Biology subjects

Zhou, G.

Publications and source records attributed to Zhou, G..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Host-derived Interleukin 1α induces an immunosuppressive tumor microenvironment via regulating monocyte-to-macrophage differentiation

Tumor-associated macrophages exhibit high heterogeneity and contribute to the establishment of an immunosuppressive tumor microenvironment (TME). Although numerous studies have demonstrated that extracellular factors promote macrophage proliferation and polarization, the regulatory mechanisms governing the differentiation process to generate phenotypically, and functionally diverse macrophage subpopulations remain largely unexplored. In this study, we examined the influence of interleukin 1 (IL-1) on the development of an immunosuppressive TME using orthotopic transplantation murine models of breast cancer. Deletion of host Il1 led to the rejection of inoculated congenic tumors. Single-cell sequencing analysis revealed that CX3CR1+ macrophage cells were the primary sources of IL-1 in the TME. The absence of IL-1 reprogrammed the monocyte-to-macrophage differentiation process within the TME, characterized by a notable decrease in the subset of CX3CR+ ductal-like macrophages and an increase in iNOS-expressing inflammatory cells. Comparative analysis of gene signatures in both human and mouse macrophage subsets suggested that IL-1 deficiency shifted the macrophage polarization from M2 to M1 phenotypes, leading to enhanced cytotoxic T lymphocyte activity in the TME. Importantly, elevated levels of IL-1 in human cancers were associated with worse prognosis following immunotherapy. These findings underscore the pivotal role of IL-1 in shaping an immune-suppressive TME through the regulation of macrophage differentiation and activity, highlighting IL-1 as a potential target for breast cancer treatment. TeaserInterleukin 1 dictates macrophage behavior, influencing an immunosuppressive microenvironment in breast cancer, suggesting it as a treatment target.

cancer biology↗

bioRxiv2024

Towards high-resolution modeling of small molecule-ion channel interactions

Ion channels are critical drug discovery targets for a wider range of pathologies, such as epilepsy, chronic pain, itch, autoimmunity, and cardiac arrhythmias. To develop effective and safe therapeutics, it is necessary to design small molecules with high potency and selectivity for specific ion channel subtypes. There has been increasing implementation of structure-guided drug design for the development of small molecules targeting ion channels. We evaluated the performance of two Rosetta ligand docking methods, RosettaLigand and GALigandDock, on structures of known ligand - cation channel complexes. Ligands were docked to voltage-gated sodium (NaV), voltage-gated calcium (CaV), and transient receptor potential vanilloid (TRPV) channel families. For each test case, RosettaLigand and GALigandDock methods were able to frequently sample a ligand binding pose within 1-2 [A] root mean square deviation (RMSD) relative to the native ligand - channel structure. However, RosettaLigand and GALigandDock scoring functions cannot consistently identify native drug binding poses as top-scoring models. Our study reveals that the proper scoring criteria for RosettaLigand and GALigandDock modeling of ligand - ion channel complexes should be assessed on a case-by-case basis using sufficient ligand and receptor interface sampling, knowledge about state specific interactions of the ion channel and inherent receptor site flexibility that could influence ligand binding.

biophysics↗

bioRxiv2024

An artificial intelligence accelerated virtual screening platform for drug discovery

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we developed a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screened multi-billion compound libraries against two unrelated targets, a novel ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. On both targets, we discover hits, including seven novel hits (14% hit rate) to KLHDC2 and four novel hits (44% hit rate) to NaV1.7 with single digit micromolar binding affinities. Screening in both cases was completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

biochemistry↗

bioRxiv2024

The BioRECIPE Knowledge Representation Format

AO_SCPLOWBSTRACTC_SCPLOWThe BioRECIPE (Biological system Representation for Evaluation, Curation, Interoperability, Preserving, and Execution) knowledge representation format was introduced to facilitate seamless human-machine interaction while creating, verifying, evaluating, curating, and expanding executable models of intra- and intercellular signaling. This format allows a human user to easily preview and modify any model component, while it is at the same time readable by machines and can be processed by a suite of model development and analysis tools. The BioRECIPE format is compatible with multiple representation formats, natural language processing tools, modeling tools, and databases that are used by the systems biology community. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/579694v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@76a130org.highwire.dtl.DTLVardef@505cb4org.highwire.dtl.DTLVardef@1f64e68org.highwire.dtl.DTLVardef@195bd03_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

Spatial Transcriptomics Sequencing of Mouse Liver at 2 Micron Resolution Using a Novel Spatial DNA Chip

