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Convergence of retrotransposons in oomycetes and plants
Kirill Ustyantsev ORCID: orcid.org/0000-0003-4346-38681,
Alexandr Blinov1 &
Georgy Smyshlyaev2
Mobile DNA volume 8, Article number: 4 (2017) Cite this article
Retrotransposons comprise a ubiquitous and abundant class of eukaryotic transposable elements. All members of this class rely on reverse transcriptase activity to produce a DNA copy of the element from the RNA template. However, other activities of the retrotransposon-encoded polyprotein may differ between diverse retrotransposons. The polyprotein domains corresponding to each of these activities may have their own evolutionary history independent from that of the reverse transcriptase, thus underlying the modular view on the evolution of retrotransposons. Furthermore, some transposable elements can independently evolve similar domain architectures by acquiring functionally similar but phylogenetically distinct modules. This convergent evolution of retrotransposons may ultimately suggest similar regulatory pathways underlying the lifecycle of the elements.
Here, we provide new examples of the convergent evolution of retrotransposons of species from two unrelated taxa: green plants and parasitic protozoan oomycetes. In the present study we first analyzed the available genomic sequences of oomycete species and characterized two groups of Ty3/Gypsy long terminal repeat retrotransposons, namely Chronos and Archon, and a subgroup of L1 non-long terminal repeat retrotransposons. The results demonstrated that the retroelements from these three groups each have independently acquired plant-related ribonuclease H domains. This process closely resembles the evolution of retrotransposons in the genomes of green plants. In addition, we showed that Chronos elements captured a chromodomain, mimicking the process of chromodomain acquisition by Chromoviruses, another group of Ty3/Gypsy retrotransposons of plants, fungi, and vertebrates.
Repeated and strikingly similar acquisitions of ribonuclease H domains and chromodomains by different retrotransposon groups from unrelated taxa indicate similar selection pressure acting on these elements. Thus, there are some major trends in the evolution of the structural composition of retrotransposons, and characterizing these trends may enhance the current understanding of the retrotransposon life cycle.
Retrotransposons are "copy-and-paste" mobile elements transferred via an RNA intermediate through the process of reverse transcription. Generally, retrotransposons are further subdivided in two major groups: long terminal repeat retrotransposons (LTR-RTs), with their viral descendants (retroviruses), and non-LTR retrotransposons (non-LTR-RTs). The only general structural feature shared between autonomous elements from both groups is the reverse transcriptase (RT) domain, a key enzyme responsible for reverse transcription. In contrast, the set of other encoded activities could largely vary and rely on the life cycle organization and insertion strategy of the retrotransposon [1–3]. Each of these additional domains can have an evolutionary history independent from that of the RT domain. There are multiple examples of independent acquisitions of domains with the same enzymatic activity by the diverse retrotransposons, suggesting the importance of the domain-encoded function for the performance of each element [4–10]. One of these examples is the ribonuclease H (RNH) domain, which has been captured by diverse retrotransposons on different occasions [4–6, 8, 11–14].
RNH activity is required for the removal of an RNA template from a cDNA/RNA hybrid generated during reverse transcription. Retrotransposons rely on either the host genome-encoded RNH enzyme or encode their own RNH domains [4]. For example, non-LTR-RTs often rely on host genome-encoded RNH activity, as the reverse transcription of these transposons occurs directly in the nucleus where the host cellular RNH enzyme is naturally present [4, 15]. Nevertheless, some non-LTR-RTs encode their own RNH. For example, some non-LTR-RTs of oomycetes and plants have acquired RNH closely related to the Archaea-like RNHs (aRNH). Interestingly, these two groups of non-LTR-RTs independently acquired aRNHs [6, 11]. In case of the LTR-RTs, the presence of the element-encoded RNH is obligatory, as reverse transcription occurs in the cytoplasm where no host-encoded enzyme is available [4]. Accordingly, the RNH domain has been detected in all LTR-RTs, and the evolution of the domains follows that of the RT [5]. However, some retroelements, such as retroviruses, have captured additional RNH domains, resulting in a 'dual' RNH [4, 5, 16]. Strikingly similar to retroviruses, the Tat LTR-RTs of green plants have acquired an additional RNH domain, aRNH, indicating structural and functional convergence between plant Tat LTR-RTs and vertebrate retroviruses [5].
In the present study, we mined all aRNH-containing retrotransposons from oomycete genomes and provided new examples of convergence in retrotransposons between plants and oomycetes. We identified and characterized two groups of Ty3/Gypsy LTR-RTs, Chronos and Archon, and a subgroup of L1 non-LTR-RTs in the genomes of oomycetes, which to our knowledge has not previously been described. These retrotransposons captured aRNH in the same manner as plant retrotransposons. In addition, we showed that Chronos LTR-RTs also captured a chromodomain (CHD), resembling the evolution of plant Chromoviruses and Ty1/Copia CoDi-I LTR-RTs from the free-living Stramenopiles Phaeodactylum tricornutum [7, 17–19].
Diversity of aRNH-containing retrotransposons in oomycete genomes
aRNH is a subgroup of the type I RNH, which also includes Fungi/Metazoa-like RNHs (fmRNH) and LTR-RT RNH. While fmRNHs and aRNHs are characterized by the presence of histidine or arginine residues respectively in the active site, LTR-RTs RNHs lack any conserved residues in that position [4, 16]. aRNHs were originally described in the archaeal genomes and were also identified as cellular genes in the genomes of plants and some bacteria [20]. Furthermore, RNH domains that were found in Ty3/Gypsy Tat LTR-RTs and Ta11 L1 non-LTR-RTs of higher plants [12–14] were shown to be phylogenetically related to cellular-like aRNHs [5, 6]. In addition, Kojima and Jurka [11] identified a subgroup of aRNH-containing non-LTR-RTs of the Utopia group in oomycete genomes.
To determine the presence of the aRNH in other retroelements, we screened for aRNH sequences in Repbase Update (RU, v. 20.08), the database of eukaryotic transposable elements [21, 22]. Consistent with previous data, all retrotransposons predicted to have an aRNH domain (see Methods for details) were detected in either the genomes of higher plants or the parasitic protozoans oomycetes. Surprisingly, in addition to the previously described Utopia non-LTR-RTs [11], some oomycete Ty3/Gypsy LTR-RTs and L1 non-LTR-RTs also encode aRNH (for the RU accession numbers see Additional file 1: Table S1).
Since the variability of the oomycete retrotransposons annotated and deposited in RU 20.08 was restricted only to retrotransposons from seven species, of which retrotransposons from only four species contained aRNH (Additional file 1: Table S1), to provide comprehensive insight into the diversity of the identified elements, we further analyzed oomycete genomic sequences for the presence of aRNH-containing retrotransposons. This mining resulted in an overall set of 2899 distinct retrotransposon sequences from 21 out of 25 analyzed oomycete genomes. We initially classified the identified elements into the three groups, Ty3/Gypsy, L1 and Utopia, based on homology to the ORF2 amino acid sequences of aRNH-containing retrotransposons identified in RU. When possible, full-length copies were retrieved as representatives for each genome, and their structure and domain composition were analyzed (Fig. 1a, Additional file 1: Table S2).
Diversity of aRNH-containing retrotransposons in oomycetes. a Schematic structural composition of the elements from the identified groups: ORFs are shown as horizontal ovals (ORFs 1 are shaded); PR – protease; gRH – RNH of Ty3/Gypsy LTR-RTs; aRH – aRNH (in red); IN – integrase; CHD – chromodomain (in blue); EN – apurinic/apyrimidinic endonuclease-like endonuclease, RLE – restriction-like endonuclease; CCHC Zn finger motif indicated as vertical gray line; gray arrows, LTRs – long terminal repeats. b Consensus of Maximum-likelihood and Bayesian trees based on the amino acid sequences of RT domain of LTR-RTs. Approximate likelihood-ratio test (aLRT) statistical support values (unit fractions) are shown at the corresponding nodes of the tree; the values are highlighted in red if the corresponding node was additionally supported by more than 60 of 100 bootstrap replicates. Groups of aRNH-containing retrotransposons of oomycetes and plants are emphasized in bold and highlighted in blue and green, respectively. CHD-containing retrotransposons without aRNH of Chromoviruses (ChromoVir) group are emphasized in bold. On the right from the tree schemes of the consensus structures of the ORF2 of the corresponding groups are shown; cRH – RNH of Tc1/Copia LTR-RTs. The complete Maximum-likelihood and Bayesian phylogenetic trees with accession numbers, the names of the elements, and all the statistical support values are presented in Additional file 2: Figure S1. c Consensus of Maximum-likelihood and Bayesian trees based on the amino acid sequences of RT domain of non-LTR-RTs. Approximate likelihood-ratio test (aLRT) statistical support values (unit fractions) are shown at the corresponding nodes of the tree; the values are highlighted in red if the corresponding node was additionally supported by more than 60 of 100 bootstrap replicates. On the right of the tree, the schemes of the consensus structures of the corresponding groups are shown; RH – RNH domain of non-LTR-RTs. The complete Maximum-likelihood and Bayesian phylogenetic trees with accession numbers, the names of the elements, and all the statistical support values are presented in Additional file 2: Figure S1
Based on the RT phylogeny and comparative structural analysis, we identified two groups of aRNH-containing Ty3/Gypsy LTR-RTs in oomycetes. The first group, designated here as Archon, is specific for Saprolegniales genomes, and its members have an aRNH next to the original Ty3/Gypsy RNH domain. Interestingly, this RNH-aRNH junction resembles the 'dual' RNH domains of Tat LTR-RTs and retroviruses [5]. The second group, named Chronos, comprises elements detected in the Peronosporales and Pythiales genomes. In addition, a single copy of a Chronos element was identified in Aphanomyces astaci (Saprolegniales). These retrotransposons also have 'dual' RNH domains. However, in contrast to all other known aRNH-containing elements, these transposons possess a CHD in the 3′ end of their pol next to the INT domain (Fig. 1b, Additional file 2: Figure S1, Additional file 1: Table S2). Previously, the presence of a CHD was shown only for two groups of LTR-RTs: Chromoviruses (a group of Ty3/Gypsy LTR-RTs [7, 9, 18, 23]) and CoDi-I elements (a group of Ty1/Copia LTR-RTs from the free-living Stramenopiles, pennate diatom, Phaeodactylum tricornutum [17]). Although Archon and Chronos LTR-RTs share similar structural organization with Tat LTR-RTs and Chromoviruses, they seem to be only distantly related to these elements (Fig. 1b, Additional file 2: Figure S1).
Identified in most of the Peronosporales and Pythiales genomes and undetectable in the Saprolegniales genomes (Additional file 1: Table S2), oomycete aRNH-containing L1 elements are similar in general organization to aRNH-containing Ta11 L1 of plants (Fig. 1c). In both groups, the aRNH domain is positioned at the C-terminal end of ORF2. Notably, both groups are also characterized by a CCHC cysteine motif located upstream of the aRNH. In other non-LTR-RTs harboring an RNH, the CCHC is positioned downstream of the RNH in ORF2 [24]. However, despite the similarities in the general organization of ORF2 (Fig. 1c, Additional file 3: Figure S2), oomycete and plant L1s do not form a monophyletic clade within the L1 group.
