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A Journey to the West: The Ancient Dispersal of Rice Out of East Asia
Robert N. Spengler, Sören Stark, Xinying Zhou, Daniel Fuks, Li Tang, Basira Mir-Makhamad, Rasmus Bjørn, Hongen Jiang, Luca M. Olivieri, Alisher Begmatov, Nicole Boivin
Rice is one of the most culturally valued and widely grown crops in the world today, and extensive research over the past decade has clarified much of the narrative of its domestication and early spread across East and South Asia. However, the timing and routes of its dispersal into West Asia and Europe, through which rice eventually became an important ingredient in global cuisines, has remained less clear. In this article, we discuss the piecemeal, but growing, archaeobotanical data for rice in West Asia. We also integrate written sources, linguistic data, and ethnohistoric analogies, in order to better understand the adoption of rice outside its regions of origin. The human-mediated westward spread of rice proceeded gradually, while its social standing and culinary uses repeatedly changing over time and place. Rice was present in West Asia and Europe by the tail end of the first millennium BC, but did not become a significant crop in West Asia until the past few centuries. Complementary historical, linguistic, and archaeobotanical data illustrate two separate and roughly contemporaneous routes of westward dispersal, one along the South Asian coast and the other through Silk Road trade. By better understanding the adoption of this water-demanding crop in the arid regions of West Asia, we explore an important chapter in human adaptation and agricultural decision making.
Agricultural intensification
Archaeobotany
Crop exchange
Paddy farming
Dive into the research topics of 'A Journey to the West: The Ancient Dispersal of Rice Out of East Asia'. Together they form a unique fingerprint.
West Asia Agriculture & Biology 100%
East Asia Agriculture & Biology 92%
rice Earth & Environmental Sciences 72%
Asia Earth & Environmental Sciences 67%
crop Earth & Environmental Sciences 29%
crops Agriculture & Biology 22%
cuisine Agriculture & Biology 19%
Europe Earth & Environmental Sciences 18%
Spengler, R. N., Stark, S., Zhou, X., Fuks, D., Tang, L., Mir-Makhamad, B., Bjørn, R., Jiang, H., Olivieri, L. M., Begmatov, A., & Boivin, N. (2021). A Journey to the West: The Ancient Dispersal of Rice Out of East Asia. Rice, 14(1), [83]. https://doi.org/10.1186/s12284-021-00518-4
Spengler, Robert N. ; Stark, Sören ; Zhou, Xinying et al. / A Journey to the West : The Ancient Dispersal of Rice Out of East Asia. In: Rice. 2021 ; Vol. 14, No. 1.
@article{fa07788c31b3431c82db643cb1504b4e,
title = "A Journey to the West: The Ancient Dispersal of Rice Out of East Asia",
abstract = "Rice is one of the most culturally valued and widely grown crops in the world today, and extensive research over the past decade has clarified much of the narrative of its domestication and early spread across East and South Asia. However, the timing and routes of its dispersal into West Asia and Europe, through which rice eventually became an important ingredient in global cuisines, has remained less clear. In this article, we discuss the piecemeal, but growing, archaeobotanical data for rice in West Asia. We also integrate written sources, linguistic data, and ethnohistoric analogies, in order to better understand the adoption of rice outside its regions of origin. The human-mediated westward spread of rice proceeded gradually, while its social standing and culinary uses repeatedly changing over time and place. Rice was present in West Asia and Europe by the tail end of the first millennium BC, but did not become a significant crop in West Asia until the past few centuries. Complementary historical, linguistic, and archaeobotanical data illustrate two separate and roughly contemporaneous routes of westward dispersal, one along the South Asian coast and the other through Silk Road trade. By better understanding the adoption of this water-demanding crop in the arid regions of West Asia, we explore an important chapter in human adaptation and agricultural decision making.",
keywords = "Agricultural intensification, Archaeobotany, Crop exchange, Paddy farming, Rice, West Asia",
author = "Spengler, {Robert N.} and S{\"o}ren Stark and Xinying Zhou and Daniel Fuks and Li Tang and Basira Mir-Makhamad and Rasmus Bj{\o}rn and Hongen Jiang and Olivieri, {Luca M.} and Alisher Begmatov and Nicole Boivin",
note = "Funding Information: Research funds and support were provided by the Max Planck Institute for the Science of Human History and the European Research Council, grant number 851102, Fruits of Eurasia: Domestication and Dispersal (FEDD), and the NSFC (41672171). Publisher Copyright: {\textcopyright} 2021, The Author(s).",
journal = "Rice",
publisher = "Springer Publishing Company",
Spengler, RN, Stark, S, Zhou, X, Fuks, D, Tang, L, Mir-Makhamad, B, Bjørn, R, Jiang, H, Olivieri, LM, Begmatov, A & Boivin, N 2021, 'A Journey to the West: The Ancient Dispersal of Rice Out of East Asia', Rice, vol. 14, no. 1, 83. https://doi.org/10.1186/s12284-021-00518-4
A Journey to the West : The Ancient Dispersal of Rice Out of East Asia. / Spengler, Robert N.; Stark, Sören; Zhou, Xinying et al.
In: Rice, Vol. 14, No. 1, 83, 01.12.2021.
T1 - A Journey to the West
T2 - The Ancient Dispersal of Rice Out of East Asia
AU - Spengler, Robert N.
AU - Stark, Sören
AU - Zhou, Xinying
AU - Fuks, Daniel
AU - Tang, Li
AU - Mir-Makhamad, Basira
AU - Bjørn, Rasmus
AU - Jiang, Hongen
AU - Olivieri, Luca M.
AU - Begmatov, Alisher
AU - Boivin, Nicole
N1 - Funding Information: Research funds and support were provided by the Max Planck Institute for the Science of Human History and the European Research Council, grant number 851102, Fruits of Eurasia: Domestication and Dispersal (FEDD), and the NSFC (41672171). Publisher Copyright: © 2021, The Author(s).
N2 - Rice is one of the most culturally valued and widely grown crops in the world today, and extensive research over the past decade has clarified much of the narrative of its domestication and early spread across East and South Asia. However, the timing and routes of its dispersal into West Asia and Europe, through which rice eventually became an important ingredient in global cuisines, has remained less clear. In this article, we discuss the piecemeal, but growing, archaeobotanical data for rice in West Asia. We also integrate written sources, linguistic data, and ethnohistoric analogies, in order to better understand the adoption of rice outside its regions of origin. The human-mediated westward spread of rice proceeded gradually, while its social standing and culinary uses repeatedly changing over time and place. Rice was present in West Asia and Europe by the tail end of the first millennium BC, but did not become a significant crop in West Asia until the past few centuries. Complementary historical, linguistic, and archaeobotanical data illustrate two separate and roughly contemporaneous routes of westward dispersal, one along the South Asian coast and the other through Silk Road trade. By better understanding the adoption of this water-demanding crop in the arid regions of West Asia, we explore an important chapter in human adaptation and agricultural decision making.
AB - Rice is one of the most culturally valued and widely grown crops in the world today, and extensive research over the past decade has clarified much of the narrative of its domestication and early spread across East and South Asia. However, the timing and routes of its dispersal into West Asia and Europe, through which rice eventually became an important ingredient in global cuisines, has remained less clear. In this article, we discuss the piecemeal, but growing, archaeobotanical data for rice in West Asia. We also integrate written sources, linguistic data, and ethnohistoric analogies, in order to better understand the adoption of rice outside its regions of origin. The human-mediated westward spread of rice proceeded gradually, while its social standing and culinary uses repeatedly changing over time and place. Rice was present in West Asia and Europe by the tail end of the first millennium BC, but did not become a significant crop in West Asia until the past few centuries. Complementary historical, linguistic, and archaeobotanical data illustrate two separate and roughly contemporaneous routes of westward dispersal, one along the South Asian coast and the other through Silk Road trade. By better understanding the adoption of this water-demanding crop in the arid regions of West Asia, we explore an important chapter in human adaptation and agricultural decision making.
KW - Agricultural intensification
KW - Archaeobotany
KW - Crop exchange
KW - Paddy farming
KW - Rice
KW - West Asia
JO - Rice
JF - Rice
Spengler RN, Stark S, Zhou X, Fuks D, Tang L, Mir-Makhamad B et al. A Journey to the West: The Ancient Dispersal of Rice Out of East Asia. Rice. 2021 Dec 1;14(1):83. doi: 10.1186/s12284-021-00518-4


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NFLS Sponsors Pulitzer Prize Nominee Nigel Poor at WLA Fall Conference
Earlier this year, the NFLS Board agreed to sponsor one of the keynote speakers, Nigel Poor, at the upcoming WLA Fall Conference in Green Bay.
Poor is a volunteer at San Quentin Prison and co-hosts the podcast "Ear Hustle." The podcast is a partnership between Poor, a Bay Area visual artist, and Earlonne Woods, formerly incarcerated at San Quentin. The podcast was also co-founded with former San Quentin resident Antwan Williams. Poor currently co-hosts with Rahsaan "New York" Thomas, an inmate at the prison.
On May 4, 2020, Ear Hustle was nominated for a Pulitzer Prize for Audio Reporting. Audio Reporting is a new category for 2020 given the rise in the number and popularity of podcasts. The new prize was awarded for "a distinguished example of audio journalism that serves the public interest, characterized by revelatory reporting and illuminating storytelling.''
The Ear Hustle team works in the San Francisco Bay Area, both in San Quentin State Prison's media lab and from offices on the outside, to produce stories that are sometimes difficult, often funny and always honest. Episodes offer a nuanced view of people involved with the American prison system and those reintegrating into society after serving time.
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You can also check out an episode recorded in Wisconsin at a juvenile facility in Madison: https://www.earhustlesq.com/episodes/2020/5/13/catch-a-kite-5

