text:
An ancient history of gene duplications, fusions and losses in the evolution of APOBEC3 mutators in mammals
- First Online:
- Received:
- Accepted:
- Citations
- 4.4k Downloads
The APOBEC3 (A3) genes play a key role in innate antiviral defense in mammals by introducing directed mutations in the DNA. The human genome encodes for seven A3 genes, with multiple splice alternatives. Different A3 proteins display different substrate specificity, but the very basic question on how discerning self from non-self still remains unresolved. Further, the expression of A3 activity/ies shapes the way both viral and host genomes evolve.
We present here a detailed temporal analysis of the origin and expansion of the A3 repertoire in mammals. Our data support an evolutionary scenario where the genome of the mammalian ancestor encoded for at least one ancestral A3 gene, and where the genome of the ancestor of placental mammals (and possibly of the ancestor of all mammals) already encoded for an A3Z1-A3Z2-A3Z3 arrangement. Duplication events of the A3 genes have occurred independently in different lineages: humans, cats and horses. In all of them, gene duplication has resulted in changes in enzyme activity and/or substrate specificity, in a paradigmatic example of convergent adaptive evolution at the genomic level. Finally, our results show that evolutionary rates for the three A3Z1, A3Z2 and A3Z3 motifs have significantly decreased in the last 100 Mya. The analysis constitutes a textbook example of the evolution of a gene locus by duplication and sub/neofunctionalization in the context of virus-host arms race.
Our results provide a time framework for identifying ancestral and derived genomic arrangements in the APOBEC loci, and to date the expansion of this gene family for different lineages through time, as a response to changes in viral/retroviral/retrotransposon pressure.
KeywordsAPOBEC Cytydine deaminase Gene duplication Subfunctionalisation Virus/host arms race
Cytidine deaminases of the APOBEC3 (A3) gene family have a broad antiviral activity against retroviruses, can inhibit LTR- and non-LTR-retrotransposons, parvoviruses, hepadnaviruses, flaviviruses and paramyxoviruses, and might repress also TT-viruses, papillomaviruses and herpesviruses [1, 2, 3, 4, 5, 6, 7, 8, 9]. Under the already tested additional viruses, adenoviruses, poxviruses and influenza viruses replicate well irrespective of A3s [3, 10, 11]. The hunt is on for the identification of additional viral targets [12]. The APOBEC3 gene family encodes a characteristic zinc (Z)-coordinating catalytic motif (His-X-Glu-X23–28-Pro-Cys-X2–4-Cys) [13] and the A3 proteins can be classified according to the presence of an A3Z1, A3Z2 or A3Z3 motif [14, 15, 16]. A3s act by deaminating cytidine into uridine (EC 18.104.22.168) using single-stranded DNA as a substrate. This DNA editing results in the introduction of mutations that eventually render the target genome inactive. However, uncontrolled chemical editing of DNA sequences puts at risk the genomic information in the cell [17, 18] and many mechanistic questions regarding A3 activity and substrate specificity remain open.
During retroviral budding, A3 molecules incorporated into progeny retroviral particles are carried over within the virion and counteract the new infection upon release in the cytoplasm of the newly infected cell. Retroviruses have evolved mechanisms to prevent encapsidation of A3s into viral particles. The Vif protein in lentiviruses, the Bet protein in foamyviruses and the nucleocapsid protein in Human T-cell lymphotrophic virus accomplish this anti-antiviral activity [19, 20, 21, 22, 23, 24]. The expression of A3s is not restricted to the immune system [25], and different cell types may express different A3 repertoires, and even different variants may present different subcellular location [26]. Thus, some retroviruses without vif or bet genes might escape A3-mediated antiviral inhibition by a restriction of their cellular tropisms, as it is discussed for the Equine infectious anemia virus[27]. Several aspects on how retroviruses cope with the deaminase activity of the A3s encoded by their host species are still a matter of debate [28, 29]. Thus, and despite several studies, it remains unknown which strategies the Moloney murine leukemia virus or the Mouse mammary tumor virus have evolved to prevent inhibition by the murine A3 [30, 31, 32, 33, 34, 35, 36].
In mammals the A3 locus appears always flanked by the CBX6 and CBX7 genes but there is ample variation across species, regarding the number and arrangement of A3 individual genes, presence of fused genes, expression pattern, splice alternatives and read-through mechanisms and substrate specificity, even in related species. Thus, although Primates and Rodents are relatives and belong together within Euarchontoglires, the human genome contains seven A3 genes, four of them resulting from the fusion of two A3 domains, while the mouse genome encodes for a single A3Z2-A3Z3 fused gene [37]. Additionally the dog genome presents two A3 genes, while there are four A3 genes in cats, two to three in pigs, sheep and cow and six in horses [15, 16, 27], and all these species belong together within Laurasiatheria. The finding of A3Z1, A3Z2, and A3Z3 genes in these two mammalian lineages strongly indicates that their ancestor (Boreoeutheria) was probably equipped with a single copy of each gene.
One important mechanism of genome evolution and for the appearance of novel gene functions is gene duplication [38]. Genes within a genome that are descendants of gene duplications are paralogs, while two genes in different species that derive from a single gene in the last common ancestor of both species are orthologs. Paralogs derive either from an ancestral duplication ("outparalogs") or they derive from a lineage-specific duplication ("inparalogs"), giving rise to co-orthologous relationships [39]. Several models for the emergence, maintenance and evolution of gene copies have been proposed (for a review see [40]). The duplication of genes may in some cases have no immediate consequence for the host, but in other cases can be deleterious or linked to disease [41], or confer an selective advantage [42]. For the antiviral A3 genes evolutionary solutions reflect the trade off between a potential self-toxicity against cellular DNA -in cases of an A3 exacerbated response- and the emergence of viral pathogens if low A3 activity and/or diversity allow for restriction escape variants. Fixation of duplicated A3 genes and the subsequent preservation in certain population is likely driven by a strong selective advantage for the individuals carrying additional copies of the gene/s. Any subsequent acquisition of genetic differences between the gene copies can alter the chances of both copies being preserved and might change the function of the encoded proteins and result either in gene loss, 'neo-functionalization' [38] or 'sub-functionalization' [43].
We present here a time scale of the evolution of the A3 loci, arising from their common origin with other cytidine deaminases. According to our results, the track of duplications in the A3 locus started with the ancestral gene itself. The appearance of the three clades within the A3 family dates back to emergence of placental mammals. The evolutionary history of A3s has since then been landmarked by duplication events especially in Primates, but also in Perissodactyla and Carnivora, as well as by deletions events, such as in Rodentia. Three main trends can be observed: first, a sustained decrease in evolutionary rate for the A3 subfamilies in the last 100 Mya; second, duplication events have occurred in the A3Z1 and A3Z2 subfamilies, but not in the A3Z3; third, duplication events accumulate in the last 50 Mya. The molecular mechanisms that generate the tandem duplication of A3, and the evolutionary pressures that drive the sub/neofunctionalisation and eventually selection of the duplicated genes still need to be identified.
There are three main clades of A3 genes
The most recent common ancestor (MRCA) of the A1s, A3s and AICDA sequences can be dated 441 Mya (95% HPD, 431–456 Mya). The MRCA of the A3s and AICDA sequences can be dated 424 Mya (95% HPD, 417–438 Mya), while the MRCA of extant A3 sequences can be dated 247 Mya (95% HPD 193–351 Mya) (Figure 1). The three members of the A3 family radiated later at comparable time points: 112 Mya (95% HPD 109–113 Mya) for A3Z1; 111 Mya (95% HPD 106–113 Mya) for A3Z2; and 109 Mya (95% HPD 102–113 Mya) for A3Z3. The relationships among the three A3 subfamilies could not be solved with certainty, but both ML (44% bootstrap support) and Bayesian inference (0.52 Bayesian posterior probability) suggested a sisterhood relationship between A3Z1 and A3Z2, with a MRCA dated 199 Mya (95% HPD 164–251 Mya). Only more quality data from Afrotheria, or the discovery of A3 genes in Monotremes, Marsupials and/or Xenarthra may help us define and date these basal nodes with confidence.
Evolution of the A3 loci in Euarchontoglires
A3Z1 genes in primates
The A3Z1 gene appears in humans in three copies, named Z1a, Z1b and Z1c, and we will name the corresponding orthologs according to the nomenclature in humans (Figure 2). The MRCA of A3Z1 genes can be dated 71 Mya (95% HPD 60–84), which grossly corresponds to the basal divergence time of primates, some 73–87 Mya. Orthologs of the A3Z1 genes can be found in the genomes of several Haplorrhini species, including Catarrhini (Old World monkeys and apes) and Platyrrhini (New World monkeys). We could only detect one A3Z1 gene in the platyrrhines genomes we have browsed, and these sequences are the outgroup of the Z1a, Z1b and Z1c found in catarrhines. Phylogenetic relationships and inferred divergence times among the Z1c orthologs grossly correspond to those among the corresponding species. Regarding the Z1a and Z1b genes, we could identify the orthologs of both human genes in the genomes of the common chimpanzee, western gorilla, northern white-cheeked gibbon and Rhesus macaque. The ortholog of the Z1b human gene could be identified in the Sumatran orangutang. The duplication event that generated the Z1a and Z1b genes can be dated some 27 Mya (95% HPD 22–32). This timing could be compatible with the Hylobatidae/Hominidae split some 18–24 Mya, although it matches well the Cercopithecidae/Hominoidea split some 26–34 Mya [44].
A plausible scenario for the evolution of the A3Z1 loci in primates would include a basal split, generating the ancestors of the Z1a/Z1b (hereafter Z1ab) and the Z1c genes. Whether this first duplication event predates the early split Haplorrhini (tarsiers, monkeys and apes)/Strepsirrhini (non-tarsier prosimians) or is rather exclusive to the Haplorrhini lineage is not clear and must still be solved, since there are no sequence data available for Strepsirrhini. In Catarrhini, the ancestral Z1c gene may have evolved without further duplication event. The ancestral Z1ab gene underwent duplication and generated the ancestral Z1a and Z1b genes. Orthologs of both Z1a and Z1b are present in all hominids [45], as the MRCA to both genes predates well the split Ponginae/Homininae. Future identification of Z1ab genes in hylobatids and in cercopithecoids will clear whether the Z1a/Z1b duplication event is basal to catarrhini or to Hominoidea.
A3Z2 genes in primates
Our fragmentary information on the A3 gene content makes it impossible to reconstruct with confidence the evolutionary relationships among the A3Z2 genes in primates. Additionally, a number of A3Z2 sequences arise from genomic material while others belong to cDNA, and the complex alternative splicing of the A3 genes may disturb further the algorithms for phylogenetic inference. Finally, in this locus gene conversion may have played a key role, given the large number of gene duplication events in this A3 subfamilly -up to six in humans- and the nature of the locus, where the copies generated remain in a tandem arrangement. A proper phylogenetic reconstruction will thence need to wait until genomic sequences with better coverage are available for a larger number of primate species, especially if encompassing Strepsirrhini and Tarsiidae. A basal split event some 94 Mya (95% HPD 89–99) generated the two main lineages Z2bdf and Z2aceg. This timing could match the basal strepsirrhini/haplorrhini diversification time in primates (some 87 Mya), although it would be more compatible with one duplication exclusive to haplorrhini (MRCA some 85 Mya) or even to simiiformes. In either case our interpretation implies that the ancestral simiiformes already carried two copies of the A3Z2 genes in their genomes. The Z2bdf group comprises exclusively sequences from catarrhines, with an estimated MRCA around 43 Mya (95% HPD 35–54). Sequences here cluster into two groups: Z2b and Z2df. Both clusters contain sequences from cercopithecoids and from hominoids. It can thus be inferred that the duplication event that generated both groups took place before the divergence between cercopithecoids and hominoids some 29 Mya. This timing is compatible with the estimated MRCAs around 28 Mya (95% HPD 23–34) for Z2b and 32 Mya (95% HPD 28–34) for Z2df. Within the Z2aceg group, phylogenetic inference cannot solve the fine relationships. Only a major, monophyletic group containing the Z2g genes can be clearly defined, containing sequences of both catarrhines and platyrrhines. Phylogenetic relationships among Z2g genes grossly correspond to those of the corresponding species: platyrrhines are basal to the cluster, with calculated split time 39 Mya (95% HPD 34–45), and cercopithecoids and hominids cluster separately, with a MRCA around 24 Mya (95% HPD 23–28).
A plausible scenario for the evolution of the A3Z2 genes in primates would include a first basal split Z2aceg/Z2bdf, previous to the split between platyrrhini and catarrhini. The ancestral Z2aceg underwent a second duplication Z2ace/Z2g also before this split event. Modern Z2a, Z2c and Z2e human genes appeared after subsequent duplication events. On the other hand, the ancestral Z2bdf underwent a first duplication Z2b/Z2df at least before the divergence between cercopithecoids and hominoids. The basal position of human Z2d with respect to Z2f sequences suggests also that the duplication event Z2d/Z2f predates diversification within catarrhines, but a larger repertoire of sequences is needed here to reconstruct phylogenetic relationships with confidence.
A3Z3 genes in primates
Orthologs of the A3Z3 gene can be found in the genomes of the catarrhines chimpanzee, bonobo, human, gorilla, Bornean and Sumatran orangutan, gibbon and macaque (Figure 1). The MRCA of these A3Z3 genes dates back to 31 Mya (95% HPD 26–34). In all these species the A3Z3 gene appears as a single copy, with no evidences of gene duplication. Additionally, the phylogenetic relationships among these genes and the inferred timing for the nodes perfectly match those of the corresponding species. The most parsimonious explanation for the absence of A3Z3 in platyrrhines and in other primates is therefore our lack of information about the locus, and it can be anticipated that the missing A3Z3 genes should be identified in the future, as genome coverage increases.
