Elucidating chromatin and nuclear domain architecture with electron spectroscopic imaging

Electron microscopy has been the ‘gold standard’ of spatial resolution for studying the structure of the cell nucleus. Electron spectroscopic imaging (ESI) offers advantages over conventional transmission electron microscopy by eliminating the need for heavy-atom contrast agents. ESI also provides mass-dependent and element-specific information at high resolution, permitting the distinguishing of structures that are primarily composed of protein, DNA, or RNA. The technique can be applied to understand the structural consequences of epigenetic modifications, such as modified histones, on chromatin fiber morphology. ESI can also be applied to elucidate the multifunctional behavior of subnuclear ‘organelles’ such as the nucleolus and promyelocytic leukemia nuclear bodies. The authors dedicate this paper to the memory of Ying Ren (1961–2007). We all benefited from knowing her. Our research advanced through the technical creativity she provided.     

Plasma membrane-mediated nuclear uptake and chromatin binding of insulin in tumor cell lines

Analysis of different cellular fractions after incubation of SW 948 and SW 707 colorectal carcinoma cells or WM 266-4 melanoma cells with 125I-insulin revealed the nondegraded hormone in the chromatin of these cells. Nuclear 125I-insulin was bound to specific fragments of EcoRI-, HaeIII-, and HincII-digested chromatin. A 45-kDa chromatin protein species that binds 125I-insulin was identified. Actinomycin D and cycloheximide inhibited the insulin-stimulated expression of chromatin receptors. Uptake of 125I-insulin by isolated nuclei occurred only in the presence of plasma membranes. Thus, at least some effects of insulin on target cells can be explained by direct gene regulation instead of "second messenger" action.     

Measurement of lymphocyte activation by a chromatin topo-optical reaction

Summary A rapid inexpensive method is presented for detecting peripheral blood lymphocyte chromatin activation by the neutral red topo-optical reaction, which causes strong and easily measurable birefringence in the lymphocyte nuclei. This reaction can be enhanced by fixing the cells with 150 mM/l NaCl in 70% ethanol and/or by treating the unfixed cellular suspensions with 0.2 M/l HCl to remove histones. In histone-removed preparations, 30 min DNase I treatment almost completely abolished the birefringent reaction, whereas RNase treatment resulted in only 18% loss.Chromatin activation induced by enzyme inhibition increased chromatin birefringence significantly. The same phenomenon could be induced in sensitive subjects' lymphocytes by specific antigens or haptens much more rapidly. The monocytes were not activated to a significant extent. In non-sensitive subjects different kinetics of antigen or hapten-dependent activation and no cytotoxic effects have been observed. Depletion of T-lymphocytes in vivo in SLE patients or by in vitro treatment with 0.5 mM/l KCN as well as with 0.02% trypsin has caused a significant drop in the mean chromatin birefringence. The effect of trypsin was reversible.     

Chain length heterogeneity of nucleosomal DNA in mouse liver after dimethylnitrosamine administration

The effect of dimethylnitrosamine on the nucleosomal structure of mouse liver chromatin was studied. After a single oral dose of dimethylnitrosamine (2–75 mg/kg body weight 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcus nuclease. Nucleosomes were separated on sucrose density gradients. There were no differences in nucleosomal sedimentation velocities between preparations from control and dimethylnitrosamine treated animals. The supernatant obtained after centrifugation of the lysed nuclei (2 min at 4,000 g _av) and nucleosomal peak fractions were used for isolation of DNA. DNA was heat denatured in 7 M urea or formamide. After electrophoresis on polyacrylamide gels areas under mononucleosomal DNA and smaller fragments were measured and compared with the total DNA area. The increase in DNA fragmentation was dimethylnitrosamine dose response dependent. When expressed as per cent of controls it amounted to 106% for 2 mg; 115% for 10 mg; 127% for 25 mg; 164% for 75 mg dimethylnitrosamine/kg body weight. A good correlation between mobility and log of chain length of 174 RF DNA-Hae III digest was obtained in nondenaturing 5% polyacrylamide gels and denaturing non-aqueous formamide polyacrylamide gels but not in 12% polyacrylamide gels containing 7 M urea. DNA of mononucleosomal peak fractions contained 200 and that of dinucleosomal peak fractions 400 nucleotides. Fragmentation of DNA was closely related to in vivo dimethylnitrosamine treatment but was not detected in measurements of protein-DNA complexes in the chromatin. It was disclosed on denaturation of DNA followed by polyacrylamide gel electrophoresis.Abbreviations DMN dimethylnitrosamine - SDS sodium dodecyl sulfate The work was supported by Grant Number 1 R0 1 CA26642-01, awarded to A.v.d.D. by the National Cancer Institute, DHEW     

