Biology subjects

Gibson, S.

Publications and source records attributed to Gibson, S..

15 recordsLinked to original sources

bioRxiv2026

Chronic Electrophysiology Captures Progressive Variability in DNA-Damaged Cardiac Networks

Electrophysiological recordings provide a key functional readout of cardiac health. Most in vitro studies rely on acute measurements rather than recording evolving functional changes. Here, we implement a microelectrode array (MEA) based system for chronic electrophysiological monitoring of human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) monolayers. Electrophysiology recordings were maintained for up to 40 days, enabling longitudinal assessment of field potential amplitude, field potential duration, beat frequency, conduction velocity, and waveform morphology. Untreated iPSC-CM monolayers exhibited stable electrophysiological behavior throughout chronic culture. Following exposure to the cardiotoxic chemotherapeutic doxorubicin, chronic recordings captured progressive deterioration in electrical activity, including reduced field potential amplitude, altered repolarization dynamics, increased beat-to-beat variability, and reduced conduction that persisted after drug removal. Electrode-resolved analyses further revealed pronounced progressive spatial variability, highlighting localized responses that would be obscured by population-averaged measurements. Endpoint assays indicated reduced calcium transient amplitude and altered calcium reuptake, decreased mitochondrial membrane potential, and loss of connexin 43, findings consistent with the impaired electrical activity observed in chronic recordings. Together, these results establish chronic electrophysiological monitoring as a powerful approach for resolving the spatiotemporal progression of cardiotoxicity and other dynamic cardiac processes in vitro.

bioengineering↗

bioRxiv2026

A combined program of induced stemness and differentiation in response to interferon gamma drives acute myeloid leukaemia growth

How inflammation shapes acute myeloid leukaemia (AML) has come under scrutiny, as it may explain the disease resistance to immunotherapy approaches. IFNg has emerged as a key cytokine with paradoxical roles in suppressing and supporting AML growth, and the fundamental question of how leukemic stem cells (LSCs) respond to IFNg and whether IFNg signaling influences LSC quiescence and their capacity to regenerate disease over the long term remains unanswered. Here, we study primary human AML cells and murine models and combine bioinformatics analyses and functional assays to show that AML hierarchical heterogeneity is responsive to IFNg challenge. We uncover that IFNg triggers parallel stemness and differentiation programs; HSC/MPP-like cells enter deeper stemness, associated with quiescence and high leukaemia regeneration potential, while the surviving pool of progenitor-like cells divides faster but produces progeny that is quickly lost. Finally, with murine models we show that exposure to inflammation in vivo results in only transient impairment of leukaemia propagating capacity. This mechanism is driven by a previously unrecognised intraclonal fate bifurcation, relevant for the development of more effective therapeutic approaches.

cancer biology↗

bioRxiv2026

Screening metatranscriptomes for ultrastable RNA secondary structures reveals hidden bacteriophages and novel capsid nanomaterials

Metatranscriptomics has transformed our view of RNA bacteriophage diversity, revealing vast numbers of single-stranded RNA (ssRNA) phages whose protein capsids can be engineered for biotechnology applications. However, many ssRNA phages remain hidden from current detection methods, which require protein-level similarity to known phages. Here we show that RNA structure provides an additional signal for the detection of ssRNA phages in metatranscriptomes, including hidden phages missed by prior protein-based methods. By computationally folding each contig and screening for exceptionally stable RNA secondary structures, we find evidence of thousands of previously unrecognized phages encoding novel coat proteins. We express a library of 12,000 such coat proteins in E. coli and find that most assemble into nuclease-resistant capsids. We determine the 3D structure of one such capsid by cryo-electron microscopy and demonstrate that it can be disassembled and reassembled in vitro to package heterologous RNA--a key step toward repurposing these particles as RNA delivery vehicles. We compile the newly discovered ssRNA phages with previously known ones into a database that contains sequence and structural information for over 460,000 unique RNA molecules and over 100,000 distinct coat proteins, providing a comprehensive resource for microbiology and nanomaterials research.

biochemistry↗

bioRxiv2024

Synthetic G-quadruplex components for predictable, precise two-level control of mammalian recombinant protein expression

Control of mammalian recombinant protein expression underpins the in vitro manufacture and in vivo performance of all biopharmaceutical products. However, routine optimization of protein expression levels in these applications is hampered by a paucity of genetic elements that function predictably across varying molecular formats and host cell contexts. Herein, we describe synthetic genetic components that are specifically built to simplify bioindustrial expression cassette design processes. Synthetic G-quadruplex elements with varying sequence feature compositions were systematically designed to exhibit a wide-range of regulatory activities, and inserted into identified optimal positions within a standardized, bioindustry compatible core promoter-5UTR control unit. The resulting library tuned protein production rates over two orders of magnitude, where DNA and RNA G-quadruplexes could be deployed individually, or in combination to achieve synergistic two-level regulatory control. We demonstrate these components can predictably and precisely tailor protein expression levels in i) varying gene therapy and biomanufacturing cell hosts, and ii) both plasmid DNA and synthetic mRNA contexts. As an exemplar use-case, a vector design platform was created to facilitate rapid optimization of polypeptide expression ratios for difficult-to-express multichain products. Permitting simple, predictable titration of recombinant protein expression, this technology should prove useful for gene therapy and biopharmaceutical manufacturing applications.

