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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.
Genome analysis of three Frankia sp. strains has revealed a high number of transposable elements in two of the strains. Twelve out of the 20 major families of bacterial Insertion Sequence (IS) elements are represented in the 148 annotated transposases of Frankia strain HFPCcI3 (CcI3) comprising 3% of its total coding sequences (CDS). EAN1pec (EAN) has 183 transposase ORFs from 13 IS families comprising 2.2% of its CDS. Strain ACN14a (ACN) differs significantly from the other strains with only 33 transposase ORFs (0.5% of the total CDS) from 9 IS families.
Insertion sequences in the Frankia genomes were analyzed using BLAST searches, PHYML phylogenies and the IRF (Inverted Repeat Finder) algorithms. To identify putative or decaying IS elements, a PSI-TBLASTN search was performed on all three genomes, identifying 36%, 39% and 12% additional putative transposase ORFs than originally annotated in strains CcI3, EAN and ACN, respectively. The distribution of transposase ORFs in each strain was then analysed using a sliding window, revealing significant clustering of elements in regions of the EAN and CcI3 genomes. Lastly the three genomes were aligned with the MAUVE multiple genome alignment tool, revealing several Large Chromosome Rearrangement (LCR) events; many of which correlate to transposase clusters.
Analysis of transposase ORFs in Frankia sp. revealed low inter-strain diversity of transposases, suggesting that the majority of transposase proliferation occurred without recent horizontal transfer of novel mobile elements from outside the genus. Exceptions to this include representatives from the IS3 family in strain EAN and seven IS4 transposases in all three strains that have a lower G+C content, suggesting recent horizontal transfer. The clustering of transposase ORFs near LCRs revealed a tendency for IS elements to be associated with regions of chromosome instability in the three strains. The results of this study suggest that IS elements may help drive chromosome differences in different Frankia sp. strains as they have adapted to a variety of hosts and environments.
The genus Frankia consists of actinobacteria that form root nodule symbioses with non-leguminous plants wherein they fix N2 to ammonia that is assimilated by the plant [1-4]. The genomes of three Frankia sp. strains show a complex pattern of deleted, duplicated and hypothetical genes plus many transposable elements suggesting a high degree of plasticity[5]. Despite having 16S rRNA sequences that are greater than 97% identical to each other, the strains have genome sizes that range from five to nine Mbp in size. Their genome sizes reflect the diversity of plants infected [5]. HFPCcI3 (CcI3), with the smallest genome (5.4 Mbp) of the three, infects plants from one family whereas EAN1pec (EAN), with the largest genome (9 Mbp), infects plants in five families. Strain ACN14a (ACN) has a moderate genome size (7.4 Mbp) and infects plants from two families [1].
The number of transposase open reading frames (ORFs) in the Frankia genomes is not proportional to their sizes, contrary to some models suggesting that larger genomes are likely to contain more mobile genetic elements than smaller genomes [6]. Initial annotations have indicated that transposase ORFs, associated with insertion sequences (ISs), are highly duplicated and diverse in strains CcI3 (148 orfs) and EAN1 (183 orfs), but less so in strain ACN14a (33 orfs).
IS elements are mobile genetic elements that lack a selectable marker gene and insert into the genome of a host without the need for extensive DNA homology at a target site (for a review see [7-10]). Most bacterial IS elements consist of one, or more, transposase ORF(s) that catalyze excision from, and reinsertion into, a genome. They are classified into major families based on amino acid sequence similarity, structure of ORFs devoted to transposition or the presence of flanking repeat sequences [11]. Inverted repeats (IRs) are commonly found flanking the transposase ORF(s) of IS elements, but are notably absent in the IS200/IS605 [12-14] and IS110 families [15]. IRs can contain promoter elements for the flanked ORF(s) and serve as targets for the active transposase [11,16,17]. Transposition often creates small direct repeats (DR) beside the IR sequences of the element, which may be used to identify prior insertion points of that element in the genome.
IS-associated transposase genes are generally poorly expressed [18]. The insertion, excision, or duplication of IS elements can cause insertion mutations or lead to genome rearrangements often, but not always, to the detriment of the host [7,19]. Point mutations that make IS elements hyperactive can be lethal to their host [20-22]. If many ISs are maintained within a genome, they may confer a degree of genome plasticity allowing for rapid adaptation to new environments [6,23,24].
