Biology subjects

Allen, R. S.

Publications and source records attributed to Allen, R. S..

7 recordsLinked to original sources

bioRxiv2026

Environmentally relevant depleted uranium exposure damages mitochondria, decreases cytosolic reductive capacity, and increases global DNA damage accumulation through a ROS-independent mechanism involving slingshot protein phosphatase 1b enrichment.

Depleted uranium (DU) is an environmental contaminant with a 30 {micro}g/L (ppb; parts per billion) EPA maximum contaminant level (MCL) for drinking water. The mining of uranium and use of DU in modern weapons underly human exposure that disproportionally impacts military and tribal communities in the United States. Uraniums radiotoxic characteristics are understood, but its chemical hazards much less so. In zebrafish (Danio rerio) and human cell cultures we test the hypothesis that exposure to DU negatively impacts cellular function and development through disruption of mitochondrial metabolism. Using a novel shrapnel model with TEM/SEM+EDS, we showed uranium microparticles caused proximity-dependent mitochondrial disruption. In waterborne exposure paradigms, larval movement was reduced and hatching delayed as a result of reduced movement and not enzyme deficiencies in response to 18 ppb DU, below the MCL. Increased DNA damage accumulation was detected in exposed larva and cells. DNA-damage quantitative PCR of DU-exposed larvae showed increased damage in the ahr1 locus (nuclear gene) and decreased mitochondrial DNA (mtDNA) copy number, but mtDNA damage levels varied across experiments. Mitochondrial function was assessed using a resazurin-based assay in the presence and absence of antioxidants and showed diminished cytoplasmic reductive capacity. DU exposure alone did not enrich antioxidant gene expression, contrasting with arsenic exposure, a known ROS-inducer and Nrf2-activator. Sulforaphane (SFN), a potent Nrf2-activator, did not blunt the effects of DU exposure, despite activation of antioxidant response element (ARE) genes (gstp and gss), but did blunt the effects of arsenic exposure. The most enriched transcript in DU-exposed larvae coded for slingshot protein phosphatase (ssh), further exploration revealed ssh1b as the zebrafish-specific ortholog activated in response to DU, and inhibition using an identified SSH1 inhibitor, Sennoside A, partially rescued the metabolic and hatching defects observed. Our data points to a cytotoxic mechanism in which DU disrupts mitochondrial function through ssh1b enrichment that impairs normal mitophagy, leading to decreased cellular reductive potential independent of either ROS production or ARE-activation. Our results suggest that health impacts from DU exposure may be directly linked to impaired mitochondrial functions.

pharmacology and toxicology↗

bioRxiv2026

Transcriptomic data of larval zebrafish exposed to continuous sub- and supra-MCL sodium arsenite and uranyl nitrate.

Uranium (U) and arsenic (As) are both ubiquitous contaminants in the American southwest, posing risks to humans, animals, and the environment. Depleted uraniums (DU) chronic effects and mechanisms of toxicity are incompletely understood. Differential gene expression of concomitant exposures to identify markers of toxicity have not been undertaken until now. Continuous low-dose, high-dose, and concomitant exposures are investigated using the larval zebrafish (Danio rerio), with exposure paradigms lasting from embryo collection until sampling at 5 days post fertilization (dpf). Herein, we describe overall differential gene expression with counts and pathway enrichment statistics using both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The raw dataset has been deposited in NCBIs Gene Expression Omnibus (GEO) repository [1] under the accession number GSE319292 [2]. O_TBL View this table: org.highwire.dtl.DTLVardef@9b121aorg.highwire.dtl.DTLVardef@c17073org.highwire.dtl.DTLVardef@1bdc2b9org.highwire.dtl.DTLVardef@13b130aorg.highwire.dtl.DTLVardef@15f1d22_HPS_FORMAT_FIGEXP M_TBL C_TBL VALUE OF THE DATAO_LIUranyl nitrate (UN), a water-soluble depleted uranium species, and sodium arsenite (As) are both ubiquitous contaminants in the American southwest, posing risks to humans, animals, and the environment. The United States Environmental Protection Agency (EPA) has set maximum contaminant limits (MCL) of 30 ppb U atoms and 10 ppb As atoms, respectively. C_LIO_LIThese data show differentially expressed genes (DEGs) from larval zebrafish exposed to 1 or 10 {micro}M As, 30 or 300 {micro}g/L UN, or 1 {micro}M As and 30 {micro}g/L UN in combination. Concentrations were specifically chosen based on environmental relevance. C_LIO_LIGene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of up- and down-regulated DEGs are provided to understand the molecular mechanisms of uranium toxicity and inform future studies. C_LIO_LIThese data should be used for biomarker identification and mechanistic interrogation of single and combinatorial exposures of environmentally relevant compounds at realistic exposure levels. C_LI

