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Transcriptome screen for fast evolving genes by Inter-Specific Selective Hybridization (ISSH)
Methodology article
Juan I Montoya-Burgos1,
Aurélia Foulon1 &
Ilham Bahechar1
Fast evolving genes are targets of an increasing panel of biological studies, from cancer research to population genetics and species specific adaptations. Yet, their identification and isolation are still laborious, particularly for non-model organisms. We developed a method, named the Inter-Specific Selective Hybridization (ISSH) method, for generating cDNA libraries enriched in fast evolving genes. It utilizes transcripts of homologous tissues of distinct yet related species. Experimental hybridization conditions are monitored in order to discard transcripts that do not find their homologous counterparts in the two species sets as well as transcripts that display a strong complementarity between the two species. Only heteroduplexes that disanneal at low stringency are used for constructing the resulting cDNA library.
We demonstrate the efficiency of the ISSH method by generating a brain cDNA library enriched in fast evolving transcripts of a non-model catfish species as well as a control, non-enriched library. Our results indicate that the enriched library contains effectively more fast evolving sequences than the control library. Gene annotation analyses also indicate enrichment in genes with low expression levels and non-ubiquitously expressed genes, both categories encompassing the majority of fast evolving genes. Furthermore, most of the identified transcripts show higher sequence divergence between two closely related catfish species as compared to recognized fast evolving DNA markers.
The ISSH method offers a simple, inexpensive and efficient way to screen the transcriptome for isolating fast evolving genes. This method opens new opportunities in the investigation of biological mechanisms that include fast evolving genes, such as the evolution of lineage specific processes and traits responsible for species adaptation to their environment.
Fast evolving DNA sequences are used for answering a broad range of biological questions relative to population processes and phylogeography [e.g. [1]], species diversification [e.g. [2, 3]], conservation biology [4] and also genome or phenotype mapping [e.g. [5]]. However, due to the very same intrinsic quality for which they are looked for, i.e. their high evolutionary rate, fast evolving DNA sequences display "lineage specific" changes and therefore require de novo development each time a new group of non-model organisms is being investigated. Despite various methodologies targeted toward the isolation of unspecific polymorphic DNA fragments [e.g. [6–8]] the identification and the isolation of fast evolving DNA sequences in non-model organisms is still laborious and expensive, making it a major impediment to the routine analysis of multiple loci on many taxa.
The isolation of fast evolving genes has gained new motivation and attention as genes involved in several actively investigated processes display high substitution rates: the evolution of species specific traits such as the human brain [e.g. [9, 10]], speciation genes [e.g. [11, 12]], reproduction genes [e.g. [13, 14]] or genes governing the evolution of adaptive traits [e.g. [15]]. Theoretical approaches suggest that adaptation genes should be fast evolving so that selection could have a substrate on which to act [16]. Furthermore, speciation genes, those that are directly or indirectly involved in the establishment of the genetic barrier between closely related species, consistently displayed high divergence rates [11]. At present, fast evolving genes which often evolve under positive selection can be identified either through large genomic comparisons which are feasible only for model organisms like Drosophila species [e.g. [17, 18]] or human-chimpanzee comparisons [19] or via long term experimental approaches such as in the discovery of the hybrid inviability gene Hmr in Drosophila [20]. The increasing interest in biological mechanisms driven by fast evolving genes appeals to the development of a more efficient and cost effective method for the isolation of such genes across closely related species and which would not imply the prior knowledge of genetic or genomic information.
Here we describe a simple and efficient experimental approach for enriching a cDNA library in fast evolving transcript fragments. Our method, named Inter-Specific Selective Hybridization (ISSH) is based primarily on the principles of the widely used subtractive hybridization (SH) procedure developed more than two decades ago for isolating cDNAs of differentially expressed genes [21–23]. In the original SH protocol, the hybridization of cDNAs versus mRNAs of different cell lines belonging to the same organism distinguishes transcripts that are equally expressed in both cell lines from those that are uniquely or differentially expressed in the cell line of interest. In the ISSH approach (Figure 1), the transcriptome of the species of interest ("probed" species) is reverse transcribed into single stranded (ss) cDNA and then hybridized against the biotinylated mRNA pool of a distinct yet close relative ("selector" species). During hybridization, three populations of transcripts of the species of interest can be found: (1) transcripts that never find their selector counterparts due to differential expression or gene loss; (2) fast evolving transcripts that find their homologous counterparts but the resulting heteroduplexes are unstable due to numerous nucleotide mispairings; and (3) conserved transcripts that form stable heteroduplexes. The second population of transcripts is in fact composed by sequences of varying divergence. The transcripts of interest are those that disanneal at the lowest stringencies and are therefore rescued for constructing the cDNA library enriched in fast evolving transcripts. The ISSH method was applied for isolating fast evolving transcript fragments of the non-model catfish species Ancistrus temminckii (family Loricariidae). We used the zebrafish genome as a reference for assessing the sequence divergence of the isolated transcripts and for transcript annotation and characterization.
Schematic representation of the ISSH method. The cDNA pool of the species of interest, whose fast evolving transcripts are to be isolated, is called the "probed" while the mRNA pool of the species used as a template is called the "selector". Thick lines, probed transcripts; thin lines, selector mRNAs; small black dot, biotin; small opened or dashed bars at the donor transcript ends, tails of the short-tailed random primers A and B; grey ball, magnetic beads coated with streptavidin; magnet shape, magnetic separator; grey bars at the ends of short-tailed random primers, double strand adapters; arrows, PCR primers. Fast evolving transcripts which are isolated with the ISSH method are shown at the bottom of the chart.
The ISSH method (Figure 1) confronts in solution complementary transcriptomes of two closely related species with the aim of rescuing transcripts of fast evolving genes. The property of evolving fast implies that such transcripts will disanneal at low stringencies from the heteroduplexes formed by homologous complementary sequences of the two species. Our method was applied to build a cDNA library enriched in fast evolving transcript fragments of brain tissue of the catfish Ancistrus temminckii. We used as the selector species its close relative Ancistrus dolichopterus. To assess the efficiency of the ISSH method we prepared a non-enriched control cDNA library of brain tissue of A. temminckii using standard protocols. The two libraries were sequenced with the FLX Genome Sequencer technology (Roche). We then "blasted" the enriched and control libraries against the complete genome of the zebrafish and analyzed the differences. We also annotated the transcripts producing significant matches and examined their characteristics to highlight the effectiveness of our method. As the zebrafish is not a close relative to our catfish and because the sequences of interest display high sequence divergence, a substantial proportion of the enriched library yielded no significant Blast matches. Therefore, we prepared an EST library of a close catfish relative, Hypostomus gr. plecostomus, belonging to the same subfamily (Loricariidae: Hypostominae), for refining the analyses.
Analysis of sequence divergence
High-throughput sequencing and reads assembly yielded 2429 and 1255 contigs for the enriched and control libraries respectively. We blasted the contigs against the zebrafish genome using parameters suitable for comparing divergent sequences. Only Blast results with E-values lower than 10e-5 were considered for the analyses; they represented 45.7% and 40.6% of all contigs of the enriched and control libraries, respectively. Among the contigs producing non-significant Blast matches about half were low-complexity sequences as indicated by RepeatMasker and were excluded from further analyses (20.7% and 22% of the enriched and control libraries, respectively). Significant Blast alignments were classified by size in order to produce a finer analysis of the differences that exist between the enriched and control libraries. The sequence divergence comparisons (Table 1) consistently show that the enriched library displays more divergent sequences (higher mean) than the control library using as reference the zebrafish genome. Applying a mean t-test unambiguously indicates that the mean divergence per category is significantly higher in the enriched library than in the control library, and this for all size categories (Table 1).
Table 1 Analysis of sequence divergence for the enriched and the control libraries.
When using the zebrafish genome as reference, the fastest evolving sequences may not find their homologous counterparts due to the distant evolutionary relationship between the zebrafish and our non-model catfish. Thus, performing the same analysis yet using an evolutionary closer reference - our EST database of the catfish Hypostomus gr. plecostomus - may allow a better understanding of the efficiency of ISSH method. The sequence divergence comparisons (Table 1) show again a systematic and significant enrichment in fast evolving sequences in the enriched library as compared to the control library. The difference between the two libraries is generally higher than when using the zebrafish as reference. This is likely explained by the inclusion of a set of faster evolving genes which can now find their homologues in the evolutionary closer Hypostomus reference.
Characteristics of the sequences retained by the ISSH method
In order to better assess the usefulness of the ISSH method we annotated the contigs of the enriched and control libraries according to the information collected from their translated best Blast hit in the Swissprot/Uniprot database with a minimum threshold of E-score ≤ 10e-8. In this way only 60 contigs were characterized in the enriched library (2.5% of all contigs) and 39 in the control library (3.1% of all contigs). The analysis of the annotated sequences will serve to test three predictions that have to be fulfilled if the method achieves its goal. First, as mitochondrial genes evolve significantly faster than the vast majority of nuclear genes they should be more numerous in the enriched library than in the control. The results indicate that mitochondrial genes represent 22.5% versus 8.6% of the annotated contigs of the enriched and control libraries, respectively, fulfilling the prediction. The second expectation concerns the overall correlation between the expression level and gene sequence conservation, where conserved genes are generally expressed at higher rates than fast evolving genes [24]. The expression level of the annotated contigs were approximated by using Unigene database information on the expression level of their orthologous genes in nervous system tissues of the zebrafish or, alternatively, of the mouse or human when the data was not available. Annotated contigs were classified into four categories of gene expression levels (Figure 2). We discarded here the mitochondrial genes which are fast evolving yet possess a high expression level. As expected, the enriched library is essentially composed by genes belonging to the category with the lowest expression-level (58% of the total versus 26% in the control library). The enriched library also shows a depletion of genes in the highly expressed gene categories, which are generally the more conserved ones. The third prediction refers to the observation that genes with tissue-specific expression evolve generally faster than genes with ubiquitous expression, a category in which most housekeeping genes are found [25, 26]. As predicted, our method resulted in an enrichment of non-ubiquitously expressed genes totaling 57% of the annotated contigs of the enriched library versus 35% in the control library. The non-ubiquitously expressed genes are defined as those expressed in less than four tissues according to Unigene expression information.