Insertion sequences have also been implicated as causes of large chromosome rearrangements through intra-chromosomal recombination [7,25,26]. Such changes might allow some pathogens to evade or adapt to host defences [27,28]. Large numbers of IS elements have been shown to induce genome deletions and rearrangements in the pathogens B. pertussis and B. parapertussis [29]. Similarly, a hostile or changing soil environment could select for microbes that can quickly adapt to new conditions. It is also possible that IS elements confer no such selective advantage on their hosts. This study focused on annotated transposase ORFs in Frankia sp. and the identification of fragmentary IS elements present in three strains of Frankia sp. Their positioning and diversity suggest roles in driving genome size and strain differences that may have contributed to the adaptation of different Frankia lineages to their hosts and soils.
A total of 364 IS-associated transposase ORFs have been annotated among the three Frankia genomes. Frankia alni strain ACN has 33, Frankia sp. strain CcI3 has 148 and Frankia sp. strain EAN has 183 ORFs distributed among 13 IS families plus several unclassified transposases (Table (Table11 and [5]). This diversity of transposase ORFs is the greatest yet found in bacterial genomes as of this time. The 33 ISs in ACN are in nine families plus the unclassified ORFs. The 148 ORFs in CcI3 are in 12 of the 13 Frankia IS families. Four paralog groups of transposases in CcI3 contain ORFs that, within each group, have >99% amino acid sequence identity; the largest group contains 14 IS4 transposase ORFs that share 100% amino acid identity. These paralog groups reflect probable recent duplication [30]. The 183 ORFs in EAN are even more diverse with members in all families with few that are identical. Inverted repeats were found flanking 16 transposases in EAN and 41 transposases in CCI3 (see Additional File 1: Papersupplemental1.xls).
For identifying transposases from ancient or horizontal transfer [31] we conducted BLAST searches of the non-redundant (nr) database using the amino acid sequences of all annotated Frankia transposases. Most of the ORFs (258, 70.8%) hit transposase ORFs in at least one other Frankia strain (Table (Table1).1). Of these, 103 ORFs had BLAST hits among all three Frankia strains, with 18 (55%) of such ORFs in strain ACN, 35 (23%) in CcI3 and 50 (27%) in EAN. Only 8, 29 and 69 ORFs limited to ACN, CcI3, or EAN respectively (Figure (Figure1).1). Fifty-five of those ORFs hit transposases (Evalue < 10-15) in other bacteria but not in the other strains of Frankia; 42 of those ORFs belong to strain EAN. A complete listing of the results of the BLAST searches against the nr database can be found in Additional File 1: papersupplemental1.xls.
Interestingly, the largest shared group was composed of 80 transposase sequences in CcI3 that retrieved sequences in EAN (Figure (Figure1).1). The second largest group was comprised of 62 ORFs in EAN that hit ORFs in CcI3, making 142 total ORFs shared by CcI3 and EAN. Altogether, more than 67% of all Frankia sp. transposases are present in groups shared between strains CcI3 and EAN (Figure (Figure2).2). The ACN genome contains only 9% of the total number of Frankia sp. transposases but shares most (18/33; 55%) of those ORFs with the other two strains. Eight (24.4%) transposase ORFs in ACN are unique to the strain, compared with 38% (69) of the transposase content of EAN. ACN also lacks the diversity of IS content of the other strains with members of only nine IS families. The majority of novel transposase ORFs found in strain ACN are putative; suggesting that they may be remnant members of other IS families. Ten of the eleven members of the IS3 family in strain EAN have no external BLAST hits at the 10-15 cut-off value.
In order to screen for transposases that may have been more recently horizontally transferred, we compared transposase ORF G+C% to the results of the BLAST searches. The three Frankia genomes have a combined average G+C% of 71.28% [5], so we considered a G+C% of less than 65% to be significantly below the average. Using this cut-off value, we identified 5, 6 and 23 transposase ORFs that had significantly lower G+C content in strains CcI3, ACN and EAN respectively. Members of the IS3 family that are novel to strain EAN make up 39% (9) of the 23 low G+C% transposase ORFs identified in its genome. This fact supports the notion that a majority of the IS3 family transposases were a horizontal acquisition by strain EAN. Excluding the IS3 family in strain EAN, relatively few strain specific transposase ORFs had low G+C percentages. Only three other transposases (one per strain) had a low G+C percentage in addition to no external BLAST hits. Seven of the 23 low G+C transposases were identified as being shared by all three strains, with 1, 2, and 4 ORFs present in strains CcI3, ACN and EAN respectively. All seven ORFs were annotated as IS4 transposases with an average length of 390 amino acids.
