The Effect of Varying Preoperative Hemoglobin Levels on the Risk of Major Complications and Surgical Site Infections After Single Level Lumbar Fusion.

INTRODUCTION: Blood transfusions are associated with an increased risk of complications after lumbar fusion, and current anemia hemoglobin thresholds are not surgery specific. We aimed to calculate single-level lumbar fusion-specific preoperative hemoglobin strata that observe the likelihood of 90-day transfusion and evaluate whether these strata are associated with increased risk of 90-day complications and 2-year infections. METHODS: A national database identified patients undergoing primary single-level lumbar fusion with preoperative hemoglobin values (g/dL). Stratum-specific likelihood ratio analysis calculated sex-based hemoglobin strata associated with the risk of 90-day transfusion. Incidence and risk of 90-day major complications and 2-year infections were observed between strata. RESULTS: Three female (hemoglobin strata, likelihood ratio [<10.9, 2.41; 11.0 to 12.4, 1.35; 12.5 to 17.0, 0.78]) and male (<11.9, 2.95; 12.0 to 13.4, 1.46; 13.5 to 13.9, 0.71) strata were associated with varying likelihood of 90-day blood transfusion. Increased 90-day complication risk was associated with two female strata (hemoglobin strata, relative risk [11.0 to 12.4, 1.52; <10.9, 3.40]) and one male stratum (<11.9, 2.02). Increased 2-year infection risk was associated with one female (<10.9, 3.67) and male stratum (<11.9, 2.11). CONCLUSION: Stratum-specific likelihood ratio analysis established sex-based single-level lumbar fusion-specific hemoglobin strata that observe the likelihood of 90-day transfusion and the risk of 90-day major complications and 2-year infections. These thresholds are a unique addition to the literature and can assist in counseling patients on their postoperative risk profile and in preoperative patient optimization. LEVEL OF EVIDENCE: Level III.

Promoter dependent RNA polymerase II bypass of the epimerizable DNA lesion, Fapy•dG and 8-Oxo-2'-deoxyguanosine.

Formamidopyrimidine (Fapy•dG) is a major lesion arising from oxidation of dG that is produced from a common chemical precursor of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). In human cells, replication of single-stranded shuttle vectors containing Fapy•dG is more mutagenic than 8-OxodGuo. Here, we present the first data regarding promoter dependent RNA polymerase II bypass of Fapy•dG. 8-OxodGuo bypass was examined side-by-side. Experiments were carried out using double-stranded shuttle vectors in HeLa cell nuclear lysates and in HEK 293T cells. The lesions do not significantly block transcriptional bypass efficiency. Less than 2% adenosine incorporation occurred in cells when the lesions were base paired with dC. Inhibiting base excision repair in HEK 293T cells significantly increased adenosine incorporation, particularly from Fapy•dG:dC bypass which yielded ∼25% adenosine incorporation. No effect was detected upon transcriptional bypass of either lesion in nucleotide excision repair deficient cells. Transcriptional mutagenesis was significantly higher when shuttle vectors containing dA opposite one of the lesions were employed. For Fapy•dG:dA bypass, adenosine incorporation was greater than 85%; whereas 8-OxodGuo:dA yielded >20% point mutations. The combination of more frequent replication mistakes and greater error-prone Pol II bypass suggest that Fapy•dG is more mutagenic than 8-OxodGuo.

Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

Biochemical and structural characterization of Fapy•dG replication by Human DNA polymerase ß.

N6-(2-deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG.

Using emerging science to inform risk characterizations for wildlife within current regulatory frameworks.

Many jurisdictions have regulatory frameworks that seek to reduce the effects of environmental exposures of anthropogenic chemicals on terrestrial wildlife (i.e., mammals, birds, reptiles, and amphibians). The frameworks apply for new and existing chemicals, including pesticides (prospective assessments), and to environmental contamination from releases (retrospective risk assessments). Relatively recently, there have been many scientific advances that could improve risk estimates for wildlife. Here, we briefly describe current regulations from North America (United States and Canada) and from Europe that include risk assessments for wildlife to ascertain whether they are conducive to the use of emerging science and new methods. We also provide examples where new and emerging science may be used to improve wildlife risk characterization and identify areas in need of future research. Integr Environ Assess Manag 2024;20:765-779. © 2024 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management © 2024 Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Molecular Mechanism of RNA Polymerase II Transcriptional Mutagenesis by the Epimerizable DNA Lesion, Fapy·dG.