Number of annotated contigs per category of gene expression level for the enriched and control libraries. Using Unigene database information, gene expression level is calculated as the number of ESTs of the gene under consideration in the studied tissue divided by the total ESTs of the tissue library, multiplied by 10'000.
In an attempt to better characterize the fast evolving transcripts isolated in Ancistrus temminckii and which show a putative orthologous sequence in the Hypostomus gr. plecostomus EST dataset, we search for a tentative annotation by Blastn comparisons against the mRNA reference sequences (mRNA refseq) database of NCBI, limited to teleost sequences (a threshold of E < 1e-8 was used). In this way, 26 transcripts present in the catfishes Ancistrus and Hypostomus, as well as in the teleost mRNA refseq database were annotated and their sequence divergence was calculated based on the overlapping region of the sequence alignment, allowing a direct comparison (Table 2). The tentatively annotated transcript fragments show an overall high sequence divergence between the two Hypostominae catfishes (mean = 0.28 ± 0.09), not much different from the divergence between Ancistrus and the closest teleost orthologous sequence of the mRNA refseq database (mean = 0.39 ± 0.08), which do not include catfish sequences. Interestingly, about half of the transcript fragments encompasses coding sequence (cds) (Table 2). To estimate whether the sequence divergence is higher than in recognized fast evolving coding and non-coding DNA markers, we calculated the sequence divergence of the mitochondrial cytochrome oxydase I gene (COI), used by the Barcode of Life Initiative for characterizing species http://www.dnabarcodes.org/, and the two introns of the reticulon 4 (RTN4) gene, used to infer fish phylogenies at the specific level [2]. Most of the tentatively annotated transcripts display higher sequence divergence between Ancistrus temminckii and Hypostomus gr. plecostomus, as compared to the two reference markers (Table 2), validating once again the ISSH method. Likewise, most of the transcripts show higher sequence divergence between Ancistrus temminckii and Danio rerio as compared to the COI sequence. However, the intronic sequences of the RTN4 are much more divergent than the annotated transcripts. This is likely explained by the frequent insertion/deletion events in non-coding sequences and which enhance drastically the sequence divergence between distantly related species.
Table 2 Tentatively annotated fast evolving transcript fragments and their sequence divergence as compared to the closest ortholog in the teleost mRNA refseq database and in the Hypostomus gr. plecostomus EST dataset.
We emphasize that the sequences of the transcripts annotated using the mRNA refseq database likely represent the most conserved regions of the isolated transcripts dataset, as faster evolving regions will not find their sequence counterparts in the refseq database, which comprises no closely related catfish sequences.
The isolation of fast evolving genes can be easily accomplished on model organisms for which abundant genomic and transcriptomic knowledge exist. Bioinformatic routines and experimental procedures (micro-array technology) are available for this purpose. At present, however, there is no efficient method for doing so in non-model organisms. The ISSH method presented here is a fast and cost-effective procedure for enriching a cDNA library in fast evolving genes. The various tests we have performed resulted in a convincing demonstration of the efficiency of our method. We have shown that the overall sequence divergence was significantly increased in the enriched library as compared to the control when blasting these libraries against the zebrafish genome or against our Hypostomus catfish EST library. Moreover, the results of the ISSH method fulfilled the three predictions made upon the knowledge of general properties of fast versus slowly evolving genes. Briefly, the enriched library displayed (1) a higher proportion of fast evolving mitochondrial genes, (2) a higher fraction of genes with low expression level, and (3) proportionally more non-ubiquitously expressed genes. Furthermore, the fast evolving transcripts with orthologous sequences in the two catfish species and in the mRNA refseq fish database displayed generally higher sequence divergence than recognized fast evolving DNA markers.
A complementary support of these results comes from the Gene Ontology (GO) classification. The GO annotation (Figure 3) showed that genes involved in metabolic processes were less abundant in the enriched library than in the control (30% versus 64% of all annotated contigs in the enriched and control libraries, respectively). This is consistent with the observation that the set of housekeeping genes is generally rich in metabolic genes, for example in human [27]. Besides indicating that the enriched library is depleted in housekeeping genes, the GO annotation also shows that this library is enriched in nervous system tissue-specific genes (Neuronal activity, Figure 3), highlighting once more the ability of the method in isolating genes with tissue-specific expression that generally evolve faster than ubiquitously expressed genes.
Gene ontology classification of the fraction of annotated transcripts belonging to the library enriched in fast evolving genes and the control library. Only the major categories of biological processes are used, according to Panther database. Dotted bars indicate biological processes involved in metabolism.
The proportion of annotated contigs via Uniprot/Swissprot comparisons is rather small, particularly in the enriched library. This can be explained firstly by the relatively poor representation of fish genes in the Uniprot/Swissprot database combined with the likely high sequence divergence between the genes of the non-fish organism in the database and our catfish. Secondly, not all contigs may contain coding sequence; they may be composed mainly of UTR sequence. However, the enriched library shows no marked bias toward UTR sequences, which evolved generally faster than their contiguous coding sequences. Indeed, about 68% of contigs longer that 240 bp display putative open reading frames (ORF) longer than 80 aa (criterion of the H-invitational annotation project), and 51% of contigs longer that 300 bp display putative ORFs longer than 100 aa (criterion of the Functional Annotation of Mouse (FANTOM) project), which corresponds to four and five times the calculated sequence length without stop codons in non-coding sequence using the same base frequencies, respectively. Similar proportions are observed in the control library (70% and 54%, respectively) indicating no strong enrichment in UTR sequences. Furthermore, a significant part of the isolated transcripts may be non-coding RNAs. It has been shown, for instance, that non-coding RNAs constitute more than half of the mammalian transcriptome [28]. As the annotation of the isolated fast evolving transcripts is difficult due to the lack of sequence similarity with distant reference species, we are unable for the moment to assess the proportion of fast evolving non-coding RNAs in our dataset.
Our method has the advantage of being theoretically very versatile in terms of evolutionary divergence relating the species of interest and its selector species. The faster evolving genes will already show detectable sequence divergence between closely related species while using a more distant selector species will allow the isolation of a wider set of fast evolving genes. Likewise, one can modify the hybridization temperature to fine-tune the degree of sequence divergence one is looking for between the species of interest and the selector species. Our method may also be applicable for screening intra-species gene-associated polymorphism. Only in that objective the ISSH method may be compared with the In-Gel Competitive Reassociation and EST Array Hybridization method [29], which exploits the property that the vast majority of RFLP fragments between two strains or populations share the same electrophoretic size. Deviation from this property generates false positives and, therefore, the method of Gotoh and Oishi (2003) looses its interest if more distantly related groups are used.
Interestingly, the ISSH method can also be used for isolating the fraction of highly conserved genes between species. This is achieved by rescuing the fraction of ss cDNA that disanneal only at very stringent conditions, which guarantees an almost perfect complementation between the probed and selector pool of transcripts. Moreover, the species from which the selector pool of mRNA is extracted may be selected in order to increase the level of conservation of the enriched cDNA library: the more evolutionary distant the selector species will be, the more conserved the isolated transcripts will be.
The ISSH method is not linked to a specific sequencing technology. In this study we used the long-read 454 FLX technology (Roche) to ensure a minimum sequence length for downstream sequence analyses. However, this argument is currently less valid as the Illumina short-read sequencing technology, which produces many more reads at a lower cost per base, has been recently shown to be useful and accurate in de novo transcriptome assembly of non-model organisms [30]. Traditional Sanger sequencing can also be used providing that the PCR amplified fast evolving transcripts are cloned before sequencing.
We demonstrated that the ISSH method efficiently enriches a cDNA library in fast evolving genes. As this new method does not rely on the previous knowledge of sequence information, it can be performed on every non-model organism, and is therefore of wide use. Although the improvements and reduced cost of next-generation sequencing technologies may lead to ever more complete transcriptomes assemblies, and may have the potential to be used for identifying fast evolving transcripts with bioinformatic tools, the ISSH method will still have and interesting role to play. First, the ISSH method is inexpensive, of little labor, and leads directly to the set of transcripts of interest. Second, as the fast evolving genes are often expressed at low level, they may be hard to retrieve using next-generation sequencing technologies unless very deep sequence coverage is performed, at high cost. Therefore, the ISSH method opens new possibilities in screening transcriptomes in search of genes involved in lineage specific processes and traits, a field of growing interest in evolutionary biology.
RNA extraction and preparation of the control library
Total RNA was extracted from fresh brain tissue of Ancistrus temminckii (probed species) and its close relative Ancistrus dolichopterus (selector species) using TRIzol reagent (Gibco). We also extracted total RNA from our catfish outgroup reference Hypostomus gr. plecostomus. After quantification and quality verification of the total RNA, mRNA was isolated using the mRNA Isolation Kit (Roche Diagnostics). The SuperScript double-stranded cDNA synthesis kit (Invitrogen) was used to prepare the brain control library of Ancistrus temminckii and also the outgroup reference Hypostomus gr. plecostomus, starting with 1 μg of brain mRNA and the option of oligo(dT) anchor priming for the first strand synthesis step.
The ISSH protocol
