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Aquaporins (Aqps) are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. Among vertebrate species, Aqps are highly conserved in both gene structure and amino acid sequence. These proteins are vital for maintaining water homeostasis in living organisms, especially for aquatic animals such as teleost fish. Studies on teleost Aqps are mainly limited to several model species with diploid genomes. Common carp, which has a tetraploidized genome, is one of the most common aquaculture species being adapted to a wide range of aquatic environments. The complete common carp genome has recently been released, providing us the possibility for gene evolution of aqp gene family after whole genome duplication.
In this study, we identified a total of 37 aqp genes from common carp genome. Phylogenetic analysis revealed that most of aqps are highly conserved. Comparative analysis was performed across five typical vertebrate genomes. We found that almost all of the aqp genes in common carp were duplicated in the evolution of the gene family. We postulated that the expansion of the aqp gene family in common carp was the result of an additional whole genome duplication event and that the aqp gene family in other teleosts has been lost in their evolution history with the reason that the functions of genes are redundant and conservation. Expression patterns were assessed in various tissues, including brain, heart, spleen, liver, intestine, gill, muscle, and skin, which demonstrated the comprehensive expression profiles of aqp genes in the tetraploidized genome. Significant gene expression divergences have been observed, revealing substantial expression divergences or functional divergences in those duplicated aqp genes post the latest WGD event.
Citation: Dong C, Chen L, Feng J, Xu J, Mahboob S, Al-Ghanim K, et al. (2016) Genome Wide Identification, Phylogeny, and Expression of Aquaporin Genes in Common Carp (Cyprinus carpio). PLoS ONE 11(12): e0166160. https://doi.org/10.1371/journal.pone.0166160
Editor: Graça Soveral, Universidade de Lisboa Faculdade de Farmacia, PORTUGAL
Received: May 11, 2016; Accepted: October 24, 2016; Published: December 9, 2016
Copyright: © 2016 Dong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Sequence data and primer information are available in the supplementary files.
Funding: This research was supported by grants from the National Natural Science Foundation of China (No. 31422057), the National High-Technology Research and Development Program of China (863 program; 2011AA100401), and the Special Scientific Research Funds for Central Non-profit Institutes of Chinese Academy of Fishery Sciences (2014A03YQ01 and 2016GH02). The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research (No. RG 1435-012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Aquaporins (Aqps) are a large superfamily of major intrinsic proteins (MIP), which selectively control the flow of water and other small molecules through biological membranes [1]. Therefore, Aqps play an important role in maintaining body osmotic balance for many organisms, especially for aquatic organisms. The first water channel protein was reported as Aqp1, which plays diverse roles in the mammalian erythrocytes [1, 2]. Since then, more and more genome-wide analyses of aqps have been published, and deposited thousands of aqp genes into public databases. For instance, it was reported that zebrafish (Danio rerio) have up to 20 aqp genes [3] in the diploid genome. There are 42 aqp paralogs in the Atlantic salmon (Salmo salar) genome, which has experienced an additional round of whole genome duplication (WGD) compared with most other diploid teleost fish [3]. Therefore, the aqp genes are in the duplication manners in the tetraploidized salmon genome.
Vertebrate aqps used to be classified into 13 classes. However, recent study has reported a total of 17 classes in aqps gene family in various vertebrates by Finn et al. Besides of 13 aqp classes retained in human genome, there are four additional classes, including aqp13 in aquaglyceroporins, aqp14 and aqp15 in classical aquaporins, and aqp16 in aquaporin-8 in various vertebrate genomes. Therefore, the vertebrate aqps were classified as follows: classical aquaporins (aqp 0, -1, -2, -4, -5, -6, -14, and -15), aquaglyceroporins (aqp 3, -7, -9, -10, and -13), aquaporin-8 (aqp 8, and -16), and unorthodox aquaporins (aqp 11 and -12) [3]. Although overall primary sequences are not well conserved (approximately 30% identity), all Aqps share a relatively conserved molecular structure, containing six membrane-spanning segments (TM1–TM6) with five connecting loops (LA–LE) [4]. Each Aqp half contains a conserved asparagine–proline–alanine (NPA) motif, located at LB and LE, that form short hydrophobic helices and dip halfway into the membrane from opposite sides, facing each other and participating in substrate selectivity [5]. A cysteine residue at position 189 in LE of human Aqp1 and 181 of Aqp2 is responsible for conferring mercury sensitivity [6]. In the biological membrane, Aqps are grouped as homotetramers embedded in the lipid bilayer and each monomer functions independently as a single pore channel [5].
Cyprinids are one of the most important teleost families in the world. Many species are domesticated as important aquaculture fish for food and ornamental purpose. Despite of the importance of Aqps for teleost fish, limited studies have been performed on aqp gene family in cyprinid species, except model species zebrafish. Common carp (Cyprinus carpio) originated in Europe and Asia. The species has been domesticated and introduced into various environments worldwide. It is an important economic and model species for various studies on ecology, environmental toxicology, developmental biology, nutrition, physiology, immunology, and evolutionary genomics. Therefore, significant genome resources have been developed in the past decade, including vast amount of genetic markers [7–10], genetic maps [11–13], BAC libraries and physical maps [14–16], expressed sequence tags (ESTs) [17], and transcriptome sequences [8, 18]. Recently, the common carp genome has been completely sequenced and assembled [19]. The evidence has shown that common carp is a species with an allotetraploidized genome, which had experienced an additional round of whole genome duplication (WGD). It has been hypothesized that the duplicated genome provides the basis for its enhanced adaptation to varied environments [20]. Therefore, it is of interest to determine if the number of aqp genes is doubled comparing with that of other diploid teleost fish, and elucidate Aqp functional evolution post the most recent WGD event.
In this study, by utilizing all available common carp genomic resources, we identified 37 aqp genes across the genome. Further phylogenetic analysis confirmed the gene annotation and nomenclature. Moreover, we examined the tissue distribution of aqp genes in common carp. The expression patterns of each gene, together with the results from comparative study with other vertebrate species, were used to infer the potential functions of aqp genes in common carp. Our study on examining the aqp gene family in common carp provides insights into the evolutionary and physiological aspects of post-WGD adaptation in common carp.
Results and Discussion
Aqp gene identification and characterization
We have identified a total of 20 aqp genes from zebrafish (D. Rerio) genome. Therefore, we used the 20 aqp genes of zebrafish as query to screen their orthologs in common carp genome. A total of 37 putative members of the aqp gene family have been identified from common carp genome. The 37 genes were distributed on 19 chromosomes and 18 scaffolds in the common carp genome, which are significantly more abundant than that in most other vertebrate genomes. For instance, there are 19 aqp genes in human (H. Sapiens), and 15 aqp genes in clawed frog (X. Tropicalis) and 12 aqp genes in medaka (O. Latipes) genome. Detailed information of their location, corresponding genomic sequences, coding sequences and DDBJ database accession number are summarized in Table 1.
The functional domains of aqp genes were predicted based on their protein sequences. As shown in S1 Fig, all Aqps possess one conserved domain (MIP) except for Aqp8ab-2, which is consistent with previous reports on Aqp protein structure [21], indicating high conservation of aqps. Additionally, there are four Aqps (Aqp7-2, 11b-1, 11b-2 and 15–2) contain the transmembrane domain (TM) at the N-terminal or C-terminal end of the protein, and eight Aqps (Aqp3b-1, 3b-2, 4b-1, 8ab-2, 9a-1, 9a-2, 10a-1 and 10a-2) exhibited low complexity (LW) in the domain structure prediction.
