigenesis remains elusive. Here, using a ubiquitously overexpressing transgenic mouse model, we show that Cep55 overexpression causes spontaneous tumorigenesis and accelerates Trp53+/- induced tumours in vivo. At the cellular level, using mouse embryonic fibroblasts (MEFs), we demonstrate that Cep55 overexpression induces proliferation advantage by modulating multiple cellular signalling networks including the PI3K/AKT pathway. Notably, the Cep55 overexpressing MEFs demonstrate high level of mitotic chromosomal instability (CIN) due to stabilized microtubules. Interestingly, Cep55 overexpressing MEFs have a compromised Chk1-dependent S-phase checkpoint, causing increased replication speed and DNA damage, resulting in a prolonged aberrant mitotic division. Importantly, this phenotype was rescued by pharmacological inhibition of Pi3k/Akt or expression of mutant Chk1 (S280A), that is insensitive to regulation by active AKT, in Cep55 overexpressing cell. Collectively, our data demonstrates causative effects of deregulated Cep55 on genome stability and tumorigenesis which have potential implications for tumour initiation and therapy.

cancer biology↗

bioRxiv2019

Dynamic analysis of pulsed cisplatin identifies effectors of resistance in lung adenocarcinoma

Identification of clinically viable strategies for overcoming resistance to platinum chemotherapy in lung adenocarcinoma has been hampered by inappropriately tailored in vitro assays of drug response. Therefore, using a pulse model that closely recapitulates the in vivo pharmacokinetics of platinum therapy, we profiled cisplatin-induced signalling, DNA damage and apoptotic responses across a panel of lung adenocarcinoma cell lines. By coupling this data with real-time, single cell imaging of cell cycle and apoptosis, we show that TP53 mutation status influenced the mode of cisplatin induced cell cycle arrest, but could not predict cisplatin sensitivity. In contrast, P70S6K-mediated signalling promoted resistance by increasing p53/p63 and p21 expression, reducing double-stranded DNA breaks and apoptosis. Targeting P70S6K sensitised both TP53 wildtype and null lines to cisplatin, but not TP53 mutant lines. In summary, using in vitro assays that mimic in vivo pharmacokinetics identified P70S6K as a robust mediator of cisplatin resistance and highlighted the importance of considering somatic mutation status when designing patient-specific combination therapies.

cancer biology↗

bioRxiv2016

The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia formation via ubiquitylation of CSPP-L.

Primary cilia are crucial for signal transduction in a variety of pathways, including Hedgehog and Wnt. Disruption of primary cilia formation (ciliogenesis) is linked to numerous developmental disorders (known as ciliopathies) and diseases, including cancer. The Ubiquitin-Proteasome System (UPS) component UBR5 was previously identified as a putative modulator of ciliogenesis in a functional genomics screen. UBR5 is an E3 Ubiquitin ligase that is frequently deregulated in tumours, but its biological role in cancer is largely uncharacterised, partly due to a lack of understanding of interacting proteins and pathways. We validated the effect of UBR5 depletion on primary cilia formation using a robust model of ciliogenesis, and identified CSPP1, a centrosomal and ciliary protein required for cilia formation, as a UBR5-interacting protein. We show that UBR5 ubiquitylates CSPP1, and that UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery. Hence, we have established a key role for UBR5 in ciliogenesis that may have important implications in understanding cancer pathophysiology.

cell biology↗