The selector pool of mRNA, extracted in this work from Ancistrus dolichopterus, is biotinylated to allow subsequent separation by magnetic particles coated with streptavidin. Biotinylation of 5 μg mRNA was done using the BIO-ULS labeling kit (Kreatech); the final volume was reduce to 7 μl using a Speedvac concentrator. The probed pool of mRNAs extracted from the species of interest Ancistrus temminckii is reverse-transcribed into ss cDNA using a short-tailed random hexamer primer (5'-AGGA-(N)6-3'). We used 1 μg of mRNA (one fifth of the selector's mRNA amount) and 200 ng of the short-tailed random primer in a total volume of 12 μl. The reverse transcription was performed using the SuperScript II RT (Invitrogen) following the manufacturer's protocol for random priming; the final volume was 20 μl. The RNA template is destroyed by alkaline hydrolysis (0.35 N NaOH; 0.35 M EDTA) at 65°C for 15 min. The solution is then neutralized with 0.35 N HCl and first strand cDNAs are purified using the Mini Elute PCR Purification Kit (Qiagen) following the manufacturer's protocol but with an additional washing step and two rounds of elution. The final volume was reduced to 7 μl using a Speedvac concentrator.
Inter-Specific Selective Hybridization
The pool of biotinylated selector mRNA (7 μl) and the pool of first strand cDNA of the species of interest (7 μl) are mixed and the total volume is adjusted to 15 μl. An equal volume (15 μl) of 2× hybridization buffer is added (10 mM EDTA pH8, 1.5 M NaCl, 40 mM sodium phosphate buffer pH 7.2, 10× Denhardt's, 0.2% SDS). The solution is heated at 90°C for 2 min and quickly placed in a rotary shaker located inside a preheated hybridization oven at 55°C. The hybridization is carried on during 60 hours at 55°C. At the end of the hybridization step, 75 μl of NaCl 1 M is added to the hybridization mixture, which is kept at RT.
Separation of the fraction enriched in fast evolving cDNAs
The selector-probed hybridization mix is sequentially denatured to separate two fractions of cDNAs with increasing denaturation stringencies, the first fraction containing the non-hybridized or non-specifically hybridized probed cDNAs and the second fraction is the one enriched in fast evolving transcripts. First, streptavidin magnetic particles (Roche Diagnostics) are prepared according to the manufacturer's instructions (1200 μg) and resuspended in 100 μl of TEN 1000 buffer. The hybridization mixture is then transferred in to the tube containing the streptavidin magnetic particles and placed in a rotary shaker for 45 min at RT. In this step the biotinylated selector mRNAs, which may be hybridized or not with a complementary probed ss cDNA, are linked to the streptavidin magnetic particles. The non-hybridized probed cDNAs are discarded by placing the tube in a magnetic separator (Qiagen) and by removing the supernatant. The magnetic particles with their attached molecules are washed three times at 55°C for 15 min, in 600 μl of preheated 5× SSC, then resuspended in 50 μl of 0.1× SSC and incubated at 65°C for 15 min. In this last step the fast evolving probed cDNAs will disanneal from their selector counterpart and this fraction of interest is recovered in the supernatant after a magnetic separation. This step is repeated once. The fraction enriched in fast evolving cDNAs is purified by ethanol precipitation in presence of ammonium acetate and glycogen. The pellet is rinsed once in 70% ethanol and resuspended in 20 μl water.
Second strand synthesis and adapter ligation
The ss cDNAs are transformed into double stranded (ds) cDNAs using short-tailed random hexamer primers (CCAC-(N)6) and the DNA polymerase I Klenow fragment (Promega), according to the manufacturer's random priming protocol. cDNAs are then blunt ended using T4 DNA Polymerase (Promega), extracted with phenol/chloroform/isoamylalcohol (25:24:1) and recovered by ethanol precipitation with ammonium acetate. Double strand EcoRI adapters (Invitrogen) are ligated to the ds cDNA ends according to the manufacturer's instructions. The final volume is adjusted to 100 μl with water and the cDNAs are purified using the High Pure PCR Product Purification Kit (Roche Diagnostics).
PCR amplification of the fraction enriched in fast evolving cDNAs
A first PCR amplification is performed using a single primer (5'-GTCGACGCGGCCGCGAATT-3') targeted toward the EcoRI adapter ligated at both ends. The PCR reaction is done in 50 μl final volume with 10 μl of cDNA as template and with the following profile: 1 min initial denaturation at 94°C followed by 35 cycles with 30 s at 94°C, 30 s at 62°C, 2.5 min at 72°C and a final elongation step of 5 min at 72°C. The PCR product is checked on 1,5% agarose gel. A nested PCR is performed using specific primers overlapping the end of the EcoRI adapters and the tails of the two short-tailed random primers used for the synthesis of the first strand and then for the synthesis of the second strand (EcoRI-AGGA: 5'-TCGCGGCCGCGTCGACAGGA-3'; EcoRI-CCAC: 5'-TCGCGGCCGCGTCGACCCAC-3'). The PCR conditions are as described above but the amount of template DNA is adjusted according to the result of the first PCR. The PCR products are checked on 1,5% agarose gel and then purified using the High Pure PCR Product Purification Kit (Roche Diagnostics).
High-throughput sequencing
For the Ancistrus control and the outgroup reference Hypostomus gr. plecostomus, shotgun DNA libraries were prepared with a starting amount of 4 μg DNA. The mean fragment size was of about 500 bp, obtained using nebulizers and chemicals from the GS DNA Library Preparation Kit (Roche Diagnostics) according to the manufacturer's manual. This step was not needed for the Ancistrus library enriched in fast evolving transcripts as the ISSH method results in a PCR product containing fragmented transcripts, generally in the range of 300 to 1000 bp. After DNA purification, the DNA end repair step and the ligation of the barcoding adaptors were performed following established protocols [31]. The adapter-ligated DNA from each of the three libraries were pooled and prepared for the 454 sequencing according to standard protocols [32], using the GS DNA Library Preparation Kit with Titanium reagents, and following the instructions of the GS FLX manuals (Roche Diagnostics). The library was sequenced on one 16th region of a full GS FLX sequencing plate with a prior titration run. Upon completion, sequences were screened for primer concatemers, week signal, poly A/T sequences, and barcodes for assigning them to one of the three samples. The average lengths of the reads were 180 bp. cDNA assemblies were performed with the SeqMan software from DNAStar. The cDNA library enriched in fast evolving genes and the control library of Ancitrus temminkii were deposited in the Short Read Archive (SRA) of NCBI under the accession number SRA009346.1
Blast search and transcript annotation
The Blast search against the zebrafish sequences of all the EMBL sub-divisions (Expressed Sequence Tag; High Throughput cDNA sequencing; High Throughput Genome sequencing; mRNA of Standard; Whole Genome Shotgun) were performed on the Vital-IT high-performance computing facility of the Swiss Institute of Bioinformatics http://www.vital-it.ch. We used blast parameter values suitable for comparing divergent sequences (word size = 7; match score = +1; mismatch score = -1; initial penalty for opening a gap = 1; penalty for extending a gap = 2). The local Blast search against our Hypostomus gr. plecostomus brain EST database was performed using the software blast-2.2.19 developed by NCBI. Perl scripts for parsing the blast outputs were built using Eclipse SDK 3.4.1.
The proportion of contigs with low complexity sequences or sequence repeats was assessed using RepeatMasker open-3.2.8 (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-3.0. 1996-2004; http://www.repeatmasker.org). Transcripts were annotated according to their best Blast hit against Swissprot/Uniprot databases, with a minimal E-score of 10e-8. The translations into the six frames were performed using BCM Search Launcher [33] and blasting was done with Blastp at NCBI. Expected frequency of stop codons in non-coding sequences was calculated by multiplying the three single nucleotide frequencies taken from the sequence data of the corresponding library, and summing the frequency of the three possible stop codons. Gene transcription levels in specific tissues were taken from the Unigene database and are expressed in number of ESTs of the gene under consideration divided by the total ESTs of the tissue library, multiplied by 10'000. Gene ontology classification was performed on Panther (Protein ANalysis THrough Evolutionary Relationships; http://www.pantherdb.org), complemented with ontology information given in Uniprot database. We used only the top categories of the classification hierarchy, as given in Panther. Fast evolving transcripts found in Ancistrus temminckii, Hypostomus gr. plecostomus EST, and in the mRNA reference sequences database of NCBI, restricted to the Teleostei (Blastn threshold E-score < 10e-8), were used to asses the sequence divergence. A tentative annotated was given according to the best hit against the mRNA refseq database. For direct comparison purposes, sequence divergence was calculated on the sequence region present in all three taxa. Sequences of the reference fast evolving markers were obtained from GeneBank: Ancistrus brevipinnis COI: EU359402; Hypostomus boulengeri COI: EU359422; Danio rerio complete genome: NC_002333; Ancistrus cirrhosus RTN4 introns: EU817562; Hypostomus boulengeri RTN4 introns: EU817560. The RTN4 introns from Danio rerio were retrieved from Ensembl http://www.ensembl.org/, locus: chromosome: Zv8:1:42092991:42094205:1.
Raw read data is available at the Short Read Archive (SRA) of NCBI under the accession number SRA009346.1
No ethical approval was required for this study.
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Nielsen R, Bustamante C, Clark AG, Glanowski S, Sackton TB, Hubisz MJ, Fledel-Alon A, Tanenbaum DM, Civello D, White TJ: A scan for positively selected genes in the genomes of humans and chimpanzees. PLoS Biol. 2005, 3 (6): 976-985. 10.1371/journal.pbio.0030170.
Barbash DA, Siino DF, Tarone AM, Roote J: A rapidly evolving MYB-related protein causes species isolation in Drosophila. Proc Natl Acad Sci USA. 2003, 100 (9): 5302-5307. 10.1073/pnas.0836927100.
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Zhang LQ, Li WH: Mammalian housekeeping genes evolve more slowly than tissue-specific genes. Mol Biol Evol. 2004, 21 (2): 236-239. 10.1093/molbev/msh010.
Tuller T, Kupiec M, Ruppin E: Evolutionary rate and gene expression across different brain regions. Genome Biology. 2008, 9 (9): R142-10.1186/gb-2008-9-9-r142.
Eisenberg E, Levanon EY: Human housekeeping genes are compact. Trends Genet. 2003, 19 (7): 362-365. 10.1016/S0168-9525(03)00140-9.
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Gotoh K, Oishi M: Screening of gene-associated polymorphisms by use of in-gel competitive reassociation and EST (cDNA) array hybridization. Genome Res. 2003, 13 (3): 492-495. 10.1101/gr.434103.
Gibbons JG, Janson EM, Hittinger CT, Johnston M, Abbot P, Rokas A: Benchmarking Next-Generation Transcriptome Sequencing for Functional and Evolutionary Genomics. Mol Biol Evol. 2009, 26 (12): 2731-2744. 10.1093/molbev/msp188.
Meyer M, Stenzel U, Hofreiter M: Parallel tagged sequencing on the 454 platform. Nature Protocols. 2008, 3 (2): 267-278. 10.1038/nprot.2007.520.
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We thank Dr. L. Falquet who performed the Blast calculations against the zebrafish transcript databases on the Vital-IT high-performance computing facility of the Swiss Institute of Bioinformatics http://www.vital-it.ch. We acknowledge P.-Y. Pettina and A. Coulot for their help in preliminary laboratory tests of feasibility. We thank Dr. Y. Surget-Groba for bioinformatic advises and for revising the manuscript, and two anonymous reviewers for helpful comments and suggestions. This work was supported by funds from the Canton de Genève; the Swiss National Research Fund [grant number 3100A0-122303/1]; and the G & A Claraz Foundation.
Department of Zoology and Animal Biology, University of Geneva, 30 quai Ernest Ansermet, 1211, Geneva 4, Switzerland
Juan I Montoya-Burgos, Aurélia Foulon & Ilham Bahechar
Juan I Montoya-Burgos
Aurélia Foulon
Ilham Bahechar
Correspondence to Juan I Montoya-Burgos.
JIMB conceived the project and designed the analyses. JIMB and AF performed the bioinformatic and sequence analyses. JIMB and IB performed the experimental work. The manuscript was prepared by JIMB with assistance from AF and IB. All authors have read and approved the final version of the manuscript.
Montoya-Burgos, J.I., Foulon, A. & Bahechar, I. Transcriptome screen for fast evolving genes by Inter-Specific Selective Hybridization (ISSH). BMC Genomics 11, 126 (2010). https://doi.org/10.1186/1471-2164-11-126
Zebrafish Genome
Enrich Library
High Sequence Divergence
Selector Species
Control Library