Phylogenetic analysis and nomenclature of aqp gene family in common carp
In the evolution of higher eukaryotes, WGDs followed by polyploidization, as well as gene loss, have been an important recurrent process. Ancient WGDs, inferred from analyzed sequenced genomes and comparative genomics, are prevalent and recurring throughout the evolutionary history of higher eukaryotic lineages [22]. To examine phylogenetic relationships of aqp genes in the teleosts and representative higher organisms, we collected a total of 103 aqp genes from five species, including human, clawed frog, medaka, zebrafish, and common carp. Also, phylogenetic analysis can be used to support the gene annotation, especially for non-model species [23], we investigated the molecular phylogeny of these aqp genes to validate the orthology of the common carp aqps.
Two phylogenetic dendrograms constructed based on alignments of the amino acid sequences of the Aqp proteins using both neighbor-joining (NJ, Fig 1) and maximum likelihood (ML, S2 Fig) showed high topological consistency, indicating the reliability of the phylogenetic relationships of the aqp genes. As shown in these two figures, the phylogenetic analysis results showed that each of common carp aqp genes clustered with its respective counterpart from other species, indicating all genes are highly conserved.
The aqp gene family is separated into four clades. The Aqp amino acid sequences are collected from the following vertebrates: human (Hs), zebrafish (Dr), medaka (Ol), frogs (Xt), and common carp (Cc).
For aqp gene family, as revealed by phylogenetic tree, all of the aqps were categorized into four clades. Also, we speculated that these genes falling into one clade may be derived from a very ancient lineage of vertebrate. Similar relationships of these genes have been reported in mammalian genes, which therefore are divided into four subfamilies [24]. The 103 vertebrate Aqps formed four distinct monophyletic groups orresponding to four subfamilies. The four subfamilies were characterized based on the topology: subfamily I (classical aquaporins), which contained Aqp 0, 1, 2, 4, 5, 6, 14 and 15; subfamily II (unorthodox aquaporins), which contained Aqp 11, and 12; subfamily III (aquaglyceroporin) which contained Aqp 3, 7, 9 and 10; and subfamily IV (aquaporin-8), which contained Aqp8.
The nomenclature of these genes was based on their identity to zebrafish orthologs and their phylogenetic position. Additionally, all the Aqps of common carp were categorized into a teleost subclade with members of the medaka and zebrafish. We therefore annotated each of the common carp paralogs with the postscript "-1" or "-2" following the name of zebrafish orthologs to reflect the 4R WGD [3, 25]. According to the naming strategy, none of aqp genes in common carp were presented as single copy, while aqp4, 7, 11, 12, 14 and 15 (Cc aqp4b-1, 4b-2, 7–1, 7–2, 11b-1, 11b-2, 12–1, 12–2, 14–1, 14–2, 15–1 and 15–2) have two copies, aqp0, 1, 3, -9 and 10 (Cc aqp0a-1, 0a-2, 0b-1, -0b-2, 1aa-1, 1aa-2, 1ab-1, 1ab-2, 3a-1, 3a-2, 3b-1, 3b-2, 9a-1, 9a-2, 9b-1, 9b-2, 10a-1, 10a-2, 10b-1 and 10b-2) have four copies, and aqp8 (Cc aqp8aa-1, 8aa-2, 8ab-1, 8ab-2, 8bb) has five copies, respectively (Table 2).
As previous studies reported, two rounds of WGD have occurred in the ancestor of vertebrates, plus two in the lineage of common carp, of which, the 3R WGD is known as teleost-specific (TS) WGD, and the 4R WGD is only occurred in some tetraploid teleost, such as salmonids and some cyprinids [26]. Common carp genome had been previously confirmed as allotetraploidized genome based on comparative genomic studies [19]. Significant gene duplications are presented in the Aqp topologies of common carp, which are clearly consistent with previous findings [27–29].
Gene duplications and losses in common carp
WGD is one of the major drivers that shaped the evolutionary history of many vertebrates. Ohno has suggested that two rounds of large-scale gene duplication had occurred early in vertebrate evolution [30], and a number of studies of comparative analysis of various gene clusters provided solid evidence in support of Ohno's hypothesis [31–33]. An additional round of duplication, also named teleost-specific (TS) WGD, or the 3R WGD [34, 35], took place in the common ancestor of all extant teleosts.
As a result of genome duplication, teleost fish usually have two paralogous copies for many genes, while only one ortholog is present in tetrapods. Also, it is generally-accepted hypothesized that, comparing to other teleost, salmonids and some cyprinids such as common carp and goldfish had undergone additional whole genome duplication (the 4R WGD) [27], Microsatellite analysis [28] and comparing common carp linkage map to zebrafish genome [29] provided critical evidence in support of the 4R WGD event in common carp [36]. The comprehensive estimation based on whole genome datasets suggests that the latest WGD event occurred around 8.2 MYA [37]. Therefore, the significant expansion of aqp genes in the common carp genome may be the result of this additional WGD, which could have caused a sudden doubling of the aqp genes. As shown in Table 2, common carp retained double or more than double the aqp copies of the zebrafish aqp genes, except aqp8bb, which strongly suggests that the 4RWGD event was the major contributor to aqp gene family expansion in common carp. Similar results were observed when to the common carp aqp genes were compared with the aqp genes in other teleost genomes.
Although mammals and teleosts last shared a common ancestor many hundred million years ago, a growing number of studies have reported extensive conserved synteny between the chromosomes of teleosts and mammals, which favors the rule of additional genome duplication in fishes [38]. In this study, syntenic blocks of aqp1 genes were constructed as shown in Fig 2. Clearly, in zebrafish, the two aqp genes are on chromosomes 2 and, in common carp, the four genes were distribute on chromosomes 2 and 4. Here, we consider a new evolutionary scenario to explain the gene duplication event in common carp. Assuming that the putative teleost ancestor had the aqp1 aa/ab genes on two different chromosomes, then, when genome duplication was finished in common carp, the four orthologs (aqp1 aa-1/aa-2/ab-1/ab-2) were distributed on four different chromosomes. However, with the cis and trans mechanisms, in the common carp genome, the two aqp genes on other chromosomes moved to chromosome 4, resulting in three aqp genes on chromosome 2 and one aqp on chromosome 4 (Fig 2). This hypothesis can explain the distribution of aqp in the common carp genome reasonably.
Vertical lines denote orthologous relationships.
After duplication, one of the two redundant copies of a gene should theoretically be free to degenerate and become lost from the genome without consequence [39, 40]. Most gene pairs formed by a WGD have only a brief lifespan before one copy becomes deleted, leaving the other to survive as a single-copy locus. We observed that there are only one aqp8bb in common carp and two copies in zebrafish, which is different with other gene number comparison. The Aqp8bb protein sequences were found to be highly conserved across all the vertebrate species, suggesting that the conserved aqp8bb gene is critical for survivability and very little change is allowed in its coding sequence and copy number in common carp. Abundant copies of a single gene might accumulate detrimental mutations due to relaxed selection on one of the duplicates. Gradually, they will become pseudogenized and they will either be deleted from the genome or become so diverged from the parental genes that they are not identifiable any longer [41, 42]. In addition, we have not identified that aqp 2, 5, and 6 are absence in all surveyed teleost fish but retained in other vertebrates, which is consistent with previous report [3], suggests the gene losses occurred in the common ancestor of teleost fish post the divergence of teleost fish and tetrapods. Regarding gene losses, it may occur in aqp gene family in common carp post the latest WGD as those identified 37 aqp genes are much less than our expectation, however, we also suspect another possibility that imperfect genome assembly and annotation lead to the "gene losses", especially on such a tetraploidized genome of common carp [43].
