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1.  The Genomes of Oryza sativa: A History of Duplications 
Yu, Jun | Wang, Jun | Lin, Wei | Li, Songgang | Li, Heng | Zhou, Jun | Ni, Peixiang | Dong, Wei | Hu, Songnian | Zeng, Changqing | Zhang, Jianguo | Zhang, Yong | Li, Ruiqiang | Xu, Zuyuan | Li, Shengting | Li, Xianran | Zheng, Hongkun | Cong, Lijuan | Lin, Liang | Yin, Jianning | Geng, Jianing | Li, Guangyuan | Shi, Jianping | Liu, Juan | Lv, Hong | Li, Jun | Wang, Jing | Deng, Yajun | Ran, Longhua | Shi, Xiaoli | Wang, Xiyin | Wu, Qingfa | Li, Changfeng | Ren, Xiaoyu | Wang, Jingqiang | Wang, Xiaoling | Li, Dawei | Liu, Dongyuan | Zhang, Xiaowei | Ji, Zhendong | Zhao, Wenming | Sun, Yongqiao | Zhang, Zhenpeng | Bao, Jingyue | Han, Yujun | Dong, Lingli | Ji, Jia | Chen, Peng | Wu, Shuming | Liu, Jinsong | Xiao, Ying | Bu, Dongbo | Tan, Jianlong | Yang, Li | Ye, Chen | Zhang, Jingfen | Xu, Jingyi | Zhou, Yan | Yu, Yingpu | Zhang, Bing | Zhuang, Shulin | Wei, Haibin | Liu, Bin | Lei, Meng | Yu, Hong | Li, Yuanzhe | Xu, Hao | Wei, Shulin | He, Ximiao | Fang, Lijun | Zhang, Zengjin | Zhang, Yunze | Huang, Xiangang | Su, Zhixi | Tong, Wei | Li, Jinhong | Tong, Zongzhong | Li, Shuangli | Ye, Jia | Wang, Lishun | Fang, Lin | Lei, Tingting | Chen, Chen | Chen, Huan | Xu, Zhao | Li, Haihong | Huang, Haiyan | Zhang, Feng | Xu, Huayong | Li, Na | Zhao, Caifeng | Li, Shuting | Dong, Lijun | Huang, Yanqing | Li, Long | Xi, Yan | Qi, Qiuhui | Li, Wenjie | Zhang, Bo | Hu, Wei | Zhang, Yanling | Tian, Xiangjun | Jiao, Yongzhi | Liang, Xiaohu | Jin, Jiao | Gao, Lei | Zheng, Weimou | Hao, Bailin | Liu, Siqi | Wang, Wen | Yuan, Longping | Cao, Mengliang | McDermott, Jason | Samudrala, Ram | Wang, Jian | Wong, Gane Ka-Shu | Yang, Huanming
PLoS Biology  2005;3(2):e38.
We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000–40,000. Only 2%–3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.
Comparative genome sequencing of indica and japonica rice reveals that duplication of genes and genomic regions has played a major part in the evolution of grass genomes
doi:10.1371/journal.pbio.0030038
PMCID: PMC546038  PMID: 15685292
2.  Whole-genome sequencing of Oryza brachyantha reveals mechanisms underlying Oryza genome evolution 
Nature Communications  2013;4:1595-.
The wild species of the genus Oryza contain a largely untapped reservoir of agronomically important genes for rice improvement. Here we report the 261-Mb de novo assembled genome sequence of Oryza brachyantha. Low activity of long-terminal repeat retrotransposons and massive internal deletions of ancient long-terminal repeat elements lead to the compact genome of Oryza brachyantha. We model 32,038 protein-coding genes in the Oryza brachyantha genome, of which only 70% are located in collinear positions in comparison with the rice genome. Analysing breakpoints of non-collinear genes suggests that double-strand break repair through non-homologous end joining has an important role in gene movement and erosion of collinearity in the Oryza genomes. Transition of euchromatin to heterochromatin in the rice genome is accompanied by segmental and tandem duplications, further expanded by transposable element insertions. The high-quality reference genome sequence of Oryza brachyantha provides an important resource for functional and evolutionary studies in the genus Oryza.
