Next-generation sequencing methodologies to detect low-frequency mutations: "Catch me if you can".

Mutations, the irreversible changes in an organism's DNA sequence, are present in tissues at a variant allele frequency (VAF) ranging from ∼10-8 per bp for a founder mutation to ∼10-3 for a histologically normal tissue sample containing several independent clones - compared to 1%- 50% for a heterozygous tumor mutation or a polymorphism. The rarity of these events poses a challenge for accurate clinical diagnosis and prognosis, toxicology, and discovering new disease etiologies. Standard Next-Generation Sequencing (NGS) technologies report VAFs as low as 0.5% per nt, but reliably observing rarer precursor events requires additional sophistication to measure ultralow-frequency mutations. We detail the challenge; define terms used to characterize the results, which vary between laboratories and sometimes conflict between biologists and bioinformaticists; and describe recent innovations to improve standard NGS methodologies including: single-strand consensus sequence methods such as Safe-SeqS and SiMSen-Seq; tandem-strand consensus sequence methods such as o2n-Seq and SMM-Seq; and ultrasensitive parent-strand consensus sequence methods such as DuplexSeq, PacBio HiFi, SinoDuplex, OPUSeq, EcoSeq, BotSeqS, Hawk-Seq, NanoSeq, SaferSeq, and CODEC. Practical applications are also noted. Several methods quantify VAF down to 10-5 at a nt and mutation frequency (MF) in a target region down to 10-7 per nt. By expanding to > 1 Mb of sites never observed twice, thus forgoing VAF, other methods quantify MF < 10-9 per nt or < 15 errors per haploid genome. Clonal expansion cannot be directly distinguished from independent mutations by sequencing, so it is essential for a paper to report whether its MF counted only different mutations - the minimum independent-mutation frequency MFminI - or all mutations observed including recurrences - the larger maximum independent-mutation frequency MFmaxI which may reflect clonal expansion. Ultrasensitive methods reveal that, without their use, even mutations with VAF 0.5-1% are usually spurious.

Chemiexcitation and melanin in photoreceptor disc turnover and prevention of macular degeneration.

Age-related macular degeneration, Stargardt disease, and their Abca4-/- mouse model are characterized by accelerated accumulation of the pigment lipofuscin, derived from photoreceptor disc turnover in the retinal pigment epithelium (RPE); lipofuscin accumulation and retinal degeneration both occur earlier in albino mice. Intravitreal injection of superoxide (O2•-) generators reverses lipofuscin accumulation and rescues retinal pathology, but neither the target nor mechanism is known. Here we show that RPE contains thin multi-lamellar membranes (TLMs) resembling photoreceptor discs, which associate with melanolipofuscin granules in pigmented mice but in albinos are 10-fold more abundant and reside in vacuoles. Genetically over-expressing tyrosinase in albinos generates melanosomes and decreases TLM-related lipofuscin. Intravitreal injection of generators of O2•- or nitric oxide (•NO) decreases TLM-related lipofuscin in melanolipofuscin granules of pigmented mice by ~50% in 2 d, but not in albinos. Prompted by evidence that O2•- plus •NO creates a dioxetane on melanin that excites its electrons to a high-energy state (termed "chemiexcitation"), we show that exciting electrons directly using a synthetic dioxetane reverses TLM-related lipofuscin even in albinos; quenching the excited-electron energy blocks this reversal. Melanin chemiexcitation assists in safe photoreceptor disc turnover.

Chemiexcited Neurotransmitters and Hormones Create DNA Photoproducts in the Dark.

In DNA, electron excitation allows adjacent pyrimidine bases to dimerize by [2 + 2] cycloaddition, creating chemically stable but lethal and mutagenic cyclobutane pyrimidine dimers (CPDs). The usual cause is ultraviolet radiation. Alternatively, CPDs can be made in the dark (dCPDs) via chemically mediated electron excitation of the skin pigment melanin, after it is oxidized by peroxynitrite formed from the stress-induced radicals superoxide and nitric oxide. We now show that the dark process is not limited to the unusual structural molecule melanin: signaling biomolecules such as indolamine and catecholamine neurotransmitters and hormones can also be chemiexcited to energy levels high enough to form dCPDs. Oxidation of serotonin, dopamine, melatonin, and related biogenic amines by peroxynitrite created triplet-excited species, evidenced by chemiluminescence, energy transfer to a triplet-state reporter, or transfer to O2 resulting in singlet molecular oxygen. For a subset of these signaling molecules, triplet states created by peroxynitrite or peroxidase generated dCPDs at levels comparable to ultraviolet (UV). Neurotransmitter catabolism by monoamine oxidase also generated dCPDs. These results reveal a large class of signaling molecules as electronically excitable by biochemical reactions and thus potential players in deviant mammalian metabolism in the absence of light.