pharmacology and toxicology↗

bioRxiv2025

Specific cell states underlie complex tissue regeneration in spiny mice

SUMMARY STATEMENTComparing regenerative vs. fibrotic healing, we identify injury-induced cell states associated with persistent cell cycle progression and complex tissue regeneration in mammals. Cell proliferation is an elemental feature of epimorphic regeneration in vertebrate taxa. We previously reported that in contrast to fibrotic repair observed in laboratory mouse (Mus) strains, highly regenerative spiny mice (Acomys spp.) exhibit cell cycle progression and cell proliferation to faithfully replace missing tissue. However, little is known about proliferation dynamics, and specific cell types and states that may contribute to complex tissue regeneration in mammals. Using temporal pulse-chase experiments, we show that stromal cells in Acomys dimidiatus rapidly re-enter the cell cycle in response to injury and maintain tight spatiotemporal control of cell cycle progression to restrict the proliferative population to a distal area relative to the injury. Conversely, Mus stromal cells incorporate thymidine analogs without cell division supporting an S-phase arrest after D10. Deploying immunostaining and scRNA-seq, we identify several key cell types (CRABP1+, SMA+) differentially associated with regenerating versus scar tissue. Importantly, our single cell data revealed distinct gene expression profiles for cross-species stromal cell types, identifying cell states specific for regenerative or fibrotic healing. While CRABP1+ fibroblasts are enriched in Acomys ears before and after injury, similar fibroblasts enriched in young, postnatal Mus ears remain unable to promote regeneration. Our data underscore the finely regulated dynamics of proliferating cells during regeneration and emphasize that regeneration depends on multiple factors including the presence of specific cell types and the ability of cells to acquire specific states. Key Conclusions-Differentiated cells in Acomys, Mus and Danio re-enter the cell cycle in response to injury, while homeostatic cycling cells contribute to blastema formation in Ambystoma -Pulse-chase thymidine analog labeling shows tight spatiotemporal control of proliferating stromal cells during regeneration in Acomys. -Following injury, CRABP1 and SMA are expressed in distinct stromal cell populations in Acomys but are co-expressed in Mus stromal cell populations. -Species-specific cell states underlie regenerative and fibrotic repair -CRABP1+ cells are lost during embryonic development in Mus ear pinna but are retained in Acomys to adulthood. -Young neonatal Mus with abundant CRABP1+ cells still fail to execute regenerative healing

developmental biology↗

bioRxiv2023

Neural crest cells give rise to non-myogenic mesenchymal tissue in the adult murid ear pinna

Despite being a major target of reconstructive surgery, development of the external ear pinna remains poorly studied. As a craniofacial organ highly accessible to manipulation and highly conserved among mammals, the ear pinna represents a valuable model for the study of appendage development and wound healing in the craniofacial complex. Here we provide a cellular characterization of late gestational and postnatal ear pinna development in Mus musculus and Acomys cahirinus and demonstrate that ear pinna development is largely conserved between these species. Using Wnt1-cre;ROSAmT/mG mice we find that connective tissue fibroblasts, elastic cartilage, dermal papilla cells, dermal sheath cells, vasculature, and adipocytes in the adult pinna are derived from cranial crest. In contrast, we find that skeletal muscle and hair follicles are not derived from neural crest cells. Cellular analysis using the naturally occurring short ear mouse mutant shows that elastic cartilage does not develop properly in distal pinna due to impaired chondroprogenitor proliferation. Interestingly, while chondroprogenitors develop in a mostly continuous sheet, the boundaries of cartilage loss in the short ear mutant strongly correlate with locations of vasculature-conveying foramen. Concomitant with loss of elastic cartilage we report increased numbers of adipocytes, but this seems to be a state acquired in adulthood rather than a developmental abnormality. In addition, chondrogenesis remains impaired in the adult mid-distal ear pinna of these mutants. Together these data establish a developmental basis for the study of the ear pinna with intriguing insights into the development of elastic cartilage.

