Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down‐regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase‐independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4‐Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi‐BRCT‐domain (MBD) module that recapitulates the action of Slx4‐Rtt107 in checkpoint down‐regulation. MBD mimics the damage‐induced Dpb11‐Slx4‐Rtt107 complex by synergistically interacting with lesion‐specific phospho‐sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11‐Slx4‐Rtt107 or MBD via a cooperative ‘two‐site‐docking’ mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio‐temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT‐domains interactions.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.     

钙离子振荡对肝糖磷酸化酶激活的随机效应研究

在复杂生化系统的研究过程中,仿真与建模变得越来越重要.对于参与分子数量比较大的生化系统,通常可以采用常微分方程来解决这一问题.对于分子数量比较小的系统,离散粒子基础上的随机模拟方法更精确.然而目前还没有明确的理论方法来确定,对于实际问题用哪种方法能得到更合理的结果.因此需要在一个普遍研究的体系中,通过Ca~(2+)振荡传导信号来研究从随机行为到确定行为的过渡过程.本文以肝细胞中Ca~(2+)振荡对肝糖磷酸化酶激活随机效应为例,讨论了利用随机微分方程来解决分子数量比较小的生化系统的仿真与建模问题,利用细胞内Ca~(2+)有关的Li-Rinzel随机模型,研究了在磷酸化酶降解肝糖的磷酸化-去磷酸化作用循环过程中,三磷酸肌醇受体通道(IP_3R)释放Ca~(2+)的调控作用.结果表明,肝糖磷酸化酶的激活率随受体通道IP_3R的总数增大而减弱,而且三磷酸肌醇浓度比较小时出现相干共振.     

新疆焉耆盆地人类活动与气候变化的效应机制

通过对新疆焉耆盆地及其周边近40a(1973—2014)的气候变化趋势检测、LUCC和生物量估算,探讨气候变化和人类活动的生态效应机制,研究区域陆地生态系统演变及其归因。分析结果表明:(1)焉耆盆地山区和平原区降水变化都有明显的突变点,并呈现增加趋势,蒸发量在山区减少,在平原区波动性减少趋势;(2)LUCC分析表明,山区裸地面积减少5.40%,冰川面积减少3.36%,高地植被面积增加8.76%;同时平原区天然绿洲面积增加1.96%,沙漠面积减少1.62%,水域面积减少1.30%,人工绿洲面积增加15.41%,湿地面积增加1.27%;(3)山区陆地生态系统对区域气候变化非常敏感,其中降水变化是决定山区地表植被生存状态和分布的重要因素;(4)人类活动的推动作用和有益气候变化的支撑是绿洲平原区生态系统好转的原因,其中人口急剧增加和社会经济快速发展,导致绿洲平原区生态系统结构及其时空分布的主要因素。焉耆盆地及其周围区域陆地生态系统的演变对气候变化和人类活动有明显的时空尺度效应,其反应程度各不相同。     

The Occurrence,Sources and Spatial Characteristics of Soil Salt and Assessment of Soil Salinization Risk in Yanqi Basin,Northwest China

In order to evaluate the soil salinization risk of the oases in arid land of northwest China, we chose a typical oasis-the Yanqi basin as the research area. Then, we collected soil samples from the area and made comprehensive assessment for soil salinization risk in this area. The result showed that: (1) In all soil samples, high variation was found for the amount of Ca²⁺ and K⁺, while the other soil salt properties had moderate levels of variation. (2) The land use types and the soil parent material had a significant influence on the amount of salt ions within the soil. (3) Principle component (PC) analysis determined that all the salt ion values, potential of hydrogen (pHs) and ECs fell into four PCs. Among them, PC1 (C1^-, Na⁺, SO₄ ^2-, EC, and pH) and PC2 (Ca²⁺, K⁺, Mg²⁺and total amount of salts) are considered to be mainly influenced by artificial sources, while PC3 and PC4 (CO₃ ^- and HCO₃ ^2-) are mainly influenced by natural sources. (4) From a geo-statistical point of view, it was ascertained that the pH and soil salt ions, such as Ca²⁺, Mg²⁺ and HCO₃ ^-, had a strong spatial dependency. Meanwhile, Na⁺ and Cl^- had only a weak spatial dependency in the soil. (5) Soil salinization indicators suggested that the entire area had a low risk of soil salinization, where the risk was mainly due to anthropogenic activities and climate variation. This study can be considered an early warning of soil salinization and alkalization in the Yanqi basin. It can also provide a reference for environmental protection policies and rational utilization of land resources in the arid region of Xinjiang, northwest China, as well as for other oases of arid regions in the world.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress

Relocalization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein reorganization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by subcellular destination enables the identification of pathways that respond to replication stress. We analysed pairwise combinations of GFP fusions and gene deletion mutants to define and order two previously unknown DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways.     

Rumen digestion and microbial protein synthesis by growing lambs fed high-concentrate diets: Effects of cereal processing and animal age

Rumen digestion and microbial protein synthesis were studied using 36 growing lambs given either free choice access to separate feeders with whole barley grain and a pelleted soybean meal-based supplement (treatment WB) or a pelleted compound feed (treatment C) made by combining ground barley (0.65) with the same protein supplement (0.35). Free access to barley straw was offered in treatment C but not in WB. While both treatments were imposed before and after weaning (at 42 days), for a third treatment the compound feed was replaced after weaning by whole barley grain and the protein supplement without access to straw (treatment CWB). Six lambs from each treatment were slaughtered at 10 and 30 days post-weaning after ¹⁵N-labelling of microbial N and abomasal digesta flows were estimated using C₃₁ alkane as marker. Processing of barley grain increased (P<0.05) the apparent digestibility of dry matter in the rumen irrespective of age (0.53, 0.48 and 0.43 (S.E. 0.027) for C, CWB and WB). Ruminal pH averaged 5.5 (S.E. 0.06) regardless of cereal processing. The molar ratio of acetate to propionate decreased with age reflecting a higher proportion of grain in the mixed diets (WB and CWB) at 30 than 10 days post-weaning. Even with similar dietary proportions of grain and protein supplement, the acetate to propionate ratio was lower for lambs fed the free-choice (WB and CWB) vs. those fed the compound (C) diet (1.3 vs. 2.2 (S.E.D. 0.37)). Ruminal ammonia concentration increased with age and was lower for treatment C compared with treatments WB and CWB (24 vs. 127 (S.E.D. 22.2) mg/L), reflecting the higher protein intake of the latter two treatments. However, recovery of N intake as abomasal non-ammonia N was low for all treatments due to high protein intake and inefficient microbial growth compared to values reported for mixed diets. The efficiency of microbial N synthesis did not differ between treatments but it was negatively correlated (r = −0.73) with the organic matter apparently digested in the rumen, resulting in similar microbial yields in spite of the lower digestion in the rumen of whole barley diets. Feeding whole barley is thus a useful strategy to modify the site of digestion in intensive lamb fattening, allowing to reduce ruminal fermentation without depressing microbial N yield.