Biology subjects

Jones, E. M.

Publications and source records attributed to Jones, E. M..

16 recordsLinked to original sources

bioRxiv2025

Accelerated memory T cell decline and tolerogenic recall responses to SARS-CoV-2 vaccination in diabetes

Type 1 and type 2 diabetes are associated with increased severity and mortality from respiratory virus infections, including SARS-CoV-2. Vaccination in the general population significantly reduces the risk of severe respiratory viral infection and triggers a strong, polyfunctional and lasting T cell response in healthy individuals. However, vaccine effectiveness in people with diabetes is unclear. Here we studied the magnitude and functional characteristics of vaccine-specific CD4+ and CD8+ T cell responses to the full vaccination protocol, and the recall response after a third booster dose of SARS-CoV-2 vaccine in people with type 1 and type 2 diabetes, and compared them to those of people without diabetes. We found defects in both CD4+ and CD8+ T cell memory maintenance and the functionality of the vaccine specific T cells in people with diabetes compared to people without. In those individuals with diabetes that harbored detectable vaccine-specific T cells, they displayed an unfocused, tolerogenic phenotype characterized by increased expression of IL-13 and IL-10 in T1D and T2D compared to people without diabetes. These results have implications for vaccination strategies for people with diabetes.

immunology↗

bioRxiv2024

High resolution deep mutational scanning of the melanocortin-4 receptor enables target characterization for drug discovery

Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6,600 single amino acid substitutions on MC4Rs function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gs- and Gq-signaling pathways, which could be used to design drugs that selectively bias MC4Rs activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.

synthetic biology↗

bioRxiv2023

LanTERN: a fluorescent sensor that specifically binds lanthanides

Lanthanides, a series of fifteen f-block elements, are crucial in modern technology, and their purification by conventional chemical means comes at a significant environmental cost. Synthetic biology offers promising solutions. However, the lack of biochemical tools to measure lanthanide binding is a bottleneck to progress. Here, we introduce LanTERN, a lanthanide-responsive fluorescent protein rationally engineered from the lanmodulin-binding protein, LanM. LanTERN was designed based on GCaMP, a genetically encoded calcium indicator that couples the ion binding of four EF hand motifs to increased GFP fluorescence. We engineered seven mutations across the parent constructs four EF hand motifs to switch specificity from calcium to lanthanides. The resulting protein, LanTERN, directly converts the binding of 10 measured lanthanides to 14-fold or greater increased fluorescence. LanTERN development opens new avenues for creating improved lanthanide-binding proteins and bio-sensing systems.

synthetic biology↗

bioRxiv2023

Loss of cholesterol in Junctional Epidermolysis Bullosa skin identifies a key role for Laminin-332 in actomyosin mediated cholesterol transport

Individuals with Junctional Epidermolysis Bullosa (JEB), a rare genetic skin disease characterised by loss of function mutations in the Laminin332 (Lam332), do not survive beyond their first birthday. Here we report that loss of Lam332 leads to absence of cholesterol lipid from the epidermis in vitro and in vivo. Using 3D skin equivalents, a JEB mouse model and JEB patient samples we confirmed changes in epidermal lipid synthesis, which was further explored using lipidomics. Cholesterol biosynthesis genes were increased with loss of Laminin-332 in vitro, however a decrease in immunofluorescence lipid staining was observed. Cholesterol transport in Laminin-332 knockdown keratinocytes was revealed to be disrupted, which in keratinocytes is dependent on the actomyosin network. In conclusion these findings suggest a role for the basement membrane protein Laminin-332 in lipid metabolism in the skin, and a broader role for epidermal homeostasis and barrier formation. Restoration of cholesterol transport in epidermal keratinocytes of JEB patients offers the potential to improve their skin barrier.

cell biology↗

bioRxiv2023

A systematic global review of mammalian carnivore responses to production forests