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Esports Games And Trends That Will Define The Year 2022 Are Set To Start
by Alisha J. Perez.
Alisha J. Perez - E-Sports expert.
What Can We Anticipate From The World Of Esports In The Next Months?
Mobile Gaming Is Becoming More Popular
Sony's PlayStation 5 And Microsoft's Xbox Series X/S
Demographics Of Esports In 2022
In recent years, the gaming industry has made significant progress, particularly in terms of new breakthroughs and inventions.
The meteoric rise of esports in recent years is transforming the business, as seen by the sector's growing general popularity and new patterns, notably in 2021.
Over Twitch, YouTube, Instagram, Facebook, and other real-time media, we've witnessed massive growth in viewership and socioeconomics in esports like CS:GO, Overwatch, Dota 2, Fortnite, and League of Legends.
While most businesses were impacted by COVID-19 by 2020, the popularity of esports grew as those locked inside looked for ways to make the most of their time.
The real world remained tranquil, but gaming awoke and offered a hopeful alternative to gamers.
Despite the fact that, to be fair, the esports industry was benefiting from a temporary uptick even before the lockout.
As 2021 fades into memory, esports are poised for a year of growth in terms of revenue and audience. The industry is expected to generate $1194.8 million in 2022 alone.
To begin with, it should go without saying that the major events will continue to be main subjects. The enormous, multi-faceted ecosystems of games like CSGO and Dota 2 ensure that they will continue to attract the interest of millions of enthusiasts.
Sure, there's been talk of CSGO "dying" in recent months, but as esports pundit Jacob "Pimp" Winneche put it, "Counter Strike has been dying for the past 20 years and will most likely continue for the next 20 years."
In 2022, we can anticipate Valorant to continue to grab some of the spotlight from CSGO.
Last year, Riot Games' FPS was launched to radioactive levels of excitement, quickly encroaching on the area of its predecessor. "Valorant was specifically made for people who love CSGO but are also bored with it," YouTuber Adrix remarked.
The goal now is to turn Valorant into the next esports powerhouse, with the Valorant Champions Tour set to debut in 2022, featuring regional competitions leading up to a global championship event
Mobile esports have exploded in popularity, especially in Southeast Asia and Brazil, thanks to the availability of light applications.
In 2022, mobile esports will be given development freedoms as one of the fastest growing serious gaming areas.
Industry insiders and players have long discussed how and when mobile esports would captivate followers in the West on a comparable scale.
It's safe to say that League of Legends: Wild Rift stands a good chance of shattering stereotypes and expectations.
The mere heritage of the League of Legends moniker is enough to pique interest.
Riot Games is putting forth a lot of effort to promote Wild Rift as an esports title, with events planned for Thailand and Malaysia.
When the game is released in places like Europe and North America, it will undoubtedly make PC gamers sit up and take mobile esports seriously.
PUBG Mobile is another game that will bring attention to the rise of mobile esports in 2022.
Last year, it was announced that the PUBG Mobile ecosystem would be significantly extended in 2022, with additional Pro Leagues in Western Europe, North America, and Latin America.
This is all part of a concerted effort to generate the type of engagement that PUBG Mobile now has in Asia.
The overall prize pool of $14 million in 2022 is sure to pique interest.
We may also expect further linkages between esports and iGaming to form. Even before the Covid-19 shutdowns, there was a massive increase in esports betting in 2021. Between March 2020 and March 2021, esports bets in the United Kingdom increased by 2922 % year on year.
Unsurprisingly, betting sites have been eager to invest in the market. Many bookies have struck sponsorship partnerships with esports organizations, with Betsafe being the major sponsor of Swedish esports company GODSENT's CSGO branch in November 2020.
There are surely exciting times ahead for esports in 2022 and beyond, with the introduction of new games, the concentration on mobile, and the rising relevance of iGaming.
Until now, augmented reality (AR) has altered the esports scene, and it will continue to do so in 2022.
As gamers want more immersive experiences, ground-breaking AR innovation will be critical to the future of esports.
Players may have a chosen in-game experience where they feel soaked on the inside with VR.
As we begin a new year, gamers' perceptions of virtual reality will improve as they strive to converse with their own avatar through virtual continuing conversations.
Despite the epidemic, Sony and Microsoft figured out how to get their cutting-edge gaming conveniences out there, effectively transforming the gaming experience.
Many titles are supported by these new consoles, which also have significant graphics improvements.
Laptops are driving esports interest, but with these cutting-edge conveniences, we expect console usage to increase and soon narrow the gap.
The cutting-edge consoles will encourage engineers to design esports titles that are supported by these stages, making them greater, sleeker, more dazzling, and exceeding everyone's expectations.
With the 5G organization promising a year of progress and fervor in the next year, esports will be one of the sectors to benefit from this turn of events.
Download rates, streaming capabilities, and continuously continuing engagement encounters are all expected to alter with 5G.
As a result, esports may enable players to support their own experience without having to install a new framework.
Virtualization and the construction of organization cuts are encouraged in 5G enterprises, allowing each cut to be allocated to a specific purpose.
Observers on competition fields may enjoy fluid 3D augmented reality experiences, get instant replays on their devices, and obtain 360-degree views of their continuing interactions thanks to organization cutting.
We expect viewership and demographics to expand as portable esports and contests become more widely available.
According to statistics, esports socioeconomics will expand by 18% by the end of 2022, with over 550 million esports followers worldwide.
While the majority of esports enthusiasts are young, male, and well-off, these demographics are likely to shift slightly in 2022.
As players remember it as a professional game, we expect more girls and young adults to join the growing following.
The possibilities are endless as long as innovation continues to progress and develop.
As esports acquires these top 5 patterns and highlights formerly prominent in the actual universe of sports, the combination of virtual and actual games will continue to gravitate toward.
In 2022, developing partnerships, advertising earnings, and wagering will aid in the growth of the firm.
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Valorant Patch Notes - AFK Detection Will Be Improved With Valorant Patch 4.02
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text:
in: 1814 births, 1881 deaths, Confederate States Army generals,
People from Philadelphia, Pennsylvania
People from Fauquier County, Virginia
Members of the Aztec Club of 1847
People of Mississippi in the American Civil War
American Civil War prisoners of war
John C. Pemberton
For other people named John Pemberton, see John Pemberton (disambiguation).
John Clifford Pemberton
[[Image:File:JCPembertonBLY.jpg|center|200px|border]]John Clifford Pemberton
August 10, 1814(1814-08-10)
July 13, 1881 (aged 66)
Brev. Major (USA)
Lieutenant General (CSA)
Army of Mississippi
Seminole Wars
John Clifford Pemberton (August 10, 1814 – July 13, 1881), was a career United States Army officer who fought in the Seminole Wars and with distinction during the Mexican–American War.
He also served as a Confederate general during the American Civil War, noted for his defeat and surrender in the critical Siege of Vicksburg in the summer of 1863. After the war he took up farming.
1.1 Mexican War
2 Civil War service
2.1 Vicksburg
3 Postbellum
Early life and career[]
Pemberton was born in Philadelphia, Pennsylvania in the fall of 1814. He entered the United States Military Academy at West Point in 1833, and graduated four years later, standing 27th out of 50 cadets.[1] He was commissioned a second lieutenant in the 4th U.S. Artillery Regiment on July 1, 1837. He participated with the 4th during the U.S. actions against the Seminoles in 1837 and 1838, fighting in Florida at the Battle of Locha-Hatchee on January 24, 1838.[2]
Pemberton and the 4th Artillery served in garrison duty at Fort Columbus, New York, in 1838 and into 1839, and then at the Camp of Instruction located near Trenton, New Jersey, in 1839. He then served along the northern U.S. frontier during the Canada Border Disturbances. Pemberton and the 4th were next stationed in Michigan, serving at Detroit in 1840, at Fort Mackinac in 1840 and 1841, and at Fort Bradyin in 1841. He then served in Buffalo, New York, in 1841 to 1842, and was promoted to first lieutenant on March 19, 1842. Pemberton and the 4th returned to garrison duty at Fort Monroe, Virginia in 1842, then were stationed at the U.S. Army Cavalry School at Carlisle Barracks, Pennsylvania, in 1842 and 1843, and returned to Fort Monroe from 1844 to 1845.[2]
Mexican War[]
From 1845 to 1846, Pemberton and the 4th Artillery were part of the U.S. Military Occupation of Texas, and then the 4th was sent to Mexico at the start of the Mexican-American War. He fought at the Battle of Palo Alto on May 8, 1846, and at the Battle of Resaca de la Palma the next day. He then fought well at the Battle of Monterrey in that fall, and was brevetted to the rank of captain[3] on September 23.[2]
Pemberton then fought in the Army's 1847 actions in Mexico, including the Siege of Vera Cruz in March, the Battle of Cerro Gordo in April, the skirmish near Amazoque in May, the capture of San Antonio and the Battle of Churubusco in August, and most notably in the Battle of Molino del Rey that September. Pemberton was appointed a brevet major[4] for his performance at Molino del Rey on September 8. He then was part of the storming of Chapultepec Castle on September 13, and the Battle for Mexico City that day and the next,[2] where Pemberton was wounded.[1] Pemberton held the position of aide-de-camp to Brev. Brig. Gen. William J. Worth from August 4, 1846, to May 1, 1849.[2]
After the war with Mexico ended, Pemberton and the 4th Artillery served in garrison duty at Fort Pickens near Pensacola, Florida, in 1849. He then fought in Florida during hostilities against the Seminoles in 1849 and 1850. The 4th returned to garrison duty at New Orleans Barracks in Louisiana in 1850, and Pemberton was promoted to captain on September 16. He next served in Fort Washington, Maryland, in 1851 and 1852, at Fort Hamilton, New York, in 1852 to 1856. He and the 4th fought again in Florida during hostilities against the Seminoles from 1856 to 1857.[2]
Pemberton and the 4th was then on frontier duty at Fort Leavenworth, Kansas, from 1857 to 1858, and participated in the Utah War in 1858. He was the stationed at Fort Kearny in the New Mexico Territory in 1859, at Fort Ridgely in present-day Minnesota from 1859 to 1861, and in garrison duty at the Washington Arsenal in Washington, D.C., in 1861.[2]
Civil War service[]
At the start of the American Civil War in 1861, Pemberton chose to resign his commission and join the Confederate cause, despite his Northern birth. He resigned his commission, effective April 29.[1] His decision was due the influence of his Virginia-born wife and many years of service in the southern states before the war.[5] He was appointed a lieutenant colonel in the Confederate Army on March 28, and was made assistant adjutant general of the forces around and in the Southern capital of Richmond, Virginia, on April 29. He was promoted to colonel on May 8, and the next day was assigned to the Virginia Provisional Army Artillery, with the rank of lieutenant colonel. Pemberton was then appointed a major in the Confederate Army Artillery on June 15 and quickly promoted to brigadier general two days later. His first brigade command was in the Department of Norfolk, leading its 10th Brigade from June to November.[1]
Pemberton was promoted to major general on January 14, 1862, and given command the Confederate Department of South Carolina and Georgia, an assignment lasting from March 14 to August 29,[1] with his headquarters in Charleston. As a result of Pemberton's abrasive personality, his public statement that if he had to make the choice he would abandon the area rather than risk the loss of his outnumbered army,[6] and the distrust of his Northern birth, the governors of both states in his Department petitioned Confederate President Jefferson Davis for his removal. Davis needed a commander for a new department in Mississippi and also a command for Gen. P.G.T. Beauregard, so he sent Pemberton west and assigned the more popular Beauregard to Charleston.[7]
Vicksburg[]
File:VicksburgSiege.png
Siege of Vicksburg; positions from June 23–July 4, 1863
On October 10, 1862, Pemberton was promoted to the rank of lieutenant general,[1] and assigned to defend the fortress city of Vicksburg, Mississippi, and the Mississippi River, known as the Department of Mississippi and West Louisiana. Davis gave him the following instructions regarding his new assignment: "...consider the successful defense of those States as the first and chief object of your command." Pemberton arrived at his new headquarters in Jackson, Mississippi, on October 14.[8]
His forces consisted of fewer than 50,000 men under the command of Maj. Gens. Earl Van Dorn and Sterling Price, with around 24,000 in the permanent garrisons at Vicksburg and Port Hudson, Louisiana.[7] Historina John D. Winters describes the men under Pemberton as "a beaten and demoralized army, fresh from the defeat at Corinth, Mississippi.[9] Pemberton faced his former Mexican War colleague,[10] the aggressive Union commander Maj. Gen. Ulysses S. Grant and over 100,000 Union soldiers in the Vicksburg Campaign.
In an attempt to carry out his orders from both Davis and Gen. Joseph E. Johnston, Pemberton and his Army of Mississippi set out east to combine with Johnston's forces gathering around Jackson, while remaining in contact and covering Vicksburg.[11] Another order from Johnston changing their proposed meeting location caused Pemberton to turn around, and when he did he accidentally collided with Grant's army at the Battle of Champion Hill on May 16 and suffered a major defeat. Pemberton retreated to the Big Black River, where he fought and suffered even more heavy losses on May 17. Pemberton resolved to defend Vicksburg and led his defeated men back into its defenses on May 18. Although advised by Johnston to escape with his army, sacrificing the city, Pemberton held firm for over six weeks, while soldiers and civilians were starved into submission. (Pemberton, well aware of his reputation as a Northerner by birth, was probably influenced by his fear of public condemnation as a traitor if he abandoned Vicksburg.)
On the evening of July 2, 1863, Pemberton asked in writing his four division commanders if they believed their men could "make the marches and undergo the fatigues necessary to accomplish a successful evacuation" after 45 days of siege. With four votes of no, the next day Pemberton asked the Federals for an armistice to allow time for the discussion of terms of surrender, and at 10:00 a.m. on July 4 he surrendered the city and his army to Grant. The written terms (which in the first talks were simply unconditional surrender) were negotiated so that the Confederate soldiers would be paroled and:[12]
...be allowed to march out of our lines, the officers taking with them their side-arms and clothing, and the field, staff, and cavalry officers one horse each. The rank and file will be allowed all their clothing, but no other property.[12]
File:John Pemberton Statue.jpg
Pemberton's statue in Vicksburg
Pemberton surrendered 2,166 officers and 27,230 men, 172 cannon, and almost 60,000 muskets and rifles to Grant.[12] This, combined with the successful Siege of Port Hudson on July 9, gave the Union complete control over the Mississippi River, resulting in a major strategic loss for the Confederacy, and cutting off Lt. Gen. Edmund Kirby Smith's command and the Trans-Mississippi Theater from the Confederacy for the rest of the war.
After his surrender, Pemberton was exchanged as a prisoner on October 13, 1863, and he returned to Richmond. There he spent some eight months without an assignment. At first Gen. Braxton Bragg thought he could use Pemberton, but after conferring with his own ranking officers he advised Davis that taking on the discredited lieutenant general "would not be advisable." Pemberton finally wrote Davis directly, asking he be returned to duty "in any capacity in which you think I may be useful." Davis replied that his own personal confidence in his remained unshaken, saying:[13]
I thought and still think that you did right to risk an army for the purpose of keeping command of even a section of the Mississippi River. Had you succeeded none would have blamed; had you not made the attempt, few if any would have defended your course.[13]
Pemberton resigned as a general officer on May 9, 1864, and Davis offered him a commission as a lieutenant colonel of artillery three days later,[1] which he accepted, a testimonial of his loyalty to the South and the Confederate cause.[14] He commanded the artillery of the defenses of Richmond until January 9, 1865. He was appointed inspector general of the artillery as of January 7,[1] and held this position until he was captured in Salisbury, North Carolina, on April 12. Along with Pemberton and his 14 remaining guns, the Federals rounded up about 1,300 men and nearly 10,000 small arms.[15] There is no record of his parole after his capture.[1]
File:JCPembertonCiv.jpg
Pemberton in later life
Postbellum[]
After the war, John Pemberton lived on his farm near Warrenton, Virginia, from 1866 to 1876, and then returned to Pennsylvania. He died in the village of Penllyn, Pennsylvania, five years later and is buried in the Laurel Hill Cemetery in Philadelphia.
A statue depicting Pemberton was erected in the Vicksburg National Military Park.
He was not related to John Pemberton, a Confederate soldier who later invented Coca-Cola.
buried, Laurel Hill Cemetery, 3822 Ridge Avenue, Philadelphia, Pennsylvania [16]
↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Eicher, p. 423.
↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 ""Military biography of John C. Pemberton"". www.library.ci.corpus-christi.tx.us. http://www.library.ci.corpus-christi.tx.us/MexicanWar/pembertonjc.htm. Retrieved 2008-09-04.
↑ "for Gallant Conduct in the several Conflicts at [Monterrey]"
↑ "for Gallant and Meritorious Conduct in the Battle of Molino del Rey"
↑ ""US National Park Service biography of Pemberton"". www.nps.gov. http://www.nps.gov/archive/vick/cs_cmnd/pembertn.htm. Retrieved 2008-09-07.
↑ ""Civil War Home biography of Pemberton"". www.civilwarhome.com. http://www.civilwarhome.com/pembertonbio.htm. Retrieved 2008-09-07.
↑ 7.0 7.1 Foote Vol. I, pp. 776-78.
↑ John D. Winters, The Civil War in Louisiana, Baton Rouge: Louisiana State University Press, 1963, ISBN: 0-8071-0834-0, p. 171
↑ Winters, p. 1971
↑ Grant and Pemberton were staff lieutenants in the same division in Mexico
↑ This was more or less conflicting orders, as Pemberton could not do both, but he tried to.
↑ 12.0 12.1 12.2 Foote Vol. II, pp. 606-13.
↑ 13.0 13.1 Foote Vol. II, p. 645.
↑ Foote, Vol II. p. 646. "In this capacity Pemberton served out the war, often in the thick of battle, thereby demonstrating a greater devotion to the cause he had adopted than did many who had inherited it as a birthright."
↑ Foote Vol. III, p. 967.
Eicher, John H., and Eicher, David J., Civil War High Commands, Stanford University Press, 2001, ISBN 0-8047-3641-3.
Foote, Shelby, The Civil War: A Narrative: Vol. I Fort Sumter to Perryville, Vintage Books, 1986, ISBN 0-394-74623-6.
Foote, Shelby, The Civil War: A Narrative: Vol. II Fredericksburg to Meridian, Vintage Books, 1986, ISBN 0-394-74621-X.
Foote, Shelby, The Civil War: A Narrative: Vol. III Red River to Appomattox, Vintage Books, 1986, ISBN 0-394-74622-8.
Civil War Home biography of Pemberton
Online military biography of John C. Pemberton
US National Park Service biography of Pemberton
40x40px Wikimedia Commons has media related to: John Clifford Pemberton
NAME Pemberton, John C.
SHORT DESCRIPTION Confederate Army general
DATE OF BIRTH August 10, 1814
PLACE OF BIRTH Philadelphia, Pennsylvania
DATE OF DEATH July 13, 1881
PLACE OF DEATH Penllyn, Pennsylvania
da:John C. Pemberton de:John C. Pemberton fr:John C. Pemberton ko:존 C. 펨버턴 it:John Clifford Pemberton ja:ジョン・C・ペンバートン ru:Пембертон, Джон Клиффорд fi:John C. Pemberton sv:John C. Pemberton