Vilsmeir法合成头孢曲松钠的工艺研究

目的　探讨头孢曲松钠的Vilsmeir法合成工艺。方法　用二氯硫酰代替二氯亚硫酰来制备得到改进的Vilsmeir试剂 ,用vilsmeir试剂活化的氨噻肟乙酸同 7-ACT缩合反应制备头孢曲松钠。结果与讨论　Vilsmeir法合成工艺简单、便宜、收率达 85 %。     

盐酸索他洛尔合成工艺条件的分析与优化

本文利用正交实验方法系统地研究了合成盐酸索他洛尔过程中各工艺参数对产品收率的影响 ,得到了最佳工艺条件 ,在此条件下 ,盐酸索他洛尔的收率可达 80 %以上     

Chromatin remodelling and DNA repair genes are frequently mutated in endometrioid endometrial carcinoma

In developed countries, endometrial carcinoma is the most common cancer that affects the female genital tract. Endometrial carcinoma is divided into two main histological types, type I or endometrioid and type II or non‐endometrioid, each of which have characteristic, although not exclusive, molecular alterations and mutational profiles. Nevertheless, information about the implication and relevance of some of these genes in this disease is lacking. We sought here to identify new recurrently mutated genes in endometrioid cancers that play a role in tumourigenesis and that influence the clinical outcome. We focused on low‐grade, non‐ultramutated tumours as these tumours have a worse prognosis than the ultramutated POLE‐positive endometrioid endometrial carcinomas (EECs). We performed exome‐sequencing of 11 EECs with matched normal tissue and subsequently validated 15 candidate genes in 76 samples. For the first time, we show that mutations in chromatin remodelling‐related genes (KMT2D, KMT2C, SETD1B and BCOR) and in DNA‐repair‐related genes (BRCA1, BRCA2, RAD50 and CHD4) are frequent in this subtype of endometrial cancer. The alterations to these genes occurred with frequencies ranging from 35.5% for KMT2D to 10.5% for BRCA1 and BCOR, with some showing a tendency toward co‐occurrence (RAD50‐KMT2D and RAD50‐SETD1B). All these genes harboured specific mutational hotspots. In addition, the mutational status of KMT2C, KMT2D and SETD1B helps to predict the degree of myometrial invasion, a critical prognostic feature. These results highlight the possible implication of these genes in this disease, creating opportunities for new therapeutic approaches.     

Hydrophilic reentrant SLIPS enabled flow separation for rapid water harvesting

Water harvesting from air is desired for decentralized water supply wherever water is needed. When water vapor is condensed as droplets on a surface the unremoved droplets act as thermal barriers. A surface that can provide continual droplet-free areas for nucleation is favorable for condensation water harvesting. Here, we report a flow-separation condensation mode on a hydrophilic reentrant slippery liquid-infused porous surface (SLIPS) that rapidly removes droplets with diameters above 50 μm. The slippery reentrant channels lock the liquid columns inside and transport them to the end of each channel. We demonstrate that the liquid columns can harvest the droplets on top of the hydrophilic reentrant SLIPS at a high droplet removal frequency of 130 Hz/mm². The sustainable flow separation without flooding increases the water harvesting rate by 110% compared to the state-of-the-art hydrophilic flat SLIPS. Such a flow-separation condensation approach paves a way for water harvesting.