A3 genes in rodents
In the rodent Cavia porcellus, as in rat and mouse, the Z2Z3 genes are fused and the A3Z1 gene is missing. Considering the presence of A3Z1 in Primates, the last common ancestor of Muridae and Caviidae probably possessed already 50–85 Mya the genome organization found in the extant rat and mouse genomes. Precise answers on the timing for the A3Z1 deletion event and for the Z2Z3 fusion event will need to wait until new genomic information is available. Interesting sequences could come from the genomes of squirrels, which are basal to Muridae/Caviidae, or from rabbits, which constitute together with rodents the Glires taxon, sister to Primates.
Evolution of the A3 loci in Laurasiatheria
A3genes in cetartiodactyla
In the group of Cetartiodactyla (even-toed ungulates) the evolution of the individual A3 genes matches the evolution of the host genomes (Figure 1), although there is controversy about the phylogenetic relationships among mammalian orders here [46]. The genomes of cow and sheep present single copies of the A3Z1, A3Z2 and A3Z3 genes, with no evidences for gene duplications. In the A3 locus in the pig, the A3Z1 gene however seems to have been lost during evolution [15, 47]. The presence of a A3Z1 gene in the genomes of cow and sheep suggests that the loss of the A3Z1 gene in pig genome occurred after the split that generated the Suidae lineage.
A3genes in horses
a3genes in carnivora
Evidences of A3 genes can be found in the genome of canids and felids. The A3Z3 gene is present in all carnivores genomes analysed, but the A3Z1 gene is missing in Felidae and the A3Z2 gene is missing in Canidae (Figure 13b). Both gene loss events must have occurred independently after the split between cat and dog lineages, some 43–64 Mya. In the genome of the domestic cat (F. catus) we identified four A3 genes (Z2a, Z2b, Z2c, and Z3), while only one expressed A3Z2 and one A3Z3 gene could be found in diverse species in the Panthera genus [16]. The duplication events that generated the three A3Z2 genes in the genome of the cat date 30 Mya (95% HPD 13–48) and 14 Mya (95% HPD 4.7-30) (Figure 3b). Although fine details in the evolution of felids are a matter of debate, the MRCA of Pantherinae and Felinae could have lived some 12 Mya [49]. The fact that we have found the A3Z2 gene in lion, tiger and leopard –all of them Pantherinae- basal to the A3Z2 genes in cat, puma and lynx –all of them Felinae- suggests that the split between Pantherinae and Felinae predated the duplication events that generated the A3Z2 diversity in cats. Further, three different mRNA sequences were retrieved from lion and two from tiger [16]. If these mRNA sequences originated from different genes this would imply that independent gene duplications could have occurred in the Panthera lineage. However, considering again the potential for multiple alternative splicing in the A3 genes, the analysis of genomic sequences is required to determine whether panthers and cats have found similar, convergent evolutionary answers to a selective pressure that we still need to identify.
Evidences for selection in the evolution of A3 loci
Statistics for theKa/Ksvalues obtained either with the Mechanistic Empirical model (MEC) or with Random Effects Likelihood (REL) estimates
All sequences
Excluding primates sequences
MEC ka/ks
quantil 25%
quantil 90%
maximum value
REL dN‒dS
percentage of sites under positive|puryfying selection
Evolutionary rate has decreased in the three A3 subfamilies
Evolutionary rates for the different large clades described measured as substitutions per site per million of years, as inferred from the Bayesian time analyses
Evolutionary rate (substitutions per site per My)
Evolution of the evolutionary rate (Δ evolutionary rate per My)
Huber M‒estimator
Median absolute deviation
95% confidence interval of the mean
Standard error
1.62 10-3
1.07 10-3
1.97 10-3
4.31 10-4 – 6.44 10-3
5.86 10-4
1.15 10-3
4.49 10-4 – 2.29 10-3
5.15 10-6
1.7 10-6
2.74 10-4
7.99 10-4
4.06 10-4 – 1.53 10-3
9.88 10-4
2.14 10-3
7.49 10-4 – 6.50 10-3
4.15 10-5
4.4 10-6
1.28 10-3
2.60 10-3
4.52 10-4 – 7.61 10-3
3.34 10-5
9.4 10-6
1.38 10-3
2.15 10-3
4.17 10‒4 – 4.40 10-3
2.61 10-5
4.7 10-6
A3 genes belong to a large superfamily of deaminases that edit nucleic acids and constitute the sister taxa to AICDA, as previous studies suggested [37, 54]. Certain members of the deaminase family, such as A1, target RNA as a substrate, but it has been proposed that the ancestral activity may have been to target DNA [54]. Our results show that the MRCA of AICDA and A3, ca 420 Mya, predates the split between the lineages of zebra fish and humans. The subsequent duplication events of the ancestral A3 gene to generate the three extant A3Z1, A3Z2 and A3Z3 genes could not be dated with precision and overlap largely. The first one occurred ca. 247 Mya (95% HPD 193–351 Mya) and the second one ca. 199 Mya (95% HPD 164–251). Representatives of Laurasiatheria, Euarchontoglires and Afrotheria are found in all A3Z1, A3Z2 and A3Z3 subtrees. For Afrotherians, a few sequences were found basal to the A3Z1 subgroup (only sequences from the African bush elephant Loxodonta Africana), to the A3Z2 subgroup (the N-terminus from L. Africana) or to the A3Z3 subgroup (an in silico prediction from genomic DNA from the rock hyrax Procavia capensis and the C-terminus from one L. africana sequence). On the view of the evolutionary relationships and time divergences, we can conclude that the duplication events that originated the three ancestral A3 loci had already occurred well before diversification within placental mammals, which took place at some point between 95–120 Mya [55]. The presence of A3Z1 and A3Z3 representatives in Afrotheria and of A3Z1, A3Z2 and A3Z3 in Boreoeutheria sustains also this view. The estimated appearance of the most recent common ancestor of all A3s predates the split between placental mammals and marsupials 125–150 Mya, and also the split of monotremes at the base of the crown clade of modern mammals, debated between 160–200 Mya [56], and 203–238 Mya [57]. Thus, our data support an evolutionary scenario where the genome of the MRCA of all mammals already encoded for at least an ancestral A3, and where the genome of the MRCA of all placental mammals already encoded for a A3Z1-A3Z2-A3Z3 arrangement. This genomic arrangement is conserved in all placental mammals, with the A3 genes flanked by the CBX6 and CBX7 genes, as in chromosome 22 q13.1 in humans. This conserved chromosomic location could be used for targeting and sequencing of the A3 locus of selected species in the future. Unfortunately, the sequence gap between CBX6 and CBX7 is not resolved with certainty in the opossum and in the platypus genomes, and we cannot confirm whether the last common ancestor of Eutheria and Metatheria already possessed a single ancestral A3, the intermediate stage with two loci, or already three loci, as the ancestor of all placental mammals. A note of caution should be nevertheless stated here, since our results cannot go beyond the sequence dataset that we have been able to gather. In a number of genomes we have not been able to recover any relative of the A3 sequences. This holds true for Monotremes, Marsupials and Xenarthra. For other organisms we have not been able to detect certain A3 genes, such as the A3Z1 gene in the cat genome and in Rodentia, or the A3Z2 gene in the dog genome. Since for humans the existence of copy number variation in the A3A and A3B genes is documented in a good number of cases (Additional file 4: Figure S3), a similar situation can be detected in the future in other species. It is important to note here that obtaining genomic sequences of the A3 locus is not trivial, as it evolves under strong selective pressures [45]. In our current analyses we have opted for the most parsimonious explanation: e.g. the presence of an A3Z2 gene in Euarchontoglires and in Felidae and the absence in Canidae has been interpreted as the Boroeutherian ancestor carrying a copy of the ancestral A3Z2 gene, which may had got lost in the Canidae lineage. The hypothetical finding in the future of an A3Z2 gene in Canidae would not affect our timing results as all calibrations have been chosen on subtrees whose structure matched that of the corresponding species (Additional file 5: Table S2).
The results here presented show that the A3 loci in mammalian genomes are extremely variable and have undergone independent events of gene duplication followed by fixation, gene loss and gene fusion. The outcome of these events has lead to parallel evolution, as is the case of the loss of A3Z1 gene in rodents, dogs and pigs. The most striking result, however, is that the amplification of A3 genes and the fixation of duplicated genes has been selected and expanded in the population, independently in several taxa, such as the A3Z2 gene in Primates (Figure 2), horses (Figure 3a) and cats (Figure 3b) and the A3Z1 gene in Primates and horses. Further, an increase in the frequency of duplication events in the last 50 Mya can be observed in these three lineages, i.e. human, horse, cat (labelled with star symbols in Figure 1). Gene duplications have occurred in both A3Z1 and A3Z2, but not in the A3Z3 clade. Duplication of the A3Z3 gene seems thus to be unfavoured, possibly reflecting the trade-off between the advantages of an increased antiviral repertoire and the increased self-toxicity by gene dosage effect [18]. Also pointing in this direction, there is evidence for independent events of A3Z3 activity loss in human alleles [58]. Finally, further data that exemplify the broad variability in the A3 locus come from copy number variation studies in humans, with several reports on the loss of the A3A (A3Z1a) and/or the A3B (A3Z2a-A3Z1b) genes (Additional file 4: Figure S3).
Different taxa have selected similar solutions through independent events in the A3 locus in terms of gene duplications and/or deletions. Such parallel evolution may be the response to similar environmental changes, e.g. increased retroviral activity either exogenous or endogenous, which could account for the parallel expansion of the A3 family in different mammalian clades. The fixation of similar duplication events in different lineages suggests that the preservation of the duplicated genes has a positive effect in fitness. Further, the main activity of all A3 genes seems to be ssDNA editing, and the differences among them are rather related to substrate specificity [59, 60], expression pattern [61, 62], and virus specificity [2]. The fate of new A3 copies may thus be sub-functionalisation by broadening the spectrum of molecular targets that can be edited by the A3 proteins, and that this fine tuning of the edited DNA substrates results in an increase in fitness. Such shift in substrate specificity is also supported by the evidence of positive selection in A3s in primates, rodents, felids, horses [16, 50, 51, 52, 53, 63], and our results confirm as well the presence of residues under positive selection in all A3Z1, A3Z2 and A3Z3 clades (Table 1). However, the antiviral relevance of the differential deaminase activity as a function of the ssDNA sequence still needs to be explored.
Our results show that residues evolving under neutrality co-occur with residues under positive and under purifying selection in the A1 and in the A3 genes, and that this holds true for all sequences considered together even after accounting for the excess of Primates sequences (Table 1 and Figure 4). For all these genes, there is a clear peak and a substantial proportion of sites evolving close to neutrality. Additionally, a substantial amount of area below the curve is located in the two tails of the multimodal distribution, corresponding to positions evolving under strict purifying selection and intermediate values of 0.5 < Ka/Ks < 0.8, and under positive selection. Previous descriptions had rather focused on specific genes in specific taxa, i.e. primates, rodents, horses, felids and pigs [16, 50, 51, 52, 53, 63]. Our analyses have addressed the three A3 gene clusters in all mammals and show that multiple evolutionary pressures coexist in the same gene sequence. Similar cases of positive selection observed as an increase of Ka/Ks during subfunctionalisation of duplicated genes has been also documented in other gene families [64]. The biological interpretation of the significance of individual residues under positive selection needs to be analysed in the context of the evolution of the gene in which they reside and in the biochemical context of the exon combination present in the actually expressed proteins. Methods for detecting positive selection based on Ka/Ks ratios may result in false positives, as a relaxation of evolutionary constraints could also lead to an increase in this parameter [65]. Such constraint relaxation is specially expected to occur in genes that have undergone duplication events, as it is the case in the A3 genes. Thus, the actual meaning of sites under positive selection in the three A3Z1, A3Z2 and A3Z3 clades requires experimental confirmation. Additionally, gene duplication could be the result of the genomic arms race between target viruses and their hosts, as novel A3 proteins can counteract anti-antiviral activity raised in a virus that evolves anti-A3 mechanisms, such as the Vif or the Bet protein [19, 20, 21, 22, 24]. This combination could correspond to certain models among those proposed by Innan and Kondrashov [40] to classify the evolution of duplicated genes, such as modified duplication or diversifying selection. The evolution of the A3Z2 locus in primates, horse and cat, with several rounds of successive amplification events, suggests however that gene duplication may have occurred here through an adaptive radiation model, as proposed by Francino [66]. The new A3 copies could explore the sequence space that expands the family of substrates for the action of deaminases, typically by modifying the sequence context of the C- > U edition. Similarity among duplicated genes could also allow for gene conversion and/or recombination [67], further enlarging the repertoire of substrates. In analogy to gene conversion at the DNA level, similarity in DNA sequence between tandem duplicated genes may be responsible for the read-through mRNA species that encompass exons from different genes, and that have been described in cats [16, 68], horses [27] and pigs [15, 47] and have also been communicated in humans [69] (Additional file 6: Figure S4). Currently more than 500 ESTs in the databases map onto the A3 human loci, with a number of them containing spliced exons from different A3 genes (Additional file 6: Figure S4). This additional generation of diversity adds to the high number of alternatively spliced mRNAs originated from each individual A3 gene, especially of those composed of two fused "mono" A3s, either A3Z2-A3Z1 or A3Z2-A3Z2. In these cases we face fused genes that retain their individual coding capacity, can generate multiple splice alternatives and show potential to encode for read-through mRNAs. Finally, an additional level of spatiotemporal complexity to the regulation of this antiviral activity arises from A3 expression heterogeneity linked to cell type, tissue, developmental time or exposure to foreign DNA.
