PCAF-Mediated Histone Acetylation Promotes Replication Fork Degradation by MRE11 and EXO1 in BRCA-Deficient Cells.

Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.

Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity.

Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.

PCAF promotes R-loop resolution via histone acetylation.

R-loops cause genome instability, disrupting normal cellular functions. Histone acetylation, particularly by p300/CBP-associated factor (PCAF), is essential for maintaining genome stability and regulating cellular processes. Understanding how R-loop formation and resolution are regulated is important because dysregulation of these processes can lead to multiple diseases, including cancer. This study explores the role of PCAF in maintaining genome stability, specifically for R-loop resolution. We found that PCAF depletion promotes the generation of R-loop structures, especially during ongoing transcription, thereby compromising genome stability. Mechanistically, we found that PCAF facilitates histone H4K8 acetylation, leading to recruitment of the a double-strand break repair protein (MRE11) and exonuclease 1 (EXO1) to R-loop sites. These in turn recruit Fanconi anemia (FA) proteins, including FANCM and BLM, to resolve the R-loop structure. Our findings suggest that PCAF, histone acetylation, and FA proteins collaborate to resolve R-loops and ensure genome stability. This study therefore provides novel mechanistic insights into the dynamics of R-loops as well as the role of PCAF in preserving genome stability. These results may help develop therapeutic strategies to target diseases associated with genome instability.

R-loops are harmful DNA-RNA hybrid structures that cause genome instability, disrupting normal cell functions. This study explored the role of the protein PCAF in resolving R-loops to maintain genome stability. The researchers found that depleting PCAF leads to increased R-loop formation, especially during transcription, compromising the genome. Mechanistically, PCAF facilitates histone acetylation, recruiting proteins like MRE11, EXO1, FANCM and BLM to R-loop sites. These proteins collaborate to resolve R-loop structures. The findings suggest that PCAF, histone acetylation, and these repair proteins work together to untangle R-loops and preserve genome integrity. Understanding this process provides insights into R-loop dynamics and PCAF's role in genome maintenance, potentially leading to therapeutic strategies for diseases associated with genome instability, such as cancer.

USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing.

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.

Clinical and Mechanistic Implications of R-Loops in Human Leukemias.

Genetic mutations or environmental agents are major contributors to leukemia and are associated with genomic instability. R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and a non-template single-stranded DNA. These structures regulate various cellular processes, including transcription, replication, and DSB repair. However, unregulated R-loop formation can cause DNA damage and genomic instability, which are potential drivers of cancer including leukemia. In this review, we discuss the current understanding of aberrant R-loop formation and how it influences genomic instability and leukemia development. We also consider the possibility of R-loops as therapeutic targets for cancer treatment.

Preserving genome integrity and function: the DNA damage response and histone modifications.

Modulation of chromatin templates in response to cellular cues, including DNA damage, relies heavily on the post-translation modification of histones. Numerous types of histone modifications including phosphorylation, methylation, acetylation, and ubiquitylation occur on specific histone residues in response to DNA damage. These histone marks regulate both the structure and function of chromatin, allowing for the transition between chromatin states that function in undamaged condition to those that occur in the presence of DNA damage. Histone modifications play well-recognized roles in sensing, processing, and repairing damaged DNA to ensure the integrity of genetic information and cellular homeostasis. This review highlights our current understanding of histone modifications as they relate to DNA damage responses (DDRs) and their involvement in genome maintenance, including the potential targeting of histone modification regulators in cancer, a disease that exhibits both epigenetic dysregulation and intrinsic DNA damage.

Effects of the Trifluoromethyl Group on the Emission Efficiency of Red Phosphorescent Iridium(III) Complexes.

In this work, three novel phosphorescent iridium(III) complexes, namely (PT-TFP)2Ir(tmd), (PT-P)2Ir(tmd), and (MN-TFP)2Ir(tmd), were synthesized. All three complexes were phosphorescent red-emitting diode materials. The main ligands were synthesized by the Suzuki coupling reaction, and comprised an electron donor and an electron acceptor group. Subsequently, the iridium(III) complexes were synthesized by the Nonoyama reaction and their photochemical luminescence properties were investigated by ultraviolet-visible and photoluminescence spectroscopy. The manufactured devices were characterized by current density-voltage-luminance, power efficiency, external quantum efficiency, as well as their electroluminescence spectra. Finally, the effects of the trifluoromethyl group on the emission efficiency of the organic light-emitting diodes were investigated by comparing the energy levels and luminescence efficiency of the three iridium complexes.

