Effect of Guanine Adduct Size, Shape, and Linker Type on the Conformation of Adducted DNA: A DFT and Molecular Dynamics Study.

DNA is damaged through various exogenous sources (e.g., automobile exhaust, tobacco smoke, and processed foods), which can yield diverse C8-dG bulky aryl adducts. Adducts are known to induce structural changes to DNA that can lead to various biological outcomes, ranging from cell death to diseases such as cancer. Unfortunately, the relationship between the chemical composition of the damaged product, the adducted DNA structure, and the biological consequences is not well understood, which limits the development of disease detection and prevention strategies. The present study uses density functional theory (DFT) calculations and quintuplicate 1 µs molecular dynamics (MD) simulations to characterize the structure of DNA containing 21 model C8-dG adducts that systematically differ in size (phenyl to pyrenyl), shape (α (2,3), ß (3,4) fusion, or ring substitution), and nucleobase-aryl group linkage (N, O, and C-linked). DFT calculations reveal that the inherent structural features of the G nucleobase adducts are impacted by linker type and bulky moiety shape, but not size, with the conformational flexibility reducing with α-ring fusion and linker composition as N > O > C. These structural properties are maintained in nucleoside models, which also reveal an increased propensity for anti-to-syn rotation about the glycosidic bond with N < O < C linker type. Although these diverse chemical features do not influence the global structure of adducted DNA, the adducts differentially impact the conformation local to the adducted site, including the relative populations of structures with the bulky moiety in the major groove (B conformer) and intercalated (stacked) into the helix (S conformer). Specifically, while the smallest phenyl adducts favor the B conformation and the largest pyrenyl-derived adducts stabilize the S conformation, the B/S ratio decreases with an increase in ring size and N > O > C linker composition. The shape and size (length) of the adduct can further finetune the B/S ratio, with ß-fused naphthyl or α-fused phenanthryl N-linked adducts and O or C-linked adducts containing ring substitution increasing the prevalence of the S adducted DNA conformation. Overall, this work uncovers the significant effect of bulky moiety size and linker type, as well as the lesser impact of aryl group shape, on adducted DNA structure, which suggests differential replication and repair outcomes, and thereby represents an important step toward rationalizing connections between the structure and biological consequences of diverse DNA adducts.

Downregulation of SF3B2 protects CNS neurons in models of multiple sclerosis.

OBJECTIVE: Neurodegeneration induced by inflammatory stress in multiple sclerosis (MS) leads to long-term neurological disabilities that are not amenable to current immunomodulatory therapies. METHODS AND RESULTS: Here, we report that neuronal downregulation of Splicing factor 3b subunit 2 (SF3B2), a component of U2 small nuclear ribonucleoprotein (snRNP), preserves retinal ganglion cell (RGC) survival and axonal integrity in experimental autoimmune encephalomyelitis (EAE)-induced mice. By employing an in vitro system recapitulating the inflammatory environment of MS lesion, we show that when SF3B2 levels are downregulated, cell viability and axon integrity are preserved in cortical neurons against inflammatory toxicity. Notably, knockdown of SF3B2 suppresses the expression of injury-response and necroptosis genes and prevents activation of Sterile Alpha and TIR Motif Containing 1 (Sarm1), a key enzyme that mediates programmed axon degeneration. INTERPRETATION: Together, these findings suggest that the downregulation of SF3B2 is a novel potential therapeutic target to prevent secondary neurodegeneration in MS.

Multiscale computational investigations of the translesion synthesis bypass of tobacco-derived DNA adducts: critical insights that complement experimental biochemical studies.

