Biology subjects

Hurley, A.

Publications and source records attributed to Hurley, A..

15 recordsLinked to original sources

bioRxiv2026

Squamous-state excursions activate APOBEC3A in cancer

The cytidine deaminase APOBEC3A is a major endogenous mutagen in human cancer, yet is rarely captured in bulk tumor RNA or protein profiles despite the prominent mutational scars it leaves in cancer genomes. The origin of these episodic mutational bursts has remained unclear, with prevailing models emphasizing sustained inflammatory signaling. Here, we show that APOBEC3A is induced in a rare subpopulation of cancer cells engaging a transient squamous differentiation program, linking APOBEC3A mutagenesis to lineage-state plasticity rather than persistent inflammatory signaling. Across breast and lung cancer cell lines and patient tumors, keratinocyte differentiation markers, including the stress keratins KRT6A and KRT16, are the strongest correlates of endogenous APOBEC3A expression. These days-long squamous-state excursions explain how APOBEC3A can leave durable mutational scars while remaining largely invisible to bulk tumor RNA and protein profiling. APOBEC3A catalytic activity reinforces selected components of this program through uracil excision and JNK-AP-1 signaling. The squamous differentiation transcription factor ZNF750 promotes APOBEC3A induction during squamous-state engagement in breast and lung cancer models. In established human squamous tumors, however, ZNF750 loss-of-function is associated with elevated APOBEC3A expression and APOBEC mutagenesis, revealing lineage-context-dependent regulation. These findings identify a transient differentiation state as a mutagenic intermediate, coupling cell-state plasticity to cancer genome evolution.

cancer biology↗

bioRxiv2024

Co-zorbs: Motile, multispecies biofilms aid transport of diverse bacterial species

Biofilms are three-dimensional structures containing one or more bacterial species embedded in extracellular polymeric substances. Although most biofilms are stationary, Flavobacterium johnsoniae forms a motile spherical biofilm called a zorb, which is propelled by its base cells and contains a polysaccharide core. Here, we report formation of spatially organized, motile, multispecies biofilms, designated "co-zorbs," that are distinguished by a core-shell structure. F. johnsoniae forms zorbs whose cells collect other bacterial species and transport them to the zorb core, forming a co-zorb. Live imaging revealed that co-zorbs also form in zebrafish, thereby demonstrating a new type of bacterial movement in vivo. This discovery opens new avenues for understanding community behaviors, the role of biofilms in bulk bacterial transport, and collective strategies for microbial success in various environments. Significance StatementThis paper reports the discovery of co-zorbs, which are spherical aggregates of bacteria that move and transport other bacteria. Zorbs move toward other bacteria and collect them in a manner reminiscent of phagocytes. Once inside the zorb, the new species form a striking, organized core. The discovery of co-zorbs introduces an entirely new type of bacterial movement and transport involving cooperation among bacterial species. Co-zorbs have potential for engineering microbial systems for biotechnology applications and for managing spread of bacterial pathogens in their hosts.

microbiology↗

bioRxiv2024

Covalently linked adenovirus-AAV complexes as a novel platform technology for gene therapy

Adeno-associated virus (AAV) has found immense success as a delivery system for gene therapy, yet the small 4.7 kb packaging capacity of the AAV sharply limits the scope of its application. In addition, high doses of AAV are frequently required to facilitate therapeutic effects, leading to acute toxicity issues. While dual and triple AAV approaches have been developed to mitigate the packaging capacity problem, these necessitate even higher doses to ensure that co-infection occurs at sufficient frequency. To address these challenges, we herein describe a novel delivery system consisting of adenovirus (Ad) covalently linked to multiple adeno-associated virus capsids as a new way of more efficiently co-infecting cells with lower overall amounts of AAVs. We utilize the DogTag-DogCatcher (DgT-DgC) molecular glue system to construct our AdAAVs and we demonstrate that these hybrid virus complexes achieve enhanced co-transduction of cultured cells, including physiologically relevant primary cells. On this basis, AdAAV technology may eventually facilitate therapeutic co-delivery of multiple transgenes at low virus doses for treating complex ailments.

synthetic biology↗

bioRxiv2024

Surface colonization by Flavobacterium johnsoniae promotes its survival in a model microbial community