It is always an exciting time when a couple announcing that they are tying the knot. Whether the wedding is in a few months or a few years, at some point a plan for their big day will need to be made and executed in a timely manner if they want everything to go smoothly. Because this big day is such an important event in the lives of the couple, as well as their family and friends, there are certain things that need to be considered when they actually start to plan this celebration.
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It is no secret that weddings can be quite costly. Between paying for the venue, caterer, flowers and DJ, the cost of your wedding can quickly add up. It is possible to have a wedding for an affordable price, no matter what your budget may be, but you will still want to be realistic about it. You may not have enough money to spend $100,000 on your wedding, but having a budget of less than $5,000 might prove to be difficult to stick to.
Are there any DIY wedding projects?
A lot of people like to do things themselves to save a bit of money. For example, centerpieces can be designed and put together by you, rather than purchasing centerpieces that are made by a professional. With a couple of items from Target or another store with crafts and arts supplies, you can have beautiful centerpieces for fairly cheap.
Is your guest list too big?
It is understandable that you want everyone you've ever met to be there for such a special occasion, but the more people you have at your wedding, the more it is going to cost you. After you have written out your guest list, it would be wise to make a few passes and eliminate some of the people on the list to help you save some money.
Planning a wedding can be fun, but it is also stressful. You will be spending your hard earned money on this special day, so will want everything to go as planned. If you take the time to consider certain things before and during the planning process, you will be well on your way to having a beautiful wedding.


