Base Damage Research Articles

Comparisons between the Direct and Indirect Effect of 1.5 keV X-rays and 0-30 eV Electrons on DNA: Base Lesions, Stand Breaks, Cross-Links, and Cluster Damages.

The interaction of low energy electrons (LEEs; 1-30 eV) with genomic material can induce multiple types of damage that may cause the loss of genetic information, mutations, genome instability, and cell death. For all damages measurable by electrophoresis, we provide the first complete set of G-values (yield of a specific product per energy deposited) induced in plasmid DNA by the direct and indirect effects of LEEs (GLEE) and 1.5 keV X-rays (GX) under identical conditions. Low energy photoelectrons are produced via X-rays incident on a tantalum (Ta) substrate covered with DNA and placed in a chamber filled with nitrogen at atmospheric pressure, under four different humidity levels, ranging from dry conditions to full hydration (Γ = 2.5 to Γ = 33, where Γ is the number of water molecules/nucleotide). Damage yields are measured as a function of X-ray fluence and humidity. GLEE values are between 2 and 27 times larger than those for X-rays. At Γ = 2.5 and 33, GLEE values for double strand breaks are 27 and 16 times larger than GX, respectively. The indirect effect contributes ∼50% to the total damage. These G-values allow quantification of potentially lethal lesions composed of strand breaks and/or base damages in the presence of varying amounts of water, i.e., closer to cellular conditions.

Effect of Ultrahigh Dose Rate on Biomolecular Radiation Damage.

Dose rate is one of the important parameters in radiation-induced biomolecular damage. The effects of dose rate have been known to modify radiation toxicity in biological systems. The rate and extent of sublethal DNA damage (e.g., base damage and single-strand breaks) repair and those of cell proliferation have been manifested by dose rate. However, the recent preclinical application of ultrahigh dose rate [(UHDR) ca. 40 Gy/s and higher] radiation modalities have been shown to lower the type and extent of radiation damage to biological systems. At these UHDR, radiation-induced physicochemical and chemical processes are expected to differ from those observed after irradiation at conventional dose rates (CONV). It is unclear whether these UHDR conditions can affect the quality (type) and quantity (extent) of biomolecular damage such as DNA lesions. Here, we comparatively study the influence of indirect effects of CONV and UHDR on the formation of DNA strand breaks and clustered damage including densely accumulated lesions in an aerated and an anoxic dilute aqueous solution of a plasmid DNA model under low and high hydroxyl radical (•OH) scavenging conditions. Aqueous solutions of purified supercoiled plasmid DNA (pUC19) were prepared in either air- or nitrogen-saturated conditions, with Tris buffer added as the radiation-produced •OH scavenger at low and high scavenging capacities. These DNA samples were irradiated using kV X-ray systems at CONV (0.1 Gy/s) and high dose rate (HDR, 25 Gy/s) as well as UHDR (55 and 125 Gy/s) under different scavenging and environmental conditions. DNA lesions including strand breaks and clustered damage including densely accumulated lesions were quantified by gel electrophoresis and the yields of these lesions were calculated from the dose-response curve. Non-DSB clustered damage including densely accumulated lesions were evaluated by treating DNAs using bacterial endonuclease enzymes (Fpg and Nth) prior to gel electrophoresis. UHDR of 55 and 125 Gy/s induced lower amounts of both isolated strand breaks and clustered DNA damage including densely accumulated lesions at doses >40 Gy in the presence of oxygen, compared to the abundance of these lesions induced by 0.1 and 25 Gy/s irradiation under the same dose conditions. Overall, the strand break and clustered damage including densely accumulated lesions yields decreased by factors of 1.3-3.5 after UHDR. We did not observe these differences either via •OH scavenging or by removing oxygen from the solution. In addition, our results point out that the inter-track recombination reactions did not contribute to the observed dose-rate effects on DNA damage. The effects of dose rate on DNA damage are highly dependent on the total dose, as expected, but also on the •OH scavenging capacity that is employed in the aqueous DNA solutions. These important variables may be relevant in biological systems as well. On a practical level, our in vitro plasmid DNA model, which permits to precisely vary the scavenging capacity and gassing conditions (air saturated vs. N2 saturated) can help to differentiate dose-rate effects on biomolecular damage. Our results indicate that the radical-radical reactions are important in understanding the dose-rate effect on DNA damage.