Chemiexcitation: Mammalian Photochemistry in the Dark†.

Light is one way to excite an electron in biology. Another is chemiexcitation, birthing a reaction product in an electronically excited state rather than exciting from the ground state. Chemiexcited molecules, as in bioluminescence, can release more energy than ATP. Excited states also allow bond rearrangements forbidden in ground states. Molecules with low-lying unoccupied orbitals, abundant in biology, are particularly susceptible. In mammals, chemiexcitation was discovered to transfer energy from excited melanin, neurotransmitters, or hormones to DNA, creating the lethal and carcinogenic cyclobutane pyrimidine dimer. That process was initiated by nitric oxide and superoxide, radicals triggered by ultraviolet light or inflammation. Several poorly understood chronic diseases share two properties: inflammation generates those radicals across the tissue, and cells that die are those containing melanin or neuromelanin. Chemiexcitation may therefore be a pathogenic event in noise- and drug-induced deafness, Parkinson's disease, and Alzheimer's; it may prevent macular degeneration early in life but turn pathogenic later. Beneficial evolutionary selection for excitable biomolecules may thus have conferred an Achilles heel. This review of recent findings on chemiexcitation in mammalian cells also describes the underlying physics, biochemistry, and potential pathogenesis, with the goal of making this interdisciplinary phenomenon accessible to researchers within each field.

Cyclobutane Pyrimidine Dimer Hyperhotspots as Sensitive Indicators of Keratinocyte UV Exposure†.

The dominant DNA damage generated by UV exposure is the cyclobutane pyrimidine dimer (CPD), which alters skin cell physiology and induces cell death and mutation. Genome-wide nucleotide-resolution analysis of CPDs in melanocytes and fibroblasts has identified "CPD hyperhotspots", pyrimidine-pyrimidine sites hundreds of fold more susceptible to the generation of CPDs than the genomic average. Identifying hyperhotspots in keratinocytes could enable measuring individual past UV exposure in small skin samples and predicting future skin cancer risk. We therefore exposed neonatal human epidermal keratinocytes to narrowband UVB and quantified CPDs using the adductSeq high-throughput DNA sequencing method. Keratinocytes contained thousands of CPD hyperhotspots, with a UVB-sensitivity up to 550 fold greater than the genomic average. As with melanocytes, the most sensitive sites were located in promoter regions at ETS-family transcription factor binding sequence motifs, near RNA processing genes. Moreover, they lay at sequence motifs bound to ETS1 in CpG islands. These genes were specifically upregulated in skin and the CPD hyperhotspots were mutated in a fraction of keratinocyte cancers. Crucially for their biological importance and practical application, CPD hyperhotspot locations and UV-sensitivity ranking demonstrated high reproducibility across experiments and across skin donors. CPD hyperhotspots are therefore sensitive indicators of UV exposure.

Slip versus Slop: A Head-to-Head Comparison of UV-Protective Clothing to Sunscreen.

Ultraviolet radiation (UVR) exposure is the most important modifiable risk factor for skin cancer development. Although sunscreen and sun-protective clothing are essential tools to minimize UVR exposure, few studies have compared the two modalities head-to-head. This study evaluates the UV-protective capacity of four modern, sun-protective textiles and two broad-spectrum, organic sunscreens (SPF 30 and 50). Sun Protection Factor (SPF), Ultraviolet Protection Factor (UPF), Critical Wavelength (CW), and % UVA- and % UVB-blocking were measured for each fabric. UPF, CW, % UVA- and % UVB-blocking were measured for each sunscreen at 2 mg/cm2 (recommended areal density) and 1 mg/cm2 (simulating real-world consumer application). The four textiles provided superior UVR protection when compared to the two sunscreens tested. All fabrics blocked erythemogenic UVR better than the sunscreens, as measured by SPF, UPF, and % UVB-blocking. Each fabric was superior to the sunscreens in blocking full-spectrum UVR, as measured by CW and % UVA-blocking. Our data demonstrate the limitations of sunscreen and UV-protective clothing labeling and suggest the combination of SPF or UPF with % UVA-blocking may provide more suitable measures for broad-spectrum protection. While sunscreen remains an important photoprotective modality (especially for sites where clothing is impractical), these data suggest that clothing should be considered the cornerstone of UV protection.

Triplet-Energy Quenching Functions of Antioxidant Molecules.

