The emergence of the primitive streak, representing an organizing center for gastrulation, marks the mesendodermal lineage specification from epiblast, in which the epiblast cells undergo highly organized collective behaviors to form mesendodermal cells properly. Cell death is observed at the peri-gastrulation stage, especially in the primitive streak region. However, the dynamic and regulatory mechanism of cell death in the primitive streak formation is unclear. Here, we observed that a quick inhibition of the fast elevated cell death is coinciding with an accumulation of β-catenin during the early stage of primitive streak induction from human embryonic stem cells (hESCs). Deficiency of β-catenin in hESCs does not affect their self-renewal but cause robust cell death after primitive streak induction, while neuroectodermal differentiation remains unchanged. Overexpression of full-length β-catenin in β-catenin-deficient hESCs restores the cell death restriction during induction of primitive streak. Mechanistically, the β-catenin-restricted cell death during primitive streak is transcription-independent. The accumulated β-catenin traps casein kinase-1 in β-catenin destruction complex following WNT activation via its ARM repeat domain, resulting in the inhibition of mTORC1 by stabilizing DEPTOR, subsequently attenuates mitochondrial translocation of p53 and enhances mitophagy to promote cell survival. Consistently, mTORC1 inhibition by rapamycin or RAD001 attenuates the cell death in β-catenin-deficient cells during induction of primitive streak. In addition, only the β-catenin retains activations of cell death restriction and transcriptional activity can promote hESCs to successfully differentiate into primitive streak and cardiomyocytes, suggesting that β-catenin-restricted cell death safeguards the fate transition during the primitive streak induction via offering a crucial window for the accumulation of β-catenin to induce lineage-specific genes. These findings provide new insights into the function and mechanisms by which β-catenin coordinates the cell death and early lineage commitment.

针对现有高铁桥梁施工风险评估模型存在的主观性强、环节繁琐及需要大量样本等不足,提出基于层次分析法(AHP法)+BP神经网络的高铁桥梁安全风险评估模型.首先对高铁桥梁施工安全进行风险因素的识别,建立风险评估体系;然后采用AHP法对专家经验评估法获取的样本数据进行预处理,结合BP神经网络对处理后的样本进行训练,构建评估模型;最后将该评估模型应用于某高铁桥梁施工安全风险评估.结果表明:该模型能够较全面地评估主要风险因素对高铁桥梁施工的影响,预测得到的风险等级与专家评审结果一致,且该方法更加简洁实用,便于编制标准化的算法程序,可为高铁工程项目的施工安全风险等级评估提供参考.

目前缺少地震断裂带对高速铁路跨越工程影响的系统性研究.以徐连高铁跨越郯庐断裂带为例,结合现有研究成果和规范要求,研究了郯庐断裂带对徐连高铁的影响,并采取针对性的工程措施.研究认为,郯庐断裂带潍坊—嘉山段(又称沂沭断裂带),仅安丘—莒县断裂(f5)为全新世活动断层,推测如果未来发生地震将产生地表水平、垂直断错,同时蠕滑变形将超出无砟轨道的可调节范围.为减少地震断裂带对工程的影响,线路采用十字交叉方案跨越地震断裂带,影响范围内采用更易修复的路基结构和有砟轨道,范围外桥梁结构进行抗震加强设计,优先选择简支梁结构,大跨度结构进行隔震设计;并对新沂南站进行安全距离避让选址.同时建议在该区域内建立地震预警系统加强地震监测,为高速铁路运营提供安全保障.

Background Mesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. TATA box-binding protein (TBP) and TBP-like factors are general transcription factors responsible for the transcription initiation by recruiting the preinitiation complex to promoter regions. However, the role of TBP family members in the regulation of mesendodermal specification remains largely unknown. Methods We used an in vitro mesendodermal differentiation system of human embryonic stem cells (hESCs), combining with the microarray and quantitative polymerase chain reaction (qRT-PCR) analysis, loss of function and gain of function to determine the function of the TBP family member TBP-related factor 3 (TRF3) during mesendodermal differentiation of hESCs. The chromatin immunoprecipitation (ChIP) and biochemistry analysis were used to determine the binding of TRF3 to the promoter region of key mesendodermal genes. Results The mesendodermal differentiation of hESCs was confirmed by the microarray gene expression profile, qRT-PCR, and immunocytochemical staining. The expression of TRF3 mRNA was enhanced during mesendodermal differentiation of hESCs. The TRF3 deficiency did not affect the pluripotent marker expression, alkaline phosphatase activity, and cell cycle distribution of undifferentiated hESCs or the expression of early neuroectodermal genes during neuroectodermal differentiation. During the mesendodermal differentiation, the expression of pluripotency markers decreased in both wild-type and TRF3 knockout (TRF3 −/− ) cells, while the TRF3 deficiency crippled the expression of the mesendodermal markers. The reintroduction of TRF3 into the TRF3 −/− hESCs rescued inhibited mesendodermal differentiation. Mechanistically, the TRF3 binding profile was significantly shifted to the mesendodermal specification during mesendodermal differentiation of hESCs based on the ChIP-seq data. Moreover, ChIP and ChIP-qPCR analysis showed that TRF3 was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN , BRACHYURY , mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of hESCs. Conclusions These results reveal that the TBP family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation. However, it directs mesendodermal lineage commitment of hESCs via specifically promoting the transcription of key mesendodermal transcription factors. These findings provide new insights into the function and mechanisms of the TBP family member in hESC early lineage specification.