Expression profiling of aqp genes in common carp and potential functional inferences
Exploring expression profiling of aqps could help to speculate their functions. The relative expression of the common carp aqp genes in adult tissues was evaluated by RT-PCR employing isoform-specific oligonucleotide primers. As shown in Fig 3, the aqp gene family exhibited unique tissue-specific expression. In general, most of the aqp genes were widely expressed, but has a relatively high expression levels in brain, spleen and intestine and relative irregularity expression levels in other tissues. Also, we observed that aqp genes were almost no expressed in muscle, implying their unimportant roles in muscle organ development. Aqp8aa-1 and aqp14-1 were highly expressed in skin, suggesting their specific expression and special functions in the development of skin in common carp. As expected, we do observed significant difference on aqp expression profiles (Fig 3), which implied the functional divergence of duplicated aqp genes. For instance, we observed some consistent expression patterns in two copies of aqp genes in common carp, including aqp 0a, 1ab, 3b and aqp 8aa. Moreover, distinct expression patterns in two copies of 15 aqp genes, including aqp 0b, 1aa, 3a, 4b, 7, 8ab, 9a, 9b, 10a, 10b, 11b, 12, 14 and 15 were also obsevered. The two copies of aqp 0b exhibit an almost complementary expression pattern. For the rest of the 14 aqp genes, one of the two copies have broad expression profiles in surveyed tissues and the other one have relatively narrow expression. The spatial expression difference of the two copies of genes suggested quick functional divergence of these newly emerged aqp copies. It has been recommended that unless the presence of an extra gene product is of advantage, two genes with identical functions are unlikely to be stably maintained in the genome [42]. As the results, the duplicates would develop difference in some functional aspect, such as subfunctionalization, which could be stablely maintained in the genome. The expression profiles of aqp gene family in common carp suggested that aqp duplicates evolve quickly and subfunctionalization is commonly occurred in the tetraploidized genome.
β-actin was used as an internal control, gene names are indicated on the left of the panel. The eight tissues are brain, heart, spleen, liver, intestine, muscle, gill and skin.
We also observed significant gene expression differences compared with previous studies on model species. For instance, comparing the expression patterns with other vertebrate species, like zebrafish and human, conservation/divergence patterns were revealed as expected. In common carp, three of the four aqp1 copies showed tissue-wide expression patterns, while the remaining aqp1aa-2 had a tissue-specific expression pattern. Similar cases occurred in zebrafish, where one of the aqp1 genes the most ubiquitously expressed aqps, while another aqp otholog only expressed in several specific tissues [44], consistent with the presence of the human aqp1 ortholog [45]. Furthermore, aqp3 are mostly ubiquitously expressed in human and common carp tissues, however, there are no gene expression in the liver of zebrafish which are different with its orthologs in both common carp and human. These phenomenon maybe indicated that the expression pattern in different genes is different and has its own species-specific [46]. Obviously, these significant expression differences in those duplicated aqp genes, providing evidence for gene subfunctionalization post-WGD event. Most likely, the ancestral gene was capable of performing all functions and was expressed broadly in the tissues, while the descendant duplicate genes only perform partial functions and are specifically expressed in certain tissues. The functional divergence of duplicated genes may avoid potential adaptive conflicts [45].
In this study, we identified a total of 37 aqp genes in tetraploidized common carp genome. Phylogenetic and syntenic analysis as well as comparative genomic study revealed comprehensive understanding of aqp gene family and their distribution in the genome. Our analyses revealed extensive gene duplications in common carp which result from the additional WGD in common carp. Expression profiles of the complete set of aqp genes in common carp were assessed, which revealed extensive gene functional divergence in aqps in common carp. Our study provides essential genomic resources for future biochemical, toxicological, physiological, and evolutionary studies in common carp.
Materials and Methods
Ethic statement
This study was approved by the Animal Care and Use committee of Centre for Applied Aquatic Genomics at Chinese Academy of Fishery Sciences. The methods were carried out in accordance with approved guidelines. Adult common carp were collected from the Breeding Station of Henan Academy of Fishery Research, Zhengzhou, Henan province, China. Euthanasia is performed by immersion fish in MS-222 solution, and all efforts were made to minimize suffering.
Aqp identification and sequence analysis
All available aqp gene sequences and Aqp amino acid sequence from four species (human, clawed frog, medaka, zebrafish) were downloaded from public database Ensembl (http://asia.ensembl.org/), GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and ZFIN (http://zfin.org/). The genomes of these four species have been well-characterized and annotated previously. Amino acid sequences of Aqps in zebrafish were used as queries to search against all available common carp genomic resources by BLAST tools, with an E-value cutoff of 1e-5 to acquire the candidate aqp genes. Then reciprocal BLAST searches were conducted by using the candidate common carp aqp genes as queries to verify the veracity of candidate genes. The predicted sequences were extracted, analyzed, and confirmed by BLASTP searches against the NCBI non-redundant protein sequence database (nr).
The simple modular architecture research tool (SMART) was used to predict the conserved domains in common carp AQPs. The simple modular architecture research tool (SMART, http://smart.embl-heidelberg.de/) was used to predict the conserved domains based on sequence homology and further confirmed by "conserved domains" prediction software (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?) in NCBI.
Phylogenetic analysis
To annotate the aqp genes, phylogenetic analysis was conducted with reference Aqp proteins from zebrafish, and other representative vertebrate species. First of all, the Aqp protein sequences of four surveyed species were downloaded from the Ensemble databases. Then, the translated protein sequences of the common carp aqp orthologous genes and the Aqp protein sequences from the four other species (a total of 103 sequences) were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The sequences were then manually trimmed of all sites that were not unambiguously aligned. The deduced Aqp protein sequences were used for phylogenetic analysis in conjunction with reference Aqp proteins from zebrafish, medaka, frog, and human. We performed neighbor-joining (NJ) analysis in MEGA7 [47] with the p-distance model/method. Also, a maximum likelihood (ML) tree with default parameters was constructed using the MEGA7 to verify the accuracy of the toplogy of NJ tree. A total of 1000 bootstrap replicates were conducted for each calculation.
Gene nomenclature
Zebrafish aqp genes were named in accordance with previous studies [44, 48]. The aqp orthologous genes in common carp were named based on their phylogenetic topologies as well as BLAST result with their most related zebrafish genes. First, the subfamilies and gene members were determined for each common carp aqp orthologs based on the phylogenetic clades and the result of BLAST (for instance, aqp 0, aqp 1, etc.). Then, the closely related zebrafish aqp genes were assigned to each common carp aqp ortholog and the aqp genes were named after their most closely related zebrafish gene. When more than one copy of a common carp aqp gene was clustered with a certain zebrafish aqp gene, latin numbers suffixes were added to each copy (for instance, aqp 0a1, aqp 0a2, aqp 0b1, aqp 0b2, etc.). The names of each aqp gene in common carp and other surveyed species are listed in Tables 1 and 2.
Syntenic analysis
Syntenic analyses were performed on selected aqp genes across the human, zebrafish, and common carp chromosomes by identifying the positions of aqp neighboring genes. The organization of the genes on the chromosomes of the model species was obtained from the Ensemble databases, while the gene organization of common carp was based on the draft sequences of the common carp genome assembly. Syntenic maps were then drawn based on the gene locations in the surveyed species.
Expression profiling of aqp genes
Total RNA from various adult common carp tissues (brain, heart, spleen, liver, intestine, gill, muscle, and skin) was extracted with TRIzol® reagent (Life Technologies, NY, USA). The cDNA, which was used for PCR to examine the aqp expression patterns, was synthesized by RT-PCR using the SuperScript® III Synthesis System (Life Technologies). The β-actin gene was used as an internal positive control, with forward primer (5′ 9-TGCAAAGCCGGATTCGCTGG-3′ 9) and reverse primer (5′ 9-AGTTGGTGACAATACCGTGC-3′ 9). The whole PCR process was designed as follows: denaturation step for 5min at 94°C, 35cycles of denaturation (30 sec at 94°C), annealing (30 s), the temperature of which differed according various primers, and extension (30s at 72°C), and a final elongation step of 5 min at 72°C. The PCR products were separated by gel electrophoresis (1.5% agarose gel at 150 V) in the presence of ethidium bromide and visualized under ultraviolet light.
Supporting Information
S1 Fig. Schematic of the domain architecture of Aqp protein in common carp.
S2 Fig. Maximum-likelihood-based phylogenetic tree of 103 Aqp protein sequences.