The wild rice species can be used as germplasm resources for this crop’s genetic improvement. Here Chen and colleagues report the de novo sequencing of the O. brachyantha genome, and identify the origin of genome size variation, the role of gene movement and its implications on heterochromatin evolution in the rice genome.
doi:10.1038/ncomms2596
PMCID: PMC3615480  PMID: 23481403
3.  Whole-genome analyses resolve early branches in the tree of life of modern birds 
Jarvis, Erich D. | Mirarab, Siavash | Aberer, Andre J. | Li, Bo | Houde, Peter | Li, Cai | Ho, Simon Y. W. | Faircloth, Brant C. | Nabholz, Benoit | Howard, Jason T. | Suh, Alexander | Weber, Claudia C. | da Fonseca, Rute R. | Li, Jianwen | Zhang, Fang | Li, Hui | Zhou, Long | Narula, Nitish | Liu, Liang | Ganapathy, Ganesh | Boussau, Bastien | Bayzid, Md. Shamsuzzoha | Zavidovych, Volodymyr | Subramanian, Sankar | Gabaldón, Toni | Capella-Gutiérrez, Salvador | Huerta-Cepas, Jaime | Rekepalli, Bhanu | Munch, Kasper | Schierup, Mikkel | Lindow, Bent | Warren, Wesley C. | Ray, David | Green, Richard E. | Bruford, Michael W. | Zhan, Xiangjiang | Dixon, Andrew | Li, Shengbin | Li, Ning | Huang, Yinhua | Derryberry, Elizabeth P. | Bertelsen, Mads Frost | Sheldon, Frederick H. | Brumfield, Robb T. | Mello, Claudio V. | Lovell, Peter V. | Wirthlin, Morgan | Schneider, Maria Paula Cruz | Prosdocimi, Francisco | Samaniego, José Alfredo | Velazquez, Amhed Missael Vargas | Alfaro-Núñez, Alonzo | Campos, Paula F. | Petersen, Bent | Sicheritz-Ponten, Thomas | Pas, An | Bailey, Tom | Scofield, Paul | Bunce, Michael | Lambert, David M. | Zhou, Qi | Perelman, Polina | Driskell, Amy C. | Shapiro, Beth | Xiong, Zijun | Zeng, Yongli | Liu, Shiping | Li, Zhenyu | Liu, Binghang | Wu, Kui | Xiao, Jin | Yinqi, Xiong | Zheng, Qiuemei | Zhang, Yong | Yang, Huanming | Wang, Jian | Smeds, Linnea | Rheindt, Frank E. | Braun, Michael | Fjeldsa, Jon | Orlando, Ludovic | Barker, F. Keith | Jønsson, Knud Andreas | Johnson, Warren | Koepfli, Klaus-Peter | O’Brien, Stephen | Haussler, David | Ryder, Oliver A. | Rahbek, Carsten | Willerslev, Eske | Graves, Gary R. | Glenn, Travis C. | McCormack, John | Burt, Dave | Ellegren, Hans | Alström, Per | Edwards, Scott V. | Stamatakis, Alexandros | Mindell, David P. | Cracraft, Joel | Braun, Edward L. | Warnow, Tandy | Jun, Wang | Gilbert, M. Thomas P. | Zhang, Guojie
Science (New York, N.Y.)  2014;346(6215):1320-1331.
To better determine the history of modern birds, we performed a genome-scale phylogenetic analysis of 48 species representing all orders of Neoaves using phylogenomic methods created to handle genome-scale data. We recovered a highly resolved tree that confirms previously controversial sister or close relationships. We identified the first divergence in Neoaves, two groups we named Passerea and Columbea, representing independent lineages of diverse and convergently evolved land and water bird species. Among Passerea, we infer the common ancestor of core landbirds to have been an apex predator and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence and high levels of incomplete lineage sorting that occurred during a rapid radiation after the Cretaceous-Paleogene mass extinction event about 66 million years ago.
doi:10.1126/science.1253451
PMCID: PMC4405904  PMID: 25504713
4.  GSK3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime inhibit microRNA maturation in mouse embryonic stem cells 
Scientific Reports  2015;5:8666.