redpajama_set_name: RedPajamaCommonCrawl


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Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity
Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity Zheng, Dianpeng; Ye, Sichao; Wang, Xiuyun; Zhang, Yongjun; Yan, Daoyu; Cai, Xiangsheng; Gao, Weihong; Shan, Hongbo; Gao, Yang; Chen, Juanjuan; Hu, Zhiming; Li, Hongwei; Li, Jinlong 2017-06-06 00:00:00 www.nature.com/scientificreports OPEN Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity Received: 21 February 2017 1 1 1 1 1 1 Dianpeng Zheng , Sichao Ye , Xiuyun Wang , Yongjun Zhang , Daoyu Yan , Xiangsheng Cai , 1 2,3 1 1 1 1 Accepted: 24 April 2017 Weihong Gao , Hongbo Shan , Yang Gao , Juanjuan Chen , Zhiming Hu , Hongwei Li & Published: xx xx xxxx Jinlong Li MCM7, a subunit of mini-chromosome maintenance proteins (MCM) complex, plays an important role in initiating DNA replication during the G1 phase and extending DNA strands during the S phase. Here, we demonstrated that MCM7 is not only sustained but maintains association with chromatin during M phase. Remarkably, MCM7 siRNA can accelerate mitotic exit. MCM7 depletion leads to CDK1 inactivation and promotes subsequent cohesin/RAD21 cleavage, which eventually leads to sister chromatin segregation. Moreover, MCM7 is co-localized with tubulin in the mitotic cells and MCM7 depletion results in aberrant mitosis. Our results indicate that MCM7 may exert certain functions on spindle formation to prevent cytokinesis during early mitosis by regulating CDK1 activity. Eukaryotic DNA replication is a methodical and strictly controlled process evolved to ensure accurate replication. Many mistakes that occur during DNA replication can cause genomic instability. The pre-replicative complex (Pre-RC), which plays an important role in initiating DNA replication, consists of the origin recognition complex (ORC), CDC6, CDT1 and mini-chromosome maintenance proteins (MCMs). MCM2-7 is a heterohexamer com- plex which contains six MCM homologues. MCMs are determined to be DNA helicases involved in the initiation of DNA replication. MCM subunits are evolutionarily conserved in organisms ranging from yeast to human 1, 2 including features such as the ATPase motifs . Recent studies have determined that there are more MCMs bound onto chromatin than required by replication origins, and the phenomenon is coined as the "MCM para- dox". The mechanism of the paradox is still unknown. Recent cancer studies have also suggested that MCM2-7 3–12 complex overexpression is relative to proliferation and malignancy of various cancers . MCM7, a key component of the MCM complex, forms a double trimeric complex with MCM4 and MCM6. e do Th uble trimeric complex initiate DNA replication through unwinding DNA double strand. During S phase, the complex participates in elongating DNA stands. However, a growing body of evidence has demonstrated that the function of MCM7 is not limited to starting DNA replication. Recent studies suggest that MCM7 inter- act with the polo-box domain (PBD) of endogenous PLK1, a kinase expressed during S, G2 and M phases of the cell cycle and plays an important role in dissociating the cohesion from chromosome . MCM7 also inter- act with ATR-interacting protein (ATRIP)-ATR, which is activated in the presence DNA replication damage . Furthermore, the MCM7 gene codes for an important microRNA cluster miR-106b-25, and contribute to the induction of megakaryocytes polyploidy . In vitro, MCM7 is highly expressed in multiple malignancies. Immunohistochemical studies in a variety of tissues show increased expression of MCM7 in several solid tumors, 16 17 18 19 including melanoma , endometrial carcinoma , esophageal adenocarcinoma , oral squamous cell carcinoma , 20 21 22 colorectal adenocarcinoma , glioblastoma and thyroid cancer . In this study, mRNA copy number and expression of MCMs were examined during cell cycle progression. We have also explored the correlation between MCM7 depletion and the M phase process. From this we have discov- ered that MCM7 depletion accelerates the M phase by promoting CDK1 phosphorylation and cleaving Cohesin/ RAD21, allowing sister chromatids to segregate. Institute of Biotherapy, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, Guangdong, China. Department of Endoscopy, Sun Yat-sen University Cancer Center, Guangzhou, China. State Key Laboratory of Oncology in South China and Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China. Correspondence and requests for materials should be addressed to H.L. (email: hongwei1@yahoo.com) or J.L. (email: lijinlong@smu.edu.cn) Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 1 www.nature.com/scientificreports/ Figure 1. MCMs' mRNA express in HepG2 and UMUC-3 Cells. (A) Cells were synchronized with double thymine and incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. Cell cycle was analyzed by FACS. Data are shown as mean ± SD of three independent experiments. N: asynchronized cells. (B) P-Histone H3 was detected by western-blot. (C) MCMs' mRNA levels were examined by real-time RCR. Data are normalized to the beta-actin level in each sample. These experiments were performed in triplicate, and the values are expressed as mean ± SD. Results MCMs' mRNA express in a cell cycle. To identify the mRNA expression levels of MCMs in a single cell cycle, HepG2 cells and UMUC-3 cells were synchronized at G1 phase by double thymidine block. Cells were then incubated in fresh medium and collected at 0 h, 3 h, 6 h, 9 h, 12 h, and 15 h. HepG2 cells were successfully synchronized in G1 phase (G1: 86.34% ± 0.62% at 0 h) and slowly entered the S phase at 6 h. Aer in ft cubation in fresh medium at 9 h, most cells progressed into the G2/M phase (G2/M: 60.30% ± 2.93%) and entered the next cell cycle by the 15 h timepoint. Compared to HepG2, UMUC-3 demonstrated quicker cell cycle progression. UMUC-3 were arrested in G1 (G1: 63.11% ± 2.13%) at 0 h. Aer in ft cubation in fresh medium, at about 6 h, most cells had entered the G2/M phase (G2/M: 40.61% ± 3.87%) and entered the next cell cycle aer 9 ft h (Fig. 1A). Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 2 www.nature.com/scientificreports/ Figure 2. MCM7 keep binding with chromatin in G2/M of HepG2 and UMUC-3 Cells. Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. The chromatin binding (chromatin) and non-chromatin binding (supernatant) MCMs in HepG2 cells (A) and UMUC-3 cells (B) were examined by Chromatin-binding assays. Histone H3 and GAPDH were used as loading control for chromatin- binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments. N: asynchronized cells. To confirm the cell cycle progression of the above cells, the mitotic marker p-Histone (Thr3) was examined by Western Blotting. In HepG2 cells, p-histone H3 was detected aer r ft eleasing 9 h and deceased aer r ft eleasing 12 h, which was consistent with FCM result. Similar results were observed in UMUC-3 cells (Fig. 1B). To determine MCMs' mRNA expression in one cell cycle, MCMs' mRNA isolated in these time points were detected by QPCR (Fig. 1C). We found that all the six MCMs' mRNA gradually decreased during G2/M phase (HepG2: 9 h and UMUC-3: 6 h–9 h). Interestingly, there was slight increase of MCMs' mRNA during S phase in HepG2 cells. M C M 4 , M C M 6 and MC M 7 maint aining associat ion wit h chromat in during G 2 / M phase. Chromatin-binding assay were performed to identify distribution of MCMs corresponding to cell cycle progression. In HepG2 cells, chromatin-binding MCM5 were decreased gradually as cells progressed into the G2/M phase. However, large amount of MCM4, MCM6 and MCM7 remained in chromatin in G2/M cells. MCM7 differed from the other MCMs in that it maintained binding to chromatin through the cell cycle (Fig. 2A). In UMUC-3 cells, MCM7 were also observed to be bound to chromatin in G2/M phase (Fig. 2B). Subsequently, we performed the chromatin-binding assay in human lung carcinoma SPC-A1 cells. SPC-A1 cells were synchronized by double thymine block in G1 (G1: 45.23% ± 1.20%). Cells entered G2/M phase after incubation in fresh medium at 6 h (G2/M: 44.51%). Most cells exit M phase aer in ft cubation in fresh medium at 9 h (Fig. 3A). The presence of p-Histone was consistent with FACS results (Fig. 3B). Like HepG2 cells, SPA-A1 Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 3 www.nature.com/scientificreports/ Figure 3. MCM7 constantly bind chromatin throughout cell cycle in SPC-A1 Cells. SPC-A1 Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. (A) SPC-A1 cell cycle was analyzed by FACS. (B) P-Histone H3 was detected by western-blot. (C) Chromatin binding and non- chromatin binding MCM4, MCM5, MCM6 and MCM7 were analyzed by chromatin binding assay. Histone H3 and GAPDH were used as loading control for chromatin-binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments. cells also exhibited sustained MCM7 binding to chromatin through the cell cycle. The chromatin-binding abili- ties of other MCMs in SPC-A1 cells were similar to that of HepG2 cells, except for MCM5 which was present on chromatin in G2/M SPC-A1 cells (Fig. 3C). MCM7 protein present in high level in mitotic cells. To observe MCM7 expression directly in the cells, MCM7 was detected via immunohistochemistry (ICH). MCM7 has high levels of expression throughout the whole M phase, including prophase, metaphase, anaphase and telophase (Fig. 4B). MCM7 depletion promotes mitotic exit. Based on the latter results, it is confirmed that MCM7 main- tains presence and association with chromatin during G2/M phase. To examine its function in the G2/M phase, MCM7 was depleted by RNAi in nocodazole-blocked G2/M cells. After being treated with 0.2 µ g/ml nocoda- zole for 24 h, most HepG2 were blocked in the G2/M phase (G2/M: 89.33% ± 2.01%). After transferring into fresh medium from 0 h to 4 h, cell gradually exited M phase. The RNAi transfection successfully reduced MCM7 expression (Fig. 5A). In the MCM7 siRNA cells, the speed of mitotic exit was quicker than control group. Most cells exited mitosis 2 h after transferring (Fig. 5B,C). To further confirm this phenomenon in no-cancer cells, normal hepatocytes LO2 cells were used to explore MCM7 depletion effect on mitotic progression. The same phenomenon was observed in LO2 cells (Fig. 5B,C). During M phase, CDK1 inactivation is the key event for activating separase for cleaving Cohesin/RAD21, allowing sister chromatids to segregate. Therefore, we monitored changes in RAD21 and p-Cdk1 levels (p-Cdk1 is a conserved tyrosine phosphorylation (Tyr15 in humans), leading to inhibition of Cdk1 activity). In the control Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 4 www.nature.com/scientificreports/ Figure 4. MCM7 proteins highly presents in mitosis phase in HepG2 Cells. (A) HepG2 Cells were synchronized by double thymine and then releasing for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. Western blot analysis of MCM7 (left). Data are expressed as optical density (OD) fold difference related to GAPDH from 3 duplicate experiments (right). N: asynchronized cells. (B) and (C) Immunohistochemistry analysis of MCM7 in HepG2 Cells. HE: H.E staining, IHC: immunohistochemistry. group, aer n ft ocodazole release, the p-CDK1 levels were higher in the earlier time points (0, 1, and 2 h) and the RAD21 was gradually cleaved during mitosis exiting. Interestingly, the MCM7 levels were gradually increased. In MCM7 siRNA group, the p-CDK1 levels were kept much higher levels than in the control group indicating that Cdk1 activity was inhibited. Correspondingly, the RAD21 levels were decreased significantly (Fig. 5D,E). These results demonstrated that MCM7 depletion led to inactivation of CDK1 and cohesin/RAD21 degradation. MCM7 depletion results in aberrant mitosis. To further characterize MCM7 in mitosis, HepG2 cells were synchronized by nocodazole and then incubated in fresh medium for 0, 0.5, 1 and 2 h, immunouo fl res- cence was performed to detect the localization of tubulin and MCM7. In the control, cells were retained in mito- sis from 0 to 2 h. Compared to control group, cells exited from mitosis at 2 h after MCM7 depletion (Fig. 6A). Moreover, aberrant mitosis was observed in MCM7 depletion groups (Fig. 6B). In the interphase cells, MCM7 was restricted in nuclear. In mitotic cells, when nuclear envelope breaks down, MCM7 was distributed through cell and co-localized with tubulin (Fig. 6C). Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 5 www.nature.com/scientificreports/ Figure 5. MCM7 depletion promotes mitotic exit. (A) HepG2 cells were transfected with MCM7 siRNA or negative control siRNA (Control) for 24 h or 72 h. MCM7 protein level was analyzed by Western blot. GAPDH was used as the loading control. (B) HepG2 cells and LO2 cells were transfected with MCM7 siRNA (siRNA) or negative control siRNA (Control) for 12 h, then 0.2 μg/ml nocodazole was added to cell culture for further 12 h and then incubated in fresh medium for 0 h, 1 h, 2 h, 3 h and 4 h. Cell cycle was analyzed by FACS and data are shown as mean ± SD of three independent experiments (C), *P < 0.05. (D) MCM7, p-CDK1 and RAD21 were detected by western-blot. GAPDH was used as the loading control. Data are expressed as optical density (OD) fold difference related to GAPDH from 3 duplicate experiments (E), *P < 0.05. Discussion In our study, we have found that MCM2-7 mRNA decreased during a cell cycle. More intriguingly, MCM2-7 mRNA was lowest in the G2/M phase. We also found that the reduction was not gradual. MCM2-7 mRNA Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 6 www.nature.com/scientificreports/ Figure 6. MCM7 depletion contributes to aberrant mitosis. (A,B) HepG2 cells were transfected with MCM7 siRNA or negative control siRNA. Aer b ft eing transfected for 12 h, cells treated with 0.2 μg/ml nocodazole for further 12 h and then incubated in fresh medium for 0 h, 0.5 h, 1 h and 2 h. Immunouo fl rescence staining was performed to detect MCM7 (red) and β-Tubulin (green) in the cells, and nucleuses were visualized by DAPI staining (blue). (C) HepG2 cells were treated with or without 0.2 μg/ml nocodazole for 12 h to block cell in M phase, above immunouo fl rescence staining was performed and observed under confocal microscopy. increased in the S phase and then gradually decreased. This phenomenon was also detected in HeLa cells . e M Th CM complex plays an important role in initiating DNA replication in the G1 phase and extending DNA strains in S phase. Therefore, it was assumed that MCMs will disassociate from chromatin in the G2/M phase to avoid DNA reduplication. However, many studies claim that the function of MCMs were not limited to the G1 and S phase. In this research, MCM4, MCM6 and MCM7 were found to be still associated with chromatin in G2/M phase. Previous studies have shown that phosphorylated MCM4 binds to chromatin in G2/M phase in order to prevent MCM complex from regaining association with chromatin to avoid DNA reduplication . Our Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 7 www.nature.com/scientificreports/ study is the first to show that MCM6 and MCM7 are still bound to chromatin in the G2/M phase (Figs 2 and 3). e c Th hromatin-bound MCMs' functions are still unclear. It is possible that MCMs have depolymerized in late G2 phase and there are some MCMs' subunits still associating with chromatin to regulate M phase events. To confirm MCM7 expression in a cell cycle, we analyzed protein expression by western-blot and immunohis- tochemistry (ICH) and demonstrated that MCM7 is always expressed throughout the whole cell cycle. Directly inferring from ICH results, it is surprising that MCM7 have continued expression throughout the entire M phase (Fig. 4). Immunouo fl rescence results also found that MCM7 maintained expression in mitosis (Fig. 6A). Next, we determined the function of MCM7 in M phase through the action of MCM7 siRNA. When com- pared to the negative control, MCM7 depletion accelerated mitotic exit. Moreover, increased CDK1 inhibitory phosphorylation and Cohesin/RAD21 cleavage were observed in MCM7 siRNA group. It is well known that spindle assembly checkpoint (SAC) is the key to control cytokinesis. When spindle assemble completely, SAC will be inactive and ultimately leads to CDK1 inactivity. CDK1 can inhibit separase before anaphase. Separase triggers anaphase by cleaving its substrate cohesin, which is the protein that bind sister chromatids. The inactivation of Cdk1 will activate separase and then trigger cohesin degradation, and ultimately initiates the final separation of sister chromatids . In this research, we demonstrate that MCM7 depletion will lead to CDK1 inactivation and accelerate mitotic exit (Fig. 5). In addition, MCM7 is co-localized with tubulin and MCM7 depletion lead to aberrant mitosis (Fig. 6). Thus, it is possible that MCM7 participates in SAC. MCM7 may help to assemble the spindle and prevent mitosis premature exit. However, the mechanisms underlying MCM7 functions on mitosis events still need further explorations. Based on this study, we conclude that MCM7 roles are more than just assembling pre-RC complex for launc- ing DNA replication. MCM7 maintains expression and association with chromatin in M phase to prevent prema- ture exit from mitosis. Materials and Methods Cell lines. Hepatocellular carcinoma HepG2, human lung carcinoma SPC-A1 and normal hepatocytes LO2 cells were routinely maintained in DMEM medium (Gibco BRL, Grand Island, NY, USA). Human bladder cancer UMUC-3 was routinely maintained in MEM medium (Gibco BRL, Grand Island, NY, USA). The medium con- tains 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were cultivated at 37 °C in a balanced air humidified incubator with an atmosphere of 5% CO . Cell cycle synchronization. Cells were synchronized at G1/S border by double thymidine block . Briefly, cells were treated with 2 mM thymidine (purchased from Sigma-Aldrich) for 19 h, incubated in fresh medium containing 10% FBS for 9 h and then synchronized for an additional 18 h with 2 mM thymidine. After washing three times with PBS, the blocked cells were incubated in fresh medium containing 10% FBS. Cells were harvested at 0, 3, 6, 9, 12 and 21 h time points. The cell-cycle progression was detected by flow cytometric analysis. For mito- sis block, cells were treated with 0.2 μg/ml nocodazole (purchased from Sigma-Aldrich) for 12 h. Aer ft washing three times with PBS, the blocked cells were incubated in fresh medium containing 10% FBS. Cells were harvested at 0, 1, 2, 3 and 4 h. The cell-cycle progression was detected by flow cytometric analysis. Western blot. Total proteins from cells were extracted using ice-cold lysis buffer (50 mM Tris-HCL pH 7.5, 150 mM NaCl, 1% NP40, 1 mM PMSF, and 10 units/ml aprotinin) for 5–10 min, then centrifuged at 12000 rpm form 10 min at 4 °C to obtain the whole cell lysate. Proteins (about 20 μg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes and incubated overnight at 4 °C with antibody against