bioengineering↗

bioRxiv2024

Discovery of orally active SARS-CoV-2 papain-like protease (PLpro) inhibitors

Vaccines and first-generation antiviral therapeutics have provided important protection against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease (Mpro) have demonstrated clinical efficacy, known PLpro inhibitors have to date lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Herein, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.

microbiology↗

bioRxiv2024

Subcellular localization of truncated MAGEL2 proteins: insight into the molecular pathology of Schaaf-Yang syndrome

Schaaf-Yang syndrome (SYS) is an ultra-rare neurodevelopmental disorder caused by truncating mutations in MAGEL2. Heterologous expression of wild-type (WT) or a truncated (p.Gln638*) C-terminal HA-tagged MAGEL2 revealed a shift from a primarily cytoplasmic to a more nuclear localization for the truncated protein variant. We now extend this analysis to six additional SYS mutations on a N-terminal FLAG-tagged MAGEL2. Our results replicate and extend our previous findings, showing that all the truncated MAGEL2 proteins consistently display a predominant nuclear localization, irrespective of the C-terminal or N-terminal position and the chemistry of the tag. The variants associated with arthrogryposis multiplex congenita (AMC) display a more pronounced nuclear retention phenotype, suggesting a correlation between clinical severity and the degree of nuclear mis-localization. These results point to a neomorphic effect of truncated MAGEL2, which might contribute to the pathogenesis of SYS.

molecular biology↗

bioRxiv2024

Noxious stimulation induces self-protective behaviour in bumblebees

Self-grooming directed towards a noxiously-stimulated body part is one indicator that an animal may feel pain. In insects, the lack of evidence for such behaviour has been widely argued to reflect the absence of pain experiences. Here, we tested whether bumblebees (Bombus terrestris) selectively groom one of their antennae that was touched with a noxiously-heated (65 {square}C) probe. In the first two minutes after being touched with the noxiously-heated probe, bees groomed their touched antenna a) more than their untouched antenna, b) more than bees that were touched on the antenna with an unheated probe, and c) more than control (untouched) bees groomed either of their antennae. Our results clearly show that bumblebees can direct grooming towards a site of noxious stimulation. Our findings thus refute arguments that claim that insects do not feel pain because of their lack of displaying this behaviour.

animal behavior and cognition↗

bioRxiv2023

Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have also revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and the astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity across transgenes and in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

neuroscience↗

bioRxiv2023

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development

In the past decade, Zika virus (ZIKV) emerged as a global public health concern. While adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of symptoms caused by this virus including microcephaly. In this study, we generated a toolkit to ectopically express Zika viral proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We use this toolkit to identify phenotypes and host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins cause scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size, and neuronal function defects. We further use this system to identify strain-dependent phenotypes that may contribute to the increased pathogenesis associated with the more recent outbreak of ZIKV in the Americas. Our work demonstrates Drosophilas use as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

developmental biology↗

bioRxiv2022

Surveillance for SARS-CoV-2 in Ohio's wildlife, companion, and agricultural animals

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly to become a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g. shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g. pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n=76), mink (n=57), multiple species of bats (n=65), and other wildlife in addition to domestic cats (n=275) and pigs (n= 184). In total, we tested 800 animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, due to lack of validated tests for animals, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing re-emergence of SARS-CoV-2 in humans or animals.

microbiology↗

bioRxiv2021

SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus)

Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer (Odocoileus virginianus) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies. One-Sentence SummaryA significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.

molecular biology↗

bioRxiv2021

MFN2 Influences Amyotrophic Lateral Sclerosis Pathology

ObjectiveTo better understand the pathology of amyotrophic lateral sclerosis, we used sequence data from patients seen at the University of Utah to identify novel disease-associated loci. We utilized both in vitro and in vivo studies to determine the biological effect of patient mutations in MFN2. MethodSequence data for a total of 140 patients were run through VAAST and Phevor to determine genes that were more burdened with rare, nonsynonymous variants compared to control longevity cohort. Variants identified in MFN2 were expressed in Mfn2 knockout cells to determine if mutant MFN2 could rescue mitochondrial morphology defects. We identified additional rare, nonsynonymous variants in MFN2 in ALSdb that were expressed in knockout mouse embryonic fibroblasts (MEFs). Membrane potential was measured to quantify mitochondrial health upon mutant MFN2 expression. mfn2 knockout zebrafish were used to examine movement compared to wildtype and protein aggregation in brain. ResultsMFN2 mutations identified in ALS patients from our University of Utah cohort and ALSdb were defective in rescuing morphological defects in Mfn2 knockout MEFs. Selected mutants showed decreased membrane potential compared to wildtype MFN2 expression. Zebrafish heterozygous and homozygous for loss of mfn2 showed increased TDP-43 levels in their hindbrain and cerebellum. ConclusionIn total, 21 rare, deleterious mutations in MFN2 were tested in Mfn2 knockout MEFs. Mutant MFN2 expression was not able to rescue the knockout phenotype, though at differing degrees of severity. Decreased membrane potential also argues for inhibited mitochondrial function. Increased TDP-43 levels in mutant zebrafish illustrates MFN2s function in ALS pathology. MFN2 variants influence ALS pathology and highlight the importance of mitochondria in neurodegeneration.