The architecture of several tandem copies of paralogs in a genome facilitates non-homologous recombination among paralog copies resulting in gene conversion, as has been suggested for the A3G gene in humans [67]. A high degree of gene conversion is expected to result in an increased degree of sequence similarity through concerted evolution [70, 71], with the undesired outcome of rendering younger divergence times [72]. Our dataset is unfortunately not suited for an in-depth analysis of gene conversion, since it includes well-characterised mRNAs, but also ESTs that may contain exons that originate from different genes and putative mRNAs inferred from genomic sequences. A proper analysis will need good quality genomic sequences with enough sampling of individuals if variations found in the human A3 locus in terms of gene copy number and indels (Additional file 4: Figure S3 and Additional file 6: S4) appear also in other species. Such analyses will need to address gene order within the locus, but also exon-intron order within the genes, and alternative splicing and mRNA read-through. Nevertheless, certain cases can be considered. The confounding role of gene conversion may have disturbed the phylogenetic reconstructions for the A3Z2 genes. In Primates, the A3Z2a, A3Z2c and A3Z2e A3Z2g paralogs have appeared after two duplication events from their common ancestor around 73 Mya (Figure 2). The topology for the A3Z2g subtree matches well the phylogeny of the corresponding species (Additional file 2: Figure S1), and gene conversion may have had a limited impact here. The evolution of the A3Z2a, A3Z2c and A3Z2e genes however cannot be reconstructed with confidence, and appears as a series of small branches with small support values, which could be interpreted as a signature of gene conversion. The same holds true for the rest of the A3Z2 paralogs, A3Z2b/d/f. Finally, a second candidate for gene conversion to have occurred is the A3Z2a/b gene tandem in the cat genome [16].
The AICDA genes show the lowest evolutionary rate among the five clades studied, 1.11 10-3 substitutions per site per My, significantly different from the values inferred for the three A3 genes, which are around three times higher (Table 2). Very interestingly, we have found that these evolutionary rates have decreased for the A3 genes in the last 100 My, whereas for the AICDA and the A1 genes there are no significant variations in the evolutionary rate with time (Table 2). We interpret the different outcome for the A3 genes and for their sister taxa as an evidence for our results being genuine rather than an artefact from the evolutionary inference. The simultaneous identification of positions evolving under positive selection in the three A3 genes and the finding of a trend towards decrease with time of the evolutionary rate in the same genes are not contradictory. Our calculations for Ka/Ks have been performed separately for each position, while the values inferred for the evolutionary rates refer to the corresponding nodes in the phylogenetic reconstruction. In our scenario of gene duplication and gene family expansion, we interpret that the episodes of gene duplication have lowered the restrictions on the duplicated copies of the genes, thus allowing for increased evolutionary rates. Subfunctionalisation of the sister copies may have yielded one population of conserved sites evolving under purifying pressures (e.g. those sites that are indispensable for the deaminase activity), one population of sites positively selected sites (e.g. those that are responsible for the differential substrate recognition) and a third large majority of sites evolving close to neutrality. The decrease in evolutionary rate possibly reflects a plateau in the fixation of the novel function. In the A3Z2 genes, which have undergone the largest number of duplication events, the large variation in evolutionary rate for the different nodes (Table 2) and the lower proportion of the decrease in evolutionary rate that is explained by the independent variable time alone (less than 15%; Figure 6b) supports further the idea that gene duplication fosters a transient increase in evolutionary rate.
We have not been able to identify in the databases sequences that could be orthologs of the A3 genes in Marsupials or in Monotremes. We have dated the MRCA of the A3 genes around 246 Mya (95% HPD 192–351 Mya), and the second split that generated the MRCA of A3Z1 and A3Z2 around 199 Mya (95% HPD 164–250). The timing for the crown clade of mammals (i.e. the split between Monotremata and Theria) as well as the timing for the split between marsupials and placentals are controversial, ranging between 160 and 240 Mya [55, 56, 57]. Three explanations could thence account for the absence of extant A3 genes in monotremes and marsupials: i) we simply lack information and further sequencing will provide us with the missing genes; ii) members of one or of both groups may indeed have lost the A3 genes; and iii) genes may be exclusive to placentals if the MRCA of placental mammals predates the appearance of the MRCA of all A3s. Our interpretation implies in any case that the genome of the placental ancestor already encoded for an A3 locus with the arrangement A3Z1-A3Z2-A3Z3. The absence of A3 genes in Xenarthra, the fourth large clade within placental mammals, must therefore imply either gene loss or incomplete coverage of the A3 locus in the two species analysed. Since we have described that loss of certain A3 genes has occurred in parallel in different lineages, as in Rodents and in Artyodactyla, it is conceivable that in certain lineages the loss of all A3 genes may have been selected. The adaptive value of an enlarged armoury against viruses is obvious, and evidences supporting positive selection of the A3 genes in different branches of the mammalian tree are strong. The intriguing hypothesis of the total local loss of A3 genes might imply that the constraints and pressures imposed by viral infections can largely vary among different taxa. Certain host lineages may thus either be less exposed to (certain) viruses, and/or may have evolved alternative antiviral strategies. An exciting question that arises from our dating results is the relative coincidence in time between speciation events and gene duplication events, as exemplified in Figures 2 and 3. It could be speculated that gene duplication of the A3 genes may trigger speciation, possibly through the differential fitness against viral infections that the additional A3 gene copy provides. Further genetic and functional research will be required to elucidate the fitness landscapes integrating viral pressures, expansion of the A3 repertoire and concomitant risks for the own genetic information.
The A3 gene family appeared together with the ancestral mammals. Independently in certain lineages, the A3 locus was expanded through a series of tandem duplications, best exemplified in Primates. The repertoire of A3 proteins is additionally expanded through splice alternatives and read-through mechanisms, resulting in broader substrate specificity and finer regulation of DNA modification potential. We have shown that this diversity has been generated by series of tandem duplication in the A3 locus probably followed by positive selection and/or relaxation of constraints and resulting in sub/neo-functionalisation. Such evolutionary solution has been independently selected in several lineages: Primates, felids and equids. Our findings constitute a paradigm of genomic parallel evolutionary solutions in the framework of the arms race between viruses and their hosts.
Annotated sequences were retrieved from GenBank and Ensembl Genome Browser, the data set was completed with cDNA sequences and with genomic sequences putatively encoding for products similar to A3 after iterative BLAT, tBLASTn and PSI-BLAST searches. Our final dataset included sequences from within Laurasiatheria (seven Carnivora, seven Cetartiodactyla, one Perissodactyla and one Chiroptera species), Euarchontoglires (three Rodentia and seventeen Primates species) and Afrotheria (one Hyracoidea and one Proboscidea species). Fused genes as in human A3Z2a-A3Z1b were split and analysed as two sequences.
A selection of 24 APOBEC1 (A1) sequences (21 Eutheria species, two Metatheria and one Aves species) and 34 Activation induced cytidine deaminases (AICDA) sequences (23 Eutheria species, three Metatheria, one Prototheria, two Aves, two Amphibia and three Actinopterygii species) and were used as outgroups. The final sequence set comprised 202 sequences. A list of species and accession numbers is given in Additional file 1: Table S1.
Phylogenetic inference
Sequences were aligned with mafft (http://mafft.cbrc.jp/alignment/software/) at the amino acid level and back-translated into nucleotides for phylogenetic analyses. This matrix is available as Additional file 7 and from IGB on request. Maximum likelihood (ML) inference was performed with raxml_v7.2.8 (http://www.exelixis-lab.org/) [73] using 5000 bootstrap cycles with the GTR + G4 model and introducing three partitions, one per codon position (number of patterns 290, 290 and 298, respectively) and allowing for different total tree length for each partition. Bayesian inference was performed with phylobayes v3.3 (http://www.phylobayes.org) [74] using the same dataset, under the GTR model, removing constant sites, running two independent chains and checking for convergence comparing discrepancies among partitions. Trees obtained after ML and Bayesian reconstructions were compared regarding topological congruence as well as pairwise distances using K-TreeDist [75], with the following result: Robinson-Foulds distance, 67/401 (16.7%); K-score, 1.07; scale factor, 0.542. Both ML and Bayesian topologies rendered A3Z1, A3Z2 and A3Z3 as monophyletic, and A1 as the outgroup to A3s and AICDA. Twenty-five local topologies, highly supported in both analyses (above 90% bootstrap support and above 0.98 Bayesian posterior probability) and consistent with the phylogenetic relationships of the hosts were identified and used for molecular dating, using truncated uniform priors based on fossil calibration dates, as suggested by Benton and Donaghue [56, 76]. Calibration dates are listed in Additional file 5: Table S2. All discussion on the results obtained refers to the dates and analyses proposed by TimeTree (http://www.timetree.org/index.php) [77]. The backbone tree for time inference was constructed with RAxML v7.2.8 with the same settings as described above, further forcing monophyly for each of the nodes used for calibration (−g option), plus for the sequences belonging to the crowngroups Afrotheria, Laurasiatheria and Euarchontoglires within each of the clades A1, AICDA, A3Z1, A3Z2 and A3Z3, respectively. This tree was not significantly worse than the original one, under the maximum-likelihood framework, as evaluated with a Shimodaira-Hasegawa test [78] implemented in RAxML v7.2.8. The comparison between the unconstrained and the constrained maximum likelihood trees rendered the following values: Robinson-Foulds distance, 71/401 (17.7%); K-score; 0.780; scale factor, 0.823. Using this tree, Bayesian time inference was performed with phylobayes v3.3 using a discrete gamma distribution with eight categories under the GTR matrix of exchange rates, using a log-normal autocorrelated relaxed clock together with a uniform prior on divergence times. A gamma prior of mean 550 and standard deviation 200 Mya was specified for the age of the root. The results from three 50-million steps independent chains were combined and analysed.
Positive selection analysis
Evidence for positive selection was analysed in a Bayesian framework using selecton v2.4 (http://selecton.tau.ac.il/) and datamonkey (http://www.datamonkey.org/). The alignments for the A1, AICDA, A3Z1, A3Z2and A3Z3 genes were analysed separately for the whole sequence set, and those for A3Z1, A3Z2 and A3Z3 were additionally analysed after excluding the Primates sequences, to discard that the results were driven by the behaviour of sequences from this clade. With selectonv2.4, the Mechanistic Empirical Model (MEC) was tested against the M8a model [79]. In both cases the topology of the best-known maximum likelihood tree previously obtained was used as scaffold for the calculation of synonymous Ks and non-synonymous Ka mutations. Briefly, MEC expands an empirical amino acid replacement matrix –in our case the WAG matrix [80], the best scoring one as determined by ProtTest [81] among those available in the selecton algorithm- into a codon replacement matrix. This way, the chemical similarity between amino acids is incorporated when calculating non-synonymous substitutions [79]. The likelihood of this model was tested against the M8a model, which does not allow for positive selection, using the Akaike information criterion [82]. For all of the sequence sets the MEC model was preferred over the M8a. For each position, the value for Ka/Ks was calculated. The variation of Ka/Ks along the corresponding sequence was analysed using a slide-window analysis of width five and step one. With datamonkey, the Random Effects Likelihood (REL) method was applied, which assumes an underlying nucleotide substitution model and allows for rate variation in both Ka and Ks substitution rates [83]. All analyses were repeated after excluding the Primates sequences from the dataset, in order to exclude putative biases inherent to the overrepresentation of this clade in terms of sequence composition and presence of the different genes in the genome. Statistic analyses were performed with R v1.40 and with JMP v7.0.2.
We thank Dieter Häussinger for continuous support. The authors thank Francisco Codoner for technical help with initial sequence retrieval. This work was supported by the disappeared Spanish Ministry for Science and Innovation (MICINN) [Programa Ramón y Cajal, BFU2009-06702-E/BMC and CGL2010-16713]; the Generalitat Valenciana [FPA/2011/002]; and the Heinz-Ansmann Foundation for AIDS Research.
Supplementary material
Copyright information
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|Chris Fraschetti <frasche...@gmail.com>
|deleting with an open writer.
|Mon, 08 Nov 2004 04:36:55 GMT
part of my indexing process is determining if an older instance of the current document bring indexed exists.. and if it does, delete it. which required me to have an IndexWriter open .. and then an IndexReader... when I call the delete of a document, I of course get an io exception caused by the lucene lock. Is there any way around this? I've tried to simply close the writer, delete, and then reopen, but that seems to not have much of an affect. anyone have any luck with deleting and writing at the same time? -- ___________________________________________________ Chris Fraschetti e firstname.lastname@example.com --------------------------------------------------------------------- To unsubscribe, e-mail: email@example.com For additional commands, e-mail: email@example.com
|View raw message

