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ChemMedChem Sep 2014The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject.... (Review)
Review
The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject. Herein, we report and discuss the formation of 5-methylcytosine-osmium complexes that are used as the basis for a bisulfite-free chemical assay for DNA methylation analysis. Osmium in the oxidized state reacts with C5-methylated pyrimidines in the presence of a bipyridine ligand to give a stable ternary complex. On the basis of this reaction, an adenine derivative with a tethered bipyridine moiety has been designed for sequence-specific osmium complex formation. Osmium complexation is then achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and results in the formation of a cross-linked structure. This novel concept of methylation-specific reaction, based on a straightforward chemical process, expands the range of methods available for the analysis of epigenetic modifications. Advantages of the described method include amplification-insensitive detection, 5-hydroxymethylcytosine complexation, and visualization through methylation-specific in situ hybridization.
Topics: Animals; DNA; DNA Methylation; Epigenesis, Genetic; Epigenomics; Humans; Osmium Compounds
PubMed: 24989699
DOI: 10.1002/cmdc.201402114 -
Methods in Molecular Biology (Clifton,... 2017Enzymatic electrodes are becoming increasingly common for energy production and sensing applications. Research over the past several decades has addressed a major issue...
Enzymatic electrodes are becoming increasingly common for energy production and sensing applications. Research over the past several decades has addressed a major issue that can occur when using these biocatalysts, i.e., slow heterogeneous electron transfer, by incorporation of a redox active species to act as an electron shuttle. There are several advantages to immobilizing both the enzyme and mediator at the enzyme surface, including increased electron transfer rates, decreased enzyme leaching, and minimized diffusion limitations. Redox polymers consisting of a redox active center attached to a polymer backbone are a particularly attractive option because they have high self-exchange rates for electron transfer and tunable redox potential. Osmium (Os) polymers are the most well studied of this type of polymer for bioelectrocatalysis. Here, we describe the methods to synthesize one of the most common Os redox polymers and how it can be used to fabricate glucose oxidase electrodes. Procedures are also outlined for evaluating the enzymatic electrodes.
Topics: 2,2'-Dipyridyl; Aspergillus niger; Biosensing Techniques; Electrochemical Techniques; Electrodes; Electron Transport; Enzymes, Immobilized; Glucose; Glucose Oxidase; Imidazoles; Models, Molecular; Osmium; Oxidation-Reduction; Polymers; Polyvinyls
PubMed: 27770421
DOI: 10.1007/978-1-4939-6499-4_13 -
Journal of Bone and Mineral Research :... Aug 2017The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be...
The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be attenuated via exercise and whether this correlates with gains in bone quantity and quality. C57BL/6 mice were divided into diet-induced obesity (DIO, n = 14) versus low-fat diet (LFD, n = 14). After 3 months, 16-week-old mice were allocated to an exercise intervention (LFD-E, DIO-E) or a control group (LFD, DIO) for 6 weeks (4 groups, n = 7/group). Marrow adipocyte area was 44% higher with obesity (p < 0.0001) and after exercise 33% lower in LFD (p < 0.0001) and 39% lower in DIO (p < 0.0001). In LFD, exercise did not affect adipocyte number; however, in DIO, the adipocyte number was 56% lower (p < 0.0001). MAT was 44% higher in DIO measured by osmium-μCT, whereas exercise associated with reduced MAT (-23% in LFD, -48% in DIO, p < 0.05). MAT was additionally quantified by 9.4TMRI, and correlated with osmium-µCT (r = 0.645; p < 0.01). Consistent with higher lipid beta oxidation, perilipin 3 (PLIN3) rose with exercise in tibial mRNA (+92% in LFD, +60% in DIO, p < 0.05). Tibial µCT-derived trabecular bone volume (BV/TV) was not influenced by DIO but responded to exercise with an increase of 19% (p < 0.001). DIO was associated with higher cortical periosteal and endosteal volumes of 15% (p = 0.012) and 35% (p < 0.01), respectively, but Ct.Ar/Tt.Ar was lower by 2.4% (p < 0.05). There was a trend for higher stiffness (N/m) in DIO, and exercise augmented this further. In conclusion, obesity associated with increases in marrow lipid-measured by osmium-μCT and MRI-and partially due to an increase in adipocyte size, suggesting increased lipid uptake into preexisting adipocytes. Exercise associated with smaller adipocytes and less bone lipid, likely invoking increased ß-oxidation and basal lipolysis as evidenced by higher levels of PLIN3. © 2017 American Society for Bone and Mineral Research.
