Methotrexate (MTX), an antifolate drug, is widely used for clinical treatment of malignancies and ectopic pregnancy. Many studies have documented that MTX has strong side-effects on rapidly dividing somatic cells. However, its side-effects on female reproductive cells have not been widely reported. Combined with in vitro culture, two-photon fluorescence imaging and three-dimensional reconstruction, this study analyzed the effects of MTX on oocyte maturation time, chromosome arrangement, karyotype, spindle morphology, and the localization of microtubule organizing centers (MTOCs). Compared with a control group (84%), the rate of germinal vesical breakdown in the MTX group dropped to 73% (P < 0.05). The rate of the first polar body extrusion in the MTX group (53%) was also below the control group (63%; P < 0.05). The rate of abnormal chromosomal arrangement in the MTX group was 60%, but 24% in the control group (P < 0.05). The matured oocyte karyotypes showed 20 univalents in both control and MTX groups, while point-shaped DAPI signals were detected in the MTX group. The rate of abnormal spindle in the MTX group was 49%, but 17% in the control group (P < 0.05). MTOCs in oocytes with normal spindles concentrated at the poles, while MTOCs in oocytes with abnormal spindles were scattered around the poles or in the ooplasm. MTX changes the structures of chromosomes and spindles, potentially by interfering with DNA methylation. The above results indicate a basis for understanding negative effects of MTX on oocyte maturation quality, and provide information for the clinical application of MTX in female patients.

Many studies have suggested a link between the spatial organization of genomes and fundamental biological processes such as genome reprogramming, gene expression, and differentiation. Multicolor fluorescence in situ hybridization on three-dimensionally preserved nuclei (3D-FISH), in combination with confocal microscopy, has become an effective technique for analyzing 3D genome structure and spatial patterns of defined nucleus targets including entire chromosome territories and single gene loci. This technique usually requires the simultaneous visualization of numerous targets labeled with different colored fluorochromes. Thus, the number of channels and lasers must be sufficient for the commonly used labeling scheme of 3D-FISH, "one probe-one target". However, these channels and lasers are usually restricted by a given microscope system. This paper presents a method for simultaneously delineating multiple targets in 3D-FISH using limited channels, lasers, and fluorochromes. In contrast to other labeling schemes, this method is convenient and simple for multicolor 3D-FISH studies, which may result in widespread adoption of the technique. Lastly, as an application of the method, the nucleus locations of chromosome territory 18/21 and centromere 18/21/13 in normal human lymphocytes were analyzed, which might present evidence of a radial higher order chromatin arrangement.

Summary. --The effect of weight loss on the dimensional stability of some addition-polysiloxanes used as impression materials in dentistry is studied. We show that polymerization is not the only factor affecting the shrinkage of these materials. An explanation for the time-dependent weight functions is given.PACS 81.70 -Materials testing. PACS 87.90 -Other topics in biophysics, medical physics, and biomedical engineering. Polysiloxane-based impression materials, which cure by addition reaction, are considered materials with superior properties in dentistry [1]. Several works report that they show little dimensional change with time [2][3][4], when compared with the polysulphides and condensation silicones. This is because the addition polymerization does not produce volatile by-products [5,6]. Recently, it has been suggested [7] that the shrinkage is a complicated function of time: the early shrinkage, that proceeds exponentially for very few hours, is affected by the polymerization reaction. However, depending on the sample thickness, the long-term shrinkage, which is not exponential, finishes after one or more days. The knowledge of all the factors affecting the dimensional variation is closely related to the accuracy of the impressions and is a condition required to improve the material quality.In this work the possible evaporation of volatile components was tested by monitoring the weight of addition impression silicones. Their weight loss with time can be ascribed to evaporation. To confirm this phenomenon, we tested the weight variation of samples having similar shapes but different thicknesses. In contrast with the polymerization, which does not depend on thickness, the ratio W/W1 (where W = = W1 -W2, W1 is the sample weight measured at zero-time, W2 is the weight after t time) is larger for thin samples, compared with the thick ones, in the early stages of the evaporation.The influence of evaporation on the dimensional stability was assessed by examining the linear shrinkage as a function of time, for materials showing different

This study aimed to show that the polymerization contraction of dental methacrylate-based materials, when used as adhesives on hard substrate, produces voids at the material-substrate interface. This phenomenology is closely related with the nanoleakage and the sealing ability of these materials. One prime/bond system, three restorative composite resins, and one orthodontic bonding system were cured by using mirror-like glass slides as a compliance-free reference substrate. The adhesive surface was analyzed by atomic force microscopy, and the polymerization contraction of bulk material was tested by laser beam-scanning method. Nanoperiodic structure of three-dimensional (3D) images, section analysis, and roughness characteristics (R(a) and R(z)) indicated that polymerization contraction produced voids at the interface. When the adhesive surface was exposed to oral simulating fluids (water, ethanol, and lactic acid solutions), hydrolytic degradation involved some hundreds of nanometers in depth. In visible light-cured (VLC) materials, the interface porosity decreased when an irradiation pause ( approximately 2 min) was carried out during gelation.

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