Oomycete Utopia elements were identified in most Peronosporales and Pythiales genomes, while only one copy was detected in Saprolegnia diclina (Saprolegniales) (Additional file 1: Table S2). Utopia is one of the "old" clades of non-LTR-RTs (such as R2, R5, and CRE) and its elements have sequence-specific restriction-like endonuclease domain (RLE), which guides their insertion to U2 small nuclear RNA genes [11]. The Utopia elements identified in our study did not differ in organization from the original Utopias identified by Kojima and Jurka [11] (Fig. 1c, Additional file 3: Figure S2).
The distinct positions of the oomycete Chronos, Archon, L1, and Utopia groups on the RT phylogenetic trees from all previously known aRNH-containing retrotransposons and from each other suggested that aRNH was independently acquired by each of these groups. However, to further elaborate on this idea, we performed a comparative analysis of the aRNHs from genomes of oomycetes, plants and other organisms.
Diversity of aRNH in oomycetes
After screening the oomycete genomic sequences, we detected aRNHs that were not associated with RT (individual aRNHs) and could therefore represent potential cellular genes. To obtain reference cellular RNH sequences, we additionally screened for fmRNHs using a set of sequences from a previous study [5]. Table 1 summarizes the results of the analysis comparing the distribution of individual aRNHs and fmRNHs to that of the RT-associated aRNH domains. We identified individual aRNHs in 21 out of 25 oomycete genomes. Notably, we previously identified aRNH-containing retrotransposons in these same 21 genomes. In contrast, fmRNH was identified in all studied genomes. For a majority of the genomes there was only single copy of an individual aRNH, while other genomes contained up to eleven copies of an individual aRNH. The copy number of fmRNHs per genome was also relatively low, varying from one to seven (Table 1), suggesting that due to its ubiquity and low copy number, fmRNH is the most likely candidate for the cellular RNH gene in oomycetes. However, the functions and origins of the individual aRNHs in oomycetes remain elusive.
Table 1 Diversity, distribution, and the number of aRNH and fmRNH domains in the studied oomycete species
To unveil the origin of both RT-associated aRNHs and individual aRNHs in oomycetes we performed a comparative analysis of RNH genes and domains from various sources (Figs. 2 and 3, Additional file 4: Figure S3, Table 1). L1, Archon, Chronos, and Utopia oomycete aRNH domains and aRNHs of plant retrotransposons form distinct clades on the tree (Fig. 2). The identified individual aRNHs were split into three clades on the tree: aRNH 1, aRNH 2, and aRNH 3. Two clades, aRNH 1 and aRNH 3, clustered together with the aRNH domains from oomycete retrotransposons Archon and L1, respectively, although this clustering was not supported by the bootstrap. aRNH 2 formed a distinct clade that did not show any significant clustering with any RT-associated aRNHs (Fig. 2, Additional file 4: Figure S3). Notably, multiple copies of both aRNH 1 and aRNH 3 were detected in the studied oomycete genomes (Table 1). Thus, together with the potential relationship between the two aRNH groups and the RT-associated aRNHs of oomycetes, these results may suggest that aRNH 1 and aRNH 3 may represent remnants of Archon and L1 retrotransposons. In contrast, aRNH 2 was not related to RT-associated aRNHs (Fig. 2, Additional file 4: Figure S3). Therefore, it is likely that aRNH 2, in addition to fmRNH, could be a cellular RNH gene in oomycetes. This finding is also supported by the wide distribution and low copy number of aRNH 2 (Table 1).
Maximum-likelihood representative tree based on the amino acid sequences of different types of type I RNHs. Approximate likelihood-ratio test (aLRT) statistical support values (unit fractions) are shown at the corresponding nodes of the tree; the values are highlighted in red if the corresponding node was additionally supported by more than 60 of 100 bootstrap replicates. Comparison of Maximum-likelihood and Bayesian reconstructions and bootstrap values are presented in Additional file 4: Figure S3. RNH lineages specific for oomycetes and plants are highlighted in blue and green gradient blocks, respectively. RTV – retroviruses. The names of oomycete non-LTR-RT and LTR-RT RNH sequences identified in the present study correspond to those in Additional file 1: Table S2. Names of RNHs of other LTR-RTs and non-LTR-RTs correspond to those in GyDB [39] and Repbase Update [21], respectively. NCBI accession numbers are indicated to the right of other RNH sequences. Schemes of the secondary structures of three subtypes of RNH with the corresponding active site residues are shown at the right of the tree. The α-helices are depicted as helices, and the β-sheets are shown as arrows. The conserved R/H residue of the active site, which varies between different RNH subtypes, is highlighted in red. *The D-E-D-D catalytic residues are not conserved in the gRNHs of Archon, Chronos and Tat LTR-RTs
Multiple amino acid sequence alignment of different types of RNHs. The names of RNH sequences corresponding to oomycete and plant lineages are emphasized in bold and highlighted in blue and green, respectively. Archaeal RNHs, Fungi/Metazoa RNHs, and original RNHs of LTR retrotransposons are designated as aRNH, fmRNH, and LTR-RTs, respectively. Apart from RIRE2 and Ogre gRNH that were retrieved from GyDB, all the sequences are available in the Additional file 8. Conserved catalytic residues (D-E-D-R/H-D) are indicated by asterisks at the top of the alignment. The semiconservative (R/H)-residue varying between the aRNH and fmRNH is additionally denoted by the bigger font at position 166 of the alignment. The conserved residues are highlighted in shades of gray. The secondary structure of Escherichia coli fmRNH (PDB: 1g15_A) is shown at the bottom of the alignment. The secondary structures of oomycete Chronos-1_PInfe LTR gRNH (predicted, this study) and Sulfolobus tokodaii aRNH (PDB: 3aly_A) are shown at the top of the alignment. The α-helices are depicted as helices, and the β-sheets are shown as arrows
To shed more light on the evolution of both aRNH and fmRNH in oomycetes, we mined aRNH and fmRNH homologs from the free-living Stramenopiles taxa, the closest relatives of oomycetes available in databases (Additional file 1: Table S3) using a tBLASTn search against NCBI WGS and TSA databases with oomycete aRNH and fmRNH amino acid domain sequences as queries (Fig. 2, Additional file 4: Figure S3) [25]. The results revealed aRNHs in the Stramenopiles genomes but did not detect fmRNHs (Additional file 1: Table S3). The aRNH domains of free-living Stramenopiles form a monophyletic clade on the Maximum-likelihood RNH tree (only weakly supported by the bootstrap) and a paraphyletic clade on the Bayesian tree. In addition, these RNH sequences did not show any significant clustering with other studied aRNHs (Additional file 4: Figure S3).
Potential origin of aRNH and fmRNH in oomycetes
While searching for homologs of aRNH and fmRNH in oomycete genomes, we identified aRNH in both free-living Stramenopiles and oomycete taxa, while fmRNH was detected only in oomycetes (Table 1). In addition, aRNH is absent in some groups of oomycetes, likely reflecting its loss in small genome parasitic lineages, such as Albuginales [26]. One possibility is that aRNH was present in the ancestor of the Stramenopiles lineage and was vertically transmitted to oomycetes. Alternatively, aRNH might have been horizontally transferred from green plants, onto which most of the oomycete taxa examined in the present study typically parasitize [25, 27, 28]. The lack of aRNH in some oomycete genomes can be explained by the redundancy of aRNH and fmRNH functions.
The lack of fmRNH in the free-living Stramenopiles most likely indicates that oomycetes acquired this gene after the divergence from the Stramenopiles stem. The horizontal transfer of genes from fungi to oomycetes as an adaptation to parasitism on algae and plants has been previously proposed [27, 28]. Fungal genomes encode fmRNHs, which are responsible for the precise removal of RNA primers of Okazaki fragments during DNA replication and are critical for the maintenance of genome integrity (Fig. 2, Additional file 4: Figure S3) [29, 30]. Thus, it could be hypothesized that oomycetes might have acquired fmRNH through horizontal transfer together with other genes from ancient fungal lineages. However, in our phylogenetic reconstruction oomycete fmRNHs are only distantly related to fungal fmRNHs, which contradicts this hypothesis (Additional file 4: Figure S3).
Convergence between oomycete and plant retrotransposons
In the present study we showed that based on RT phylogeny, the identified aRNH-containing oomycete L1 non-LTR-RTs, and Chronos and Archon LTR-RTs are only distantly related to the previously described aRNH-containing Ta11 L1 non-LTR-RTs and Tat LTR-RTs of green plants (Fig. 1, Additional file 2: Figure S1, and Additional file 3: Figure S2). The distinct phylogenetic positions of the elements contradict the possibility of a single origin of all aRNH-containing LTR and non-LTR retroelement from plants and oomycetes. We therefore suggest that presence of aRNH in Tat, Chronos, and Archon LTR-RTs and Ta11 L1 and oomycete L1 non-LTR-RTs could be the best explained by series of independent aRNH acquisitions by ancestors of these elements, reflecting their convergent evolution to the similar structural compositions. However, the single origin of all aRNH-containing LTR and non-LTR retrotransposons from plants and oomycetes could not be completely rejected by the phylogenetic reconstructions due to the low bootstrap support values (in contrast to the aLRT and Bayesian posterior probabilities supports) that we obtained for the paraphyletic origin of the aRNH-containing retrotransposons (Fig. 1, Additional file 2: Figure S1, and Additional file 3: Figure S2), leaving the alternative to convergent evolution still open for discussion.
The repeated sequestration and fixation of some functional domains during the evolution by diverse members of a certain genetic lineage may reflect a beneficial effect on the selection in the environment that this lineage inhabits. Previously, we proposed that the 'dual' RNH domains of plant Tat LTR-RTs reflected convergent evolution with vertebrate retroviruses [5]. With the discovery of Chronos and Archon LTR-RTs in oomycetes, 'dual' RNH acquisition may indicate a more general evolutionary tendency in all LTR-RTs. Indeed, the loss of the conserved catalytic residues (D-E-D-R/H-D) in the original Ty3/Gypsy RNH domain and their complete set in aRNH of Chronos and Archon representatives (Fig. 3) is similar to what was shown for Tat LTR-RTs [5], and resembles transformation of the original retroviral RNH to the connection (tether) RNH domain after the acquisition of new eukaryotic fmRNH in retroviruses [16] that is supported by the structural study of Ty3 reverse transcriptase [31]. Intriguingly, this evolutionary pathway may resemble an early stage in the transition of a Ty3/Gypsy retrotransposon into a retrovirus, preceding the acquisition of the infection-mediating envelope domain.
The beneficial effect from the RNH acquisition for non-LTR-RTs, however, is still poorly understood, as these elements typically rely on the host-encoded RNH activity. Furthermore, RNH could also be lost within some non-LTR-RT groups [32]. The finding of multiple examples of RNH acquisition in non-LTR-RTs therefore remains enigmatic.
The structural analysis of Chronos LTR-RTs revealed that apart from the aRNH domain, these elements also harbor CHD on the C-terminal end of the ORF2 next to the INT domain (INT-CHD), similar to the Ty3/Gypsy Chromoviruses from plants, fungi, and vertebrates [7, 9, 18, 19, 33]. Based on RT phylogeny, we showed that Chronos LTR-RTs and Chromoviruses are evolutionarily distinct from each other, thereby suggesting the convergent acquisition of the CHD by both groups. Interestingly, apart from Chromoviruses and Chronos LTR-RTs the INT-CHD domain was also reported for phylogenetically distant Ty1/Copia CoDi-I elements observed in the free-living Stramenopiles, pennate diatom, Phaeodactylum tricornutum [17]. See Additional file 5: Figure S4 for the multiple sequence alignment of CHDs from Chronos, Chromoviruses, and CoDI-I LTR-RTs. CHDs are widespread domains involved in chromatin remodeling in eukaryotes [34, 35]. The fusion of the CHD to the INT in LTR-RTs likely targets retrotransposon integration to the heterochromatin away from gene-rich regions [36]. Thus, multiple acquisitions of the CHD reflect the evolutionary tendency in LTR-RTs to minimize the damage to the host, while "quietly hitchhiking" its cellular machinery for retrotransposon propagation within the genome.
