Biology subjects

McConnell, M. J.

Publications and source records attributed to McConnell, M. J..

11 recordsLinked to original sources

bioRxiv2025

A new genetically engineered transplant model of glioma recapitulates key phenotypes of low- and high-grade gliomas

Immune competent animal models are essential in preclinical glioma research. The ability to investigate tumor development with key tumor microenvironment components such as immune infiltration, stromal cells and extracellular matrix allows for the investigation of these complex tumors. However, the current range of syngeneic models of glioma possess intrinsic limitations which must be acknowledged when designing a preclinical study. To address this gap, we developed genetically engineered mouse cell line models (GEM-CLeMs) by introducing common glioma driver mutations into immortalized astrocytes. A high-grade glioma model was generated by combining Pten knockdown with RAS V12 overexpression, while a low-grade glioma model was produced through p53 knockdown with mutant IDH1R132H overexpression. The RAS/Pten GEM-CLeM tumors grew rapidly in vivo, displayed necrosis, multinucleated pleomorphisms, abundant vascularization, and showed strong enrichment for extracellular matrix remodelling and mesenchymal genes, features closely aligned with human glioblastoma. Importantly, the RAS/Pten GEM-CLeM tumors showed similar survival trends and immune infiltration patterns as a corresponding KrasG12D/PtencKO GEMM, but could be grown to larger sizes, facilitating better stromal and immune analyses. In contrast, the IDH1R132H/p53 GEM-CLeM formed slow-growing tumors with distinctive immune infiltration and vascular patterns, consistent with low-grade glioma phenotypes. Compared with the commonly used cell line GL261, the GEM-CLeM tumors had higher levels of stromal integration and immune suppression, making them a more faithful model of the glioma tumor microenvironment. This system enables rapid generation of transplantable glioma models with defined driver mutations in a low-mutational background, offering a flexible platform for dissecting glioma biology and evaluating immunotherapies. Importance of the studyWe have generated a set of customizable, modular Genetically Engineered Mouse Cell Line Models (GEM-CLeMs) of "high grade" and "low grade" glioma. They reliably form tumors when transplanted intracranially into immune-competent C57BL/6 mice, and they are cost- and time-effective at capturing the important characteristics of glioma, both mutant IDH1 low grade glioma and high grade glioblastoma. Critically these characteristics include the myeloid-rich immune suppressive tumor immune microenvironment, a key weakness of existing murine glioma cell lines like GL261. These GEM-CLeM models can be used in multiple ways. The cells are amenable to further manipulation, so therapeutic targets and drug mechanism of action can be assessed. The activity of candidate genes in tumour formation and phenotype can be determined. Most importantly the models can be used to develop effective immunotherapies, including strategies to target macrophage and myeloid cell immune suppression. Key pointsO_LICombinations of driver mutations were engineered into an immortalised mouse astrocyte. C_LIO_LIEngineered cells formed tumours on intracranial transplant into immune competent mice. C_LIO_LITumors had key histological and immune suppressive features of human glioma. C_LI

cancer biology↗

bioRxiv2025

Oxidative stress facilitates a diverse adaptive response in bacteria

The adaptive oxidative stress response allows bacteria to adapt to hostile environmental conditions. We explored how the evolution of cellular ROS in oxidative stress triggers physiological and genetic changes in multiple pathogenic species. Our study posits that growth-survival tradeoffs under oxidative stress enact a diverse array of genetic and physiological changes resulting in cross-resistance to acid, antimicrobial resistance, and metabolic reprogramming in bacteria.

microbiology↗

bioRxiv2025

Hepatic Arterial Flow-Induced Portal Tract Fibrosis in Portal Hypertension: The Role of VCAM-1 and Osteopontin-Expressing Macrophages