Dear Editor, Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are of significant translational value to in vitro studies of human cardiac development, drug and cardiotoxicity testing and cardiac disease modelling.Differentiation of hPSCs to CMs,however, yields mixed cultures of atrial-, ventricular-, and pacemaker-like cells as well as non-CMs in variable proportions.1

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in biomedical applications. However, the immature state of cardiomyocytes obtained using existing protocols limits the application of hPSC-CMs. Unlike adult cardiac myocytes, hPSC-CMs generate ATP through an immature metabolic pathway-aerobic glycolysis, instead of mitochondrial oxidative phosphorylation (OXPHOS). Hence, metabolic switching is critical for functional maturation in hPSC-CMs. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is a key regulator of mitochondrial biogenesis and metabolism, which may help promote cardiac maturation during development. In this study, we investigated the effects of PGC-1α and its activator ZLN005 on the maturation of human embryonic stem cell-derived cardiomyocyte (hESC-CM). hESC-CMs were generated using a chemically defined differentiation protocol and supplemented with either ZLN005 or DMSO (control) on differentiating days 10 to 12. Biological assays were then performed around day 30. ZLN005 treatment upregulated the expressions of PGC-1α and mitochondrial function-related genes in hESC-CMs and induced more mature energy metabolism compared with the control group. In addition, ZLN005 treatment increased cell sarcomere length, improved cell calcium handling, and enhanced intercellular connectivity. These findings support an effective approach to promote hESC-CM maturation, which is critical for the application of hESC-CM in disease modeling, drug screening, and engineering cardiac tissue.

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes.

为建造“安全、舒适”高速铁路,需严格控制桥梁的刚度和变形,以满足线路平顺性要求.结合高速铁路桥梁工程技术要求,按常规跨度桥梁、车站道岔区桥梁、大跨度桥梁3类总结高速铁路桥梁主要设计原则.根据大跨度桥梁刚度、变形控制技术难点,依托工程实例研究梁端转角、结构徐变和桩基沉降控制措施.对梁端转角、墩台刚度、挠跨比等指标进行优化,提出采用曲率半径作为统一刚度指标的建议,给出连续梁固定墩纵向刚度、大跨度桥梁挠跨比的经验限值,建议进一步深入研究梁端转角限值、墩台横向刚度指标.以福厦高铁3×70 m无支座整体式连续刚构桥、昌赣客专赣州赣江特大桥和商合杭高铁裕溪河特大桥为例,介绍高速铁路桥梁刚度变形控制技术创新实践,可供类似桥梁工程参考.

Histone methyltransferases play a critical role in early human development, whereas their roles and precise mechanisms are less understood. SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase induced during early differentiation of human embryonic stem cells (hESCs), but little is known about its function in undifferentiated hESCs and in their early lineage fate decision as well as underlying mechanisms. Here, we explored the role of SMYD2 in the self-renewal and mesendodermal lineage commitment of hESCs. We demonstrated that the expression of SMYD2 was significantly enhanced during mesendodermal but not neuroectodermal differentiation of hESCs. SMYD2 knockout (SMYD2-/- ) did not affect self-renewal and early neuroectodermal differentiation of hESCs, whereas it blocked the mesendodermal lineage commitment. This phenotype was rescued by reintroduction of SMYD2 into the SMYD2-/- hESCs. Mechanistically, the bindings of SMYD2 at the promoter regions of critical mesendodermal transcription factor genes, namely, brachyury (T), eomesodermin (EOMES), mix paired-like homeobox (MIXL1), and goosecoid homeobox (GSC) were significantly enhanced during mesendodermal differentiation of SMYD2+/+ hESCs but totally suppressed in SMYD2-/- ones. Concomitantly, such a suppression was associated with the remarkable reduction of methylation at histone 3 lysine 4 and lysine 36 but not at histone 4 lysine 20 globally and specifically on the promoter regions of mesendodermal genes, namely, T, EOMES, MIXL1, and GSC. These results reveal that the histone methyltransferase SMYD2 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation, but it promotes the mesendodermal differentiation of hESCs through the epigenetic control of critical genes to mesendodermal lineage commitment. Stem Cells 2019;37:1401-1415.

创新来源于基层,并实践应用于基层,基层是科技创新的基础和源泉.结合笔者从事基层科技创新管理工作经历及调研情况就基层科技创新管理现状及如何提升基层科技创新管理能力进行探讨.

墩顶竖向变形是高速铁路桥梁安全的重要参数之一.在温度效应下,相邻桥墩高差较大时,桥梁会出现竖向变位差,造成轨道不平顺,这将影响高速铁路的舒适性和安全性.目前国内各类规范对桥墩竖向变形的限值规定不尽相同,为了研究大高差桥墩顶竖向变形的温度效应影响,以合福高铁巷坑大桥为例,对大高差桥墩在温度效应下的墩顶竖向变形进行现场测量,将测量和计算结果同各类规范进行对比,进一步对考虑温度效应时车辆通过桥梁结构时耦合响应进行仿真分析,结果表明:巷坑大桥桥墩顶竖向变形差超过规范限值,但车辆通行时的舒适性和安全性均能满足规范要求,车辆走行性偏于安全,合福高铁通车至今该桥一直运营正常.综上所述,现行规范对大跨度桥梁桥墩的变形指标要求偏严,特殊情况下无法满足规范要求时,建议参考公式,适当放松验收指标.本研究对突破规范限制,在大跨度桥梁铺设无砟轨道具有重要的意义.

烤烟移栽期适宜与否对烤烟的生长发育、成熟落黄和烟叶产质量有重要影响.本文分析了温度、降雨量、日照时数等气候条件对烤烟移栽期的影响,并对不同移栽期烤烟的产质量进行比较,并根据结果提出了临朐烟区烤烟移栽的适宜期.

To investigate the interaction effects of different planting density and nitrogen (N) application amount on tobacco leaf development and tissue structures, a split-plot field experiment with three different planting densities (main district) and two different N application amounts (deputy district) was carried out using the cultivar NC102. Through paraffin section method, the tissue structure of middle leaves with different leaf ages (10, 20, 40 and 73 d) were observed. The results showed that leaf thickness, palisade tissue thickness, spongy tissue thickness and leaf area increased with leaf aging, but palisade cell density decreased. Leaf thickness, palisade tissue thickness, spongy tissue thickness, leaf area and specific leaf weight increased but palisade cell density decreased with the decrease of planting density and increased N application rate. The action time of N application amount on palisade cell density was earlier than that of planting density, while the timing of effects of these two treatments was the opposite for palisade tissue thickness. There was a positive interaction between low planting density and high N application amount on leaf thickness, palisade tissue thickness, ratio of palisade tissue thickness and spongy tissue thickness, leaf area and specific leaf weight when leaf age was 73 d. The increase of N application amount showed more significant impact under lower planting densities. Adopting appropriate planting density and N application amount and taking advantage of their interaction could improve tissue structures of middle leaves and ultimately improve the quality of tobacco under proper yield.

Emerging evidence suggests that Ca2+ signals are important for the self-renewal and differentiation of human embryonic stem cells (hESCs). However, little is known about the physiological and pharmacological properties of the Ca2+-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca2+-handling proteins; we also used confocal Ca2+ imaging and pharmacological approaches to determine the contribution of the Ca2+-handling machinery to the regulation of Ca2+ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca2+-handling-related genes. ATP-induced Ca2+ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IP3R) blocker 2-APB or xestospongin C. In addition, Ca2+ transients were induced by a ryanodine receptor (RyR) activator, caffeine, in 10%-15% of hESCs and were blocked by ryanodine, whereas caffeine and ATP did not have additive effects. Moreover, store-operated Ca2+ entry (SOCE) but not voltage-operated Ca2+ channel-mediated Ca2+ entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca2+ concentration ([Ca2+]i). For the Ca2+ extrusion pathway, inhibition of plasma membrane Ca2+ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca2+]i, whereas the Na+/Ca2+ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca2+]i. Taken together, increased [Ca2+]i is mainly mediated by Ca2+ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca2+-signaling machinery in hESCs; maintenance of low [Ca2+]i is mediated by uptake of cytosolic Ca2+ into the ER via SERCA and extrusion of Ca2+ out of cells via NCX and PMCA in hESCs.

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8-/Y) model, we found that deletion of phf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8+/Y) and ph8-/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8-/Y by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8-/Y ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways.

The advantages of water and fertilizer coupling in flue-cured tobacco production were analyzed from aspects of reducing tobacco field disease, improving economic benefits, reducing production cost and improving soil structure, the key points of flue-cured tobacco drip ir-rigation under plastic film and fertilization technology were summarized.

波尔多液是一种广谱、持效、保护性杀菌剂,可用于防治烟草不同生育期常见的多种病害,如烟草炭疽病、猝倒病、野火病、角斑病、赤星病等.研究了波尔多液在烟草生产中的施用效果,为波尔多液在烟草生产中的更广泛推广提供依据.

Technology optimization and application of Loose-leaf Baking in Bulk Curing Barn of baking mode and roasting technology was studied by contrasting loose leaf basket with comb type tobacco clamp and tobacco stems,on the basis of the same fertility,tobacco varieties,cultivation and management level.Results showed that the capacity of Loose Leaf Baking increased 26% and 30% than that of comb type tobacco clamp and tobacco stems.The cost decreased 0.17 yuan and 0.18 yuan per kilogram.The rate of first-class tobacco leaf increased 10.1% and 13.23%,the mean price increased 2.42 yuan and 3.04 yuan per kilogram.Baking barn capacity was raised,reduce baking cost was decreased,and the income of tobacco grower was increased.