Biology subjects

O'Brien, E.

Publications and source records attributed to O'Brien, E..

16 recordsLinked to original sources

bioRxiv2025

A widespread protein misfolding mechanism is differentially rescued by chaperones based on gene essentiality

Protein misfolding involving changes in non-covalent lasso entanglement (NCLE) status has been proposed based on simulations and biochemical assays of a small number of proteins. Here, we detect hallmarks of these misfolded states across hundreds of proteins by integrating E. coli proteome-wide limited-proteolysis mass spectrometry with structural datasets of protein native structures. Proteins containing native NCLEs are twice as likely to misfold, predominantly in regions where these NCLEs naturally occur. Surprisingly, the chaperones DnaK and GroEL do not typically correct this misfolding, except in the case of essential proteins. Statistical analysis links this differential rescue activity to weaker loop-closing contacts in the NCLEs of essential proteins, suggesting misfolding involving these loops is easier to rectify by chaperones. Molecular simulations indicate a mechanism where premature NCLE loop closure, prior to proper placement of the threading segment, leads to persistent misfolded states. This mechanism explains why, in the mass spectrometry data, proteins with NCLEs are more likely to misfold and misfold in NCLE regions. These results suggest widespread NCLE misfolding, that such misfolded states in non-essential proteins can bypass the refolding action of chaperones, and that some protein sequences may have evolved to allow chaperone rescue from this class of misfolding.

biophysics↗

bioRxiv2025

High-throughput Experimental Validation of Synonymous Mutation Neutrality in the Human Genome

The assumption that synonymous mutations are fitness-neutral is central to many foundational results in the fields of genetics, genomics, evolutionary biology, and medicine. However, recent results suggest synonymous mutations have pervasive and strong fitness effects. These vigorously debated studies in non-human model systems have even suggested that the proportion of synonymous mutations and their fitness effect sizes are similar to non-synonymous mutations. To systematically test synonymous neutrality across the human genome we utilize recent advances in base editing to probe the fitness effects of 8,558 potential synonymous mutations in 128 highly essential genes. Combined with novel base editing controls and extensive individual sgRNA guide-level validation experiments that quantify false positive rates, we find that synonymous mutations rarely have fitness effects on growth, occurring 165-fold (95% CI, 35-2929 fold) less frequently than non-synonymous fitness-altering mutations. Thus, despite a decade of high-profile controversies, the neutrality assumption for spontaneous synonymous mutations is valid.

genetics↗

bioRxiv2025

Bioinspired Oxidized mRNA Lipid Nanoparticles for Ex Vivo Engineering of Chimeric Antigen Receptor Macrophages Targeting Solid Tumors

Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The composition of the top LNP was subsequently optimized using an orthogonal design of experiments (DoE) and physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding a particle that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E (ApoE) independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform can potentially be utilized to engineer a range of human CAR-M immunotherapies to treat various types of solid tumors.

bioengineering↗

bioRxiv2024

Effects of injury size on local and systemic immune cell dynamics in volumetric muscle loss

We took a systems approach to the analysis of macrophage phenotype in regenerative and fibrotic volumetric muscle loss outcomes in mice together with analysis of systemic inflammation and of other leukocytes in the muscle, spleen, and bone marrow. Macrophage dysfunction in the fibrotic group occurred as early as day 1, persisted to at least day 28, and was associated with increased numbers of leukocytes in the muscle and bone marrow, increased pro-inflammatory marker expression in splenic macrophages, and changes in the levels of pro-inflammatory cytokines in the blood. The most prominent differences were in muscle neutrophils, which were much more abundant in fibrotic outcomes compared to regenerative outcomes at day 1 after injury. However, neutrophil depletion had little to no effect on macrophage phenotype or on muscle repair outcomes. Together, these results suggest that the entire system of immune cell interactions must be considered to improve muscle repair outcomes.

bioengineering↗

bioRxiv2024

CPI203, a BET inhibitor, down-regulates a consistent set of DNA synthesis genes across a wide array of glioblastoma lines.

