DNA Cleavage Research Articles

Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET.

CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.

Synthesis, crystal structure, magnetic studies, DNA binding, molecular docking studies, cytotoxicity and luminescence properties of a new coordination polymer copper(II) complex with 4,4′-trimethylenedipyridine and 1,10-phenanthroline

In the present work, a novel coordination polymer {[Cu(phen)(Tmdipy)(NO3)H2O](NO3) 2.5(H2O)}n (phen = 1,10-phenanthroline, Tmdipy = 4,4′- Trimethylenedipyridine), has been prepared and structurally determined by elemental analysis, IR, EPR, UV–visible and single-crystal X-ray crystallography. The complex crystallized in triclinic space group P -1, and the copper(II) shows a distorted octahedral geometry, with Jahn-Teller distortion occurring in the (4 + 2) CuN4O2 coordination sphere. The crystal structure evidences that the CP1 is formed of dinuclear [Cu(phen)(Tmdipy)(NO3)(H2O)]2 units weakly connected by nitrate anions giving rise to 1D polymeric chain structure. EPR measurement confirms about the distorted octahedral geometry of the complex. By using UV–Vis absorption, fluorescence spectroscopy, and cyclic voltammetric techniques, the binding studies of copper(II) complex with calf thymus DNA (CT-DNA) were investigated. Additionally, the gel electrophoresis method was used to examine the cleavage of pBR322 DNA by copper(II) complex and exhibited potent cytotoxic effects against human cell line (HepG2). Finally, magnetic properties, molecular docking studies and photoluminescence characteristics have been evaluated.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Cas1 mediates the interference stage in a phage-encoded CRISPR-Cas system.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are prokaryotic adaptive immune systems against invading phages and other mobile genetic elements. Notably, some phages, including the Vibrio cholerae-infecting ICP1 (International Center for Diarrheal Disease Research, Bangladesh cholera phage 1), harbor CRISPR-Cas systems to counteract host defenses. Nevertheless, ICP1 Cas8f lacks the helical bundle domain essential for recruitment of helicase-nuclease Cas2/3 during target DNA cleavage and how this system accomplishes the interference stage remains unknown. Here, we found that Cas1, a highly conserved component known to exclusively work in the adaptation stage, also mediates the interference stage through connecting Cas2/3 to the DNA-bound CRISPR-associated complex for antiviral defense (Cascade; CRISPR system yersinia, Csy) of the ICP1 CRISPR-Cas system. A series of structures of Csy, Csy-dsDNA (double-stranded DNA), Cas1-Cas2/3 and Csy-dsDNA-Cas1-Cas2/3 complexes reveal the whole process of Cas1-mediated target DNA cleavage by the ICP1 CRISPR-Cas system. Together, these data support an unprecedented model in which Cas1 mediates the interference stage in a phage-encoded CRISPR-Cas system and the study also sheds light on a unique model of primed adaptation.

Trimethyltin(IV) (p-fluoro-/p‑hydroxy-/p-nitro-)benzoates and their benzimidazole complexes: X-ray structures of [Me3Sn(p-NO2C6H4CO2)]n, and [Me3Sn(p-NO2C6H4CO2). bz], DFT calculations, DNA binding, and DNA cleavage studies

A series of trimethyltin(IV) carboxylates with Sn‒O bonds: [Me3Sn(p-FC6H4CO2)] (1), [Me3Sn(p-HOC6H4CO2)] (2), [Me3Sn(p-NO2C6H4CO2)]n (3), and their benzimidazole complexes with Sn‒N bond: [Me3Sn(p-FC6H4CO2).bz] (4), [Me3Sn(p-HOC6H4CO2).bz] (5), and [Me3Sn(p-NO2C6H4CO2).bz] (6) (bz = benzimidazole) have been synthesized and characterized by elemental analysis, FT-IR, 1H, 13C, 119Sn NMR, and electrospray ionization mass spectrometric (ESI-MS) studies. The crystal structures for (3) and (6) have been authenticated by X-ray diffraction. In the crystal structure, polymeric coordination compound (3) and the monocrystal system (6) have trigonal bipyramidal geometry around the Sn centre, where most electronegative atoms occupied the axial positions, and three methyl groups occupied the equatorial positions. The gas-phase tetrahedral geometries of (1) and (2), asymmetric unit (Sc-XRD) of (3), and trigonal bipyramidal geometries of (4–6) have been optimized using the B3LYP function and LanL2Z basis, and obtained ∠C-Sn-C bond angles agree with those obtained from NMR/X-ray studies. The interaction of (1–6) with calf thymus DNA (ctDNA) has been investigated by spectroscopic titrations such as UV–visible, fluorescence, and circular dichroism (CD) titrations, and calculated binding constant values found in the 104–105 M−1 range which suggest a groove binding or electrostatic interaction between complexes and ctDNA. The gel electrophoresis studies suggest that (1–6) effectively cleaved the plasmid pBR322 DNA into three bands: the supercoiled, nicked/circular, and linear structures. The intriguing biophysical findings from the study indicate that these complexes have properties that make them strong contenders for additional research as potential anti-cancer agents.

