Biology subjects

Stone, A.

Publications and source records attributed to Stone, A..

8 recordsLinked to original sources

bioRxiv2025

CRISPRi-Linked Multi-Module Negative Feedback Loops to Address Winner-Take-All Resource Competition

Cellular resource limitations create unintended interactions among synthetic gene circuit modules, compromising circuit modularity. This challenge is particularly pronounced in circuits with positive feedback, where uneven resource allocation can lead to Winner-Takes-All (WTA) behavior, favoring one module at the expense of others. In this study, we experimentally implemented a Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi) and evaluated its effectiveness in mitigating WTA behavior in two gene circuits: dual self-activation and cascading bistable switch. We chromosomally integrated a tunable dCas9 gene and designed module-specific gRNAs, with each module encoding its own gRNA to self-repress via competition for limited dCas9. This configuration introduces strong negative feedback to the more active module while reallocating resources to the less active one, promoting balanced module activation. Compared to the control group lacking dCas9-mediated repression, the NCR controller significantly increased module coactivation and suppressed WTA behavior. Our quantitative results demonstrate that NCR provides an effective strategy for regulating resource competition and improving the modularity of synthetic gene circuits.

synthetic biology↗

bioRxiv2023

Adaptations to water stress and pastoralism in the Turkana of northwest Kenya

The Turkana people inhabit arid regions of east Africa--where temperatures are high and water is scarce--and they practice subsistence pastoralism, such that their diet is primarily composed of animal products. Working with Turkana communities, we sequenced 367 genomes and identified 8 regions putatively involved in adaptation to water stress and pastoralism. One of these regions includes a putative enhancer for STC1--a kidney-expressed gene involved in the response to dehydration and the metabolism of purine-rich foods such as red meat. We show that STC1 is induced by antidiuretic hormone in humans, is associated with urea levels in the Turkana themselves, and is under strong selection in this population (s[~]0.041). This work highlights that partnerships with subsistence-level groups can lead to new models of human physiology with biomedical relevance.

evolutionary biology↗

bioRxiv2022

Human bone marrow organoids for disease modelling, discovery and validation of therapeutic targets in hematological malignancies

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from iPSCs committed to mesenchymal, endothelial and hematopoietic lineages. These 3-dimensional structures capture key features of human bone marrow - stroma, lumen-forming sinusoidal vessels and myeloid cells including pro-platelet forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGF{beta} stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate discovery and prioritization of novel targets for bone marrow disorders and blood cancers. Significance StatementWe present a 3D, vascularised human bone marrow organoid that supports growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment, and provides a much-needed, ex vivo tool for prioritization of new therapeutics.

cancer biology↗

bioRxiv2022

Identification of CD317-Positive Pro-inflammatory Immune Stromal Cells in Human Mesenchymal Stromal Cell Preparations

Mesenchymal stromal cell (MSC) heterogeneity clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterised by CD317 expression (CD317pos (29.77{+/-}3.00% of the total MSC population), comprising CD317dim (28.10{+/-}4.60%) and CD317bright (1.67{+/-}0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarisation towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

cell biology↗

bioRxiv2021

Double-edged role of resource competition in gene expression noise and control

Despite extensive investigation demonstrating that resource competition can significantly alter the circuits deterministic behaviors, a fundamental issue is how resource competition contributes to the gene expression noise and how the noise can be controlled. Utilizing a two-gene circuit as a prototypical system, we uncover a surprising double-edged role of resource competition in gene expression noise: the competition decreases noise through a resource constraint but generates its own type of noise which we name as "resource competitive noise." Utilization of orthogonal resources enables retaining the noise reduction conferred by resource constraint while removing the added resource competitive noise. The noise reduction effects are studied using three negative feedback controller types: negatively competitive regulation (NCR), local, and global controllers, each having four placement architectures in the protein biosynthesis pathway (mRNA or protein inhibition on transcription or translation). Our results show that both local and NCR controllers with mRNA-mediated inhibition are efficacious at reducing noise, with NCR controllers demonstrating a superior noise-reduction capability. We also find that combining negative feedback controllers with orthogonal resources can improve the local controllers. This work provides deep insights into the origin of stochasticity in gene circuits with resource competition and guidance for developing effective noise control strategies.