OPTIMIZATION OF THE SYNERGISTIC ANTIOXIDANT EFFECT OF SELECTED PHENOLIC COMPOUNDS (GALLIC ACID, ROSMARINIC ACID and CAFFEIC ACID) AND INVESTIGATION OF THEIR ABILITY TO PREVENT FORMATION OF DNA BASE DAMAGE

Considering the areas of use of phenolic compounds, it is important to determine the concentrations at which they show synergistic and antagonistic interactions for their integration into various systems and their correct use. In this study, the synergistic interaction concentration of rosmarinic acid, gallic acid, and caffeic acid was determined by Folin–Ciocalteu and FRAP methods. The central composite design–response surface methodology was used to determine the optimum concentration for synergistic interaction. As a result of the optimization, caffeic acid, rosmarinic acid, and gallic acid showed synergistic interaction at 7.87 μM, 6.75 μM and 9.42 μM concentrations for Folin–Ciocalteu method; 8.03 μM, 9.34 μM and 6.00 μM concentration for FRAP method respectively. The capacity of phenolic compounds to prevent the formation of DNA base damage products was evaluated by GC–MS/MS. As a result, the synergistic concentration of three phenolics reduces the DNA damage products at 37.17% (FOLIN) and 40.17% (FRAP).

Monte Carlo investigation of the nucleus size effect and cell’s oxygen content on the damage efficiency of protons

Living tissues could suffer different types of DNA damage as a result of being exposed to ionizing radiations. Monte Carlo simulations of the underlying interactions have been instrumental in predicting the damage types and the processes involved. In this work, we employed Geant4-DNA and MCDS for extracting the initial DNA damage and investigating the dependence of damage efficiency on the cell’s oxygen content. The frequency-mean lineal ( y¯F ) and specific ( z¯F ) energies were derived for a spherical volume of water of various diameters between 2 and 11.1 μm. This sphere would serve as the nucleus of a cell of 100 μm diameter, engulfed by a homogeneous beam of protons. These microdosimetric quantities were calculated assuming spherical samples of 1 μm diameter in MCDS. The simulation results showed that for 230 MeV protons, an increase in the oxygen content from 0 by 10% raised the frequency of single- and double-strand breaks and lowered the base damage frequency. The resulting damage frequencies appeared to be independent of nucleus diameter. For proton energies between 2 and 230 MeV, y¯F showed no dependence on the cell diameter and an increase of the cell size resulted in a decrease in z¯F. An increase in the proton energy slowed down the decreasing rate of z¯F as a function of nucleus diameter. However, the ratio of y¯F values corresponding to two proton energies of choice showed no dependence on the nucleus size and were equal to the ratio of the corresponding z¯F values. Furthermore, the oxygen content of the cell did not affect these microdosimetric quantities. Contrary to damage frequencies, these quantities appeared to depend only on direct interactions due to deposited energies. Our calculations showed the near independence of DNA damages on the nucleus size of the human cells. The probabilities of different types of single and double-strand breaks increase with the oxygen content.

Activation of multifunctional DNA repair APE1/Ref-1 enzyme by the dietary phytochemical Ferulic acid protects human neuroblastoma SH-SY5Y cells against Aβ(25–35)-induced oxidative stress and inflammatory responses

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder associated with the amyloid beta (Aβ) and tau hallmarks. The molecular insights into how neuroinflammation is initially triggered and how it affects neuronal cells are yet at the age of infancy. In this study, SH-SY5Y cells were used as a model for neurons by differentiating and were co-cultured with differentiated THP1 cells (microglia model) as well as treated with Aβ(25–35) and with antioxidant FA to study inflammatory, oxidative stress responses and their effects on co-cultured neurons. Neurons co-cultured with microglial cells showed pronounced increase in ROS levels, NOS expression, truncated N-terminal form (34 kDa) of APE1 expression and AIF’s translocation in the nucleus. The pre-treatment of FA, on the other hand reversed these effects. It was further evaluated how FA/Aβ treatment altered microglial phenotype that in turn affected the neurons. Microglial cells showed M1 phenotype upon Aβ(25–35) stress, while FA induced M2 phenotype against Aβ stress, suggesting that FA alleviated Aβ induced phenotype and its associated effects in the co-cultured neurons by altering the phenotype of microglial cells and induced expression of full length (37 kDa) APE1 enzyme and inhibiting AIF’s nuclear translocation, thus inhibiting apoptosis. This is the first study that revealed Aβ induced cleavage of APE1 enzyme in differentiated neurons, suggesting that APE1 may be the potential early target of Aβ that loses its function and exacerbates AD pathology. FA activated a fully functional form of APE1 against Aβ stress. The impaired function of APE1 could be the initial mechanism by which Aβ induces oxidative and inflammatory responses and dietary phytochemical FA can be a potential therapeutic strategy in managing the disease by activating APE1 that not only repairs oxidative DNA base damage but also maintains mitochondrial function and alleviates neuroinflammatory responses.