The current understanding of the diversity of retrotransposons and other mobile elements increases with an increasing number of sequenced genomes from a broad taxa range. In the present study, we identified and characterized several groups of retrotransposons from oomycete genomes, which to our knowledge has not previously been described. Importantly, the similar patterns of acquisitions of aRNH and CHD by unrelated retrotransposon groups from oomycetes and plants suggest that these events may represent a major evolutionary trend in retroelement evolution. This trend is likely independent of the retrotransposon host genome and may reflect similarities in the fundamental organization of retrotransposon life cycle, suggesting a beneficial role for the acquired domains in this cycle.
Computational mining for aRNH-containing repeats in Repbase update
The complete database of prototypic repetitive sequences Repbase Update (RU, v. 20.08) [21] was downloaded and analyzed for the presence of aRNH-containing repeats. Based on a hidden Markov model profile (HMM profile), aRNH domains were mapped using hmmsearch tool of the HMMER package [37] in translations of the retrieved RU sequences. The HMM profile was constructed from the amino acid alignment of aRNH sequences from the Ustyantsev et al. [5]. Repeats without the predicted similarity to aRNH were filtered out. The remained RU repeats were initially grouped according to the taxon of origin and subsequently grouped according to repeat type.
Computational mining for aRNH-containing retrotransposons, individual aRNH and fmRNH domains in oomycete genomes
The oomycete genomic sequences used in the present study were retrieved from public databases, as listed in Additional file 1: Table S2. To identify all retrotransposons harboring aRNH, the following algorithm was implemented using the UGENE workflow designer [38]. First, based on the aRNH HMM profile, aRNH domains were mapped using the hmmsearch tool of the HMMER [37] package in translations of the genomic DNA sequences. Second, sequences surrounding the regions of significant similarity to the aRNH profile were expanded, when possible, to 10,000 bp in both directions. Third, the enlarged sequences were screened for the presence of significant similarity to RT domains of non-LTR-RTs and LTR-RTs HMM profiles using hmmsearch. The non-LTR-RTs HMM profile was generated from the RT alignment of Repbase [21] non-LTR-RTs amino acid sequences available in the RTclass1 [12] server output. The corresponding HMM profile for LTR-RTs was constructed from the RT alignment of LTR-RTs amino acid sequences available in Gypsy Database [39]. Fourth, RT-positive sequences were divided into two groups corresponding to either non-LTR-RTs or LTR-RTs, and RT-negative sequences were filtered out, and identified aRNH sequences were retained for a further separate analysis as individual aRNHs. For each dataset, representative sequences were retrieved, and the number of elements belonging to each group (Ty3/Gypsy, L1, and Utopia) was counted by repeated BLAST [40], using ORF2 amino acid sequences of the previously identified RU aRNH-containing retrotransposons of oomycetes (Gypsy_18_PIT_I Ty3/Gypsy LTR-RT, L1-5_PI L1 non-LTR-RT, and R2I-1_PI Utopia non-LTR-RT) as seeding quires in the tBLASTn search.
Fungi/Metazoa RNHs (fmRNH) were mined using the HMM profile reconstructed based on the alignment of fmRNH amino acid sequences from Ustyantsev et al. [5] with hmmsearch, and the flanking sequences were expanded 1,000 bp in both directions.
Characterization of the structural composition of aRNH-containing retrotransposons
For each of the identified representative retrotransposons, a detailed analysis of the structural composition was performed. We used NCBI ORFfinder [41] to identify ORFs and NCBI CD-search [42] and HHpred [43] for a subsequent homology-based mining of conserved retrotransposon-specific domains. For LTR-RT representatives, when possible, the sequences of their LTRs were predicted by aligning 5′ upstream and 3′ downstream sequences flanking ORF1 and ORF2 using BLAST [40]. Secondary structure prediction for Chronos-1_PInfe aRNH was performed using Quick2D from the MPI bioinformatics toolkit [44].
Comparative and phylogenetic analysis
The RT amino acid sequences of the LTR-RT and non-LTR-RT representatives were aligned using hmmalign tool from the HMMER package to the corresponding HMM profiles [37]. The amino acid sequences of RNH are less conservative than RT, and a profile multiple alignment with the predicted local structures and 3D constraints (PROMALS3D) server was used to produce the alignment [45]. The alignments (refer to Additional files 6, 7, and 8 for corresponding LTR-RTs RT, non-LTR-RTs RT, and RNH alignments) were manually curated, and the phylogenetic trees were reconstructed using the maximum-likelihood and Bayesian algorithms implemented in the PhyML [46] and MrBayes [47] program tools. The best model for phylogenetic reconstruction, LG + G, was suggested using the ProtTest stand-alone tool [48] based on the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) for each of the alignments. In PhyML, an optimal tree topology was searched among 100 random starting trees under the subtree pruning and regrafting (SPR) algorithm, from which the tree with the largest log-likelihood value was taken, and its robustness was estimated using a Bayesian-like transformation of approximate likelihood-ratio test (aLRT, aBayes) and 100 bootstrap replicates [49]. In MrBayes, 10 split Markov chain Monte Carlo (MCMC) chains were run for 2,500,000 generations with sampling each 250 generations and discarding the first 5000 samples prior to consensus tree estimation.
aRNH:
RNH of archaeal and plant origin
CHD:
Chromodomain
fmRNH:
RNH of Fungi/Metazoa origin
INT:
LTR-RTs:
Long terminal repeat retrotransposons
non-LTR-RTs:
Non-long terminal repeat retrotransposons
ORF:
Open reading frame
RLE:
Restriction-like endonuclease
RNH:
Ribonuclease H
Repbase update database
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Smyshlyaev GA, Blinov AG. Evolution and biodiversity of L1 retrotransposons in angiosperm genomes. Russian J Genetics. 2012;2:72–8.
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Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.
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Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59:307–21.
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012;61:539–42.
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The authors are grateful to everyone who made the data freely available for the present study. The authors would also like to thank the American Journal Experts (AJE) for English language editing.
This work was financially supported by the Russian Foundation for Basic Research (Project No. 14-04-01498a) and the State scientific project (Project No. 0324-2016-0008).
Availability of the data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
KU performed all the bioinformatics assays and data analyses. KU and GS conceived and directed the study. AB provided computational resources and helped with the manuscript editing and writing. All authors contributed to the manuscript review. All authors read and approved the final manuscript.
Institute of Cytology and Genetics, Laboratory of Molecular Genetic Systems, Prospekt Lavrentyeva 10, 630090, Novosibirsk, Russia
Kirill Ustyantsev & Alexandr Blinov
Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117, Heidelberg, Germany
Georgy Smyshlyaev
Kirill Ustyantsev
Alexandr Blinov
Correspondence to Kirill Ustyantsev.
Diversity and distribution of aRNH-containing repetitive elements identified in the Repbase Update v. 20.08 (08-30-2015) database [21]. Table S2. Diversity, distribution and selected representatives of identified aRNH-containing retrotransposons in the studied oomycete genomes. Table S3. Individual aRNHs identified in the free-living Stramenopiles species. (XLSX 40 kb)
Additional file 2: Figure S1.
The complete Maximum-likelihood and Bayesian phylogenetic trees reconstructed based on the amino acid sequences of RT domain of LTR-RTs (see Additional file 6 for the alignment). Statistical support was evaluated using aBayes aLRT (unit fractions) and 100 bootstrap replicates (% after a slash), and MCMC runs (%) in Maximum-likelihood and Bayesian reconstructions, respectively, and are shown at the corresponding nodes of the tree. Bootstrap values are shown only for the main indicated clusters. Chromodomain-containing clade names are underlined, and the names of the aRNH-containing clades are indicated in blue and green for plant and oomycete LTR-RTs, respectively. The names of the oomycete LTR-RT sequences identified in the present study correspond to those in Additional file 1: Table S2. Unless otherwise stated, the names of other LTR-RTs correspond to those in GyDB [39]. (PDF 779 kb)
The complete Maximum-likelihood and Bayesian phylogenetic trees reconstructed based on the amino acid sequences of RT domain of non-LTR-RTs (see Additional file 7 for the alignment). Statistical support was evaluated using aBayes aLRT (unit fractions) and 100 bootstrap replicates (% after a slash), and MCMC runs (%) in Maximum-likelihood and Bayesian reconstructions, respectively, and the results are shown at the corresponding nodes of the tree. Bootstrap values are shown only for the main indicated clusters. The names of the aRNH-containing clades are indicated in blue and green for plant and oomycete non-LTR-RTs, respectively. The names of oomycete non-LTR-RT sequences identified in the present study correspond to those in Additional file 1: Table S2. The names of other non-LTR-RTs correspond to those in Repbase Update [21]. (PDF 366 kb)
The complete Maximum-likelihood and Bayesian trees reconstructed based on different type I RNH amino acid sequences (see Additional file 8 for the alignment). Statistical support was evaluated using aBayes aLRT (unit fractions) and 100 bootstrap replicates (% after a slash), and MCMC runs (%) in Maximum-likelihood and Bayesian reconstructions, respectively, and the results are shown at the corresponding nodes of the tree. Bootstrap values are shown only for the main indicated clusters. The names of the RNH clades from plant and oomycete genomes are highlighted in green and blue, respectively. The names of oomycete non-LTR-RT and LTR-RT RNH sequences identified in the present study correspond to those in Additional file 1: Table S2. Names of RNHs of other LTR-RTs and non-LTR-RTs correspond to those in GyDB [39] and Repbase Update [21], respectively. NCBI accession numbers are indicated to the right of other RNH sequences. (PDF 863 kb)
Multiple amino acid sequence alignment of CHDs from LTR-RTs and human Chromodomain Protein Y-Like 2 (PDB accession number 5JJZ_A). Additional information about the amino acid conservation is shown as a sequence Logo generated from the alignment, which is positioned at the bottom. (PDF 1096 kb)
Multiple amino acid sequence alignment of RT domains from diverse LTR-RTs constructed and used for the phylogenetic reconstruction in the present study. (TXT 58 kb)
Multiple amino acid sequence alignment of RT domains from diverse non-LTR-RTs constructed and used for the phylogenetic reconstruction in the present study. (TXT 83 kb)
Multiple amino acid sequence alignment of RNH genes and domains from diverse taxa constructed and used for the phylogenetic reconstruction in the present study. (TXT 45 kb)
Ustyantsev, K., Blinov, A. & Smyshlyaev, G. Convergence of retrotransposons in oomycetes and plants. Mobile DNA 8, 4 (2017). https://doi.org/10.1186/s13100-017-0087-y
Received: 19 December 2016
Accepted: 07 March 2017
Convergent evolution
Retrotransposons