subset: commoncrawl


text: Medgenics, Inc. (NYSE MKT: MDGN)(AIM: MEDU)(AIM: MEDG) (the "Company"), developer of the novel Biopump™ technology for the sustained production and delivery of therapeutic proteins in patients using their own tissue, today announced receipt of clearance from the U.S. Food and Drug Administration ("FDA" or the "Agency") to proceed with a Phase IIb clinical trial of EPODURE™ for the treatment of anemia in patients undergoing renal dialysis. EPODURE is intended to produce and deliver erythropoietin ("EPO") on a sustained basis. This multi-center, randomized clinical trial will be the first study in the U.S. using the Company's proprietary Biopump™ technology, and will evaluate the safety and efficacy of sustained EPO therapy delivered via Medgenics' EPODURE Biopump. Treatment of anemia in dialysis patients with EPODURE is intended to replace frequent injections of EPO or other erythropoietic stimulating agents ("ESAs"). "This timely acceptance of our first Investigational New Drug ("IND") to the FDA marks a significant milestone for our Biopump protein platform. We are very pleased with our U.S. regulatory progress and with the interactions with the FDA, allowing us to proceed with the launch of our first U.S. trial," stated Andrew L. Pearlman, Ph.D., Chief Executive Officer of Medgenics. "This trial is significant for Medgenics as a company as well as for patients with anemia, as we anticipate EPODURE could provide a better treatment for anemia in the future." "We are hopeful that the positive interaction with the FDA for EPODURE bodes well for the Agency's approach to other future indications for our Biopump protein delivery platform technology," added Dr. Pearlman. The FDA cleared the Phase IIb study protocol based on results from the Company's prior Phase I/II anemia study of EPODURE in Israel, which reported safety and maintenance of hemoglobin for months from a single EPODURE treatment in pre-dialysis patients without requiring injections of EPO or ESAs, as well as on the complete preclinical package. The Company was particularly encouraged that clearance was received within the 30-day required regulatory response period from submission of the IND application and without major issues being raised. Medgenics views this as an early, second validation from an independent critical review in the U.S. of its Biopump platform. The Company announced in March that the National Institutes of Health's Recombinant DNA Advisory Committee had given positive responses to the Company's prior scientific and clinical data and its proposed Phase IIb study protocol.