text:
package org.springframework.cloud.sleuth.instrument.zuul;

import java.util.ArrayList;

import org.junit.After;
import org.junit.Before;
import org.junit.Test;
import org.junit.runner.RunWith;
import org.mockito.Mock;
import org.mockito.runners.MockitoJUnitRunner;
import org.springframework.cloud.netflix.ribbon.SpringClientFactory;
import org.springframework.cloud.netflix.zuul.filters.route.RibbonCommandContext;
import org.springframework.cloud.netflix.zuul.filters.route.RibbonCommandFactory;
import org.springframework.cloud.sleuth.Span;
import org.springframework.cloud.sleuth.TraceKeys;
import org.springframework.cloud.sleuth.Tracer;
import org.springframework.cloud.sleuth.instrument.web.HttpTraceKeysInjector;
import org.springframework.cloud.sleuth.trace.TestSpanContextHolder;
import org.springframework.http.HttpHeaders;
import org.springframework.util.LinkedMultiValueMap;

import com.netflix.niws.client.http.RestClient;
import com.netflix.zuul.context.RequestContext;

import static org.mockito.BDDMockito.given;
import static org.mockito.Matchers.any;
import static org.mockito.Matchers.anyString;
import static org.springframework.cloud.sleuth.assertions.SleuthAssertions.then;

/**
 * @author Marcin Grzejszczak
 */
@RunWith(MockitoJUnitRunner.class)
public class TraceRibbonCommandFactoryTest {

	@Mock Tracer tracer;
	@Mock SpringClientFactory springClientFactory;
	HttpTraceKeysInjector httpTraceKeysInjector;
	@Mock RibbonCommandFactory ribbonCommandFactory;
	TraceRibbonCommandFactory traceRibbonCommandFactory;
	Span span = Span.builder().name("name").spanId(1L).traceId(2L).parent(3L)
			.processId("processId").build();

	@Before
	@SuppressWarnings({ "deprecation", "unchecked" })
	public void setup() {
		this.httpTraceKeysInjector = new HttpTraceKeysInjector(this.tracer, new TraceKeys());
		this.traceRibbonCommandFactory = new TraceRibbonCommandFactory(
				this.ribbonCommandFactory, this.tracer,
				httpTraceKeysInjector);
		given(this.springClientFactory.getClient(anyString(), any(Class.class)))
				.willReturn(new RestClient());
		given(this.tracer.getCurrentSpan()).willReturn(span);
		given(this.tracer.isTracing()).willReturn(true);
	}