IntroductionGlioblastomas utilize malignant gene expression pathways to drive growth. Many of these gene pathways are not directly accessible with molecularly targeted pharmacological agents. Chromatin-modifying compounds can alter gene expression and target glioblastoma growth pathways. In this study, we utilize a systematic screen of chromatin-modifying compounds on a panel of patient-derived glioblastoma lines to identify promising compounds and their associated gene targets. MethodsFive glioblastoma cell lines were subjected to a drug screen of 106 chromatin-modifying compounds representing 36 unique drug classes to determine the twelve most promising drug classes and the best candidate inhibitors in each class. These twelve drugs were then tested with a panel of twelve patient-derived gliomasphere lines to identify growth inhibition and corresponding gene expression patterns. Overlap analysis and weighted co-expression network analysis (WCGNA) were utilized to determine potential target genes and gene pathways. ResultsThe initial drug screen identified twelve candidate pharmacologic agents for further testing. Drug sensitivity testing indicated an overall high degree of variability between gliomasphere lines. However, CPI203 was the most consistently effective compound, and the BET inhibitor class was the most consistently effective class of compounds across the gliomasphere panel. Correspondingly, most of the compounds tested had highly variable effects on gene expression between gliomasphere lines. CPI203 stood out as the only compound to induce a consistent effect on gene expression across different gliomasphere lines, specifically down-regulation of DNA-synthesis genes. Amongst the twelve tested cell lines, high expression of CDKN2A and CDKN2B distinguished more drug sensitive from more drug resistant lines. WCGNA identified two oncogenic gene modules (FBXO5 and MELK) that were effectively downregulated by CPI203 (FBXO5) and ML228 (FBXO5 and MELK). ConclusionsThe bromodomain inhibitor CPI203 induced relatively consistent effects on gene expression and growth across a variety of glioblastoma lines, specifically down-regulating genes associated with DNA replication. We propose that clinically effective BET inhibitors have the potential to induce consistent beneficial effects across a spectrum of glioblastomas.

cancer biology↗

bioRxiv2023

Conformational dynamics of the μ-opioid receptor determine ligand intrinsic efficacy

The -opioid receptor (OR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the OR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of {beta}-arrestin-1 with the OR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi. One-Sentence SummaryLigand-dependent conformational dynamics of the -opioid receptor determine downstream signaling efficacy.

biochemistry↗

bioRxiv2023

Extensive re-modelling of the cell wall during the development of Staphylococcus aureus bacteraemia

Introductory Paragraph / AbstractThe bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

microbiology↗

bioRxiv2022

Negative allosteric modulation of the glucagon receptor by RAMP2

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The glucagon receptor (GCGR), a Family B GPCR responsible for maintenance of proper blood sugar levels, interacts with RAMP2, though the purpose and consequence of this interaction is poorly understood. Using a series of biochemical and cell-based assays, we show that RAMP2 interacts with and broadly inhibits GCGR-induced downstream signaling. Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) demonstrates that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) as well as at a key region in the 6th transmembrane helix, while single-molecule fluorescence resonance energy transfer (smFRET) experiments show that this RAMP2-induced ECD disorder results in inhibition of active and intermediate states of the intracellular face of the receptor. Using cryo-electron microscopy (cryoEM), we determined the structure of the GCGR-Gs complex at 2.9 [A] resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner, yet the ECD of GCGR is indeed largely disordered in the presence of RAMP2. This disorder is accompanied by rearrangements of several key areas of the receptor, resulting in the formation of a likely unproductive complex. Together, our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

biochemistry↗

bioRxiv2022

PDX models of relapsed pediatric AML preserve global gene expression patterns and reveal therapeutic targets

As patient-derived xenograft (PDX) models of acute myeloid leukemia (AML) become increasingly common tools for preclinical evaluation of targeted therapies it becomes important to consider the fidelity with which this system recapitulates the disease state found in patients. Gene expression profiling of patient blasts has been successfully used to identify distinct subtypes of AML to uncover sub-type specific vulnerabilities and to predict response to therapy and outcomes. Currently, there is little information regarding how well PDX models of AML mimic global gene expression patterns found in patients. In order to address this point, we performed detailed RNA-Seq analysis of data obtained from a diverse series of pediatric AML PDXs, separately and compared to primary patient data. When unsupervised clustering was applied to the PDX sample dataset, we found grouping associated with KMT2A (MLL) gene status. Additionally, in combined analysis, PDX samples were found to align with primary patient samples harboring similar genetics. We found a strong correlation of expression levels of nearly all expressed transcripts in PDX and patient datasets thus demonstrating faithful recapitulation of gene expression signatures. Furthermore, paired patient/PDX samples showed strong concordance, suggesting retention of sample-specific gene expression in immune deficient mice. Comparisons of PDX models propagated in NOD/SCID/IL2rg -/- (NSG) mice compared to NSG mice with transgenic expression of human SCF, GM-CSF, and IL-3 (NSGS) revealed minimal differences related to increased JAK/STAT and macrophage activation pathways in NSGS. Additionally, a unique RAM immunophenotype associated expression signature pointed to discovery of cryptic CBFA2T3-GLIS2 rearrangement as the mechanistic driver mutation in two PDX models. Based on the relatively high BCL2 mRNA in these models, we tested the efficacy of venetoclax in combination with CPX-351 which resulted in reduced leukemia burden and prolonged survival. These results validate the PDX system as surrogate of the molecular signatures in high-risk pediatric AML and highlight this systems utility for pre-clinical therapeutic discovery, especially in very rare subtypes of disease.