synthetic biology↗

bioRxiv2018

The Medical Genome Reference Bank: Whole genomes and phenotype of 2,570 healthy elderly

Population health research is increasingly focused on the genetic determinants of healthy ageing, but there is no public resource of whole genome sequences and phenotype data from healthy elderly individuals. Here we describe the Medical Genome Reference Bank (MGRB), comprising whole genome sequence and phenotype of 2,570 elderly Australians depleted for cancer, cardiovascular disease, and dementia. We analysed the MGRB for single-nucleotide, indel and structural variation in the nuclear and mitochondrial genomes. Individuals in the MGRB had fewer disease-associated common and rare germline variants, relative to both cancer cases and the gnomAD and UK BioBank cohorts, consistent with risk depletion. Pervasive age-related somatic changes were correlated with grip strength in men, suggesting blood-derived whole genomes may also provide a biologic measure of age-related functional deterioration. The MGRB provides a broadly applicable reference cohort for clinical genetics and genomic association studies, and for understanding the genetics of healthy ageing. This research has been conducted using the UK Biobank Resource under Application Number 17984.

genomics↗

bioRxiv2018

MicroRNA-196a is regulated by ER and is a prognostic biomarker in ER+ Breast Cancer

MicroRNAs are potent post-transcriptional regulators involved in all hallmarks of cancer. Mir-196a is transcribed from two loci and has been implicated in a wide range of developmental and pathogenic processes, with targets including Hox, Fox, Cdk inhibitors and annexins. Genetic variants and altered expression of miR196a are associated with risk and progression of multiple cancers including breast cancer, however little is known about the regulation of the genes encoding this miRNA, nor the impact of variants therein. Here we demonstrate that MIR196A displays complex and dynamic expression patterns, in part controlled by long range transcriptional regulation between promoter and enhancer elements bound by ER. Expression of this miRNA is significantly increased in models of hormone receptor positive disease resistance. The expression of MIR196A also proves to be a robust prognostic factor for patients with advanced and post-menopausal ER+ disease. This work sheds light on the normal and abnormal regulation of MIR196A and provides a novel stratification method for therapeutically resistant breast cancer.

cancer biology↗

bioRxiv2018

The Medical Genome Reference Bank: a whole-genome data resource of 4,000 healthy elderly individuals. Rationale and cohort design

Allele frequency data from human reference populations is of increasing value for filtering and assignment of pathogenicity to genetic variants. Aged and healthy populations are more likely to be selectively depleted of pathogenic alleles, and therefore particularly suitable as a reference populations for the major diseases of clinical and public health importance. However, reference studies of the healthy elderly have remained under-represented in human genetics. We have developed the Medical Genome Reference Bank (MGRB), a large-scale comprehensive whole-genome dataset of confirmed healthy elderly individuals, to provide a publicly accessible resource for health-related research, and for clinical genetics. It also represents a useful resource for studying the genetics of healthy aging. The MGRB comprises 4,000 healthy, older individuals with no reported history of cancer, cardiovascular disease or dementia, recruited from two Australian community-based cohorts. DNA derived from blood samples will be subject to whole genome sequencing. The MGRB will measure genome-wide genetic variation in 4,000 individuals, mostly of European decent, aged 60-95 years (mean age [≥] 75 years). The MGRB has committed to a policy of data sharing, employing a hierarchical data management system to maintain participant privacy and confidentiality, whilst maximizing research and clinical usage of the database. The MGRB will represent a dataset of international significance, broadly accessible to the clinical and genetic research community.

genomics↗