	@After
	public void cleanup() {
		RequestContext.getCurrentContext().unset();
		TestSpanContextHolder.removeCurrentSpan();
	}

	@Test
	public void should_attach_trace_headers_to_the_span() throws Exception {
		this.traceRibbonCommandFactory.create(ribbonCommandContext());

		then(this.span).hasATag("http.method", "GET");
		then(this.span).hasATag("http.url", "http://localhost:1234/foo");
	}

	private RibbonCommandContext ribbonCommandContext() {
		return new RibbonCommandContext("serviceId", "GET", "http://localhost:1234/foo",
				false, new HttpHeaders(), new LinkedMultiValueMap<>(), null, new ArrayList<>());
	}
}

redpajama_set_name: RedPajamaGithub


The ultimate objective of Buddhism is to attain Nirvana, which is a state of total spiritual satisfaction. It is this mental framework that is believed to bring the Taoist practitioner into alignment with the rhythms of the Tao, and be an expression of someone who has accomplished this. Taoists do not believe in a personal God. Marriage It is not a religious duty to marry. To attain enlightenment and be released from the cycle of rebirth and death, thus attaining Nirvana.
This essay will reflect my past experience, motivation and why I want to be a social worker, values, beliefs and theory of practice. In The Doctrine of the Mean , harmony with the Absolute is equivalent to integrity and sincerity. He only rarely speaks of the t'ien Dao Way of Heaven. Nothing exists apart from the Principle of Heaven in Neo-Confucianism. And lastly either path we have to have a right concentration that refers to the mental capacity and force that focuses on the ultimate goals of Buddhism which involves the practice and meditation which makes the mind pure and develop the right need for concentration. Taoism, also referred to as Daoism, first blossomed in China and has been in existence for more than 2000 years. The other culture, in contrast, is Taoism which refers to a way or a path.
Hindu prince of southern Nepal. There exist five precepts in this religion that are pre-determined training rules to lead a happy and better life. Buddhism is a philosophical way of living your life, not a religion that dictates your life like Christianity. It seems more likely to me that people like Buddha and Jesus were simply sharing their experience and the wisdom they attained via a community and like so often happens with people, when something blows our minds, we want to share it with everyone, whether they are ready or not to hear it. It includes health and healing, dietary theory, martial arts tai chi, bagua, hsing yi , geomancy, sorcery and spell casting, etc.
Teachings about General Conduct Abstain from evil, strive for nirvana, constantly cleanse the mind. In a sense where we compare to Taoism which illustrates that life is about being right, Buddhism describes of illness and sickness and trouble as the natural way of life the way life here is meant to be. These are two philosophical trends and traditions that people rely on for their religious beliefs. The flaw with becoming an immortal is that even if you can extend your life, in the end, we all still die - so it's good, but not ultimate. And through the continual existence of the soul, what else is there to look forward to but the infinite source, the first cause of the universe, the Tao.
Keller considers it similar to the of Western scholars, but the Tao is rarely an object of direct worship, being treated more like the concepts of or than as a divine object. It meets with no death. Related words in a few modern Indo-European languages are Russian doroga way, road , Polish droga way, road , Czech dráha way, track , Serbo-Croatian draga path through a valley , and Norwegian dialect drog trail of animals; valley. In total, the Dao is equated with the Absolute. Much of East Asian philosophical writing focuses on the value of adhering to the principles of the Tao and the various consequences of failing to do so. The first has a sour look on his face, the second wears a bitter expression, but the third man is smiling. It is an extension in the direction in which you already growing.
To me this is a positive side of confucious teaching in that people essentially follow the golden rule. He explicitly paired 'Dao' and 'De', focusing on humane nature and righteousness. It does not mean complacency. The former is the oldest surviving branch and is widely popular in Southeast Asia and Sri Lanka. Confucianism emphasized a reiteration of current moral values and Taoism developed a system of based upon a harmonization of man with the natural order. In order to attain the state of mindfulness one must understand some of the concepts of Buddhism: one must break free from the ties to the current world, that everything in the world changes and that all actions have consequences.
I recall that the perspective of each being is respected in taoism. Other small minorities exist in other countries. In the Buddhist understanding, it is a natural phenomenon, and not different than heterosexualiy. Doing so is very much a human experience, and we pretty much have to do that to learn lessons and to make progress. Confucianism has waxed and waned in influence over the centuries in China.
China, Korea, to lesser extent Vietnam and Japan. Confucianism was based on early teachings of Taoism, and many folk religions derive from Taoism. Perhaps the bodhisattva path is similar but that path is very vast and more flexible in some sense. He didn't realize the importance of the Eightfold Path until he obtained the wisdom that went into understanding it. There is no single central text that is referred to by all traditions. The main goal, here, is to attain Nirvana, enlightenment and happiness, in life.
It is related to the idea of , the essential energy of action and existence. The rest are its manifestations. How Taoism, or Daoism, was shaped and influenced by Chinese culture, as well as presenting how Daoism has formed and prospered in Chinese civilization with both Confucianism and Buddhism as an influence. The expression on each man's face shows his individual reaction. The Tao, Emptiness, Buddha nature and the unborn is all the same thing conceived of differently by the way I see it. I just find it useful to practice radical acceptance sometimes, interspersed with Buddhist behaviour modification.


The team behind Vegan Life - the UK's premier vegan magazine - invites you to embrace the ultimate cruelty-free lifestyle with this amazing two day event in February 2018.
Our specifically selected exhibitors will prove that choosing veganism doesn't mean denying yourself anything; from delicious food and drink, to incredible clothing, and cruelty-free skincare - there's something for everyone. With cooking demonstrations, product presentations, and a full schedule of workshops, you'll be surrounded by experts who are on hand to provide advice and guidance.


Read the latest articles from the DJ City blog.
Keeping up to date with all the newest products, latest promotions and helpful advice.
DJ City is a proud local family business supplying professional sound, lighting and audio visual equipment to DJ's, entertainers, club, bar and restaurant owners, zumba instructors and more across Australia. Our DJ Store stocks the major DJ gear and equipment brands including Pioneer, JBL, Electro Voice, Skytec, Chauvet DJ, LD Systems, Reloop, Novation, Shure and Behringer, making us one of the biggest suppliers of DJ gear. We have several DJ Shop locations across Australia with our head office located in Dandenong, Melbourne.


Scientists at the School of Pharmacy, University of Colorado at Denver studied the effects of Grape Seed Extract (GSE) against colorectal cancer. In the first part of the study, exposing various colon cancer cell lines to GSE slowed the growth of colon cancer cells and also caused the cancer cells to die.
In the second part of the study mice lacking immune function were given human colon cancer in a surgical procedure called a xenograft. High doses of Grape Seed Extract shrunk the size of the cancerous tumors by 44% after 8 weeks of treatment without causing any toxicity. The GSE both inhibited cell growth and caused the death of the cancer cells within the tumor. The study is published in the October 15th, 2006 issue of the journal Clinical Cancer Research.
Grape Seed Extract lowered the blood pressure of patients with metabolic syndrome who had high blood pressure according to the UC Davis cardiovascular researchers. The patients were riddled with health concerns including high cholesterol, elevated blood glucose, being overweight and of course suffering with high blood pressure. The one month study included twenty-four patients who had their pressure monitored for the next twelve hours each day after supplementation. Giving the patients from 150mg to 300mg of Grape Seed Extract daily decreased their blood pressure by 12 points on the systolic or upper figure and by 8 points on the diastolic or lower figure in their blood pressure reading. The study?s lead researcher C T Kappagoda, professor of cardiovascular medicine and director of the Preventive Cardiology Program at UC Davis states that patients who were supplemented with 300mg of GSE also saw a stabilization of their LDL-cholesterol; it was less likely to turn rancid and contribute to heart disease progression. Three previous studies in animal models by this team have indicated that GSE may also prevent hardening of the arteries. The findings were presented at the March 26th 2006, American Chemical Society Meeting in Atlanta. For more on these studies visit the UC Davis Health System website.