Position specific scoring matrices (PSSM's) were generated for the five major IS families found in the original annotation of the three Frankia genomes. Amino acid sequences of all IS110, IS4, IS605, IS630 and IS66 transposases found in the Frankia genomes were used to create five separate PSSM's for each major family. To reduce potential false positives, only two iterations of the PSI-BLAST algorithm were run against the non-redundant database for each PSSM. Each PSSM was then used in separate TBLASTN searches against the nucleotide sequence of each genome. The results of these searches uncovered 36% (53) and 42% (77) more transposases or their remnants in strains CcI3 and EAN, respectively (Table (Table2).2). Only four putative transposase remnants were identified in strain ACN.
Several transposase remnants were identified in intergenic regions, including 21 fragments in CcI3 and 32 in EAN. The remaining putative transposases were previously annotated ORFs that were reclassified as transposases by the PSI-TBLASTN search, with 45 in EAN and 33 in CcI3. One ORF reclassification of note is the reassignment of a family of cytosine-5-methyltransferases as transposases of the IS605 family in EAN (see Additional File 2: PsiBlastSupplemental.xls). Conserved domain feature identification using pre-recorded data on the NCBI site reveals that this family of methyltransferases contains two transposase-associated protein domains and no methyltransferase domains. The presence of transposase protein domains in these ORFs supports their reclassification as IS605 transposases.
Due to the stringency of the search, each novel putative transposase was identified by only one PSSM; however, some hits from the search gave different nucleotide coordinates for the same putative transposase ORF that varied by as much as 100 bp. The hit with the highest percent identity to the query sequence was retained in those situations. False positives were identified as sequences having less than 50 bp of length plus less than 40% amino acid identity to the query sequence. Five false positives were identified in strain EAN and six in strain CcI3. All originally annotated transposases of the five major IS families used in the PSI-TBLASTN search were identified, resulting in no false negatives. Altogether, a total of 37 transposase ORFs plus remnants were identified in ACN, 201 were identified in CcI3 and 261 in EAN. The complete list of reannotated ORFs and truncated derivatives can be found in Additional File 2: PsiBlastSupplemental.xls.
Using the MAUVE genome alignment program [32] we identified 8 and 13 Large Chromosome Rearrangements (LCRs) in strains CcI3 and EAN respectively using ACN as the reference (Additional File 3: PaperSupplemental3.xls). We defined the LCRs as regions greater than 5 kb common to all three strains with a maximum of a 100 bp gap that were out of order or inverted compared to homologous regions in strain ACN. A preliminary GRIMM (Genome Rearrangement In Man and Mouse) analysis [33] of our original MAUVE alignment projected that ACN had the fewest rearrangement events from the node of the trifurcation (data not shown).
Interestingly, nearly all LCRs in strains CcI3 and EAN occur either near the origin of replication of the chromosome or at its terminus. Inversions at the terminus can be explained by recombinase-mediated homologous recombination [34], however, overlays of transposable element density maps on the genomes revealed IS clustering near these regions as well, potentially linking terminus inversions to intra-chromosomal recombination of mobile genetic elements (Figure (Figure3).3). Despite evidence of clustering in these regions, identical transposase ORFs do not flank any identified LCRs as was found with a large genome inversion mediated by IS905 in Lactococcus lactis [35]. Chi square analysis of transposase gene content within each defined LCR segment revealed no significant difference from expected numbers of ORFs for a region of the same size (Additional File 3: PaperSupplemental3.xls). This suggests that the LCRs themselves do not serve as hotspots for IS element insertion, despite their rearrangement with respect to strain ACN. Instead, they appear to be relatively stable genomic islands that have simply moved to new loci within each genome.