Evaluation of doxorubicin and β-lapachone analogs as anticancer agents, a biological and computational study.

We have conducted an experimental and computational evaluation of new doxorubicin (4a-c) and β-lapachone (5a-c) analogs. These novel anticancer analogs were previously synthesized, but had not been tested or characterized until now. We have evaluated their antiproliferative and DNA cleavage inhibition properties using breast (MCF-7 and MDA-MB-231) and prostate (PC3) cancer cell lines. Additionally, cell cycle analysis was performed using flow cytometry. Computational studies, including molecular docking, pharmacokinetic properties, and an analysis of DFT and QTAIM chemical descriptors, were performed to gain insights into the electronic structure and elucidate the molecular binding of the new β-lapachone and doxorubicin analogs with a DNA sequence and Topoisomerase II (Topo II)α. Our results show that 4a analog displays the highest antiproliferative activity in cancer cell lines by inducing cell death. We observed that stacking interactions and hydrogen bonding are essential to stabilize the molecule-DNA-Topo IIα complex. Moreover, 4a and 5a analogs inhibited Topo's DNA cleavage activity. Pharmacodynamic results indicated that studied molecules have favorable adsorption and permeability properties. The calculated chemical descriptors indicate that electron accumulation in quinone rings is relevant to the reactivity and biological activity. Based on our results, 4a is a strong candidate for becoming an anticancer drug.

Bioactive benzhydrazone derived Schiff base and its metal(II) complexes: Synthesis, characterisation, cytotoxicity, DNA binding/cleavage, antimicrobial and antioxidant activity

Bivalent mononuclear transition metal complexes of cobalt, nickel, copper and zinc were synthesized from (E)-2-(1-(2-(2-hydroxybenzoyl)hydrazono)ethoxy)benzoic acid. The FTIR, 1H NMR, 13C NMR, HR-Mass, UV–Vis., EPR, Powder XRD and SEM analysis were employed to investigate the structural features of synthesized compounds. The spectral data confirmed the square planar geometry of the complexes. The EPR spectra of the Cu(II) complex supported the proposed geometry. Powder X-ray diffraction (PXRD) analysis and scanning electron microscopy (SEM) imaging revealed that the complexes consist of nano-sized grains exhibiting a polycrystalline structure. The in vitro antimicrobial efficacy of the compounds was evaluated against various bacterial and fungal strains using the well diffusion method. The in vitro antibacterial activity demonstrated that all synthesized compounds were biologically active, and Zn(II) complex exhibited the highest inhibitory activity against S. aureus (gram-positive bacteria). For antioxidant activity, the free ligand and its metal chelates were evaluated using the standard DPPH method, revealing that the Co(II) complex displayed least inhibitory concentration value (IC50 = 16 ± 2.3 μg/mL) indicating the highest antioxidant activity. DNA interaction studies showed intercalation between complexes and DNA, and the Cu(II) complex exhibited a higher binding affinity of 2.78 ± 0.34 × 105 M−1. The DNA cleavage ability of the compounds was assessed using gel electrophoresis, demonstrating the effective cleavage of the pBR322 plasmid DNA. The Schiff base and its complexes were subjected to an anticancer evaluation against the human breast cancer cell line (MCF-7) using the MTT assay method. The percentage cell inhibition of the compounds was calculated.