MCM4, MCM5, MCM6, and MCM7 (Santa Cruz), p-histone H3 (Thr3) (Cell Signaling technology), histone H3 (Cell Signaling technology), p-Cdk1 (Abcam) or GAPDH (Bioworld technology). After washing with Tris-buffered saline with 0.1% Tween 20, the membranes were incubated with HRP-conjugated IgG at room temperature for 1 h. Signal detection was carried out with an ECL system (Millipore, Billerica, MA, USA). WB band were quantified with gel-pro software and expressed as optical density fold difference related to GAPDH (relative OD). Chromatin binding assay. Cells were harvested and resuspended in tubes with EB buffer (100 mM KCl, 50 mM HEPES-KOH pH 7.5, 2.5 mM MgCl , 50 mM Na P O , 0.1 mM NaVO , 0.5% triton X-100) containing 2 4 2 7 3 protease inhibitors, then set on ice for 5–10 min for incubation. e Th tubes were flicked to mix the solution every 2–3 min during incubation. Subsequently, 30% ice-cold sucrose containing protease inhibitors was added to the bottom of the tubes. The tubes were then spinned at 12–15 rcf, 10 min, 4 °C and the supernatants were transferred to new tubes. The pellets were washed with EB buffer and flicked to dislodge the pellets from the wall of the tubes and vibrated briefly for resuspension, followed by spinning in a microfuge, 12–15 rcf, 5 min, 4 °C. Combined the supernatants from the two steps (this is the non-chromosomal fraction). The pellets were resuspended with EB buffer (the pellets are the chromatin binding fraction) and analyzed by Western blot. Immunohistochemistry. Cells grown on 6-well plates were fixed for 15 min in 4% (w/v) paraformaldehyde (PFA)/PBS aer t ft reatment and then permeabilized for 15 min in 0.25% (v/v) Triton X-100/PBS. Aer fixa ft tion and permeabilization, cells were washed three times in PBS and then blocked with goat serum for 1 h. Cells were incu- bated with antibodies against MCM7 (Santa Cruz) as required for 4 °C overnight, followed by three times wash with PBS and a 60 min incubation with goat anti-mouse HRP-conjugated secondary antibody. Positive signals were visualized using 3, 3′-diaminobenzidine. Immunofluorescence. Cells grown on 6-well plates were fixed for 15 min in 4% (w/v) para-formaldehyde (PFA)/PBS after treatment and then permeabilized for 15 min in 0.25% (v/v) Triton X-100/PBS. After fixation and permeabilization, cells were washed three times in PBS and then blocked with goat serum for 1 h. Cells were Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 8 www.nature.com/scientificreports/ incubated with antibodies against MCM7, β-Tubulin (Santa Cruz) as required for 4 °C overnight, followed by three times wash with PBS and a 60 min incubation with goat anti-mouse FITC secondary antibody. Fluorescence images were taken under an Olympus fluorescent microscope. Flow cytometry. Cells were fixed in 70% ethanol overnight at 4 °C. Fixed cells were then washed once in ice-cold PBS and stained with propidium iodide (PI) staining solution (50 μg/ml PI, 100 μg/ml RNase, 0.05% Triton X-100 in ddH2O) for 20–30 min. PI-stained cells were then analyzed for their DNA content by using FACS (BD biosciences, San Jose, CA, USA). SiRNA knockdown. Three Cdc6-targeting siRNAs (si-MCM7: 5′GAGT TGGTGGACTCAAT T T3′) were purchased from Guangzhou Ribobiotech. For the transfection of siRNA, cells (5 × 10 ) were seeded into 6-well plates and then were transfected with siRNA in diluted Lipofectamine containing Opti-MEM Medium (Invitrogen) according to manufacturer's protocol. Non-targeting siRNA was used as the negative control. Statistical analysis. SPSS version 13.0 for Windows was used for all statistical analyses. Average values were expressed as mean ± standard deviation (S.D.) and statistical significance between different groups was determined by the Student's t-test. Statistical comparisons were made using one-way ANOVA. P values < 0.05 were considered to be significant. References 1. Nieduszynski, C. A., Blow, J. J. & Donaldson, A. D. The requirement of yeast replication origins for pre-replication complex proteins is modulated by transcription. Nucleic Acids Research 33, 2410–2420, doi:10.1093/nar/gki539 (2005). 2. Schaarschmidt, D., Ladenburger, E. M., Keller, C. & Knippers, R. Human Mcm proteins at a replication origin during the G1 to S phase transition. Nucleic Acids Res 30, 4176–4185 (2002). 3. Ren, B. et al. MCM7 amplification and overexpression are associated with prostate cancer progression. Oncogene 25, 1090–1098, doi:10.1038/sj.onc.1209134 (2006). 4. Giaginis, C. et al. Clinical significance of MCM-2 and MCM-5 expression in colon cancer: association with clinicopathological parameters and tumor proliferative capacity. Dig Dis Sci 54, 282–291, doi:10.1007/s10620-008-0305-z (2009). 5. Saydam, O. et al. 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Separase is recruited to mitotic chromosomes to dissolve sister chromatid cohesion in a DNA-dependent manner. Cell 137, 123–132, doi:10.1016/j.cell.2009.01.040 (2009). 26. Chiang, P. C. et al. Antroquinonol displays anticancer potential against human hepatocellular carcinoma cells: a crucial role of AMPK and mTOR pathways. Biochem Pharmacol 79, 162–171, doi:10.1016/j.bcp.2009.08.022 (2010). Acknowledgements This work was supported by the grants from, National Natural Science Foundation of China Grants 81672915 (J.L.), 81373122 (Z.M.), and in part from Science and Technology Plan Projects of Guangdong Province of China (No. 2012B031800282) and Sun Yat-sen University young teachers training projects (No. 13ykpy45). Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 9 www.nature.com/scientificreports/ Author Contributions J.L., H.L. and Z.H. designed experiments; D.Z., S.Y., X.W., D.Y., Y.G., J.C. and Y.Z. carried out experiments; D.Z., X.C., W.G. and H.S. analyzed experiments results; J.L. and D.Z. wrote the manuscript. All authors reviewed the manuscript. Additional Information Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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Zheng, Dianpeng; Ye, Sichao; Wang, Xiuyun; Zhang, Yongjun; Yan, Daoyu; Cai, Xiangsheng; Gao, Weihong; Shan, Hongbo; Gao, Yang; Chen, Juanjuan; Hu, Zhiming; Li, Hongwei; Li, Jinlong
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www.nature.com/scientificreports OPEN Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity Received: 21 February 2017 1 1 1 1 1 1 Dianpeng Zheng , Sichao Ye , Xiuyun Wang , Yongjun Zhang , Daoyu Yan , Xiangsheng Cai , 1 2,3 1 1 1 1 Accepted: 24 April 2017 Weihong Gao , Hongbo Shan , Yang Gao , Juanjuan Chen , Zhiming Hu , Hongwei Li & Published: xx xx xxxx Jinlong Li MCM7, a subunit of mini-chromosome maintenance proteins (MCM) complex, plays an important role in initiating DNA replication during the G1 phase and extending DNA strands during the S phase. Here, we demonstrated that MCM7 is not only sustained but maintains association with chromatin during M phase. Remarkably, MCM7 siRNA can accelerate mitotic exit. MCM7 depletion leads to CDK1 inactivation and promotes subsequent cohesin/RAD21 cleavage, which eventually leads to sister chromatin segregation. Moreover, MCM7 is co-localized with tubulin in the mitotic cells and MCM7 depletion results in aberrant mitosis. Our results indicate that MCM7 may exert certain functions on spindle formation to prevent cytokinesis during early mitosis by regulating CDK1 activity. Eukaryotic DNA replication is a methodical and strictly controlled process evolved to ensure accurate replication. Many mistakes that occur during DNA replication can cause genomic instability. The pre-replicative complex (Pre-RC), which plays an important role in initiating DNA replication, consists of the origin recognition complex (ORC), CDC6, CDT1 and mini-chromosome maintenance proteins (MCMs). MCM2-7 is a heterohexamer com- plex which contains six MCM homologues. MCMs are determined to be DNA helicases involved in the initiation of DNA replication. MCM subunits are evolutionarily conserved in organisms ranging from yeast to human 1, 2 including features such as the ATPase motifs . Recent studies have determined that there are more MCMs bound onto chromatin than required by replication origins, and the phenomenon is coined as the "MCM para- dox". The mechanism of the paradox is still unknown. Recent cancer studies have also suggested that MCM2-7 3–12 complex overexpression is relative to proliferation and malignancy of various cancers . MCM7, a key component of the MCM complex, forms a double trimeric complex with MCM4 and MCM6. e do Th uble trimeric complex initiate DNA replication through unwinding DNA double strand. During S phase, the complex participates in elongating DNA stands. However, a growing body of evidence has demonstrated that the function of MCM7 is not limited to starting DNA replication. Recent studies suggest that MCM7 inter- act with the polo-box domain (PBD) of endogenous PLK1, a kinase expressed during S, G2 and M phases of the cell cycle and plays an important role in dissociating the cohesion from chromosome . MCM7 also inter- act with ATR-interacting protein (ATRIP)-ATR, which is activated in the presence DNA replication damage . Furthermore, the MCM7 gene codes for an important microRNA cluster miR-106b-25, and contribute to the induction of megakaryocytes polyploidy . In vitro, MCM7 is highly expressed in multiple malignancies. Immunohistochemical studies in a variety of tissues show increased expression of MCM7 in several solid tumors, 16 17 18 19 including melanoma , endometrial carcinoma , esophageal adenocarcinoma , oral squamous cell carcinoma , 20 21 22 colorectal adenocarcinoma , glioblastoma and thyroid cancer . In this study, mRNA copy number and expression of MCMs were examined during cell cycle progression. We have also explored the correlation between MCM7 depletion and the M phase process. From this we have discov- ered that MCM7 depletion accelerates the M phase by promoting CDK1 phosphorylation and cleaving Cohesin/ RAD21, allowing sister chromatids to segregate. Institute of Biotherapy, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, Guangdong, China. Department of Endoscopy, Sun Yat-sen University Cancer Center, Guangzhou, China. State Key Laboratory of Oncology in South China and Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China. Correspondence and requests for materials should be addressed to H.L. (email: hongwei1@yahoo.com) or J.L. (email: lijinlong@smu.edu.cn) Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 1 www.nature.com/scientificreports/ Figure 1. MCMs' mRNA express in HepG2 and UMUC-3 Cells. (A) Cells were synchronized with double thymine and incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. Cell cycle was analyzed by FACS. Data are shown as mean ± SD of three independent experiments. N: asynchronized cells. (B) P-Histone H3 was detected by western-blot. (C) MCMs' mRNA levels were examined by real-time RCR. Data are normalized to the beta-actin level in each sample. These experiments were performed in triplicate, and the values are expressed as mean ± SD. Results MCMs' mRNA express in a cell cycle. To identify the mRNA expression levels of MCMs in a single cell cycle, HepG2 cells and UMUC-3 cells were synchronized at G1 phase by double thymidine block. Cells were then incubated in fresh medium and collected at 0 h, 3 h, 6 h, 9 h, 12 h, and 15 h. HepG2 cells were successfully synchronized in G1 phase (G1: 86.34% ± 0.62% at 0 h) and slowly entered the S phase at 6 h. Aer in ft cubation in fresh medium at 9 h, most cells progressed into the G2/M phase (G2/M: 60.30% ± 2.93%) and entered the next cell cycle by the 15 h timepoint. Compared to HepG2, UMUC-3 demonstrated quicker cell cycle progression. UMUC-3 were arrested in G1 (G1: 63.11% ± 2.13%) at 0 h. Aer in ft cubation in fresh medium, at about 6 h, most cells had entered the G2/M phase (G2/M: 40.61% ± 3.87%) and entered the next cell cycle aer 9 ft h (Fig. 1A). Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 2 www.nature.com/scientificreports/ Figure 2. MCM7 keep binding with chromatin in G2/M of HepG2 and UMUC-3 Cells. Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. The chromatin binding (chromatin) and non-chromatin binding (supernatant) MCMs in HepG2 cells (A) and UMUC-3 cells (B) were examined by Chromatin-binding assays. Histone H3 and GAPDH were used as loading control for chromatin- binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments. N: asynchronized cells. To confirm the cell cycle progression of the above cells, the mitotic marker p-Histone (Thr3) was examined by Western Blotting. In HepG2 cells, p-histone H3 was detected aer r ft eleasing 9 h and deceased aer r ft eleasing 12 h, which was consistent with FCM result. Similar results were observed in UMUC-3 cells (Fig. 1B). To determine MCMs' mRNA expression in one cell cycle, MCMs' mRNA isolated in these time points were detected by QPCR (Fig. 1C). We found that all the six MCMs' mRNA gradually decreased during G2/M phase (HepG2: 9 h and UMUC-3: 6 h–9 h). Interestingly, there was slight increase of MCMs' mRNA during S phase in HepG2 cells. M C M 4 , M C M 6 and MC M 7 maint aining associat ion wit h chromat in during G 2 / M phase. Chromatin-binding assay were performed to identify distribution of MCMs corresponding to cell cycle progression. In HepG2 cells, chromatin-binding MCM5 were decreased gradually as cells progressed into the G2/M phase. However, large amount of MCM4, MCM6 and MCM7 remained in chromatin in G2/M cells. MCM7 differed from the other MCMs in that it maintained binding to chromatin through the cell cycle (Fig. 2A). In UMUC-3 cells, MCM7 were also observed to be bound to chromatin in G2/M phase (Fig. 2B). Subsequently, we performed the chromatin-binding assay in human lung carcinoma SPC-A1 cells. SPC-A1 cells were synchronized by double thymine block in G1 (G1: 45.23% ± 1.20%). Cells entered G2/M phase after incubation in fresh medium at 6 h (G2/M: 44.51%). Most cells exit M phase aer in ft cubation in fresh medium at 9 h (Fig. 3A). The presence of p-Histone was consistent with FACS results (Fig. 3B). Like HepG2 cells, SPA-A1 Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 3 www.nature.com/scientificreports/ Figure 3. MCM7 constantly bind chromatin throughout cell cycle in SPC-A1 Cells. SPC-A1 Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. (A) SPC-A1 cell cycle was analyzed by FACS. (B) P-Histone H3 was detected by western-blot. (C) Chromatin binding and non- chromatin binding MCM4, MCM5, MCM6 and MCM7 were analyzed by chromatin binding assay. Histone H3 and GAPDH were used as loading control for chromatin-binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments. cells also exhibited sustained MCM7 binding to chromatin through the cell cycle. The chromatin-binding abili- ties of other MCMs in SPC-A1 cells were similar to that of HepG2 cells, except for MCM5 which was present on chromatin in G2/M SPC-A1 cells (Fig. 3C). MCM7 protein present in high level in mitotic cells. To observe MCM7 expression directly in the cells, MCM7 was detected via immunohistochemistry (ICH). MCM7 has high levels of expression throughout the whole M phase, including prophase, metaphase, anaphase and telophase (Fig. 4B). MCM7 depletion promotes mitotic exit. Based on the latter results, it is confirmed that MCM7 main- tains presence and association with chromatin during G2/M phase. To examine its function in the G2/M phase, MCM7 was depleted by RNAi in nocodazole-blocked G2/M cells. After being treated with 0.2 µ g/ml nocoda- zole for 24 h, most HepG2 were blocked in the G2/M phase (G2/M: 89.33% ± 2.01%). After transferring into fresh medium from 0 h to 4 h, cell gradually exited M phase. The RNAi transfection successfully reduced MCM7 expression (Fig. 5A). In the MCM7 siRNA cells, the speed of mitotic exit was quicker than control group. Most cells exited mitosis 2 h after transferring (Fig. 5B,C). To further confirm this phenomenon in no-cancer cells, normal hepatocytes LO2 cells were used to explore MCM7 depletion effect on mitotic progression. The same phenomenon was observed in LO2 cells (Fig. 5B,C). During M phase, CDK1 inactivation is the key event for activating separase for cleaving Cohesin/RAD21, allowing sister chromatids to segregate. Therefore, we monitored changes in RAD21 and p-Cdk1 levels (p-Cdk1 is a conserved tyrosine phosphorylation (Tyr15 in humans), leading to inhibition of Cdk1 activity). In the control Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 4 www.nature.com/scientificreports/ Figure 4. MCM7 proteins highly presents in mitosis phase in HepG2 Cells. (A) HepG2 Cells were synchronized by double thymine and then releasing for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. Western blot analysis of MCM7 (left). Data are expressed as optical density (OD) fold difference related to GAPDH from 3 duplicate experiments (right). N: asynchronized cells. (B) and (C) Immunohistochemistry analysis of MCM7 in HepG2 Cells. HE: H.E staining, IHC: immunohistochemistry. group, aer n ft ocodazole release, the p-CDK1 levels were higher in the earlier time points (0, 1, and 2 h) and the RAD21 was gradually cleaved during mitosis exiting. Interestingly, the MCM7 levels were gradually increased. In MCM7 siRNA group, the p-CDK1 levels were kept much higher levels than in the control group indicating that Cdk1 activity was inhibited. Correspondingly, the RAD21 levels were decreased significantly (Fig. 5D,E). These results demonstrated that MCM7 depletion led to inactivation of CDK1 and cohesin/RAD21 degradation. MCM7 depletion results in aberrant mitosis. To further characterize MCM7 in mitosis, HepG2 cells were synchronized by nocodazole and then incubated in fresh medium for 0, 0.5, 1 and 2 h, immunouo fl res- cence was performed to detect the localization of tubulin and MCM7. In the control, cells were retained in mito- sis from 0 to 2 h. Compared to control group, cells exited from mitosis at 2 h after MCM7 depletion (Fig. 6A). Moreover, aberrant mitosis was observed in MCM7 depletion groups (Fig. 6B). In the interphase cells, MCM7 was restricted in nuclear. In mitotic cells, when nuclear envelope breaks down, MCM7 was distributed through cell and co-localized with tubulin (Fig. 6C). Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 5 www.nature.com/scientificreports/ Figure 5. MCM7 depletion promotes mitotic exit. (A) HepG2 cells were transfected with MCM7 siRNA or negative control siRNA (Control) for 24 h or 72 h. MCM7 protein level was analyzed by Western blot. GAPDH was used as the loading control. (B) HepG2 cells and LO2 cells were transfected with MCM7 siRNA (siRNA) or negative control siRNA (Control) for 12 h, then 0.2 μg/ml nocodazole was added to cell culture for further 12 h and then incubated in fresh medium for 0 h, 1 h, 2 h, 3 h and 4 h. Cell cycle was analyzed by FACS and data are shown as mean ± SD of three independent experiments (C), *P < 0.05. (D) MCM7, p-CDK1 and RAD21 were detected by western-blot. GAPDH was used as the loading control. Data are expressed as optical density (OD) fold difference related to GAPDH from 3 duplicate experiments (E), *P < 0.05. Discussion In our study, we have found that MCM2-7 mRNA decreased during a cell cycle. More intriguingly, MCM2-7 mRNA was lowest in the G2/M phase. We also found that the reduction was not gradual. MCM2-7 mRNA Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 6 www.nature.com/scientificreports/ Figure 6. MCM7 depletion contributes to aberrant mitosis. (A,B) HepG2 cells were transfected with MCM7 siRNA or negative control siRNA. Aer b ft eing transfected for 12 h, cells treated with 0.2 μg/ml nocodazole for further 12 h and then incubated in fresh medium for 0 h, 0.5 h, 1 h and 2 h. Immunouo fl rescence staining was performed to detect MCM7 (red) and β-Tubulin (green) in the cells, and nucleuses were visualized by DAPI staining (blue). (C) HepG2 cells were treated with or without 0.2 μg/ml nocodazole for 12 h to block cell in M phase, above immunouo fl rescence staining was performed and observed under confocal microscopy. increased in the S phase and then gradually decreased. This phenomenon was also detected in HeLa cells . e M Th CM complex plays an important role in initiating DNA replication in the G1 phase and extending DNA strains in S phase. Therefore, it was assumed that MCMs will disassociate from chromatin in the G2/M phase to avoid DNA reduplication. However, many studies claim that the function of MCMs were not limited to the G1 and S phase. In this research, MCM4, MCM6 and MCM7 were found to be still associated with chromatin in G2/M phase. Previous studies have shown that phosphorylated MCM4 binds to chromatin in G2/M phase in order to prevent MCM complex from regaining association with chromatin to avoid DNA reduplication . Our Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 7 www.nature.com/scientificreports/ study is the first to show that MCM6 and MCM7 are still bound to chromatin in the G2/M phase (Figs 2 and 3). e c Th hromatin-bound MCMs' functions are still unclear. It is possible that MCMs have depolymerized in late G2 phase and there are some MCMs' subunits still associating with chromatin to regulate M phase events. To confirm MCM7 expression in a cell cycle, we analyzed protein expression by western-blot and immunohis- tochemistry (ICH) and demonstrated that MCM7 is always expressed throughout the whole cell cycle. Directly inferring from ICH results, it is surprising that MCM7 have continued expression throughout the entire M phase (Fig. 4). Immunouo fl rescence results also found that MCM7 maintained expression in mitosis (Fig. 6A). Next, we determined the function of MCM7 in M phase through the action of MCM7 siRNA. When com- pared to the negative control, MCM7 depletion accelerated mitotic exit. Moreover, increased CDK1 inhibitory phosphorylation and Cohesin/RAD21 cleavage were observed in MCM7 siRNA group. It is well known that spindle assembly checkpoint (SAC) is the key to control cytokinesis. When spindle assemble completely, SAC will be inactive and ultimately leads to CDK1 inactivity. CDK1 can inhibit separase before anaphase. Separase triggers anaphase by cleaving its substrate cohesin, which is the protein that bind sister chromatids. The inactivation of Cdk1 will activate separase and then trigger cohesin degradation, and ultimately initiates the final separation of sister chromatids . In this research, we demonstrate that MCM7 depletion will lead to CDK1 inactivation and accelerate mitotic exit (Fig. 5). In addition, MCM7 is co-localized with tubulin and MCM7 depletion lead to aberrant mitosis (Fig. 6). Thus, it is possible that MCM7 participates in SAC. MCM7 may help to assemble the spindle and prevent mitosis premature exit. However, the mechanisms underlying MCM7 functions on mitosis events still need further explorations. Based on this study, we conclude that MCM7 roles are more than just assembling pre-RC complex for launc- ing DNA replication. MCM7 maintains expression and association with chromatin in M phase to prevent prema- ture exit from mitosis. Materials and Methods Cell lines. Hepatocellular carcinoma HepG2, human lung carcinoma SPC-A1 and normal hepatocytes LO2 cells were routinely maintained in DMEM medium (Gibco BRL, Grand Island, NY, USA). Human bladder cancer UMUC-3 was routinely maintained in MEM medium (Gibco BRL, Grand Island, NY, USA). The medium con- tains 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were cultivated at 37 °C in a balanced air humidified incubator with an atmosphere of 5% CO . Cell cycle synchronization. Cells were synchronized at G1/S border by double thymidine block . Briefly, cells were treated with 2 mM thymidine (purchased from Sigma-Aldrich) for 19 h, incubated in fresh medium containing 10% FBS for 9 h and then synchronized for an additional 18 h with 2 mM thymidine. After washing three times with PBS, the blocked cells were incubated in fresh medium containing 10% FBS. Cells were harvested at 0, 3, 6, 9, 12 and 21 h time points. The cell-cycle progression was detected by flow cytometric analysis. For mito- sis block, cells were treated with 0.2 μg/ml nocodazole (purchased from Sigma-Aldrich) for 12 h. Aer ft washing three times with PBS, the blocked cells were incubated in fresh medium containing 10% FBS. Cells were harvested at 0, 1, 2, 3 and 4 h. The cell-cycle progression was detected by flow cytometric analysis. Western blot. Total proteins from cells were extracted using ice-cold lysis buffer (50 mM Tris-HCL pH 7.5, 150 mM NaCl, 1% NP40, 1 mM PMSF, and 10 units/ml aprotinin) for 5–10 min, then centrifuged at 12000 rpm form 10 min at 4 °C to obtain the whole cell lysate. Proteins (about 20 μg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes and incubated overnight at 4 °C with antibody against MCM4, MCM5, MCM6, and MCM7 (Santa Cruz), p-histone H3 (Thr3) (Cell Signaling technology), histone H3 (Cell Signaling technology), p-Cdk1 (Abcam) or GAPDH (Bioworld technology). After washing with Tris-buffered saline with 0.1% Tween 20, the membranes were incubated with HRP-conjugated IgG at room temperature for 1 h. Signal detection was carried out with an ECL system (Millipore, Billerica, MA, USA). WB band were quantified with gel-pro software and expressed as optical density fold difference related to GAPDH (relative OD). Chromatin binding assay. Cells were harvested and resuspended in tubes with EB buffer (100 mM KCl, 50 mM HEPES-KOH pH 7.5, 2.5 mM MgCl , 50 mM Na P O , 0.1 mM NaVO , 0.5% triton X-100) containing 2 4 2 7 3 protease inhibitors, then set on ice for 5–10 min for incubation. e Th tubes were flicked to mix the solution every 2–3 min during incubation. Subsequently, 30% ice-cold sucrose containing protease inhibitors was added to the bottom of the tubes. The tubes were then spinned at 12–15 rcf, 10 min, 4 °C and the supernatants were transferred to new tubes. The pellets were washed with EB buffer and flicked to dislodge the pellets from the wall of the tubes and vibrated briefly for resuspension, followed by spinning in a microfuge, 12–15 rcf, 5 min, 4 °C. Combined the supernatants from the two steps (this is the non-chromosomal fraction). The pellets were resuspended with EB buffer (the pellets are the chromatin binding fraction) and analyzed by Western blot. Immunohistochemistry. Cells grown on 6-well plates were fixed for 15 min in 4% (w/v) paraformaldehyde (PFA)/PBS aer t ft reatment and then permeabilized for 15 min in 0.25% (v/v) Triton X-100/PBS. Aer fixa ft tion and permeabilization, cells were washed three times in PBS and then blocked with goat serum for 1 h. Cells were incu- bated with antibodies against MCM7 (Santa Cruz) as required for 4 °C overnight, followed by three times wash with PBS and a 60 min incubation with goat anti-mouse HRP-conjugated secondary antibody. Positive signals were visualized using 3, 3′-diaminobenzidine. Immunofluorescence. Cells grown on 6-well plates were fixed for 15 min in 4% (w/v) para-formaldehyde (PFA)/PBS after treatment and then permeabilized for 15 min in 0.25% (v/v) Triton X-100/PBS. After fixation and permeabilization, cells were washed three times in PBS and then blocked with goat serum for 1 h. Cells were Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 8 www.nature.com/scientificreports/ incubated with antibodies against MCM7, β-Tubulin (Santa Cruz) as required for 4 °C overnight, followed by three times wash with PBS and a 60 min incubation with goat anti-mouse FITC secondary antibody. Fluorescence images were taken under an Olympus fluorescent microscope. Flow cytometry. Cells were fixed in 70% ethanol overnight at 4 °C. Fixed cells were then washed once in ice-cold PBS and stained with propidium iodide (PI) staining solution (50 μg/ml PI, 100 μg/ml RNase, 0.05% Triton X-100 in ddH2O) for 20–30 min. PI-stained cells were then analyzed for their DNA content by using FACS (BD biosciences, San Jose, CA, USA). SiRNA knockdown. Three Cdc6-targeting siRNAs (si-MCM7: 5′GAGT TGGTGGACTCAAT T T3′) were purchased from Guangzhou Ribobiotech. For the transfection of siRNA, cells (5 × 10 ) were seeded into 6-well plates and then were transfected with siRNA in diluted Lipofectamine containing Opti-MEM Medium (Invitrogen) according to manufacturer's protocol. Non-targeting siRNA was used as the negative control. Statistical analysis. SPSS version 13.0 for Windows was used for all statistical analyses. Average values were expressed as mean ± standard deviation (S.D.) and statistical significance between different groups was determined by the Student's t-test. Statistical comparisons were made using one-way ANOVA. P values < 0.05 were considered to be significant. References 1. Nieduszynski, C. A., Blow, J. J. & Donaldson, A. D. The requirement of yeast replication origins for pre-replication complex proteins is modulated by transcription. Nucleic Acids Research 33, 2410–2420, doi:10.1093/nar/gki539 (2005). 2. Schaarschmidt, D., Ladenburger, E. M., Keller, C. & Knippers, R. Human Mcm proteins at a replication origin during the G1 to S phase transition. Nucleic Acids Res 30, 4176–4185 (2002). 3. Ren, B. et al. MCM7 amplification and overexpression are associated with prostate cancer progression. Oncogene 25, 1090–1098, doi:10.1038/sj.onc.1209134 (2006). 4. Giaginis, C. et al. Clinical significance of MCM-2 and MCM-5 expression in colon cancer: association with clinicopathological parameters and tumor proliferative capacity. Dig Dis Sci 54, 282–291, doi:10.1007/s10620-008-0305-z (2009). 5. Saydam, O. et al. Comparative protein profiling reveals minichromosome maintenance (MCM) proteins as novel potential tumor markers for meningiomas. J Proteome Res 9, 485–494, doi:10.1021/pr900834h (2010). 6. Cobanoglu, U. et al. The expression of MCM-2 in invasive breast carcinoma: a stereologic approach. Bratisl Lek Listy 111, 45–49 (2010). 7. Lau, K. M. et al. Minichromosome maintenance proteins 2, 3 and 7 in medulloblastoma: overexpression and involvement in regulation of cell migration and invasion. Oncogene 29, 5475–5489, doi:10.1038/onc.2010.287 (2010). 8. Giaginis, C. et al. MCM-2 and MCM-5 expression in gastric adenocarcinoma: clinical significance and comparison with Ki-67 proliferative marker. Dig Dis Sci 56, 777–785, doi:10.1007/s10620-010-1348-5 (2011). 9. Nicol, A. F. et al. Evaluation of MCM-2 expression in TMA cervical specimens. PLoS One 7, e32936, doi:10.1371/journal. pone.0032936 (2012). 10. Das, M. et al. Over expression of minichromosome maintenance genes is clinically correlated to cervical carcinogenesis. PLoS One 8, e69607, doi:10.1371/journal.pone.0069607 (2013). 11. Kuijjer, M. L. et al. Identification of osteosarcoma driver genes by integrative analysis of copy number and gene expression data. Genes Chromosomes Cancer 51, 696–706, doi:10.1002/gcc.21956 (2012). 12. Zhong, X. et al. Overexpression of G9a and MCM7 in oesophageal squamous cell carcinoma is associated with poor prognosis. Histopathology 66, 192–200, doi:10.1111/his.12456 (2015). 13. Tsvetkov, L. & Stern, D. F. Interaction of chromatin-associated Plk1 and MCM7. J Biol Chem 280, 11943–11947, doi:10.1074/jbc. M413514200 (2005). 14. Cortez, D., Glick, G. & Elledge, S. J. Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc Natl Acad Sci USA 101, 10078–10083, doi:10.1073/pnas.0403410101 (2004). 15. Haldar, S., Roy, A. & Banerjee, S. Differential regulation of MCM7 and its intronic miRNA cluster miR-106b-25 during megakaryopoiesis induced polyploidy. RNA Biol 11, 1137–1147, doi:10.4161/rna.36136 (2014). 16. Gambichler, T. et al. Minichromosome maintenance proteins are useful adjuncts to die ff rentiate between benign and malignant melanocytic skin lesions. J Am Acad Dermatol 60, 808–813, doi:10.1016/j.jaad.2009.01.028 (2009). 17. Li, S. S. et al. Replicative MCM7 protein as a proliferation marker in endometrial carcinoma: a tissue microarray and clinicopathological analysis. Histopathology 46, 307–313, doi:10.1111/j.1365-2559.2005.02069.x (2005). 18. Kan, T. et al. The miR-106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim. Gastroenterology 136, 1689–1700 (2009). 19. Feng, C. J., Li, H. J., Li, J. N., Lu, Y. J. & Liao, G. Q. Expression of MCM7 and Cdc6 in oral squamous cell carcinoma and precancerous lesions. Anticancer Res 28, 3763–3769 (2008). 20. Nishihara, K. et al. Minichromosome maintenance protein 7 in colorectal cancer: implication of prognostic significance. Int J Oncol 33, 245–251 (2008). 21. Facoetti, A. et al. Minichromosome maintenance protein 7: a reliable tool for glioblastoma proliferation index. Anticancer Res 26, 1071–1075 (2006). 22. Kebebew, E. et al. Diagnostic and prognostic value of cell-cycle regulatory genes in malignant thyroid neoplasms. World J Surg 30, 767–774, doi:10.1007/s00268-005-0308-2 (2006). 23. Schulte, D., Richter, A., Burkhart, R., Musahl, C. & Knippers, R. Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm proteins. Eur J Biochem 235, 144–151 (1996). 24. Komamura-Kohno, Y. et al. Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells. FEBS J 273, 1224–1239, doi:10.1111/j.1742-4658.2006.05146.x (2006). 25. Sun, Y. et al. Separase is recruited to mitotic chromosomes to dissolve sister chromatid cohesion in a DNA-dependent manner. Cell 137, 123–132, doi:10.1016/j.cell.2009.01.040 (2009). 26. Chiang, P. C. et al. Antroquinonol displays anticancer potential against human hepatocellular carcinoma cells: a crucial role of AMPK and mTOR pathways. Biochem Pharmacol 79, 162–171, doi:10.1016/j.bcp.2009.08.022 (2010). Acknowledgements This work was supported by the grants from, National Natural Science Foundation of China Grants 81672915 (J.L.), 81373122 (Z.M.), and in part from Science and Technology Plan Projects of Guangdong Province of China (No. 2012B031800282) and Sun Yat-sen University young teachers training projects (No. 13ykpy45). Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 9 www.nature.com/scientificreports/ Author Contributions J.L., H.L. and Z.H. designed experiments; D.Z., S.Y., X.W., D.Y., Y.G., J.C. and Y.Z. carried out experiments; D.Z., X.C., W.G. and H.S. analyzed experiments results; J.L. and D.Z. wrote the manuscript. All authors reviewed the manuscript. Additional Information Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Repo R ts | 7: 2854 | DOI:10.1038/s41598-017-03148-3 10
Scientific Reports – Springer Journals
The requirement of yeast replication origins for pre-replication complex proteins is modulated by transcription
Nieduszynski, CA; Blow, JJ; Donaldson, AD
Human Mcm proteins at a replication origin during the G1 to S phase transition
Schaarschmidt, D; Ladenburger, EM; Keller, C; Knippers, R
MCM7 amplification and overexpression are associated with prostate cancer progression
Ren, B
Clinical significance of MCM-2 and MCM-5 expression in colon cancer: association with clinicopathological parameters and tumor proliferative capacity
Giaginis, C
Comparative protein profiling reveals minichromosome maintenance (MCM) proteins as novel potential tumor markers for meningiomas
Saydam, O
The expression of MCM-2 in invasive breast carcinoma: a stereologic approach
Cobanoglu, U
Minichromosome maintenance proteins 2, 3 and 7 in medulloblastoma: overexpression and involvement in regulation of cell migration and invasion
Lau, KM
MCM-2 and MCM-5 expression in gastric adenocarcinoma: clinical significance and comparison with Ki-67 proliferative marker
Evaluation of MCM-2 expression in TMA cervical specimens
Nicol, AF
Over expression of minichromosome maintenance genes is clinically correlated to cervical carcinogenesis
Das, M
Identification of osteosarcoma driver genes by integrative analysis of copy number and gene expression data
Kuijjer, ML
Overexpression of G9a and MCM7 in oesophageal squamous cell carcinoma is associated with poor prognosis
Zhong, X
Interaction of chromatin-associated Plk1 and MCM7
Tsvetkov, L; Stern, DF
Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases
Cortez, D; Glick, G; Elledge, SJ
Differential regulation of MCM7 and its intronic miRNA cluster miR-106b-25 during megakaryopoiesis induced polyploidy
Haldar, S; Roy, A; Banerjee, S
Minichromosome maintenance proteins are useful adjuncts to differentiate between benign and malignant melanocytic skin lesions
Gambichler, T
Replicative MCM7 protein as a proliferation marker in endometrial carcinoma: a tissue microarray and clinicopathological analysis
Li, SS
The miR-106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim
Kan, T
Expression of MCM7 and Cdc6 in oral squamous cell carcinoma and precancerous lesions
Feng, CJ; Li, HJ; Li, JN; Lu, YJ; Liao, GQ
Minichromosome maintenance protein 7 in colorectal cancer: implication of prognostic significance
Nishihara, K
Minichromosome maintenance protein 7: a reliable tool for glioblastoma proliferation index
Facoetti, A
Diagnostic and prognostic value of cell-cycle regulatory genes in malignant thyroid neoplasms
Kebebew, E
Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm proteins
Schulte, D; Richter, A; Burkhart, R; Musahl, C; Knippers, R
Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells
Komamura-Kohno, Y
Separase is recruited to mitotic chromosomes to dissolve sister chromatid cohesion in a DNA-dependent manner
Sun, Y
Antroquinonol displays anticancer potential against human hepatocellular carcinoma cells: a crucial role of AMPK and mTOR pathways
Chiang, PC
https://www.deepdyve.com/lp/springer_journal/pre-rc-protein-mcm7-depletion-promotes-mitotic-exit-by-inhibiting-cdk1-9H0Rs09oiT?utm_source=freeShare&utm_medium=link&utm_campaign=freeShare
Zheng, D., Ye, S., Wang, X., Zhang, Y., Yan, D., Cai, X., Gao, W., Shan, H., Gao, Y., Chen, J., Hu, Z., Li, H., & Li, J. (2017). Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity. Scientific Reports, 7(1), 1-10.
Zheng, Dianpeng, Sichao Ye, Xiuyun Wang, Yongjun Zhang, Daoyu Yan, Xiangsheng Cai, Weihong Gao, Hongbo Shan, Yang Gao, Juanjuan Chen, Zhiming Hu, Hongwei Li, and Jinlong Li. "Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity." Scientific Reports 7.1 (2017): 1-10.