Previous heuristic analyses of the three Frankia genomes found more transposase ORFs in regions of each genome lacking synteny with the others [5]. In order to analyze these IS hotspots, a sliding window was used to count the number of transposase ORFs present every 250 kb. All transposase positions found in our PSI-TBLASTN search were used in the data sets for each organism, as the data recovered from that analysis most likely indicates recent as well as current positions of IS elements in the genomes. To assess statistically the distribution of IS elements, we used a probability mass function (pmf) derived from the average number of transposases found per window for each genome. That number varied from 3, to 12 to 10 ORFs in ACN, CcI3, and EAN respectively.
Strain ACN was found to have significant clustering of transposase ORFs near 3 and 5 Mb (Figure (Figure3b).3b). Despite the relatively smaller dataset compared to the other two strains, these clusters in ACN represented 9 and 11 transposases in a 250 kb window giving their clustering a greater than 99% confidence. The ACN genome also has significant clustering of transposase ORFs near the terminus mirroring the heterogeneity of the terminus found in all three genomes.
Approximately 57% of all transposase ORFs identified in strain CcI3 are found either near the origin, or between the 2 Mb to 2.5 Mb region. Both of these regions have clusters well above a 95% confidence interval, suggesting that the clusters are not solely due to random insertion. These regions are in proximity to LCRs in the CcI3 genome, suggesting preferential IS insertion around these regions may be linked with Frankia genome rearrangement events. Again, the terminus of this genome serves as a hotspot of transposase ORFs in addition to a large peak near the 4.9 Mb region of the genome closer to the origin. This region of the genome was found to correspond to a potential deletion (Figures (Figures33 and and44).
The number of transposase ORFs found in the EAN genome dwarfs those found in the other two strains; a clear pattern of clustering was found. Two major clusters were near and symmetrically oriented around the terminus near 3.5 and 5.5 Mb. Both clusters had a confidence interval greater than 99%, and corresponded to breaks in synteny with the ACN genome. The lack of synteny near these clusters suggests that these regions may contain gene duplicates. An increased presence of the top three duplicated gene families in strain EAN near these clusters confirms this prediction (Table (Table3).3). The symmetrical appearance of these clusters was striking but their significance remains unknown.
Using a similar sliding window analysis, we mapped ORFs present in strains EAN and ACN but deleted in strain CcI3 [5]. A comparison of this map (Figure (Figure3a)3a) with the transposase cluster map (Figure (Figure3b)3b) revealed a pattern of gene deletion corresponding to some clusters of transposase ORFs in the CcI3 genome. Comparing all ORFs in these regions revealed a general lack of synteny in all three strains, with a notable reduction of both nucleotides and number of ORFs in strain CcI3 in comparison to EAN. Three such regions flanked by syntenic LCBs were selected for a more detailed comparison of gene position and content among the three strains (Figure (Figure4).4). Regions A and B have fewer ORFs in CcI3's genome compared to ACN; region C showed an increase in sequence over ACN. Only two transposase ORFs were present in strain ACN over all three regions. CcI3 had 8, 10 and 35, and EAN had 10, 7 and 14 transposase ORFs in regions A, B and C, respectively.
The termini of ACN and EAN have the largest clusters of genes deleted in CcI3; however, a general lack of synteny among all three strains in this region precluded a neighborhood analysis. In general, the largest significant cluster of transposases from the 2 Mb to 2.5 Mb region of strain CcI3 corresponds to regions of gene deletion.
Despite having transposase ORF's from thirteen major IS families, relatively few are strain-specific in Frankia. The presence of diverse and numerous IS elements in a genome is often believed to reflect horizontal gene transfer [6]. Given the different geographic origins of the three strains [1,5] and the sequence similarity of their transposase ORFs, it is unlikely that recent horizontal gene transfer is solely responsible for the proliferation of transposases in strains EAN and CcI3. Instead, it appears that the majority of transposase ORFs are descended from ones that were present in a common ancestor that diverged with the emergence of actinorhizal plant families approximately 100 million years ago [36]. Some proliferated or were maintained in lineages leading to strains EAN and CcI3, but some were lost from the ACN lineage. One family of transposases, the IS3 transposases of EAN, and a small subset of IS4 transposases are clearly horizontally acquired. These 16 ORFs only comprise ~5% of the total number of transposases annotated. The general lack of novel IS elements is further supported by the near absence of unique transposase ORFs in ACN.