text:
The panel included the conductor of the MSO's Beethoven Festival Douglas Boyd, MSO Concertmaster Wilma Smith, noted Melbourne music lovers and expert debaters Barry Jones and Julian Burnside, plus comedian and broadcaster Michael Veitch. Hear the passions and opinions fly in this special broadcast - before the concert featuring the Second and Fifth Symphonies.
Production: Alex Stinson


text: My wife and I attended a 7 day Golf school here in November and our stay was just perfect. The facilities of the hotel are wonderful and make fora very relaxing stay ( this was our fourth here). It was my wife's birthday while we were there and she was surprised by a delightful gift from the hotel management. Only... More


text:
From Conflict Actors to Architects of Peace: Promoting Human Security in Colombia and Internationally
In 2016, the Colombian government and the country's largest rebel group, the Revolutionary Armed Forces of Colombia—People's Army (FARC) signed a remarkable peace deal. It ended the longest-running armed conflict in recent global history that left more than 220,000 people dead and about 6.7 million displaced. Achieving sustainable peace, however, remains a major challenge. The ELN, the country's second largest guerrilla group, continues its fight against the government. Multiple other violent non-state groups operate in Colombia as well, many of them involved in the illicit drug trade and other forms of transnational organised crime. The cross-border effects of the unstable situation in neighbouring Venezuela further contributes to the complexities of a successful transition from war to peace.
Other countries around the world have been facing similar security challenges: post-conflict Central American states for example are now plagued by disputes and alliances among gangs, and countries such as Iraq and Sudan have proved that the security threats linked to the presence of violent non-state groups during armed conflict persist or increase in its aftermath.
Understanding Security Challenges in Transitions from War to Peace
Against this backdrop, this project explores the changes in the security landscape that come along with transitions from war to peace. In particular, it examines:
(i) how the reshuffling – rather than the disappearance – of violent non-state groups that occurs during such transitions matters for questions related to conflict, security, order and (non-state) governance;
(ii) the implications for ethics and norms in contexts where the line between armed conflict and organised crime is increasingly blurred;
(iii) how historical turning points such as the end of Colombia's armed conflict with the FARC are relevant for, and influenced by, broader geopolitical shifts and the world's evolving security landscape.
Rethinking Colombia's Security Architecture
In addition to academic research on these themes, the project explores how Colombia's security architecture needs to be adapted to these changes in order to adequately anticipate and respond to them with a view to promoting human security in Colombia and internationally. For this end, the project establishes a collaborative partnership between the Strategic Analysis Centre of the Colombian Army (CAEEF) and the University of Oxford's Changing Character of War (CCW) Programme. Drawing on a wide range of different perspectives including marginalised communities, indigenous, Afro-Colombian and other civil society leaders, guerrillas, ex-combatants, displaced people, military and police officials, government representatives and NGO as well as UN staff, the project involves applied research and knowledge exchange activities to critically rethink the Colombian Army's transformation process and provide locally grounded advice to senior stakeholders in security and defence.
Funder: The Foreign and Commonwealth Office, UK
Principal Investigator: Dr Annette Idler
Advisory Board: Lord John Alderdice, Sir General David Capewell, Prof Laurence Whitehead, Prof Eduardo Posada-Carbo, Malcolm Deas and Joaquin Villalobos


text:
Search for guests or keywords:
A window into health in California.
« Full episode for
Tue, Sep 4, 2012
There's a growing scientific consensus that heat waves are becoming longer and hotter, and they're hitting more frequently. State officials are talking about how to respond. A plan from the state Environmental Protection Agency includes recommendations to plant more trees in cities and protect key parts of the power grid from overload. Reporter: Molly Samuel
Never miss an episode of The California Report.
The California Report is a production of KQED Public Radio.
Generous support for The California Report is provided by:
Eric and Wendy Schmidt |
The James Irvine Foundation |
Barracuda Networks |
The California Endowment |
California HealthCare Foundation |
Bracamontes & Vlasak, P.C. |
Blach Construction |
The Westly Foundation |
Digital Foundry


text:
RingyDingyDingy is a lightweight app which lets you send a text message to your phone (or your Google Voice number, for phones and tablets) to make it ring (disabling silent/vibrate if on, and restoring them when ringing has stopped) and lock (on Froyo or newer).
A tool for securing DNS communications between a client and a DNS resolver.
A polyhedral extraction tool (PET).


text:
To call for technical assistance or product support in the United States or Canada, browse to the following Microsoft Web site to locate the phone number:
More Information
Contact information and hours of operation
- To contact Microsoft Customer Service and Support, telephone (800) MICROSOFT (642-7676).
- In Canada, call (877)568-2495.
- Microsoft Customer Service hours of operation:
- Monday through Sunday, 5:00 AM - 9:00 PM Pacific Time
Article ID: 319726 - Last Review: May 31, 2012 - Revision: 1
Customer Service and Support Information


text:
Parents' opt-out vote at risk
A consultation paper, due to be issued soon, will seek opinions on how to fulfil the Prime Minister pledge to expand the grant-maintained sector. While the document is understood to make no reference to enforced opting out it will invite views on options, including a "fast track" for voluntary-aided schools, allowing them to become GM without balloting parents.
This option, revealed in Mr Major's speech to GM heads in Birmingham last month, has been opposed by church leaders, who believe that singling out their schools would be divisive.
Ministers are now thought to be examining the possibility of a "fast track" for all state schools. The Government plans to introduce legislation, following consultation, in the next Parliamentary session.
Robin Squire, the education junior minister, hinted heavily to GM and voluntary-aided schools last week that a fast track for all schools was on the agenda.
"Our minds are not closed to arguments for creating easier access to self-government for other types of schools," he told the Association of Grant-Maintained and Aided Schools.
Any move to scrap parental ballots would be politically risky for a Government which has set great store in promoting parental choice. In his Birmingham speech Mr Major suggested the decision on whether to go GM could be taken solely by governors.
One way of meeting objections about depriving parents of a vote could be to allow them to overturn the decision in a "reverse" ballot.
Such an idea has long been promoted by Sir Robert Balchin, chairman of the Grant-Maintained Schools Foundation and a key player in the Government's latest GM drive. He said this week: "I would welcome legislation which would eventually give all schools the opportunity to be self-governing and all children the benefits that come from GM status."
Senior Conservative strategists fear that Anglican bishops and Roman Catholic peers may combine to wreck legislation when it reaches the House of Lords, if it singles out church schools.
The churches in the main have been opposed or neutral to their schools becoming GM and many Catholics bishops have taken the moral stance that opting out takes resources away from other schools. However, at least a fifth of the 1,000-plus GM schools are church schools.
Last week Bishop David Konstant, chair of the Catholic Education Service and Bishop David Young, chair of the Church of England board of education met Gillian Shephard, the Education and Employment Secretary, to discuss the fast track.
The bishops are not commenting on their meeting but a spokeswoman for the Catholic Education Service said: "We regard aided schools as an integral part of the system and we are concerned that any fast track which was offered just to church schools would be divisive.
"We have made the point all along if GM is such a good thing why don't all schools become GM."
Echoing remarks made by the Prime Minister, Mr Squire said he wanted all maintained schools to opt out and added: "We are looking at how this might best be done.
"Giving aided schools easier access to self-government would be an important step, but further initiatives will be required to achieve our overall aim.
"We must also look carefully at the implications of any large-scale changes, which means moving forward at a measured pace."
His comments came just days before a report in Monday's Times which said that ministers were considering a far more radical plan to take control of school funding away from education authorities.
Downing Street has refused to comment.


text:
West Paw Design Bumper Bed® with Organic Cotton, Basil
West Paw Design
12 in stock
West Paw Design
Bumper dog beds with an eco-friendly organic cotton outer fabric are filled with thick denier 100% recycled IntelliLoft® polyfill made from recycled plastic. Cushions do not bunch or flatten from extreme use. Floor-safe zippered opening so it won't scratch floors. Handcrafted in the USA; machine washable cover for easy maintenance.
You and your dog can rest easy knowing that this pet product was Certified Safe by Oeko-Tex, the same third-party certifier that deems baby carriers and children's clothes ecologically safe.
Organic cotton is grown without the use of toxic and persistent pesticides and synthetic fertilizers. Since there are no chemical fertilizers or synthetic pesticides used with organic cotton farming, there's less soil and water contamination. Growing organic cotton requires more time and a different set of skills than conventional cotton, but it has a lower impact on the environment. And that feels good to us (and your pet).
Our dog beds are so comfy your pooch will be happily sleeping through everything (so much for thinking you had a guard dog). If you (or your dog) aren't totally satisfied, we'll make it right - Guaranteed.
Handcrafted in USA - Each dog bed is sewn by hand, with care and craftsmanship in Bozeman, Montana.
Wash and Dry: These dog beds may feature luxurious, silky-soft fabrics (like Organic Cotton and Hemp) but they don't need special care—they're all easily refreshed with a single wash and dry.
Available Sizes: Small 11-20 lbs (23" x 20"), Medium 21-40 lbs (31" x 26"), Large 41-60 lbs (36" x 28"), X-Large 61-80 lbs ("42 x 34"), XX-Large 81+ lbs (48" x 39")
Returns Policy
If you are not delighted with your purchase, Canine Supply Co. offers the following option for returns: simply return the item in its original condition within 15 days for a full refund, less any original shipping costs if applicable. Customer is responsible for return shipping fees.
Please allow up to two billing cycles for refunds to appear on your credit card statement. This time period includes the transit time for us to receive your return from the shipper (5 to 10 business days), the time it takes us to process your return once we receive it (3 to 5 business days), and the time it takes your bank to process our refund request (5 to 10 business days).
If you need to return an item, simply complete the return form included with your order and email us at firstname.lastname@example.com for a Return Authorization Number (RA#) so we may expedite processing. We will then notify you via e-mail of your refund once we've received and processed the returned item.
We currently offer complimentary Standard Shipping, on all orders in the Continental USA. Extra fees do apply for shipments to Alaska and Hawaii. Please note that there are restrictions on some products, and we currently do not ship outside the USA.
Orders placed Monday-Friday before 12PM ET will begin processing that day (excluding holidays). Orders placed after 12PM ET will begin processing the next business day (excluding holidays). Orders placed after 12PM ET on Friday will begin processing the following Monday (excluding holidays).
For more detailed Shipping & Returns Policy information, please refer to links below Customer Service at bottom of page.