Texas Review, photography, review and promotional coverage for concerts, sports and other events in the Austin and San Antonio, Texas area.
Post Malone performing at the Austin360 Amphitheater in Austin, Texas on June 16, 2018.
Post Malone stirs a mix of genres into his own sonic sound that's both intoxicating and invigorating on his RIAA double-platinum certified full-length debut album, Stoney. His uncanny ability to cook up a 21st century "sauce" of genres transformed the multiplatinum Dallas, TX maverick into the "#1 Debut Hip-Hop Artist of 2016." Upon release, Stoney bowed at #1 on the iTunes Overall Top Albums Chart and Top 10 on the Billboard Top 200.
Post Malone was the main act at the Austin360 Amphitheater with guests 21 Savage and SOB x RBE. The band continues across the United States, Europe, and Mexico with the last stop at the Dos Equis Pavilion in Dallas, Texas on October 28, 2018.
blink-182 performing at the AT&T Center in San Antonio, Texas on July 30, 2016, with Mark Hoppus, Travis Barker, and Matt Skiba.
2Cellos performing at the Frank Erwin Center in Austin, Texas on January 18, 2018, with Stjepan Hauser and Luka Sulic.
Breaking Benjamin performing at ACL Live in Austin, Texas on March 4, 2016.
Calum Scott performing at the Dos Equis Pavilion in Dallas, Texas on July 26, 2018.