O_LIUnmodified forests are increasingly rare worldwide, with forestry a major contributor to habitat modification. Extending conservation practices beyond protected areas is important to conserve forest ecosystems. C_LIO_LIWe investigate the response of native mammalian carnivores (both Order Carnivora and Family Dasyuridae) to production forests globally, including harvested native forest and timber plantations. We examine how carnivores recorded in production forests use these forests versus other land uses, particularly native and unharvested forest; how habitat use relates to threatened status, body size, diet, and harvesting method; carnivore responses to habitat features within production forests; and carnivore denning, breeding, and predation behaviour in production forests. C_LIO_LIWe review 294 studies recording 132 carnivore species in production forests. Carnivores generally show higher use of native and unharvested forests and lower use of agricultural land than production forests. Threatened species and large carnivores respond more negatively to production forests than non-threatened species and small carnivores respectively. Hypercarnivores respond more negatively than omnivores to plantations compared to native forest, but there was no difference in the use of harvested and unharvested native forest between these dietary groups. C_LIO_LISurprisingly, a high proportion of carnivore species use clearfelled more than unharvested native forest. In forest with partial harvesting or reduced-impact logging, most species show no difference in use between harvested and unharvested forest. C_LIO_LICarnivores generally respond positively to habitat features such as riparian areas and coarse woody debris. Several carnivores were recorded denning and breeding in production forests. Production forests often influence the prey availability, hunting success, and diet of carnivores. C_LIO_LIWe show that many carnivores use production forests, and how they respond to production forestry varies with species traits. We recommend that production forests are managed as valuable carnivore habitat, and highlight strategies to enhance the use of these forests by carnivores. C_LI

ecology↗

bioRxiv2023

Portable X-ray fluorescence for identifying harmful lead exposure in wild mammals through hair analysis

Lead exposure threatens scavenging wildlife globally. For inexpensive estimation of lead concentration in bones from avian scavengers, portable X-ray fluorescence (XRF) devices have been trialed. However, portable XRF has not been validated for tissue lead measurement in non-human mammalian scavengers. We evaluated portable XRF for hair lead measurement in the endangered Tasmanian devil (Sarcophilus harrisii). We first analyzed large ([~]1.0 g) hair samples from 39 deceased animals from southeastern Tasmania via portable XRF and then inductively-coupled plasma mass spectrometry (ICP-MS) (validation study). We then measured lead concentrations via portable XRF only in 61 small ([~]0.1 g) hair samples from live devils from a plantation landscape (plantation study). Portable XRF measures of hair lead levels were positively correlated with ICP-MS values (R2 = 0.61). In the validation study, 95% of sampled Tasmanian devils had relatively low lead levels (< 2 mg/kg), but the remaining two showed elevated lead levels (> 15 mg/kg). Mean lead levels in the plantation study and validation study were not significantly different. Our preliminary results suggest that portable XRF can provide a useful measure of differences in lead levels in wildife hair over a coarse scale. We provide recommendations for further research and refinement of this method. SynopsisPortable XRF can provide inexpensive and non-destructive analysis of environmental contaminants in wildlife. We present the first evaluation of portable XRF for analysis of lead contamination in mammalian scavenger hair.

ecology↗

bioRxiv2023

Movements and habitat selection of a marsupial carnivore in a modified landscape

Landscape modification is a major threat to carnivores worldwide, but modified landscapes can also provide important habitat for these species, as protected areas alone are insufficient. Understanding how carnivores use modified landscapes, such as production forests, can inform management strategies to improve the value of these landscapes to carnivores. Little is known about habitat selection by marsupial carnivores in production forests, where they occupy a similar ecological niche to their more well-studied eutherian counterparts. We used GPS tracking, Hidden Markov Models, and Manlys selection ratios to identify the habitat selected in 3 behavioral states by the largest marsupial carnivore, the Tasmanian devil (Sarcophilus harrisii), in a timber plantation-dominated landscape. Behavioral states were approximated as: state 1, resting or feeding at a carcass; state 2, foraging; and state 3, travel. Devils did not show preferences for any of native forest, native grassland, and plantation in any behavioral state. Within plantations, devils preferred a plantation age of 4-7 years (selection ratio [wi] = 1.52). Devils preferred roads (state 1: wi = 2.71, state 2: wi = 2.48, state 3: wi = 2.97) and plantation edges (state 1: wi = 2.38, state 2: wi = 2.24, state 3: wi = 2.78) in all behavioral states, and moved faster on roads and edges than away from them. Together, our results indicate devils use road and edges for foraging (scavenging and hunting) and travel. No measured habitat variables influenced devil home range size. To support devils in plantation landscapes, we recommend maintaining a heterogeneous landscape of different plantation ages and native remnants and reducing the risk of vehicle collisions by minimizing forestry traffic at night. Tasmanian devils share similar adaptable traits to generalist eutherian carnivore species in their use of modified landscapes. Plantations can provide valuable habitat for this and other threatened predator species.