Wnt/β-catenin signalling plays a prominent role in maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). microRNAs (miRNAs) have critical roles in maintaining pluripotency and directing reprogramming. To investigate the effect of GSK3 inhibitors on miRNA expression, we analysed the miRNA expression profile of J1 mESCs in the absence or presence of CHIR99021 (CHIR) or 6-bromoindirubin-3′-oxime (BIO) by small RNA deep-sequencing. The results demonstrate that CHIR and BIO decrease mature miRNAs of most miRNA species, 90.4% and 98.1% of the differentially expressed miRNAs in BIO and CHIR treated cells were downregulated respectively. CHIR and BIO treatment leads to a slight upregulation of the primary transcripts of the miR-302–367 cluster and miR-181 family of miRNAs, these miRNAs are activated by Wnt/β-catenin signalling. However, the precursor and mature form of the miR-302–367 cluster and miR-181 family of miRNAs are downregulated by CHIR, suggesting CHIR inhibits maturation of primary miRNA. Western blot analysis shows that BIO and CHIR treatment leads to a reduction of the RNase III enzyme Drosha in the nucleus. These data suggest that BIO and CHIR inhibit miRNA maturation by disturbing nuclear localisation of Drosha. Results also show that BIO and CHIR induce miR-211 expression in J1 mESCs.
doi:10.1038/srep08666
PMCID: PMC4345320  PMID: 25727520
5.  Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity 
Protein & Cell  2014;6(1):42-54.
ABSTRACT
Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-014-0102-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-014-0102-8
PMCID: PMC4286133  PMID: 25311840
HDAC6; substrate; lysine acetylation; quantitative proteomics; interaction
6.  Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity 
Protein & Cell  2014;6(1):42-54.
ABSTRACT
Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-014-0102-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-014-0102-8
PMCID: PMC4286133  PMID: 25311840
HDAC6; substrate; lysine acetylation; quantitative proteomics; interaction
7.  CistromeFinder for ChIP-seq and DNase-seq data reuse 
Bioinformatics  2013;29(10):1352-1354.
Summary: Chromatin immunoprecipitation and DNase I hypersensitivity assays with high-throughput sequencing have greatly accelerated the understanding of transcriptional and epigenetic regulation, although data reuse for the community of experimental biologists has been challenging. We created a data portal CistromeFinder that can help query, evaluate and visualize publicly available Chromatin immunoprecipitation and DNase I hypersensitivity assays with high-throughput sequencing data in human and mouse. The database currently contains 6378 samples over 4391 datasets, 313 factors and 102 cell lines or cell populations. Each dataset has gone through a consistent analysis and quality control pipeline; therefore, users could evaluate the overall quality of each dataset before examining binding sites near their genes of interest. CistromeFinder is integrated with UCSC genome browser for visualization, Primer3Plus for ChIP-qPCR primer design and CistromeMap for submitting newly available datasets. It also allows users to leave comments to facilitate data evaluation and update.
Availability: http://cistrome.org/finder.
Contact: xsliu@jimmy.harvard.edu or henry_long@dfci.harvard.edu
doi:10.1093/bioinformatics/btt135
PMCID: PMC3654708  PMID: 23508969
8.  Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro 
Tanshinone I (Tan-I) is a diterpene quinone extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge. Recently, Tan-I has been reported to have anti-tumor effects. In this study, we investigated the growth inhibition and apoptosis inducing effects of Tan-I on three kinds of monocytic leukemia cells (U937, THP-1 and SHI 1). Cell viability was measured by MTT assay. Cell apoptosis was assessed by flow cytometry (FCM) and AnnexinV/PI staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR–enzyme-linked immunosorbent assay (ELISA) were used to detect human telomerase reverse transcriptase (hTERT) expression and telomerase activity before and after apoptosis. The activity of caspase-3 was determined by Caspase colorimetric assay kit and Western blot analysis. Expression of the anti-apoptotic gene Survivin was assayed by Western blot and Real-time RT-PCR using the ABI PRISM 7500 Sequence Detection System. The results revealed that Tan-I could inhibit the growth of these three kinds of leukemia cells and cause apoptosis in a time- and dose-dependent manner. After treatment by Tan-I for 48 h, Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17-kD subunit, and a 89-kD cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also found clearly. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently in a dose-dependent manner. Moreover, Real-time RT-PCR and Western blot revealed a significant down-regulation of Survivin. We therefore conclude that the induction of apoptosis by Tan-I in monocytic leukemia U937 THP-1 and SHI 1 cells is highly correlated with activation of caspase-3 and decreasing of hTERT mRNA expression and telomerase activity as well as down-regulation of Survivin expression. To our knowledge, this is the first report about the effects of Tan-I on monocytic leukemia cells.