cancer biology↗

bioRxiv2022

MutSigCVsyn: Identification of Thirty Synonymous Cancer Drivers

Synonymous mutations, which change only the DNA sequence but not the encoded protein sequence, can affect protein structure and function, mRNA maturation, and mRNA half-lives. The possibility that synonymous mutations can act as cancer drivers has been explored in several recent studies. However, none of these studies control for all three levels (patient, histology, and gene) of mutational heterogeneity that are known to affect the accurate identification of non-synonymous cancer drivers. Here, we create an algorithm, MutSigCVsyn, an adaptation of MutSigCV, to identify synonymous cancer drivers based on a novel non-coding background model that takes into account the mutational heterogeneity across these levels. Examining 2,572 PCAWG cancer whole-genome sequences, MutSigCVsyn identifies 30 novel synonymous drivers that include mutations in promising candidates like BCL-2. By bringing the best practices in non-synonymous driver identification to the analysis of synonymous drivers, these are promising candidates for future experimental study.

bioinformatics↗

bioRxiv2021

Self-sensitisation of Staphylococcus aureus to the antimicrobial factors found in human blood.

Introductory ParagraphFor opportunistic pathogens, the switch from a commensal to an invasive lifestyle is often considered an accidental event. But with plentiful opportunity, what leads one accidental event to result in an invasive infection, and another not to? And how much of this apparent stochasticity is driven by bacterial factors? To answer these questions, here we focussed on the major human pathogen Staphylococcus aureus, which can both reside asymptomatically as a member of our respiratory microbiome, or become invasive and cause infections as severe as bacteraemia. Survival upon exposure to the antibacterial factors found in serum is a critical aspect of their ability to cause bacteraemia, and across a collection of 300 clinical isolates we found there to be significant variability in this capability. Utilising a GWAS approach we have uncovered the genetic basis of much of this variability through the identification and functional verification of a number of new polymorphic loci that affect serum survival: tcaA, tarK, gntR, ilvC, arsB, yfhO, and pdhD. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, while simultaneously enhancing the sensitivity of S. aureus to serum through a process involving the ligation of wall teichoic acids into the cell wall. As blood-stage infections are a transmission dead-end for the bacteria, that S. aureus actively responds to serum to produce a protein which specifically limits their ability to survive in this environment demonstrates that the switch from the commensal to the invasive lifestyle is complex, and that TcaA may contribute to the long-term success of S. aureus by restricting the bacteria to their more readily transmissible commensal state.

microbiology↗

bioRxiv2019

A small membrane protein critical to both the offensive and defensive capabilities of Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, where the widespread emergence of antibiotic resistance is making infections more challenging to treat. Toxin induced tissue damage and resistance to the hosts immune system are well established as critical to its ability to cause disease. However, recent attempts to study S. aureus pathogenicity at a population level have revealed significant complexity and hierarchical levels of regulation. In an effort to better understand this we have identified and characterized a principle effector protein, MasA. The inactivation of this small highly-conserved membrane protein simultaneously disrupts toxin production and impairs S. aureus ability to resist several aspects of the innate immune system. These pleiotropic effects are mediated by both a change in the stability of the bacterial membrane and the dysregulation of iron homeostasis, which results in a significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and a sepsis model of infection. That proteins with such major effects on pathogenicity remain unidentified in a bacterium as well studied as S. aureus demonstrates how incomplete our understanding of their ability to cause disease is, an issue that needs to be addressed if effective control and treatment strategies are to be developed.

microbiology↗

bioRxiv2018

Matrilineal Transmission of Familial Excess Longevity (mtFEL): Effects on Cause-specific Mortality in Utah, 1904-2002

The heritable component to a long and healthy life is likely to involve the actions and interactions of both nuclear and mitochondrial genetic variants. Using computerized genealogical records with accompanying cause of death information from the Utah population, we previously reported cause-specific mortality rate distributions associated with the nuclear genetic component of familial exceptional longevity. Here we identify Utah matrilineages (mitochondrial lineages) in which overall survival is better than expected, and compare cause-specific mortality rates in those matrilineages to cause-specific mortality rates in the general population. We also examine the effects on cause-specific mortality of interactions between the nuclear and mitochondrial components of familial excess longevity (nuclear FEL and mtFEL). Among individuals from the bottom quartile of nuclear FEL, those who were also in the top quartile for mtFEL had lower all-cause, heart disease, cancer, stroke, and diabetes mortality rates than those in the bottom quartile of mtFEL. In contrast, among individuals from the top quartile of nuclear FEL, the mortality rates from these diseases were similar for those also in the top quartile of mtFEL vs. those also in the bottom quartile of mtFEL, with the exception of diabetes mortality, which was dramatically suppressed in the high nuclear FEL + high mtFEL group as compared to the high nuclear FEL + low mtFEL group. Moreover, the highest mortality rates from diabetes were found in individuals aged 90 years or older who were members of both the high nuclear FEL and low mtFEL quartiles. These results support the hypothesis that some nuclear genetic variants contributing to long life carry an increased risk of dying from diabetes that is strongly ameliorated by some mitochondrial DNA variants.