text:
Matthew McConaughey Editor's Picks
The Dark Tower Review
For a film that's trying to be grand and epic, The Dark Tower doesn't amount to anything more than a series of fantasy and young adult clichés.
IT is NOT a remake!
susanna finn
Top 5 Sci-Fi films of 2017 so far - and the 5 we can't wait to see...
As we come to an end of our "half-time" report on all things film in 2017, we take a look back at some of the sci-fi spectaculars we have seen so far, whether it's in a galaxy far, far away, here on earth or in the distant future we have seen it all – and there's still so much to see.
The Dark Tower - Trailer
The Gunslinger, Roland Deschain, roams an Old West-like landscape where "the world has moved on" in pursuit of the man in black. Also searching for the fabled Dark Tower, in the hopes that reaching it will preserve his dying world.
Matthew McConaughey Movie News
Stephen King explains disappointing The Dark Tower adaptation
Yeah I know, just like you, we were at Flickreel felt a pinch of excitement when we heard Stephen King's fantasy series The Dark Tower was getting a movie adaptation.
The Dark Tower is being destroyed by critics
Sony's film adaptation of Stephen King's The Dark Tower is being hammered by critics, following its first wave of reviews.
The Dark Tower's first reactions are in
The first reactions are in for Sony's film adaptation of Stephen King's The Dark Tower.
The Dark Tower's production "was plagued with problems"
Sony's upcoming film adaptation of Stephen King's The Dark Tower reportedly had all kinds of problems during production.
Matthew McConaughey Movie Reviews
Gold Review
Though undoubtedly a flawed endeavour, McConaughey turns in a staggeringly impressive performance to ensure this narrative is easy to follow and adhere to.
Sing Review
Sing is an infectiously fun family picture that plays out like Pitch Perfect meets Zootopia.
Free State of Jones - Review
Though Free State of Jones has a remarkable story that lends itself so well to a silver screen retelling, Ross's clumsy direction makes for a film unsure of its focus.
Matthew McConaughey Movie Previews
New Sing photos show the cast alongside their characters
Universal has released new promotional photos from their forthcoming animation Sing, which shows each actor pose with the character they have voiced.
Matthew McConaughey Movie Features
The Rise of Matthew McConaughey
"It all began with The Lincoln Lawyer. Up until that point in Matthew McConaughey's career, he was renowned predominantly for appearing in cliched, tedious romantic comedies, and being the butt of several jokes in Family Guy. Then, in 2011, Brad Furman took a punt on him"…
Interstellar building a black hole
With our time on Earth coming to an end, a team of explorers undertakes the most important mission in human history: traveling beyond this galaxy to discover whether mankind has a future among the stars.
Flickreel's 2014 Oscar Predictions
It's that time of year again to celebrate the very best of the past year in film, with the prestigious 86th Academy Awards soon to be upon us. As we all prepare to settle in for the night, and ridicule the red carpet coverage and then open up the dolly mixture and marvel in the ceremony itself, beforehand let's run through some of our 2014 Oscar predictions…
Matthew McConaughey Movie Trailers
The Dark Tower - Trailer 2
Gold - UK Trailer
An unlikely pair venture to the Indonesian jungle in search of gold.
Sing - Final Trailer
Matthew McConaughey Movie Interviews
Interstellar Christopher Nolan interview