Among the numerous agents that damage DNA, tobacco products remain one of the most lethal and result in the most diverse set of DNA lesions. This perspective aims to provide an overview of computational work conducted to complement experimental biochemical studies on the mutagenicity of adducts derived from the most potent tobacco carcinogen, namely 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosaminoketone or NNK). Lesions ranging from the smallest methylated thymine derivatives to the larger, flexible pyridyloxobutyl (POB) guanine adducts are considered. Insights are obtained from density functional theory (DFT) calculations and molecular dynamics (MD) simulations into the damaged nucleobase and nucleoside structures, the accommodation of the lesions in the active site of key human polymerases, the intrinsic base pairing potentials of the adducts, and dNTP incorporation opposite the lesions. Overall, the computational data provide atomic level information that can rationalize the differential mutagenic properties of tobacco-derived lesions and uncover important insights into the impact of adduct size, nucleobase, position, and chemical composition of the bulky moiety.

Assessment of the Accuracy of DFT-Predicted Li+-Nucleic Acid Binding Energies.

Understanding how lithium interacts with complex biosystems is crucial for uncovering the roles of this alkali metal in biology and designing extraction techniques for battery production and environmental remediation. In this light, fundamental information about Li+ binding to nucleic acids is required. Herein, a new database of Li+-nucleic acid interactions is presented that contains CCSD(T)/CBS benchmark energies for all nucleobase and phosphate binding locations. Furthermore, the performance of 54 DFT functionals in combination with three triple-zeta (TZ) basis sets (6-311+G(3df,2p), aug-cc-pVTZ, and def2-TZVPP) is tested. The results identify a range of functionals across different families (B2-PLYP, PBE-QIDH, ωB97, ωB97X-D, MN15, B3PW91, B97-2, TPSS, BP86-D3(BJ), and PBE) that can accurately describe coordinated Li+-nucleic acid interactions, with the average mean percent error (AMPE) across binding positions and basis sets being below 2%. Nevertheless, only three functionals tested (B2-PLYP, PBE-QIDH, and ωB97X-D) preserve this accuracy for metal cation-π interactions, suggesting that caution is warranted when choosing a functional to describe a diverse range of Li+-nucleic acid complexes. Removal of counterpoise corrections has very little impact on the reliability of most functionals, while the effect of empirical dispersion corrections varies depending on the functional choice and interaction type. While increasing the basis set to quadruple-zeta quality had little impact on the AMPE, the accuracy of double-zeta basis sets varies with family. Importantly, DFT methods reproduce the CCSD(T)/CBS trend in the preferred binding position for a given nucleic acid component and the global trend across components (phosphate ≫ G > C ≫ A ∼ T = U), as well as the geometries of the metal-nucleic acid complexes. The overall top performing functional is PBE-QIDH, which results in deviations from CCSD(T)/CBS values as small as ∼0.1 kcal/mol for nucleobase contacts and ∼1 kcal/mol for phosphate interactions. The most accurate DFT methods identified in the present work are recommended for future investigations of lithium interactions in larger nucleic acid systems to provide insights into the biological roles of this metal and the design of novel biosensing strategies.

Microstructures and Mechanical Properties of Deposited Fe-8Cr-3V-2Mo-2W on SCM420 Substrate Using Directed Energy Deposition and Effect of Post-Heat Treatment.

In this study, the Fe-8Cr-3V-2Mo-2W tool steel powder was deposited on the SCM420 substrate through the directed energy deposition (DED) process. This study focuses on the mechanical properties of the deposited Fe-8Cr-3V-2Mo-2W and the effect of heat treatment on it. The changes in the microstructural characteristics of the deposited region due to heat treatment after deposition were observed. The influence of heat treatment on the mechanical properties was then analyzed accordingly and hence, the hardness, wear, impact and tensile tests were conducted on the deposited material. These properties were compared with those of the commercial tool steel powder M2-deposited material and the carburized specimen. In the deposited Fe-8Cr-3V-2Mo-2W layer, an increased martensite phase fraction was obtained through post-heat treatment and the amount of precipitated carbides was also increased. This increased the hardness from 48 to 62 HRc after heat treatment and the wear resistance was significantly improved as well. The amount of impact energy absorbed decreased from 11 J before heat treatment to 6 J after heat treatment, but the tensile strength significantly increased from 607 to 922 MPa. When compared with the M2-deposited surface, the Fe-8Cr-3V-2Mo-2W deposits had 3% lower surface hardness and 76% lower fracture toughness but exhibited 56% higher tensile strength. When compared with the carburized SCM420, the Fe-8Cr-3V-2Mo-2W deposits exhibited 3% higher surface hardness and wear resistance, 90% lower fracture toughness and 5% higher tensile strength. This study shows that surface hardening through DED can exhibit similar or superior mechanical properties when compared to carburizing.