Remnant transposases, as detected through the PSI-TBLASTN method, indicate past IS activity. The low ratio of annotated IS elements to newly discovered fragments in strain ACN suggests that IS movement was selected against in this lineage. By contrast, the EAN genome has at least 81 transposase remnants, suggesting that transposition and/or duplication are common and ongoing. Transposase ORFs are associated with heterogeneity and expansion at the terminus of the EAN genome, as compared to the ACN genome, as well as the large number of LCRs identified by the MAUVE alignment. IS elements have long been known to facilitate chromosome rearrangements [7,28,37,38] so breaks in synteny near identifiable IS clusters are likely due to IS-mediated rearrangements. Support for such activity outside of observed proximity would require direct observation of chromosome rearrangement.
With only 33 identifiable transposase ORFs in a genome of 7 Mb, the ACN genome has fewer transposases than the other two Frankia genomes. Given the potentially deleterious nature of IS elements [6,7], one would expect that transposition events would be fixed infrequently in a population. In phylogenetic analyses, CcI3 and ACN are more closely related than either is to EAN [5]. Thus, the presence of nearly identical transposase ORFs in strains EAN and CcI3, but not in ACN suggests that the common ancestor of all three strains contained elements from many IS families that were retained and duplicated in lineages leading to EAN and CcI3 but lost in ACN. All lineages continued to acquire novel ISs through horizontal transfer.
In contrast to strain ACN, 77 remnant and 182 annotated transposases are found in the EAN genome. The transposase ORFs identified in this study amount to approximately 3% of the EAN genome, and nearly 4% of the total CDS. The high numbers of IS elements may help strains in the EAN lineage adapt quickly to new niches. A similar observation has been made in clinical isolates of Enterococcus faecium in which IS elements were implicated in developing new subspecies that are better adapted to a hospital environment [23]. Of the three sequenced strains, members of the EAN lineage infect the broadest range of host plant families [1]. This broad host range may be enabled by gene duplications, and rearrangements, driven by IS element insertion and cointegrate formation. At present, 123 pseudogenes have been identified in the finished assembly of the EAN genome. Similar pseudogene and IS element content is found in Yersinia pestis, another dynamic genome [39].
IS clusters determined by our sliding window analysis may indicate genome instability in certain regions of the EAN genome. At least two statistically significant IS clusters were identified; one is almost entirely composed of transposases identified by the PSI-TBLASTN search (red bars in Figure Figure5).5). We also identified regions of the EAN genome that have expanded by gene duplication (Figure (Figure4).4). An increased number of ORFs and other nucleotide sequence in regions that are comparatively deleted in strain CcI3 suggest that such regions in the Frankia genomes have been subject to major genome polymorphisms.
The genome of CcI3 appears to be undergoing genome ratcheting coincident with specialization within a narrow range of plant hosts and soil types [5]. IS clusters, consisting largely of those present in the common ancestor of the three Frankia strains, may have promoted the deletion of large regions around the terminus during homologous recombination (Figure (Figure3).3). This is evidenced by the paucity of non-transposase gene duplicates in strain CcI3 compared to strain EAN and ACN [5], as well as by the presence of 53 fragmentary transposases identified by the PSI-TBLASTN analysis. Analysis of regions of the CcI3 genome that have lost ORFs with respect to the other two strains has shown a loss of DNA in two regions of the genome identified from the sliding window plot (Figure (Figure4).4). The presence of clusters of transposase ORFs in these regions suggest that the IS elements were either responsible for the deletion events, or capitalized on the fragility of this hotspot. Analysis of the Frankia proteome using LC MS/MS has recovered peptide fragments associated with five transposases present in deletion windows A (3) and B (2). Peptides of nine other transposase present at the terminus of the CcI3 genome were also identified (J.E. Mastronunzio and Y. Huang. personal communication). Given that transposases can have numerous barriers to expression during transcription and translation [17,40,41], finding transposase peptides suggests that transposases remain active in the Frankia genomes.
Most transposase ORFs in CcI3 have homologs in the other strains, so the emergence of a strain of Frankia that infects plants from a single family appears to have been accomplished primarily through the activity of native mobile genetic elements. Whereas EAN enjoys a large host plant family range and a geographically diverse distribution, CcI3's original native range incorporates parts of Australia and Pacific islands [42]. Without diverse selective pressures, intramolecular recombination may have led to the reduction of the CcI3 genome and a narrower host range [43].