Endogenous base damage as a driver of genomic instability in homologous recombination-deficient cancers

Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway. Both familial and somatic loss of function mutation(s) in various HR genes predispose to a variety of cancer types, underscoring the importance of error-free repair of DSBs in human physiology. While environmental sources of DSBs have been known, more recent studies have begun to uncover the role of endogenous base damage in leading to these breaks. Base damage repair intermediates often consist of single-strand breaks, which if left unrepaired, can lead to DSBs as the replication fork encounters these lesions. This review summarizes various sources of endogenous base damage and how these lesions are repaired. We highlight how conversion of base repair intermediates, particularly those with 5′or 3′ blocked ends, to DSBs can be a predominant source of genomic instability in HR-deficient cancers. We also discuss how endogenous base damage and ensuing DSBs can be exploited to enhance the efficacy of Poly (ADP-ribose) polymerase inhibitors (PARPi), that are widely used in the clinics for the regimen of HR-deficient cancers.

Effects of low-dose gamma radiation on DNA measured using a quartz tuning fork sensor

Despite the expected direct effects of radiation on DNA through its direct interaction with the biomolecule, research indicates that radiation also interacts with the DNA’s environment, resulting in indirect effects. Therefore, in this study, we explored the feasibility of using the quartz tuning fork (QTF) sensor system in biomedical applications, specifically in detecting DNA damage caused by low doses of gamma radiation, directly and indirectly. We differentiated between direct and indirect damage by analyzing the fork’s resonance frequency changes. This experiment was divided into three stages: before, during, and after irradiation. Each stage involved samples of pure DNA and DNA in a 60-µL aqueous solution, evaluated under identical conditions. Before irradiation, we measured frequency shifts (Δf) over a 20-min period, resulting in values of 19.34, 20.25, and 7.6 Hz for water, DNA, and DNA in water, respectively. Subsequently, the samples were irradiated with cesium-137 for the specified duration, resulting in frequency shifts of ∼ 39.21, 28.37, and 41.23 Hz for the same conditions. Our investigations showed an increase in Δf from 20.25 to 28.3 Hz at doses ranging from 7.5 to 30 µGy for pure DNA. Interestingly, DNA in aqueous solution exhibited hypersensitivity to radiation, with frequency shifts ranging from 7.6 to 41.23 Hz. Furthermore, we observed a significant difference in frequency shift after irradiation between pure DNA and DNA in water, with shifts of ∼ 70.75–98.45 Hz and 56.32–79.28 Hz for DNA and DNA in water, respectively. This result indicates a significant increase in DNA damage in aqueous environments, driven by the generation of active hydroxyl radicals (OH−), resulting in base damage and an associated increase in strand breaks. Consequently, our research indicates a lack of substantial direct impact on DNA repair owing to the absence of a conducive postirradiation environment. Therefore, QTF is a valuable biomarker for radiation sensitivity and is promising for future applications as a mass-sensitive biosensor.

DNA Base Damage Repair Crosstalks with Chromatin Structures to Contract Expanded GAA Repeats in Friedreich's Ataxia.