genetics↗

bioRxiv2021

Sequence Determinants of Human Junctophilin-2 Protein Nuclear Localization and Phase Separation

Junctophilin-2 (JPH2) was conventionally considered as a structural membrane binding protein. Recently, it was shown that proteolytically truncated mouse JPH2 variants are imported into nucleus to exert alternative functions. However, the intranuclear behaviors of human JPH2 (hJPH2) and underlying molecular determinants have not been explored. Here, we demonstrate that full-length hJPH2 is imported into nucleus in human cells by two nuclear localization signals (NLSs), including a newly discovered one at the C-terminus. Importantly, unlike the JPH2 N-terminal truncation which diffuses throughout the nucleus, full-length hJPH2 forms nuclear bodies behaving like liquid-liquid phase separated droplets that are separated from chromatin. The C-terminal transmembrane domain is required for the formation of hJPH2 droplets. Oxidation mimicking substitution of residues C678 and M679 augments the formation of hJPH2 nuclear droplets, suggesting nuclear hJPH2 liquid-liquid phase separation could be modulated by oxidative stress. Mutation A405D, which introduces a negatively charged residue into an intrinsic disordered region (IDR) of hJPH2, turns liquid-like droplets into amyloid-like aggregates. Depletion of an Alanine Rich Region in the IDR recapitulates the liquid-amyloid phase transition. The MORN repeat regions of hJPH2 encodes intrinsic tendency to form amyloid-like structure. Together, these data for the first time revealed the novel intrinsic properties of hJPH2 to form nuclear liquid droplets, and identified critical functional domains and residues encoding these properties. We propose that hJPH2 droplets could function as membrane-less organelles participating in nuclear regulatory processes.

biochemistry↗

bioRxiv2020

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract

The human mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. Bacteriophage parasitize the prokaryotes but are not known to associate with eukaryotic cells. In the gastrointestinal tract, the interaction of these two domains influences the health of the host, especially colonization with invasive pathobionts. Antibiotics may be used but they also kill protective commensals and lack the physio-chemical properties to be specifically and optimally active in this complex milieu. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The enhanced activity is encoded in its tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, thereby positioning it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing epithelial-targeting phage to selectively remove invasive pathobiont species from mucosal surfaces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/212829v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@13219f6org.highwire.dtl.DTLVardef@469d7borg.highwire.dtl.DTLVardef@d31e0org.highwire.dtl.DTLVardef@458e14_HPS_FORMAT_FIGEXP M_FIG C_FIG Model showing (1) mucins from the intestinal mucus layer inhibit phage infection, (2) phage ES17 can bind to mucin and utilize other intestinal glycans as a receptor to infect and kill mucus-coated bacteria, and (3) phages like ES17 can be utilized to coat the intestinal epithelium by binding heparan sulfate glycans to protect from invasive pathogen infection.

microbiology↗

bioRxiv2019

Pregnant womens understanding and conceptualisations of the harms from drinking alcohol: a qualitative study

BackgroundDespite womens awareness that drinking alcohol in pregnancy can lead to lifelong disabilities in a child, it appears that an awareness alone does not discourage some pregnant women from drinking. ObjectivesTo explore influences on pregnant womens decision making around alcohol use in a population with frequent and heavy peer drinking (i.e. in two Indigenous Australian communities) and another of non-Indigenous pregnant women attending antenatal care in a range of socioeconomic settings. MethodsIndividual and group discussions were held with both Indigenous Australian and non-Indigenous pregnant women attending a variety of antenatal care models, including two Indigenous maternity services in Australia. Data were analysed using inductive content analysis. ResultsA total of 14 Indigenous Australian and 14 non-Indigenous pregnant women participated in this study. Analysis identified five main influences on pregnant womens alcohol use: the level and detail of womens understanding of harm; womens information sources on alcohol use in pregnancy; how this information influenced their choices; how women conceptualised their pregnancy; and whether the social and cultural environment supported abstinence. Conclusions for practiceThis study provides insight into how Indigenous Australian and non-Indigenous pregnant women understand and conceptualise the harms from drinking alcohol, including how their social and cultural environments impact their ability to abstain. Strategies for behaviour change need to: correct misinformation about supposed safe timing, quantity and types of alcohol; develop a more accurate perception of Fetal Alcohol Spectrum Disorder; reframe messages about harm to messages about optimising the childs health and cognitive outcomes; and develop a holistic approach encompassing womens social and cultural context.

scientific communication and education↗