The Aqp gene family is separated into four clades. The Aqp amino acid sequences are collected from the following vertebrates: human (Hs), zebrafish (Dr), medaka (Ol), frogs (Xt), and common carp (Cc).
S1 Table. Protein sequences of Aqps in common carp.
This research was supported by grants from the National Natural Science Foundation of China (No. 31422057), the National High-Technology Research and Development Program of China (863 program; 2011AA100401), and the Special Scientific Research Funds for Central Non-profit Institutes of Chinese Academy of Fishery Sciences (2014A03YQ01 and 2016GH02). The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research (No. RG 1435–012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions
- Conceptualization: PX.
- Data curation: CD LC.
- Formal analysis: CD LC.
- Funding acquisition: PX SM KAG.
- Investigation: CD LC JX JF.
- Methodology: CD LC.
- Project administration: PX.
- Resources: PX SM KAG.
- Software: LC XL.
- Supervision: PX.
- Validation: CD LC.
- Visualization: CD LC.
- Writing – original draft: CD JF PX.
- Writing – review & editing: CD LC PX.
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- 27. Wang JT, Li JT, Zhang XF, Sun XW. Transcriptome analysis reveals the time of the fourth round of genome duplication in common carp (Cyprinus carpio). Bmc Genomics. 2012;13(4):96.
- 28. David L, Blum S, Feldman MW, Lavi U, Hillel J. Recent duplication of the common carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci. Molecular Biology & Evolution. 2003;20(9):1425–34.
- 29. Zhang X, Zhang Y, Zheng X, Kuang Y, Zhao Z, Zhao L, et al. A Consensus Linkage Map Provides Insights on Genome Character and Evolution in Common Carp (Cyprinus carpio L.). Marine Biotechnology. 2013;15(3):275–312. pmid:23073608
- 30. Soukup SW. Evolution by gene duplication. S. Ohno. Springer-Verlag, New York. 1970. 160 pp. Teratology. 1974;9(2):250–1.
- 31. Gong Z. Vertebrate genome evolution and the zebrafish gene map. Nature Genetics. 1998;18(3):303.
- 32. Mclysaght A, Hokamp K, Wolfe KH. Extensive genomic duplication during early chordate evolution. Nature Genetics. 2002;31(2):200–4. pmid:12032567
- 33. Dehal P, Boore JL. Two rounds of whole genome duplication in the ancestral vertebrate. Plos Biology. 2005;3(10):e314. pmid:16128622
- 34. Cordell J. Whole-Genome Shotgun Assembly and Analysis of the Genome of Fugu rubripes. Science. 2002;297(5585):1301–10. pmid:12142439
- 35. Aparicio S. Vertebrate evolution: recent perspectives from fish. Trends in Genetics Tig. 2000;16(2):54–6. pmid:10652527
- 36. Zhao L, Zhang Y, Ji P, Zhang X, Zhao Z, Hou G, et al. A dense genetic linkage map for common carp and its integration with a BAC-based physical map. Plos One. 2013;8(5):e63928–e. pmid:23704958
- 37. Xu P, Zhang X, Wang X, Li J, Liu G, Kuang Y, et al. Genome sequence and genetic diversity of the common carp, Cyprinus carpio. Nature Genetics. 2014;46(11):1212–9. pmid:25240282
- 38. Gates MA, Kim L, Egan ES, Cardozo T, Sirotkin HI, Dougan ST, et al. A genetic linkage map for zebrafish: comparative analysis and localization of genes and expressed sequences. Genome Research. 1999;9(4):334–47. pmid:10207156
- 39. Dong C, Jiang L, Peng W, Xu J, Mahboob S, Al-Ghanim KA, et al. Phylogenetic and Evolutionary Analyses of the Frizzled Gene Family in Common Carp (Cyprinus carpio) Provide Insights into Gene Expansion from Whole-Genome Duplications. Plos One. 2015;10(12).
- 40. Loh YH, Christoffels A, Brenner S, Hunziker W, Venkatesh B. Extensive Expansion of the Claudin Gene Family in the Teleost Fish, Fugu rubripes. Genome Research. 2004;14(7):1248–57. pmid:15197168
- 41. Kimura M. The neutral theory of molecular evolution. Scientific American. 1979;241(5):98–100, 2, 8 passim. Epub 1979/11/01. pmid:504979
- 42. Zhang JZ. Evolution by gene duplication: an update. Trends Ecol Evol. 2003;18(6):292–8.
- 43. Jiang L, Zhang S, Dong C, Chen B, Feng J, Peng W, et al. Genome-wide identification, phylogeny, and expression of fibroblast growth genes in common carp. Gene. 2015;578(2):225–31. pmid:26691502
- 44. Tingaudsequeira A, Calusinska M, Finn RN, Chauvigné F, Lozano J, Cerdà J. The zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals. Bmc Evolutionary Biology. 2010;10(1):91–6.
- 45. Conrad B, Antonarakis SE. Gene duplication: a drive for phenotypic diversity and cause of human disease. Annual Review of Genomics & Human Genetics. 2007;8:17–35.
- 46. Abascal F, Irisarri I, Zardoya R. Diversity and evolution of membrane intrinsic proteins ☆. Biochimica et Biophysica Acta (BBA)—General Subjects. 2014;1840(5):1468–81.
- 47. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology & Evolution. 2016.
- 48. Abascal F, Irisarri I, Zardoya R. Diversity and evolution of membrane intrinsic proteins ☆. Biochimica Et Biophysica Acta. 2014;1840(5):1468–81. pmid:24355433


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Australia Funeral Expo- Sydney 2014
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Life Cycle Urns proudly exhibited our wholesale cremation jewelry at the Australian Funeral Expo. The AFE show was held at the Sydney Olympic park. The event was a terrific event to showcase our range of cremation jewelry to Australian Funeral Directors. It was here at the Australian Funeral expo that Life Cycle Urns began designing their latest range of cremation beads designed to fit Pandora-Style charm bracelets.


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HOMPSON, Terry Milton - June 29, 1946 - August 16, 2014 On Saturday, August 16, 2014, Mr. Terry Milton Thompson of Osoyoos, BC passed away suddenly but peacefully at his home at the age of 68 years. He was predeceased by his mother Ruth Thompson. Terry will be fondly remembered by his wife Wendy Thompson; children Tara Morissette (Innerkip), Chris Thompson (London); grandchildren Jessica-Lyn, Madison & Nathan; siblings Hank (Barb) Thompson, Tom Thompson & Connie Thompson as well as many extended family and friends. Terry enjoyed a long and distinguished career as a Firefighter with the City of Toronto Fire Department and retired as a Captain after 31 years. He also liked fishing and loved vacationing in Puerto Vallarta. Donations are gratefully accepted for the Canadian Cancer Society. Condolences and tributes may be directed to the family by visiting www.nunes-pottinger.com Cremation arrangements entrusted to Nunes-Pottinger Funeral Service & Crematorium, Oliver & Osoyoos, BC.
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cubicweb #3007241 drop ETYPE_NAME_MAP [open]
this is an attempt to support renaming of entity types while rql and other things (eg schema lookup) using the old entity types would still work.
While this is somewhat working, this is undocumented, has many fallbacks and introduce complexity. We should probably not support this at all.
NB: rename_entity migration primitive seems to rely on this for type merging (ie renaming an entity type to an already existing entity type). It would be nice to keep this though.
|done in
|<not specified>
|closed by
|<not specified>


text:
Front-load Sentence Examples
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text: Hey guys, first post..been lurking around the forums here for awhile now. I own a 2011 metallic silver GT/CS with a couple mods done. Finally bought a tuner and Steeda intake and have heard nothing but great reviews for a tune through Shaun@AED. I sent him some data logs and he replied that i have a cyl #8 failure coming and to not do anymore WOT with it. He wants me to get a compression check done for obvious reasons and that will be happening tomorrow on base. He said depending on how the results look he could correct the problem. My question is if anyone here is good at checking out how the engine looks with the data logs i have? i don't doubt Shaun i just want a second opinion as i've never tuned my vehicle in the past and its never been to the track. I have never thrown a code or check engine either. I've uploaded them to mediafire below.


