Biology subjects

Hart, D.

Publications and source records attributed to Hart, D..

9 recordsLinked to original sources

bioRxiv2026

Leukemic Stem Cell Subtypes Drive Distinct Niche Remodeling in Acute Myeloid Leukemia

Leukemic stem cells (LSCs) sustain acute myeloid leukemia (AML) and are implicated in therapy resistance and relapse. Yet, it remains unknown how LSCs remodel the bone marrow niche. AML is known to alter stromal and vascular microenvironments, but these effects are difficult to separate from bulk leukemic burden and immune inflammation. Here, we use isogenic human AML xenografts with distinct LSC characteristics but comparable engraftment to define LSC-associated niche remodeling in vivo. Single-cell profiling revealed that LSC-high AML shifts the mesenchymal niche toward fibro-inflammatory states, expanding Fmod+ fibroblasts and Cd34+ perivascular fibroblast-like cells while suppressing osteolineage differentiation. The leukemic compartment remained heterogeneous, with a specific MEP-like LSC population expressing niche-remodeling ligands including TGFB1, IL1B, and ANGPT1. LSC-high AML activated a TGF{beta}-responsive, CREB3L1-controlled fibroblast trajectory, and perturbing TGF{beta} signaling or CREB3L1 activation reduced stromal support for AML cells. These findings identify a specific LSC subtype as a source of niche-remodeling cues that shape specialized leukemia-supportive niches.

molecular biology↗

bioRxiv2026

Synthetic cell-cell adhesion provides benefits of proximity without diffusion-related costs

Multicellularity provides many advantages over unicellularity, but accessing those benefits with synthetic biology approaches is challenging due to potential tradeoffs. The same diffusion limitations that exclude toxins can limit nutrient transport, and small intercellular spaces that facilitate sharing also increase local competition. To isolate when and how cell aggregation, an essential feature of multicellularity, is useful, we compared yeast strains differing only in expression of a single adhesion gene. Unexpectedly, aggregates containing thousands of cells showed no signs of diffusion limitations, growing as well as unicellular strains in both rich and toxic media, likely because reversible cell adhesion allows cells to exchange positions. Aggregating cells also grew better than unicellular strains in the limiting carbon conditions expected to increase diffusion limitations. In mixtures of different genotypes, these benefits were specific to cells expressing the adhesion protein. Together, these results suggest key design principles for engineering aggregates: reformable bonds provide a mechanism to sidestep the fitness costs of increased size, and the benefits within such groups may arise more from nutrient sharing than from diffusion limitations.

synthetic biology↗

bioRxiv2025

Compositional Analysis of Mouse Sperm Subpopulations During Capacitation In Vitro

Mammalian sperm must undergo capacitation to become competent for fertilization, yet this process is marked by substantial phenotypic heterogeneity among sperm cells. How such variability emerges and how it relates to fertilizing potential remain unresolved, in part because sperm subpopulations are typically analyzed independently despite being intrinsically interdependent. Here, we combine large-scale single-cell spectral flow cytometry with compositional statistical modeling to quantify how sperm population structure responds to controlled capacitation signals in vitro. Using cauda epididymal mouse sperm, we implemented a two-dimensional assay that systematically varies extracellular bicarbonate and free calcium--key regulators of capacitation--and classified millions of individual cells into four irreversible physiological states defined by cell viability and acrosome reaction status. We show that bicarbonate and calcium interact nonlinearly to redistribute sperm across these subpopulations, revealing structured responses that would be otherwise obscured in measurements lacking single-cell resolution. Elevated intracellular calcium was associated with increased cell death, while the highest proportions of live, acrosome-reacted sperm occurred under relatively low extracellular calcium conditions. To enable subpopulation-level analysis, we applied a hierarchical Dirichlet-multinomial regression model that accounts for multinomial sampling noise and between-male variability, yielding posterior probability surfaces that describe how sperm subpopulations reallocate across functional states as microenvironmental signaling conditions change. Together, these results demonstrate that capacitation is a stochastic, cell-population level process shaped by structured phenotypic heterogeneity. This framework provides a quantitative foundation for linking sperm subpopulation composition to measures of fertility competence and for improving existing interpretation of flow cytometry-based assessments of male fertility.