Effects of the Methyl Group on the Emission Efficiency of the Red Phosphorescent Iridium(III) Complexes for OLEDs.

Novel red phosphorescent iridium(III) complexes, namely (MN-Q)2Ir(tmd), (MN-MQ)2Ir(tmd), (PT-P)2Ir(tmd) and (PT-MP)2Ir(tmd) were synthesized for the red phosphorescent organic lightemitting diodes (phOLEDs). The ligands have sites of both the electron donor and acceptor in a molecule. The main ligands were synthesized by the Suzuki coupling reaction, and comprised an electron donor and an electron acceptor group. Subsequently, the iridium(III) complexes were synthesized by the Nonoyama reaction and their photochemical luminescence properties were investigated by ultraviolet-visible and photoluminescence spectroscopy. The manufactured devices were characterized by current density-voltage-luminance, power efficiency, external quantum efficiency, as well as their electroluminescence spectra. Finally, the effects of the trifluoromethyl group on the emission efficiency of the organic light-emitting diodes were investigated by comparing the energy levels and luminescence efficiency of the three iridium complexes.

Persimmon leaf bio-waste for adsorptive removal of heavy metals from aqueous solution.

The aim of this study was to investigate heavy metal removal using waste biomass adsorbent, persimmon leaves, in an aqueous solution. Persimmon leaves, which are biomaterials, have a large number of hydroxyl groups and are highly suitable for removal of heavy metals. Therefore, in this study, we investigated the possibility of removal of Cu, Pb, and Cd in aqueous solution by using raw persimmon leaves (RPL) and dried persimmon leaves (DPL). Removal of heavy metals by RPL and DPL showed that DPL had a 10%-15% higher removal than RPL, and the order of removal efficiency was found to be Pb > Cu > Cd. The pseudo-second order model was a better fit to the heavy metal adsorption experiments using RPL and DPL than the pseudo-first order model. The adsorption of Cu, Pb, and Cd by DPL was more suitable with the Freundlich isothermal adsorption and showed an ion exchange reaction which occurred in the uneven adsorption surface layer. The maximum adsorption capacity of Cu, Pb, and Cd was determined to be 19.42 mg/g, 22.59 mg/g, and 18.26 mg/g, respectively. The result of the adsorption experiments showed that the n value was higher than 2 regardless of the dose, indicating that the heavy metal adsorption on DPL was easy. In the thermodynamic experiment, ΔG° was a negative value, and ΔH° and ΔS° were positive values. It can be seen that the heavy metal adsorption process using DPL was spontaneous in nature and was an endothermic process. Moreover, as the temperature increased, the adsorption increased, and the affinity of heavy metal adsorption to DPL was very good. This experiment, in which heavy metals are removed using the waste biomass of persimmon leaves is an eco-friendly new bioadsorbent method because it can remove heavy metals without using chemicals while utilizing waste recycling.

A Comparison Between the Conventional and the Laryngoscope-Assisted Lightwand Intubation Techniques in Patients With Cervical Immobilization: A Prospective Randomized Study.

BACKGROUND: Positioning of a lightwand in the midline of the oral cavity can be challenging in patients with cervical immobilization. Direct laryngoscopy may permit the lightwand tip to more easily access the glottic opening. We tested our hypothesis that a laryngoscope-assisted lightwand technique allows more successful endotracheal intubation than does a conventional lightwand approach. METHODS: A total of 162 patients requiring cervical immobilization during intubation for cervical spine surgery were allocated randomly to 2 groups. The conventional lightwand technique (group C, n = 80) or the laryngoscope-assisted lightwand technique (group L, n = 82) was used for endotracheal intubation. In the group L, a Macintosh laryngoscope was inserted into the oral cavity, advanced until the epiglottis tip was visible, but not used to lift the epiglottis tip. The lightwand tip was placed below the epiglottis under direct view of the epiglottis tip. The primary outcome (the initial intubation success rate) and secondary outcomes (intubation time, hemodynamic changes, and postoperative airway complications) were evaluated. RESULTS: The initial intubation success rate was significantly lower (75% vs 89%; relative risk [95% confidence interval]: 1.2 [1.0-1.4]; P = .034) in group C than group L. The intubation time (22 ± 13 vs 24 ± 12 seconds; mean difference [98.33% confidence interval]: 2.4 [-2.3 to 7.2]; P = .217) did not differ between groups. Postoperative sore throat score, incidences of hypertension and tachycardia, postoperative oral mucosal bleeding, and hoarseness also did not differ between groups. CONCLUSIONS: Laryngoscope-assisted lightwand intubation did not increase intubation time, and it increased first attempt intubation rates compared with traditional lightwand intubation in patients requiring cervical immobilization for cervical spine surgery.