Trinucleotide repeat (TNR) expansion is the cause of over 40 neurodegenerative diseases, including Huntington's disease and Friedreich's ataxia (FRDA). There are no effective treatments for these diseases due to the poor understanding of molecular mechanisms underlying somatic TNR expansion and contraction in neural systems. We and others have found that DNA base excision repair (BER) actively modulates TNR instability, shedding light on the development of effective treatments for the diseases by contracting expanded repeats through DNA repair. In this study, temozolomide (TMZ) was employed as a model DNA base damaging agent to reveal the mechanisms of the BER pathway in modulating GAA repeat instability at the frataxin (FXN) gene in FRDA neural cells and transgenic mouse mice. We found that TMZ induced large GAA repeat contraction in FRDA mouse brain tissue, neurons, and FRDA iPSC-differentiated neural cells, increasing frataxin protein levels in FRDA mouse brain and neural cells. Surprisingly, we found that TMZ could also inhibit H3K9 methyltransferases, leading to open chromatin and increasing ssDNA breaks and recruitment of the key BER enzyme, pol β, on the repeats in FRDA neural cells. We further demonstrated that the H3K9 methyltransferase inhibitor BIX01294 also induced the contraction of the expanded repeats and increased frataxin protein in FRDA neural cells by opening the chromatin and increasing the endogenous ssDNA breaks and recruitment of pol β on the repeats. Our study provides new mechanistic insight illustrating that inhibition of H3K9 methylation can crosstalk with BER to induce GAA repeat contraction in FRDA. Our results will open a new avenue for developing novel gene therapy by targeting histone methylation and the BER pathway for repeat expansion diseases.

Clustered DNA Damage and its Complexity: Tracking the History.

The concept of radiation-induced clustered damage in DNA has grown over the past several decades to become a topic of considerable interest across the scientific disciplines involved in studies of the biological effects of ionizing radiation. This paper, prepared for the 70th anniversary issue of Radiation Research, traces historical development of the three main threads of physics, chemistry, and biochemical/cellular responses that led to the hypothesis and demonstration that a key component of the biological effectiveness of ionizing radiation is its characteristic of producing clustered DNA damage of varying complexities. The physics thread has roots that started as early as the 1920s, grew to identify critical nanometre-scale clusterings of ionizations relevant to biological effectiveness, and then, by the turn of the century, had produced an extensive array of quantitative predictions on the complexity of clustered DNA damage from different radiations. Monte Carlo track structure simulation techniques played a key role through these developments, and they are now incorporated into many recent and ongoing studies modelling the effects of radiation. The chemistry thread was seeded by water-radiolysis descriptions of events in water as radical-containing "spurs," demonstration of the important role of the hydroxyl radical in radiation-inactivation of cells and the difficulty of protection by radical scavengers. This led to the concept and description of locally multiply damaged sites (LMDS) for DNA double-strand breaks and other combinations of DNA base damage and strand breakage that could arise from a spur overlapping, or created in very close proximity to, the DNA. In these ways, both the physics and the chemistry threads, largely in parallel, put out the challenge to the experimental research community to verify these predictions of clustered DNA damage from ionizing radiations and to investigate their relevance to DNA repair and subsequent cellular effects. The third thread, biochemical and cell-based research, responded strongly to the challenge by demonstrating the existence and biological importance of clustered DNA damage. Investigations have included repair of a wide variety of defined constructs of clustered damage, evaluation of mutagenic consequences, identification of clustered base-damage within irradiated cells, and identification of co-localization of repair complexes indicative of complex clustered damage after high-LET irradiation, as well as extensive studies of the repair pathways involved in repair of simple double-strand breaks. There remains, however, a great deal more to be learned because of the diversity of clustered DNA damage and of the biological responses.

Comparison of mutation spectra induced by gamma-rays and carbon ion beams.

The ionizing radiation with high linear energy transfer (LET), such as a heavy ion beam, induces more serious biological effects than low LET ones, such as gamma- and X-rays. This indicates a difference in the DNA damage produced by low and high LET radiations and their biological effects. We have been studying the differences in DNA damage produced by gamma-rays and carbon ion beams. Therefore, we analyze mutations induced by both ionizing radiations to discuss the differences in their biological effects in this study. pUC19 plasmid DNA was irradiated by carbon ion beams in the solution containing 1M dimethyl sulfoxide to mimic a cellular condition. The irradiated DNA was cloned in competent cells of Escherichia coli. The clones harboring some mutations in the region of lacZα were selected, and the sequence alterations were analyzed. A one-deletion mutation is significant in the carbon-irradiated DNA, and the C:G↔T:A transition is minor. On the other hand, the gamma-irradiated DNA shows mainly G:C↔T:A transversion. These results suggest that carbon ion beams produce complex DNA damage, and gamma-rays are prone to single oxidative base damage, such as 8-oxoguanine. Carbon ion beams can also introduce oxidative base damage, and the damage species is 5-hydroxycytosine. This was consistent with our previous results of DNA damage caused by heavy ion beams. We confirmed the causal DNA damage by mass spectrometry for these mutations.

