Biology subjects

Gordon, E.

Publications and source records attributed to Gordon, E..

17 recordsLinked to original sources

bioRxiv2026

Focused Ultrasound Blood-Tumor Barrier Opening Rapidly Augments Intratumor CD4 and CD8 T Cell Representation in a Genetically Engineered Mouse Model of Glioma

Glioblastoma (GBM) is a highly aggressive primary brain tumor that remains difficult to treat due in part to its disorganized and heterogeneous vasculature, known as the blood-tumor barrier (BTB), which limits therapeutic delivery and beneficial immune cell infiltration. Focused ultrasound (FUS) with microbubbles (MBs) can transiently disrupt the BTB to enhance drug delivery and may induce sterile inflammation (SI) that can beneficially remodel the tumor immune landscape. However, this concept has only been explored in implanted tumor models with modest immune effects. Here, we utilized a physiologically relevant genetically engineered mouse model (GEMM) generated via in utero electroporation targeting Nf1, Tp53, and Pten to study tumor-vascular-immune interactions. This 3x CRISPR-Cas9 GEMM recapitulates key features of human glioma, including infiltrative growth, histopathology, molecular alterations, and stage-dependent blood-brain barrier disruption. FUS+MBs were applied to transiently disrupt the BTB, and MRI confirmed increased vascular permeability in treated tumors. Flow cytometry revealed robust increases in tumor-infiltrating CD4+ helper and CD8+ effector T cells three days post-FUS treatment, without altering the CD8/Treg ratio. These findings were supported by immunofluorescence imaging. Double-negative and double-positive T cells were detected, but they were not significantly altered by FUS. Ki67 analysis indicated that increased T-cell accumulation was not driven by local proliferation. By seven days post-treatment, immune differences were no longer observed. Collectively, these results demonstrate that FUS-mediated BTB disruption selectively and rapidly enhances lymphocyte infiltration in a clinically relevant glioma model, supporting its potential as a temporally controlled immunomodulatory strategy for GBM.

cancer biology↗

bioRxiv2025

Artemis Regulates Homology-Independent Prime Editing (PRINS) for Enhanced Genomic Insertions

Nuclease-based prime editing (PEn) offers enhanced genomic insertion efficiency compared to nickase-based prime editors, but its reliance on double-strand break (DSB) repair leads to complex and often unpredictable on-target indel distributions. PRINS editing, a PEn variant utilizing springRNAs, uniquely relies on non-homologous end joining (NHEJ) for insertions, providing an insertion-only strategy ideal for functional protein tagging or serine integrase landing pad insertions, yet it suffers from inherent imprecision. Here, we identify the DNA repair factor Artemis (DCLRE1C) as a key regulator of PEn-generated indel profiles, particularly in springRNA-mediated PRINS editing. Through a targeted genetic screen, we show that the absence of Artemis significantly shifts indel distributions away from deletions and shorter truncations towards longer, functionally acceptable insertions. Our data indicates that Artemis cleaves PEn-generated 3-overhangs in a length-dependent manner, with its impact increasing for longer reverse-transcribed overhangs. This understanding reveals that regulating Artemis activity can improve insertion frequency specifically for PRINS. We develop and validate robust epigenetic (CRISPRoff) and antisense oligonucleotide (ASO) strategies to effectively silence/knockdown Artemis expression, successfully recapitulating the beneficial PRINS editing outcomes observed in Artemis-deficient cells. Leveraging these insights, we show that Artemis modulation can enhance endogenous protein tagging in cells, including challenging hiPSC-derived non-dividing cardiomyocytes. Our findings support Artemis as a key regulator of PRINS editing outcomes and present a tunable strategy to optimize insertion efficiency for diverse genomic engineering and therapeutic applications.

molecular biology↗

bioRxiv2025

Surface avidity of anionic polypeptide coatings target nanoparticles to cancer-associated amino acid transporters