Spatial transcriptomics enables analysis of gene expression that is spatially resolved within a tissue section, making it possible to elucidate the relationship between individual cells within the context of the tissue. This transformative technology enables researchers to better understand gene function within the context of health tissue, developmental processes, and disease. In this study, we present an innovative spatial transcriptomics technology and data using a high-resolution DNA chip with a total capture region size of 6.5 x 6.5 mm containing 2 x 2 {micro}m features for spatial barcoding with no gaps between the features, thereby maximizing the capture area. These chips are manufactured at wafer scale using photolithography and are transferred to hydrogels, making them compatible with existing workflows for fresh frozen or paraffin-embedded samples. Herein, we examined a fresh frozen sample from an adult mouse liver. To analyze the data, we binned 10 x 10 features to represent a 20 {micro}m x 20 {micro}m capture area. We obtained 1.3 billion unique mapped reads, 68.78% sequencing saturation, with a median of 16,967 unique reads per region, indicating the potential for more unique reads with deeper sequencing. This high-resolution mapping of liver cell types and the visualization of gene expression patterns illustrate significant advancements in spatial sequencing technology.

genomics↗

bioRxiv2023

A Bayesian Approach to Correcting the Attenuation Bias of Regression Using Polygenic Risk Score

Polygenic risk score (PRS) has become increasingly popular for predicting the value of complex traits. In many settings, PRS is used as a covariate in regression analysis to study the association between different phenotypes. However, measurement error in PRS causes attenuation bias in the estimation of regression coefficients. In this paper, we employ a Bayesian approach to accounting for the measurement error of PRS and correcting the attenuation bias in linear and logistic regression. Through simulation, we show that our approach is able to obtain approximately unbiased estimation of coefficients and credible intervals with correct coverage probability. We also empirically compare our Bayesian measurement error model to the conventional regression model by analyzing real traits in the UK Biobank. The results demonstrate the effectiveness of our approach as it significantly reduces the error in coefficient estimates.

genetics↗

bioRxiv2023

Leveraging genetic correlations and multiple populations to improve genetic risk prediction for non-European populations

Genetic prediction accuracy for non-European populations is hindered by the limited sample size of Genome-wide association studies (GWAS) data in these populations. Additionally, it is challenging to tune model parameters with a small tuning dataset for methods that require tuning data, which is often the case for non-European samples. To address these challenges, we propose JointPRS, a novel, data-adaptive framework that simultaneously models multiple populations using GWAS summary statistics. JointPRS incorporates genetic correlation structures into the prediction framework, enabling accurate performance even without individual-level tuning data. Additionally, it uniquely employs a data-adaptive approach, providing a robust solution when only a small tuning dataset is available. Through extensive simulations and real data applications to 22 quantitative traits and four binary traits in five continental populations (European (EUR); East Asian (EAS); African (AFR); South Asian (SAS); and Admixed American (AMR)) evaluated using the UK Biobank (UKBB) and All of Us (AoU), we demonstrate that JointPRS outperforms six other state-of-art methods across three different data scenarios (no tuning data, tuning and testing data from the same cohort, and tuning and testing data from different cohorts) for most traits in non-European populations, while maintaining model simplicity and computational efficiency.

genetics↗

bioRxiv2023

Molecular mechanism of NAD+ binding to the Nudix homology domains of DBC1

DBC1 (deleted in breast cancer 1) is a human nuclear protein that modulates the activities of various proteins. NAD+ (oxidized form of nicotinamide adenine dinucleotide) is thought to potentially bind to the Nudix homology domains (NHDs) of DBC1, thereby regulating DBC1-PARP1 [poly (adenosine diphosphate-ribose) polymerase] interactions, the modulation of which may restore DNA repair to protect against cancer, radiation, and aging. Therefore, our study comprehensively employed methods including NMR (Nuclear Magnetic Resonance), ITC (isothermal titration calorimetry), genetic mutation, and computer biology to thoroughly investigate the molecular mechanism of the binding interaction between NAD+ and its precursor NMN with the NHD domain of DBC1 (DBC1354-396). The results from NMR and ITC indicate that NAD+ likely interacts with DBC1354-396 through hydrogen bonding, with a binding affinity nearly twice that of NMN. The key binding sites are primarily E363 and D372. Molecular Docking further revealed the importance of conventional hydrogen bonds and carbon-hydrogen bonds in the binding process. These findings may lead to a better understanding of how NAD+ regulates the physiological functions of DBC1, thereby offering guiding principles for the development of targeted therapies and drug research focused on tumor diseases associated with DBC1.