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News Sections
Bedford Man Injured After Crashing Vehicle Into Utility Pole
Updated September 17, 2014 10:41 AM | Filed under: Accident
(SPRINGVILLE) - A Bedford man was injured after he crashed his vehicle on Seiboldt Quarry Road Tuesday morning.
According to a Lawrence County Police report, 29-year-old Philip Herbert of Bedford was traveling west on Sieboldt Quarry Road and told police he was watching a deer that was close to the road when he drove his 2001 Chevrolet Venture off the roadway and struck a utility pole and then into a tree stump.
Herbert complained of face pain following the accident.
Have a question or comment about a news story? Send it to email@example.com
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Discussion in 'Weapon Creator!' started by Terro, Apr 12, 2014.
Biggusupia= Big spear in Japanese
Should i Color this if so what colors do you guys think?
Try blue on the tip black on the inner portion white on the side spoke things and brown on the handle Maybe white or blue tip
how did you come up with the concept?
Was kind of a mistake tbh, i was making a shield and extended it to much and kept going with it lol
Separate names with a comma.


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Karlsson Huber posted an update 1 year ago
Cited consistently by recruiters and hiring managers as among the most sought-after certifications inside the IT industry, the CCNA is probably the most valuable certifications you will get on the resume. Moreover, it's essential for most higher-level Cisco certifications like the CCNP and CCDP (and, by extension, the CCIE). Earning the CCNA signifies that there is an capacity to configure and support a selection of Cisco network devices, as well as a strong general knowledge of networking, network security, and wireless networking – that are required to support the modern enterprise network.
But before you'll be able to turned into a CCNA, you'll want to pass Cisco exam 100-105 Interconnecting Cisco Networking Devices Part 1 (ICND1), that's required by order to generate certification. The CCNA exam is challenging, and passing it will be requires a lots of work and effort. Though the best focus and preparation, passing the CCNA exam is definitely an achievable goal. To obtain started, here are some ideas to make use of in get yourself ready for your CCNA examination.
Set a Course of Study
The first order of commercial should be to set the direction for the individual study. Cisco offers a syllabus for that CCNA certification, using a list of topics which can be covered. Review this list, print out and post it, and use it as your guide in crafting your personal course of study. Remember – when not about the syllabus, it's not about the exam, so limit college to the topics that Cisco highlights.
Identify Your Weaknesses with web practice exams by NWExam.com
A great second step is to identify those locations you're weakest (hint: get a practice exam to help you identify those areas) and earn them the focus of your study and exercise.
Use a firm practice on NWexam.com making use of their online practice exams for Cisco CCNA certification. You'll be able to identify your weak areas with result history. You'll be able to highlight those weak areas, and set a specific goal towards gaining a great understanding of each. Don't necessarily neglect your areas of strength completely (you don't want to forget what you've already learned!), but by turning your weaknesses into strengths you are able to dramatically increase your probability of passing the CCNA exam.
Have the complete list of CCNA certification having its online practice exams and syllabus at given URL:
Make Time for Study
The CCNA isn't a simple exam to pass through, plus it covers a great deal of ground. And, like all technical discipline, if you don't work on it on a consistent basis, knowing about it and skills will fade. Reserve a regular, regular time for study, and ensure that you maintain it. Granted, it can be hard to keep this time blocked out, particularly with each of the daily responsibilities and distractions that we all handle. The answer to passing the CCNA is frequent and consistent study and practice, so it's crucial that you determine this time around aside, limit your distractions, and adhere to the task available.
Focus on the Details
It's too few to know the theory behind the concepts presented inside the CCNA curriculum. To actually pass the CCNA exam, you'll want to how you can complete tasks and know the way things get done in the realm of Cisco. That's a crucial point because general networking concepts and how Cisco does everything is not always the identical – so it's imperative that you view the details and certain methods and procedures for implementing different networking technologies, within the Cisco environment.
Obtain access to Gear
This point can not be stressed enough. A large element of the CCNA exam contains completing tasks on simulated routers and switches, equally as you'll do them in person.
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WinZip System Utilities Suite 188.8.131.52 Crack
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Changing Gears – The art of leading yourself and others
We cannot lead others until we can lead ourselves. This workshop explores the keys to self-leadership and leading others. Understanding the importance of purpose, taking responsibility, owning our actions, overcoming sabotage and procrastination and being clear on the results are some of the areas investigated in this thought provoking and insightful workshop.
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"Sarah's ability to challenge the leadership thought process was every impressive. Identifying some of the most simple and effective personal changes to make to become a better leader was great. Great vibe, lots of laughs and plenty of food for thought." Kyle, Rip Curl Australia


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In today's market, there is a mixed perception of bulk broadband services. Some Owners and Developers want no affiliation with the video and Internet selections that their residents make. Some Owners believe in providing choice options of different providers for residents to choose from. These two schools of thought were very popular in recent years past. However, the market's perception of bulk is changing based on segment. Markets including student housing and senior housing are somewhat predisposed to bulk services. While markets such as market rate and affordable housing are beginning to see the value. What's true of all segments of the market is that Owners, Developers, Managers, and residents are curious about the Internet of Things (IoT) and connectivity.
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You may have read or heard the term "managed WiFi" in the news or from a colleague, as it has been a growing topic of discussion lately. You may be wondering what exactly is managed WiFi and why does it matter?
Topics: Managed WiFi, Telecommunication, Internet, Unmanaged WiFi, Access Point
You've probably heard the term Fiber to the Home, or perhaps your community is currently or has recently been upgraded to fiber. What exactly does Fiber to the Home mean and why is it such a big deal?
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Malware Museum Lets You Run Old MS-DOS Viruses
The Internet Archive has published a collection of old viruses that you can run under emulation to see their effects.
The Malware Museum is a collection of malware programs, usually viruses, that were distributed in the 1980s and 1990s on home computers. Once they infected a system, they would sometimes show animation or messages that you had been infected. Through the use of emulations, and additionally removing any destructive routines within the viruses, this collection allows you to experience virus infection of decades ago with safety.


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Egyptian Eye of Horus Pendant
The Eye of Horus is one of the most powerful protection symbols from Ancient Egypt - here as a pendant to protect you.
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Sr. Gracy SSpS
Provincial Leader
Hostel: A need for a hostel for both boys and girls whose homes were far away, was felt and so it was started as soon as the school building was ready.
The hostelites stayed in some class rooms in the new school. Later on a new hostel was built close to the new Convent.
They need love and care day and night and they are also helped in their studies. They are also trained to care for themselves and to learn to do the different tasks and responsibilities.
Today, in carrying out the educational ministry, our Sisters have contacts with many families and the values taught in the school reach their homes, which are visited frequently and regularly. The children of the poor are encouraged to come to school. Girl children are given preference during admission. Helping the weak students through tuition classes and a follow up of their studies bring the sisters close to them and their various needs are taken care of as much as possible.
In the year 2006, the Pre-primary section was shifted to the new school. As the school needed more classrooms to accommodate the increasing number of students, on august 16 th 2008, a new extension of the school was inaugurated and .in July 2009 the total number of students has risen to around 1200.
Since June 2007. Valigonda has become an independent parish, bifurcated from Velvarthy.and the Sisters in this community take care of Valigonda parish and a sub-station Thumbelagudem also known as Lourde Nagar
As mentioned earlier, on 16 th August 2008, a centre called Jeevitasha-a counseling centre for HIV-AIDS patients - was started by Velverthy community.in the old pre-primary school as the children had been already shifted from there to the new premises in Valigonda. The vacant school was repaired and is used as the Jeevitasha counseling centre. But now due to its proximity to Pavitratma Convent in Valigonda, the sisters who are working in that centre belong to Valigonda community and hence it has become one of the apostolates of Valigonda community since June 2009.