doi:10.3390/ijms11062267
PMCID: PMC2904915  PMID: 20640151
Tanshinone I (Tan-I); telomerase; survivin; leukemia
9.  Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands 
Scientific Reports  2016;6:20657.
Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription- activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.
doi:10.1038/srep20657
PMCID: PMC4745098  PMID: 26853907
10.  Autologous cytokine-induced killer cell transfusion increases overall survival in advanced pancreatic cancer 
Background
Advanced pancreatic cancer (PC) has very poor prognosis with present treatments, thus necessitating continued efforts to find improved therapeutic approaches. Both preclinical and preliminary clinical data indicate that cytokine-induced killer (CIK) cells are an effective tool against various types of solid tumors. Here, we conducted a study to determine whether CIK cell-based therapy (CBT) can improve the outcomes of advanced PC.
Methods
Eighty-two patients with advanced PC, whose predicted survival time was longer than 3 months, were analyzed retrospectively. Of all the patients, 57 individuals were receiving chemotherapy, while the remaining 25 individuals were treated with CBT.
Results
The overall survival analysis was based on 48 deaths in the 57 patients in the chemotherapy group (84.2 %) and 18 deaths in the 25 patients in the CBT group (72.0 %). In the CBT group, the median overall survival time was 13.5 months, as compared to 6.6 months in the chemotherapy group (hazard ratio for death, 0.39; 95 % confidence interval, 0.23 to 0.65; p < 0.001). The survival rate was 88.9 % in the CBT group versus 54.2 % in the chemotherapy group at 6 months, 61.1 % versus 12.5 % at 12 months, and 38.9 % versus 4.2 % at 18 months. The disease control rate was 68.0 % in the CBT group and 29.8 % in the chemotherapy group (p < 0.001).
Conclusions
These results from this retrospective analysis appeared to imply that CBT might prolong survival in these high-risk PC patients. Prospective study is needed to corroborate this observation.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-016-0237-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13045-016-0237-6
PMCID: PMC4740990  PMID: 26842696
Cytokine-induced killer cells; Immunotherapy; Pancreatic cancer; Overall survival
11.  Combined Value of Red Blood Cell Distribution Width and Global Registry of Acute Coronary Events Risk Score for Predicting Cardiovascular Events in Patients with Acute Coronary Syndrome Undergoing Percutaneous Coronary Intervention 
PLoS ONE  2015;10(10):e0140532.
Global Registry of Acute Coronary Events (GRACE) risk score and red blood cell distribution width (RDW) content can both independently predict major adverse cardiac events (MACEs) in patients with acute coronary syndrome (ACS). We investigated the combined predictive value of RDW and GRACE risk score for cardiovascular events in patients with ACS undergoing percutaneous coronary intervention (PCI) for the first time. We enrolled 480 ACS patients. During a median follow-up time of 37.2 months, 70 (14.58%) patients experienced MACEs. Patients were divided into tertiles according to the baseline RDW content (11.30–12.90, 13.00–13.50, 13.60–16.40). GRACE score was positively correlated with RDW content. Multivariate Cox analysis showed that both GRACE score and RDW content were independent predictors of MACEs (hazard ratio 1.039; 95% confidence interval [CI] 1.024–1.055; p < 0.001; 1.699; 1.294–2.232; p < 0.001; respectively). Furthermore, Kaplan–Meier analysis demonstrated that the risk of MACEs increased with increasing RDW content (p < 0.001). For GRACE score alone, the area under the receiver operating characteristic (ROC) curve for MACEs was 0.749 (95% CI: 0.707–0.787). The area under the ROC curve for MACEs increased to 0.805 (0.766–0.839, p = 0.034) after adding RDW content. The incremental predictive value of combining RDW content and GRACE risk score was significantly improved, also shown by the net reclassification improvement (NRI = 0.352, p < 0.001) and integrated discrimination improvement (IDI = 0.023, p = 0.002). Combining the predictive value of RDW and GRACE risk score yielded a more accurate predictive value for long-term cardiovascular events in ACS patients who underwent PCI as compared to each measure alone.