redpajama_set_name: RedPajamaCommonCrawl


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I also found a mystery Duck (probably just a Mallard but the supercilium looked off).


Shad Shakar Mi Ooder, a short experimental film.
2009-2010 Writing Studio Visits art column for Mes Armenies blog.
Wrote art reviews for Review Magazine and other publications.
Translated The Rosy Future of War.
Dance Critic for The New York Press, 2005-2006.


text:
Business News›Internet›India to become Airbnb's key revenue generator: Brian Chesky
India to become Airbnb's key revenue generator: Brian Chesky
Anumeha Chaturvedi
Madhav Chanchani
, ET Bureau Last Updated: Mar 20, 2017, 07:44 AM IST
Chesky decried the protectionist stance adopted by US President Donald Trump and his recent attempts to ban immigrants from specific nations
Airbnb CEO Brian Chesky sees India rising to become one of the company's key revenue generators with the country's young and rapidly expanding middle class spending big on travel.
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Chesky , who teamed up with Nathan Blecharczyk and Joe Gebbia in 2008 to launch the San Francisco-based company , was on his maiden visit to India to launch a new service in the country, Trips. It is designed to offer unique local experiences such as Samurai training near Tokyo and surfing in Cape Town. Airbnb has been present in India since 2012 and last year inked a strategic partnership with The Times Group, publisher of The Economic Times.
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"We started by localising our product for India and growing quickly to where the (Indians) want to travel to. Second was making sure inbound was strong. Final is domestic -Indians (travelling) within India ," Chesky said.
Airbnb had earlier said that its guests spent an estimated $60 billion in its top 200 cities last year on both accommodations and daily expenses.
The company has homes in 68,000 cities and 191 countries. The company expects alternative accommodations will be a minor part of its revenues in the future as it launches Trips and also explores a foray into aviation, which it regards as a bottleneck for growing the business.
Brian CheskyDonald TrumpAmanpreet BajajAirbnb


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text:
Q: Is there a way to generate java classes from MS-Dataset XSD files? Given a Dataset XSD file, is it possible to generate java classes, that don't make use of System.Data?
I've tried running a test using JAXB's XJC tool, but it doesn't produce anything terribly useful.
Update: I've tried XmlBeans also, following Fernando's suggestion, and it generates something similar to the XJC output - that is, class representations of the tables, but without any columns, constraints, or rows. I can add these things in a facade, but ideally they would be generated by whatever XSD compiler was used.

A: You could try XMLBeans instead of JAXB API. You can invoke it be means of command line command or just use an ANT script. I past the part of a build.xml file that invokes XMLBeans to compile XSD to a jar file.
<taskdef name="xmlbean" classname="org.apache.xmlbeans.impl.tool.XMLBean" classpathref="classpath"/>

<!-- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ -->
<!--  Crea y compila las clases del modelo                             -->
<!-- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ -->

<target name="build" description="--> crea las clases a partir del schema">
    <!-- Borra los fuentes del modelo -->
    <delete quiet="true" dir="${build.dir}/src" />

    <mkdir dir="${build.classes}"/>

    <!-- Construye fuentes asociadas y crea las clases -->
    <xmlbean srcgendir="${build.dir}/src" classpathref="classpath" classgendir="${build.classes}">
        <fileset dir="${src.dir}" excludes="**/*.xsd"/>
        <fileset dir="${schemas.dir}" includes="**/*.*"/>
    </xmlbean>

    <javac srcdir="${src.dir}"
                destdir="${build.classes}"
                encoding="Windows-1252"
                debug="${debug}"
                debuglevel="${debuglevel}"
                deprecation="${deprecation}"
                verbose="${verbose}"
                optimize="${optimize}"
                source="${source}"
                target="${target}">
                <classpath refid="classpath" />
    </javac>
</target>

redpajama_set_name: RedPajamaStackExchange


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Often when I am invited for dinner to a Turkish home the spread will include a very simple bow tie pasta dish which is extremely delicious. It is super easy to make and can be eaten alone or it can accompany other fare.
There really isn't a lot to say about this pasta dish. If you like cheese and you like butter then you are more than half way there!
Sometimes the beauty really is in the simplicity. I have to say that often the Turks just hit the nail on the head and they certainly have with this one. Their cuisine follows some pretty cool rules. Generally it is easy to create, does not mix lots of flavours together and requires fresh produce. It's no more difficult than that.
I hope you will try this dish. It does take four times longer than 2 minute noodles but there is a vast chasm between the two.
I will definitely give that a go. Just adore walnuts and it sounds like a great idea. Thank you Angela.


This platform scale features a unique chain link design for its weight sensors. This allows the full load to be placed on the Weigh Bar weight sensor while minimising side and end loads, which improves accuracy. The chain links also absorb shock loads rather than transfer them to the Weigh Bar.
The DS floor scale is suited for any industrial weighing application. It is trade approved, meaning it can be used in situations where product is sold by weight.
It also has an FM-approved junction box and Weigh Bars, and when paired with an FM-approved weight indicator, is safe for explosive atmospheres.
The carbon steel deck scales are finished with either a high-grade polyurethane or a powder paint finish, depending on the platform size. Stainless steel construction is also available.


We have service hours available for you 24 hours 7 days a week, as well as live operators when you call the office at anytime. Services are available on an hourly basis as well as live-ins on weekdays, nights, weekends, and holidays, from just a couple hours per day to a full 7-day week. We have registered nurses, licensed practical nurses, certified home health aids, and companions available. Our coordinating staff will always work closely with you to find the best service for you.
Senior companion services are a great way to mix day care and night care and help reduce boredom, anxiety, loneliness, and fear of being left alone. Live In Companions provide peace of mind for clients and families.
Home Health Aides provide care in the client's home. This makes it possible for clients with limitations to remain at home in a comfortable, familiar environment.
Home Health Aides can provide all aspects of homemaker/companion services as well as provide clients with personal care services. Personal care is assistance with activities of daily living. Some examples are bathing, grooming, medication assistance and other daily care needs.
For clients who need a little more "hands on" care we provide Home Health Aides. Home Health Aides go through training and are able to offer bathing, grooming, dressing, medication assistance as well as companionship.
Skilled Nursing can be performed in the home by both Registered Nurses (RNs) and Licensed Practical Nurses (LPNs). They educate the patient and family members on the patient's medical condition and monitor the patient for important changes.
The goal of skilled home care is to restore or maintain client's level of functioning and health. These services are rendered in lieu of hospitalization, confinement in an extended care facility, or going outside of the home for the services.
The skilled nurse works under the direction of the client's primary care physician, keeping them informed regarding the client's status and progress.