UV-like DNA damage is created in the dark by chemiexcitation, in which UV-activated enzymes generate reactive oxygen and nitrogen species that create a dioxetane on melanin. Thermal cleavage creates an electronically excited triplet-state carbonyl whose high energy transfers to DNA. Screening natural compounds for the ability to quench this energy identified polyenes, polyphenols, mycosporine-like amino acids, and related compounds better known as antioxidants. To eliminate false positives such as ROS and RNS scavengers, we then used the generator of triplet-state acetone, tetramethyl-1,2-dioxetane (TMD), to excite the triplet-energy reporter 9,10-dibromoanthracene-2-sulfonate (DBAS). Quenching measured as reduction in DBAS luminescence revealed three clusters of 50% inhibitory concentration, ~50 µM, 200-500 µM, and >600 µM, with the former including sorbate, ferulic acid, and resveratrol. Representative triplet-state quenchers prevented chemiexcitation-induced "dark" cyclobutane pyrimidine dimers (dCPD) in DNA and in UVA-irradiated melanocytes. We conclude that (i) the delocalized pi electron cloud that stabilizes the electron-donating activity of many common antioxidants allows the same molecule to prevent an electronically excited species from transferring its triplet-state energy to targets such as DNA and (ii) the most effective class of triplet-state quenchers appear to operate by energy diversion instead of electron donation and dissipate that energy by isomerization.

Bruce Nathan Ames - Paradigm shifts inside the cancer research revolution.

Dr. Bruce Ames turned 92 on December 16, 2020. He considers his most recent work linking adequate consumption of 30 known vitamins and minerals with successful aging to be his most important contribution. With the passage of time, it is not uncommon for the accomplishments of a well-known scientist to undergo a parsimonious reductionism in the public mind - Pasteur's vaccine, Mendel's peas, Pavlov's dogs, Ames' test. Those of us in the research generation subsequent to Dr. Ames' are undoubtedly affected by our own unconscious tendencies toward accepting the outstanding achievements of the past as commonplace. In doing so, seminal advances made by earlier investigators are often inadvertently subsumed into common knowledge. But having followed Ames' work since the mid-1970s, we are cognizant that the eponymous Ames Test is but a single chapter in a long and rich narrative. That narrative begins with Ames' classic studies on the histidine operon of Salmonella, for which he was elected to the National Academy of Sciences. A summary of the historical progression of the understanding of chemical carcinogenesis to which Ames and his colleagues contributed is provided. Any summary of a topic as expansive and complex as the ongoing unraveling of the mechanisms underlying chemical carcinogenesis will only touch upon some of the major conceptual advances to which Ames and his colleagues contributed. We hope that scientists of all ages familiar with Ames only through the eponymous Ames Test will further investigate the historical progression of the conceptualization of cancer caused by chemical exposure. As the field of chemical carcinogenesis gradually moves away from primary reliance on animal testing to alternative protocols under the rubric of New Approach Methodologies (NAM) an understanding of where we have been might help to guide where we should go.

Rethinking Causation for Data-intensive Biology: Constraints, Cancellations, and Quantized Organisms: Causality in complex organisms is sculpted by constraints rather than instigators, with outcomes perhaps better described by quantized patterns than rectilinear pathways.

Complex organisms thwart the simple rectilinear causality paradigm of "necessary and sufficient," with its experimental strategy of "knock down and overexpress." This Essay organizes the eccentricities of biology into four categories that call for new mathematical approaches; recaps for the biologist the philosopher's recent refinements to the causation concept and the mathematician's computational tools that handle some but not all of the biological eccentricities; and describes overlooked insights that make causal properties of physical hierarchies such as emergence and downward causation straightforward. Reviewing and extrapolating from similar situations in physics, it is suggested that new mathematical tools for causation analysis incorporating feedback, signal cancellation, nonlinear dependencies, physical hierarchies, and fixed constraints rather than instigative changes will reveal unconventional biological behaviors. These include "eigenisms," organisms that are limited to quantized states; trajectories that steer a system such as an evolving species toward optimal states; and medical control via distributed "sheets" rather than single control points.

Defective postreplication repair of UV photoproducts in melanoma: a mutator phenotype.

In this issue, the Gabrielli laboratory and collaborators address the bulky CPD lesions created in DNA when UV joins two adjacent pyrimidines (thymine or cytosine), leading to skin cancers such as melanoma (Pavey S et al. (2019) Mol Oncol). Our understanding of postreplication repair mechanisms for bulky lesions has lagged, and the newly reported predominance of translational control in the UV response has important implications. Image taken from Creative Commons Blacklight bulb in ultraviolet by brx0, licensed under CC BY-SA 2.0. Commentary on.

Genomic sites hypersensitive to ultraviolet radiation.

If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequent than ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170-fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within 2 motifs. One motif occurs at ETS family transcription factor binding sites, known to be UV targets and now shown to be among the most sensitive in the genome, and at sites of mTOR/5' terminal oligopyrimidine-tract translation regulation. The second occurs at A2-15TTCTY, which developed "dark CPDs" long after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ∼20 targeted cell pathways, letting hyperhotspots act as epigenetic marks that create phenome instability; high prevalence favors cooccurring mutations, which would allow tumor evolution to use weak drivers.