epidemiology↗

bioRxiv2018

Mitochondrial genetics of exceptional longevity in multigeneration matrilineages

Some heritable mitochondrial DNA (mtDNA) sequence variants may slow the rate of aging. The European mitochondrial haplogroup K has previously been reported to be increased in frequency in centenarians and nonagenarians relative to its frequency in younger individuals, by standard case/control study designs. To select for mitochondrial genomes likely to carry beneficial genetic variants, we screened a large genealogical database (the Utah Population Database, UPDB) for mitochondrial lineages in which the frequency of survival past 90 years was significantly higher than in the general population, and also significantly higher than in close non-matrilineal relatives. We ranked 14,900 distinct matrilineages by the strength of their association with longevity. Full sequencing of the mtDNAs from a single individual from each of 53 matrilineages in the top longevity ranks and each of 374 control matrilineages from the general Utah population, followed by analyses of the mtDNA haplogroup frequencies, identified haplogroup K2 as the haplogroup most enriched in frequency by the longevity selection (Odds Ratio = 23.05). We then analyzed overall survival and cause-specific mortality in the several thousand individuals aged 40 years or older whose mtDNA genotypes could be imputed from the 374 fully sequenced control mtDNAs. In these control matrilineages Haplogroup K2 individuals (n=332) enjoyed a significantly lower all-cause mortality risk than the general population (HR=0.81), attributable in part to a significantly lower risk of dying from heart disease (HR=0.50), as well as lower (though not significantly lower) risks of dying from cancer (HR=0.72) and diabetes (HR=0.74). Furthermore, K2 was the only haplogroup in which mortality was reduced for all three of these common causes of death.

genetics↗

bioRxiv2018

Model-driven design and evolution of non-trivial synthetic syntrophic pairs

Synthetic microbial communities are attractive for applied biotechnology and healthcare applications through their ability to efficiently partition complex metabolic functions. By pairing auxotrophic mutants in co-culture, nascent E. coli communities can be established where strain pairs are metabolically coupled. Intuitive synthetic communities have been demonstrated, but the full space of cross-feeding metabolites has yet to be explored. A novel algorithm, OptAux, was constructed to design 66 multi-knockout E. coli auxotrophic strains that require significant metabolite cross-feeding when paired in co-culture. Three OptAux predicted auxotrophic strains were co-cultured with an L-histidine auxotroph and validated via adaptive laboratory evolution (ALE). Time-course sequencing revealed the genetic changes employed by each strain to achieve higher community fitness and provided insights on mechanisms for sharing and adapting to the syntrophic niche. A community model of metabolism and gene expression was utilized to predict the relative community composition and fundamental characteristics of the evolved communities. This work presents a novel computational method to elucidate metabolic changes that empower community formation and thus guide the optimization of co-cultures for a desired application.

systems biology↗

bioRxiv2016

Familial heterogeneity in breast cancer predisposition: a study of 22 Utah families

The problem of \"missing heritability\" in genome-wide analyses of complex diseases is thought to be attributable to some combination of: rare variants of moderate to large effect, common variants of very small effect, and epigenetic, epistatic, or shared environmental effects. Rare variants do not affect large numbers of people by definition, but identified genes and pathways frequently lead to important insights into pathogenesis, and become targets of chemoprevention or therapy. Family studies remain an efficient way to identify rare variants with sizable effects on disease risk. We present a genome-wide study of breast cancer in 22 large high-risk families including 154 women diagnosed with breast cancer. Appropriate marker spacing was achieved by simulation studies of founder haplotypes to reduce the chance that linkage disequilibrium produced spurious linkage peaks. For each family, we generated 100 simulations of null linkage genome-wide to estimate the probability that individual results were due to chance. We identified a total of 12 putative susceptibility regions with per-family genome-wide probability < 0.05. These regions were located on 10 chromosomes; 10 of the 22 families showed linkage at these locations; two or more families showed linkage to 6 regions on 5 chromosomes (4q, 5q, 6p, 14q, 18p, and 18q). These results indicate that there is considerable heterogeneity among families in genomic regions and thus variants predisposing to breast cancer. Moreover, they suggest that uncommon high- or medium-risk genetic variants remain to be found, and that family designs can be an efficient way to identify them.

cancer biology↗