Tumor-targeted drug delivery enhances therapeutic efficacy while minimizing toxicity. Layer-by-layer nanoparticles (LbL-NPs) coated with anionic polypeptides selectively bind to cancer cells, though the mechanisms have been unclear. Here, we integrated in silico and in vitro approaches--including gene expression analysis, receptor inhibition, and AI-based protein modeling--to show that poly(L-glutamate) (PLE)-coated LbL-NPs bind with high avidity to SLC1A5, a glutamine transporter overexpressed in cancer. We also discovered that PLE clusters SLC1A5 on the cell membrane, promoting prolonged cell surface retention. Poly(L-aspartate) (PLD)-coated NPs similarly bind SLC1A5 but also interact with faster internalizing transporters of anionic amino acids. Correlation analyses across cancer cell lines confirmed a strong link between transporter expression and nanoparticle association. These findings demonstrate that dense glutamate or aspartate presentation through electrostatically adsorbed polypeptides enables selective targeting of overexpressed transporters, providing a mechanistic framework for receptor-targeted delivery that leverages metabolic characteristics of a range of solid tumor types.

bioengineering↗

bioRxiv2025

Optimised genome editing for precise DNA insertion and substitution using Prime Editors in zebrafish

CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase-and a nuclease-based PE to perform programmed short DNA substitution and insertion in various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA. Prime editing, CRISPR/Cas9, zebrafish, PE2, PEn

molecular biology↗

bioRxiv2024

'Target-and-release' nanoparticles for effective immunotherapy of metastatic ovarian cancer

Immunotherapies such as checkpoint inhibitors (CPI) are effective in treating several advanced cancers, but these treatments have had limited success in metastatic ovarian cancer (OC). Here, we engineered liposomal nanoparticles (NPs) carrying a layer-by-layer (LbL) polymer coating that promotes their binding to the surface of OC cells. Covalent anchoring of the potent immunostimulatory cytokine interleukin-12 (IL-12) to phospholipid headgroups of the liposome core enabled the LbL particles to concentrate IL-12 in disseminated OC tumors following intraperitoneal administration. Shedding of the LbL coating and serum protein-mediated extraction of IL-12-conjugated lipids from the liposomal core over time enabled IL-12 to disseminate in the tumor bed following rapid NP localization in tumor nodules. Optimized IL-12 LbL-NPs promoted robust T cell accumulation in ascites and tumors in mouse models, extending survival compared to free IL-12 and remarkedly sensitizing tumors to CPI, leading to curative treatments and immune memory.

bioengineering↗

bioRxiv2024

Endothelial c-Src mediates neovascular tuft formation in Oxygen-Induced Retinopathy

IntroductionVascular retinopathy, characterised by abnormal blood vessel growth in the retina, frequently results in vision impairment or loss. Neovascular tufts, a distinctive pathological feature of this condition, are highly leaky blood vessel structures, exacerbating secondary complications. Despite their clinical significance, the mechanisms underlying tuft development are not fully elucidated, posing challenges for effective management and treatment of vascular retinopathy. In this study, we investigate the role of c-Src in neovascular tuft formation. Although c-Src has been acknowledged as a pivotal regulator in developmental angiogenesis within the retinal vasculature, its specific role in governing pathological retinal angiogenesis remains to be fully understood. MethodsWe utilised the Oxygen-Induced Retinopathy (OIR) model to induce the formation of neovascular tufts in both Cre-mediated vascular specific c-Src knockout mice and their wildtype littermates. Subsequently, we conducted high-resolution imaging and analysis of isolated retinas, to elucidate the precise role of c-Src in the formation of vascular tufts. Resultsc-Src depletion demonstrated a significant reduction in the formation of neovascular tufts within the OIR model, underscoring the pivotal role of c-Src in pathological retinal angiogenesis. Notably, this decrease in tuft formation was observed independently of any alterations in cell death, cell proliferation or cell adhesion and the absence of c-Src did not impact tuft pericyte coverage and junctional morphology. ConclusionThese findings underscore the critical role of c-Src in the pathogenesis of neovascular tufts in vascular retinopathy. Understanding the molecular mechanisms involving c-Src may offer valuable insights for the development of targeted therapies aimed at mitigating vision-threatening complications associated with retinopathy.

pathology↗

bioRxiv2024

Improved nuclease-based prime editing by DNA repair modulation and pegRNA engineering