In the three sequenced Frankia genomes, IS proliferation has occurred largely without the recent input of IS elements from horizontal gene transfer. Clustering of existing transposases in the genomes is statistically significant and corresponds to regions lacking synteny with the other two strains. This finding suggests that such sequences within strains CcI3 and EAN have contributed to genome contraction and gene duplication events respectively. While it may seem contradictory to propose that IS elements have catalyzed both expansion and reduction within the Frankia genomes, selective pressures in conjunction with environmental niche availability appear to be nudging the genomes in opposite directions.
The annotations and amino acid sequences for the transposase genes for Frankia strains CcI3 [genbank:CP000249], EAN [genbank:CP000820] and ACN [genbank:CT573213] were obtained from the NCBI Genbank website http://www.ncbi.nlm.nih.gov/. A useful resource for IS classification and nomenclature is the ISfinder website http://www-is.biotoul.fr/ but Frankia ISs are only partially classified in the most recent version. FASTA files of Frankia transposase amino acid sequences obtained from Genbank were used in a BLAST [44] search of the non-redundant (nr) protein database. The E-value cutoff was set at 10-15in order to account for smaller transposase sequences. Reciprocal hits of the smaller transposase amino acid sequences often generated E-values of less than 10-20.
Only the amino acid sequence of each transposase ORF was used due to the actinobacterial tendency to replace the third base pair of each codon with a guanine or cytosine residue [1,4]. This tendency also prevented a dn/ds analysis of the transposase orfs as is traditionally used in studies of this type to determine purifying selection against transposable elements [45]. Only annotated transposase orfs were used in this analysis. Inverted repeats were also omitted from the analysis as they do not contribute to the amino acid sequence of the transposase protein.
IS element inverted repeats were identified using the Inverted Repeat Finder (IRF) program version 2.17 http://tandem.bu.edu/cgi-bin/irdb/irdb.exe[46]. Initial IRF processing was performed on the genomes of the three Frankia strains CcI3, ACN14a and EAN1 using the parameters 2, 3, 5, 40 (match, mismatch, indel, minimum score). The k-tuple values used in this initial processing corresponded to 500 for the T4 tuple class, 2000 for the T5 tuple class and 10000 for the T7 tuple class. The "lookahead test" and the "third alignment going inwards" options were also selected for the genome processing in order to more stringently identify IR sequences.
Fragmented IS elements were identified using a PSI-TBLASTN approach [47,48]. Position specific scoring matrices (PSSM's) were created using the amino acid sequences of annotated Frankia sp. transposases as queries in BLAST searches against the nr database in two PSI-BLAST iterations. Separate PSSM's were made for each annotated IS family. Searches were then performed on a six-frame translation of the nucleotide sequence of each respective genome using the TBLASTN program. Results were sorted based on sequence percent identity to the query. Hits with percent identities lower than 30% were discarded, as were hits with less than 50 bp in sequence length.
Genomes were aligned using the MAUVE genome alignment program version 2.1.1 [32]. Original alignment of the genomes was done with a minimum backbone setting of 100 bp. Local colinear block (LCB) weight of this alignment was increased until only 29 LCB's were identified by the program. LCB order numbers were then input to the GRIMM web server for rearrangement modeling [33] to determine which genome had fewer rearrangement events from the node of the trifurcation. Strain ACN was determined to have fewer LCR events and was used as an alignment standard. Subsequent MAUVE alignment of the genomes used a minimum backbone of 1200 bp, with a 500 bp minimum island value to eliminate spurious LCB assignments. Backbone and island files were visualized using the Genvision plugin for Adobe Illustrator©.
A 250 kb sliding window was used to determine the total number of elements present within each window of the genomes (accounting for their circular chromosomes). The nucleotide mid-point of each element was used as the position of the element. For ease of manipulation, only window positions where there were changes in the number total of features were used to generate the distribution figures. Using the average number of elements per window, the probability of finding particular number of elements under a Poisson distribution was determined by using the probability mass function (pmf).
Where "λ" is the average number of elements per window and "k" is the number of elements in a given interval. Genes that were predicted to have been deleted in strain CcI3 yet were still present in both ACN and EAN were mapped using another 250 kb sliding window. Windows in which the number of CcI3 deleted genes rose above the upper 99th percentile in either ACN or EAN were compared against MAUVE alignments of all three genomes. If any region had a significant cluster of deleted genes and was found between syntenic LCB's, it was depicted in Figure Figure4.4. Three such regions were identified, despite other regions of significant clustering of deleted genes.