Human Motor Neurons With SOD1-G93A Mutation Generated From CRISPR/Cas9 Gene-Edited iPSCs Develop Pathological Features of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by gradual degeneration and elimination of motor neurons (MNs) in the motor cortex, brainstem, and spinal cord. Some familial forms of ALS are caused by genetic mutations in superoxide dismutase 1 (SOD1) but the mechanisms driving MN disease are unclear. Identifying the naturally occurring pathology and understanding how this mutant SOD1 can affect MNs in translationally meaningful ways in a valid and reliable human cell model remains to be established. Here, using CRISPR/Cas9 genome editing system and human induced pluripotent stem cells (iPSCs), we generated highly pure, iPSC-derived MNs with a SOD1-G93A missense mutation. With the wild-type cell line serving as an isogenic control and MNs from a patient-derived iPSC line with an SOD1-A4V mutation as a comparator, we identified pathological phenotypes relevant to ALS. The mutant MNs accumulated misfolded and aggregated forms of SOD1 in cell bodies and processes, including axons. They also developed distinctive axonal pathologies. Mutants had axonal swellings with shorter axon length and less numbers of branch points. Moreover, structural and molecular abnormalities in presynaptic and postsynaptic size and density were found in the mutants. Finally, functional studies with microelectrode array demonstrated that the individual mutant MNs exhibited decreased number of spikes and diminished network bursting, but increased burst duration. Taken together, we identified spontaneous disease phenotypes relevant to ALS in mutant SOD1 MNs from genome-edited and patient-derived iPSCs. Our findings demonstrate that SOD1 mutations in human MNs cause cell-autonomous proteinopathy, axonopathy, synaptic pathology, and aberrant neurotransmission.

Cisplatin induced neurotoxicity is mediated by Sarm1 and calpain activation.

Cisplatin is a commonly used chemotherapy agent with significant dose-limiting neurotoxicity resulting in peripheral neuropathy. Although it is postulated that formation of DNA-platinum adducts is responsible for both its cytotoxicity in cancer cells and side effects in neurons, downstream mechanisms that lead to distal axonal degeneration are unknown. Here we show that activation of calpains is required for both neurotoxicity and formation of DNA-platinum adduct formation in neurons but not in cancer cells. Furthermore, we show that neurotoxicity of cisplatin requires activation of Sarm1, a key regulator of Wallerian degeneration, as mice lacking the Sarm1 gene do not develop peripheral neuropathy as evaluated by both behavioral or pathological measures. These findings indicate that Sarm1 and/or specific calpain inhibitors could be developed to prevent cisplatin induced peripheral neuropathy.

DFT Study on the Deglycosylation of Methylated, Oxidized, and Canonical Pyrimidine Nucleosides in Water: Implications for Epigenetic Regulation and DNA Repair.

Density functional theory (B3LYP) was used to characterize the kinetics and thermodynamics of the (nonenzymatic) deglycosylation in water for a variety of 2'-deoxycytidine (dC) and 2'-deoxyuridine (dU) nucleoside derivatives that differ in methylation and subsequent oxidation of the C5 substituent. A range of computational models are considered that combine implicit and explicit solvation of the nucleophile and nucleobase. Regardless of the model implemented, our calculations reveal that the glycosidic bond in dC is inherently more stable than that in dU. Furthermore, C5 methylation of either pyrimidine and subsequent oxidation of the methyl group yield overall small changes to the Gibbs reaction energy profiles and thereby preserve lower deglycosylation barriers for the dC compared to those for the dU nucleoside derivatives. However, hydrolytic deglycosylation becomes significantly more energetically favorable when 5-methyl-dC (5m-dC) undergoes two or three rounds of oxidation, with the Gibbs energy barrier decreasing and the reaction becoming more exergonic by up to 40 kJ/mol. In fact, two or three oxidation reactions from 5m-dC result in a deglycosylation barrier similar to that for dU, as well as those for the associated C5-methylated (2'-deoxythymidine) and oxidized (5-hydroxymethyl-dU) derivatives. These predicted trends in the inherent deglycosylation energetics in water directly correlate with the previously reported activity of thymine DNA glycosylase (TDG), which cleaves the glycosidic bond in select dC nucleosides as part of epigenetic regulation and in dU variants as part of DNA repair. Thus, our data suggests that fundamental differences in the intrinsic reactivity of the pyrimidine nucleosides help regulate the function of human enzymes that maintain cellular integrity.