text:
GovTech 100 Investors Data Subscribe
Tyler's Latest Buyout Moves It Toward Federal, Platform Work
Tyler Technologies, no stranger to mergers and acquisitions, has purchased MicroPact for $185 million. The company works a lot with the federal government, and its state and local efforts lean toward larger customers.
by Ben Miller / February 1, 2019
MicroPact
Tyler Technologies, one of the largest and oldest software companies focused on the public sector, is all about pre-built software for state and local government.
So it might come as a bit of a surprise that the company just spent $185 million to acquire a company, MicroPact, that makes an app development platform and does half its business with the federal government. But Brian Miller, Tyler's chief financial officer, says the companies are a better fit than they first appear to be.
That's primarily because MicroPact's philosophy is all about being "low-code." They develop apps specific to customers' needs, but they mostly spring from apps the company has already created. It's meant to be a streamlined approach to solving unique needs.
Tyler Technologies Acquires SocrataTyler Acquires Emergency Responder Firm SceneDocOracle Launches Community Development Solution, Competing with Tyler and Accela
"My understanding is that it's a pretty easy platform to work with. The configuration and implementation, there's not a lot of coding that takes place," Miller said. "It's a pretty nimble platform."
Still, the move represents something of a shift in the way Tyler does business. Its acquisition of Socrata last year moved the company into some federal work and inched it closer to the platform development model of doing business. The MicroPact deal extends that trend.
"Really the first federal business we've had of any note came through the Socrata deal we had last year, and it was on the order of $5 million in revenues," Miller said.
Outside the federal government, MicroPact's customer base leans toward the larger customers. Its customer base of 350 public-sector clients includes agencies in 49 of the 50 states such as the California Department of Consumer Affairs, the Florida Department of Business and Professional Regulation and the Tennessee Department of Health.
MicroPact's development platform, entellitrak, is geared toward case management and business process management. Its software portfolio includes software for courts, licensing and permitting, health and human services and workers' compensation. A lot of those are areas Tyler has worked in for a long time, but Miller said there isn't a lot of customer overlap.
"Our courts and justice [work] is primarily oriented at trial courts at the county level or traffic courts at the municipal level," he said. "They've done a lot more at the state and the federal level, so their application is used for the Department of the Navy to manage their trials."
MicroPact's delivery model often involves selling its platform to an agency with a specific use case in mind, and then the company will deliver the needed solution through some combination of development services, internal expertise and partnerships. The company operates a partner network, called the MicroPact Global Alliance, that includes partners such as Accenture, Amazon Web Services and Booz Allen Hamilton.
The buyout is the second-largest acquisition in Tyler's history behind its $670 million purchase of New World Systems in 2015. It's also the latest in a hot streak of merger and acquisition activity of the company — aside from Socrata, Tyler has also acquired CaseloadPRO, MobileEyes, Sage Data Security and SceneDoc in the past year.
MicroPact's staff shouldn't expect big personnel changes as a result of the buyout, Miller said.
"The management team as well as the staff will be pretty much intact," he said. "That's a big part of [MicroPact's] value as a company."
Ben Miller Associate Editor of GT Data and Business
Ben Miller is the associate editor of data and business for Government Technology. His reporting experience includes breaking news, business, community features and technical subjects. He holds a Bachelor's degree in journalism from the Reynolds School of Journalism at the University of Nevada, Reno, and lives in Sacramento, Calif.
Cask Spins Off Digital Transformation into Separate Business Tyler Technologies Acquires Socrata Tyler Acquires Emergency Responder Firm SceneDoc
MORE FROM GovTech Biz


From time to time as a marketer you come up with an idea that is unique and exciting. I'm really excited about this chiropractic marketing idea, but I'm not practicing right now to test it out. From the results I've seen in my own business today I know it's a winner.
A few months ago I had a message come to me that really impressed me. I had sent a mentor of mine an email asking him if he would coach us in our business. I hoped that I would hear back from him in a few days, because he's a very busy guy.
I clicked the button and there he was in crystal clear sound giving my wife and I an explanation of how he felt he could help us. He recorded a 3 minute audio for us which made us feel very special. He was able to send us a great deal of personalized information in just a few minutes. It saved him time and was much more powerful than a simple email would have been.
Today about 75% of the email messages I send to my clients are in audio form. I very often get compliments on how cool it is, and it's impressed a few new clients enough to get them to join my service.
So How Can You Use This?
I think that this would be an awesome system for patient reactivations. In a few minutes each day you could easily send out personalized audio messages to patients that you haven't seen for a while. If I were a patient in your office I would be very impressed that you cared to do that. Even if I didn't come in right then I would certainly choose your office in the future.
I think this is a patient reactivation game changer.


The inside was nothing like I expected, because the only interior photo I'd seen before featured bare log walls.
Once the Civilian Conservation Corps crews had a roof on every house, the colonists themselves worked on the interior of their own houses, covering the logs with sheets of plywood, installing trim and cabinetry, until they had a place they would be proud to call home.
How fun that you were a guest speaker at Fireside Books – it's a wonderful store. In fact, the entire town of Palmer is pretty darn wonderful. You'll have to come back during summer when the farmer's market is in full swing and the flowers are in bloom. It's a great place to call home.
I love Fireside Books too, and agree that Palmer is a lovely town, summer and winter!


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Dan Chan will steal your smartwatch and break into your iPhone, using some old-fashioned sleight of hand.
Dan Chan, a magician to the stars of Silicon Valley, has a trick that makes top software engineers shudder. He'll take a volunteer's iPhone, show the crowd it's locked, and then, after some hocus pocus — studying the volunteer's fingerprints, say, or counting dramatically — he'll hand the phone back, unlocked.
The iPhone trick has stumped Apple employees, Chan says. At one recent holiday party thrown by a major tech company, a guest whose iPhone was broken into called the experience "a little scary." At another party, hosted by a tech company that works for the federal government, observers of the iPhone trick threatened to torture Chan, he recalled. This was a joke, obviously, but Chan was nevertheless relieved when, a little later, one of the engineers figured out how he'd done it, defusing the tension.
Chan, 39, who expects to perform at more than 20 corporate events this holiday season, is fluent in traditional party magic, making coins teleport and cards appear in impossible places. But he also has a suite of tricks updated for the modern age, in which he conjures magic from a volunteer's iPhone. These tricks can make Chan appear like an ace hacker, exploiting some arcane vulnerability in the software, but in fact the mechanics behind them are surprisingly low-tech. Chan relies on classic magician's tradecraft — distraction, deception, and preparation — to befuddle the brainiest engineers.
"Engineers particularly like to reverse-engineer every trick," Chan told BuzzFeed News. "I pride myself on being able to fool the most skeptical of audiences, the guys who have those theories."
Chan, who grew up in San Francisco and got an undergraduate business degree at UC Riverside, was once part of the industry he now entertains. He joined PayPal in 2000, before its IPO, in an entry-level customer service role. He was doing magic on the side, and his PayPal interview, he said, "was me showing them tricks for 15 minutes."
"I said, 'If you hire me, I'll make sure every Friday afternoon at the office is a little bit fun,'" Chan recalled. "I was like the company glue, besides the ping pong table, the free buffet and the free vending machine."
Those early days at PayPal, when the company was constantly under attack by fraudsters and had to devise ingenious ways to fight back, have become legend in Silicon Valley; executives like Max Levchin and Peter Thiel went on to even greater success and fame. But to Chan, working at PayPal was boring. He was laid off a little more than a year after he started, and he dove into magic full-time, studying tricks and seeking mentors to develop his technique.
After a stint doing children's parties, Chan shifted his focus to corporate events about six years ago, and he has since spent a small fortune on targeted ads on Facebook and Google. To win over tech clients, he studies industry news and absorbs key trivia. When he meets an executive, he said, he'll ask to take a picture of their library, and then he'll read a book or two from the shelf.
In the process, Chan has absorbed some of the tech industry's aggressive business philosophy. "You fail, and fail fast — the Silicon Valley motto? I've done that," Chan said.
Chan has benefited from a multi-year boom in Silicon Valley, where a flood of venture capital and an intoxicating sense of optimism have pushed tech companies to outdo each other with ever more extravagant holiday parties. Even a year ago, when it seemed the industry was headed for a slowdown, Facebook threw a 1920s-themed bash with a trapeze performer, Yahoo treated its employees to champagne towers, and Google hosted "neon-costumed dancers and a trippy jellyfish electro-garden," according to Bloomberg News.
One memorable feature of a party during the boom, Chan recalled, was an ice sculpture through which guests would pour shots, drinking them chilled on the other end. But this year, he said, the parties seem more subdued, and the magic budgets have seemingly shrunk. In years past, Chan would perform alongside two or three other magicians; this year, he's usually the only one.
Software engineers, who perform wizardry with lines of code, are a tough crowd for a party magician, but Chan delights in getting under the techies' skin.
In one of his routines, he asks a volunteer to open their Safari app and then hand him the iPhone. While typing something into the device, Chan asks the volunteer to think of a city they'd like to travel to, after, say, making a fortune on a stock tip. He puts the phone on a nearby table, face down. He then asks them to name that city — it could be any city. When they flip their phone over, the Safari app displays the city they chose, on a map.
The trick does not use voice recognition, or any bleeding-edge technology. The real magic stems from the way Chan sets it up. This doesn't make the experience any less unnerving, and Chan likes to amp up the discomfort.
"When I wave my hand over the phone, all your banking information goes to my servers in the Cayman Islands," he told a guest at a recent tech company party, to nervous laughter.
Chan's repertoire includes pickpocketing — a skill he uses to procure smartwatches and smartphones from party guests, displaying them theatrically before returning them to their disoriented owners. He used to get laughs, he said, when he revealed that employees at Google parties were carrying iPhones instead of Android devices. At parties hosted by Apple and Google, he said, he has stolen top-secret versions of smartphones that haven't yet been released.
"That creates a lot of tension," he said, grinning.
His creepiest tech trick, breaking into iPhones, relies largely on finesse and some carefully concealed preparation. Chan has multiple methods of creating the illusion, switching among them to stay ahead of skeptical audiences. One of his signature moves, in a pre-show routine or even during the show itself, is to appear to make a mistake, gathering information that he might use later.
"They'll ask me, 'Do the trick again,' and I'll do it again, but do it by a different method, and then they're totally stumped," he said. "They think they know, and then they don't know."