Oxidative DNA lesions cause significant detrimental effects on a living species. Two major DNA lesions resulting from dG oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo) and formamidopyrimidine (Fapy·dG), are produced from a common chemical intermediate. Fapy·dG is formed in comparable yields under oxygen-deficient conditions. Replicative bypass of Fapy·dG in human cells is more mutagenic than that of 8-OxodGuo. Despite the biological importance of transcriptional mutagenesis, there are no reports of the effects of Fapy·dG on RNA polymerase II (Pol II) activity. Here we perform comprehensive kinetic studies to investigate the impact of Fapy·dG on three key transcriptional fidelity checkpoint steps by Pol II: insertion, extension, and proofreading steps. The ratios of error-free versus error-prone incorporation opposite Fapy·dG are significantly reduced in comparison with undamaged dG. Similarly, Fapy·dG:A mispair is extended with comparable efficiency as that of the error-free, Fapy·dG:C base pair. The α- and ß-configurational isomers of Fapy·dG have distinct effects on Pol II insertion and extension. Pol II can preferentially cleave error-prone products by proofreading. To further understand the structural basis of transcription processing of Fapy·dG, five different structures were solved, including Fapy·dG template-loading state (apo), error-free cytidine triphosphate (CTP) binding state (prechemistry), error-prone ATP binding state (prechemistry), error-free Fapy·dG:C product state (postchemistry), and error-prone Fapy·dG:A product state (postchemistry), revealing distinctive nucleotide binding and product states. Taken together, our study provides a comprehensive mechanistic framework for better understanding how Fapy·dG lesions impact transcription and subsequent pathological consequences.

Biochemical and Structural Characterization of Fapy•dG Replication by Human DNA Polymerase ß.

N6-(2-deoxy-α,ß-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG.

The Effect of Socioeconomic Deprivation on Radiographic Deformities in Children With Blount Disease.

BACKGROUND: Blount disease can occur at any time during the growth process, primarily with a bimodal distribution in children younger than 4 years old and adolescents. The disease process most commonly presents in Black adolescents, with disease severity positively correlated with obesity. Given the known associations among race, obesity, and socioeconomic status, we investigated the relationship between the degree of social deprivation and severity of lower extremity deformities among a community-based cohort with Blount disease. METHODS: A retrospective review of hospital records and radiographs of patients with previously untreated Blount disease was conducted. Patients were classified as having early-onset or late-onset Blount disease based on whether the lower limb deformity was noted before or after the age of 4 years. The area deprivation index (ADI), a nationally validated measure that assesses socioeconomic deprivation by residential neighborhood, was calculated for each patient as a surrogate for socioeconomic status. Higher state (range: 1 to 10) or national (range: 1 to 100) ADI corresponds to increased social deprivation. Full-length standing radiographs from index clinic visits were evaluated by 2 reviewers to measure frontal plane deformity. The association of ADI with various demographic and radiographic parameters was then analyzed. RESULTS: Of the 65 patients with Blount disease, 48 (74%) children were Black and 17 (26%) were non-black children. Nineteen children (32 limbs) had early-onset and 46 children (62 limbs) had late-onset disease. Black patients had significantly higher mean state (7.6 vs. 5.4, P =0.009) and national (55.1 vs. 37.4, P =0.002) ADI values than non-black patients. Patients with severe socioeconomic deprivation had significantly greater mechanical axis deviation (66 mm vs. 51 mm, P =0.008). After controlling demographic and socioeconomic factors, the results of multivariate linear regression showed that only increased body mass index (ß=0.19, 95% CI: 0.12-0.26, P <.001) and state ADI (ß=0.021, 95% CI: 0.01-0.53, P =.043) were independently associated with greater varus deformity. CONCLUSIONS: Socioeconomic deprivation was strongly associated with increased severity of varus deformity in children with late-onset Blount disease. Our analysis suggests that obesity and socioeconomic factors are the most influential with regard to disease progression. LEVEL OF EVIDENCE: Level III.

Atraumatic Polycompartment Syndrome Secondary to Cardiogenic Shock: A Case Report.

