Stuart S. Levine

DNA labelling fluorescent dyes such as ethidium bromide have long been considered to be highly mutagenic during DNA replication. While recent studies have pushed back on this narrative, the intercalative nature of these dyes continues to raise the possibility that these dyes can induce mutations. The iconPCR instrument by n6tec uses fluorescent dyes to measure amplification in real time and to adjust cycling conditions. However, since this use of qPCR is preparative and not analytical, mutations introduced by fluorescent dyes would be propagated into the sequencing reaction. To address the impact of these dyes on downstream analyses, we have performed routine mutation calling as well as mutational signature analysis on samples amplified using the iconPCR in the presence of either SYBR or EvaGreen. Sequence analysis revealed very minimal impacts of dyes on the reactions, largely within the noise regimen with only subtle changes in mutation rates seen. Mutational signature analysis was unable to identify any key signatures assignable to the dyes in either substitutions or indel domains. The mutational impact of intercalating dyes during fluorescence-guided amplification is therefore minimal and can be disregarded in all but the most sensitive NGS applications.

DNA labelling fluorescent dyes such as ethidium bromide have long been considered to be highly mutagenic during DNA replication. While recent studies have pushed back on this narrative, the intercalative nature of these dyes continues to raise the possibility that these dyes can induce mutations. The iconPCR instrument by n6tec uses fluorescent dyes to measure amplification in real time and to adjust cycling conditions. However, since this use of qPCR is preparative and not analytical, mutations introduced by fluorescent dyes would be propagated into the sequencing reaction. To address the impact of these dyes on downstream analyses, we have performed routine mutation calling as well as mutational signature analysis on samples amplified using the iconPCR in the presence of either SYBR or EvaGreen. Sequence analysis revealed very minimal impacts of dyes on the reactions, largely within the noise regimen with only subtle changes in mutation rates seen. Mutational signature analysis was unable to identify any key signatures assignable to the dyes in either substitutions or indel domains. The mutational impact of intercalating dyes during fluorescence-guided amplification is therefore minimal and can be disregarded in all but the most sensitive NGS applications.

N-Nitrosodimethylamine (NDMA) is a probable human carcinogen found in contaminated pharmaceuticals and drinking water, yet the impact of age on NDMA susceptibility remains poorly understood. Using DNA repair-deficient (Aag^-/-;Mgmt^-/-) and wild-type mice, we systematically compare the effects of NDMA exposure in juveniles and adults. Juvenile Aag^-/-;Mgmt^-/- mice are profoundly more vulnerable, exhibiting persistent DNA damage, inflammation, and mutations that lead to liver pathology and tumorigenesis, particularly in males. Adults, by comparison, are resistant to NDMA. Wild-type mice show similar, attenuated trends. NDMA-induced DNA adduct levels are comparable across age groups, implicating proliferation-dependent responses to adducts, rather than adduct formation, as the primary driver of age-related risk. Supporting this mechanism, triiodothyronine-stimulated cell proliferation in adults partially recapitulates juvenile sensitivity, linking cell division to NDMA genotoxicity. Our findings identify developmental stage, sex, and DNA repair capacity as key modifiers of NDMA-induced carcinogenesis, with potential implications for environmental risk assessment and regulatory policy.