SYNTHESIS, SPECTRAL STUDIES AND EVALUATION OF DNA BINDING, PHOTOCLEAVAGE, ANTICANCER ACTIVITY OF RU(II) POLYPYRIDYL COMPLEXES OF 2-(3,4,5-TRIMETHOXY-PHENYL)-H-IMIDAZOLE[4,5-F][1,10]PHENANTHROLINE

The two novel Ru(II) polypyridyl complexes of the type [Ru(NN)2L](ClO4)2.2H2O,where NN is dmp=4,4-dimethyl-1,10 ortho phenanthroline and dmb=4,4-dimethyl 2,2-bipyridine and L is Intercalator ligand, TMIP=2-(3,4,5-Trimethoxy-phenyl)-H-imidazole[4,5-f][1,10]phenanthroline were synthesized and characterized by different spectroscopic techniques. The binding ability of Ru (II) complexes with CT-DNA was investigated by Biophysical studies – UV-Visible absorption studies, Fluorescence studies (Emission & Quenching) and Viscosity studies. The binding constants of these complexes from absorption studies are 1.2x105 M-1and 8x104M-1respectively.These values are less as compared to the existing TMIP complex [Ru(phen)2TMPIP]+2, indicative of the effect of substitution over the ancillary ligand which reduce the binding capacity of metal complex to CT-DNA. The results from the viscosity studies suggest an intercalative binding mechanism. The DNA cleavage studies show that the Ru (II) complexes successfully cleaved the pBR322 DNA. Both the complexes exhibit not notable anti cancer acivity.

Implications of Genetic Factors Underlying Mouse Hydronephrosis: Cautionary Considerations on Phenotypic Interpretation in Genetically Engineered Mice.

Hydronephrosis, the dilation of kidneys due to abnormal urine retention, occurs spontaneously in certain inbred mouse strains. In humans, its occurrence is often attributed to acquired urinary tract obstructions in adults, whereas in children, it can be congenital. However, the genetic factors underlying hydronephrosis pathogenesis remain unclear. We investigated the cause of hydronephrosis by analyzing tetraspanin 7 (Tspan7) gene-modified mice, which had shown a high incidence of hydronephrosis-like symptoms. We found that these mice were characterized by low liver weights relative to kidney weights and elevated blood ammonia levels, suggesting liver involvement in hydronephrosis. Gene expression analysis of the liver suggested that dysfunction of ornithine transcarbamylase (OTC), encoded by the X chromosome gene Otc and involved in the urea cycle, may contribute as a congenital factor in hydronephrosis. This OTC dysfunction may be caused by genomic mutations in X chromosome genes contiguous to Otc, such as Tspan7, or via the genomic manipulations used to generate transgenic mice, including the introduction of Cre recombinase DNA cassettes and cleavage of loxP by Cre recombinase. Therefore, caution should be exercised in interpreting the hydronephrosis phenotype observed in transgenic mice as solely a physiological function of the target gene.

Antibacterial, Antidiabetic, Antioxidant and DNA cleavage studies of novel N(3-(bis(pyridine-2ylmethyl)amino)-2-hydroxypropyl) benzene sulphonamide ligand

The novel N (3-(bis(pyridine-2ylmethyl)amino)-2-hydroxypropyl) benzene sulphonamide ligand have been synthesized from 1,3-Diamino-propan-2-ol. The structure of the ligand was confirmed by Mass spectroscopy and 1H NMR spectroscopy. The synthesized Schiff base ligand was tested for their in vitro anti bacterial action with Gram-positive and negative bacteria. The anti-diabetic activities have been tested with α-amylase inhibition method and antioxidant effect of the compound was established using DPPH method. Thus all the studies reveals that the synthesised Schiff base compound has significant antibacterial, antidiabetic and antioxidant activities. The DNA cleavage studies shown that, the DNA binding nature of the ligand was appreciably good. The Molecular docking characteristics were supported that the binding approach of the effective inhibitors with the active site of enzymes.

The positional and numerical effect of N 6-methyladenosine in tracrRNA on the DNA cleavage activity of Cas9.

Post-transcriptional modifications on the guide RNAs utilized in the Cas9 system may have the potential to impact the activity of Cas9. In this study, we synthesized a series of tracrRNAs containing N 6-methyadenosine (m6A), a prevalent post-transcriptional modification, at various positions. We evaluated the effect of these modifications on the DNA cleavage activity of Cas9. Our results show that multiple m6As in the anti-repeat region of tracrRNA reduce the DNA cleavage activity of Cas9. This suggests that the m6A-modified tracrRNA can be used for Cas9 only when the number and the position of the modified residue are properly chosen in tracrRNA.

Placental apoptosis increased by hypoxia inducible factor-1 stabilization is counteracted by leptin†.