We report the case of a 53-year-old male who developed polycompartment syndrome (PCS) secondary to cardiogenic shock. After suffering a cardiac arrest, a self-perpetuating cycle of intra-abdominal hypertension (IAH) and vital organ damage led to abdominal compartment syndrome (AbCS), which then contributed to the precipitation of extremity compartment syndrome (CS) in bilateral thighs, legs, forearms, and hands. This report is followed by a review of the literature regarding the pathophysiology of this rare sequela of cardiogenic shock. While the progression from cardiogenic shock to AbCS and ultimately to PCS has been hypothesized, no prior case reports demonstrate this. Furthermore, this case suggests more generally that IAH may be a risk factor for extremity CS. Future studies should examine the potential interplay between IAH and extremity CS in patients at risk, such as polytrauma patients with tibial fractures.

DNA-Histone Cross-Link Formation via Hole Trapping in Nucleosome Core Particles.

Radical cations (holes) produced in DNA by ionizing radiation and other oxidants yield DNA-protein cross-links (DPCs). Detailed studies of DPC formation in chromatin via this process are lacking. We describe here a comprehensive examination of DPC formation within nucleosome core particles (NCPs), which are the monomeric component of chromatin. DNA holes are introduced at defined sites within NCPs that are constructed from the bottom-up. DPCs form at DNA holes in yields comparable to those of alkali-labile DNA lesions that result from water trapping. DPC-forming efficiency and site preference within the NCP are dependent on translational and rotational positioning. Mass spectrometry and the use of mutant histones reveal that lysine residues in histone N-terminal tails and amino termini are responsible for the DPC formation. These studies are corroborated by computational simulation at the microsecond time scale, showing a wide range of interactions that can precede DPC formation. Three consecutive dGs, which are pervasive in the human genome, including G-quadruplex-forming sequences, are sufficient to produce DPCs that could impact gene expression.

Synergistic effects on mutagenicity of tandem lesions containing 8-oxo-7,8-dihydro-2'-deoxyguanosine or Fapy•dG flanked by a 3' 5-formyl-2'-deoxyuridine in human cells.

Modified nucleotides often hinder and/or decrease the fidelity of DNA polymerases. Tandem lesions, which are comprised of DNA modifications at two contiguous nucleotide positions, can be even more detrimental to genome stability. Recently, tandem lesions containing 5-formyl-2'-deoxyuridine (5fdU) flanked at the 5'-position by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo) or N-(2-deoxy-α,ß-D-erythropentofuranosyl)-N-(2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy•dG) were discovered. We examined the replication of 5'- 8-OxodGuo-5fdU and 5'-Fapy•dG-5fdU tandem lesions in HEK 293T cells and several polymerase deficient variants by transfecting single-stranded vectors containing them. The local sequence of the tandem lesions encompasses the 273 codon of the p53 gene, a mutational hot-spot. The bypass efficiency and mutation spectra of the tandem lesions were compared to those of the isolated lesions. Replication of weakly mutagenic 5-fdU is little changed when part of the 5'- 8-OxodGuo-5fdU tandem lesion. G → T transversions attributable to 8-OxodGuo increase > 10-fold when the tandem lesion is bypassed. 5'-Fapy•dG-5fdU has a synergistic effect on the error-prone bypass of both lesions. The mutation frequency (MF) of 5'-Fapy•dG-5fdU increases 3-fold compared to isolated Fapy•dG. In addition, a 5'-adjacent Fapy•dG significantly increases the MF of 5fdU. The major mutation, G → T transversions, decrease by almost a third in hPol κ- cells, which is the opposite effect when isolated Fapy•dG in the same sequence context is replicated in HEK 293T cells in the same sequence. Steady-state kinetics indicate that hPol κ contributes to greater G → T transversions by decreasing the specificity constant for dCTP compared to an isolated Fapy•dG. The greater conformational freedom of Fapy•dG compared to 8-OxodGuo and its unusual ability to epimerize at the anomeric center is believed to be the source of the complex effects of 5'-Fapy•dG-5fdU on replication.

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding.

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

Photochemical and Single Electron Transfer Generation of 2'-Deoxycytidin-N4-yl Radical from Oxime Esters.