PL performed the sliding window analysis of deleted genes and transposase ORFs and provided technical support in the use of several bioinformatics programs. JPG, LST, PN and DRB provided the intellectual framework of the study and recommended several bioinformatics analyses. DRB and DMB performed the bioinformatics analyses, sorted data from those analyses and wrote the manuscript. All authors read and approved the final manuscript.
Results of a BLASTP search of all annotated Frankia transposases. Excel workbook containing formatted data from a BLASTP search of the non-redundant database using all annotated transposase amino acid sequences from the three Frankia strains as a query. Transposase ORFs that had a G+C% content less than 65% have a listing of their G+C percentage in the "G+C%" column. Inverted Repeats identified by the IRF program http://tandem.bu.edu/cgi-bin/irdb/irdb.exe are listed in the "Left IR" and "Right IR" columns.
Results of a PSI-TBLASTN search of the three Frankia genomes using five transposase PSSMs. Excel workbook containing PSI-TBLASTN search results from all three Frankia genomes.
Positions of Large Chromosome Rearrangements in EAN and CcI3. Excel workbook containing LCR coordinates and transposase contents in strains CcI3 and EAN1.
We kindly thank Greg Fournier of the University of Connecticut for his advice on statistical analysis. We would also like to thank the Biotech Center of the University of Connecticut for their computational support. This work was supported by Grant #EF-0333173 from the National Science Foundation Microbial Genome sequencing program to DRB.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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I tried to schedule a delivery with A-1 Seltzer in North Hollywood, and the phone number was disconnected. A drive by revealed their warehouse "available."
Anyone know what happened? Looks like they're out of business.
I don't suppose there's another place that does that same old-fashioned seltzer delivery in the LA area?

subset: commoncrawl


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Selena Gomez doesn't regret not having a normal childhood. In fact, the "Hands to Myself" singer simply doesn't care she had an unusual childhood.
During a candid interview with GQ, Gomez, now 23, recalled growing up in the spotlight as a child star.
"I didn't have an opportunity to figure out my life without people having an opinion every step of the way," the Barney and Friends alum explained.
That said, Gomez isn't looking for any pity or making excuses.
"I chose this," she added. "So I'm not gonna sit here and say, 'Oh, my God, poor me, I didn't have a normal childhood.' I don't give a f–k about that."
The "Revival" songstress, who is currently the most-followed person on Instagram, has clearly grown up. In the upcoming May 2016 issue, Gomez drops F-bombs and poses topless in just a pair of jean shorts.
"It's been such a transitional year for me," Gomez previously told Into the Gloss in October 2014. "Sometimes, I'll be honest, I don't handle all of this as well as people think, so it's been important to really figure out what makes me feel grounded."
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Cowboys approaching ride horses into a stable. Cattle graze. Oil wells and military airfield from overhead. Distant view of Dallas Texas city skyline. Night view of streets and signs in Amarillo Texas. Daytime view of a tall skyscraper building and wide street in Houston Texas with cars and traffic View of the Hotel Nimitz in Fredericksburg Texas. Downtown Fort Worth Texas city street with shops, offices, and cars. Downtown area in Pampa Texas. Street corner view in Kerrville Texas. Two different views of The Alamo and surroundings in San Antonio, Texas. A water tower and oil rigs in Kilgore Texas. Downtown area of El Paso, Texas from elevated view. Street view in downtown Beaumont, Texas. Downtown view in Wichita Falls, Texas. Downtown San Angelo Texas views. Distant view of the city of Austin Texas from an elevated position, and then in downtown Austin featuring the State House Capitol building. Cattle, sheep, and sorghum farming views. Rice fields and Turkey ranches. Many oil rigs and oil wells. A busy shipyard in Texas making ships for World War II war effort. A sprawling aircraft factory. Oil rigs pumping oil. Farmer on tractor plows wheat field. View of rice harvesting (Massey Harris Self Propelled Harvest Brigade). Men stack magnesium bars. Magnesium factory next to ocean in Texas. Hand cotton picking and view of a mechanical cotton picker. Tractor pulling sorghum harvester. Sulfur mining and Helium plant in Amarillo. Black clouds in sky from burning natural gas in making synthetic rubber for war effort. Cliff of sulfur dynamited and loaded by train crane. Cowboys herd cattle and view of Fort Worth Stockyards and Exchange. Angora goat farm and turkey farming. War time workers manufacturing. Alabama and Coushatta Native American Indian village with flag of service stars for men in the war. Indian children with elder and Indian logging camp. Campus of University of Texas and State Capitol in Austin. Well dressed women exit Dallas department store.