During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed toward the relevance of hypoxia as modulator of trophoblast cell death. Previous reports have shown that leptin, a placental cytokine, promotes cell survival in both cell culture and placental explant models. The aim of this work is to establish the role of leptin in apoptosis under hypoxic condition in trophoblast cells. In this study, we evaluated the effect of cobalt chloride, a hypoxia mimicking agent that stabilizes the expression of hypoxia-inducible factor-1 alpha, on Swan-71 and human placental explants. Hypoxia chamber was also used to generate 2% oxygen. Apoptosis was determined by the presence of apoptotic nucleus, fragmentation of DNA and Caspase-3 and PARP-1 cleavage. The pro-apoptotic proteins BAX, BID, BAD, and BAK and the anti-apoptotic effectors BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1 were also analyzed. We found that hypoxia-inducible factor-1 alpha stabilization increased the appearance of apoptotic nucleus, fragmentation of DNA, and Caspase-3 and PARP-1 cleavage. Hypoxia mimicking conditions enhanced the expression of pro-apoptotic effectors BAX, BID, BAD, and BAK. Hypoxia-inducible factor-1 alpha stabilization also downregulated the level of BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1. All these apoptotic parameters changes were reversed with leptin treatment. Moreover, we showed that leptin action on apoptosis modulation involves PI3K and MAPK signaling pathways. Obtained data demonstrate that hypoxia-inducible factor-1 alpha stabilization induces apoptosis in human placenta and leptin counteracts this effect, reinforcing its role as a survival cytokine.

TnpB homologues exapted from transposons are RNA-guided transcription factors.

Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Casadaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.

A programmable sensitive platform for pathogen detection based on CRISPR/Cas12a -hybridization chain reaction-poly T-Cu

Rapid and sensitive detection of pathogenic bacteria is crucial for disease prevention and control. The CRISPR/Cas12a system with the DNA cleavage capability holds promise in pathogenic bacteria diagnosis. However, the sensitivity of CRISPR-based assays remains a challenge. Herein, we report a versatile and sensitive pathogen sensing platform (HTCas12a) based on the CRISPR/Cas12a system, hybridization chain reaction (HCR) and Poly T-copper fluorescence nanoprobe. The sensitivity is improved by HCR and the Poly-T-Cu reporter probe reduces the overall experiment cost to less than one dollar per sample. Our results demonstrate the specific recognition of target nucleic acid fragments from other pathogens. Furthermore, a good linear correlation between fluorescence intensity and target quantities were achieved with detection limits of 23.36 fM for Target DNA and 4.17 CFU/mL for S.aureus, respectively. The HTCas12a system offers a universal platform for pathogen detection in various fields, including environmental monitoring, clinical diagnosis, and food safety.

Elucidating the Interaction of Indole-3-Propionic Acid and Calf Thymus DNA: Multispectroscopic and Computational Modeling Approaches.

Indole-3-propionic acid (IPA) is a plant growth regulator with good specificity and long action. IPA may be harmful to human health because of its accumulation in vegetables and fruits. Therefore, in this study, the properties of the interaction between calf thymus DNA (ctDNA) and IPA were systematically explored using multispectroscopic and computational modeling approaches. Analysis of fluorescence spectra showed that IPA binding to ctDNA to spontaneously form a complex was mainly driven by hydrogen bonds and hydrophobic interaction. DNA melting analysis, viscosity analysis, DNA cleavage study, and circular dichroism measurement revealed the groove binding of IPA to ctDNA and showed that the binding did not significantly change ctDNA confirmation. Furthermore, molecular docking found that IPA attached in the A-T rich minor groove region of the DNA. Molecular dynamics simulation showed that DNA and IPA formed a stable complex and IPA caused slight fluctuations for the residues at the binding site. Gel electrophoresis experiments showed that IPA did not significantly disrupt the DNA structure. These findings may provide useful information on the potential toxicological effects and environmental risk assessments of IPA residue in food at the molecular level.