Singer-songwriter Alice Merton has launched her single No Roots in the UK. The track became a European hit, reaching number 2 in the official German charts and claiming Gold status, and has now received over 21 million views on YouTube.
No Roots is a track about Merton's upbringing and how she never stayed in one place long enough to settle and put down roots. She was raised in Canada, finished high school in Germany and then moved to England with her family. When asked if living in multiple countries and experiencing a wealth of cultures from a young age has inspired her song-writing Merton says "I wouldn't say the places themselves have inspired my song writing but much more the experiences I've had on each country and the music I was listening to at the time." That can definitely be felt in No Roots.
The track opens with an ultra-cool, deep baseline and then Merton's luxurious and crystal clear vocal bursts onto the scene. Lyrics such as "I build a home and wait for someone to tear it down, then pack it up in boxes and head for the next town" could evoke a sense of frustration at this way of life but they're sung in a way that suggests that this was just Merton's version of normal. The track is kept fresh and catchy by the undulating baseline and backing vocal emphasis on "roots." The chorus of "I've got no roots but my home was never on the ground" is sung like a chant and is undeniably addictive. The standout part of the track is definitely the electronic breakdown where it descends into a whirlwind of synths. This splash of electronica lifts the track and blends superbly with the guitars and baseline.
Find out more about Alice Merton here and have a listen to No Roots below.