cell biology↗

bioRxiv2025

The hyaluronan receptor CD44 drives COVID-19 severity through its regulation of neutrophil migration

The novel respiratory disease COVID-19 caused by the coronavirus SARS-CoV-2 continues to be a public health emergency worldwide, and there is a need for more effective therapy for patients. The relationship between the extracellular matrix and the host immune response to infection is severely understudied. Deposition of the polysaccharide hyaluronan (HA) into the lungs is associated with more severe COVID-19 disease outcomes. HA is a major component of the extracellular matrix in connective tissues and is abundant in many parts of the body, including cartilage, skin, brain, and vitreous body. CD44 is the primary receptor for HA and is found on almost all immune cells in the lung. Known functions of CD44 include mediation of immune cell migration, activation, and differentiation. We hypothesized that increased HA deposition during COVID-19 increases CD44-mediated immune cell infiltration into lungs and results in more severe pathology. Here, we report that in mice infected with a mouse-adapted strain of SARS-CoV-2, treatment with a combination of two anti-CD44 monoclonal antibodies confers a significant survival benefit and reduces weight loss and clinical score of the mice on Day 4 post infection. We show that anti-CD44 treatment decreases many key cytokines and chemokines in the bronchoalveolar lavage fluid on Day 4. With flow cytometry, we show that anti-CD44 reduces the numbers of neutrophils in infected lungs. We also show through immunofluorescence that treatment with anti-CD44 antibodies reduces colocalization of HA and CD45 in lung sections, indicating that HAs interaction with immune cells contributes to pathology. Our findings demonstrate that disruption of HA-receptor interactions is a way to prevent inflammatory pathology in pulmonary infection.

immunology↗

bioRxiv2023

Discovery and engineering of hypercompact epigenetic modulators for durable gene activation

Programmable epigenetic modulators provide a powerful toolkit for controlling gene expression in novel therapeutic applications, but recent discovery efforts have primarily selected for potency of effect rather than contextual robustness or durability thereof. Current CRISPR-based tools are further limited by large cargo sizes that impede clinical delivery and, in gene activation contexts, by brief activity windows that preclude transient, single-dose strategies such as lipid nanoparticle (LNP) delivery. To address these limitations, we perform high-throughput screening to discover novel classes of transcriptional modulators derived from thousands of human, viral, and archaeal proteomes. We identify high-potency activators capable of mitotically stable gene activation in a multitude of cellular contexts and leverage machine learning models to rationally engineer variants with improved activities. In liver and T-cells, novel hypercompact activators (64 to 98 amino acids) derived from vIRF2 core domain (vCD) achieve superior potency and durable activation lasting weeks beyond the current large activators ([~]five-fold larger). In a humanized mouse model, we target a human hypercholesterolemia susceptibility gene and achieve activation persisting five weeks after a single dose by LNP delivery. Our discovery pipeline provides a predictive rubric for the development of contextually robust, potent, and persistent activators of compact size, broadly advancing the therapeutic potential of epigenetic gene activation.

synthetic biology↗

bioRxiv2023

Naked mole rats have distinctive cardiometabolic and genetic adaptations to their underground low-oxygen lifestyles

The naked mole-rat Heterocephalus glaber is a eusocial mammal exhibiting extreme longevity (37-year lifespan), extraordinary resistance to hypoxia and absence of cardiovascular disease. To identify the mechanisms behind these exceptional traits, RNAseq and metabolomics of cardiac tissue from naked mole-rats was compared to other African mole-rat genera. We identified metabolic and genetic adaptations unique to naked mole-rats including elevated glycogen, thus enabling glycolytic ATP generation during cardiac ischemia. Elevated normoxic expression of HIF-1 was observed while downstream hypoxia responsive-genes were down regulated, suggesting adaptation to low oxygen environments. Naked mole-rat hearts showed reduced succinate build-up during ischemia and negligible tissue damage following ischemia-reperfusion injury. These adaptive evolutionary traits reflect a unique hypoxic and eusocial lifestyle that collectively may contribute to their longevity and health span. One Sentence SummaryNaked mole-rats have metabolic adaptations distinct from other subterranean genera rendering them resistant to cardiovascular pathology.