Flavobacterium johnsoniae is a ubiquitous soil and rhizosphere bacterium, but despite its abundance, the factors contributing to its success in communities are poorly understood. Using a model microbial community, The Hitchhikers Of the Rhizosphere (THOR), we determined the effects of colonization on fitness of F. johnsoniae in the community. Insertion sequencing (INSeq), a massively parallel transposon mutant screen, on sterile sand identified 25 genes likely to be important for surface colonization. We constructed in-frame deletions of nine candidate genes predicted to be involved in cell membrane biogenesis, motility, signal transduction, and transport of amino acids and lipids. All mutants poorly colonized sand, glass, and polystyrene and produced less biofilm than the wild type, indicating the importance of the targeted genes in surface colonization. Eight of the nine colonization-defective mutants were also unable to form motile biofilms, or zorbs, thereby suggesting that the affected genes play a role in group movement and linking stationary and motile biofilm formation genetically. Furthermore, we showed that deletion of colonization genes in F. johnsoniae affected its behavior and survival in THOR on surfaces, suggesting that the same traits are required for success in a multispecies microbial community. Our results provide insight into the mechanisms of surface colonization by F. johnsoniae and form the basis for further understanding its ecology in the rhizosphere. IMPORTANCEMicrobial communities direct key environmental processes through multispecies interactions. Understanding these interactions is vital for manipulating microbiomes to promote health in human, environmental, and agricultural systems. However, microbiome complexity can hinder our understanding of the underlying mechanisms in microbial community interactions. As a first step towards unraveling these interactions, we explored the role of surface colonization in microbial community interactions using THOR, a genetically tractable model community of three bacterial species, Flavobacterium johnsoniae, Pseudomonas koreensis, and Bacillus cereus. We identified F. johnsoniae genes important for surface colonization in solitary conditions and in the THOR community. Understanding the mechanisms that promote success of bacteria in microbial communities brings us closer to targeted manipulations to achieve outcomes that benefit agriculture, the environment, and human health.

microbiology↗

bioRxiv2023

Encapsulation of AAV into protein vault nanoparticles as a novel solution to gene therapy's neutralizing antibody problem

Although adeno-associated virus (AAV) has enjoyed enormous success as a delivery modality for gene therapy, it continues to suffer from the high prevalence of preexisting neutralizing antibodies in human populations, limiting who can receive potentially life-saving treatments. In this regard, AAV therapies generally also must be administered as a single dose since neutralizing antibodies develop in patients who receive the virus. Strategies for circumventing these issues remain limited. As a novel solution, we employed SpyTag-SpyCatcher molecular glue technology to facilitate packaging of AAVs inside of recombinant protein vault nanoparticles. Vaults are endogenous particles produced by mammalian cells. We therefore hypothesized that they may shield packaged molecules from neutralizing antibodies. Vaults have previously been utilized to deliver drugs and proteins into cells, but our study represents the first time anyone has packaged an entire virus inside of a vault. We showed that our vaultAAV (VAAV) delivery vehicle transduces cells in the presence of anti-AAV neutralizing serum. VAAV is positioned as a new gene therapy delivery platform with potential to overcome the neutralizing antibody problem and perhaps even allow administration of multiple doses, expanding the scope of AAV treatments.

bioengineering↗

bioRxiv2023

In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene

Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested "Repair Drive", a novel method for improving targeted gene insertion in the liver by selectively expanding correctly repaired hepatocytes in vivo. Our system consists of transient conditioning of the liver by knocking down an essential gene, and delivery of an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene. Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing.

genetics↗

bioRxiv2022

Microbial Community Interactions on a Chip

Multispecies microbial communities drive most ecosystems on Earth. Chemical and biological interactions within these communities can affect survival of individual members and the entire community. However, the prohibitively high number of possible interactions within a microbial community has made the characterization of factors that influence community development challenging. Here we report a Microbial Community Interaction (CI) device to advance the systematic study of chemical and biological interactions within a microbial community. The CI creates a combinatorial landscape made up of an array of triangular wells interconnected with circular wells, which each contains either a different chemical or microbial strain, generating chemical gradients and revealing biological interactions. Bacillus cereus UW85 containing GFP provided the "target" readout in the triangular wells, and antibiotics or microorganisms in adjacent circular wells are designated the "variables". The CI device revealed that gentamicin and vancomycin are antagonistic to each other in inhibiting the target B. cereus UW85, displaying weaker inhibitory activity when used in combination than alone. We identified three-member communities constructed with isolates from the plant rhizosphere that increased or decreased growth of B. cereus. The CI device enables both strain-level and community-level insight. The scalable geometric design of the CI device enables experiments with high combinatorial efficiency, thereby providing a simple, scalable platform for systematic interrogation of three-factor interactions that influence microorganisms in solitary or community life.