Publisher Correction: A call for a better understanding of causation in cell biology.

In the above article, the name of the first author was spelled incorrectly. This has been corrected in the HTML and PDF versions of the article.

Accelerating cancer without mutations.

Mice get melanoma faster when they have common, inherited variants in a few genes that control cell-wide changes but also respond to the environment.

Chemiexcitation and Its Implications for Disease.

Quantum mechanics rarely extends to molecular medicine. Recently, the pigment melanin was found to be susceptible to chemiexcitation, in which an electron is chemically excited to a high-energy molecular orbital. In invertebrates, chemiexcitation causes bioluminescence; in mammals, a higher-energy process involving melanin transfers energy to DNA without photons, creating the lethal and mutagenic cyclobutane pyrimidine dimer that can cause melanoma. This process is initiated by NO and O2- radicals, the formation of which can be triggered by ultraviolet light or inflammation. Several chronic diseases share two properties: inflammation generates these radicals across the tissue, and the diseased cells lie near melanin. We propose that chemiexcitation may be an upstream event in numerous human diseases.

Fluorouracil Enhances Photodynamic Therapy of Squamous Cell Carcinoma via a p53-Independent Mechanism that Increases Protoporphyrin IX levels and Tumor Cell Death.

Photodynamic therapy (PDT), using 5-aminolevulinic acid (ALA) to drive synthesis of protoporphryin IX (PpIX) is a promising, scar-free alternative to surgery for skin cancers, including squamous cell carcinoma (SCC) and SCC precursors called actinic keratoses. In the United States, PDT is only FDA approved for treatment of actinic keratoses; this narrow range of indications could be broadened if PDT efficacy were improved. Toward that goal, we developed a mechanism-based combination approach using 5-fluorouracil (5-FU) as a neoadjuvant for ALA-based PDT. In mouse models of SCC (orthotopic UV-induced lesions, and subcutaneous A431 and 4T1 tumors), pretreatment with 5-FU for 3 days followed by ALA for 4 hours led to large, tumor-selective increases in PpIX levels, and enhanced cell death upon illumination. Several mechanisms were identified that might explain the relatively improved therapeutic response. First, the expression of key enzymes in the heme synthesis pathway was altered, including upregulated coproporphyrinogen oxidase and downregulated ferrochelatase. Second, a 3- to 6-fold induction of p53 in 5-FU-pretreated tumors was noted. The fact that A431 contains a mutant form p53 did not prevent the development of a neoadjuvantal 5-FU effect. Furthermore, 5-FU pretreatment of 4T1 tumors (cells that completely lack p53), still led to significant beneficial inductions, that is, 2.5-fold for both PpIX and PDT-induced cell death. Thus, neoadjuvantal 5-FU combined with PDT represents a new therapeutic approach that appears useful even for p53-mutant and p53-null tumors. Mol Cancer Ther; 16(6); 1092-101. ©2017 AACR.

Unanticipated role of melanin in causing carcinogenic cyclobutane pyrimidine dimmers.

Ultraviolet radiation (UVR) instantaneously generates cyclobutane pyrimidine dimers (CPDs). Paradoxically, we recently observed that UV enables the protective pigment melanin to create CPDs in the dark long after the exposure ends. UV-induced reactive oxygen species (ROS) oxidize melanin to create melanin carbonyls in a high-energy quantum state. These energetic melanin carbonyls transfer their energy to DNA in the dark, creating CPDs in the absence of UVR.

Chemical excitation of electrons: A dark path to melanoma.

Sunlight's ultraviolet wavelengths induce cyclobutane pyrimidine dimers (CPDs), which then cause mutations that lead to melanoma or to cancers of skin keratinocytes. In pigmented melanocytes, we found that CPDs arise both instantaneously and for hours after UV exposure ends. Remarkably, the CPDs arising in the dark originate by a novel pathway that resembles bioluminescence but does not end in light: First, UV activates the enzymes nitric oxide synthase (NOS) and NADPH oxidase (NOX), which generate the radicals nitric oxide (NO) and superoxide (O2(-)); these combine to form the powerful oxidant peroxynitrite (ONOO(-)). A fragment of the skin pigment melanin is then oxidized, exciting an electron to an energy level so high that it is rarely seen in biology. This process of chemically exciting electrons, termed "chemiexcitation", is used by fireflies to generate light but it had never been seen in mammalian cells. In melanocytes, the energy transfers radiationlessly to DNA, inducing CPDs. Chemiexcitation is a new source of genome instability, and it calls attention to endogenous mechanisms of genome maintenance that prevent electronic excitation or dissipate the energy of excited states. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin.