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提 要 本研究以四篇不同文本複雜度(Text complexity)之短文為實驗材料, 共31位日籍華語學習者以紙筆施測與有聲思考(Think Aloud), 探究華語學習者之二語程度(L2 proficiency)與文本複雜度對詞彙猜測和閱讀理解的影響。結果顯示: (一)各文本之文意理解和詞義選擇達顯著差異(F = 8.290, p = .000; F = 28.226, p = .000), 閱讀理解則否; (二)二語能力中的詞彙知識和句法知識分別與閱讀理解及詞彙猜測表現皆達顯著正相關(r = .586, p = .001; r = .666, p = .000; r = .364, p = .044; r = 0.535, p = .002), 句法知識可代表解釋44.4%的閱讀理解變異及28.6%的詞彙猜測表現變異; (三)不論詞義透明度高低, 猜測最常用知識來源皆為文本線索。

Triple-negative breast cancer lacks significant expression of the estrogen receptor, the progesterone receptor, and of human epidermal growth factor receptor. It is the most aggressive and malignant of all breast cancers, and for which there are currently no effective targeted therapies. We have shown previously that the RecQ helicase family member RECQL5 is essential for the proliferation and survival of TNBC cells; however, the mechanism of its involvement in cell viability has not been shown. Here we report that the expression of RecQ family helicases, including RECQL5, are regulated by the deubiquitinase USP28. We found using genetic depletion or a small molecule inhibitor that like RECQL5, USP28 is also essential for TNBC cells to proliferate in vitro and in vivo. Compromising the function of USP28 by shRNA knockdown or the inhibitor caused TNBC cells to arrest in S/G2 phases, concurrent with DNA damage checkpoint activation. We further showed that the small molecule inhibitor of USP28 displayed anti-tumor activity against xenografts derived from TNBC cells. Our results suggest that USP28 could be a potential therapeutic target for triple negative breast cancer.

Background: Triple-negative breast cancer lacks significant expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. It is the more aggressive and malignant kind of breast cancers and is currently without any effective targeted therapies.Methods: We have screened for genes in the ubiquitin-proteasome system that are essential for the proliferation and survival of TNBC cells via CRISPR/Cas9-mediated gene editing. Growth of TNBC cells were assayed using cell and tumor xenograft models to validate the vital role of USP28. We employed cell biology and biochemical methods to uncover the mechanisms underlying the requirement of USP28 for the proliferation of TNBC cells.Results: USP28, a deubiquitin enzyme, is an essential gene for TNBC cells in vitro and in vivo. Compromising the function of USP28 causes TNBC cells to arrest in S/G2 phases with DNA damage checkpoint activation. We show that RecQ family helicases are regulated by USP28, which is more important in TNBC cells than in other breast cancer cells. We further showed that a small molecule inhibitor of USP28 displayed anti-tumor activities against xenografts derived from TNBC cells.Conclusion: Our data establish a critical role played by USP28 in supporting the proliferation and viability of triple negative breast cancer cells through stabilizing RecQ family helicases and support USP28 as a therapeutic target for TNBC.

Triple‐negative breast cancer (TNBC) is a malignancy that currently lacks targeted therapies. The majority of TNBCs can be characterized as basal‐like and has an expression profile enriched with genes involved in DNA damage repair and checkpoint response. Here, we report that TNBC cells are under replication stress and are constantly generating DNA double‐strand breaks, which is not seen in non‐TNBC cells. Consequently, we found that RECQL5, which encodes a RecQ family DNA helicase involved in many aspects of DNA metabolism including replication and repair, was essential for TNBC cells to survive and proliferate in vitro and in vivo. Compromising RECQL5 function in TNBC cells results in persistence of DNA damage, G2 arrest, and ultimately, cessation of proliferation. Our results suggest RECQL5 may be a potential therapeutic target for TNBC.

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APCCdh1 plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1. We show that the removal of USP1 depends on APCCdh1 and requires Chk1 activation known to be catalysed by ssDNA-RPA-ATR signalling at the ends designated for HDR, linking the status of end processing to RIF1 or BRCA1 recruitment., The choice between homologous recombination and non-homologous end-joining is largely influenced by cell cycle. Here the authors show that APCCdh1 promotes homologous recombination by removing USP1, allowing polyubiquitinated histones to recruit BRCA1.

Cdh1 is one of the two adaptor proteins of anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase controlling mitosis and DNA replication. To date, the in vivo functions of Cdh1 have not been fully explored. In order to characterize Cdh1 in liver regeneration, we generated a conditional knock-out mouse model. Our data showed that loss of Cdh1 leads to increased and extended S phase progression possibly due to the upregulation of cyclin D1. Moreover, the increased DNA replication resulted in activated DNA damage response. Interestingly, the final liver weight after partial hepatectomy in the Cdh1 depleted mice did not differ from that of the controls, implying that Cdh1 is not required for the competence of hepatocytes to regenerate itself.

Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

The CRISPR-Cas9 genome editing system has been widely used in multiple cells and organisms. Here we developed a CRISPR-Cas9 based in vitro large DNA vector editing system, using the Ad5-based vector as an example. We demonstrate use of this system to generate targeted mutations, in-frame gene deletion, and gene replacement. This in vitro CRISPR editing system exhibits high efficiency and accuracy. We believe this system can be applied in a variety of experimental settings.

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). The choice between these two pathways is largely influenced by cell cycle phases. HDR can occur only in S/G2 when sister chromatid can provide homologous templates, whereas NHEJ can take place in all phases of the cell cycle except mitosis. Central to NHEJ repair is the Ku70/80 heterodimer which forms a ring structure that binds DSB ends and serves as a platform to recruit factors involved in NHEJ. Upon completion of NHEJ repair, DNA double strand-encircling Ku dimers have to be removed. The removal depends on ubiquitylation and proteasomal degradation of Ku80 by the ubiquitin E3 ligases RNF8. Here we report that RNF8 is a substrate of APC(Cdh1) and the latter keeps RNF8 level in check at DSBs to prevent premature turnover of Ku80.

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). The choice between these two pathways is largely influenced by cell cycle phases. HDR can occur only in S/G2 when sister chromatid can provide homologous templates, whereas NHEJ can take place in all phases of the cell cycle except mitosis. Central to NHEJ repair is the Ku70/80 heterodimer which forms a ring structure that binds DSB ends and serves as a platform to recruit factors involved in NHEJ. Upon completion of NHEJ repair, DNA double strand-encircling Ku dimers have to be removed. The removal depends on ubiquitylation and proteasomal degradation of Ku80 by the ubiquitin E3 ligases RNF8. Here we report that RNF8 is a substrate of APCCdh1 and the latter keeps RNF8 level in check at DSBs to prevent premature turnover of Ku80.

Climate financing is a key issue in current negotiations on climate protection. This study establishes a climate financing model based on a mechanism in which donor countries set up funds for climate financing and recipient countries use the funds exclusively for carbon emission reduction. The burden-sharing principles are based on GDP, historical emissions, and consumptionbased emissions. Using this model, we develop and analyze a series of scenario simulations, including a financing program negotiated at the Cancun Climate Change Conference (2010) and several subsequent programs. Results show that sustained climate financing can help to combat global climate change. However, the Cancun Agreements are projected to result in a reduction of only 0.01°C in global warming by 2100 compared to the scenario without climate financing. Longer-term climate financing programs should be established to achieve more significant benefits. Our model and simulations also show that climate financing has economic benefits for developing countries. Developed countries will suffer a slight GDP loss in the early stages of climate financing, but the longterm economic growth and the eventual benefits of climate mitigation will compensate for this slight loss. Different burden-sharing principles have very similar effects on global temperature change and economic growth of recipient countries, but they do result in differences in GDP changes for Japan and the FSU. The GDP-based principle results in a larger share of financial burden for Japan, while the historical emissions-based principle results in a larger share of financial burden for the FSU. A larger burden share leads to a greater GDP loss.

Liver sinusoidal endothelial cells (LSECs) have great capacity for liver regeneration, and this capacity can easily switch to profibrotic phenotype, which is still poorly understood. In this study, we elucidated a potential target in LSECs for regenerative treatment that can bypass fibrosis during chronic liver injury. Proregenerative LSECs can be transformed to profibrotic phenotype after 4 weeks of carbon tetrachloride administration or 10 days of bile duct ligation. This phenotypic alternation of LSECs was mediated by extracellular regulated protein kinases 1 and 2 (Erk1/2)-Akt axis switch in LSECs during chronic liver injury; Erk1/2 was normally associated with maintenance of the LSEC proregenerative phenotype, inhibiting hepatic stellate cell (HSC) activation and promoting tissue repair by enhancing nitric oxide (NO)/reactive oxygen species (ROS) ratio and increasing expression of hepatic growth factor (HGF) and Wingless-type MMTV integration site family member 2 (Wnt2). Alternatively, Akt induced LSEC profibrotic phenotype, which mainly stimulated HSC activation and concomitant senescence by reducing NO/ROS ratio and decreasing HGF/Wnt2 expression. LSEC-targeted adenovirus or drug particle to promote Erk1/2 activity can alleviate liver fibrosis, accelerate fibrosis resolution, and enhance liver regeneration. This study demonstrated that the Erk1/2-Akt axis acted as a switch to regulate the proregenerative and profibrotic phenotypes of LSECs, and targeted therapy promoted liver regeneration while bypassing fibrosis, providing clues for a more effective treatment of liver diseases.

