We present Model-based Analysis of ChIP-Seq data, MACS, which analyzes data generated by short read sequencers such as Solexa's Genome Analyzer. MACS empirically models the shift size of ChIP-Seq tags, and uses it to improve the spatial resolution of predicted binding sites. MACS also uses a dynamic Poisson distribution to effectively capture local biases in the genome, allowing for more robust predictions. MACS compares favorably to existing ChIP-Seq peak-finding algorithms, and is freely available.

Three cDNA sequences encoding the gonadotropin subunits, common glycoprotein alpha subunit (GTHalpha), FSHbeta and LHbeta subunits were isolated from marbled eel. The cDNA of GTHalpha encodes 116 amino acids with a signal peptide of 24 amino acids and a mature peptide of 92 amino acids. The FSHbeta subunit consists of 127 amino acids with a 22 amino acid signal peptide and a 105 amino acid mature peptide, while the LHbeta subunit consists of 140 amino acids with a 24 amino acid signal peptide and a 116 amino acid mature peptide. Comparison of the deduced amino acid sequences of marbled eel GTHalpha, FSHbeta, and LHbeta with that of other fishes shows a high degree of conservation in the number of cysteine residues and potential N-linked glycosylation sites. The mRNA of GTHalpha, FSHbeta and LHbeta were not only detected in pituitary, but also in ovary and testes by RT-PCR. Quantitative realtime PCR analysis revealed that the GTHalpha and LHbeta transcriptional levels in pituitaries of female and male eels gradually increased during the artificially inducing gonadal development, and peaked at late vitellogenic stage and spermiation stage, respectively. FSHbeta mRNA in the pituitaries of female eels maintained a high level at previtellogenic stage, early vitellogenic stage as well as mid-vitellogenic stage but declined sharply at late vitellogenic stage and migratory nucleus stage. In male eels, the mRNA levels of FSHbeta in the pituitaries were higher at early spermatogenesis stage than at both late spermatogenesis stage and spermiation stage. These results suggested that FSH would be in control of initiation and maintenance of gonadal growth and gametogenesis, whereas LH would be involved in the final gonadal maturation and spermiation/ovulation in the tropic eel Anguilla marmorata.

MicroRNAs (miRNAs) are endogenous non-coding genes that participate in post-transcription regulation by either degrading mRNA or blocking its translation. It is considered to be very important in regulating insect development and metamorphosis. We conducted a large-scale screening for miRNA genes in the silkworm Bombyx mori using sequence-by-synthesis (SBS) deep sequencing of mixed RNAs from egg, larval, pupal, and adult stages. Of 2,227,930 SBS tags, 1,144,485 ranged from 17 to 25 nt, corresponding to 256,604 unique tags. Among these non-redundant tags, 95,184 were matched to the silkworm genome. We identified 3,750 miRNA candidate genes using a computational pipeline combining RNAfold and TripletSVM algorithms. We confirmed 354 miRNA genes using miRNA microarrays and then performed expression profile analysis on these miRNAs for all developmental stages. While 106 miRNAs were expressed in all stages, 248 miRNAs were egg- and pupa-specific, suggesting that insect miRNAs play a significant role in embryogenesis and metamorphosis. We selected eight miRNAs for quantitative RT-PCR analysis; six of these were consistent with our microarray results. In addition, we searched for orthologous miRNA genes in mammals, a nematode, and other insects and found that most silkworm miRNAs are conserved in insects, whereas only a small number of silkworm miRNAs has orthologs in mammals and the nematode. These results suggest that there are many miRNAs unique to insects.

The three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence.

Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, is normally mild but can have life-threatening manifestations. It can be caused by enteroviruses, particularly Coxsackieviruses and human enterovirus 71 (HEV71) with highly variable clinical manifestations. In the spring of 2008, a large, unprecedented HFMD outbreak in Fuyang city of Anhui province in the central part of southeastern China resulted in a high aggregation of fatal cases. In this study, epidemiologic and clinical investigations, laboratory testing, and genetic analyses were performed to identify the causal pathogen of the outbreak. Of the 6,049 cases reported between 1 March and 9 May of 2008, 3023 (50%) were hospitalized, 353 (5.8%) were severe and 22 (0.36%) were fatal. HEV71 was confirmed as the etiological pathogen of the outbreak. Phylogenetic analyses of entire VP1 capsid protein sequence of 45 Fuyang HEV71 isolates showed that they belong to C4a cluster of the C4 subgenotype. In addition, genetic recombinations were found in the 3D region (RNA-dependent RNA polymerase, a major component of the viral replication complex of the genome) between the Fuyang HEV71 strain and Coxsackievirus A16 (CV-A16), resulting in a recombination virus. In conclusion, an emerging recombinant HEV71 was responsible for the HFMD outbreak in Fuyang City of China, 2008.

After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing lysine trimethylation modifications before and after the maternal-zygotic transition in zebrafish. Histone H3 lysine 27 trimethylation (H3K27me3), which is repressive, and H3K4me3, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by H3K4me3, including many inactive developmental regulatory genes that were also marked by H3K27me3. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed. Furthermore, we found many inactive genes that were uniquely marked by H3K4me3. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published data sets revealed similar monovalent domains in embryonic stem cells. Moreover, H3K4me3 marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA polymerase II, as indicated by the analysis of an inducible transgene. These results indicate that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal-zygotic transition.

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

MicroRNAs (miRNAs) are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Our previous studies have revealed that miR-148a and miR-152 are significantly down-regulated in gastrointestinal cancers. Interestingly, miR-148b has the same "seed sequences" as miR-148a and miR-152. Although aberrant expression of miR-148b has been observed in several types of cancer, its pathophysiologic role and relevance to tumorigenesis are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in gastric cancer.

Acquisition and quantitative analysis of high resolution images of dendritic spines are challenging tasks but are necessary for the study of animal models of neurological and psychiatric diseases. Currently available methods for automated dendritic spine detection are for the most part customized for 2D image slices, not volumetric 3D images. In this work, a fully automated method is proposed to detect and segment dendritic spines from 3D confocal microscopy images of medium-sized spiny neurons (MSNs). MSNs constitute a major neuronal population in striatum, and abnormalities in their function are associated with several neurological and psychiatric diseases. Such automated detection is critical for the development of new 3D neuronal assays which can be used for the screening of drugs and the studies of their therapeutic effects. The proposed method utilizes a generalized gradient vector flow (GGVF) with a new smoothing constraint and then detects feature points near the central regions of dendrites and spines. Then, the central regions are refined and separated based on eigen-analysis and multiple shape measurements. Finally, the spines are segmented in 3D space using the fast marching algorithm, taking the detected central regions of spines as initial points. The proposed method is compared with three popular existing methods for centerline extraction and also with manual results for dendritic spine detection in 3D space. The experimental results and comparisons show that the proposed method is able to automatically and accurately detect, segment, and quantitate dendritic spines in 3D images of MSNs.

Protein kinase C (PKC) is involved in a wide array of cellular processes such as cell proliferation, differentiation and apoptosis. Phosphorylation of both turn motif (TM) and hydrophobic motif (HM) are important for PKC function. Here, we show that the mammalian target of rapamycin complex 2 (mTORC2) has an important function in phosphorylation of both TM and HM in all conventional PKCs, novel PKCepsilon as well as Akt. Ablation of mTORC2 components (Rictor, Sin1 or mTOR) abolished phosphorylation on the TM of both PKCalpha and Akt and HM of Akt and decreased HM phosphorylation of PKCalpha. Interestingly, the mTORC2-dependent TM phosphorylation is essential for PKCalpha maturation, stability and signalling. Our study demonstrates that mTORC2 is involved in post-translational processing of PKC by facilitating TM and HM phosphorylation and reveals a novel function of mTORC2 in cellular regulation.

In vivo positioning and covalent modifications of nucleosomes play an important role in epigenetic regulation, but genome-wide studies of positioned nucleosomes and their modifications in human still remain limited.

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.