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Nanuet distance to The Bronx
How far is Nanuet from The Bronx?
Shortest distance between Nanuet and The Bronx is 18.2 miles (29.29 km).
Flight distance from Nanuet, NY to The Bronx, NY is 18.2 miles. Estimated flight time is 00 hours 03 minutes.
The driving distance from Nanuet, New York to The Bronx, New York is: 29.45 miles (47.4 km) by car.
Driving from Nanuet to The Bronx will take approximately 00 hours 40 minutes.
#1 Nanuet
City in Clarkstown, New York, United States of America
41°5'19"N 74°0'48"W
#2 The Bronx
Borough of New York City, United States
Starting point in Nanuet: South Middletown Road (-74.013629,41.088761)
Ending point in The Bronx: Cruger Avenue (-73.866845,40.849859)
Estimated Travel Time Between Nanuet and The Bronx
The distance between Nanuet and The Bronx is 47 km if you choose to drive by road. You can go 00 hours 31 minutes if you drive your car at an average speed of 90 kilometers / hour. For different choices, please review the avg. speed travel time table on the below.
There is no time difference between Nanuet and The Bronx. The current time is 08:51:00.
A car with a fuel efficiency of 8.3 l/100 km will need 3.93 liters (1.04 gallon) of gas to cover the route between Nanuet and The Bronx.
The estimated cost of gas to go from Nanuet to The Bronx is $3.44 (diesel $3.87).
The place names are translated into coordinates to approximate the distance between Nanuet and The Bronx (latitude and longitude). Cities, states, and countries each have their own regional center. The Haversine formula is used to measure the radius.
Driving distance from Albany, NY to Nanuet 124.13 miles (200 km)
Driving distance from Astoria, NY to Nanuet 28.56 miles (46 km)
Driving distance from Babylon, NY to Nanuet 69.41 miles (112 km)
Borough Park, NY
Driving distance from Borough Park, NY to Nanuet 39.06 miles (63 km)
Driving distance from Brooklyn, NY to Nanuet 38.3 miles (62 km)
Driving distance from Buffalo, NY to Nanuet 363.27 miles (585 km)
Bushwick, NY
Driving distance from Bushwick, NY to Nanuet 35.21 miles (57 km)
Driving distance from East Flatbush, NY to Nanuet 42.72 miles (69 km)
Driving distance from Elmhurst, NY to Nanuet 31.41 miles (51 km)
Gravesend, NY
Driving distance from Gravesend, NY to Nanuet 40.41 miles (65 km)
Driving distance from Jamaica, NY to Nanuet 37.21 miles (60 km)
Driving distance from Manhattan, NY to Nanuet 26.87 miles (43 km)


The Cling On Pickup is a compact and elegant passive piezo pickup with a patented ultra-quick and easy magnetic attachment. It requires absolutely no modification, nor any lengthy installation. The detachable portable cable and a built-in volume control are valuable extra features. Designed primarily for acoustic and classical guitars, the Cling On Pickup works equally well on banjos, ukuleles, cajons and other acoustic instruments with accessible flat soundboards.
Ultra QUICK and EASY magnetic attachment.
The patented magnetic design allows the pickup to securely attach and detach to the instrument in a fraction of a second. No cumbersome or messy installations necessary.
There is no need to drill or modify your instrument in any way. There are no permanently attached, glued or screwed-on parts. The Cling On Pickup Is plug and play ready right out of the box.
The constant magnetic pressure on the soundboard, contributes to more accurate transfer of the vibrations from the soundboard to the pickup. The result is a very natural and balanced sound with high signal output. No pre-amp is required.
Each pickup comes with a very small and very light magnetic base that is attached underneath the soundboard on the inside of the guitar via tacky putty. This ONE-TIME non-permanent discreet installation prepares the instrument for the pickup. It's removable, reusable and can be easily re-positioned.
Find the "SWEET SPOT" of your instrument every time.
Once the best location on the sound board is found, the magnetic base stays in place ready for the pickup to dock at the same place every time. There is no more guessing where the "sweet spot" of your instrument is.
Unobtrusive thumb wheel volume knob design gives the performer control of the sound level at all times.
THE CLING-ON PICKUP IS MY NEW FAVORITE. INSTALLATION TAKES ABOUT THREE MINUTES. APPLY THE INCLUDED PUTTY TO THE "COIN" (YOU CAN PURCHASE EXTRAS FROM CLING-ON), REACH INSIDE THE SOUNDHOLE OF YOUR GUITAR AND STICK IT TO THE SWEET-SPOT. DONE. THE PICKUP STAYS ON MAGNETICALLY. THE SOUND IS FANTASTIC! CLEAR, NATURAL, RESONANT, AND AWESOME!
WOW! I AM IMPRESSED. THIS PICKUP WAS SUPER EASY TO INSTALL (I'M NO TECH). IT'S HELD TO THE TOP OF THE GUITAR MAGNETICALLY, AND IS EASILY REMOVED WHEN NOT IN USE. THE SOUND IS ABSOLUTELY INCREDIBLE. IT'S THE PURE UNALTERED TONE OF MY GUITAR, ONLY LOUDER! IT WORKS EVERY BIT AS WELL ON MY NYLON STRING GUITAR AS WELL. ALL THIS FOR SIXTY BUCKS!
WHAT A GREAT PICKUP! THE INSTALLATION COULD NOT BE EASIER. IT TOOK ME LESS THAN FIVE MINUTES, AND I AM NOT A TECH. THE SOUND IS SPECTACULAR. WHAT I HEAR FROM THE AMP IS THE UNALTETRED, BEAUTIFUL SOUND OF MY GUITAR.
The Cling-On Pickup rocks. The sound quality is crystal clear with no feedback and the connections to my amp are secure. I used the mini cord as well as my own amp cords to see if there were any issues with sound quality or feedback and everything worked fine. What a wonderful product. I can't wait to use it on a regular basis.


We imagine and then create beautiful experiences for our clients, for the good of their business, their customers and anyone who is fortunate enough to come into contact with them.
BIG CHALLENGES need BIG IDEAS so not only do we dare to imagine. We strive to create and then we go on to support. Our work is not done in projects, we start a journey with you and then support you on that journey. it may sound poetic, but it happens to be true.
Always an idea, a concept, a thought no matter what or how developed it is, this is where we step in. We help you develop and envisage your idea breathing life into it.
The results are a testament to the what goes on in the space between idea and delivery. Not always the same process, not always the same people. Always the same results.
As they say failing to plan is planning to fail. Having said that it's always a very fluid thing, it has to be. We live in the creative realm and for that to flourish we must have the flexibility to pivot, adapt and improve whenever needed in order to take it from concept to not just reality but a truly great experience.
The plan? Well, that's a set of strategic goals and milestones to ensure expectations are met and regularly surpassed.
Between us, we need to be working within a framework of expectations, this creates a healthy industrious environment where full collaboration and honest straight up communication are key.
Honest and sometimes brutal feedback always welcome.
The journey never ends at delivery.
It's a fast-paced digital world so even at delivery you need to continue to support and develop the experiences you are providing for your clients to increase engagement and improve retention which will ultimately enhance conversion. This is where we differ from most. We see support as being just as important as the idea and the delivery.
How many times have you come across glitchy websites, dated or totally irrelevant content? That last tweet that says tweeted 2 years ago. How can you expect your customer [users] to be engaged when you are not even engaged in your own website or platform.
HUB is in our name for a reason. We are your hub 4 everything digital.
We Build Great Digital Experiences.


No one is immune from health challenges, and our 24/7 culture doesn't help.
Everyone has age-related health conditions. Your family medical history can frequently point to potential risks before anything develops. Having a risk factor does not mean you will develop a disease, especially if you minimize things within your control such as diet, environmental factors and taking nutritional supplements. This makes it all the more important to understand lifestyle choices you can make to stay healthy.
Regular eye exams, screenings and treatments can help glaucoma. This disease is a leading cause of vision loss in the US. Another vision risk is age-related macular degeneration that's associated with certain genes. Early signs of Macular Degeneration are usually detectable in an eye exam even before the disease starts to affect vision. Your doctor will examine the eye with special lenses, which help to show the interior of the eyeball through the pupil, the opening in the center of the iris through which light rays enter the eye.
Even if you're not facing either of these conditions, regular exams are important because you may need computer, reading or distance glasses.
Lutein, a supplement related to vitamin A and beta-carotene, is used to help prevent cataracts, and age-related macular degeneration. It's also used to prevent heart disease and type 2 diabetes, There are lower doses of Lutein, but the 20 mg softgel is a one-a-day supplement, or as directed by healthcare practitioner.
The supplement combined with delicious lutein-rich foods, such as orange pepper, tomatoes, broccoli, spinach, corn, kiwi fruit, grapes, zucchini, carrots, cantaloupe, dried apricots, and squash translate into a nutrition-rich powerhouse.
Another possibility is boosting the macular pigments, such as lutein, which have antioxidant properties and may help some people with intermediate or advanced macular degeneration.
When choosing sunglasses, look for UV-protection details on the product labels. Choose sunglasses that block 99 to 100 % of both UVA and UVB rays. Skip sunglasses that neglect to offer details about their UV protection. Keep in mind that the color and degree of darkness sunglasses provide have nothing to do with the ability of the lens to block UV rays. Also, opt for wraparound sunglasses or close-fitting sunglasses with wide lenses that protect your eyes from every angle.
Can you honestly say that you're not one of the two-thirds of Americans who don't consume enough water every day? It's a much needed source of nutrition for our joints, metabolism, organs and tissues.
Coffee, caffeinated drinks, and alcohol may taste wonderful, but these drinks also serve as diuretics, which cause water loss. Start to change your habits and try for at least 8 to 12 cups of water daily.
If you spend 45 minutes on the treadmill or mowing the lawn on a sunny day, your body needs more water based on your activity.
This one's for the ladies because the guys will probably just start laughing: Ladies, put your feet up, relax and treat yourself to a beautiful crystal goblet filled with cool water and a few thin lemon slices.
For those not at the gym at least three times a week, what are you doing to keep fit? Is it kick boxing, swimming, golf, basketball, or tennis?
Walking to the water cooler for a computer break is highly recommended. However, according to exercise guidelines from Mayo Clinic, a daily plan should include a minimum of 30 minutes of activity. If weight loss is your goal, aim for 300 minutes a week.
If you can't manage a 30-minutes brisk walk, try smaller units of 10 minutes each. Even without a gym membership, creative alternatives include mall walking and hall walking during the winter for many apartment dwellers.
Premium supplements are essential for a health-smart regimen designed to improve and maintain health. Start with a multiple vitamin and build out from there.
You may need added B vitamins if you're under a lot of stress, digestive enzymes, or added vitamin D3 if you live in an area with challenging winter weather.
Today, most physicians check D levels when running blood workups, but just in case, ask your doctor to check your D levels. Low D levels is a cause of many health problems.
According to an (NSF) National Sleep Foundation survey conducted from 1999-2004, over 58 % of adults report having sleep problems. Furthermore, an even higher percentage of children cope with poor sleep.
Today, there are natural sleep-aid solutions such as Sleep Tonight.
Avoid coffee after 5 PM.
No electronic devices after 9:30 or 10 o'clock at night.
Awareness is key to combat these tendencies which can harm your health. Habits are our friends—when they're good ones. Try to slowly make each suggestion into a routine to help you repeat safe and effective behaviors, and build consistency into your life.