A 2'-deoxycytidin-N4-yl radical (dC·), a strong oxidant that also abstracts hydrogen atoms from carbon-hydrogen bonds, is produced in a variety of DNA damaging processes. We describe here the independent generation of dC· from oxime esters under UV-irradiation or single electron transfer conditions. Support for this σ-type iminyl radical generation is provided by product studies carried out under aerobic and anaerobic conditions, as well as electron spin resonance (ESR) characterization of dC· in a homogeneous glassy solution at low temperature. Density functional theory (DFT) calculations also support fragmentation of the corresponding radical anions of oxime esters 2d and 2e to dC· and subsequent hydrogen atom abstraction from organic solvents. The corresponding 2'-deoxynucleotide triphosphate (dNTP) of isopropyl oxime ester 2c (5) is incorporated opposite 2'-deoxyadenosine and 2'-deoxyguanosine by a DNA polymerase with approximately equal efficiency. Photolysis experiments of DNA containing 2c support dC· generation and indicate that the radical produces tandem lesions when flanked on the 5'-side by 5'-d(GGT). These experiments suggest that oxime esters are reliable sources of nitrogen radicals in nucleic acids that will be useful mechanistic tools and possibly radiosensitizing agents when incorporated in DNA.

8-Oxo-2'-deoxyguanosine Replication in Mutational Hot Spot Sequences of the p53 Gene in Human Cells Is Less Mutagenic than That of the Corresponding Formamidopyrimidine.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo) is a ubiquitous DNA damage formed by oxidation of 2'-deoxyguanosine. In this study, plasmid DNA containing 8-OxodGuo located in three mutational hot spots of human cancers, codons 248, 249, and 273 of the Tp53 tumor suppressor gene, was replicated in HEK 293T cells. 8-OxodGuo was only a weak block of replication, and the bypass was largely error-free. The mutations (1-5%) were primarily G → T transversions, and the mutation frequency was generally lower than that of the chemically related Fapy·dG. A unique 8-OxodGuo mutation spectrum was observed at each site, as reflected by replication in translesion synthesis (TLS) polymerase- or hPol λ-deficient cells. In codon 248 (CG*G) and 249 (AG*G), where G* denotes 8-OxodGuo, hPol η and hPol ζ carried out largely error-free bypass of the lesion, whereas hPol κ and hPol ι were involved mostly in error-prone TLS, resulting in G → T mutations. 8-OxodGuo bypass in codon 273 (CG*T) was unlike the other two sites, as hPol κ participated in the mostly error-free bypass of the lesion. Yet, in all three sites, including codon 273, simultaneous deficiency of hpol κ and hPol ι resulted in reduction of G → T transversions. This indicates a convincing role of these two TLS polymerases in error-prone bypass of 8-OxodGuo. Although the dominant mutation was G → T in each site, in codon 249, and to a lesser extent in codon 248, significant semi-targeted single-base deletions also occurred, which suggests that 8-OxodGuo can initiate slippage of a base near the lesion site. This study underscores the importance of sequence context in 8-OxodGuo mutagenesis in human cells. It also provides a more comprehensive comparison between 8-OxodGuo and the sister lesion, Fapy·dG. The greater mutagenicity of the latter in the same sequence contexts indicates that Fapy·dG is a biologically significant lesion and biomarker on par with 8-OxodGuo.

Pain Self-Efficacy (PSEQ) score of <22 is associated with daily opioid use, back pain, disability, and PROMIS scores in patients presenting for spine surgery.

BACKGROUND CONTEXT: Pain self-efficacy, or the belief that one can carry out activities despite pain, has been shown to be associated with back and neck pain severity. However, the literature correlating psychosocial factors to opioid use, barriers to proper opioid use, and Patient-Reported Outcome Measurement Information System (PROMIS) scores is sparse. PURPOSE: The primary aim of this study was to determine whether pain self-efficacy is associated with daily opioid use in patients presenting for spine surgery. The secondary aim was to determine whether there exists a threshold self-efficacy score that is predictive of daily preoperative opioid use and subsequently to correlate this threshold score with opioid beliefs, disability, resilience, patient activation, and PROMIS scores. PATIENT SAMPLE: Five hundred seventy-eight elective spine surgery patients (286 females; mean age of 55 years) from a single institution were included in this study. STUDY DESIGN/SETTING: Retrospective review of prospectively collected data. OUTCOME MEASURES: PROMIS scores, daily opioid use, opioid beliefs, disability, patient activation, resilience. METHODS: Elective spine surgery patients at a single institution completed questionnaires preoperatively. Pain self-efficacy was measured by the Pain Self-Efficacy Questionnaire (PSEQ). Threshold linear regression with Bayesian information criteria was utilized to identify the optimal threshold associated with daily opioid use. Multivariable analysis controlled for age, sex, education, income, and Oswestry Disability Index (ODI) and PROMIS-29, version 2 scores. RESULTS: Of 578 patients, 100 (17.3%) reported daily opioid use. Threshold regression identified a PSEQ cutoff score of <22 as predictive of daily opioid use. On multivariable logistic regression, patients with a PSEQ score <22 had two times greater odds of being daily opioid users than those with a score ≥22. Further, PSEQ <22 was associated with lower patient activation; increased leg and back pain; higher ODI; higher PROMIS pain, fatigue, depression, and sleep scores; and lower PROMIS physical function and social satisfaction scores (p<.05 for all). CONCLUSIONS: In patients presenting for elective spine surgery, a PSEQ score of <22 is associated with twice the odds of reporting daily opioid use. Further, this threshold is associated with greater pain, disability, fatigue, and depression. A PSEQ score <22 can identify patients at high risk for daily opioid use and can guide targeted rehabilitation to optimize postoperative quality of life.