Hence now the vision, mission and goals of Valigonda community include healthcare dimension as well:
Vision: Provide care and support for people suffering from HIV/AIDS with love and compassion
Mission: to care for patients affected by HIV/AIDS irrespective of caste, creed, race, or language and to take up preventive programmes of awareness among staff and students of various institutions and by training animators for collaborating with other staff in this apostolate.
1. To identify the patients affected by HIV/AIDS
2. To counsel them and to pray with them and for them and to be supportive and
3. To conduct awareness programme wherever and whenever possible.
4. To prevent and eradicate the dreaded diseases like HIV/AIDS, T.B. etc, and to
remove the stigma and discrimination attached them.
Convent of the Holy Spirit
S O S Post
Banergutta Road
Bangalore - 81
Phone: 080-26583430


text:
In occupied Kashmir, veteran Kashmiri Hurriyet leader, Syed Ali Gilani has urged India to give up its traditional policy of force and intimidation to muzzle the voice of Kashmiris, struggling for securing their just right to self-determination. According to Kashmir Media Service, Syed Ali Gilani in a statement issued in Srinagar said that New Delhi had failed to suppress the Kashmir liberation movement through use of brute force as the people of Kashmir had pledged to continue their liberation struggle till its logical end. He said that the Indian Home Ministry was running the affairs of the territory and the sole purpose of the puppet administration was to provide cover to India's atrocities in Kashmir and hoodwink the world community. Reiterating the call for one hour protest after Juma prayers on January 14 and complete strike on January 15, the veteran Kashmiri Hurriyet leader said that protest was only way to attract attention of the world community towards the decades-long Kashmir dispute.
Our Staff Reporter
1:45 PM | January 13, 2011


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2nd T20I Live: Dominant India Eye Series Win Against England
An emphatic win in the opening game of the T20 International series against England has set the tone for the rest of India's long-awaited tour as the visitors would look to ensure that the spin-wary hosts remain on the back-foot when the two sides clash in the second T20I at Sophia Gardens in Cardiff on Friday. India's star spinner Kuldeep Yadav recorded his best figures as he took five wickets giving away just 24 runs, while KL Rahul scored an unbeaten second T20I century as the Men in Blue produced a fine all-round display to stun Eoin Morgan's men by eight wickets in Manchester for an early lead on this tour. Another win would seal the three-match series for Virat Kohli and his team. In fact, India stand on the cusp of a sixth successive T20I series win – a run stretching back to their 2-1 win over New Zealand at home in November 2017. In all, they have been unbeaten in bilateral T20I series since September last year. (Live Scorecard: India vs England)
2nd T20I, Sophia Gardens, Cardiff, Jul 06, 2018
Match yet to begin
Live Updates Between India vs England 2nd T20, straight from Cardiff
20:15 IST: The men blue have left their hotel and are in the team bus heading to the stadium.
It's time for the 2nd T20I against England at Cardiff.#ENGvIND pic.twitter.com/ZJcNkYLfqq
— BCCI (@BCCI) July 6, 2018
20:05 IST: Look who is back in the England team!
Ben Stokes will be at Sophia Gardens for our second Vitality IT20 against India following his appearance for @DurhamCricket in the @VitalityBlast.
He is expected to be available for selection for our final IT20 on Sunday. #ENGvIND pic.twitter.com/85WZeK6uc9
— England Cricket (@englandcricket) July 6, 2018
20:01 IST: Here's a look at the training session that team India earlier had ahead of the second encounter.
The boys look all geared up for the 2nd T20I against England.
Will they go up 2-0 today in the three-match T20I series?#ENGvIND pic.twitter.com/I2VwhOWCK6
— BCCI (@BCCI) July 6, 2018
20:00 IST: Hello and welcome to the live updates of the second T20I match between India and England.
In terms of ICC rankings, a 2-0 series lead will allow India to close the gap with second-placed Australia, while a 3-0 whitewash will help them leapfrog to second behind top-ranked Pakistan. Australia will have to win both their next two games in the ongoing tri-series in Zimbabwe to stay above India. A loss on Friday will see England drop down to rank seven below New Zealand, South Africa and West Indies, respectively. It will be a surprising downturn for the high-flying English team, one of the better in limited-overs' cricket at the moment. It is a stark reminder of their poor winning ratio nevertheless, having won only 5 out of their last 10 T20 internationals. The big question facing England is how to contend with the unorthodox wrist spin of Kuldeep Yadav. In the aftermath of their loss on Tuesday, both skipper Eoin Morgan and ace batsman Jos Buttler have urged their teammates to show more patience at the crease and watch the ball carefully.
But with such a short turnaround in the fixtures' list, it is easier said than done. The English camp will deploy the services of spin-bowling machine 'Merlyn' at Cardiff. A scarcity of unorthodox wrist spinners to practice against is one of the key reasons for this move, as the spin-bowling machine is capable of replicating every style of delivery. An English team last used it ahead of the 2005 Ashes, when they were practicing to contend with Shane Warne. A key reason underlined by Morgan after the first defeat was that the middle-order batsmen, Jonny Bairstow and Joe Root, who both got stumped off successive googlies, needed more time to settle in. Despite the loss, one of the key strengths for England is their top-order's ability to handle pace and they would like another fast start at Cardiff. It is perhaps the only real concern for India emanating from that first game as the enforced new-ball pairing of Bhuvneshwar Kumar and Umesh Yadav was proven ineffective at Old Trafford.


text:
Chapter Seven
Bruce's hand closed upon a marker in the desk drawer. "Yellow highlighter, that's another color."
Stephanie held out her hand for it. "Sunshine color!" She kicked her feet as she lay on her stomach.
He stepped around the little girl and her coloring project on the floor and stopped at the television. Selina nodded and he turned off Summer Gleeson's recap of today's events before joining Selina on the couch again. She chuckled as she laid her head against his shoulder. "What?" he asked.
"You remember the first night at your mansion?"
"You showing off your legs as you flipped out the window?"
"Second night then, when you were flirting so badly. Remember what you asked me to do?"
"Stay with me and help me spend my money on Gotham." The corner of his mouth lifted. "I never saw that happening like this."
She snorted as he put his arm around her shoulders. "Who would? Did you finish Leslie's paperwork?"
He nodded. "I put it back in our bag while you napped."
A cheering--muted by four stories--shook the glass doors. It startled Montoya and her heavy-set partner who had been talking by the elevators. Stephanie dropped her pen and rolled upright. "What was that?"
"Everybody's cheering that Commissioner Gordon's back." Bruce uncurled from Selina and sat up straighter.
Gordon came up the elevator and dismissed Montoya and Bullock before entering the Mayor's office. "I have to put in a supply order with Wayne Enterprises."
"Give us a few weeks. I don't know where the employees are, I still have to get my shares back, and the current CEO was part of the terrorist organization that almost blew up the city."
Gordon grimaced. "The commander of the National Guard troops is on his way in as soon as they reopen Gotham Bridge. Do you want to be a part of that debriefing?"
"There's no reason why I would be. If they have questions about the reactor or how Miranda Tate ended up in charge of Wayne Enterprises, I'll be happy to answer them, but let them think of the questions first." Bruce glanced at Selina and steeled himself for what had to be asked. "What about her record?"
Selina rolled her eyes. He turned back to Gordon's twitching mustache. "Guess you didn't hear how Police Headquarters was destroyed. We may never get all the records straightened out." He headed into the kitchenette and returned drinking from a bottle of water. "But the FBI has jurisdiction over Congressman Gilly's kidnapping."
Selina rolled her eyes again. Bruce didn't even need to look. "He consented to everything," she said. "Why don't you question him without his wife present? Stop worrying about the feds and worry about Stephanie instead."