Prime editing is a genome engineering tool that allows installation of small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing nuclease (PEn) utilizes a fully active Cas9 instead of the nickase employed in conventional prime editors. PEn is capable of editing sites resistant to nickase-based prime editors but induces more undesired editing events. In this work, we introduce two strategies to enhance PEn precision and efficiency. First, we apply a small molecule approach, selectively modulating DNA repair pathways, to improve PEn precision up to 9.8-fold and reduce off-target editing by 90%. Second, through pegRNA engineering, we devise a strategy that mitigates unintended pegRNA scaffold integration, which is a common prime editing by-product, enhancing precision up to 3.5-fold. We apply this approach to a specific type of PEn editing mediated through non-homologous end joining and use it to achieve efficient and precise prime editing in multiple human cell lines, primary human hepatocytes, and mouse embryos. Together, this work presents two general strategies to improve prime editing, overcomes the limitations of current PEn editors, and provides reliable and precise genome editing outcomes, a pivotal requirement for therapeutic applications.

bioengineering↗

bioRxiv2023

c-Src induced vascular malformations require localised matrix degradation at focal adhesions

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

cell biology↗

bioRxiv2022

memento: Generalized differential expression analysis of single-cell RNA-seq with method of moments estimation and efficient resampling

Differential expression analysis of scRNA-seq data is central for characterizing how experimental factors affect the distribution of gene expression. However, it remains challenging to distinguish biological and technical sources of cell-cell variability and to assess the statistical significance of quantitative comparisons between groups of cells. We introduce memento to address these limitations and enable accurate and efficient differential expression analysis of the mean, variability, and gene correlation from scRNA-seq. We used memento to analyze 70,000 tracheal epithelial cells to identify interferon response genes with distinct variability and correlation patterns, 160,000 T cells perturbed with CRISPR-Cas9 to reconstruct gene-regulatory networks that control T cell activation, and 1.2 million PMBCs to map cell-type-specific cis expression quantitative trait loci (eQTLs). In all cases, memento identified more significant and reproducible differences in mean expression but also identified differences in variability and gene correlation that suggest distinct modes of transcriptional regulation imparted by cytokines, genetic perturbations, and natural genetic variation. These results demonstrate memento as a first-in-class method for the quantitative comparisons of scRNA-seq data scalable to millions of cells and thousands of samples.

bioinformatics↗

bioRxiv2022

Comparative analyses of the N-glycoproteomes in HCT116 cancer cells and their non-tumorigenic DNMT1/3b double knockout (DKO1) cells and insight into the Mannose-6-phosphate pathway

N-glycoproteomic analyses provide valuable resources for investigation of cancer mechanisms, biomarkers, and therapeutic targets. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 display hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells.

biochemistry↗

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

Cortical actin flow activates an alpha-catenin clutch to assemble adherens junctions

Adherens junctions allow cell contact to inhibit epithelial migration. But a long-standing puzzle is how locomotion is downregulated when E-cadherin adhesions form at surfaces perpendicular, but not those parallel, to the direction of migration. We now show that this arises from coupling between E-cadherin adhesions and the retrograde cortical flows of leader cells in migrating epithelia. At interfaces perpendicular to the direction of motion, such flows are antiparallel, which generates a tensile signal that induces the actin-binding domain of -catenin to promote lateral growth of nascent adhesions and inhibit the lamellipodial activity necessary for migration. At interfaces parallel to the direction of motion, by contrast, cortical flows are aligned and no such mechanical inhibition takes place. Therefore, -catenin mechanosensitivity in the clutch between E-cadherin and cortical F-actin allows cells to interpret the direction of motion via cortical flows and trigger the first signal for contact to inhibit locomotion.

cell biology↗

bioRxiv2021

Tissue-Specific Biochemical Differences Between Chronic Wasting Disease Prions Isolated From Free Ranging, White-Tailed Deer (Odocoileus virginianus)