text:
Brow Serum Plus – The wonderful solution to have the eyebrows of your dreams!
Why are eyebrows important? Beyond their basic function as filter of sweat, dirt, and other particles that might get into your eyes, eyebrows are your most significant facial features. Eyebrows play huge roles in facial recognition and emotional expressions. Eyebrows make your face standout among others! You may have the perfect nose, eyes, and lips, but without the perfect eyebrows, your beauty won't make lasting impression. Hence, healthy and beautiful eyebrows are important elements of a beautiful face. However, it is a sad fact that not all of us are endowed with lush and beautiful eyebrows at birth. But, with Brow Serum Plus everyone can have amazing eye brows!
What makes Brow Serum Plus so amazing?
Brow Serum Plus is a powerful combination of effective natural oils and herbs that can give you beautiful and healthy brows. The serum's rich natural oils effectively moisturize and rejuvenate your eye brows. The serum aids brittleness of the brows making it softer like that of a baby. The best thing about Brow Serum Plus is that the effect is permanent. The powerful formula moisturizes the follicles of your eyebrows to keep them stronger and longer.
Brow Serum Plus is made from 100% natural herbs and oils so you won't have to worry about negative side effects that you can get from other brow enhancing products like:
- Discoloration of eyebrows
- Darkening of the skin underneath the eyebrows
- Skin Irritation
- Hair Loss
What can Brow Serum Plus do for you?
- Gives you fuller-looking and healthy eyebrows – Brow Serum Plus contains natural herb extracts that are proven effective in preventing and aiding hair loss. Hence, it provides the needed nutrients for healthy and fuller-looking eyebrows.
- Prevents breakage – The natural oils of this serum moisturize and nourish your follicles to treat brittleness. This helps prevent breakage resulting to stronger and longer brows.
- Conditions the skin underneath the eyebrows. – Brow Serum Plus does not only protect and moisturize your eyebrows but as well as the skin underneath. It contains natural moisturizers and antioxidants to promote healthy skin.
- Makes eyes more expressive – With healthy eye brows, your eyes can be more expressive.
- Boosts your self-confidence – Brow Serum Plus can make you look better. If you look better, you will definitely feel better about yourself!
Claim your trial bottle of Brow Serum Plus by clicking the link below!
Eyebrows are important beauty factors. This is evident among models, superstars, and other women who are famous for their extraordinary beauty. Researchers even found out that men are more attracted to women with beautiful eyebrows than to those with beautiful eyes. Hence, beautiful eyebrows can make a difference. Gone are the days where your eyebrows are just filters of dirt and dust. Today, your eyebrows can give you the beauty that can catch everyone's eyes! Hence, don't be contented with your unpleasant eyebrows. Level up your beauty with Brow Serum Plus. Click here and start your eyebrow transformation today!


text:
Listen to iFriendly audio.
Native Jazz Quartet pianist Reuel Lubag and percussionist Ed Littlefield will perform a CD release concert (7:30 PM Wed Jul 17, Odess Theater, $15 at the door) with fellow musicians Christian Fabian on bass, and Jason Marsalis on vibes. The concert is a part of this year's Native Jazz Workshop happening now at the Sitka Fine Arts Camp.


text:
Those are the magic numbers Kevin Lashus, the Managing Partner of the Austin, Texas office of Jackson Lewis LLP, says employers need to know when processing Forms I-9.
In this interview, he also discusses employer responses to DREAM Act-lite, the Obama Administration's recent action to allow young undocumented immigrants an opportunity to avoid deportation and obtain work permits.
Kevin will be hosting the Austin Human Resource Management Association's September 20th Legal and Regulatory Roundtable.
Also, I'd be remiss if I didn't mention that Imperative Information Group offers an electronic Form I-9 and E-Verify enhancement product that can take away some of the headaches Kevin mentions during the interview.
The 10 Commandments of Employment Verification (Form I-9) Compliance from Imperative Information Group


text:
Great Forest Blue Floral Nature Modern Formal Area Rug
$ 74.99
$ 39.99
- Plush 0.4" pile height. 100% polypropylene power loomed in Turkey. Stain resistant and fade resistant. 100% jute backing is safe for wood floors. Extremely durable and very easy to clean, made to last for years.
- Elegant floral nature pattern paired with modern, geometric boxes give a contemporary, transitional feel.
- Color: Light Blue, beige, red, and green.
- Luxury, handmade feel at machine made prices.
This collection is fresh and modern at is finest. Contemporary, geometric designs are paired with warm, light blue, green, and blue jewel tones are accented by neutral tones of ivory and beige. The heat set polypropylene pile is a plush 0.4" that is soft, stain resistant, and doesn't shed or fade over time. The 100% jute backing is safe for wood floors and all four sides of the rug are serged for improved durability. A wide range of shapes, sizes, and designs means this collection has something for rooms of every size and style.


text:
Bloomingdale, GA -- (ReleaseWire) -- 07/17/2006 --GetYourAssistant.com, a new virtual assistant directory, invites all virtual assistants to boost their income, not their hours. Virtual assistants can do so by taking advantage of a free three-month trial membership listing in this VA directory located at http://www.getyourassistant.com.
GetYourAssistant.com is owned by the publisher of The Effective Admin, a successfully established two-year old website for administrative professionals who work on-site for employers. GetYourAssistant.com will be the one-stop shopping source for small business owners and home based business owners that are searching for off-site administrative support help -- a virtual assistant.
By joining this VA directory virtual assistants benefit by reaching a larger target of prospective clients. VAs who currently don't have a big pool of clients work 24/7 because they can't afford to pick and choose their clients, or even say "no" sometimes to new clients who don't match their skills or job rates. GetYourAssistant.com markets globally to encourage small business owners and home based business owners to visit its site repeatedly to fill its virtual assistant needs. This extensive marketing is something the individual virtual assistant cannot do alone on a small business budget. By joining GetYourAssistant.com virtual assistants impressively broaden their business' marketing efforts and marketing budget for a very low cost.
With the increasing popularity of the VA industry as a viable home-business, it is more important than ever that virtual assistants market their services in smart ways. The competition is growing in the VA industry. It's not enough for even the most experienced or talented virtual assistant to just "set up shop" and wait for more business (or any business) to arrive. Virtual assistants must be proactive with marketing endeavors and choose wise marketing outlets with their marketing budgets. And they must market their businesses. Period.
Virtual assistants can learn more about the free three-month trial membership listing in this VA directory by visiting http://www.getyourassistant.com/more-business.htm.
Contact: Karen Fritscher-Porter is the publisher of GetYourAssistant.com, a global directory of virtual assistants available to small business owners and home based business owners, located at http://www.getyourassistant.com.


text:
When applying a NAT rule, it stops our internet traffic and doesn't work
Hi all, In our HQ we have a WAN 2800 router, its LAN Eth0 address is 220.127.116.11 which is the gateway to all remote offices. Also HQ we have a firewall connected to the internet, its LAN Eth0 address is 18.104.22.168. All users on the HQ LAN have their Gateway pointing to he WAN 2800 188.8.131.52. I need to allow all users on the LAN and WAN to use an internet service on TCP port 5631. I need to forward a TCP port 5631 from the WAN 2800 Router 126.96.36.199 to re-direct to the firewall 126.96.36.199. When I applied a NAT rule on the 2800 it completed stops internet traffic, all help will be appreciated.
Kind Regards,


text:
Becky Brisco Leather Moleskin Journal Cover
Becky Brisco Leather Moleskin Journal Cover
This journal cover is made from Italian leather. It fits over any Moleskine pocket sized note book sized 3.5" x 5.5". It fastens with a wrap-around leather cord. A pen can slide through a loop created on the outer front cover that is part of the wrap-around cord. Comes with Moleskin notebook.
100% Leather.
The Boutique
A Size Too Small
Port Coquitlam, BC
Developed by two friends who shared the same idea of how they wanted their kids to dress. Adult style clothing for small humans.
Do you have questions about this product? We're here to help!


text:
Fondant Vs Gumpaste
Decorating By Smckchat Updated 26 Oct 2007 , 8:53pm by JoAnnB
When making figurines do you prefer Fondant or Gumpaste and why. I see flowers being done in gumpaste but more of the figurines done in fondant.
Thanks for any replies!
Gumpaste is more often used for flowers, it can roll thinner and dry harder and faster than fondant.
Fondant allows more modeling time and doesn't dry as hard and fast.
50/50 gp and fondant is often used for a little of both, dries a little faster and a little firmer.
Quote by @%username% on %date%


text:
Do you want to make Ohio.com your homepage?
Vettel wins F1 title again; Button takes Brazil GP
Red Bull driver Sebastian Vettel (center) of Germany, celebrates with members of his team after the Formula One Brazilian Grand Prix at the Interlagos race track in Sao Paulo, Brazil, Sunday. Vettel overcame a first-lap crash to clinch his third straight Formula One championship title on Sunday, finishing sixth in an incident-filled Brazilian Grand Prix won by Jenson Button under pouring rain. (AP Photo/Andre Penner)
Read the story
More Photo Galleries


text:
Report: Honda Boss Says Acura Doesn't Need a Conventional Rear-Drive, V8 Sedan
Acura will not introduce a rear-drive, V8-powered luxury sedan as the automaker doesn't need one. At least that's what Honda president Takanobu Ito thnks.
Ito told Automotive News that, "I don't think we need a classic front-engine, rear-drive car." Instead Ito believes Honda should chart its own course for Acura, which includes more fuel-efficient models, including hybrids. Acura's use of all-wheel drive, however, seems somewhat contrary to its fuel-economy goals.
Ito did say that Acura has been experimenting with hybrid technology on mid- to large-size vehicles, but did not say when we might see a production model
Traditional German luxury automakers like BMW and Mercedes set the benchmark for luxury sedans with front-engine, rear-drive vehicles like the 7 Series and S-Class and so far the only Japanese luxury automaker to truly establish itself at tier-one has been Lexus, which offers the front-engine, rear-drive LS. That being said, for Acura to establish itself as a top-level contender without such a vehicle seems unlikely.
[Source: Automotive News via LeftlaneNews]