BackgroundThe liver undergoes significant hemodynamic changes during surgery, transplantation, or cirrhosis with portal hypertension(PH). The hepatic artery buffer response(HABR), which compensates for reduced portal venous flow by increasing hepatic artery(HA) flow, is hypothesized to induce pathological portal tract remodeling. This study investigates the molecular mechanisms underlying this process. MethodsPH was induced in Sprague-Dawley rats via partial portal vein ligation(PPVL). Structural evaluation(microCT), immune cell profiling, hemodynamic measurements, and transcriptomic analysis in macrophages(M{phi}) from sham or PPVL rats were conducted. ResultsMicroCT revealed decreased portal vein flow and increased HA flow correlated with portal pressure(r=0.799, p<0.01). A 2-fold increase in portal tract fibrosis(p<0.001) was observed with increased -SMA+ myofibroblasts in PPVL rats. CD68+ M{phi} peaked at 10 days post-PPVL, and their depletion significantly reduced fibrosis(p<0.001), indicating critical roles of M{phi} in portal tract remodeling. VCAM-1 was elevated in HA endothelium and portal fibroblasts (PFs); VCAM-1 neutralization reduced collagen accumulation(p<0.05), CD68+ M{phi}(46.3%, p<0.01), and CD3+ T cells(18%, p<0.05). M{phi}-conditioned medium increased VCAM-1 in PFs(8-fold, p<0.001) and enhanced PF migration, while VCAM-1 knockdown reduced this effect (p<0.01). Single-cell RNA sequencing data(GSE171904) and RNA-FISH revealed increased interactions between osteopontin (Spp1)+ M{phi} and PFs, with Spp1+ M{phi} driving fibrosis. Spp1 knockdown in M{phi} co-culture reduced PF fibrogenic markers, while recombinant Spp1 upregulated Col1a1, Fn1, and Acta2 expression in PFs. ConclusionIncreased VCAM-1 in arterial endothelial cells and PFs facilitates the recruitment of Spp1+ M{phi}, which drive HA flow-mediated vascular remodeling and portal tract fibrosis. These findings highlight arterial flow-induced fibrosis as a key mechanism in PH, potentially contributing to disease progression and decompensation. SynopsisLiver hemodynamic changes in portal hypertension drive extracellular matrix accumulation and portal tract remodeling via Spp1+ macrophages. This study highlights how altered blood flow induces fibrosis, and its potential role in decompensation, and identifies therapeutic targets for advanced liver disease.

pathology↗

bioRxiv2025

Restoration of mitochondrial function to metabolically stressed astrocytes by fusion with mesenchymal stromal cells.

Mitochondrial networks in eukaryotic cells are dynamic, undergoing fusion, fission, biogenesis and transfer between cells to manage cellular requirements for energy production and biosynthetic components. Mitochondrial transfer has been observed in response to cellular injury and can happen by a variety of mechanisms. To examine whether mitochondria could be restored to injured astrocytes, we established a model of discrete astrocyte-specific, mitochondrial-specific injury in a mixed culture model. Immortalized {rho}0 astrocytes were generated, then put under lethal metabolic stress by withdrawal of pyruvate and uridine. When cells stressed in this way were grown in the presence of mesenchymal stromal cells, enhanced survival of the {rho}0 cells was seen, accompanied by the rapid acquisition of an entire mitochondrial networks. Gap-fill ligation and rolling circle amplification for cell-specific mitochondrial genome SNP confirmed that the mitochondrial networks were derived from the mesenchymal stromal donor cells, and both mitochondrial DNA and mitochondrial function were restored to the p0 cells long-term. The presence of two nuclei in the {rho}0 cells in the early stages of mitochondrial transfer and recovery was observed. This demonstrated that in this instance, the mechanism of mitochondrial rescue was fusion of the {rho}0 astrocyte and the mesenchymal stromal cell. In support of this, other neural cells were similarly shown to fuse with stressed {rho}0 astrocytes, suggesting that whole organelles may be donated to restore critical mitochondrial function.