doi:10.1371/journal.pone.0140532
PMCID: PMC4607415  PMID: 26468876
12.  Elucidation of Echovirus 30's Origin and Transmission during the 2012 Aseptic Meningitis Outbreak in Guangdong, China, through Continuing Environmental Surveillance 
An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses.
doi:10.1128/AEM.03200-14
PMCID: PMC4357961  PMID: 25616804
13.  Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells 
Cell Research  2014;24(10):1181-1200.
The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP+ cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.
doi:10.1038/cr.2014.118
PMCID: PMC4185345  PMID: 25190258
gene labeling; pancreatic β cell; directed differentiation; embryonic stem cell; SUSD2
14.  Ultraviolet light triggers the conversion of Cu2+-bound Aβ42 aggregates into cytotoxic species in a copper chelation-independent manner 
Scientific Reports  2015;5:13897.
Increasing evidence indicates that abnormal Cu2+ binding to Aβ peptides are responsible for the formation of soluble Aβ oligomers and ROS that play essential roles in AD pathogenesis. During studying the Cu2+-chelating treatment of Cu2+-bound Aβ42 aggregates, we found that UV light exposure pronouncedly enhances cytotoxicity of the chelator-treated and -untreated Cu2+-bound Aβ42 aggregates. This stimulated us to thoroughly investigate (1) either the chelation treatment or UV light exposure leads to the increased cytotoxicity of the aggregates, and (2) why the chelator-treated and -untreated Cu2+-bound Aβ42 aggregates exhibit the increased cytotoxicity following UV light exposure if the latter is the case. The data indicated that the controlled UV exposure induced the dissociation of Cu2+-free and -bound Aβ42 aggregates into SDS-stable soluble oligomers and the production of ROS including H2O2 in an UV light intensity- and time-dependent, but Cu2+ chelation-independent manner. Although we can’t fully understand the meaning of this finding at the current stage, the fact that the UV illuminated Aβ42 aggregates can efficiently kill HeLa cells implies that the aggregates after UV light exposure could be used to decrease the viability of skin cancer cells through skin administration.
doi:10.1038/srep13897
PMCID: PMC4563556  PMID: 26350232
15.  Lysophosphatidic Acid Inhibits Apoptosis Induced by Cisplatin in Cervical Cancer Cells 
BioMed Research International  2015;2015:598386.
Cervical cancer is the second most common cause of cancer death in women worldwide. Lysophosphatidic acid (LPA) level has been found significantly increased in the serum of patients with ovarian, cervical, and colon cancers. LPA level in cervical cancer patients is significantly higher than in healthy controls. LPA receptors were found highly expressed in cervical cancer cells, suggesting LPA may play a role in the development of cervical cancer. The aim of this study is to investigate the effect of LPA on the apoptosis induced by cisplatin (DDP) in cervical cancer cell line and the underlying changes in signaling pathways. Our study found that cisplatin induced apoptosis of Hela cell through inhibiting expression of Bcl-2, upregulating the expression of Bax, Fas-L, and the enzyme activity of caspase-3 (p < 0.05); LPA significantly provided protection against the apoptosis induced by cisplatin by inhibiting the above alterations in apoptotic factor caused by cisplatin (p < 0.05). Moreover, PI3K/AKT pathway was found to be important for the LPA antiapoptosis effect, and administration of PI3K/AKT partially reversed the LPA-mediated protection against cisplatin-induced apoptosis (p < 0.05). These findings have shed new lights on the LPA bioactivity in cervical cancer cells and pointed to a possible sensitization scheme through combined administration of PI3K inhibitor and cisplatin for better treatment of cervical cancer patients, especially those with elevated LPA levels.
doi:10.1155/2015/598386
PMCID: PMC4558435  PMID: 26366416
16.  Molecular Characterization of NF1 and Neurofibromatosis Type 1 Genotype-Phenotype Correlations in a Chinese Population 
Scientific Reports  2015;5:11291.