Condensation is a common phase-change phenomenon that is widely used in water harvesting (1–5). Dropwise condensation can form discrete droplets on a low-surface-energy substrate and promote the heat transfer coefficient up to 10 times higher than filmwise condensation due to the rapid removal of condensates (6, 7). However, the displacement of air inside the structures can lead to a higher pinning force on the droplets, resulting in flooding on the superhydrophobic surface (8, 9). By replacing the air trapped inside the surface structures with liquid lubricant, researchers developed the slippery liquid-infused porous surface (SLIPS) to further promote heat transfer performance (1, 10–13). When condensed droplets grow to a certain diameter, they shed off a vertical surface due to the negligible contact angle hysteresis (12). To accelerate the droplet removal, directional droplet movements were developed to regulate the removal of water droplets on SLIPS (2, 5). However, these surfaces rely on gravity to remove large droplets that remain on the surface as thermal barriers before shedding during condensation.Therefore, surfaces with partially hydrophilic and partially hydrophobic domains were developed to remove droplets from the condensing surface to the structures underneath. Such an amphiphilic surface has a hydrophobic top with a hydrophilic porous structure underneath (14, 15). During the condensation, droplets on the hydrophobic top are absorbed by the wetted hydrophilic structures underneath. However, the liquid film within the hydrophilic structures leads to partial or complete flooding at elevated heat fluxes due to the high pinning forces (9, 16, 17). Unlike the radiative cooling-induced dew harvesting in nature, droplets are directly condensed on the reentrant SLIPS by conduction and convection (18), where condensed droplets act as thermal barriers (19–21). Based on the condensation models (6, 22–24), each droplet contributes to the total thermal resistance, while smaller droplets have a lower thermal resistance. When a surface is only covered by small droplets (e.g., at the beginning of the condensation), the heat transfer coefficient is higher than that of a surface with larger droplets (25). Thus, rapid droplet nucleation and removal are desired to achieve a high heat transfer coefficient. Microchannels are applied to transport condensates as the flow resistances can be much smaller than those of micropillars. Hydrophilic slippery channels with or without biphilic coatings are used to enhance water harvesting, but they failed to separate the vapor and liquid as droplets emerged out of the channels or completely wetted the surfaces (1, 26–28). To suppress condensates from emerging out of the channel, the liquid column must be locked inside. The reentrant channels show a potential to lock the liquid inside due to their overhang structures. With a surface engineering approach, the reentrant structure has been modified to keep highly nonwetting liquids inside (29). However, the reentrant structures are wetted by liquids during condensation as the condensates are pinned inside the channels (30). Thus, the surface will transition to a complete wetting state at elevated heat fluxes.To address those challenges, on a hydrophilic reentrant SLIPS, we present a flow-separation condensation mode that sustains rapid droplet removal on top and transports liquid columns underneath. Our approach is to make reentrant channels with hydrophilic and slippery boundary lubrication. Once droplets are condensed on the top hydrophilic slippery surface, they are removed immediately to the reentrant channels underneath, resulting in sustainable flow separation. Such a new condensation mode could significantly reduce the thermal resistance by rapidly removing droplets with diameters above 50 µm, leading to an exceptional water harvesting rate. This work not only proposes a flow separation approach to enhance water harvesting but also provides a universal concept to design surfaces for condensation heat transfer.     

垂直加压根管充填技术的临床评价

目的:对垂直加压根管充填技术进行临床疗效评价.方法:选择到本院牙体牙髓科就诊的牙髓病或根尖周病患者60例,按就诊顺序排列随机分为两组.分别采用垂直加压根管充填技术(VC组)和冷牙胶侧方加压充填技术(LC组)进行根管充填.对两组的根管充填时间、术中疼痛情况和根管充填效果进行比较.术后3个月和6个月进行复查.并拍摄X线片进行记录.结果:以牙为单位,VC组适充27例,超充2例,欠充1例;LC组中,适充25例,超充3例,欠充2例.VC组术中不适5例,LC组2例.以根管为单位,VC根管充填时间组平均62.6 s/根管,LC组平均198.9 s/根管(P<0.05).结论:垂直加压充填技术临床操作简便有效,操作时间短.     

2－芳基丙酸类消炎镇痛药物的研究5 羟醛缩合法合成酮基布洛芬

以环己酮为起始原料，经Ｓｔｏｒｋ烯胺反应、羟醛缩合、消除、芳构化制得酮基布洛芬，总收率４３．２％。