Bruce frowned. "Why?"
"We're not Child Protection Services."
Stephanie abandoned her drawing and went to Selina. "You want me to go away?"
She scooped the little girl onto her lap and hugged her. "No, I don't want Child Protection Services to take you away."
Gordon snorted. "What Child Protection Services? Every other government agency shut down to protect their workers. The few orphanages running are swamped. She's better off with you until her parents come forward."
"What if the Evermonds come?" Stephanie pressed against Selina and put her fingers into her mouth.
"We'll send them away and you'll stay with us," Bruce said as he peeked at her face. Stephanie nodded and he smiled.
"You might have some trouble getting to the Palisades. The National Guard is keeping Gotham locked down while we get the government and aid functioning again." Gordon finished his water.
"We're going to my penthouse as soon as our ride gets here."
"Assuming it isn't trashed?" Gordon asked.
"I installed really good locks."
Selina smirked. "Did you remember the keys this time?"
Blake knocked on the glass before Bruce responded. "Ready to go?"
Gordon smiled. "Take your family home, Bruce."
He had a family again. There were still gaping holes in the idea, but the ones his parents and Rachel had left had duller edges now. He could think of them as he greeted the police officers who wanted to shake his hand. The hole Alfred left--he jerked away from that one as Stephanie panicked and they concentrated on her distress. They finally loaded into the SUV. "Ready to go home?" he asked.
Blake slid behind the wheel. "I remember the penthouse was on Midtown."
"Cale Street, two blocks down from the Midtown Bridge."
"What's a penthouse?" Stephanie hugged her doll as she sat between Bruce and Selina on the back bench.
"It's the apartment at the top of the building," Selina answered.
Bruce looked out the window at the city glowing in the setting sun's light. The details revealed the work remaining: broken windows, empty streets, over-flowing garbage bins, and non-functioning light signals. Bruce glanced away from the window. Selina stared out hers with a blank expression.
Blake found an empty parking space in the garage of the apartment building between two beaten and burnt car remains. "I hope the arsonists have moved on. I'd like to return this car from the guy I commandeered it from."
Selina smirked. "I want details." Blake shook his head as he unloaded the suitcases.
Stephanie held Bruce's hand when Blake and Selina refused to let him carry anything. He led the way through the abandoned cars to the elevator up to the apartment floors. The electricity still worked and it opened for them. He pressed the button for the top floor and leaned his eye into the security camera above the buttons. A green light flashed and a panel next to the camera slid open revealing a numeric keypad. He typed in the code and the elevator lifted as the keypad closed.
"Didn't look like anyone had tampered with that," Selina said.
"As long as the power stays on tonight, I can set up entry codes for you two."
"The power's been stable in Midtown and Uptown," Blake said, "along with gas and water. Downtown got the worse with the explosions, so more blackouts. How did you let people in for parties?"
"Scan the bar code included on their invitations or call up if they neglected to bring one. The only time it didn't work was when we had police officers escort the guests for the Dent fundraiser." Bruce's lips twisted in distaste. "A dirty cop pretended to have been taken hostage."
The elevator opened on the first floor of the penthouse. The last of the sunlight streamed through the walls of glass, but most of it fell on the covered patio and kitchen beyond the open fireplace.
Stephanie darted out of the elevator first; turned; and stared at the dust cloth-covered furniture, the curved staircase, and the helicopter pad outside. "Who lives here?"
"We do," Bruce said. He stopped at the fireplace and knelt down to turn on the natural gas. The pilot light ignited and flames danced around the ceramic logs in the waist-high pit. Selina folded the white dust cloth off the orange rectangular couches surrounding the fireplace.
"But it's huge! For just us?" Stephanie spun around again.
Bruce straightened and looked at his penthouse. It was bigger than Selina's walk-up. She smirked at him. It was probably closer in square-footage to St. Swithin's' Boys Home, but four people didn't compare to the children the boys home sheltered. Blake gave Bruce a lopsided smile. Bruce cleared his throat. "I'll go check on the electricity."
The electrical panel was hidden in the hallway behind the kitchen that also held the elevator to the second floor. He turned it on, set the thermostat, and opened the water main. He returned to the kitchen where Selina unpacked the MREs and bottled water onto the dining bar. Stephanie pressed against the glass wall to look down at the street. Blake uncovered the seats and the television at the other end of the penthouse.
Bruce turned on the water faucet at the sink before Selina brushed up against him. "Your ego okay?"
"I didn't know I needed to have the glib playboy out so soon."
"Don't, that'll just confuse the poor kid. Where's the dining room?"
He pointed to the stool along the counter. "I ate out often while living here."
"To see and be seen, poor you. So what's upstairs, all the bedrooms?"
"And the study and the gym."
She nodded as Blake sat down at the end of the dining bar and slumped over his elbow on the counter. His brown eyes closed involuntarily. Selina opened an MRE and started heating the contents. Bruce moved around her. "John?"
He jerked his head off his hand and blinked at Bruce. "What now?"
"Eater supper, pick a bedroom, and go to sleep."
He twisted on the stool and looked at the twilight descending on the city. "You don't need me on the night shift?"
"Not tonight. The ones who might have tried something are locked up and the National Guard will back up Gordon. Besides, how effective would you be as exhausted as you are?"
Blake smirked as Selina set a plate and warming entrée in front of him. "Setting the Tumbler on automatic and napping not a viable option?"
"Trust me; the seats aren't comfortable enough for that." Bruce reached for the glasses while Selina opened another MRE.
"In that case, thanks for the night off. Are you okay with kids saying the darndest things?" Blake nodded his head at Stephanie.
"I'm more worried that she'll be the only kindergartner in the city who knows what mass consumption is."
"Nah," Blake said as he opened the heater bag. "Kids know what it is even if they don't know what to call it."
"She'll be fine," Selina said.
"Based on what?" Blake asked.
"She's a Gothamite. We are survivors."
"Can't argue with that," Bruce said.
Supper was quiet; it had been a long day for everyone. Bruce swept the MRE debris into the garbage and loaded the dishwasher. Selina watched him for any signs of pain. Once the kitchen was clean, he led them around the corner to a second elevator that only traveled between the two penthouse floors. They went down a small hallway on the second floor that ended in a bedroom that spanned the entire end of the building with a king-sized bed in the left corner and a television sitting area on the right. "This is the master bedroom," Bruce said.
"Who had the thing against doors and curtains, you or your decorator?" Selina asked.
"It's one-way glass," Bruce said.
"Yeah." Blake dropped Bruce's duffle bag and parked Selina's suitcase next to it. "I'll take a bedroom at the other end of the hall. With a door."
"On the right, across from the study, I'll show you." Bruce moved out the door and Blake followed him.
Selina shook her head. Stephanie pulled the dust cloths covering the modern chairs around the television. "Come on, let's find you a bedroom."
"I get my own bedroom?" Stephanie caught Selina's hand. "I can sleep on one of the couches."
"You didn't have a bed at the Evermonds?"
"They usually had people sleeping from Mr. Evermonds' medicine. The couch was better and I could hide in the closet."
One of them needed to pay the Evermonds a visit, and it looked like it would fall on Nightwing. Selina had to convince Blake he needed the bullwhip. "You get to have your very own bedroom with us." She opened the first door on the right and flicked on the left and flicked on the lights. "This isn't it."
Stephanie peered around Selina's legs at the large mat in the center of the room surrounded by free weights and high-end isolation exercise machines. "What is it?"
"It's a gym, where you exercise." Selina turned off the lights and shut the door.
"Do I need to exercise?"
"Maybe when you're older. Right now, you should play, but not in there. There's stuff in there that can hurt you." Selina opened the next door on the right. She didn't think it was originally supposed to be a bedroom since another hallway ran between the room's windows and the outer glass wall. But there was a queen-size bed and a wall of wardrobe cabinets in it now.