Topics: Adipocytes; Adipose Tissue; Animals; Bone Marrow Cells; Dietary Fats; Female; Lipolysis; Mice; Obesity; Physical Conditioning, Animal; X-Ray Microtomography
PubMed: 28436105
DOI: 10.1002/jbmr.3159 -
Dalton Transactions (Cambridge, England... Nov 2015Eight new ruthenium and five new osmium p-cymene half-sandwich complexes have been synthesized, characterized and evaluated for antimalarial activity. All complexes...
Eight new ruthenium and five new osmium p-cymene half-sandwich complexes have been synthesized, characterized and evaluated for antimalarial activity. All complexes contain ligands that are based on a 4-chloroquinoline framework related to the antimalarial drug chloroquine. Ligands HL(1-8) are salicylaldimine derivatives, where HL(1) = N-(2-((2-hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine, and HL(2-8) contain non-hydrogen substituents in the 3-position of the salicylaldimine ring, viz. F, Cl, Br, I, NO2, OMe and (t)Bu for HL(2-8), respectively. Ligand HL(9) is also a salicylaldimine-containing ligand with substitutions in both 3- and 5-positions of the salicylaldimine moiety, i.e. N-(2-((2-hydroxy-3,5-di-tert-butylphenyl)methyl-imino)ethyl)-7-chloroquinolin-4-amine, while HL(10) is N-(2-((1-methyl-1H-imidazol-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine) The half sandwich metal complexes that have been investigated are [Ru(η(6)-cym)(L(1-8))Cl] (Ru-1-Ru-8, cym = p-cymene), [Os(η(6)-cym)(L(1-3,5,7))Cl] (Os-1-Os-3, Os-5, and Os-7), [M(η(6)-cym)(HL(9))Cl2] (M = Ru, Ru-HL(9); M = Os, Os-HL(9)) and [M(η(6)-cym)(L(10))Cl]Cl (M = Ru, Ru-10; M = Os, Os-10). In complexes Ru-1-Ru-8 and Ru-10, Os-1-Os-3, Os-5 and Os-7 and Os-10, the ligands were found to coordinate as bidentate N,O- and N,N-chelates, while in complexes Ru-HL(9) and Os-HL(9), monodentate coordination of the ligands through the quinoline nitrogen was established. The antimalarial activity of the new ligands and complexes was evaluated against chloroquine sensitive (NF54 and D10) and chloroquine resistant (Dd2) Plasmodium falciparum malaria parasite strains. Coordination of ruthenium and osmium arene moieties to the ligands resulted in lower antiplasmodial activities relative to the free ligands, but the resistance index is better for the ruthenium complexes compared to chloroquine. Overall, osmium complexes appeared to be less active than the corresponding ruthenium complexes.
Topics: Animals; Antimalarials; Chloroquine; Ligands; Models, Molecular; Molecular Structure; Osmium Compounds; Plasmodium falciparum; Ruthenium Compounds; Structure-Activity Relationship; X-Ray Diffraction
PubMed: 26491831
DOI: 10.1039/c5dt02410b -
Microscopy (Oxford, England) Jun 2024The two-dimensional observation of ultrathin sections from resin-embedded specimens provides an insufficient understanding of the three-dimensional (3D) morphological... (Review)
Review
The two-dimensional observation of ultrathin sections from resin-embedded specimens provides an insufficient understanding of the three-dimensional (3D) morphological information of membranous organelles. The osmium maceration method, developed by Professor Tanaka's group >40 years ago, is the only technique that allows direct observation of the 3D ultrastructure of membrane systems using scanning electron microscopy (SEM), without the need for any reconstruction process. With this method, the soluble cytoplasmic proteins are removed from the freeze-cracked surface of cells while preserving the integrity of membranous organelles, achieved by immersing tissues in a diluted osmium solution for several days. By employing the maceration method, researchers using SEM have revealed the 3D ultrastructure of organelles such as the Golgi apparatus, mitochondria and endoplasmic reticulum in various cell types. Recently, we have developed new SEM techniques based on the maceration method to explore further possibilities of this method. These include: (i) a rapid osmium maceration method that reduces the reaction duration of the procedure, (ii) a combination method that combines agarose embedding with osmium maceration to elucidate the 3D ultrastructure of organelles in free and cultured cells and (iii) a correlative immunofluorescence and SEM technique that combines cryosectioning with the osmium maceration method, enabling the correlation of the immunocytochemical localization of molecules with the 3D ultrastructure of organelles. In this paper, we review the novel osmium maceration methods described earlier and discuss their potential and future directions in the field of biology and biomedical research.