biochemistry↗

bioRxiv2023

Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions

Oxygen (O2) tension plays a key role in tissue function and pathophysiology. O2-controlled cell culture, in which the O2 concentration in an incubators gas phase is controlled, is an indispensable tool to study the role of O2 in vivo. For this technique, it is presumed that the incubator setpoint is equal to the O2 tension that cells experience (i.e., pericellular O2). We discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0.0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O2 tensions, we developed a reaction-diffusion model that predicts pericellular O2 tension a priori. This model revealed that the effect of cellular O2 consumption is greatest in smaller volume culture vessels (e.g., 96-well plate). By controlling pericellular O2 tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs. hypoxia. MCF7 also expressed higher levels of HIF2A, CD73, NDUFA4L2, etc. and lower levels of HIF1A, CA9, VEGFA, etc. in response to hypoxia vs. anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs. anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O2 tension in cell culture incubators is insufficient to control O2 in cell culture and introduce the concept of pericellular O2-controlled cell culture.

bioengineering↗

bioRxiv2023

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists

GPCRs transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors to determine GPCR agonist localization in the whole brain. We previously engineered integrator sensors for the mu and kappa opioid receptor agonists called M- and K-SPOTIT, respectively. Here, we show a new integrator sensor design platform called SPOTall that we used to engineer sensors for the beta-2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. For multiplexed imaging of SPOTIT and SPOTall, we engineered a red version of the SPOTIT sensors. Finally, we used M-SPOTIT and B2AR-SPOTall to detect morphine, isoproterenol, and epinephrine in the mouse brain. The SPOTIT and SPOTall sensor design platform can be used to design a variety of GPCR integrator sensors for unbiased agonist detection of many synthetic and endogenous neuromodulators across the whole brain.

bioengineering↗

bioRxiv2023

Novel Pan-RAS Inhibitor ADT-007 Induces Tumor Regression in Mouse Models of GI Cancer

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RASWT cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RASWT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, while insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases expressed in RASWT and normal cells but repressed in RAS mutant cancer cells. ADT-007 binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 displayed unique advantages over mutant-specific KRAS and pan-KRAS inhibitors, as well as other pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms leading to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for RAS-driven cancers. SIGNIFICANCEADT-007 has unique pharmacological properties with distinct advantages over other RAS inhibitors by circumventing resistance and activating antitumor immunity. ADT-007 prodrugs and analogs with oral bioavailability warrant further development for RAS-driven cancers.

cancer biology↗

bioRxiv2023

Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery both in vitro and in vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significant in vivo recombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-B activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease. Graphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-B) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/535834v4_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@d0da37org.highwire.dtl.DTLVardef@1a6514corg.highwire.dtl.DTLVardef@235c9aorg.highwire.dtl.DTLVardef@194bbfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

Vascular endothelium forms a restrictive barrier to defend the underlying tissue against uncontrolled influx of circulating protein and immune cells. Mechanisms that mediate the transition from restrictive to leaky endothelium, a hallmark of tissue injury exemplified by acute lung injury (ALI), remain elusive. Using endothelial cell (EC)-Fak-/-mice, we show that FAK sensing and transmission of mechanical tension to the EC nucleus governs cell fate. In FAK- deleted EC, increased EC tension induced by Rho kinase caused tyrosine phosphorylation of nuclear envelope protein, emerin at Y74/Y95, and its localization in a nuclear cap. Activated emerin stimulated DNMT3a activity and methylation of the KLF2 promoter, impairing the restrictive EC transcriptome, including S1PR1. Inhibiting emerin phosphorylation or DNMT3a activity enabled KLF2 transcription of S1PR1, rescuing the restrictive EC phenotype in EC-Fak-/- lungs. Thus, FAK sensing of tension transmission to the nucleus is crucial for maintaining a restrictive EC fate and lung homeostasis.