Be part of Australia's most loved and iconic world-class event, the Melbourne Cup Carnival.
Experience this incredible event that combines sport, entertainment, fashion and food – all in one place, the stunning Flemington Racecourse. Glamorous, refined and full of history, the Melbourne Cup Carnival is a must-do event for Melburnians and visitors alike.
With four big race days and a range of ticketing options, there is so many different ways to experience the Carnival - attend one or all of the racedays knowing you will have an amazing time at the most anticipated event on the calendar.
Take an unexpected turn. Duck down a famed Melbourne laneway, skip up the stairs and take a tipple in a bar with a distinct personality.


Many years ago, when I was a financially strapped, single mother I purchased a yoga video hoping to bring some "zen" into my life. It was a form of fitness I hadn't tried and I wanted to see what benefits there really were. I didn't know much about yoga then, and I must say I did find it a challenge trying to learn while watching a video on a 27 inch TV, in downward dog.
Since then, I've joined a gym, and had the opportunity to take many different yoga classes over the last five years. Downward dog, Vinyasa and Savasana may sound like veterinary problems, but are now part of my regular vocabulary. I can go through a class with nary a glance at the instructor as I know exactly what she is asking us to do.
Despite being familiar with the terms and movements, I am by no means an expert. I still struggle with certain poses, in my tree pose I look like a tree during a wind storm. Balance is definitely not my strong suit, but what I lack in ability I make up for in enthusiasm. I see continued progress as I become stronger and more flexible with regular practice and even my balance is improving.
There are many different styles of yoga, but three main techniques are used: breathing, postures and meditation. Yoga is an opportunity to leave behind the worldly cares and stress we each have and to focus on the here and now(being present) instead of where we need to be in an hour. We are encouraged to do this by focusing on our breath, which some would say may be the most important aspect of yoga.
By bringing our awareness to our breath, our minds become more relaxed and we can focus on our postures and the moment we are in. The postures and breathing techniques can also help us with meditation. All these working together help to focus our bodies and minds on the energy within and around us.
One of my favourite parts of the yoga class is the savasana at the end. It is essentially like playing dead, you are laying down, completely relaxed and just focusing on the breath. In fact, it is so relaxing I have caught myself on the verge of starting to snore as I'm about to fall asleep. This pose allows your body and nervous system to relax and rejuvenate before heading back into the regular world.
Some of the benefits of yoga include improved mood, improved ability to fight the effects of stress, increased stability, improved lung capacity and improvement in insomnia. There are many other benefits, these are just to name a few and encourage you to give it a try if you haven't already. As form is so important I encourage you to join a class with a qualified instructor.
So in the end my verdict is to yoga. Namaste.


We want more efficiency in our entire field of work. We have adapted our transport system, which makes us more flexible. And we can achieve the same result with less effort.
With us, every Phalaenopsis has its own unique code with its own flower characteristics. This is saved in our system and processed immediately. Each Phalaenopsis with the same flower characteristics is automatically transported to the same lane. This unique sorting process is fully automated and never gets tired. We can therefore guarantee a uniform batch of orchids.


We are located on the Southbound access road of IH 35 (Exit 262) in Georgetown, TX Traveling North - Exit 264 and take the crossover to Southbound access road. Right next to Hog Alley with plenty of parking behind the shop.
The Backyard is 120 feet by 120 feet, equal to 13,000 square feet, which is enclosed.
The drum stage is 22 feet long and 8 feet wide.
The lower, main stage is 40 feet long and 14 feet wide.
The Stage has side stairs as well as a ramp.
The Sound stage or booth is approximately half way in the yard.
There is a Chicken Shack, for cold beverages as well as a Concession stand for special events.
The Backyard also has a large bar that is under the patio.
We currently do not have any events available for this facility. Please check out their website at http://www.hardtailsbarandgrill.com for the most up to date event listing.


Page 217 – Early Defer or Deny?
Identity: How do you see yourself and how do others see you?
Intellect: How do you think and approach the acquisition of knowledge?
Ideas: What do you think and why?
Interests: What do you choose to do when you have the time and flexibility?
Inspiration: What really motivates you?
After looking within yourself, then consider the 5 most important characteristics that you want out of your college experience. Then assess colleges with these preferences in mind and through a framework I refer to as the 4 C's.
Culture: What is the history and mission of the institution. How is the mission relevant today?
Curriculum: More than a listing of majors and programs, what is the design and aim of the courses you will take over 4 years?
Community: Who are the people, the physical spaces of campus and the relationship of the campus to the local environment?
Conclusions: What should I envision for myself at the end of my college experience? What are some of the outcomes I may expect?