Single-Cell Analysis of Histone Acetylation Dynamics at Replication Forks Using PLA and SIRF.

Genome integrity is constantly challenged by various processes including DNA damage, structured DNA, transcription, and DNA-protein crosslinks. During DNA replication, active replication forks that encounter these obstacles can result in their stalling and collapse. Accurate DNA replication requires the ability of forks to navigate these threats, which is aided by DNA repair proteins. Histone acetylation participates in this process through an ability to signal and recruit proteins to regions of replicating DNA. For example, the histone acetyltransferase PCAF promotes the recruitment of the DNA repair factors MRE11 and EXO1 to stalled forks by acetylating histone H4 at lysine 8 (H4K8ac). These highly dynamic processes can be detected and analyzed using a modified proximity ligation assay (PLA) method, known as SIRF (in situ protein interactions with nascent DNA replication forks). This single-cell assay combines PLA with EdU-coupled Click-iT chemistry reactions and fluorescence microscopy to detect these interactions at sites of replicating DNA. Here we provide a detailed protocol utilizing SIRF that detects the HAT PCAF and histone acetylation at replication forks. This technique provides a robust methodology to determine protein recruitment and modifications at the replication fork with single-cell resolution.

APOBEC3B regulates R-loops and promotes transcription-associated mutagenesis in cancer.

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.

Bromodomain proteins: protectors against endogenous DNA damage and facilitators of genome integrity.

Endogenous DNA damage is a major contributor to mutations, which are drivers of cancer development. Bromodomain (BRD) proteins are well-established participants in chromatin-based DNA damage response (DDR) pathways, which maintain genome integrity from cell-intrinsic and extrinsic DNA-damaging sources. BRD proteins are most well-studied as regulators of transcription, but emerging evidence has revealed their importance in other DNA-templated processes, including DNA repair and replication. How BRD proteins mechanistically protect cells from endogenous DNA damage through their participation in these pathways remains an active area of investigation. Here, we review several recent studies establishing BRD proteins as key influencers of endogenous DNA damage, including DNA-RNA hybrid (R-loops) formation during transcription and participation in replication stress responses. As endogenous DNA damage is known to contribute to several human diseases, including neurodegeneration, immunodeficiencies, cancer, and aging, the ability of BRD proteins to suppress DNA damage and mutations is likely to provide new insights into the involvement of BRD proteins in these diseases. Although many studies have focused on BRD proteins in transcription, evidence indicates that BRD proteins have emergent functions in DNA repair and genome stability and are participants in the etiology and treatment of diseases involving endogenous DNA damage.

Emerging roles of RNA modifications in genome integrity.

Post-translational modifications of proteins are well-established participants in DNA damage response (DDR) pathways, which function in the maintenance of genome integrity. Emerging evidence is starting to reveal the involvement of modifications on RNA in the DDR. RNA modifications are known regulators of gene expression but how and if they participate in DNA repair and genome maintenance has been poorly understood. Here, we review several studies that have now established RNA modifications as key components of DNA damage responses. RNA modifying enzymes and the binding proteins that recognize these modifications localize to and participate in the repair of UV-induced and DNA double-strand break lesions. RNA modifications have a profound effect on DNA-RNA hybrids (R-loops) at DNA damage sites, a structure known to be involved in DNA repair and genome stability. Given the importance of the DDR in suppressing mutations and human diseases such as neurodegeneration, immunodeficiencies, cancer and aging, RNA modification pathways may be involved in human diseases not solely through their roles in gene expression but also by their ability to impact DNA repair and genome stability.

Use of hybrid microcapsules, chitosan-methyl esterified sericite-tannin, for the removal of harmful lake algae and nutrient.

This paper outlines the development of a novel, low-cost, hybrid material from chitosan-methyl esterified sericite-tannin. The adsorbent material is then successfully utilized for the efficient removal of lake nutrients and harmful algae. In a FT-IR analysis, peaks related to -OH stretching, carbonyl and carboxylic groups, and CH stretching were newly created or expanded, and microcapsules were found to facilitate the removal of harmful algae and nutrients. The hybrid microcapsules obtained high removal efficiencies of 98% TN, 98% TP, and 99% Chl-a from the lake water by a quantity of hybrid microcapsules of 1 g/L, pH (7-8), and 30 min contact time at (25-30)°C. In addition, the experimental data were applied to various harmful algae growth models and were most suitable for the Heldane model. Based on the above results, microcapsules can be applied in the field, and can be expected to rapidly remove nutrients and harmful algae.