cell biology↗

bioRxiv2025

Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

The liver lymphatic system plays a critical role in maintaining interstitial fluid balance and immune regulation. Efficient lymphatic drainage is essential for liver homeostasis, but its role in liver disease progression remains poorly understood. In cirrhosis, lymphangiogenesis initially compensates for increased lymph production, but impaired lymphatic drainage in advanced stages may lead to complications such as ascites and portal hypertension. This study aimed to evaluate how liver lymphatic dysfunction affects disease progression and to assess therapeutic strategies. Using a surgical model to block liver lymphatic outflow, we found that impaired drainage accelerates liver injury, fibrosis, and immune cell infiltration, even in healthy livers. Mechanistically, enhanced TGF-{beta} signaling in liver lymphatic endothelial cells (LyECs) contributed to reduced lymphatic vessel (LV) density and function in late-stage decompensated cirrhosis. This dysfunction was linked to the progression from compensated to decompensated cirrhosis, particularly in patients with primary sclerosing cholangitis (PSC). Conversely, liver-specific overexpression of VEGF-C via AAV8 improved lymphatic drainage, restored LV density, reduced fibrosis, mitigated liver injury, and alleviated portal hypertension in cirrhotic rats. These findings establish impaired liver lymphatic function as a pivotal driver of cirrhosis progression and identify VEGF-C as a promising therapeutic target to prevent decompensation.

pathology↗

bioRxiv2024

A personalized multi-platform assessment of somatic mosaicism in the human frontal cortex

Somatic mutations in individual cells create genomic mosaicism, influencing genetic disorders and cancers. While clonal mutations in cancers are well-studied, rarer somatic variants in normal tissues remain poorly characterized. This study systematically evaluates detection methods using a personalized donor-specific assembly (DSA) from a neurotypical individuals dorsolateral prefrontal cortex assessed with Oxford Nanopore, NovaSeq, linked-read sequencing, Cas9-targeted long-read sequencing (TEnCATS), and single-neuron MALBAC amplification. The haplotype-resolved DSA improved cross-platform analysis, dramatically increasing phasing rates. Germline SNVs, structural variations (SVs), and transposable elements (TEs) were recalled with 99.4%-99.7% accuracy in bulk tissue, and phased haplotype analysis reduced false positives by 15.4%-75.1% for putative somatic candidates. Long-read single-neuron sequencing detected nine somatic SV candidates, demonstrating enhanced sensitivity for rare variants, while TEnCATS identified eight low-frequency somatic TE candidates. These findings highlight advanced methodologies for precise somatic variant detection, critical for understanding mosaicisms role in health and disease.

genomics↗

bioRxiv2023

BCL6 is a context-dependent mediator of the glioblastoma response to irradiation therapy

Glioblastoma is a rapidly fatal brain cancer with no cure. The resistance of glioblastoma tumours to available therapies means that more effective treatments are desperately needed. Previous research showed that the transcriptional repressor protein BCL6 is upregulated by chemo- and radiotherapy in glioblastoma and that inhibition of BCL6 enhances the effectiveness of these therapies. Therefore, BCL6 is a promising target to improve the efficacy of available treatments for glioblastoma. BCL6 is known as a transcriptional repressor in germinal centre B cells and is an oncogene in lymphoma, as well as in other cancers. However, previous research indicated that BCL6 induced by chemotherapy or irradiation in glioblastoma may not act as a transcriptional repressor. This study aimed to clarify the role of BCL6 in the response of glioblastoma to irradiation. The effect of BCL6 inhibition on the whole proteome response of glioblastoma cells to fractionated and acute irradiation treatment was investigated. Acute irradiation appeared to cause BCL6 to switch from a repressor of the DNA damage response to a promoter of stress response signalling. Rapid immunoprecipitation mass spectrometry of endogenous proteins enabled identification of proteins associated with BCL6 in untreated and irradiated glioblastoma cells. BCL6 associated with transcriptional coregulators in untreated glioblastoma and its association with the corepressor NCOR2 was validated using proximity ligation assays. However, the association of BCL6 with transcriptional regulatory proteins was lost in response to acute irradiation. This was accompanied by the irradiation-induced association of BCL6 with synaptic and plasma membrane proteins. Overall, these results reveal that the activity of BCL6 in glioblastoma therapy responses is context-dependent and may be mediated by the intensity of cellular stress.