text:
Tag: motorized vehicle barriers
Baby Deer ~ Fawn
Pelican Preenning
The present is full of opportunity. Never before in the history of the planet has mankind been given the privileges and opportunities that are at his disposal today. A great light has been raised and is penetrating the darkness of the world, but alas, too many with dust blinded eyes have yet to catch the vision. Some of us have. That is our privilege and our responsibility.
The fate of an individual or a nation will always be determined by the degree of his or its harmony with the forces and laws of Nature and the universe. Man is not alone in the universe but is surrounded by sources of power, harmony and knowledge.
The fullness of life depends upon man's harmony with the totality of the natural cosmic laws. Our individual evolution is a job that has to be carried on day by day by each individual himself. It is a lifelong task."~Richard St. Barbe Baker
The Stewards of the Richard St. Barbe Baker Afforestation Area are very fortunate to have a very strong and effective governance over all fiscal operations. The Stewards of the Richard St. Barbe Baker Afforestation Area have partnered with the Meewasin Valley Authority, and all contributions are held in the Richard St. Barbe Baker Afforestation Area trust fund. Financial stewardship for the Richard St. Barbe Baker Afforestation Area is paramount in order to support the stewardship of the afforestation area is the main goal for the Stewards of the Richard St. Barbe Baker Afforestation Area and this goal manifests in the following avenues;
"strategic goal of quality of life"
"strategic goal of environmental leadership"
The charitable donations and dollars have, in fact, gone straight to work at the Richard St. Barbe Baker Afforestation Area. How have the very much appreciated contributions to the Richard St. Barbe Baker Afforestation Area paid off so far?
Donations supported the 2016 volunteer clean up initiative with incentives, and very importantly ensured the safety of community volunteers who assembled for clean ups of the Richard St. Barbe Baker Afforestation Area.
Funding supported the appropriate environmentally friendly recycling of 85 tires. When properly disposed of scrap tires to do adversely affect the environment. The safe removal of the tires was not only improved the visual appearance of this semi-wilderness wildlife habitat, but it also went a long ways to restoring the quality of the wetland and watershed.
The Jersey Barriers seen out at the Richard St. Barbe Baker Afforestation Area serve to define the area of the afforestation area. Further these vehicle barricades protect the newly created groomed trails. It is also hoped that the barriers will serve to mitigate illegal trespass creating a safe and healthy environment in this peri-urban area for human users. Additionally the desire is out there that the barriers would protect the modified and native grasslands, wetlands and woodlands – for the semi wilderness wildlife habitat.
Installation of gates for the Richard St. Barbe Baker Afforestation Area goes one step further and provides the protection from illegal trespass by motorized vehicles as mentioned above, while allowing egress for service and emergency vehicles; ambulance, fire and police protective services.
Bollards were placed at the north end of the South West Off Leash Recreation Area to the east adjoining the afforestation area by the Trans Gas road. These short, sturdy, vertical posts were installed to control motorized vehicular traffic and egress into the woodlands and onto the groomed pathways from that venue.
These aforementioned activities were the first to be set into place as a tangible benefit to the public at the Richard St. Barbe Baker Afforestation Area.
If the forest, or shoreline shows evidence of litter, the result is a lack of respect for the area, and the existing refuse serves as what amounts to a trash magnet. It has been proven that persons act to preserve the environment and waterways, if they are seen to be clean. Thank you kindly to all our past clean up volunteers, and those who still monitor the afforestaton area and continue to pick up trash.
With the help of contributions the efforts of the Stewards of the Richard St. Barbe Baker Afforestation area continually upgrade the afforestation and West Swale wetland's quality so they can support a thriving environment which can be truly appreciated and used for by current and future generations of the City of Saskatoon community as Saskatoon grows to 250,000 by 2025 and to 380,000 by 2035.
There is no denial that kind and generous gifts to the "Meewasin Valley Authority Richard St. Barbe Baker Afforestation Area trust fund. (MVA RSBBAA)" have truly made a profound impact on the betterment of the afforestation area as a whole. As the Stewards of the Richard St. Barbe Baker Afforestation Area strive to protect this rich and diverse environmental habitat, it is a reflection on the City of Saskatoon to have the generous financial support, caring citizen volunteers and in-kind donations from the community.
"From water and earth we came, and the future of mankind on this planet will be determined by respectful or disrespectful treatment of these basic elements." ~Richard St. Barbe Baker
Author stbarbebakerPosted on April 7, 2017 September 8, 2019 Categories Appreciation, Donations, newsTags ambulance, bollard, Clean up, contributions, environment, envrionment, fire, garbage, George Genereux Urban REgional Park, goal, gratitude, initiative, Jersey Barriers, life, Meewasin, Meewasin Valley Authority, motorized vehicle barriers, MVA, police, post, progress, protection, recycling, report, Richard St. Barbe Baker AFforestation ARea, Saskatchewan, Saskatoon, Saskatoon Saskatchewan, service vehicle, support, thank you, tires, trash, vehicle barricades, vehicle barriers, waste1 Comment on Progress Report


text:
Q: How to rename multiple text files in a local directory using python? I have multiple files with below naming. I would like to rename them. Please suggest.
Old names:
D_Punjab_12733_Config_Backup_POST_202008041532
D_Delhi_8673_Config_Backup_POST_2020080836783
D_Mumbai_112_Config_Backup_POST_374593736783

After rename:
Punjab_12733
Delhi_8673
Mumbai_112

A: Considering you have the filepaths in the paths variable
for path in paths:
  new_path = '_'.join(path.split('_')[1:3])
  os.rename(path, new_path)

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Preview: FC Seoul vs Daegu FC
Paul Neat September 13, 2018 read ( words)
It's Expat's Day at Seoul World Cup Stadium where FC Seoul will welcome Daegu FC. Seoul are without a win in four but are still just one point away from the top six. Daegu, meanwhile, head to the capital on the back of two straight victories. A win for the visitors would move them within a point of Seoul, a victory for Seoul could see Lee Eulyong's side jump to fifth.
(image via K League)
via Soccerway
Gangwon FC 0-0 FC Seoul
Prior to the international break, FC Seoul endured somewhat of a week to forget. After being put to the sword by a rampant Ulsan Hyundai side, losing by four goals to one at the Munsu, Lee Eulyong's side then played out a forgettable 0-0 draw away to Gangwon FC. Only seven shots in the entire 90 minutes hit the target in a game which Seoul seemed to try and pass their opponents to death. Seoul made 474 passes to Gangwon's 270 but lacked that cutting edge. The capital club are now on a run of four games without victory having lost three on the spin prior to the 0-0 stalemate in Chuncheon.
Highlights: here
Daegu FC 4-2 Suwon Bluewings
Daegu, meanwhile, are on a decent run of form having won two on the bounce and three in their last five. Andre's side signed off before the international break with an impressive 4-2 home win over AFC Champions League quarter finalists Suwon Bluewings. A Kim Eunseon own goal gave Daegu the lead inside eight minutes before Lee Jongsung levelled just eight minutes later. Daegu soon regained their advantage, however, as Edgar fired home on 19 minutes before a Cesinha brace gave the Sky Blues a 4-1 lead with goals on 35 and 60 minutes. Yeom Kihun pulled one back in the 90th minute to halve the deficit for the Bluewings.
The most recent meeting between these two teams ended in a 2-2 draw down in Daegu in July. In what was both teams' first match back after the World Cup break, during which FC Seoul surrendered a two-goal lead leave them just a point for the endeavours. Goals from Cho Youngwook and Anderson Lopes had Lee Eulyong's men two goals to the good inside the first 18 minutes but a first half fight back before the interval from Daegu ensured that the spoils were shared. Edgar pulled a goal back on 37 minutes before fellow Brazilian, Cesinha, converted from the spot on the stroke of half time.
Daegu's most recent visit to Sangam wasn't quite so fruitful for the North Gyeongsang outfit, though. Goals from former Daegu forward Evandro, Go Yohan, and an own goal by Kim Jinhyeok gave Seoul a 3-0 victory. Daegu's last win at Sangam dates back to May 2011, a 2-0 victory thanks to goals from Lee Sangdeok and Ahn Seongmin.
In total, FC Seoul and Daegu FC have met 33 times, Seoul have 16 victories to Daegu's eight, four of which have come at Sangam.
FC Seoul will have goalkeeper Yoon Sanghoon, defender Kim Namchoon, and striker Yun Jutae available again after the trio returned from military service with Sangju Sangmu. Central defender Hwang Hyunsoo will also be available after his participation the Asian Games in Jakarta and Palembang. However, Shin Jinho is suspended after picking up his sixth booking of the season during the 0-0 draw with Gangwon, as is club captain Go Yohan.
For Daegu, Korean international goalkeeper, Cho Hyun-woo, was forced to pull out of Paulo Bento's squad Korean National Team squad due to an unlucky injury he picked up on the Asian Games 2018. However, regardless of fitness, the Taeguk Warrior will be unavailable through suspension due to a red card picked up in Daegu's 2-0 home defeat to Ulsan on 22nd July. What's more, two other key players, Hong Jung-woon and Jung Woo-jae, are not able to feature here either because of yellow card accumulation. Elsewhere, Brazilian striker, Ze Roberto has returned to his native Brazil to receive treatment on his injury. According to the club's official channels, Ze Roberto injured his right calf and will be out until the end of the year.
Ko Seung-bum, on-loan from Suwon Bluewings, didn't travel with the first team, but finally made an appearance in a reserve game. Even still, the 24-year-old midfielder isn't expected to feature come Sunday.
Daegu Team News by Muyeol Jung
During the international break, the first team travelled down to Namhae in the hope of repeating last year's success - a run of form that resulted in Daegu finishing the season comfortably in eighth place. It is unclear yet as to what the team had been working on during this short, intensive training camp but with defensive absentees, Andre was perhaps using the time to practise tactical transition from a back three to back four as well as to maintain their physical conditioning.
Come Sunday, there will perhaps be some unfamiliar names amongst Andre's starting lineup. Between the sticks, Choi Young-eun will be positioned once again in the absence of Cho Hyun-woo. Due to limitations, Andre will likely play a back three that consists of Han Hee-hoon, Park Byung-hyun and either Kim Jin-hyuk or Kim Woo-suk. On each side, Kang Yun-ku and Jung Seung-won are likely to play with Tsubasa, Ryu Jae-moon as the central midfielders. Like the last game, Ryu Jae-moon will play a key role in this system, moving back and forth. Upfront, Cesinha, Edgar, and Kim Dae-won will form a spearhead to try to breakdown Seoul's defence.
by Muyeol Jung
Cesinha
With seven assists and four goals to his name in just 16 appearances this term, Brazilian winger Cesinha is undoubtedly one of Daegu's most important players. The Brazilian was a key component of Andre Gaspar's side that finished eighth in the league last year too, along side current FC Seoul forward Evandro and Junior Negao who departed for Ulsan during the winter.
Already this year Cesinha has shown that he is a danger to FC Seoul having notched the equalising penalty in the 2-2 draw back in July. This season Cesinha has made no fewer than 455 passes, 60 of which were considered as key - nine of those have come against Seoul this season.
ACL hopes seem to be all-but over with the gap between Seoul and third place now as wide as 12 points but despite the poor run of form, Seoul are still only a point behind the top six. Lee Eulyong has had over two weeks to work with his players on the training ground to try and right the wrongs of the last four matches. FC Seoul fans will no doubt be expecting a reaction. Daegu, though, have shown that they are a team that fears no one and can boast of victories over Suwon Bluewings, Gangwon, and Jeju United this term. Away from home, Andre's side have just two defeats in their last seven with four wins and a draw, and so will feel that a positive result at Sangam will be well within their grasp.
Prediction: FC Seoul 0-0 Daegu FC
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This is a variation of a 4 v 2 Rondo that helps players understand the importance of keeping possessing until they can find a way to move the ball to an open space and penetrate.
In the clip below Tom Mura, from World Class Coaching, animates this 4v2 Rondo drill that requires players to circulate the ball and also switch the point of attack into an open area and then play it forward to progress the ball.
In this animation Coach Mura is using a 30 x 30 playing field. He creates to "gates" along each sideline at midfield. The drill is set up with a 4 v2 at each end of the playing area. The ball is put in play at one end. The objective is for the players to utilize great spacing a technical skills to work the ball and move it to an open area so that it can be passed successfully through the gate to a teammate at the opposite end to the field.
After the ball is passed through the gate to the other end, those 4 will work the ball verses 2 defenders and try to create the necessary space and opening to pass the ball through a gate to the other end. Should a defender touch the ball then they would take the place of the attaching player. Alternatively, you could require the defender to win the ball before switching position with the attacker.
The YouTube video below has audio, so please make sure that your volume is turned up and that you have access to the site. Note that some schools may block access to YouTube.