microbiology↗

bioRxiv2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines1,2. These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known3-6. Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers3,5,6. Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection7,8. However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.

immunology↗

bioRxiv2020

Persistent Cellular Immunity to SARS-CoV-2 Infection

SARS-CoV-2 is responsible for an ongoing pandemic that affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 months after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen specific memory that could contribute to rapid recall responses. In addition, recovered individuals show enduring immune alterations in relative numbers of CD4+ and CD8+ T cells, expression of activation/exhaustion markers, and cell division. SummaryWe show that SARS-CoV-2 infection elicits broadly reactive and highly functional memory T cell responses that persist 6 months after infection. In addition, recovered individuals show enduring immune alterations in CD4+ and CD8+ T cells compartments.

immunology↗

bioRxiv2020

Evolution of Antibody Immunity to SARS-CoV-2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 4 months after coronavirus disease-2019 (COVID-19) onset, using immunofluorescence, or polymerase chain reaction, revealed persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

immunology↗

bioRxiv2020

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies

Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1A and RBD epitopes. A 3.4[A] cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on "up" RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

immunology↗

bioRxiv2020

Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

immunology↗

bioRxiv2018

Detection of circulating extracellular mRNAs by modified small RNA-sequencing analysis

Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. Here, we present a comprehensive extracellular RNA (exRNA) study in human blood circulation based on conventional small RNA-sequencing (sRNA-seq) and sRNA-seq after T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma. Applying strict criteria for read mapping and annotation, we found that compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. Based on captured ex-mRNAs from healthy individuals, we concluded that the exRNA pool is dominated by hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt long reads originated from the coding sequences, in a pattern reminiscent of ribosome-profiling studies for high abundance transcripts. Blood sample type had a considerable influence on the exRNA profile. The number of detected distinct ex-mRNA transcripts ranged from on average ~350 to 1100 in the different plasma types. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to ~2300. For EDTA and ACD, in particular, we found evidence of destabilization of mRNA and non-coding RNA ribonucleoprotein complexes. In a proof-of-concept study, we compared patients with acute coronary syndrome (ACS) to healthy controls. The improved tissue resolution of ex-mRNAs after PNK-treatment enabled us to detect a neutrophil-signature in ACS that escaped detection in an ex-miRNA analysis. Thus, ex-mRNAs provide superior resolution for the study of exRNA changes in vivo and ex vivo. They can be readily studied by sRNA-seq after T4 PNK end-treatment.

molecular biology↗

bioRxiv2018

Introducing THOR, a model microbiome for genetic dissection of community behavior

The quest to manipulate microbiomes has intensified, but many microbial communities have proven recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa: the Firmicutes, Proteobacteria, and Bacteroidetes. We chose Bacillus cereus as a model rhizosphere Firmicute and characterized twenty other candidates, including \"hitchhikers\" that co-isolated with B. cereus from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers -- Pseudomonas koreensis from the top tier of the competition network and Flavobacterium johnsoniae from the bottom of the network to represent the Proteobacteria and Bacteroidetes, respectively. The model community has several emergent properties--induction of dendritic expansion of B. cereus colonies by either of the other members and production of more robust biofilms by the three members together than individually. Moreover, P. koreensis produces a novel family of alkaloid antibiotics that inhibit growth of F. johnsoniae, and production is inhibited by B. cereus. We designate this community THOR, because the members are the hitchhikers of the rhizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior.\n\nIMPORTANCEThe manipulation and engineering of microbiomes could lead to improved human health, environmental sustainability, and agricultural productivity. However, microbiomes have proven difficult to alter in predictable ways and their emergent properties are poorly understood. The history of biology has demonstrated the power of model systems to understand complex problems such as gene expression or development. Therefore, a defined and genetically tractable model community would be useful to dissect microbiome assembly, maintenance, and processes. We have developed a tractable model rhizosphere microbiome, designated THOR, containing Pseudomonas koreensis, Flavobacterium johnsoniae, and Bacillus cereus, which represent three dominant phyla in the rhizosphere, as well as in soil and the mammalian gut. The model community demonstrates emergent properties and the members are amenable to genetic dissection. We propose that THOR will be a useful model for investigations of community-level interactions.

microbiology↗

bioRxiv2018

Gut microbiota density influences host physiology and is shaped by host and microbial factors

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohns disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics.

microbiology↗