Characterization of Cichorium intybus L. Extract in Preventing Oxidative DNA Base Damage Using Gas Chromatography–Tandem Mass Spectrometry (GC–MS/MS)

Free radicals are unstable and are known for electron deficiency. This state renders DNA, proteins, and various biomolecules susceptible to detrimental interactions, leading to damage. However, this process can be regulated by antioxidants produced naturally on-site or externally through food or herbal supplements. In recent years, interest in characterizing alternative, natural and safe antioxidants obtained from plant sources has been increasing. Within this context, Cichorium intybus L. has garnered attention for its notable antioxidant content. In our previous study, we determined optimum conditions for ultrasound-assisted extraction of antioxidants from Cichorium intybus L. using central composite design-response surface methodology. In this study, the quantitative determination of phenolics in the extract prepared under optimal conditions was undertaken employing high-performance liquid chromatography with diode array detection (HPLC-DAD). The study’s primary goal is to investigate the impact of the extract derived from Cichorium intybus on the formation of products resulting from oxidative damage to DNA bases by gas chromatography–tandem mass spectrometry (GC–MS/MS). For this purpose, different volumes of extract were added to the DNA + Fenton medium. When 25 µL of extract were added, 239.98 ± 5.81 ng of damaged product/mg DNA was present. 233.12 ± 5.63 ng of damaged product/mg DNA were obtained for 50 µL of extract and 165.76 ± 4.58 ng of damaged product/mg DNA for 100 µL. This study shows that Cichorium intybus has potential therapeutic effects on supporting antioxidant defenses and reducing oxidative DNA damage.

Strand-resolved mutagenicity of DNA damage and repair

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

Novel Approach for Predicting Vertical Electron Attachment Energies in Bulk-Solvated Molecules.

When low-energy electrons interact with molecules, they can give rise to transient anion states commonly known as resonances. These states are formed through vertical electron attachment processes and have the potential to induce various forms of DNA lesions, including base damage, single- and double-strand breaks, cross-links, and clustered lesions that are challenging to repair. So far, most experimental and theoretical studies have investigated the formation of resonances of (bio)molecules in the gas phase or in microsolvated environments. Since cellular environments are mainly composed of water molecules, it is crucial to understand how bulk water affects the resonances of (bio)molecules. Given the existing gap in studies on resonances of bulk-solvated molecules, we propose a novel theoretical-computational approach to address this void. Our approach combines the multibasis-set (time-dependent-)density functional theory and self-consistent sequential quantum mechanics/molecular mechanics polarizable electrostatic embedding methods. We apply this combined methodology to predict the vertical electron attachment energies of 1-methyl-5-nitroimidazole (1M5NI), a well-known radiosensitizer model, in bulk water. In addition, we analyze the rapid mutual polarization between the resonances (both shape- and core-excited) of 1M5NI and the surrounding bulk water environment. For comparison, we also studied the isolated and microsolvated 1M5NI. Overall, while the polarization of the environment is clearly sensitive to the solute charge, causing a significant impact on the vertical electron affinity and consequently on the attachment electron energies, it does not have a significant impact on the excitation energies of the anion.

Oxidatively-induced DNA base damage and base excision repair abnormalities in siblings of individuals with bipolar disorder DNA damage and repair in bipolar disorder

Previous evidence suggests elevated levels of oxidatively-induced DNA damage, particularly 8-hydroxy-2’-deoxyguanosine (8-OH-dG), and abnormalities in the repair of 8-OH-dG by the base excision repair (BER) in bipolar disorder (BD). However, the genetic disposition of these abnormalities remains unknown. In this study, we aimed to investigate the levels of oxidatively-induced DNA damage and BER mechanisms in individuals with BD and their siblings, as compared to healthy controls (HCs). 46 individuals with BD, 41 siblings of individuals with BD, and 51 HCs were included in the study. Liquid chromatography-tandem mass spectrometry was employed to evaluate the levels of 8-OH-dG in urine, which were then normalized based on urine creatinine levels. The real-time-polymerase chain reaction was used to measure the expression levels of 8-oxoguanine DNA glycosylase 1 (OGG1), apurinic/apyrimidinic endonuclease 1 (APE1), poly ADP-ribose polymerase 1 (PARP1), and DNA polymerase beta (POLβ). The levels of 8-OH-dG were found to be elevated in both individuals with BD and their siblings when compared to the HCs. The OGG1 and APE1 expressions were downregulated, while POLβ expressions were upregulated in both the patient and sibling groups compared to the HCs. Age, smoking status, and the number of depressive episodes had an impact on APE1 expression levels in the patient group while body mass index, smoking status, and past psychiatric history had an impact on 8-OH-dG levels in siblings. Both individuals with BD and unaffected siblings presented similar abnormalities regarding oxidatively-induced DNA damage and BER, suggesting a link between abnormalities in DNA damage/BER mechanisms and familial susceptibility to BD. Our findings suggest that targeting the oxidatively-induced DNA damage and BER pathway could offer promising therapeutic strategies for reducing the risk of age-related diseases and comorbidities in individuals with a genetic predisposition to BD.