ecology↗

bioRxiv2022

Regulation of human trophoblast gene expression by endogenous retroviruses

The placenta is a fast-evolving organ with large morphological and histological differences across eutherians, but the genetic changes driving placental evolution have not been fully elucidated. Transposable elements, through their capacity to quickly generate genetic variation and affect host gene regulation, may have helped to define species-specific trophoblast gene expression programmes. Here, we assessed the contribution of transposable elements to human trophoblast gene expression as enhancers or promoters. Using epigenomic data from primary human trophoblast and trophoblast stem cell lines, we identified multiple endogenous retrovirus families with regulatory potential that lie close to genes with preferential expression in trophoblast. These largely primate-specific elements are associated with inter-species gene expression differences, and are bound by transcription factors with key roles in placental development. Using genetic editing we demonstrated that several elements act as transcriptional enhancers of important placental genes, such as CSF1R and PSG5. We also identified an LTR10A element that regulates ENG expression, affecting secretion of soluble ENG, with potential implications for preeclampsia. Our data show that transposons have made important contributions to human trophoblast gene regulation, and suggest that their activity may affect pregnancy outcomes.

genetics↗

bioRxiv2019

Multiplexed dissection of a model human transcription factor binding site architecture

In eukaryotes, transcription factors orchestrate gene expression by binding to TF-Binding Sites (TFBSs) and localizing transcriptional co-regulators and RNA Polymerase II to cis-regulatory elements. The strength and regulation of transcription can be modulated by a variety of factors including TFBS composition, TFBS affinity and number, distance between TFBSs, distance of TFBSs to transcription start sites, and epigenetic modifications. We still lack a basic comprehension of how such variables shaping cis-regulatory architecture culminate in quantitative transcriptional responses. Here we explored how such factors determine the transcriptional activity of a model transcription factor, the c-AMP Response Element (CRE) binding protein. We measured expression driven by 4,602 synthetic regulatory elements in a massively parallel reporter assay (MPRA) exploring the impact of CRE number, affinity, distance to the promoter, and spacing between multiple CREs. We found the number and affinity of CREs within regulatory elements largely determines overall expression, and this relationship is shaped by the proximity of each CRE to the downstream promoter. In addition, while we observed expression periodicity as the CRE distance to the promoter varied, the spacing between multiple CREs altered this periodicity. Finally, we compare library expression between an episomal MPRA and a new, genomically-integrated MPRA in which a single synthetic regulatory element is present per cell at a defined locus. We observe that these largely recapitulate each other although weaker, non-canonical CREs exhibited greater activity in the genomic context.

genomics↗

bioRxiv2019

Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning

In humans, the 813 G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state, and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G-protein signal transduction. We tested 7,800 of 7,828 possible single amino acid substitutions to the beta-2 adrenergic receptor ({beta}2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for {beta}2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we resolve residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.

molecular biology↗

bioRxiv2019

Development of a drug repressible β-catenin mutant mouse to examine effect of activated β-catenin signaling on long-term maintenance of mammary tumor growth

Stabilized {beta}-catenin expression is a well described initiator of mammary tumorigenesis in the mouse and elevated nuclear expression of this protein has been observed in human triple-negative tumor samples. However, the importance of stabilized {beta}-catenin to continued tumor growth after initiation, and in the context of other driver mutations, has yet to be elucidated. To ascertain the importance of stabilized {beta}-catenin after tumor initiation, we generated a novel transgenic mouse model, utilizing the tet-off system, to control expression of a stabilized mutant of {beta}-catenin. Pups from early litters carrying one allele of regulatable stabilized {beta}-catenin at the Rosa26 locus, but not targeted by Cre, were smaller in size compared with wildtype littermates and also developed skin lesions, requiring euthanasia. Maintenance of breeding cages on doxycycline chow allowed for healthy transgenic pups to survive past 3 weeks of age and 6 animal cohorts were established. We used two different mammary Cre strains, WAP-Cre and MMTV-NLST-Cre, crossed with regulatable, stabilized {beta}-catenin (bcatFL) animals, as well as animals expressing mutated p53-R270H. While mammary tumors developed in animals expressing Cre-targeted regulatable bcatFL, the penetrance and growth kinetics were lower than observed with other mammary mouse models expressing stabilized {beta}-catenin. Since fewer animals developed tumors, we focused analysis on the effect of turning off stabilized {beta}-catenin expression following tumor initiation. In the animals examined, a delay in tumor growth, but not regression, was observed in animals expressing Cretargeted bcatFL as well as those expressing both bcatFL and p53-R270H. These results indicate that in a less aggressive {beta}-catenin model, additional mutations likely provide independence from the initiating event.

cancer biology↗

bioRxiv2018

Modular, part-based control of gene expression response time using protein degradation tags

A fundamental principle of cellular signal processing is the encoding of information within the temporal dynamics of regulatory circuits. If synthetic circuits are to achieve the versatility and effectiveness of naturally-occurring circuits, it is necessary to develop simple, effective methods for the control of the dynamical properties of genetic circuits. However, current approaches to dynamical control often require extensive rewiring of circuit architecture, which hinders their implementation in a variety of systems. Therefore, it is essential that simple, modular, genetic parts-based frameworks are created to control the dynamical properties of circuits. Here we address this need by implementing a modular, genetic parts-based system which tunes the response time of a genes expression by tuning its degradation rate via the application of protein degradation tags with various affinities to their protease. This system provides a simple, easily- applicable framework for controlling the temporal aspects of genetic circuit behavior.

bioengineering↗

bioRxiv2018

Epigenomic Profiling and Single-Nucleus-RNA-Seq Reveal Cis-Regulatory Elements in Human Retina, Macula and RPE and Non-Coding Genetic Variation.

