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Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Burgeoning evidence demonstrates that effects of environmental exposures can be transmitted to subsequent generations through the germline without DNA mutations1,2. This phenomenon remains controversial because underlying mechanisms have not been identified. Therefore, understanding how effects of environmental exposures are transmitted to unexposed generations without DNA mutations is a fundamental unanswered question in biology. Here, we used an established murine model of male-specific transgenerational obesity to show that exposure to the obesogen tributyltin (TBT) elicited heritable changes in chromatin interactions (CIs) in primordial germ cells (PGCs). New CIs were formed within the Ide gene encoding Insulin Degrading Enzyme in the directly exposed PGCs, then stably maintained in PGCs of the subsequent (unexposed) two generations. Concomitantly, Ide mRNA expression was decreased in livers of male descendants from the exposed dams. These males were hyperinsulinemic and hyperglycemic, phenocopying Ide-deficient mice that are predisposed to adult-onset, diet-induced obesity. Creation of new CIs in PGCs, suppression of hepatic Ide mRNA, increased fat mass, hyperinsulinemia and hyperglycemia were male-specific. Our results provide a plausible molecular mechanism underlying transmission of the transgenerational predisposition to obesity caused by gestational exposure to an environmental obesogen. They also provide an entry point for future studies aimed at understanding how environmental exposures alter chromatin structure to influence physiology across multiple generations in mammals.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.

In response to the coronavirus (COVID-19) pandemic, there has been an increased need for personal and environmental decontamination to aid in curbing transmission of the SARS-CoV-2 virus. Products used for this purpose include sanitizers for hands and disinfectants for surfaces. The active chemical ingredients used in these products, termed antimicrobials, can enter waste streams after application and may be emerging as more prominent environmental contaminants. Even prior to COVID-19, there was recognized need to examine their implications for aquatic biota, which is now made more pressing due to their exaggerated use in response to the pandemic. Our objectives were to identify current antimicrobial active ingredients, quantify their increased use, and determine which may be candidates for further consideration as possible aquatic contaminants. By consulting multiple sources of publicly available information in Canada, we identified current-use antimicrobials from the lists of sanitizers and surface disinfectants approved for use against SARS-CoV-2 by Health Canada and the drug registration database. To estimate the use of sanitizers and disinfectants, we evaluated import quantities and grocery store retail sales of related compounds and products (Statistics Canada) and both lines of evidence supported increased use trends. The list of identified antimicrobials was refined to include only candidates with potential to reach aquatic ecosystems, and information on their environmental concentrations and toxicity to aquatic biota was reviewed. Candidate antimicrobials (n = 32) fell into four main categories: quaternary ammonium compounds (QACs), phenols, acids, and salts. Benzalkonium chloride, a QAC, was the most prominent active ingredient used in both nonalcohol-based hand sanitizers and surface disinfectants. Four QACs followed in prevalence and the next most used antimicrobial was triclosan (hand sanitizers only), an established and regulated environmental contaminant. Little information was found on environmental concentrations of other candidates, suggesting that the majority would fall into the category of emerging contaminants if they enter aquatic systems. Several were classified as acutely or chronically toxic to aquatic biota (Globally Harmonized System), and thus we recommend empirical research begin focusing on environmental monitoring of all candidate antimicrobials as a critical next step, with detection method development first where needed. Keywords: biocide, pharmaceutical and personal care products, PPCP, pandemic, SARS-CoV-2, Canada, COVID-19, antimicrobial En reponse a la pandemie de coronavirus (COVID-19), un besoin accru de decontamination personnelle et environnementale s'est manifeste pour aider a freiner la transmission du virus SRAS-CoV-2. Les produits utilises a cette fin comprennent des assainisseurs pour les mains et des desinfectants pour les surfaces. Les ingredients chimiques actifs utilises dans ces produits, appeles antimicrobiens, peuvent entrer dans les systemes des eaux usees apres leur application et peuvent devenir des contaminants environnementaux plus importants. Avant meme l'avenement de la COVID-19, on reconnaissait qu'il etait necessaire d'examiner leurs implications pour le biote aquatique, ce qui est aujourd'hui rendu plus urgent en raison de leur utilisation exageree en reponse a la pandemie. Nos objectifs consistaient a identifier les ingredients actifs antimicrobiens actuels, a quantifier leur utilisation accrue et a determiner ceux qui pourraient etre consideres comme des contaminants aquatiques potentiels. En consultant de multiples sources d'information publiquement accessibles au Canada, nous avons pu identifieer les antimicrobiens utilises actuellement a partir des listes d'assainisseurs et de desinfectants de surface dont l'utilisation contre le SRAS-CoV-2 a ete approuvee par Sante Canada, et de la base de donnees sur les produits pharmaceutiques. Pour estimer l'utilisation des assainisseurs et des desinfectants, nous avons evalue les quantites importees et les ventes au detail dans les epiceries de composes et de produits connexes (Statistique Canada) et les deux sources de donnees ont confirme les tendances a l'augmentation de l'utilisation. La liste des antimicrobiens identifies a ete affinee pour n'inclure que les candidats susceptibles d'atteindre les ecosystemes aquatiques, et les informations sur leurs concentrations environnementales et leur toxicite pour le biote aquatique ont ete examinees. Les antimicrobiens candidats (n = 32) se repartissent en quatre grandes categories: les composes d'ammonium quaternaire (CAQ), les phenols, les acides et les sels. Le chlorure de benzalkonium, un CAQ, etait l'ingredient actif le plus utilise dans les desinfectants non alcoolises pour les mains et les desinfectants de surface. Quatre CAQ suivaient en prevalence et l'antimicrobien le plus utilise ensuite etait le triclosan (uniquement dans les desinfectants pour les mains), un contaminant environnemental avere et reglemente. Peu d'informations sur les concentrations environnementales des autres candidats etaient accessibles, ce qui suggere que la majorite d'entre eux entreraient dans la categorie des contaminants emergents s'ils penetrent dans les systemes aquatiques. Plusieurs d'entre eux ont ete classes comme presentant une toxicite aigue ou chronique pour le biote aquatique (Systeme general harmonise de classification et d'etiquetage des produits chimiques, SGH). Les auteurs recommandent donc que la recherche empirique commence a se concentrer sur la surveillance environnementale de tous les candidats antimicrobiens comme prochaine etape critique, en commencant par le developpement de methodes de detection si necessaire. Le texte integral de l'article en francais est disponible parmi les documents supplementaires. Mots-cles : biocide, produits pharmaceutiques et de soins personnels, PPSP, pandemie, SRAS-CoV-2, Canada, COVID-19, antimicrobien, 1. Introduction The use of sanitizing and disinfecting products dramatically increased during the COVID-19 pandemic (da Silva et al. 2021; Dhama et al. 2021), and more information on the impacts [...]