The aim of the present study was to examine the efficacy of tumor-targeting Salmonella typhimurium A1-R treatment following anti-vascular endothelial growth factor (VEGF) therapy on VEGF-positive human pancreatic cancer. A pancreatic cancer patient-derived orthotopic xenograft (PDOX) that was VEGF-positive and an orthotopic VEGF-positive human pancreatic cancer cell line (MiaPaCa-2-GFP) as well as a VEGF-negative cell line (Panc-1) were tested. Nude mice with these tumors were treated with gemcitabine (GEM), bevacizumab (BEV), and S. typhimurium A1-R. BEV/GEM followed by S. typhimurium A1-R significantly reduced tumor weight compared to BEV/GEM treatment alone in the PDOX and MiaPaCa-2 models. Neither treatment was as effective in the VEGF-negative model as in the VEGF-positive models. These results demonstrate that S. typhimurium A1-R following anti-angiogenic therapy is effective on pancreatic cancer including the PDOX model, suggesting its clinical potential.

Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activating ectoderm markers in mouse embryonic stem cells (mESCs). Moreover, RA modulated the DNA methylation of mESCs by altering the expression of epigenetic-associated genes such as Dnmt3b and Dnmt3l. Furthermore, H3K4me2, a pluripotent histone modification, was repressed by RA stimulation. From microRNA sequence data, we identified two downregulated microRNAs, namely, miR-200b and miR-200c, which regulated the pluripotency of stem cells. We found that miR-200b or miR-200c deficiency suppressed the expression of pluripotent genes, including Oct4 and Nanog, and activated the expression of the ectodermal marker gene Nestin. These results demonstrate that retinoid induces mESCs to differentiate by regulating miR-200b/200c. Our findings provide the landscapes of mRNA and microRNA gene networks and indicate the crucial role of miR-200b/200c in the RA-induced differentiation of mESCs.

The v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B gene (MAFB) has been associated with serum lipid levels in the Eurpean population, but little is known about such association in the Chinese population or in atherosclerosis-related patients. Therefore, the purpose of the present study was to assess the association of the single nucleotide polymorphisms (SNPs) in the MAFB and serum lipid levels and the risk of coronary artery disease (CAD) and ischemic stroke (IS) in the Chinese population.

Natural grassland productivity, which is based on an individual plant's aboveground biomass (AB) and its interaction with herbivores, can obviously affect terrestrial ecosystem services and the grassland's agricultural production. As plant traits have been linked to both AB and ecosystem success, they may provide a useful approach to understand the changes in individual plants and grassland productivity in response to grazing on a generic level. Unfortunately, the current lack of studies on how plant traits affect AB affected by herbivores leaves a major gap in our understanding of the mechanism of grassland productivity decline. This study, therefore, aims to analyze the paths of overgrazing-induced decline in the individual AB of Leymus chinensis (the dominant species of meadow-steppe grassland in northern China) on a plant functional trait scale. Using a paired-sampling approach, we compared the differences in the functional traits of L. chinensis in long-term grazing-excluded and experimental grazing grassland plots over a continuous period of approximately 20 years (located in meadow steppe lands in Hailar, Inner Mongolia, China). We found a highly significant decline in the individual height and biomass (leaf, stem, and the whole plant) of L. chinensis as a result of overgrazing. Biomass allocation and leaf mass per unit area were significantly affected by the variation in individual size. Grazing clearly enhanced the sensitivity of the leaf-to-stem biomass ratio in response to variation in individual size. Moreover, using a method of standardized major axis estimation, we found that the biomass in the leaves, stems, and the plant as a whole had highly significant allometric scaling with various functional traits. Also, the slopes of the allometric equations of these relationships were significantly altered by grazing. Therefore, a clear implication of this is that grazing promotes an asymmetrical response of different plant functional traits to variation in individual plant size, which influences biomass indirectly. Furthermore, we detected paths of individual AB decline in L. chinensis induced by grazing by fitting to a structural equation model. These results indicate that grazing causes AB decline primarily through a 'bottom-up' effect on plant height and stem traits. However, leaf traits, via the process of allometric scaling, affect plant AB indirectly.