text:
Aiseesoft Mxf Converter v7.2.6 Multilingual (Portable)
Aiseesoft Mxf Converter v7.2.6 Multilingual (Portable) | 62.9 Mb
The professional and flexible MXF/P2 MXF Transcoding software, offers perfect solution to converting MXF video to editable video formats in 2D/3D modes with no quality loss.
Convert MXF to MOV, MP4, AVI and other popular SD and HD video formats
Convert MXF files to Adobe Premiere Pro, iMovie, Final Cut Pro, Sony Vegas Pro and Magix Video Pro for professional editing
Turn 2D MXF video to 3D
Enable you to play recorded MXF video on iPhone 6, iPhone 6 Plus and any other player
Edit the video with built-in video editor
Convert MXF video to SD/HD MOV, MP4, AVI, MKV and other formats
This MXF Video Converter allows you to convert MXF files recorded by professional camcorders to popular video formats without losing the original quality. It can convert MXF to any other popular and multi-compatible video/audio formats and HD video formats, such as H.264/MPEG-4 AVC, WMV HD Video Files, HD TS, HD MTS, MOV, MP4, 3GP, AVI, MKV, WMV, FLV, SWF, VOB, DV, and MPG, etc. By adjusting the parameter settings, you are allowed to retain the original quality to the largest extent.
Convert MXF video to editable and compatible videos
It offers the best MXF converting solution for users of Panasonic HC-V720, Sony HDR PJ, Sony XDCAM, Canon Vixia HF R or any other camcorders that shoot videos in MXF format. After being converted, the recorded MXF video can be played on any possible player, including the latest iPhone 6/6 Plus, iPhone 5s/5c, iPad mini 2 and iPad Air etc. Also, it is possible if you want to edit the recorded video with popular video editing software, such as Adobe Premiere Pro, Final Cut Pro, Sony Vegas Pro and Magix Video Pro etc.
Edit and convert MXF files
It combines the conversion and editing functions together. With the multiple editing functions, this MXF Converter enables you to crop, watermark, and merge MXF recordings. You can crop the video frame to remove the blank edges, add text/image watermark and define the text color, and merge several videos into one file. Also you can adjust the video brightness, saturation, contrast, hue, and volume. What's more, you can use the 3D conversion function to turn the 2D MXF video to 3D.
Marvelous using experience
Aiseesoft MXF Converter can convert MXF files to different output video formats simultaneously, and can also convert different source file formats within only one click. With the intuitive and elegant interface, you can easily finish the converting process in just a few clicks, and the supported NVIDIA® CUDA™ and AMD APP acceleration technology completely supports multi-core processor.
What's New: official site no says about updates
All Link Download :
Aiseesoft Mxf Converter v7.2.6 Multilingual (Portable)
Feel free to post your Aiseesoft Mxf Converter v7.2.6 Multilingual (Portable) Download, torrent, subtitles, free download, quality, NFO, Uploaded.net, ul.to, FileJoker, Rapidgator, Nitroflare, Alfafile, Turbobit, Keep2Share, Mediafire, Watch HD Movies Stream Online, free premium downloads movie, game, mp3 download, crack, serial, keygen, or whatever-related comments here. use only English, Owners of this website aren't responsible for content of comments.


text:
Sweet Nothings is a brand of candy in Grand Theft Auto IV and Grand Theft Auto V.
Sweet Nothings can be seen in some vending machines and are sold in many 24-7, Limited Service and Rob's Liquor stores, but it cannot be bought by the player. Its name and the fact that some boxes have "nothing but sugar" on them could mean these sweets are really just flavourless hard sugar lumps.
They are clearly based on UK-made Love Hearts and US-made Sweethearts.


text:
Modified: logging/log4php/branches/experimental/config-adapters/src/site/xdoc/docs/appenders/console.xml URL: http://svn.apache.org/viewvc/logging/log4php/branches/experimental/config-adapters/src/site/xdoc/docs/appenders/console.xml?rev=1182331&r1=1182330&r2=1182331&view=diff ============================================================================== --- logging/log4php/branches/experimental/config-adapters/src/site/xdoc/docs/appenders/console.xml (original) +++ logging/log4php/branches/experimental/config-adapters/src/site/xdoc/docs/appenders/console.xml Wed Oct 12 12:33:23 2011 @@ -25,8 +25,8 @@
LoggerAppenderConsole writes logging events to the stdout (
php://stdout) or
- the stderr (php://stderr) stream, the former being the default target.
LoggerAppenderConsole writes logging events to the
php://stdout or
+ the
php://stderr stream, the former being the default target.
The following options are available:@@ -44,7 +44,7 @@
Apache log4php can be configured either programatically or with a file containing the configuration parameters in one of the supported formats.-
The configuration should be provided by calling
Logger::configure(...) method before any logging
- is done. Otherwise, the default configuration will be used.
If no configuration+
The configuration should be provided by calling
Logger::configure() method before any logging
+ is done. Otherwise, the default configuration will be used.
XML is the most common configuration format, and it is the most prominently featured in the + documentation and examples.+ +
Configuration is stored in a PHP array. This is the format used internally by log4php and other - formats are converted to a PHP array before being used. Because of this, the PHP format should be - used when performance is important.+
Configuration can also be stored in a PHP array. This is the format used internally by log4php. Other + formats are converted to a PHP array before being used. Because of this, the PHP configuration format + should be used when performance is important.
It is possible to pass an configuration array directly to
Hint: to translate a XML or properties configuration file to PHP, run the following code:+ +
The properties configuration format is a legacy method of configuring log4php. It was inherited from
+ Apache log4j and uses the same format. The only
+ difference is that lines begin with
log4php instead of
The properites configuration format does not support filters.+
The following is a high level overview of all options provided by this format:- + +
+# Appender named "default" +log4php.appender.default = LoggerAppenderEcho +log4php.appender.default.layout = LoggerLayoutTTCC + +# Appender named "file" +log4php.appender.file = LoggerAppenderDailyFile +log4php.appender.file.layout = LoggerLayoutPattern +log4php.appender.file.layout.conversionPattern = %d{ISO8601} [%p] %c: %m (at %F line %L)%n +log4php.appender.file.datePattern = Ymd +log4php.appender.file.file = target/examples/daily_%s.log +log4php.appender.file.threshold = warn + +# Root logger, linked to "default" appender +log4php.rootLogger = DEBUG, default + +# Logger named "foo", linked to "default" appender +log4php.logger.foo = warn, default + +# Logger named "foo.bar", linked to "file" appender +log4php.logger.foo.bar = debug, file +log4php.additivity.foo.bar = true + +# Logger named "foo.bar.baz", linked to both "file" and "default" appenders +log4php.logger.foo.bar.baz = trace, default, file +log4php.additivity.foo.bar.baz = false + +# Renderers for Fruit and Beer classes +log4php.renderer.Fruit = FruitRenderer +log4php.renderer.Beer = BeerRenderer + +# Setting base threshold +log4php.threshold = debug +
If no configuration is provided before the initial logging request is issued, log4php will configure
+ using the default configuration. This consists of a single
LoggerAppenderEcho appender,
+ using
LoggerLayoutTTCC, attached to the root logger and set to the DEBUG level.
The default configuration in PHP format is:+ +
+array( + 'rootLogger' => array( + 'appenders' => array('default'), + ), + 'appenders' => array( + 'default' => array( + 'class' => 'LoggerAppenderConsole', + 'layout' => array( + 'class' => 'LoggerLayoutSimple' + ) + ) + ) +) +-
It is possible to configure log4php fully programmatically.+
Filtering is a mechanism which allows the user to configure more precisely which logging events will be + logged by an appender, and which will be ignored.+ +
Multiple filters can be defined on any appender; they will form a filter chain. When a logging event is + passed onto an appender, the event will first pass through the filter chain. Each filter in the chain will + examine the logging event and make a decision to either:+ +
Filters are configurable in the XML and PHP configuration format. They cannot be configured using + the properties configuration format.+ +
Like appenders and layouts, depending on the class used, filters may have configurable parameters + which determine their behaviour.+ +
Here is an XML configuration example:+ +
++ +
++ + + + ++ + + ++ ++ +]]> + +
And the same configuration in PHP format:+ +
array( + 'default' => array( + 'class' => 'LoggerAppenderEcho' + 'layout' => array( + 'class' => 'LoggerLayoutSimple' + ), + 'filters' => array( + array( + 'class' => 'LoggerFilterStringMatch', + 'params' => array( + 'stringToMatch' => 'interesting', + 'acceptOnMatch' => true, + ) + ), + array( + 'class' => 'LoggerFilterLevelRange', + 'params' => array( + 'levelMin' => 'debug', + 'levelMax' => 'error', + ) + ) + ) + ) + ), + 'rootLogger' => array( + 'appenders' => array('default'), + ) +) +]]>+ + +
In this example, there are two filters defined for the default appender.+ +
The first filter
LoggerFilterStringMatch searches for the string "interesting" in the
+ logging event's message. If the string is found, the filter will ACCEPT the logging event, and the
+ event will be logged. If the string is not found, the filter will remain NEUTRAL, and the event will be
+ passed on to the next filter.
The second filter
LoggerFilterLevelRange ACCEPTS all events which have a level between
+ DEBUG and ERROR (in other words, levels DEBUG, INFO, WARN and ERROR). It DENIES all other events.
Therefore, this filter configuration will log events which which have a level between DEBUG and + ERROR, except of theose which have the string "interesting" in the message. Those will be logged + regardless of their level.+ +
The following filters are available in log4php:+ +
|Denies all logging events.+
|Filters based on logging event level.+
|Filters based on logging event level range.+
|Filters by searching for a string in the logging event message.+
This filters simply denies all logging events. It has no configurable parameters.+
This filter either accepts the specified logger level or denies it.+ +
|The level to match+
|If true, the matching log level is accepted, denied otherwise.+
The following filter configuration will deny all logging events with level DEBUG. It will remain + neutral for others.+ +
+ + + +]]>+ +
This filter accepts or denies logging events if their log level is within the specified range.+ +
|The minimum level to log. If set, levels lower than this will be denied.+
|The maximum level to log. If set, levels higher than this will be denied.+
|If true, the matching log level is accepted, denied otherwise.+
The following filter configuration denies levels greater than WARN.+ +
+ + + +]]>+ +
This filter allows or denies logging events if their message contains a given string.+ +
|The level to match+
|The level to match+
|If true, the matching log level is accepted, denied otherwise.+
The following filter configuration denies events which contain the string "not-interesting" in + their message.+ +
+ + + +]]>+ +
Download the latest source package from the download page and unpack it.+ +
The package directory structure is as follows:+ +
+ââââapidocs - API generated documentation +ââââsrc + ââââassembly - Maven assembly configuration + ââââchanges - The change log + ââââexamples - Various usage examples + ââââmain + â ââââphp - The main source code + ââââsite - Web site source + ââââtest - Unit tests ++
Most users will primarily be interested in the source code which is located in
/src/main/php. The contents of this directory may be copied to a directory within your
+ project for easier access.
Apache log4php has it's own PEAR channel.+ +
To install from the PEAR channel, execute the following commands:+ +
+pear channel-discover pear.apache.org/log4php +pear install log4php/Apache_log4php ++ +
Information about your use of this web site is collected using server access logs and a tracking cookie. + The collected information consists of the following:+ +
Part of this information is gathered using a tracking cookie set by the + Google Analytics service + and handled by Google as described in their + privacy policy. + See your browser documentation for instructions on how to disable the cookie if you prefer not to share + this data with Google.+ +
We use the gathered information to help us make our site more useful to visitors and to better understand how + and when our site is used. We do not track or collect personally identifiable information or associate gathered + data with any personally identifying information from other sources.+ +
By using this web site, you consent to the collection of this data in the manner and for the purpose described + above.+
This example covers named loggers, layouts and best practices in object-oriented programming.+ +
Create a configuration file named
log4php.xml with the following content:
++ +
+ ++]]> + + ++ + + + ++ + + ++ + +
The configuration defines two appenders: one writes to the console, and the other to a file.+ +
+ console appender doesn't have a layout defined, so it will revert to default layout
+ (
LoggerLayoutSimple). The
+ file appender uses a different layout
+ (
+ which will result in different formatting of the logging
+ events.
The console appender is linked to the root logger. The file appender is linked to the logger named
Foo, however
Foo also inherits appenders from the root logger (in this case
+ the console appender). This means that logging events sent to the
Foo logger will be
+ logged both to the console and the file.
Consider the following code snippet:+ +
log = Logger::getLogger(__CLASS__); + } + + /** Logger can be used from any member method. */ + public function go() + { + $this->log->info("We have liftoff."); + } +} + +$foo = new Foo(); +$foo->go(); +]]>+ +
This produces the following output in the console:+
INFO - We have liftoff.+ +
And the following in the log file:+
01/06/11 18:43:39,545 [5428] INFO Foo - We have liftoff.+
Note the different layout, this is because LoggerLayoutTTCC was used as layout for the file appender.