Neurofibromatosis type 1 (NF1) is an autosomal dominant hereditary disease that is primarily characterized by multiple café au-lait spots (CALs) and skin neurofibromas, which are attributed to defects in the tumor suppressor NF1. Because of the age-dependent presentation of NF1, it is often difficult to make an early clinical diagnosis. Moreover, identifying genetic alterations in NF1 patients represents a complex challenge. Currently, there are no effective detective methods, and no comprehensive NF1 mutation data are available for mainland China. We screened 109 Chinese patients from 100 families with NF1-like phenotypes (e.g., CALs, neurofibromas, etc.) using Sanger sequencing, multiplex ligation-dependent probe amplification and cDNA sequencing. NF1 mutations were identified in 97 individuals, among which 34 intragenic mutations have not previously been reported. Our exhaustive mutational analysis detected mutations in 89% (89/100) of the NF1-like probands and 93% (70/75) of subjects fulfilling the National Institutes of Health (NIH) criteria. Our findings indicate that individuals who exclusively present with multiple CALs exhibit a high possibility (76%) of having NF1 and show a significantly lower mutation rate (p = 0.042) compared with subjects who fulfill the NIH criteria, providing clinicians with the information that subjects only with multiple CALs harbor a considerable possibility (24%) of being attributed to other comparable diseases.
doi:10.1038/srep11291
PMCID: PMC4460887  PMID: 26056819
17.  Comparative genomics reveals insights into avian genome evolution and adaptation 
Zhang, Guojie | Li, Cai | Li, Qiye | Li, Bo | Larkin, Denis M. | Lee, Chul | Storz, Jay F. | Antunes, Agostinho | Greenwold, Matthew J. | Meredith, Robert W. | Ödeen, Anders | Cui, Jie | Zhou, Qi | Xu, Luohao | Pan, Hailin | Wang, Zongji | Jin, Lijun | Zhang, Pei | Hu, Haofu | Yang, Wei | Hu, Jiang | Xiao, Jin | Yang, Zhikai | Liu, Yang | Xie, Qiaolin | Yu, Hao | Lian, Jinmin | Wen, Ping | Zhang, Fang | Li, Hui | Zeng, Yongli | Xiong, Zijun | Liu, Shiping | Zhou, Long | Huang, Zhiyong | An, Na | Wang, Jie | Zheng, Qiumei | Xiong, Yingqi | Wang, Guangbiao | Wang, Bo | Wang, Jingjing | Fan, Yu | da Fonseca, Rute R. | Alfaro-Núñez, Alonzo | Schubert, Mikkel | Orlando, Ludovic | Mourier, Tobias | Howard, Jason T. | Ganapathy, Ganeshkumar | Pfenning, Andreas | Whitney, Osceola | Rivas, Miriam V. | Hara, Erina | Smith, Julia | Farré, Marta | Narayan, Jitendra | Slavov, Gancho | Romanov, Michael N | Borges, Rui | Machado, João Paulo | Khan, Imran | Springer, Mark S. | Gatesy, John | Hoffmann, Federico G. | Opazo, Juan C. | Håstad, Olle | Sawyer, Roger H. | Kim, Heebal | Kim, Kyu-Won | Kim, Hyeon Jeong | Cho, Seoae | Li, Ning | Huang, Yinhua | Bruford, Michael W. | Zhan, Xiangjiang | Dixon, Andrew | Bertelsen, Mads F. | Derryberry, Elizabeth | Warren, Wesley | Wilson, Richard K | Li, Shengbin | Ray, David A. | Green, Richard E. | O’Brien, Stephen J. | Griffin, Darren | Johnson, Warren E. | Haussler, David | Ryder, Oliver A. | Willerslev, Eske | Graves, Gary R. | Alström, Per | Fjeldså, Jon | Mindell, David P. | Edwards, Scott V. | Braun, Edward L. | Rahbek, Carsten | Burt, David W. | Houde, Peter | Zhang, Yong | Yang, Huanming | Wang, Jian | Jarvis, Erich D. | Gilbert, M. Thomas P. | Wang, Jun
Science (New York, N.Y.)  2014;346(6215):1311-1320.