Craniofacial Developmental Protein 1 (CFDP1) is a member of the evolutionarily conserved family of Bucentaur (BCNT) proteins and was originally classified as a protein required for cell survival and differentiation during tooth development. Yeast Swc5, a BCNT family member, is an essential subunit of the yeast SWR1C chromatin remodeling complex that catalyzes the deposition of histone variant H2A.Z. Direct connections between CFDP1, H2A.Z deposition, and the mammalian SWR1 homolog, Snf2-Related CREBBP Activator Protein (SRCAP), have not been identified. Here, we perform detailed biochemical reconstitution and characterization of the human SRCAP complex (SRCAP-C). We find that CFDP1 weakly interacts with SRCAP-C in a salt concentration-dependent manner. SRCAP-C purified under a high-salt condition does not co-purify with CFDP1 and is inactive in H2A.Z dimer exchange reaction, but the addition of exogeneous CFDP1 restores the H2A.Z deposition activity of SRCAP-C, demonstrating that CFDP1 is required for H2A.Z dimer exchange by SRCAP-C. We show that CFDP1 stimulates the basal ATPase activity of reconstituted SRCAP-C, suggesting a requirement for CFDP1 in regulating intrinsic catalytic ATPase activity. Consistent with this idea, CFDP1 deficiency in human induced pluripotent stem cells (hiPSCs) leads to a genome-wide reduction of H2A.Z, H3K27me3, and H3K4me3 deposition, accompanied by the upregulation of developmental genes normally marked by these modifications. Taken together, our results provide mechanistic insights into how CFDP1 regulates histone variant H2A.Z deposition by SRCAP-C. Given mutations in the SRCAP gene cause Floating-Harbor syndrome (FHS), a rare, dominant developmental disorder, our study provides an additional link between craniofacial defects and SRCAP-mediated H2A.Z deposition.

N-Nitrosodimethylamine (NDMA), a probable human carcinogen, induces toxic and mutagenic O ⁶ -methylguanine lesions that are repaired by the O ⁶ -methylguanine methyltransferase (MGMT). To elucidate mechanisms of NDMA-induced liver cancer progression, we performed longitudinal analyses of phenomic, transcriptomic, and phosphoproteomic changes in wild-type and MGMT-deficient mice, observing amplified responses in the deficient genotype. Early molecular rewiring indicative of a DNA damage response was detected by phosphoproteomic and transcriptomic profiling within days post-exposure. Transcriptomic analyses identified a persistent and robust interferon response as the dominant activated pathway. This chronic interferon signaling, which remained unresolved, correlated with extensive clonal expansion, an early hallmark of oncogenesis. Spatial transcriptomics further revealed pathway alterations favoring tumorigenesis within clonally expanded cells. These findings delineate the cascade of molecular events triggered by acute early-life NDMA exposure, culminating in cancer development months later. Our study unveils potential predictive biomarkers and strategies for disease mitigation.

Glioblastoma (GBM) is an aggressive primary brain cancer with few effective therapies. Stereotactic needle biopsies are routinely used for diagnosis; however, the feasibility and utility of investigative biopsies to monitor treatment response remains ill-defined. Here, we demonstrate the depth of data generation possible from routine stereotactic needle core biopsies and perform highly resolved multi-omics analyses, including single-cell RNA sequencing, spatial transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics on standard biopsy tissue obtained intra-operatively. We also examine biopsies taken from different locations and provide a framework for measuring spatial and genomic heterogeneity. Finally, we investigate the utility of stereotactic biopsies as a method for generating patient-derived xenograft (PDX) models. Multimodal dataset integration highlights spatially mapped immune cell-associated metabolic pathways and validates inferred cell-cell ligand-receptor interactions. In conclusion, investigative biopsies provide data-rich insight into disease processes and may be useful in evaluating treatment responses.

Mycobacteria adapt to infection stresses by entering a reversible non-replicating persistence (NRP) with slow or no cell growth and broad antimicrobial tolerance. Hypoxia and nutrient deprivation are two well-studied stresses commonly used to model the NRP, yet little is known about the molecular differences in mycobacterial adaptation to these distinct stresses that lead to a comparable NRP phenotype. Here we performed a multisystem interrogation of the Mycobacterium bovis BCG (BCG) starvation response, which revealed a coordinated metabolic shift away from the glycolysis of nutrient-replete growth to depletion of lipid stores, lipolysis, and fatty acid ß-oxidation in NRP. This contrasts with BCG’s NRP hypoxia response involving a shift to cholesterol metabolism and triglyceride storage. Our analysis reveals cryptic metabolic vulnerabilities of the starvation-induced NRP state, such as their newfound hypersensitivity to H₂O₂. These observations pave the way for developing precision therapeutics against these otherwise drug refractory pathogens.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53—the most frequently mutated gene in cancer—to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein–protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Supplementary Table from Developmental Deconvolution for Classification of Cancer Origin

Supplementary Table from Developmental Deconvolution for Classification of Cancer Origin