Novel bodipy-calix[4]arene fluorophores: DNA interaction, spectroscopic properties, electrophorese and molecular docking

This work includes the synthesis of some BODIPY-derived heterocyclic Calix[4]arene compounds and the investigation of their effects on DNA. Among the calixarene-BODIPY-based target compounds, compounds 3 and 9 were newly synthesized, and compound 7 was synthesized as given in the literature. The structures of all synthesized compounds were determined by elemental analysis and spectroscopic techniques (13C NMR, 1H NMR, FT-IR). The effects of the synthesized Calixarene/BODIPY compounds (3, 7, and 9) against ct-DNA were examined via UV–Vis spectrometric and fluorimetric methods. The obtained results indicated that compounds 3, 7, and 9 bind to DNA, and the calculated binding constants (Kb) were found 1.62 × 103M−1, 1.54 × 103M−1, and 1.14 × 102M−1 respectively. Also, the DNA cleavage activity was examined against pBR322 plasmid DNA. The results showed that all compounds (3, 7, and 9) had a quite high effect on pBR322 plasmid DNA. Molecular docking studies were also performed to gain insight into the binding affinities and key interactions of DNA-synthesized compounds. The strong binding of compounds to the DNA can be attributed to high negative values of binding free energy between -20.2 and -17.4 kcal mol−1. These results gave the idea that these molecules are candidate molecules, especially for anti-bacterial and anticancer studies.

Identification of Family-Specific Features in Cas9 and Cas12 Proteins: A Machine Learning Approach Using Complete Protein Feature Spectrum.

The recent development of CRISPR-Cas technology holds promise to correct gene-level defects for genetic diseases. The key element of the CRISPR-Cas system is the Cas protein, a nuclease that can edit the gene of interest assisted by guide RNA. However, these Cas proteins suffer from inherent limitations such as large size, low cleavage efficiency, and off-target effects, hindering their widespread application as a gene editing tool. Therefore, there is a need to identify novel Cas proteins with improved editing properties, for which it is necessary to understand the underlying features governing the Cas families. In this study, we aim to elucidate the unique protein features associated with Cas9 and Cas12 families and identify the features distinguishing each family from non-Cas proteins. Here, we built Random Forest (RF) binary classifiers to distinguish Cas12 and Cas9 proteins from non-Cas proteins, respectively, using the complete protein feature spectrum (13,494 features) encoding various physiochemical, topological, constitutional, and coevolutionary information on Cas proteins. Furthermore, we built multiclass RF classifiers differentiating Cas9, Cas12, and non-Cas proteins. All the models were evaluated rigorously on the test and independent data sets. The Cas12 and Cas9 binary models achieved a high overall accuracy of 92% and 95% on their respective independent data sets, while the multiclass classifier achieved an F1 score of close to 0.98. We observed that Quasi-Sequence-Order (QSO) descriptors like Schneider.lag and Composition descriptors like charge, volume, and polarizability are predominant in the Cas12 family. Conversely Amino Acid Composition descriptors, especially Tripeptide Composition (TPC), predominate the Cas9 family. Four of the top 10 descriptors identified in Cas9 classification are tripeptides PWN, PYY, HHA, and DHI, which are seen to be conserved across all Cas9 proteins and located within different catalytically important domains of the Streptococcus pyogenes Cas9 (SpCas9) structure. Among these, DHI and HHA are well-known to be involved in the DNA cleavage activity of the SpCas9 protein. Mutation studies have highlighted the significance of the PWN tripeptide in PAM recognition and DNA cleavage activity of SpCas9, while Y450 from the PYY tripeptide plays a crucial role in reducing off-target effects and improving the specificity in SpCas9. Leveraging our machine learning (ML) pipeline, we identified numerous Cas9 and Cas12 family-specific features. These features offer valuable insights for future experimental and computational studies aiming at designing Cas systems with enhanced gene-editing properties. These features suggest plausible structural modifications that can effectively guide the development of Cas proteins with improved editing capabilities.

Rosmarinic acid, a natural polyphenol, has a potential pro-oxidant risk via NADH-mediated oxidative DNA damage