Birds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.
doi:10.1126/science.1251385
PMCID: PMC4390078  PMID: 25504712
18.  Computational methodology for ChIP-seq analysis 
Quantitative biology  2013;1(1):54-70.
Chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) is a powerful technology to identify the genome-wide locations of DNA binding proteins such as transcription factors or modified histones. As more and more experimental laboratories are adopting ChIP-seq to unravel the transcriptional and epigenetic regulatory mechanisms, computational analyses of ChIP-seq also become increasingly comprehensive and sophisticated. In this article, we review current computational methodology for ChIP-seq analysis, recommend useful algorithms and workflows, and introduce quality control measures at different analytical steps. We also discuss how ChIP-seq could be integrated with other types of genomic assays, such as gene expression profiling and genome-wide association studies, to provide a more comprehensive view of gene regulatory mechanisms in important physiological and pathological processes.
doi:10.1007/s40484-013-0006-2
PMCID: PMC4346130  PMID: 25741452
19.  Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT 
PLoS ONE  2015;10(1):e0114423.
Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.
doi:10.1371/journal.pone.0114423
PMCID: PMC4300223  PMID: 25602959
20.  Mudskipper genomes provide insights into the terrestrial adaptation of amphibious fishes 
Nature Communications  2014;5:5594.
Mudskippers are amphibious fishes that have developed morphological and physiological adaptations to match their unique lifestyles. Here we perform whole-genome sequencing of four representative mudskippers to elucidate the molecular mechanisms underlying these adaptations. We discover an expansion of innate immune system genes in the mudskippers that may provide defence against terrestrial pathogens. Several genes of the ammonia excretion pathway in the gills have experienced positive selection, suggesting their important roles in mudskippers’ tolerance to environmental ammonia. Some vision-related genes are differentially lost or mutated, illustrating genomic changes associated with aerial vision. Transcriptomic analyses of mudskippers exposed to air highlight regulatory pathways that are up- or down-regulated in response to hypoxia. The present study provides a valuable resource for understanding the molecular mechanisms underlying water-to-land transition of vertebrates.
Mudskippers are amphibious fishes that have adapted to live on mudflats. Here, the authors sequence the genomes of four different mudskipper species and highlight genetic changes that may have had an evolutionary role in the water-to-land transition of vertebrates.
doi:10.1038/ncomms6594
PMCID: PMC4268706  PMID: 25463417
21.  Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment 
GigaScience  2014;3:27.
Background
Penguins are flightless aquatic birds widely distributed in the Southern Hemisphere. The distinctive morphological and physiological features of penguins allow them to live an aquatic life, and some of them have successfully adapted to the hostile environments in Antarctica. To study the phylogenetic and population history of penguins and the molecular basis of their adaptations to Antarctica, we sequenced the genomes of the two Antarctic dwelling penguin species, the Adélie penguin [Pygoscelis adeliae] and emperor penguin [Aptenodytes forsteri].
Results
Phylogenetic dating suggests that early penguins arose ~60 million years ago, coinciding with a period of global warming. Analysis of effective population sizes reveals that the two penguin species experienced population expansions from ~1 million years ago to ~100 thousand years ago, but responded differently to the climatic cooling of the last glacial period. Comparative genomic analyses with other available avian genomes identified molecular changes in genes related to epidermal structure, phototransduction, lipid metabolism, and forelimb morphology.
Conclusions
Our sequencing and initial analyses of the first two penguin genomes provide insights into the timing of penguin origin, fluctuations in effective population sizes of the two penguin species over the past 10 million years, and the potential associations between these biological patterns and global climate change. The molecular changes compared with other avian genomes reflect both shared and diverse adaptations of the two penguin species to the Antarctic environment.
Electronic supplementary material
The online version of this article (doi:10.1186/2047-217X-3-27) contains supplementary material, which is available to authorized users.
doi:10.1186/2047-217X-3-27
PMCID: PMC4322438  PMID: 25671092
Penguins; Avian genomics; Evolution; Adaptation; Antarctica
22.  Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a target in LKB1 Mutant Lung Cancer 
Cancer discovery  2013;3(8):870-879.