Topics: Microscopy, Electron, Scanning; Imaging, Three-Dimensional; Organelles; Animals; Osmium; Golgi Apparatus; Humans; Endoplasmic Reticulum
PubMed: 37930813
DOI: 10.1093/jmicro/dfad050 -
Metallomics : Integrated Biometal... Feb 2021The organo-osmium complex [OsII(ɳ6-p-cym)(PhAzPy-NMe2)I]+ (FY26) exhibits promising in vitro antitumour activity against mouse hepatocarcinoma Hepa1-6 and other mouse...
The organo-osmium complex [OsII(ɳ6-p-cym)(PhAzPy-NMe2)I]+ (FY26) exhibits promising in vitro antitumour activity against mouse hepatocarcinoma Hepa1-6 and other mouse or human cancer cell lines. Here, we drastically enhance water solubility of FY26 through the replacement of the PF6- counter-anion with chloride using a novel synthesis method. FY26⋅PF6 and FY26⋅Cl displayed similar in vitro cytotoxicity in two cancer cell models. We then show the moderate and late anticancer efficacy of FY26⋅PF6 and FY26⋅Cl in a subcutaneous murine hepatocarcinoma mouse model. Both efficacy and tolerability varied according to FY26 circadian dosing time in hepatocarcinoma tumour-bearing mice. Tumour and liver uptake of the drug were determined over 48 h following FY26⋅Cl administration at Zeitgeber time 6 (ZT6), when the drug is least toxic (in the middle of the light span when mice are resting). Our studies suggest the need to administer protracted low doses of FY26 at ZT6 in order to optimize its delivery schedule, for example through the use of chrono-releasing nanoparticles.
Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Coordination Complexes; Dose-Response Relationship, Drug; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Tissue Distribution
PubMed: 33595653
DOI: 10.1093/mtomcs/mfaa003 -
Dalton Transactions (Cambridge, England... Jun 20202-(Arylamino)-4,6-di-tert-butylphenols containing 4-substituted phenyl groups (RapH2) react with oxobis(ethylene glycolato)osmium(vi) in acetone to give square pyramidal...
2-(Arylamino)-4,6-di-tert-butylphenols containing 4-substituted phenyl groups (RapH2) react with oxobis(ethylene glycolato)osmium(vi) in acetone to give square pyramidal bis(amidophenoxide)oxoosmium(vi) complexes. A mono-amidophenoxide complex is observed as an intermediate in these reactions. Reactions in dichloromethane yield the diolate (Hap)2Os(OCH2CH2O). Both the glycolate and oxo complex are converted to the corresponding cis-dichloride complex on treatment with chlorotrimethylsilane. The novel bis(aminophenol) ligand EganH4, containing an ethylene glycol dianthranilate bridge, forms the chelated bis(amidophenoxide) complex (Egan)OsO, where the two nitrogen atoms of the tetradentate ligand bind in the trans positions of the square pyramid. Structural and spectroscopic features of the complexes are described well by an osmium(vi)-amidophenoxide formulation, with the amount of π donation from ligand to metal increasing markedly as the co-ligands change from oxo to diolate to dichloride. In the oxo-bis(amidophenoxides), the symmetry of the ligand π orbitals results in only one effective π donor interaction, splitting the energy of the two osmium-oxo π* orbitals and rendering the osmium-oxo bonding appreciably anisotropic.
PubMed: 32525176
DOI: 10.1039/d0dt01735c -
Nature Communications Apr 2022The limited therapeutic effect on hypoxic and refractory solid tumors has hindered the practical application of photodynamic therapy. Herein, we report our investigation...
The limited therapeutic effect on hypoxic and refractory solid tumors has hindered the practical application of photodynamic therapy. Herein, we report our investigation of an osmium-peroxo complex (Os2), which is inactive in the dark, but can release a peroxo ligand O upon light irradiation even in the absence of oxygen, and is transformed into a cytotoxic osmium complex (Os1). Os1 is cytotoxic in the presence or absence of irradiation in hypoxic tumors, behaving as a chemotherapeutic drug. At the same time, the light-activated Os2 induces photocatalytic oxidation of endogenous 1,4-dihydronicotinamide adenine dinucleotide in living cancer cells, leading to ferroptosis, which is mediated by glutathione degradation, lipid peroxide accumulation and down-regulation of glutathione peroxidase 4. In vivo studies have confirmed that the Os2 can effectively inhibit the growth of solid hypoxic tumors in mice. A promising strategy is proposed for the treatment of hypoxic tumors with metal-based drugs.