Naturally occurring hydrophilic colloids are versatile excipients in drug delivery systems. They are often used as coating materials, disintegrating agents, binders, emulsion stabilizers, and other applications. This study sought to investigate the physicochemical and emulsifying properties of gum extracted from Melia azedarach (MA). The gum was harvested, authenticated, and purified using ethanol precipitation. Physicochemical, microbial, and proximate analyses were performed on the purified gum. Oil of olive emulsions containing different amounts (5–15%w/v) of the gum as emulsifiers were prepared by homogenization. The zeta potential, creaming index, and average droplet size of products were assessed. The effects of pH changes, temperature, and monovalent and divalent electrolytes on the stability of the emulsions were also investigated. The yield of the gum after purification was 68.3%w/w. The gum has low moisture content and good swelling properties. Lead, copper, cadmium, and mercury were not detected. Emulsions containing 15%w/v of acacia or MA gum had the smallest average (Z-average) droplet size (acacia: 1.837 ± 0.420 μm; MA gum: 2.791 ± 0.694 μm) and the highest zeta potential (acacia: −30.45 mV; MA gum: −32.867 mV). Increasing the concentration of the gums increased the emulsion viscosity with MA gum emulsions being more viscous than corresponding acacia emulsions. MA gum emulsions had higher emulsion capacity and stability but lower creaming index relative to acacia gum emulsions of similar concentrations. Potassium chloride (KCl) reduced zeta potential but increased Z-average for emulsions prepared with either gum. Calcium chloride (CaCl2) produced a similar but more pronounced effect. When the pH was decreased from 10 to 2, the zeta potential of the droplets was reduced, but the droplet size of emulsions prepared from either gum was increased. Increasing temperature from 25 to 90°C produced no significant (p value >0.9999) change in droplet size. These findings suggest that MA gum is a capable emulsifying agent at 15%w/v.

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.

The discovery of drivers of cancer has traditionally focused on protein-coding genes1-4. Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium5 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers6,7, raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.

The catalog of cancer driver mutations in protein-coding genes has greatly expanded in the past decade. However, non-coding cancer driver mutations are less well-characterized and only a handful of recurrent non-coding mutations, most notably TERT promoter mutations, have been reported. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancer across 38 tumor types, we perform multi-faceted pathway and network analyses of non-coding mutations across 2583 whole cancer genomes from 27 tumor types compiled by the ICGC/TCGA PCAWG project that was motivated by the success of pathway and network analyses in prioritizing rare mutations in protein-coding genes. While few non-coding genomic elements are recurrently mutated in this cohort, we identify 93 genes harboring non-coding mutations that cluster into several modules of interacting proteins. Among these are promoter mutations associated with reduced mRNA expression in TP53, TLE4, and TCF4. We find that biological processes had variable proportions of coding and non-coding mutations, with chromatin remodeling and proliferation pathways altered primarily by coding mutations, while developmental pathways, including Wnt and Notch, altered by both coding and non-coding mutations. RNA splicing is primarily altered by non-coding mutations in this cohort, and samples containing non-coding mutations in well-known RNA splicing factors exhibit similar gene expression signatures as samples with coding mutations in these genes. These analyses contribute a new repertoire of possible cancer genes and mechanisms that are altered by non-coding mutations and offer insights into additional cancer vulnerabilities that can be investigated for potential therapeutic treatments.