Tumor-derived heat shock proteins peptide complex (HSP.PC-Tu) has been regarded as a promising antitumor agent. However, inadequate immunogenicity and low bioavailability limit the clinical uses of this agent. In a previous study, we first produced an improved HSP70.PC-based vaccine purified from dendritic cell (DC)-tumor fusion cells (HSP70.PC-Fc) which had increased immunogenicity due to enhanced antigenic tumor peptides compared to HSP70.PC-Tu. In order to increase the bioavailability of HSP70.PC-Fc, the peptide complex was encapsulated with nanoliposomes (NL-HSP70.PC-Fc) in this study. After encapsulation, the tumor immunogenicity was observed using various assays. It was demonstrated that the NL-HSP70.PC-Fc has acceptable stability. The in vivo antitumor immune response was increased with regard to T-cell activation, CTL response and tumor therapy efficiency compared to that of HSP70.PC-Fc. In addition, it was shown that DC maturation was improved by NL-HSP70.PC-Fc, which added to the antitumor immunity. The results obtained for NL-HSP70.PC-Fc, which improved immunogenicity and increases the bioavailability of HSP70.PC, may represent superior heat shock proteins (HSPs)-based tumor vaccines. Such vaccines deserve further investigation and may provide a preclinical rationale to translate findings into early phase trials for patients with breast tumors.

Explaining and predicting animal distributions is one of the fundamental objectives in ecology and conservation biology. Animal habitat selection can be regulated by top-down and bottom-up processes, and is mediated by species interactions. Species varying in body size respond differently to top-down and bottom-up determinants, and hence understanding these allometric responses to those determinants is important for conservation. In this study, using two differently sized goose species wintering in the Yangtze floodplain, we tested the predictions derived from three different hypotheses (individual-area relationship, food resource and disturbance hypothesis) to explain the spatial and temporal variation in densities of two goose species. Using Generalized Linear Mixed Models with a Markov Chain Monte Carlo technique, we demonstrated that goose density was positive correlated with patch area size, suggesting that the individual area-relationship best predicts differences in goose densities. Moreover, the other predictions, related to food availability and disturbance, were not significant. Buffalo grazing probably facilitated greater white-fronted geese, as the number of buffalos was positively correlated to the density of this species. We concluded that patch area size is the most important factor determining the density of goose species in our study area. Patch area size is directly determined by water levels in the Yangtze floodplain, and hence modifying the hydrological regimes can enlarge the capacity of these wetlands for migratory birds.

The extracellular matrix of scaly green flagellates consists of small organic scales consisting of polysaccharides and scale-associated proteins (SAPs). Molecular phylogenies have shown that these organisms represent the ancestral stock of flagellates from which all green plants (Viridiplantae) evolved. The molecular characterization of four different SAPs is presented. Three SAPs are type-2 membrane proteins with an arginine/alanine-rich short cytoplasmic tail and an extracellular domain that is most likely of bacterial origin. The fourth protein is a filamin-like protein. In addition, we report the presence of proteins similar to the integrin-associated proteins α-actinin (in transcriptomes of glaucophytes and some viridiplants), LIM-domain proteins, and integrin-associated kinase in transcriptomes of viridiplants, glaucophytes, and rhodophytes. We propose that the membrane proteins identified are the predicted linkers between scales and the cytoskeleton. These proteins are present in many green algae but are apparently absent from embryophytes. These proteins represent a new protein family we have termed gralins for green algal integrins. Gralins are absent from embryophytes. A model for the evolution of the cell surface proteins in Plantae is discussed.

Consolidated bioprocessing (CBP) of butanol production from cellulosic biomass is a promising strategy for cost saving compared to other processes featuring dedicated cellulase production. CBP requires microbial strains capable of hydrolyzing biomass with enzymes produced on its own with high rate and high conversion and simultaneously produce a desired product at high yield. However, current reported butanol-producing candidates are unable to utilize cellulose as a sole carbon source and energy source. Consequently, developing a co-culture system using different microorganisms by taking advantage of their specific metabolic capacities to produce butanol directly from cellulose in consolidated bioprocess is of great interest.