Fat mass and obesity-associated gene (FTO) is a member of the Fe (II)- and oxoglutarate-dependent AlkB dioxygenase family and is linked to both obesity and intellectual disability. The role of FTO in neurodevelopment and neurogenesis, however, remains largely unknown. Here we show that FTO is expressed in adult neural stem cells and neurons and displays dynamic expression during postnatal neurodevelopment. The loss of FTO leads to decreased brain size and body weight. We find that FTO deficiency could reduce the proliferation and neuronal differentiation of adult neural stem cells in vivo, which leads to impaired learning and memory. Given the role of FTO as a demethylase of N6-methyladenosine (m6A), we went on to perform genome-wide m6A profiling and observed dynamic m6A modification during postnatal neurodevelopment. The loss of FTO led to the altered expression of several key components of the brain derived neurotrophic factor pathway that were marked by m6A. These results together suggest FTO plays important roles in neurogenesis, as well as in learning and memory.

Cdh1 is one of the two adaptor proteins of anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase controlling mitosis and DNA replication. To date, the in vivo functions of Cdh1 have not been fully explored. In order to characterize Cdh1 in liver regeneration, we generated a conditional knock-out mouse model. Our data showed that loss of Cdh1 leads to increased and extended S phase progression possibly due to the upregulation of cyclin D1. Moreover, the increased DNA replication resulted in activated DNA damage response. Interestingly, the final liver weight after partial hepatectomy in the Cdh1 depleted mice did not differ from that of the controls, implying that Cdh1 is not required for the competence of hepatocytes to regenerate itself.

Background ADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that play roles in membrane dynamics and vesicle trafficking. AGEF-1, which is thought to act as a guanine nucleotide exchange factor of class I ARFs, is required for caveolin-1 body formation and receptor-mediated endocytosis in oocytes of Caenorhabditis elegans . This study explores additional roles of AGEF-1 in endocytic transport. Methods agef-1 expression was knocked down by using RNAi in C. elegans . Markers that allow analysis of endocytic transport in scavenger cells were investigated for studying the effect of AGEF-1 on different steps of membrane transport. Results Knockdown of AGEF-1 levels results in two apparent trafficking defects in coelomocytes of C. elegans . First, there is a delay in the uptake of solutes from the extracellular medium. Second, there is a dramatic enlargement of the sizes of lysosomes, even though lysosomal acidification is normal and degradation still occurs. Conclusion Our results suggest that AGEF-1 regulates endosome/lysosome fusion or fission events, in addition to earlier steps in endocytic transport. General significance AGEF-1 is the first identified GTPase regulator that functions at the lysosome fusion or fission stage of the endocytic pathway. Our study provides insight into lysosome dynamics in C. elegans .

Ethanol affects the formation of learning and memory in many species. However, the molecular mechanisms underlying the behavioral effects of ethanol are still poorly understood. In Caenorhabditis elegans, gustatory plasticity is a simple learning paradigm, in which animals after prolonged pre-exposure to a chemo-attractive salt in the absence of food show chemo-aversion to this salt during subsequent chemotaxis test stage. We characterized the effect of ethanol on this simple learning model. Our results showed that ethanol administration interfered with gustatory plasticity during pre-exposure or test stage in well-fed worms. Genetic analysis revealed that one mutant previously implicated involved in acute ethanol responses, slo-1, as well as two mutants with defects in serotonin synthesis, tph-1 and bas-1, failed to exhibit ethanol interference with gustatory plasticity. Furthermore, two metabotropic serotonin receptors, SER-4 and SER-7, were found to be involved in ethanol-mediated gustatory plasticity. In addition, the tph-1 and ser-4 loci were also involved in ethanol's effect on locomotion behavior. These data suggested an essential role of serotonin signaling in modulating acute effects of ethanol.