DNA lesion bypass and the stochastic dynamics of transcription-coupled repair

DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the subsequent triggering of repair processes have major roles in shaping the genome-wide distribution of mutations, clearing barriers to transcription, and minimizing the production of miscoded gene products. Despite its importance for genetic integrity, key mechanistic features of this transcription-coupled repair (TCR) process are controversial or unknown. Here, we exploited a well-powered in vivo mammalian model system to explore the mechanistic properties and parameters of TCR for alkylation damage at fine spatial resolution and with discrimination of the damaged DNA strand. For rigorous interpretation, a generalizable mathematical model of DNA damage and TCR was developed. Fitting experimental data to the model and simulation revealed that RNA polymerases frequently bypass lesions without triggering repair, indicating that small alkylation adducts are unlikely to be an efficient barrier to gene expression. Following a burst of damage, the efficiency of transcription-coupled repair gradually decays through gene bodies with implications for the occurrence and accurate inference of driver mutations in cancer. The reinitation of transcription from the repair site is not a general feature of transcription-coupled repair, and the observed data is consistent with reinitiation never taking place. Collectively, these results reveal how the directional but stochastic activity of TCR shapes the distribution of mutations following DNA damage.

DNA Damage Accelerates G-Quadruplex Folding in a Duplex-G-Quadruplex-Duplex Context.

Molecular details for the impact of DNA damage on folding of potential G-quadruplex sequences (PQSs) to noncanonical DNA structures involved in gene regulation are poorly understood. Here, the effects of DNA base damage and strand breaks on PQS folding kinetics were studied in the context of the VEGF promoter sequence embedded between two DNA duplex anchors, termed a duplex-G-quadruplex-duplex (DGD) motif. This DGD scaffold imposes constraints on the PQS folding process that more closely mimic those found in genomic DNA. Folding kinetics were monitored by circular dichroism (CD) to find folding half-lives ranging from 2 s to 12 min depending on the DNA damage type and sequence position. The presence of Mg2+ ions and G-quadruplex (G4)-binding protein APE1 facilitated the folding reactions. A strand break placing all four G runs required for G4 formation on one side of the break accelerated the folding rate by >150-fold compared to the undamaged sequence. Combined 1D 1H NMR and CD analyses confirmed that isothermal folding of the VEGF-DGD constructs yielded spectral signatures that suggest the formation of G4 motifs and demonstrated a folding dependency on the nature and location of DNA damage. Importantly, the PQS folding half-lives measured are relevant to replication, transcription, and DNA repair time frames.

Abstract 2838: ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer

Abstract Immunomodulatory agents are an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but are not fully understood in cancer. Our studies of the little-known gene, NFX1-type zinc finger-containing 1 (ZNFX1), show that it acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high-grade and endometrial serous carcinoma (OC) cell lines (N=3), transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells, show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, including in bioinformatics of RNA seq data, thus defining this gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Further pathway analysis of ZNFX1 KO vs wild-type (WT) RNAseq data suggest increased proliferation and epithelial to mesenchymal transition (EMT) that was validated by in vitro and in vivo assays. Not only is ZNFX1 a master regular of the above mt dynamics, but it is a tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, in-vivo, increased tumor growth. This work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael Topper, Stephen Baylin, Kenneth Nephew, Feyruz Rassool. ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2838.