cell biology↗

bioRxiv2023

Melanin enhances metastatic melanoma colonization by inhibiting ferroptosis

Melanoma associated death is mainly caused by metastatic disease. Increased melanin levels are associated with decreased melanoma patient survival, yet the contribution of melanin to this process is unknown. Here we show that melanin protects circulating melanoma cells from ferroptosis, enhancing their metastatic potential. We observed that melanin levels in patient-derived uveal melanoma cells as well as cutaneous and conjunctival melanoma cell lines correlate with their metastatic potential in zebrafish xenografts. We find strong associations of the melanin biosynthesis gene TYRP1, ferroptosis related enzyme GPX4 and mitochondrial anion channel (VDAC1) with reduced melanoma-specific survival in TCGA data of cutaneous melanoma. Modulation of melanin levels significantly impacts melanoma metastatic potential, increasing or decreasing in concordance with melanin levels. Furthermore, melanin depletion significantly sensitized melanoma cells to ferroptosis leading to a decreased metastatic capacity and enhanced efficacy of ferroptosis induction based anti-cancer therapeutic strategies. Collectively, our results reveal that combined inhibition of melanin biosynthetic enzymes and induction of ferroptosis has potential as a treatment strategy of metastatic melanoma. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/535376v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1fc21a5org.highwire.dtl.DTLVardef@502b6org.highwire.dtl.DTLVardef@f522beorg.highwire.dtl.DTLVardef@1bbfdfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Novel endogenous engineering platform for robust loading and delivery of functional mRNA by extracellular vesicles

Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a plethora of clinical applications. For in vivo functionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, a bioengineering platform for efficient mRNA loading and functional delivery using bionormal nanoparticles, Extracellular Vesicles (EVs), is established by expressing a highly specific RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA. The additional combination with a fusogenic endosomal escape moiety, VSVg, enables functional mRNA delivery in vivo at doses substantially lower than currently used clinically with synthetic lipid-based nanoparticles. Importantly, the application of EVs loaded with effective cancer immunotherapy proves highly effective in an aggressive melanoma mouse model. This technology addresses substantial drawbacks currently associated with EV-based nucleic acid delivery systems and is a leap forward to clinical EV applications. Graphical Abstract(Figure created using BioRender) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/533081v3_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@140058eorg.highwire.dtl.DTLVardef@2ceaccorg.highwire.dtl.DTLVardef@eafd52org.highwire.dtl.DTLVardef@19d4019_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Single-Cell Map of Dynamic Multicellular Ecosystem of Radiation-Induced Intestinal Injury

Intestine is a highly radiation-sensitive organ that could be injured during the radiotherapy for abdominal or pelvic cavity tumors. However, the dynamic change of the intestinal microenvironment related to radiation-induced intestine injury (RIII) is still unclear. Using single-cell RNA sequencing, we pictured a dynamic landscape of the intestinal microenvironment during RIII and regeneration. We showed that the multicellular ecosystem of intestine exhibited heterogeneous radiosensitivities. We revealed the distinct dynamic patterns of three subtypes of intestinal stem cells (ISCs), and the cellular trajectory analysis suggested a complex interconversion pattern among them. For the immune cells, we found that Ly6c+ monocytes can give rise to both pro-inflammatory macrophages and resident macrophages after RIII. Besides, through cellular communication analysis, we identified a positive feedback loop between the macrophages and endothelial cells, which could amplify the inflammatory response induced by radiation. Overall, our study provides a valuable single-cell map of the dynamic multicellular ecosystem during RIII and regeneration, which may facilitate the understanding of the mechanism of RIII.

cell biology↗

bioRxiv2023

Cyclic peptide structure prediction and design using AlphaFold

Deep learning networks offer considerable opportunities for accurate structure prediction and design of biomolecules. While cyclic peptides have gained significant traction as a therapeutic modality, developing deep learning methods for designing such peptides has been slow, mostly due to the small number of available structures for molecules in this size range. Here, we report approaches to modify the AlphaFold network for accurate structure prediction and design of cyclic peptides. Our results show this approach can accurately predict the structures of native cyclic peptides from a single sequence, with 36 out of 49 cases predicted with high confidence (pLDDT > 0.85) matching the native structure with root mean squared deviation (RMSD) less than 1.5 [A]. Further extending our approach, we describe computational methods for designing sequences of peptide backbones generated by other backbone sampling methods and for de novo design of new macrocyclic peptides. We extensively sampled the structural diversity of cyclic peptides between 7-13 amino acids, and identified around 10,000 unique design candidates predicted to fold into the designed structures with high confidence. X-ray crystal structures for seven sequences with diverse sizes and structures designed by our approach match very closely with the design models (root mean squared deviation < 1.0 [A]), highlighting the atomic level accuracy in our approach. The computational methods and scaffolds developed here provide the basis for custom-designing peptides for targeted therapeutic applications.

biochemistry↗

bioRxiv2023

Duplex Sequencing Provides Detailed Characterization of Mutation Frequencies and Spectra in the Bone Marrow of MutaMouse Males Exposed to Procarbazine Hydrochloride

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with >80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

genomics↗