Histone Deacetylase 1 Inhibition by Peptides Containing a DNA Damage-Induced, Nonenzymatic, Histone Covalent Modification.

Treatment of HeLa cells with the DNA damaging agent, bleomycin (BLM), results in the formation of a nonenzymatic 5-methylene-2-pyrrolone histone covalent modification on lysine residues (KMP). KMP is much more electrophilic than other N-acyllysine covalent modifications and post-translational modifications, including N-acetyllysine (KAc). Using histone peptides containing KMP, we show that this modification inhibits the class I histone deacetylase, HDAC1, by reacting with a conserved cysteine (C261) located near the active site. HDAC1 is inhibited by histone peptides whose corresponding N-acetylated sequences are known deacetylation substrates, but not one containing a scrambled sequence. The HDAC1 inhibitor, trichostatin A, competes with covalent modification by the KMP-containing peptides. HDAC1 is also covalently modified by a KMP-containing peptide in a complex milieu. These data indicate that peptides containing KMP are recognized by HDAC1 and are bound in the active site. The effects on HDAC1 indicate that KMP formation in cells may contribute to the biological effects of DNA damaging agents, such as BLM, that form this nonenzymatic covalent modification.

A Five-Year Cost-Utility Analysis Comparing Synthetic Cage Versus Allograft Use in Anterior Cervical Discectomy and Fusion Surgery for Cervical Spondylotic Myelopathy.

STUDY DESIGN: Retrospective cost-utility analysis. OBJECTIVE: To conduct a cost-analysis comparing synthetic cage (SC) versus allograft (Allo) over a five-year time horizon. SUMMARY OF BACKGROUND DATA: SC and Allo are two commonly used interbody choices for anterior cervical discectomy and fusion (ACDF) surgery. Previous analyses comparative analyses have reached mixed conclusions regarding their cost-effectiveness, yet recent estimates provide high-quality evidence. MATERIALS AND METHODS: A decision-analysis model comparing the use of Allo versus SC was developed for a hypothetical 60-year-old patient with cervical spondylotic myelopathy undergoing single-level ACDF surgery. A comprehensive literature review was performed to estimate probabilities, costs (2020 USD) and quality-adjusted life years (QALYs) gained over a five-year period. A probabilistic sensitivity analysis using a Monte Carlo simulation of 1000 patients was carried out to calculate incremental cost-effectiveness ratio and net monetary benefits. One-way deterministic sensitivity analysis was performed to estimate the contribution of individual parameters to uncertainty in the model. RESULTS: The use of Allo was favored in 81.6% of the iterations at a societal willing-to-pay threshold of 50,000 USD/QALY. Allo dominated (higher net QALYs and lower net costs) in 67.8% of the iterations. The incremental net monetary benefits in the Allo group was 2650 USD at a willing-to-pay threshold of 50,000 USD/QALY. One-way deterministic sensitivity analysis revealed that the cost of the index surgery was the only factor which significantly contributed to uncertainty. CONCLUSION: Cost-utility analysis suggests that Allo maybe a more cost-effective option compared with SCs in adult patients undergoing ACDF for cervical spondylotic myelopathy.

Covalent Modification of Bromodomain Proteins by Peptides Containing a DNA Damage-Induced, Histone Post-Translational Modification.