cancer biology↗

bioRxiv2023

Mapping the Complex Genetic Landscape of Human Neurons

When somatic cells acquire complex karyotypes, they are removed by the immune system. Mutant somatic cells that evade immune surveillance can lead to cancer. Neurons with complex karyotypes arise during neurotypical brain development, but neurons are almost never the origin of brain cancers. Instead, somatic mutations in neurons can bring about neurodevelopmental disorders, and contribute to the polygenic landscape of neuropsychiatric and neurodegenerative disease. A subset of human neurons harbors idiosyncratic copy number variants (CNVs, "CNV neurons"), but previous analyses of CNV neurons have been limited by relatively small sample sizes. Here, we developed an allele-based validation approach, SCOVAL, to corroborate or reject read-depth based CNV calls in single human neurons. We applied this approach to 2,125 frontal cortical neurons from a neurotypical human brain. This approach identified 226 CNV neurons, as well as a class of CNV neurons with complex karyotypes containing whole or substantial losses on multiple chromosomes. Moreover, we found that CNV location appears to be nonrandom. Recurrent regions of neuronal genome rearrangement contained fewer, but longer, genes.

neuroscience↗

bioRxiv2021

Predicted Impact of the Viral Mutational Landscape on the Cytotoxic Response against SARS-CoV-2

The massive assessment of immune evasion due to viral mutations that potentially increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Potential epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 HLA class I families. Allele families showed extreme differences in number of recognized epitopes, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Among them, the B27 supertype showed the lowest number of epitopes. Epitope escape mutations identified in ~118,000 NCBI isolates mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% evaded epitopes for the same supertype in the same isolate, except for B27, which reached up to 33%. In contrast to most supertypes, eight particular allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles. Collectively, these data indicate that independent escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. This study highlights the automated integration of genomic, geographical and immunoinformatic information for surveillance of SARS-CoV-2 variants potentially affecting the population as a whole, as well as minority subpopulations. AUTHOR SUMMARYThe cytotoxic T response, a type of immune response dependent upon an individual's genetics that does not require antibodies, is critical for neutralizing SARS-CoV-2 infection. The potential bypass of the cytotoxic T response by mutations acquired by the virus after one year of the pandemic is therefore of maximal concern. We have approached the complexity of human variability and more than 100.000 viral genomes in this respect using a computational strategy. We have detected numerous mutations in these genomes that mask some viral regions involved in the cytotoxic response. However, the accumulation of these changes in independent isolates is still too low to threaten the global human population. In contrast, our protocol has identified mutations that may be relevant for specific populations and minorities with cytotoxic genetic backgrounds susceptible to SARS-CoV-2 infection. Some viral variants co-existed in the same country with these human communities which warrants deeper surveillance in these cases to prevent local outbreaks. Our study support the integration of massive data of different natures in the surveillance of viral pandemics.

immunology↗

bioRxiv2020

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Single cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed base content. Through optimization of a quasi-linear genome amplification method, we achieve better targeting of the parasite genome over contaminants and generate coverage levels that allow detection of relatively small copy number variations on a single cell level. These improvements are important for expanding accessibility of single cell approaches to new organisms and for improving the study of adaptive mechanisms.

genomics↗

bioRxiv2018

CNV Neurons Are Rare in Aged Human Neocortex

Megabase-scale somatic copy number variants (CNVs) alter allelic diversity in a subset of human neocortical neurons. Reported frequencies of CNV neurons range from [~]5% of neurons in some individuals to greater than 30% in other individuals. Genome-wide and familial studies implicitly assume a constant brain genome when assessing the genetic risk architecture of neurological disease, thus it is critical to determine whether divergent reports of CNV neuron frequency reflect normal individual variation or technical differences between approaches. We generated a new dataset of over 800 human neurons from 5 neurotypical individuals and developed a computational approach that measures single cell library quality based on Bayesian Information Criterion and identifies integer-like variant segments from population-level statistics. A brain CNV atlas was assembled using our new dataset and published data from 10 additional neurotypical individuals. This atlas reveals that the frequency of neocortical CNV neurons varies widely among individuals, but that this variability is not readily accounted for by tissue quality or CNV detection approach. Rather, the age of the individual is anti-correlated with CNV neuron frequency. Fewer CNV neurons are observed in aged individuals than young individuals.

neuroscience↗