A Novel Reciprocal Crosstalk between RNF168 and PARP1 to Regulate DNA Repair Processes.

Emerging evidence has suggested that cellular crosstalk between RNF168 and poly(ADP-ribose) polymerase 1 (PARP1) contributes to the precise control of the DNA damage response (DDR). However, the direct and reciprocal functional link between them remains unclear. In this report, we identified that RNF168 ubiquitinates PARP1 via direct interaction and accelerates PARP1 degradation in the presence of poly (ADP-ribose) (PAR) chains, metabolites of activated PARP1. Through mass spectrometric analysis, we revealed that RNF168 ubiquitinated multiple lysine residues on PARP1 via K48-linked ubiquitin chain formation. Consistent with this, micro-irradiation-induced PARP1 accumulation at damaged chromatin was significantly increased by knockdown of endogenous RNF168. In addition, it was confirmed that abnormal changes of HR and HNEJ due to knockdown of RNF168 were restored by overexpression of WT RNF168 but not by reintroduction of mutants lacking E3 ligase activity or PAR binding ability. The comet assay also revealed that both PAR-binding and ubiquitin-conjugation activities are indispensable for the RNF168-mediated DNA repair process. Taken together, our results suggest that RNF168 acts as a counterpart of PARP1 in DDR and regulates the HR/NHEJ repair processes through the ubiquitination of PARP1.

Identification of the Thioredoxin-Like 2 Autoantibody as a Specific Biomarker for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC. To address this point, we utilized a human protein microarray system to identify serum autoantibodies that showed different expression patterns between TNBC and normal serum samples, and identified five autoantibodies showing TNBC-specific expression. Among them, we selected the thioredoxin-like 2 (TXNL2) autoantibody and evaluated its diagnostic relevance by dot blot analysis with the recombinant TXNL2 protein. We demonstrated that the TXNL2 autoantibody showed 2- to 6-fold higher expression in TNBC samples than in normal samples suggesting that serum TXNL2 autoantibodies are potential biomarkers for TNBC.

Effect of Prewarming during Induction of Anesthesia on Microvascular Reactivity in Patients Undergoing Off-Pump Coronary Artery Bypass Surgery: A Randomized Clinical Trial.

BACKGROUND: General anesthesia may induce inadvertent hypothermia and this may be related to perioperative cardiovascular complications. Microvascular reactivity, measured by the recovery slope during a vascular occlusion test, is decreased during surgery and is also related to postoperative clinical outcomes. We hypothesized that microvascular changes during surgery may be related to intraoperative hypothermia. To evaluate this, we conducted a randomized study in patients undergoing off-pump coronary artery bypass surgery, in which the effect of prewarming on microvascular reactivity was evaluated. METHODS: Patients scheduled for off-pump coronary artery bypass surgery were screened. Enrolled patients were randomized to the prewarming group to receive forced-air warming during induction of anesthesia or to the control group. Measurement of core and skin temperatures and vascular occlusion test were conducted before anesthesia induction, 1, 2, and 3 h after induction, and at the end of surgery. RESULTS: In total, 40 patients were enrolled and finished the study (n = 20 in the prewarming group and n = 20 in the control group). During the first 3 h of anesthesia, core temperature was higher in the prewarming group than the control group (p < 0.001). The number of patients developing hypothermia was lower in the prewarming group than the control group (4/20 vs. 13/20, p = 0.004). However, tissue oxygen saturation and changes in recovery slope following a vascular occlusion test at 3 h after anesthesia induction did not differ between the groups. There was no difference in clinical outcome, including perioperative transfusion, wound infection, or hospital stay, between the groups. CONCLUSIONS: Prewarming during induction of anesthesia decreased intraoperative hypothermia, but did not reduce the deterioration in microvascular reactivity in patients undergoing off-pump coronary artery bypass surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT02186210.

Effective therapeutic approach for head and neck cancer by an engineered minibody targeting the EGFR receptor.

Cetuximab, a chimeric monoclonal antibody developed for targeting the Epidermal Growth Factor Receptor (EGFR), has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG) antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody) that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (VH) and light (VL) region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purified MI061 significantly interferes ligand binding to EGFR and blocks EGFR's phosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. These results indicated that engineered MI061 retains both binding specificity and affinity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly significant anti-tumor effect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more effective and promising agent for in vivo targeting of solid tumors.