physiology↗

bioRxiv2022

eEF2 improves dense connective tissue repair and healing outcome by regulating cellular death, autophagy, apoptosis, proliferation and migration

Outcomes following human dense connective tissue (DCT) repair are often variable and suboptimal, resulting in compromised function and development of chronic painful degenerative diseases. Moreover, biomarkers and mechanisms that guide good clinical outcomes after DCT injuries are mostly unknown. Here, we characterize the proteomic landscape of DCT repair following human tendon rupture and its association with long-term patient-reported outcome. Moreover, the regulatory mechanisms of relevant biomarkers were assessed partly by gene silencing experiments. A Mass-Spectrometry based proteomic approach quantified a large number (769) of proteins, including 51 differentially expressed proteins among 20 good versus 20 poor outcome patients. A novel biomarker, elongation factor-2 (eEF2) was identified as being strongly prognostic of the 1-year clinical outcome. Further bioinformatic and experimental investigation revealed that eEF2 positively regulated autophagy, cell proliferation and migration, as well as reduced cell death and apoptosis, leading to improved DCT repair and outcomes. Findings of eEF2 as novel prognostic biomarker could pave the way for new targeted treatments to improve healing outcomes after DCT injuries.

cell biology↗

bioRxiv2022

Generation of a human ovarian granulosa cell model from induced pluripotent stem cells

Sex development is an intricate and crucial process in all vertebrates that ensures the continued propagation of genetic diversity within a species, and ultimately their survival. Perturbations in this process can manifest as variations/differences of sex development (VSD/DSD). Primary gonadal somatic cells - the ovarian granulosa cells (GCs) in the case of women - represent the absolute model to investigate mechanism of disease in VSDs. Collection of these cells in humans is laborious and invasive, while classical animal models fail to recapitulate the human phenotype and function. Furthermore, in patients with the most severe forms of VSD gonadal cells are totally absent. It is therefore vital to develop an alternative cell-model. In view of this, we established an efficient method to reprogram donor-derived urinary progenitor cells (UPs) and differentiate iPSCs into granulosa-like cells (GLCs). The UPs presented a less invasive and high-quality cell source, improving the clinical applicability of the model along with utilising a non-integrative reprogramming method that eliminates alteration of the original genome. This novel GLC model closely resembles human GCs in morphology and marker gene expression of GC cell-fate and essential function. These results provide the prospect to generate patient-specific personalised GC models to investigate mechanism of disease in VSDs and could improve understanding of the intricacies in female gonadal development.

developmental biology↗

bioRxiv2017

C-terminal truncation of Pik3r1 in mice models human lipodystrophic insulin resistance uncoupled from dyslipidemia

Heterodimeric class IA phosphatidylinositol-3-kinases (PI3K) transduce signals from many receptor tyrosine kinases including the insulin receptor. PI3K recruitment to phosphotyrosines is mediated by Pik3r1 gene products including the most intensely studied PI3K regulatory subunit, p85, which also binds and regulates the PIP3 phosphatase Pten, and the lipogenic transcription factor Xbp1. Mutations in human PIK3R1 cause SHORT syndrome, featuring lipodystrophy and severe insulin resistance which, uniquely, are uncoupled from fatty liver and dyslipidemia. We describe a novel mouse model of SHORT syndrome made by knock in of the Pik3r1 Y657X mutation. Homozygous embryos die at E11.5, while heterozygous mice exhibit pre-and postnatal growth impairment with diminished placental vascularity. Adipose tissue accretion on high fat feeding was reduced, however adipocyte size was unchanged and preadipocyte differentiation ex vivo unimpaired. Despite severe insulin resistance, heterozygous mice were hypolipidemic, and plasma adiponectin, liver weight, cholesterol, glycogen and triglyceride content were unchanged. Mild downregulation of lipogenic Srebp1, Srebp2 and Chrebp transcriptional activity but no suppression of Xbp1 target genes was seen after fasting. These findings give new insights into the developmental role of Pik3r1, and establish a model of lipodystrophic insulin resistance dissociated from dyslipidemia as seen in SHORT syndrome.

biochemistry↗