Caenorhabditis elegans, one of the widely studied model organisms, sense external chemical cues and perform relative chemotaxis behaviors through its simple chemosensory neuronal system. To study the mechanism underlying chemosensory behavior, a rapid and reliable method for quantitatively analyzing the worms' behaviors is essential. In this work, we demonstrated a microfluidic approach for investigating chemotaxis responses of worms to chemical gradients. The flow-based microfluidic chip was consisted of circular tree-like microchannels, which was able to generate eight flow streams containing stepwise chemical concentrations without the difference in flow velocity. Worms' upstream swimming into microchannels with various concentrations was monitored for quantitative analysis of the chemotaxis behavior. By using this microfluidic chip, the attractive and repellent responses of C. elegans to NaCl were successfully quantified within several minutes. The results demonstrated the wild type-like repellent responses and severely impaired attractive responses in grk-2 mutant animals with defects in calcium influx. In addition, the chemotaxis analysis of the third stage larvae revealed that its gustatory response was different from that in the adult stage. Thus, our microfluidic method provided a useful platform for studying the chemosensory behaviors of C. elegans and screening of chemosensation-related chemical drugs.

The propagation of intercellular calcium wave (ICW) is essential for coordinating cellular activities in multicellular organisms. However, the limitations of existing analytical methods hamper the studies of this biological process in live animals. In this paper, we demonstrated for the first time a novel microfluidic system with an open chamber for on-chip microinjection of C. elegans and investigation of ICW propagations in vivo. Worms were long-term immobilized on the side wall of the open chamber by suction. Using an external micro-manipulator, localized chemical stimulation was delivered to single intestinal cells of the immobilized worms by microinjection. The calcium dynamics in the intestinal cells expressing Ca(2+) indicator YC2.12 was simultaneously monitored by fluorescence imaging. As a result, thapsigargin injection induced ICW was observed in the intestinal cells of C. elegans. Further analysis of the ICW propagation was realized in the presence of heparin (an inhibitor for IP3 receptor), which allowed us to investigate the mechanism underlying intercellular calcium signaling. We expect this novel microfluidic platform to be a useful tool for studying cell-cell communication in multicellular organisms in vivo.

Crowding stress has been reported to play an important role in affecting physiological behaviour. To study this process, a reliable analytical method under confined space is essential. In this work, we demonstrated a microfluidic approach for investigating physiological responses of C. elegans to confined spaces. The PDMS microfluidic chip consisting of arrays of micro-columns enabled us to mimic different crowding conditions by changing the intervals among micro-columns. C. elegans were transferred into this micro-column array and the subcellular distribution of DAF-16, which is a well-known transcription factor regulating different stress responses, was monitored for analysing the physiological responses to the confined spaces. We found that the worms exhibited a gradual increase in DAF-16 nuclear localization in the micro-column array with intervals from 200 μm to 40 μm. Moreover, the results showed that the absence of food and crowding stress could cooperate to promote increased DAF-16 nuclear localization. Finally, loss-of-function mutations in mec-4 and mec-10, which are amiloride-sensitive Na(+) channel genes expressed in all six gentle touch neurons, accelerated the velocity of DAF-16 nuclear localization, induced by confined space, revealing that mec-4/mec-10 were not required for this stress response. Thus, this device will provide a versatile, reliable and controllable platform for crowding stress studies.

The priority of buyer order is a key issue in production scheduling in MTO (make to order) enterprises. In view of the deficiencies in current studies related to the assessment of the priority, a new emerging method for determining the priority in supply chain based on radial basis function (RBF) neural network is put forward which considers the constraint in supply chain and the complicated relation between the evaluation-index system and the priority. The evaluation-index system covers the constraints or determining factors not only in the enterprise and buyers but also in supply chain, and hence is a more complete and effective. A 3-layer RBF neural network model which takes form of the Gauss Function is built for determining the priority of buyer order. And finally by the testing and contradistinctive analysis of the three different methods, this study showed that the evaluation method ground on RBF neural network exceeds that on traditional AHP and BP neural network under supply chain environment, the assessment result is more accurate while the model training time is shorter using the RBF neural network in supply chain.

We developed an adenovirus-based CRISPR/Cas9 system for gene editing in vivo. In the liver, we demonstrated that the system could reach the level of tissue-specific gene knockout, resulting in phenotypic changes. Given the wide spectrum of cell types susceptible to adenoviral infection, and the fact that adenoviral genome rarely integrates into its host cell genome, we believe the adenovirus-based CRISPR/Cas9 system will find applications in a variety of experimental settings.