Cis-regulatory elements (CREs) orchestrate the dynamic and diverse transcriptional programs that assemble the human central nervous system (CNS) during development and maintain its function throughout life. Genetic variation within CREs plays a central role in phenotypic variation in complex traits including the risk of developing disease. However, the cellular complexity of the human brain has largely precluded the identification of functional regulatory variation within the human CNS. We took advantage of the retina, a well-characterized region of the CNS with reduced cellular heterogeneity, to establish a roadmap for characterizing regulatory variation in the human CNS. This comprehensive resource of tissue-specific regulatory elements, transcription factor binding, and gene expression programs in three regions of the human visual system (retina, macula, retinal pigment epithelium/choroid) reveals features of regulatory element evolution that shape tissue-specific gene expression programs and defines the regulatory elements with the potential to contribute to mendelian and complex disorders of human vision.

neuroscience↗

bioRxiv2018

A Scalable, Multiplexed Assay for Decoding Receptor-Ligand Interactions

Chemicals such as drugs, hormones, and odorants can have many potential interactions with endogenous targets, and uncovering these relationships is critical for understanding and modulating function. Mammalian olfactory receptors (ORs), a large family of G protein-coupled receptors, mediate olfaction through activation by small molecules. Each OR can respond to many odorants, and vice versa, making exploring this space one interaction at a time difficult. We developed a high-throughput receptor screening platform in human cell lines to screen libraries of chemicals against a multiplexed library of receptors using next-generation sequencing of barcoded genetic reporters. We screened three concentrations of 181 odorants, where in each well we record the activity of 39 ORs simultaneously, and identified 79 novel associations, including ligands for 15 orphan receptors. This platform allows the cost-effective mapping of large chemical libraries to receptor repertoires at scale.

biochemistry↗

bioRxiv2018

The Genetic Insulator RiboJ Increases Expression of Insulated Genes

The self-cleaving ribozyme RiboJ is an insulator commonly used in genetic circuits to prevent unexpected interactions between neighboring parts. These interactions can compromise the modularity of the circuit, impeding the implementation of predictable genetic constructs. Despite its utility as an insulator, a quantitative assessment of the effect of RiboJ on the properties of downstream genetic parts is lacking. Here, we characterized the impact of insulation with RiboJ on expression of a reporter gene driven by a promoter from a library of 24 frequently employed constitutive promoters. We show that depending on the strength of the promoters, insulation with RiboJ increased protein abundance between twofold and tenfold and increased transcript abundance by an average of twofold. This result is the first to demonstrate that genetic insulators can impact the expression of downstream genes, potentially hindering the design of predictable genetic circuits and constructs.

synthetic biology↗

bioRxiv2017

Large-scale screening of rare genetic variants in humans reveals frequent splicing disruptions

Any individuals genome contains [~]4-5 million genetic variants that differ from reference, and understanding how these variants give rise to trait diversity and disease susceptibility is a central goal of human genetics1. A vast majority (96-99%) of an individuals variants are common, though at a population level the overwhelming majority of variants are rare2-5. Because of their scarcity in an individuals genome, rare variants that play important roles in complex traits are likely to have large functional effects6,7. Mutations that cause an exon to be skipped can have severe functional consequences on gene function, and many known disease-causing mutations reduce or eliminate exon recognition8. Here we explore the extent to which rare genetic variation in humans results in near complete loss of exon recognition. We developed a Multiplexed Functional Assay of Splicing using Sort-seq (MFASS) that allows us to measure exon inclusion in thousands of human exons and surrounding intronic sequence simultaneously. We assayed 27,733 extant variants in the Exome Aggregation Consortium (ExAC)9 within or adjacent to 2,339 human exons, and found that 3.8% (1,050) of the variants, almost all of which were extremely rare, led to large-effect defects in exon recognition. Importantly, we find that 83% of these splice-disrupting variants (SDVs) are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate that loss of exon recognition is an important and underappreciated means by which rare variants exert large functional effects, and that MFASS enables their empirical assessment for splicing defects at scale.

genetics↗