Experimental and numerical analysis of power take-off control effects on the dynamic performance of a floating wind-wave combined system

The integration of wave energy converters (WEC) into floating offshore wind turbines (FOWT) is regarded as a promising approach for comprehensively harnessing deep-sea energy and reducing the levelized cost of energy. This study aims to investigate the WEC power take-off (PTO) control effects on the dynamic performance of a floating wind-wave combined system, wherein three heaving-type WECs are integrated into a semi-submersible FOWT. In particular, the hydraulic PTO is modelled as a Coulomb damping system to enable a more realistic analysis. The aero-hydro-servo-elastic-mooring coupled numerical simulations and 1:50 wave basin experimental data demonstrate good agreement. It is observed that wave power production varies significantly with different control settings, potentially reaching 24.0 % of the overall hybrid energy production with proper control parameters. Furthermore, platform pitch oscillation generally shows a decreasing trend with increasing damping forces under below-rated and rated conditions, while showing minimal influence when above-rated. Conversely, tower base damage equivalent load (DEL) tends to initially decrease and subsequently increase across all examined conditions. This consistent convexity indicates the potential use of DEL as a performance index for optimising PTO control. In summary, power increment, motion reduction, and load mitigation could be achieved concurrently with appropriate control design. For instance, an extra 0.48 MW wave power could be produced under the rated condition, while a 10.4 % reduction in tower base DEL and a 6.57 % mitigation in platform pitch oscillation could also be achieved at the same time.

Abstract PR-005: ZNFX1 is a master regulator for epigenetic reprograming of mitochondrial inflammasome signaling and pathogen mimicry in cancer cells

Abstract Immunomodulatory agents represent an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviral elements (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA leading to activation of stimulator of interferon genes (STING). We previously reported in triple negative breast cancer (TNBC) and ovarian cancer (OC) that DNMTis in combination with PARPi induce STING-dependent interferon/inflammasome signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but upstream activating events in cancer are not understood. We now show NFX1-type zinc finger–containing 1 (ZNFX1), a little-studied innate immune gene, acts as a master nucleic acid (dsRNA/DNA) sensor for this mt gateway function. Importantly, in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high grade serous (TYK-nu, OVCAR4) as well as endometrial ovarian (A2780) cancer cells, transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane, using immunofluorescence-based proximity ligation assays. In studies of mt dysfunction, we further show that dsRNA/DNA as well as above-described anti-cancer drugs increase mt reactive oxygen species (ROS), using flow cytometry of ROS dye mitoSOX. DNMTi and PARPi treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured long range PCR and 8-oxoguanine (8-oxoG) ELISA assays, respectively. Importantly, these drug treatment increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. ZNFX1 knockout robustly attenuates these dynamics, thus defining this immune gene as essential for interferon/inflammasome signaling induced by mtDNA damage. Our data indicates a master role for the little-studied dsRNA/DNA sensor ZNFX1 in initiating a pathogen mimicry response to DNMTi and PARPi combination treatment, and further highlights the critical role of the mt in cellular antiviral signaling mechanisms. Importantly, this work suggests a novel avenue for manipulation of inflammasome signaling to improve cancer therapy responses and disease outcomes. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael J. Topper, Kenneth P. Nephew, Stephen B. Baylin, Feyruz V. Rassool. ZNFX1 is a master regulator for epigenetic reprograming of mitochondrial inflammasome signaling and pathogen mimicry in cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-005.

No evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 nanoparticles in three mammalian cell lines despite the initial reduction of cellular mitochondrial activity

Abstract We studied the effects of Aeroxide P25 titanium dioxide nanoparticles (TiO2 NPs) with a diameter of 21 nm on induction of DNA damage and long-term survival of three human cell lines: hepatocellular liver carcinoma HepG2, colorectal adenocarcinoma HT29 and lung carcinoma A549. The endpoints examined were DNA breakage estimated by the comet assay and oxidative base damage recognized by formamide-pyrimidine glycosylase (FPG) estimated with the FPG+ comet assay, frequencies of histone γH2AX foci and micronuclei, apoptosis, cell metabolic activity measured by mitochondrial activity (MTT) assay and long-term survival measured by colony-forming ability. Each cell line had a different pattern of DNA breakage and base damage vs. nanoparticle (NP) concentration and treatment time. There was no increase in the frequencies of histone γH2AX foci and micronuclei as compared to those in the untreated cells. In parallel with these results, no induction of apoptosis has been found in none of the cell lines tested. The reported experiments provided no evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 NPs, despite a slight decrease in mitochondrial activity and cell survival during the first 72 h.