Stephanie's eyes widened. "All mine?"
"All yours." Selina opened the other door on the far right wall and found a bathroom of white marble and aqua-green glass tiles and fully stocked with soap, shampoo, conditioner, and towels. She decided not to think about how much it cost to keep this skyscraper mansion ready to occupy at a second's notice.
"Where will you sleep?"
"In that huge open bedroom with Bruce." She started filling the tub large enough for Stephanie to swim in. The little girl looked lost. "If you have a nightmare, you run to me, okay? Bruce won't care."
Selina finally got Stephanie into bed wrapped in a tied-to-make-it-smaller T-shirt from Bruce's closet after a bubble bath and blow drying her blonde hair. Few lights in the neighboring buildings shone against the black night and matched Selina's trepidation as she headed to the master bedroom. Their first night alone without a common enemy. Time enough for what she did to sink in. Her dragging feet crossed the threshold into the huge master suite.
The opaque glass panels dividing the bed from the rest of the room glowed from the bedside lamps Bruce had on. Bruises dotted his uncovered arms. Bruce set down his tablet with a smile. "You're wet. Come here and let me make you wetter."
"What part of no strenuous activity don't you understand?" She pulled off her wet black blouse. Now where was the bathroom to hang it up in?
Bruce swung out of the bed wearing only a pair of boxer shorts. "It won't rupture my sutures to give you an orgasm." He took her blouse and tossed it over one of the dividers where it hung and dripped. "It sounded like a good celebration to me."
She refused to walk around the issue for eight years. "You want to celebrate my lying to you."
The smile dropped off his lips, but he didn't look angry. "Did you not tell me you're pregnant because you thought I'd reject you or because of Bane?"
"Bane. I know you wouldn't reject your child. But sidelining me when they didn't consider me a threat," she clenched her fist. "And damned if you didn't try to do it again without knowing! If I hadn't decided not to listen to you...." She trailed off, feeling exposed under his serious gaze.
"I'm not about to apologize for trying to protect you," he said. "And I don't expect one from you for doing the same thing." He touched her hair. "It doesn't suit you."
"I'm not apologizing, but you don't like lying."
"But I lied too," he said. "I never had a chance to test the software patch on the autopilot. I thought it would hurt less than if I had said I was coming back and it failed."
"Great, we're already treating each other shitty. At least you have the excuse of getting better at being selfish."
"For all the good it did us." He shook his head. "So let's learn from this mess." Selina raised her eyebrows. "No secrets, no thinking what is best for the other, trust we always have each other's back, and let this go."
"You trust me that much?"
Something inside her fluttered loose and warmed her with the smoldering look he gave her. "With my life. In fact--"
She cut him off with a kiss and wrapped her arms around his neck. "I want you to get me wet now."
He caressed her hips. "We're having a baby," he murmured against her lips. "And how did you get your jeans wet too?"
"Stephanie figured her bathtub doubled as a swimming pool." Selina stepped out of them and her panties as Bruce tugged them down. She unclasped her bra while he tossed them over the next divider. "Do you have one around here?"
"Wayne Manor does." He crawled over her as she lay back on the mattress. "If she needs one, we can install one on the patio."
She pulled down his head and kissed him. "In December?"
"We can close in the patio."
His lips moved down her neck. She skimmed her hands up his arms and over his shoulders, careful not to press on his bruises. He traced over her collarbone. Her fingers combed through his thick hair. His breath warmed her skin as it shivered in his wake.
She lost all thoughts of renovation projects and considerations for the other inhabitants as Bruce parted her thighs with his gentle hands. "Yes, please." She canted her hips toward his mouth.
He drew patterns on her inner thighs before dropping a soft kiss there. "I've missed how you taste."
"You're the one who-oh!" She forgot the complaint about no bedroom in the bunker when his tongue slid along her folds and flicked her clit. Actually, it was an inspired way to forestall an argument and she planned on using the technique on him as soon as he was medically cleared. He stroked her with his fingertips and she levitated.
He coaxed out her orgasm and milked it until her brain dissolved from the sensation. The first thing she recognized as her senses returned was feather-soft kisses below her navel. Next was Bruce's heartbeat against her thigh. Her fingers were still wrapped in his hair and the heat from his scalp warmed them. Tears sprang to her eyes and she couldn't stop them from falling.
She let go of Bruce's hair and wiped her wet temples. Stop, stop! Before he notices! She pressed the heels of her hands down on her eyes. Her breathing tore out of her mouth.
"Selina?" Bruce lifted himself off her legs. "Did I hurt you?"
She rocked her head against the pillow. Her screwed-up reactions weren't his fault. And she was letting the autopilot go, damn it. She rolled away as he settled on the mattress, but his strong arms wrapped around her.
He tugged her to his chest and pinned her there. "You're safe. We're all safe. Let it go."
She was stronger than this, stronger than what she had done, stronger than what she had seen and felt. She pressed her lips together and closed her teeth down on her sob.
His hand massaged the back of her neck. "It's okay. You know I won't spoil your tough girl image."
She started to laugh at his call back to their teasing banter from a lifetime ago. The laughter morphed into a sob. He continued to rub her tense muscles with a croon under his breath. No judgment, no horror, no bewilderment. "You're safe," he whispered. "Do what you need to."
Her arms wrapped around his chest, high above his stitches, and squeezed until she felt his even breathing. The sobs shoved out of her. They shook both their bodies but Bruce's hands stroked her neck and shoulders.
The sobs finally ended, but he hummed under his breath. She eased one arm free and wiped her face. "I didn't expect that," she said.
"It's probably better than my repress everything until I have nightmares." He eased his arms back but kept his hands on her skin.
She remembered the awful yells when he was unconscious but had blamed them on his pain. "You have nightmares every night?"
"Regularly, not every night unless something else is going on." His cheek muscles moved under her hand as he smiled. "Don't worry. I've had them for years and know how to keep them quiet."
"Don't hide from me."
"I won't." He sealed it with a kiss. "How do you feel?"
Exhausted and numb, but it was a good numb unlike getting drunk to forget. "Ask me in the morning." She settled against his chest and his heart beat under her ear.


text:
On 9 August, Anji foreign trade enterprises are organized together to Aibaba in Hangzhou in order to learn and communicate with experienced researchers and makers.
Luckily, Three Primary Colors Paper Co., Ltd have the opportunity to participate in this activity.
We enjoy the environment and atmosphere there.
There are some words we need to reflect:
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text: Jesse M. Donaldson (1947–1953) - Postmaster General [cite this] ↑Harry S. Truman Home Page Jesse Monroe Donaldson was born on August 17, 1885, in Shelbyville, Illinois. Donaldson first joined the post office in 1908 as a letter carrier. He worked his way up the ranks and became the assistant postmaster general in 1945. During 1946, Postmaster General Robert Hannegan became unsatisfied with his job, and Donaldson often sat in for him during cabinet meetings. In 1947, President Truman appointed Donaldson postmaster general upon Hannegan's resignation. The appointment was unprecedented because the post was traditionally given to political leaders in the President's party, and Donaldson was the first career civil servant to hold the office. During his tenure as postmaster general (1947-1953), Donaldson modernized the post office and streamlined its service. He died on March 25, 1970. Harry S. Truman Essays Life in Brief Life Before the Presidency Campaigns and Elections Domestic Affairs Foreign Affairs Life After the Presidency Family Life The American Franchise Impact and Legacy [ print all essays ] Harry S. Truman Home Citation Information Consulting Editor Alonzo L. Hamby Professor Hamby is a Distinguished Professor of History at Ohio University. His writings include: For the Survival of Democracy: Franklin Roosevelt and the World Crisis of the 1930s (Free Press, 2004) Man of the People: A Life of Harry S. Truman (Oxford University Press, 1998) Beyond the New Deal: Harry S. Truman and American Liberalism (Columbia University Press, 1973) American President has changed! Click here to take a short survey and tell us what you think!