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Photons, such as X- or γ-rays, induce DNA damage (distributed throughout the nucleus) as a result of low-density energy deposition. In contrast, particle irradiation with high linear energy transfer (LET) deposits high-density energy along the particle track. High-LET heavy-ion irradiation generates a greater number and more complex critical chromosomal aberrations, such as dicentrics and translocations, compared with X-ray or γ irradiation. In addition, the formation of >1000 bp deletions, which is rarely observed after X-ray irradiation, has been identified following high-LET heavy-ion irradiation. Previously, these chromosomal aberrations have been thought to be the result of misrepair of complex DNA lesions, defined as DNA damage through DNA double-strand breaks (DSBs) and single-strand breaks as well as base damage within 1–2 helical turns (

Spearmint oil (SMO) has been revealed to inhibit oral cancer cells. Its activity increases when it is incorporated into nanoemulsions. However, SMO alone cannot be prepared into stable nanoemulsions without an Ostwald ripening inhibitor. This study aimed to evaluate the effect of an Ostwald ripening inhibitor, i.e., fixed oil, on the formation and characteristic of anticancer nanoemulsions. Various fixed oils, namely, virgin coconut oil (VCO; C12), palm oil (PMO; C16), olive oil (OLO; C18:1), soybean oil (SBO; C18:2), and perilla oil (PRO; C18:3), were selected, and their abilities to stabilize nanoemulsions were compared. Results indicated that the formation and characteristic of nanoemulsions were dependent on the type of fixed oils and the ratio of SMO to fixed oils. Nevertheless, the stable nanoemulsions with nearly the same droplet size could be prepared at a ratio of 80:20 regardless of the type of fixed oils. The stable nanoemulsions might be a result of specific molecular interactions among surfactants, SMO, and fixed oils. Among the nanoemulsions with fixed oils, 80:20 SMO:PRO nanoemulsion showed the most potent cytotoxic effect against oral cancer cells and therefore was selected for further study. The influence of excipients on the properties, including droplet size, carvone content, and cytotoxic activity, of the formulated nanoemulsions were comparatively evaluated after storage at 40 °C for 6 months. Both nanoemulsions had excellent stability. An apoptotic property assay was employed to probe the cell death mechanism. 80:20 SMO:PRO nanoemulsion could induce cancer cell death via the intrinsic apoptosis pathway, which was the desirable pathway for candidate anticancer agents. In conclusion, nanoemulsions containing SMO with cytotoxic effects against oral cancer cells were successfully prepared by selecting specific amounts and types of surfactants and fixed oils. This research might serve as a basis for fabricating stable nanoemulsions containing other volatile oils.

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR-induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of γH2AX foci at the chromosome boundary of chromosome 1 after carbon-ion irradiation was >4-fold higher than that after X-ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of γH2AX foci at chromosome boundaries is prevented by inhibition of DNA-PKcs activity, indicating that the DSB repair is NHEJ-dependent. Finally, we found that γH2AX foci at chromosome boundaries after carbon-ion irradiation contain DSBs undergoing DNA-end resection, which promotes repair utilizing microhomology mediated end-joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation. © 2016 Wiley Periodicals, Inc.

Binding of bile salts by dietary fiber is believed to promote their excretion and hence to reduce the serum cholesterol level in man and experimental animals. In this study, the binding efficiency of soluble pectin from various sources, i.e., apple, citrus and pomelo, was examined. Sodium deoxycholate and sodium cholate hydrate were used as a model to represent bile salt in human body. The binding efficiency was assayed by acid reaction, thin layer chromatography (TLC) and enzyme cycling method. The results demonstrated that enzyme cycling method was the most suitable for assaying the in-vitro binding of bile salts while the TLC was not very sensitive, i.e., low amount of bile salts cannot be detected by TLC. Excess pectin from binding test could also interfere the acid reaction method even though the centrifugation was used to remove the excess pectin. When the concentration of pectin was increased, the binding efficiency with sodium deoxycholate increased. However, at 1% w/w of pectin, the binding efficiency decreased. The exception is for pomelo pectin in which the binding efficiency increased when the pectin concentration increased. With sodium cholate hydrate, only slight difference in binding efficiency was observed for all types and concentrations of pectin. The results indicate that the ability to bind bile salts of pectin might be responsible for its hypocholesterolemic action observed in experimental animals and humans.

Chemical constituents as well as cytotoxic and insecticidal activity of the crude methanol extract from the leaves of Phyllanthus reticulatus Poir. (Euphorbiaceae) were investigated. (5R*,6R*)-4,6-Dimethoxycarbonyl-5-[2',3',4'-trihydroxy-6'-(methoxycarbonyl) phenyl]-5,6-dihydro-2H-pyran-2-one (1) along with 3,4,3'-tri-O-methylellagic acid, and methyl gallate were isolated from the dichloromethane extract. Determination of their structures was based on spectroscopic analysis. Compound 1 possessed a very weak insecticidal activity against Spodoptera frugiperda (Sf9) with an IC(50) value of 27.27 microg/mL.

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis., Nucleic Acids Research 37(4), e31; 2009