developmental biology↗

bioRxiv2022

Reduced GS domain serine/threonine requirements of Fibrodysplasia Ossificans Progressiva mutant type I BMP receptor ACVR1 in the zebrafish

Fibrodysplasia ossificans progressiva (FOP) is a rare human genetic condition characterized by altered skeletal development and extra-skeletal bone formation. All cases of FOP are caused by mutations in the type I BMP receptor gene ACVR1 that result in over-activation of the BMP signaling pathway. Activation of the wild-type ACVR1 kinase requires assembly of a tetrameric type I and II BMP receptor complex followed by phosphorylation of the ACVR1 GS domain by type II BMP receptors. Previous studies showed that the FOP mutant ACVR1-R206H requires type II BMP receptors and presumptive GS domain phosphorylation for over-active signaling. Structural modeling of the FOP-ACVR1 mutant kinase domain supports that FOP mutations alter the conformation of the GS domain, but it is unclear how this leads to overactive signaling. Here we show using a developing zebrafish embryo BMP signaling assay that the FOP mutant receptors ACVR1-R206H and -G328R have reduced requirements for GS domain phosphorylation sites to signal compared to wild-type ACVR1. Further, ligandindependent and ligand-dependent signaling through the FOP ACVR1 receptors have distinct GS domain phosphorylation site requirements. Moreover, ACVR1-G328 showed increased GS domain serine/threonine requirements for ligand-independent signaling compared to ACVR1-R206H, whereas it exhibited reduced serine/threonine requirements for ligand-dependent signaling. Remarkably, while ACVR1-R206H does not require the type I BMP receptor partner, Bmpr1, to signal, a ligand-dependent GS domain mutant of ACVR1-R206H could signal independently of Bmpr1 only when Bmp7 ligand was overexpressed. Of note, unlike human ACVR1-R206H, the zebrafish paralog Acvr1l-R203H does not show increased signaling activity. However, in domain-swapping studies the human kinase domain, but not the human GS domain, was sufficient to confer overactive signaling to the Acvr1l-R203H receptor. Together these results reflect the importance of GS domain activation and kinase domain functions in regulating ACVR1 signaling and identify mechanisms of reduced regulatory constraints conferred by FOP mutations.

molecular biology↗

bioRxiv2019

An experimental workflow identifies nitrogenase proteins ready for expression in plant mitochondria

Industrial nitrogen fertilizer is intrinsic to modern agriculture yet expensive and environmentally harmful. We aim to reconstitute bacterial nitrogenase function within plant mitochondria to reduce nitrogen fertilizer usage. Many nitrogen fixation (Nif) proteins are required for biosynthesis and function of the mature nitrogenase enzyme, and these will need to be correctly processed and soluble within mitochondria as a pre-requisite for function. Here we present our workflow that assessed processing, solubility and relative abundance of 16 Klebsiella oxytoca Nif proteins targeted to the plant mitochondrial matrix using an Arabidopsis mitochondrial targeting peptide (MTP). The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using bacterial nitrogenase assays. We found that despite the use of the same constitutive promoter and MTP, MTP::Nif processing and relative abundance in plant leaf varied considerably. Assessment of solubility for all MTP::Nif proteins found NifF, M, N, S, U, W, X, Y and Z were soluble, while NifB, E, H, J, K, Q and V were mostly insoluble. Although most Nif proteins tolerated the N-terminal extension as a consequence of mitochondrial processing, this extension in NifM reduced nitrogenase activity to 10% of controls. Using proteomics, we detected a [~]50-fold increase in the abundance of NifM when it contained the N-terminal MTP extension, which may account for this reduction seen in nitrogenase activity. Based on plant mitochondrial processing and solubility, and retention of function in a bacterial assay, our workflow has identified that NifF, N, S, U, W, Y and Z satisfied all these criteria. Future work can now focus on improving these parameters for the remaining Nif components to assemble a complete set of plant-ready Nif proteins for reconstituting nitrogen fixation in plant mitochondria.

synthetic biology↗

bioRxiv2019

Engineering a functional NifDK polyprotein resistant to mitochondrial degradation

To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

synthetic biology↗