Chronic wasting disease (CWD) is an invariably fatal prion disease affecting cervid species world-wide. Prions can manifest as distinct strains that can influence disease pathology and transmission. CWD is profoundly lymphotropic and most infected cervids likely shed peripheral prions replicated in lymphoid organs. However, CWD is a neurodegenerative disease and most research on prion strains has focused on neurogenic prions. Thus, a knowledge gap exists comparing prions in the brain to prions in the lymph node. In this study we compared prions from the obex and lymph node of naturally exposed white-tailed deer to identify potential biochemical strain differences. Here, we report biochemical evidence of strain differences between the brain and lymph node from these animals. Future work should examine the biological and zoonotic impact of these biochemical differences and examine more cervids from multiple locations to see if these differences are conserved across species and locations.

pathology↗

bioRxiv2021

Individual-Specific Areal-Level Parcellations Improve Functional Connectivity Prediction of Behavior

Resting-state functional MRI (rs-fMRI) allows estimation of individual-specific cortical parcellations. We have previously developed a multi-session hierarchical Bayesian model (MS-HBM) for estimating high-quality individual-specific network-level parcellations. Here, we extend the model to estimate individual-specific areal-level parcellations. While network-level parcellations comprise spatially distributed networks spanning the cortex, the consensus is that areal-level parcels should be spatially localized, i.e., should not span multiple lobes. There is disagreement about whether areal-level parcels should be strictly contiguous or comprise multiple non-contiguous components, therefore we considered three areal-level MS-HBM variants spanning these range of possibilities. Individual-specific MS-HBM parcellations estimated using 10min of data generalized better than other approaches using 150min of data to out-of-sample rs-fMRI and task-fMRI from the same individuals. Resting-state functional connectivity (RSFC) derived from MS-HBM parcellations also achieved the best behavioral prediction performance. Among the three MS-HBM variants, the strictly contiguous MS-HBM (cMS-HBM) exhibited the best resting-state homogeneity and most uniform within-parcel task activation. In terms of behavioral prediction, the gradient-infused MS-HBM (gMS-HBM) was numerically the best, but differences among MS-HBM variants were not statistically significant. Overall, these results suggest that areal-level MS-HBMs can capture behaviorally meaningful individual-specific parcellation features beyond group-level parcellations. Multi-resolution trained models and parcellations are publicly available (https://github.com/ThomasYeoLab/CBIG/tree/master/stable_projects/brain_parcellation/Kong2022_ArealMSHBM).

neuroscience↗

bioRxiv2020

Defective NETs Clearance contributes to sustained FXII Activation in COVID-19-associated Pulmonary Thrombo-Inflammation

BackgroundCoagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. MethodsWe performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced Factor XIII (FXII) activation using a chromogenic substrate assay. FindingsFXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasma FXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro. InterpretationCollectively, our study supports that the NETs/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both, NETs and FXIIa, could provide a strategy to mitigate COVID-19-induced thrombo-inflammation. FundingThis study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306).

molecular biology↗

bioRxiv2020

Multiple known and a novel parvovirus associated with an outbreak of feline diarrhea and vomiting

An unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and PCR. We characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by 8/17 affected cats and different feline bocaviruses shed by 9/17 cats. Also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. Epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease.

microbiology↗

bioRxiv2017

R2DGC: Threshold-free peak alignment and identification for 2D gas chromatography mass in R

Summary: Comprehensive two dimensional gas chromatography-mass spectrometry is a powerful method for analyzing complex mixtures of volatile compounds. This method produces a large amount of raw data that requires downstream processing to align signals of interest (peaks) across multiple samples and match peak characteristics to reference standard libraries prior to downstream statistical analysis. To address the paucity of applications addressing this need, we have developed an R package that implements retention time and mass spectra similarity threshold-free alignments, seamlessly integrates retention time standards for universally reproducible alignments, performs common ion filtering, and provides compatibility with multiple peak quantification methods. We demonstrate the packages utility on a controlled mix of metabolite standards separated under variable chromatography conditions and data generated from cell lines.\n\nAvailability and documentation: R2DGC can be downloaded at https://github.com/rramaker/R2DGC or installed via the Comprehensive R Archive Network (CRAN).\n\nContact: sjcooper@hudsonalpha.org\n\nSupplementary information: Supplementary data are available at Bioinformatics online.

bioinformatics↗