User manuals for portable battery packs list the basic and advanced functions of the computers & solutions - power - portable battery packs. They also often list contact information for warranty repairs and service. The user manual should be included with the portable battery packs you purchase.
If you have a problem with portable battery packs settings, are using a new product for the first time, or just want to learn more about what your portable battery packs can do and you don't have a printed user manual or manual on CD, you can download the manual from the manufacturer's website under computers & solutions - power - portable battery packs.
We will also help you to find links on our website for portable battery packs electronic user manuals in *.pdf or *.doc format.


There it lies, dormant and unwavering.
The summit of the lands. Is it not the most constant thing?
using the backs of it's past to climb higher into the sky.
It's been a long time since I've posted here, but I intend to be not only more regular but more professional and focused with my posts here.
I'll document my experiences definitely, most certainly that's what a blog should be, but in addition to normal free-form pieces, I'll be posting short analytical essays, poems, flash fiction and the occasional short story.
Basically, I'd like this to function as a place for expression, but also a type of public portfolio. Looking over this blog, that hasn't been touched since I was overseas in 2015, well it gives me pause to reflect on change, time and most importantly, growth.
It's a topic that I find innately interesting and very much worth of a lot of interrogation through fiction. In this current age, as technology moves forwards in leaps and bounds, as our impact on the environment becomes more and more unignorable, as the ugly underbellies of many of the platforms of our political norm are exposed; it is here that we feel like the entire world is in a fit of giant, wrathful growth.
Now, I'm not going to be commenting on that macro level of growth here. I'll leave that to those qualified to express opinion and analysis on such topics. Instead, I find myself thinking about my personal growth and how I could related it to Mt Fuji, which I was lucky enough to see on a clear day as pictured.
It was explained by our guide that Fuji has been four different volcano during its life, each of a different type and each of the previous three forming the base of the current icon of Japan.
The more I think about it, as I discover parts of this blog that make me wince with embarrassment and wonder "How did I ever think that was a good idea?", I think about Fuji and remember that you need to build on what you were, no matter how bad that seems now, to create what you are now, to become iconic.


In these areas you will have the chance to enjoy watching the colorful fish and amazing coral reefs.
Also a chance to see the famous wrecked ship location.
You may also be lucky enough to see dolphins.
This trip is all inclusive, includes lunch and soft drinks.
2. Journey will be to either Ras Mohamed National Park or the Straits of Tiran, (Ras Mohamed has more reefs and a shorter distance, Tiran is a longer sail before the first snorkeling stop) – wide sundecks on boat allowing opportunity to relax and sunbathe.


Junjou Romantica is a manga series from 2002 with anime adaptation from 2008.
The main character is innocent Misaki Takahashi – an eighteen-year-old who, as a result of certain events, lives together with ten years older Akihiko Usami – the author of… boys love novels.
There are also two side stories – Hiroki Kamijou (university professor, uke) and Nowaki Kusama (doctor, seme), the two who once met in their childhood, and later Kusama "hunted" Kamijo to teach him.
The second side story is the story of Yō Miyagi and Shinobu Takatsuki – it is different from the other pairings in Junjo and Sekaiichi, because of the age difference – it is 17 years here – what does not change the fact, that it's still an absolutely charming, romantic story about students love to his professor.
The most similar to Junjou Romantica is Sekaiichi Hatsukoi – both created by the same mangaka, Shungiku Nakamura.


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Where Are The Riders This September In NYC
Where are the riders in NYC this September
New York's a busy city, but some places are busier than others at certain times of year. At INSHUR we look for ways to help you make more money. Revealing where the crowds are is one of them. They say that good business is where you find it, but many Uber drivers will be cruising for riders in all the same old places. We think that's way too competitive. Instead, here are some busy spots that may get overlooked. Scoop up the riders that the others never see.
Brazilian Day
Event: Brazilian Independence Day street party.
Where: Little Brazil – West 46th Street.
When: Sunday, 1st September.
Times: 11.00am 'til late.
Rio comes to Manhattan in this salsa, song and good times festival. A mini-carnival that spreads across 25 city blocks from the heart of Little Brazil, it draws the crowds all day long. Hot weather is predicted. Combine that with the non-stop action and there'll be plenty of riders aching for an air-conditioned ride.
Insider's scoop: Attention night-time drivers: Check out Pier 15, as party-goers start early with a pre-parade boat ride on August 31st . They board at 9.00pm and return at 2.00am. Here is a map to help out:
Jersey City Beer Fest
Event: German beer festival.
Where: Harborside 3 Atrium, Jersey City NJ07311.
When: Saturday, 14th September.
Times: 1.30pm to 9.30pm.
If 150 different beers, good music and fine food don't bring throngs of eager beavers, then nothing will. Located on the river near the Hyatt Regency Hotel and Exchange Place PATH ferry terminal, it's just eight solid hours of drinking, dancing and eating.
Insider's scoop: Many New Yorkers will opt for the ferry from Brookfield Place in Battery Park. Expect busy foot traffic at the terminal after 9.30pm. Click below for a map of the area:
African American Day Parade
Event: Parade and street fair.
Where: 116th – 136th Streets & Adam Clayton Powell Jr Blvd.
When: Sunday, 15th September.
Now in its 50th year, the annual African American Day Parade is a celebration of diversity and talent. Crowds pack the sidewalks for 20 blocks to see musicians, marchers, pom-poms and more. Look out for groups trying to get to the staging area at 111th and 115th and crowds arriving and leaving the start and finish lines.
Insider's scoop: The VIP Reviewing Stand is at 163 West 125th Street, outside the Adam Clayton Powell Jr State Office Building. It's the ideal spot to find those who want to ride in style. See map below:
57th New York Film Festival
Event: Annual film showcase.
Where: Lincoln Center, 65th Street.
When: September 27th to October 13th.
Times: Late morning 'til night time every day.
Yes, it's back. New York's magnet for film buffs makes its 57th outing at Lincoln Center. With 29 feature films, plus documentaries, shorts and foreign flicks, it's a major draw for lovers of the silver screen. Look out for star-struck riders around Lincoln Center and on Amsterdam Ave.
Insider's scoop: The festival screens movies in several theaters. Be prepared to be flexible with your pick- up spots, see below:
 Find the riders that other Uber drivers miss.
 Brazilian Day Festival, Sunday 1st September.
 Jersey City Beer Fest, Saturday 14th September.
 African American Day Parade, Sunday 15th September.
 New York Film Festival, 27th September to 13th October.
 Maps and handy Tips in our Insider's Scoop.
SOURCES and LINKS:
Brazilian Festival: http://www.brazilianday.com/en/
Boat Ride: https://www.eventbrite.com/e/the-1-pre-brazilian-day-parade-yacht-cruise-nyc-boat-party-saturday-tickets-62180365146
Jersey City Beer Fest: http://www.jerseycitybeerfest.com/
Brookfield Place ferry: https://www.google.com/maps/@40.7157734,-74.0278368,16z?hl=en
African American Day Parade: https://africanamericandayparade.org/parade
Adam Clayton Powell Jr Building: https://en.wikipedia.org/wiki/Adam_Clayton_Powell_Jr._State_Office_Building#/map/0
NY Film Festival: https://www.filmlinc.org/nyff2019/
Lincoln Center film venues: https://www.filmlinc.org/visit-us/
Driver tips US, Events, US
Momentum Continues For Inshur With Additional $1.5M FundraiseINSHUR hires former Uber Legal Director


With its winning design and silver, red or chromium-plated trimmings, the Nevada Professional series represents the range of SICO KITCHENWARE® production ideal for the family, chefs, hunters, fishermen and small restaurants.
A powerful pump and transformer guarantee quickness and professional level performances. They are designed for continuous and intensive use.
Find out more about our Sico Triple Seal line models here.