An electrophilic 5-methylene-2-pyrrolone modification (KMP ) is produced at lysine residues of histone proteins in nucleosome core particles upon reaction with a commonly formed DNA lesion (C4-AP). The nonenzymatic KMP modification is also generated in the histones of HeLa cells treated with the antitumor agent, bleomycin that oxidizes DNA and forms C4-AP. This nonenzymatic covalent histone modification has the same charge as the N-acetyllysine (KAc ) modification but is more electrophilic. In this study we show that KMP -containing histone peptides are recognized by, and covalently modify bromodomain proteins that are KAc readers. Distinct selectivity preferences for covalent bromodomain modification are observed following incubation with KMP -containing peptides of different sequence. MS/MS analysis of 3 covalently modified bromodomain proteins confirmed that Cys adduction was selective. The modified Cys was not always proximal to the KAc binding site, indicating that KMP -containing peptide interaction with bromodomain protein is distinct from the former. Analysis of protein adduction yields as a function of bromodomain pH at which the protein charge is zero (pI) or cysteine solvent accessible surface area are also consistent with non-promiscuous interaction between the proteins and electrophilic peptides. These data suggest that intracellular formation of KMP could affect cellular function and viability by modifying proteins that regulate genetic expression.

Local Alteration of Ionic Strength in a Nucleosome Core Particle and Its Effect on N7-Methyl-2'-deoxyguanosine Depurination.

Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those imposed by post-translational modifications, impacts chromatin dynamics, protein binding, and the fate of DNA damage. There is evidence that N-terminal histone tails affect the local ionic environment within a nucleosome core particle (NCP), but this phenomenon is not well understood. Determining the modulation of the local ionic environment within an NCP by histone tails could help uncover the underlying mechanisms of their functions and effects. Utilizing bottom-up syntheses of NCPs containing wild-type or mutated histones and a fluorescent probe that is sensitive to the local ionic environment, we show that interaction with positively charged N-terminal tails increases the local ionic strength near nucleosomal DNA. The effect is diminished by replacing positively charged residues with neutral ones or deleting a tail in its entirety. Replacing the fluorescent probe with the major DNA methylation product, N7-methyl-2'-deoxyguanosine (MdG), revealed changes in the depurination rate constant varying inversely with local ionic strength. These data indicate that the MdG hydrolysis rates depend on and also inform on local ionic strength in an NCP. Overall, histone tail charge contributes to the complexity of the NCP structure and function by modulating the local ionic strength.

Structural Dynamics of a Common Mutagenic Oxidative DNA Lesion in Duplex DNA and during DNA Replication.

N6-(2-Deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido pyrimidine (Fapy•dG) is a prevalent form of genomic DNA damage. Fapy•dG is formed in greater amounts under anoxic conditions than the well-studied, chemically related 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo). Fapy•dG is more mutagenic in mammalian cells than 8-oxodGuo. A distinctive property of Fapy•dG is facile epimerization, but prior works with Fapy•dG analogues have precluded determining its effect on chemistry. We present crystallographic characterization of natural Fapy•dG in duplex DNA and as the template base for DNA polymerase ß (Pol ß). Fapy•dG adopts the ß-anomer when base paired with cytosine but exists as a mixture of α- and ß-anomers when promutagenically base paired with adenine. Rotation about the bond between the glycosidic nitrogen atom and the pyrimidine ring is also affected by the opposing nucleotide. Sodium cyanoborohydride soaking experiments trap the ring-opened Fapy•dG, demonstrating that ring opening and epimerization occur in the crystalline state. Ring opening and epimerization are facilitated by propitious water molecules that are observed in the structures. Determination of Fapy•dG mutagenicity in wild type and Pol ß knockdown HEK 293T cells indicates that Pol ß contributes to G → T transversions but also suppresses G → A transitions. Complementary kinetic studies have determined that Fapy•dG promotes mutagenesis by decreasing the catalytic efficiency of dCMP insertion opposite Fapy•dG, thus reducing polymerase fidelity. Kinetic studies have determined that dCMP incorporation opposite the ß-anomer is ∼90 times faster than the α-anomer. This research identifies the importance of anomer dynamics, a feature unique to formamidopyrimidines, when considering the incorporation of nucleotides opposite Fapy•dG and potentially the repair of this structurally unusual lesion.