BackgroundRosmarinic acid (RA) has a wide range of beneficial effects on human health. On the other hand, RA has been reported to induce metal-mediated reactive oxygen species (ROS) generation and DNA damage. However, its mechanism remains unknown. In this study, to clarify the underlying mechanism, we analyzed metal-mediated DNA damage in isolated DNA treated with RA and its analog isorinic acid.ResultsRA plus Cu(II), but not Fe(III), significantly increased 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage, in calf thymus DNA. Furthermore, a comparison of the 8-oxodG formation induced by RA and its analog isorinic acid suggested that the catechol groups in RA could be associated with their abilities to form 8-oxodG. Interestingly, the 8-oxodG formation induced by RA and isorinic acid plus Cu(II) was markedly enhanced by the addition of NADH, an endogenous reductant. To elucidate the mechanism of RA plus Cu(II)-induced oxidative DNA damage, we examined DNA damage in 32P-labeled DNA treated with RA in the presence of Cu(II). RA plus Cu(II) caused DNA cleavage, which was enhanced by piperidine treatment, suggesting that RA causes not only DNA strand breakage but also base modification. RA plus Cu(II)-induced DNA damage was inhibited by catalase (H2O2 scavenger), bathocuproine (Cu(I) chelator), and methional (scavenger of a variety of ROS other than •OH) but not by typical •OH scavengers and SOD, indicating the involvement of H2O2, Cu(I), and ROS other than •OH. DNA cleavage site analysis showing RA-induced site-specific DNA damage (frequently at thymine and some cytosine residues) supports the involvement of ROS other than •OH, because •OH causes DNA cleavage without site specificity. Based on these results, Cu(I) and H2O2 generation with concomitant RA autoxidation could lead to the production of Cu(I)-hydroperoxide, which induces oxidative DNA damage. o-Quinone and o-semiquinone radicals are likely to be again reduced to RA by NADH, which dramatically increases oxidative DNA damage, particularly at low concentrations of RA.ConclusionsIn this study, physiologically relevant concentrations of RA effectively induced oxidative DNA damage in isolated DNA through redox cycle reactions with copper and NADH.

Bithiophene‐Functionalized Infrared Two‐Photon Absorption Metal Complexes as Single‐Molecule Platforms for Synergistic Photodynamic, Photothermal, and Chemotherapy

AbstractA planar conjugated ligand functionalized with bithiophene and its Ru(II), Os(II), and Ir(III) complexes have been constructed as single‐molecule platform for synergistic photodynamic, photothermal, and chemotherapy. The complexes have significant two‐photon absorption at 808 nm and remarkable singlet oxygen and superoxide anion production in aqueous solution and cells when exposed to 808 nm infrared irradiation. The most potent Ru(II) complex Ru7 enters tumor cells via the rare macropinocytosis, locates in both nuclei and mitochondria, and regulates DNA‐related chemotherapeutic mechanisms intranuclearly including DNA topoisomerase and RNA polymerase inhibition and their synergistic effects with photoactivated apoptosis, ferroptosis and DNA cleavage. Ru7 exhibits high efficacy in vivo for malignant melanoma and cisplatin‐resistant non‐small cell lung cancer tumors, with a 100 % survival rate of mice, low toxicity to normal cells and low residual rate. Such an infrared two‐photon activatable metal complex may contribute to a new generation of single‐molecule‐based integrated diagnosis and treatment platform to address drug resistance in clinical practice and phototherapy for large, deeply located solid tumors.

Toll-Like Receptor Activation and B Cell Maturation Via MyD88-Dependent Pathway Under Hyperglycemia Condition

hyperglycemia causes a degenerative syndrome that involves an inflammatory process with an increase in certain proinflammatory cytokines and chemokines which in the process will activate B cells to produce immunoglobulins through several mechanisms. One of the interesting mechanisms is the mechanism via the MyD88 pathway. Objectives: to explore the role of MyD88 adapter protein in Toll-like receptor activation and B cell maturation under hyperglycemia conditions. Methods: a literature review was done to answer the study objectives. Results: Class switching process under hyperglycemia conditions involves activation of NFkB through the inflammatory MyD88-dependent pathway to trigger the expression of TLR and B cell maturation and proliferation as well as antibody production. The MyD88 adapter protein is a protein formed by stimulation of pro-inflammatory cytokines IL-6 and plays a role in the continuation of signals from the TLR and IL-1 pathways. Mature B cell stimulation induces 2 genetic changes in the Ig gene locus, called somatic hypermutation (SHM) and class switch recombination (CSR) to produce antibodies. Changes in immunoglobulin genes occur related to changes in certain DNA segments at the locus of genes where CSR occurs, this gene change requires the role of AID (activated-induced cytidine deaminase) in DNA cleavage. AID in mature B cells is activated by proinflammatory cytokines via induction of NfKB activation via the inflammatory MyD88-dependent pathway. Conclusion: Toll-like receptor activation plays a crucial role in B cell maturation activated by pro-inflammatory cytokine via MyD88 dependent-NFkB activation.