Discover the power of Ketones, the cleanest, highest form of energy your body produces, prefers and gets from burning fat with PRIME!
So What Exactly is Kegenix PRIME?
Kegenix Prime provides bioidentical, bioactive Ketones. Its multi-patented proprietary formula developed through years of scientific research by Dr. Dominic D'Agostino, PhD. at the University of South Florida is designed to deliver the most ketones per gram possible.
PRIME mimics the body's natural ketogenic process. Only PRIME delivers the special combination blend of ketones to both elevate and then SUSTAIN your ketone levels so you can get all the benefits of ketones.
PRIME really tastes great! This is so important for a supplement that is intended to be taken every day and multiple times a day.
HIGH POWERED KETONES THAT TASTE DELICIOUS: Kegenix PRIME, our multi-patented, advanced ketone-boosting combination technology is unique, exclusive and works to first, directly provide your body usable ketones and second, helps your body produce its own ketones. This special combination technology aids your body in achieving Ketosis. Did you know that ketones (fat) deliver 225% more energy than carbs (sugar) and is the body's preferred fuel?
NO NEED TO START A RESTRICTIVE DIET: Kegenix PRIME provides the ketones and natural ketone producing ingredients to help suppress your appetite and cravings, while providing the missing energy you need to perform your best while also preserving your muscles. Prime can be taken multiple times per day by those who just want to realize the benefits of ketones regardless of their diet or can be used as a "power tool" for those who want to easily enter into and maintain a ketogenic, paleo lifestyle.
MAXIMUM BRAIN FUNCTION, CLARITY & ENERGY LEVELS…YES: No one wants "brain fog." Ketones have been scientifically shown to enhance brain function, improve sleep, and increase energy all without causing jitters or nausea that come from other "stimulant" only products. Our proprietary ketone formula is 100% tested, proven and multi patented!
WANT TO LOSE WEIGHT & REGAIN YOUR LOST CONFIDENCE: Our dynamic and proprietary ketone formula provides the ketones your body needs to help support your own weight loss goals. Kegenix™ PRIME provides the ketones you need to help burn fat, suppress hunger, and control those terrible sugar and carb cravings.
How KEGENIX works to keep you in ketosis?
Kegenix PRIME is the first ketone supplement designed to provide CKS (Continuous Ketone Supplementation) to help your body sustain its beneficial ketone levels for consistent mitochondrial energy production.
Kegenix PRIME's multi-patented, proprietary, combination ketone formula allows for a unique phased ketone delivery process to assist in ketone and electrolyte absorption without interfering with the body's own ketone production process. We believe one of the keys to achieving greater benefits from ketones is to provide the right ketone ratios to the blood and hepatic system in maintaining beneficial ketone levels.
Ketones are the most powerful and the most preferred fuel your body produces. Ketones are produced when your body burns fat, whether that fat comes from your stored fat (greatest way to lose weight) or the fat you eat. Ketones come from burning fat while glucose is produced from burning carbs and SUGARS. Most people don't know that Ketones have 225% more energy than carbs, are clean burning in your cells (less free radicals to damage cells) and have been scientifically shown to provide superior benefits like increased mental clarity, appetite and craving suppression, sleep improvement, reduced inflammation to name a few that you just don't get from burning glucose.
So with all those benefits, how do you get Ketones into your regular daily diet? There are two ways. The first and hardest way is to deplete your body of glucose and then eat a rather strict ketogenic diet (70% fat, 25% protein, 5% carbs). The second and easiest way is to take Kegenix Prime, which supplies your body with already created bio identical ketones and can additionally help your body produce its own Ketones. Either with Kegenix Prime or with the diet you will put your body into a natural metabolic state called Ketosis, which simply means your body is now using Ketones (fat) rather than Glucose (sugar/carbs) for its primary fuel.
For those who feel maintaining a strict ketogenic diet (we absolutely recommend people reduce their carbs) is just too difficult, they should take Kegenix Prime multiple times throughout the day to keep their body refreshed with ketones (we call this CKS, Continuous Ketone Supplementation). And for those who want to support a paleo, low carb or Ketogenic lifestyle, Kegenix Prime is perfect to help them get into and maintain that lifestyle at a less strict carb level.
But don't take our word for it, just try Kegenix PRIME and feel what it does for you. With millions of servings under our belt, we know it works and customers have been enjoying the benefits of our multi-patented combination ketone technology for years now. The only thing you have to lose is weight, poor sleep, brain fog, hunger, cravings, and free radicals.
Make your favorite drinks keto with PRIME! Check out some recipes below!


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In 1983, the American cryptographer David Chaum conceived an anonymous cryptographic electronic money called ecash.[7][8] Later, in 1995, he implemented it through Digicash,[9] an early form of cryptographic electronic payments which required user software in order to withdraw notes from a bank and designate specific encrypted keys before it can be sent to a recipient. This allowed the digital currency to be untraceable by the issuing bank, the government, or any third party.
There is no company or centralized organization that controls Ethereum. Ethereum is maintained and improved over time by a diverse global community of contributors who work on everything from the core protocol to consumer applications. This website, just like the rest of Ethereum, was built - and continues to be built - by a collection of people working together.
^ "Bitcoin: The Cryptoanarchists' Answer to Cash". IEEE Spectrum. Archived from the original on 4 June 2012. Around the same time, Nick Szabo, a computer scientist who now blogs about law and the history of money, was one of the first to imagine a new digital currency from the ground up. Although many consider his scheme, which he calls "bit gold", to be a precursor to Bitcoin
The rapid price increase of Ethereum has not only attracted investors but developers too. Ethereum has tens of thousands of developers in its open source community, each contributing to the many layers of the "Ethereum stack". This includes code contributions to the core Ethereum clients, second layer scaling tech and the "decentralized applications" (dApps) that are built on top of the platform. The appeal of Ethereum to developers is unique in that it was the first platform to allow anyone in the world to write and deploy code that would run without the risk of censorship. The community of developers which have formed around these core principles have led to the creation of technologies that could not have existed without the inception of Ethereum, many of which were never predicted. Some of the major use-cases of Ethereum so far have been:
On 3 January 2009, the bitcoin network was created when Nakamoto mined the first block of the chain, known as the genesis block.[25][26] Embedded in the coinbase of this block was the text "The Times 03/Jan/2009 Chancellor on brink of second bailout for banks".[16] This note references a headline published by The Times and has been interpreted as both a timestamp and a comment on the instability caused by fractional-reserve banking.[27]:18


'African Start-Up' on CNN International meets Abiola Olaniran, a Nigerian entrepreneur who created his first game at University and then launched Gamsole, after spotting the growing demand for games worldwide.
According to PricewaterhouseCoopers, Nigeria's gaming market will reach $170 million by 2017. Olanarian and Gamsole are central to that growth as African sales make up a healthy five per cent of his company's growth.
Olanarian talks to 'African Start-Up' about of the early days of his company, recalling he received $35,000 in seed funding, which allowed him to expand. To date, Gamsole has released 35 games and generates over $30,000 a month, selling the games for free but placing advertisements in them.


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When you replace you existing software with a Helix Pharmacy Management System, a Triad Retail Pharmacy System, or install MedTablet, Mobile MedSoft converts your data for you.
With over four decades of experience in developing, installing and supporting software for pharmacies such as yours, Mobile MedSoft has the most efficient, accurate technology, and qualified personnel to make sure your business continues to run smoothly during the transition and into the future.
Standing out from other pharmacy system software developers, Mobile MedSoft does all of its data conversions in-house. This means you know the people doing the conversion and they know you.
We never outsource such an important task to a third party, and we assure a secure data conversion. None of this he said, she said rhetoric. urgent essay help One company to call who is responsible. Best of all, we guarantee your data conversion to work.


Tomorrow I'm going out to northwestern Mass. to restart my photography project. I'm heading NW so that I can take in an exhibit on Picasso and Degas at the Clark in Williamstown, but it seemed like a perfect time to restart a long dormant photo-project. First up: Rowe, a town with a population of 351 in the 2000 census. That seems auspicious for a project that involves photographing all 351 towns and cities in the Commonwealth.
And I'm thinking about rejoining my former camera club when it starts up in a few weeks.