The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung cancer, LKB1 is somatically inactivated in 25-30% of cases, often concurrently with activating KRAS mutation. Here, we employed an integrative approach to define novel therapeutic targets in KRAS-driven LKB1 mutant lung cancers. High-throughput RNAi screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling demonstrated that Lkb1-null cells had striking decreases in multiple nucleotide metabolites as compared to the Lkb1-wt cells. Thus, LKB1 mutant lung cancers have deficits in nucleotide metabolism conferring hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.
doi:10.1158/2159-8290.CD-13-0015
PMCID: PMC3753578  PMID: 23715154
LKB1; KRAS; DTYMK; CHEK1; NSCLC; GEMM-derived cell line; genome wide RNAi screen; metabolic profiling
23.  Identifying ChIP-seq enrichment using MACS 
Nature protocols  2012;7(9):10.1038/nprot.2012.101.
Model-based Analysis of ChIP-seq (MACS) is a computational algorithm that identifies genome-wide locations of transcription/chromatin factor binding or histone modification from ChIP-seq data. MACS consists of four steps: removing redundant reads, adjusting read position, calculating peak enrichment, and estimating the empirical false discovery rate. In this protocol, we provide a detailed demonstration of how to install MACS and how to use it to analyze three common types of ChIP-seq datasets with different characteristics: the sequence-specific transcription factor FoxA1, the histone modification mark H3K4me3 with sharp enrichment, and the H3K36me3 mark with broad enrichment. We also explain how to interpret and visualize the results of MACS analyses. The algorithm requires approximately 3 GB of RAM and 1.5 hours of computing time to analyze a ChIP-seq dataset containing 30 million reads, an estimate that increases with sequence coverage. MACS is open-source and is available from http://liulab.dfci.harvard.edu/MACS.
doi:10.1038/nprot.2012.101
PMCID: PMC3868217  PMID: 22936215
MACS; ChIP-seq; peak calling; transcription factor; histone modification
24.  CR Cistrome: a ChIP-Seq database for chromatin regulators and histone modification linkages in human and mouse 
Nucleic Acids Research  2013;42(Database issue):D450-D458.
Diversified histone modifications (HMs) are essential epigenetic features. They play important roles in fundamental biological processes including transcription, DNA repair and DNA replication. Chromatin regulators (CRs), which are indispensable in epigenetics, can mediate HMs to adjust chromatin structures and functions. With the development of ChIP-Seq technology, there is an opportunity to study CR and HM profiles at the whole-genome scale. However, no specific resource for the integration of CR ChIP-Seq data or CR-HM ChIP-Seq linkage pairs is currently available. Therefore, we constructed the CR Cistrome database, available online at http://compbio.tongji.edu.cn/cr and http://cistrome.org/cr/, to further elucidate CR functions and CR-HM linkages. Within this database, we collected all publicly available ChIP-Seq data on CRs in human and mouse and categorized the data into four cohorts: the reader, writer, eraser and remodeler cohorts, together with curated introductions and ChIP-Seq data analysis results. For the HM readers, writers and erasers, we provided further ChIP-Seq analysis data for the targeted HMs and schematized the relationships between them. We believe CR Cistrome is a valuable resource for the epigenetics community.
doi:10.1093/nar/gkt1151
PMCID: PMC3965064  PMID: 24253304
25.  Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows 
Nature Communications  2013;4:2565.
Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Although zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species. Here we show in bovine fetal fibroblasts that targeting ZFNickases to the endogenous β-casein (CSN2) locus stimulates lysostaphin gene addition by homology-directed repair. We find that ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete lysostaphin in their milk and in vitro assays demonstrate the milk’s ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine.
Zinc-finger nickases are programmable nucleases that can be used to generate site-specific single-strand breaks in DNA. Liu et al. use this technology to insert an antimicrobial gene into the endogenous beta-casein locus in cloned cows, with the aim of providing protection against mastitis.
doi:10.1038/ncomms3565
PMCID: PMC3826644  PMID: 24121612

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