Biology subjects

Balbo, S.

Publications and source records attributed to Balbo, S..

7 recordsLinked to original sources

bioRxiv2025

A Universal Duplex Sequencing Approach for Accurate Detection of Somatic Mutations

Ultra-accurate detection of rare somatic mutations is critical for understanding mutational processes in human disease, aging, and environmental exposures, yet current methods are limited by error rates, restricted genome coverage, and high DNA input. We present UDSeq, a duplex sequencing protocol combining random fragmentation, efficient UMI ligation, and quantitative input control to achieve near-complete genome/exome representation from as little as 100 pg DNA. Benchmarking in human sperm estimates a UDSeq error rate of [~]2.5x10-9 per base pair. UDSeq captures mutational signatures from heterogeneous populations without clonal expansion, reproduces exposure-specific patterns in cell lines and rodent models, and enables cross-species profiling. Compared with prior duplex methods, UDSeq yields up to fourfold more usable duplex molecules, improves library conversion, and remains cost-effective. We include a step-by-step protocol with quality-control checkpoints for fragment size, ligation yield, library conversion, and duplication rate. UDSeq provides a scalable, low-input platform for accurate profiling of somatic mutagenesis.

genomics↗

bioRxiv2025

Genome-scale multi-organ analysis of mutagenic effects of ethanol and acetaldehyde in Sprague Dawley rats

Alcohol consumption is a major cancer risk factor, particularly for head and neck cancers, including the oral cavity. Acetaldehyde, the primary genotoxic metabolite of ethanol, may play key roles in oral carcinogenesis, though the mechanisms remain unclear. While mutational signatures SBS16, DBS4, and ID11 have been tentatively linked to alcohol use, they are not exclusive to alcohol-related cancers. In this study, we examined the genome-wide in vivo mutagenic effects of ethanol and acetaldehyde by analyzing tumors from the cheek, Zymbal gland, larynx, forestomach, and liver of rats chronically exposed to these compounds. Signature analysis revealed exposure-specific, early-onset formation of SBS17 in [~]28% of head and neck tumors, suggesting inflammation and/or oxidative damage as potential mediators of carcinogenesis. Cancer driver gene analysis identified a relative enrichment of exposed tumors with mutations in the Tp53 and Mtor genes. No notable exposure-specific changes were observed in doublet-base substitutions, indel signatures, or copy number variants. Notably, SBS16, DBS4, and ID11 were absent. Our findings suggest direct mutagenicity may not be the main driver of alcohol-related cancer. Other harmful cellular effects, undetectable by whole genome sequencing, may be involved. Our findings suggest that SBS17 could function as a potential exposure-specific molecular marker of alcohol-related cancers in humans.

cancer biology↗

bioRxiv2025

Expanding the MR1 ligandome using chemical class-specific fragmentation and molecular networking

The identification and subsequent characterization of unknown analytes using mass spectrometry presents a long-standing challenge across many research fields, particularly when analyte levels are low and the compound class is underrepresented in mass spectral databases. We have developed a data analysis workflow for investigating classes of small molecules and demonstrated its application through the reanalysis of data collected to probe for modified nucleoside MR1-presented antigens. We reanalyzed the datasets to screen for additional classes of compounds within the MR1 ligandome using Compound Discoverer, a commercial software package designed for metabolomic analysis, featuring fragmentation filtering nodes, molecular networking, and spectral database searching. Our study identified two compound classes that bind to MR1. One class includes compounds characterized by the presence of a ribityl substructure and molecular formulas consistent with structural similarity to riboflavin, where the most abundant compound differs from riboflavin by two additional oxygen atoms and one fewer carbon atom. A second class comprises an adenosine monophosphate isomer and larger analytes that are putatively identified as consisting of di- and tri-covalently bound nucleotides. The application of our analytical approach to characterize the MR1 ligandome demonstrates the power of combining compound-class fragmentation, molecular networking, and mass spectral database searching in exploring receptor ligandomes and, more generally, identifying novel classes of compounds.

immunology↗

bioRxiv2025

The specificity and structure of DNA crosslinking by the gut bacterial genotoxin colibactin

Accumulating evidence has connected the chemically unstable, DNA-damaging gut bacterial natural product colibactin to colorectal cancer, including the identification of mutational signatures that are thought to arise from colibactin-DNA interstrand crosslinks (ICLs). However, we currently lack direct information regarding the structure of this lesion. Here, we combine mass spectrometry and nuclear magnetic resonance spectroscopy to elucidate the specificity and structure of the colibactin-DNA ICL. We find that colibactin alkylates within the minor groove of AT-rich DNA, explaining the origins of mutational signatures. Unexpectedly, we discover that the chemically unstable central motif of colibactin mediates the sequence specificity of crosslinking. By directly elucidating colibactins interactions with DNA, this work enhances our understanding of the structure and genotoxic mechanisms of this cancer-linked gut bacterial natural product.

biochemistry↗

bioRxiv2025

Tissue-specific mutagenesis from endogenous guanine damageis suppressed by Polκ and DNA repair

Knowledge of mutational patterns has expanded significantly, but a persistent challenge is to link these complex patterns to the underlying molecular mechanism or source of DNA damage, especially when that damage is endogenous and not driven by environmental mutagens. Technological advances now allow us to catalogue mutation across tissues or even closely related cell types, but the results are largely descriptive until we identify the endogenous sources of mutation and how they differ across tissues. Here, we combine mouse genetics, advanced sequencing, biochemistry and mass spectrometry to provide a detailed mechanistic understanding of endogenous mutagenesis. We reveal that endogenous guanine adducts are significant drivers of tissue-specific mutagenesis, while the interwoven actions of DNA polymerase Pol{kappa} and DNA repair mechanisms are pivotal in mitigating mutagenesis. For the first time, we use untargeted DNA adductomics to characterize new sources of endogenous DNA damage. Our novel approach to understanding endogenous mutational landscapes reveals previously unseen mutational processes and points to vast potential for new discoveries.

molecular biology↗

bioRxiv2024

Human cancer genomes harbor the mutational signature of tobacco-specific nitrosamines NNN and NNK

Tobacco usage is linked to multiple cancer types and accounts for a quarter of all cancer-related deaths. Tobacco smoke contains various carcinogenic compounds, including polycyclic aromatic hydrocarbons (PAH), though the mutagenic potential of many tobacco-related chemicals remains largely unexplored. In particular, the highly carcinogenic tobacco-specific nitrosamines NNN and NNK form pre-mutagenic pyridyloxobutyl (POB) DNA adducts. In the study presented here, we identified genome-scale POB-induced mutational signatures in cell lines and rat tumors, while also investigating their role in human cancer. These signatures are characterized by T>N and C>T mutations forming from specific POB adducts damaging dT and dC residues. Analysis of 2,780 cancer genomes uncovered POB signatures in [~]180 tumors; from cancer types distinct from the ones linked to smoking-related signatures SBS4 and SBS92. This suggests that, unlike PAH compounds, the POB pathway may contribute uniquely to the mutational landscapes of certain hematological malignancies and cancers of the kidney, breast, prostate and pancreas.

genomics↗

bioRxiv2019

The reactivity of an unusual amidase may explain colibactin’s DNA cross-linking activity

Certain commensal and pathogenic bacteria produce colibactin, a small molecule genotoxin that causes interstrand cross-links in host cell DNA. Though colibactin has been found to alkylate DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and {beta}-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line. This transformation would incorporate two electrophilic cyclopropane warheads into the final natural product scaffold. Overall, this work provides a biosynthetic explanation for colibactins DNA crosslinking activity and paves the way for further study of its chemical structure.

biochemistry↗