DNA damage accumulates and responses are engaged in human ALS brain and spinal motor neurons and DNA repair is activatable in iPSC-derived motor neurons with SOD1 mutations.

DNA damage is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, relationships between DNA damage accumulation, DNA damage response (DDR), and upper and lower motor neuron vulnerability in human ALS are unclear; furthermore, it is unknown whether epigenetic silencing of DNA repair pathways contributes to ALS pathogenesis. We tested the hypotheses that DNA damage accumulates in ALS motor neurons along with diminished DDR, and that DNA repair genes undergo hypermethylation. Human postmortem CNS tissue was obtained from ALS cases (N = 34) and age-matched controls without neurologic disease (N = 15). Compared to age-matched controls, abasic sites accumulated in genomic DNA of ALS motor cortex and laser capture microdissection-acquired spinal motor neurons but not in motor neuron mitochondrial DNA. By immunohistochemistry, DNA damage accumulated significantly in upper and lower motor neurons in ALS cases as single-stranded DNA and 8-hydroxy-deoxyguanosine (OHdG) compared to age-matched controls. Significant DDR was engaged in ALS motor neurons as evidenced by accumulation of c-Abl, nuclear BRCA1, and ATM activation. DNA damage and DDR were present in motor neurons at pre-attritional stages and throughout the somatodendritic attritional stages of neurodegeneration. Motor neurons with DNA damage were also positive for activated p53 and cleaved caspase-3. Gene-specific promoter DNA methylation pyrosequencing identified the DNA repair genes Ogg1, Apex1, Pnkp and Aptx as hypomethylated in ALS. In human induced-pluripotent stem cell (iPSC)-derived motor neurons with familial ALS SOD1 mutations, DNA repair capacity was similar to isogenic control motor neurons. Our results show that vulnerable neurons in human ALS accumulate DNA damage, and contrary to our hypothesis, strongly activate and mobilize response effectors and DNA repair genes. This DDR in ALS motor neurons involves recruitment of c-Abl and BRCA1 to the nucleus in vivo, and repair of DNA double-strand breaks in human ALS motor neurons with SOD1 mutations in cell culture.

Anti-Inflammatory Activity of Populus deltoides Leaf Extract via Modulating NF-κB and p38/JNK Pathways.

Populus deltoides, known as eastern cottonwood, has been commonly used as a medicinal plant. The aim of the present study was to investigate the mechanism underlying the anti-inflammatory activity of P. deltoides leaf extract (PLE). PLE effectively inhibited the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, but not that of cyclooxygenase-2 (COX-2) and prostaglandin E2. Proinflammatory tumor necrosis factor alpha (TNF-α) levels were also reduced by the extract. PLE inhibited the phosphorylation of nuclear factor-kappa B (NF-κB) and inhibitor of Kappa Bα (IκBα), and blunted LPS-triggered enhanced nuclear translocation of NF-κB p65. In mitogen-activated protein kinase (MAPK) signaling, PLE effectively decreased the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK), but not of extracellular signal-regulated kinase 1/2 (ERK1/2). Taken together, these results suggest that anti-inflammatory activity of P. deltoides leaf extract might be driven by iNOS and NO inhibition mediated by modulation of the NF-κB and p38/JNK signaling pathways.