text: Tweet of the Moment I just started watching "Breaking Bad." I'm not sure I'll continue to season 2. Disturbing on so many levels. Couldn't watch "Weeds" either. — Write From Karen (@writefromkaren) June 16, 2014 Advertisements Share this:FacebookTwitterPinterestTumblrMoreRedditPrintEmailGoogleLike this:Like Loading... Related Published by Karen Hi! My day job is a CMA in neurosurgery.. I'm a mom to two twenty-something sons. I've been married to the same man for 26 years. I have a lot to say about nothing. Lucky you. View all posts by Karen


text:
|Angry Birds Toons Reviews
Chuck Time · Where's My Crown? · Full Metal Chuck · Another Birthday · Egg Sounds · Pig Talent · Cordon Bleugh · True Blue? · Do As I Say! · Off Duty · Slingshot 101 · Thunder Chuck · Gardening with Terence · Dopeys on a Rope · Trojan Egg · Double Take · Crash Test Piggies · Slappy-Go-Lucky · Sneezy Does It · Run, Chuck, Run! · Hypno Pigs · Eggs' Day Out · Gate Crasher · Hog Roast · The Bird That Cried Pig · Hamshank Redemption
Now the series introduces the Chef Pig, who, like the rest of the Pigs, is dumb. I wonder why the animators decided to put the chef pig in this episode instead of a usual Helmet Pig, which would make more sense. How would cooking skills benefit theivery? This seems like an error on the producer's part.
I'm glad that there is once again some consistency in this TV show. The Blues are more intelligent than they look, since they saw though the disguise right away, but played along to bring the pig into a false sense of security. Also, the Chef Pig is dumb for not taking the eggs while the Blues are distracted. Why send someone as a diversion if they're supposed to get the eggs? Diversions attract attention. The Chef Pig completely destroys the purpose of that distraction by making the distraction get the eggs.
Overall, this episode was again, meh.


text:
Jacob J.wrote a review of Stoic Vaporshell 3L Pant - Men's on October 12, 2014
Basically I feel like a clown wearing bell bottoms, they don't run large in the waist or length, but are way to wide or baggy, the bottom of the pants drag on the ground, maybe if you were going to use rain pants for snowboarding they would be fine but why wouldn't you just buy snowboard pants? I can't understand why you would design rain pants baggier then snow pants from the 90's . Do you understand what I'm saying? In fact all your designers must be idiots or these were sown in sweat shops. I was looking for casual rain pants to walk around in the rain, but frankly I don't think they exist. Keep up the great work! Peace. Im so sick of crappy designs, I'm sure they are water proof but so are trash bags.


text:
A.N.Prahlada Rao: Crossword Centenery-3 Soragavi and Vijayakumar Music MP3
The 60th birthday of A.N.Prahlada Rao, the highest Crossword Writer in India and the Centenery Celeberations of World Crossword celebrated on 7 October ...
Ramakant Gaikwad,Live Concert @ Delhi,Raga Marwa,Bageshri,Pahadi,Bharavi Thumri MP3
Live Concert @ India Habitat Centre,Organised by Vidushi Mr.Shubha Mudgal & Pt.Aneess Pradhan. Ramakant Gaikwad-Classsical Vocal, Raga-Marwa ...
Puttaraj gawai, Pt .Mounesh kumar chavani singing vachanas at Alvas Virasat MP3
He is a disciple f puttaraj gawai. i.e he is a student of puttaraj gawai. He is all india radio 'A' grade artist. he lives at mangalore.
Jeffery Archer's Indian connection MP3
Noted author Jeffery Archer, who is in India for the third time to promote his new novel, talks about India and its importance.
O Perfect Love - Best Wedding Hymn with Lyrics - Visual Worship ! MP3
Very Blessed soothing Wedding Hymn... with beautiful lyrics explaining His Agape Love.. also encourages to live the WORD.. along with listening ! Enjoy & Keep ...
Pi Loon by Azim Sabir MP3
Azim Sabir Singing "pi loon" - Azeem Sabir Singing Karaoke Song.


text:
March 15th, 2013 - By handjobhubster
Content Quality
Overall Points
Reviewed Site Details
35 Videos
(Average Length 18-20 minutes)
Video Formats:
HD Videos:
DRM Protection:
Video Streaming:
35 Picture Sets
(w/ 30-100 Pictures Per Set)
Zip Sets:
High Res Pics:
Download Limit:
The Pricing
$2.99 for 3 days
$19.99 for 30 days
3 Months:
$59.99 for 90 days
Site Pros
- Stellar HD quality
- tons of bonus feeds
- gorgeous models
Site Cons
- no longer updating
- no bonus site access
- a few guys finish themselves off
Our Review of Tits and Tugs
Love big boobs and handjobs? What male on this planet does not, I ask you?
Well, Titsandtugs.com has them both in stunning HD quality. Tits and Tugs features super hot chicks with huge nautural and fake boobs with an immense appetite for cocks and tugging. You immediately feel invaded by vast amount of huge boobage while almost every pair has a huge dick disappearing between them. As a fan of both hand jobs and big massive boobs, we here at handjob hub recommend highly this website. Tits and Tugs is brought to you by good people of Scoreland, the same folks who pioneered the big boob niche with their flagship site Scoreland.com. Unfortunately, your membership to Tits and Tugs does not include access to Scoreland…sigh.
The tour pages of Titsandtugs.com are pretty generous, they offer you free access to pictures (captures from the videos), stories and almost one minute of every video, just to keep you interested in watching further away. Inside the members area, there are 35 movies with each one an average length of 18-25 minutes and 35 matching sets of pictures with each set having around 30-150 pictures. Navigation inside the members area is limiting, with no advanced search features or filtering options. Although with the small amount of content currently inside the members area, this doesn't seem to be that big of a problem.
The content quality is superb. Stunning high definition videos can be streamed or downloaded in a variety of formats (see format options below). With each episode comes a full length video and accompanying photo set. The great part is that the photo sets are not screen grabs, but hi resolution still shots from the episode. The handjob action is also outstanding with great scenarios and well thought our and planned dialogue make these hand-job scenes extremely hot. With an ample supply of hand-job action for us handjob enthusiasts, there is also plenty of tit fucking action too. Massive big boobs babes like Selena Star can be seen getting her massive boobs fucked, sucked and jizz upon by some very large penises. The models are extremely hot and they know what to do with a guy's joystick in order to make it rise up and then blow like a bursting volcano.
The video download speed is fast, and there is no cap on the amount of videos you may download. Whats more, there are also several hundred bonus video feeds available. These feeds are not handjob related, but general hardcore fucking, sucking and the like, so this is an added bonus to the site. On the flip side, the site is no longer updating and seems to be rotating its content which is a slight bummer, however the great quality and hotness of the content makes the lack of content an minor issue, we suggest checking it out if only for the trail or the full one month access option.
Memberships start at $2.99 for a limited 3 day trial. What this means is that you will only be able to view a limited number of hand job episodes for just three days. How many we do not know, but if you like the content and decide to upgrade, simply do nothing and be automatically billed as a full members for only $19.99 per month. This is a good deal considering the small amount of content currently. There is also an offer for paying three months of membership in a one time charge of $59.99, essentially getting one month free. Tugs and Tugs does also have a one month option for $39.99 non recurring. This is a convenient option so you wont have to cancel your membership as its just one payment and you wont be charged again.
If we sum it up, the quality and creativity of the episodes, as well as the hotness of the models on TitsansTugs.com makes up for the lack of updates, and we can safely recommend it to any hand job and big boob enthusiast. Check out the link below to visit tits and tugs.


text:
BAKER — The City Council is urging City Court Judge Kirk Williams to sign a contract with a company to collect debts owed to the court.
The council Tuesday also discussed the possibility of employing that company, Texas-based American Municipal Services, to help the city recover its own outstanding debts.
"As you know, I know every mayor in the state and those involved in the program were positive about it," Councilman Pete Heine said.
However, Mayor Harold Rideau said the city already has a contract with a debt-collection company, but he couldn't remember the particulars and said he will research the matter.
All administrative costs of collecting the outstanding debts would be covered by a 30 percent collection fee paid by the debtor, Amanda Stucker, of AMS, told the council. The fee is set by state law, which also states that parking tickets cannot be included. The company works with cities to lift warrants on debtors if they pay their debts or commit to a payment plan. The program would not cost the city anything and would likely bring in revenue from debts they are unable to collect, Stucker said.
The council voted 4-1 in favor of a resolution encouraging Williams to sign the contract. Charles Vincent cast the lone no vote, saying he wants to talk to Williams personally rather than pressure him with a resolution.
In other business, the council unanimously passed a resolution in support of proposed state constitutional amendments 3 and 10, which are designed to streamline the process of disposing of blighted or abandoned properties. The amendments will go before voters on Nov. 4. Amendment 3 would allow municipalities to hire outside collection agencies to collect delinquent property taxes. Amendment 10 would reduce the length of time local governments have to wait before selling vacant property from 36 to 18 months.
"This would make it easier to get blighted property back into where someone can use it or build on it," Rideau said.


text:
STARmeterSEE RANK
Down 517 this week
Scott Mosier
Scott Mosier was born on March 5, 1971 as Scott A Mosier. He is a producer, known for Clerks (1994), Good Will Hunting (1997) and Dogma (1999). He has been married to Alex Hilebronner since September 1, 2006. See full bio »
More at IMDbPro » Contact Info: View agent, legal and company