Topics: Animals; Antineoplastic Agents; Hypoxia; Mice; Neoplasms; Osmium; Oxygen; Photochemotherapy
PubMed: 35473926
DOI: 10.1038/s41467-022-29969-z -
Journal of the American Society For... Mar 2020The Os arene anticancer complex [(η-bip)Os(en)Cl] (Os1-Cl; where bip = biphenyl and en = ethylenediamine) binds strongly to DNA and biomolecules. Here we investigate...
The Os arene anticancer complex [(η-bip)Os(en)Cl] (Os1-Cl; where bip = biphenyl and en = ethylenediamine) binds strongly to DNA and biomolecules. Here we investigate the interaction between Os1-Cl and the model protein, BSA, using ultrahigh resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS). The specific binding location of Os1 on BSA was investigated with the use of collisionally activated dissociation (CAD) and electron capture dissociation (ECD). CAD MS/MS was found to dissociate the osmium complex from the metallo-peptide complex readily producing unmodified fragments and losing location information. ECD MS/MS, however, successfully retains the osmium modification on the peptides upon fragmentation allowing localization of metallocomplex binding. This study reveals that lysine is a possible binding location for Os1-Cl, apart from the expected binding sites at methionine, histidine, and cysteine. Using a nano liquid chromatography (nLC)-FT-ICR ECD MS/MS study, multiple binding locations, including the N-terminus and C-terminus of digested peptides, glutamic acid, and lysine were also revealed. These results show the multitargeting binding ability of the organo-osmium compound and can be used as a standard workflow for more complex systems, e.g., metallocomplex-cell MS analysis, to evaluate their behavior toward commonly encountered biomolecules.
Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Binding Sites; Cattle; Coordination Complexes; Models, Molecular; Osmium; Peptides; Protein Binding; Serum Albumin, Bovine; Tandem Mass Spectrometry
PubMed: 31967804
DOI: 10.1021/jasms.9b00054 -
Metallomics : Integrated Biometal... Apr 2021The treatment of tuberculosis (TB) poses a major challenge as frontline therapeutic agents become increasingly ineffective with the emergence and spread of...
The treatment of tuberculosis (TB) poses a major challenge as frontline therapeutic agents become increasingly ineffective with the emergence and spread of drug-resistant strains of Mycobacterium tuberculosis (Mtb). To combat this global health problem, new antitubercular agents with novel modes of action are needed. We have screened a close family of 17 organometallic half-sandwich Os(II) complexes [(arene)Os(phenyl-azo/imino-pyridine)(Cl/I)]+Y- containing various arenes (p-cymene, biphenyl, or terphenyl), and NMe2, F, Cl, or Br phenyl or pyridyl substituents, for activity towards Mtb in comparison with normal human lung cells (MRC5). In general, complexes with a monodentate iodido ligand were more potent than chlorido complexes, and the five most potent iodido complexes (MIC 1.25-2.5 µM) have an electron-donating Me2N or OH substituent on the phenyl ring. As expected, the counter anion Y (PF6-, Cl-, I-) had little effect on the activity. The pattern of potency of the complexes towards Mtb is similar to that towards human cells, perhaps because in both cases intracellular thiols are likely to be involved in their activation and their redox mechanism of action. The most active complex against Mtb is the p-cymene Os(II) NMe2-phenyl-azopyridine iodido complex (2), a relatively inert complex that also exhibits potent activity towards cancer cells. The uptake of Os from complex 2 by Mtb is rapid and peaks after 6 h, with temperature-dependence studies suggesting a major role for active transport. Significance to Metallomics Antimicrobial resistance is a global health problem. New advances are urgently needed in the discovery of new antibiotics with novel mechanisms of action. Half-sandwich organometallic complexes offer a versatile platform for drug design. We show that with an appropriate choice of the arene, an N,N-chelated ligand, and monodentate ligand, half-sandwich organo-osmium(II) complexes can exhibit potent activity towards Mycobacterium tuberculosis (Mtb), the leading cause of death from a single infectious agent. The patterns of activity of the 17 azo- and imino-pyridine complexes studied here towards Mtb and normal lung cells suggest a common redox mechanism of action involving intracellular thiols.
Topics: Antineoplastic Agents; Antitubercular Agents; Cell Proliferation; Humans; Molecular Structure; Mycobacterium tuberculosis; Neoplasms; Organometallic Compounds; Osmium; Tuberculosis; Tumor Cells, Cultured
PubMed: 33693931
DOI: 10.1093/mtomcs/mfab007