Events related to the testing of triangular parachute on Mark III Aerial Illuminating flares in San Antonio, Texas. Aerial Views of flare descending by parachute. Partially inflated parachute with expended flare casing descend and hit ground.
Twin .30 cal machine guns positioned on swivel-bar in DH-4 cockpit and fired at target during training in Antonio, Texas. Pair of .30 cal machine guns fired at target as seen from DH-4 cockpit. DH-4 on firing test stand as pairs of .30 cal machine guns fire through rotating propeller and from mount in rear cockpit. Cannon in a GA-1 airship open nose turret and gun fired. GA-1 parked as gun is fired.
The San Antonio Municipal Auditorium built at Veterans Memorial Plaza, honoring the veterans of World War One. The auditorium was opened in 1926. Views of automobiles driving near the auditorium, in 1928.
Tourist bus travels on a bridge and arrives at the 'Alamo' in San Antonio, Texas, which was a former Catholic mission and fortress compound but now a museum. View of Iron gates at the museum.
Report from Texas: Texas' contribution to World War II on both battlefield and home front. Horsemen gallop across the terrain. Man on horse with cattle. Texas increases its production of synthetic rubber, magnesium, sulphur and agricultural products. Factories and industries. Construction work underway. An airfield. Soldiers march. Views of Dallas, Houston, Fredericksburg, Hotel Nimitz, Ft. Worth, Kerrville, Pampa, San Antonio, Kilgore, El Paso, Beaumont, Wichita Falls, San Angelo and Austin.


text:
Discussion in 'Disney Resorts' started by babygirlamg, Mar 9, 2013.
does the fridge at AOA have a frezzer part???
In the suites, yes there is a small freezer section. We were able to fit about 4 water bottles in there.
In the regular rooms, the fridge does not have the freezer section.
Do you know if a box or two of eggo waffles will fit in the freezer? I plan to order them from Garden Grocer for breakfasts for the kids and bring a little toaster from home.
No, the freezer is not large enough for boxes of frozen food.
It looks something like this:
16 oz bottles of water fit as long as they were on their side. I could fit about 4 of them side by side.
Separate names with a comma.


text:
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Tyler Willis
Saddle Bronc Riding, Bull Riding
2/12/1990 Steamboat Springs, Colo.
Joined PRCA:
PRCA Career Earnings:
World Titles Won:
WNFR Qualifications:
2 (2011, 2013)
Current Residence:
Wheatland, Wyo.
2013 world standings place: 14th
2013 Wrangler NFR place: 14th
2013 Wrangler NFR earnings: $7,813
2013 earnings: $83,098
Tour Finale qualifications: 1 (2013)
Heartland ProRodeo Series Championships: 1 (2009)
RNCFR qualifications: 1 (2012)
Education: Vernon (Texas) College, farm and ranch management
Career Highlights
• 2013: Placed in two rounds of the Wrangler National Finals Rodeo. Co-champion at the Sanders County Fair & PRCA Rodeo (Plains, Mont.)
• 2012: Won Nebraska's Big Rodeo (Burwell, Neb.); the Fort Worth (Texas) Xtreme Bulls Division; the Goliad (Texas) County Fair PRCA Rodeo and the Butterfield Stage Days PRCA Rodeo (Bridgeport, Texas). Finished 19th in the world standings with $53,529
• 2011: Placed in five rounds of his Wrangler National Finals Rodeo debut to finish third in the average with 423.5 points and sixth in the final world standings with $146,558. Won the Amarillo (Texas) Tri-State Fair and Rodeo; the Silver State Stampede (Elko., Nev.) and the Magic Valley Stampede (Filer, Idaho). Co-champion at the Ram Mountain States Circuit Finals Rodeo (Rock Springs, Wyo.); the Sisters (Ore.) Rodeo and the Sanders County Fair & PRCA Rodeo (Plains, Mont.).
• 2010: Won the Days of '47 Rodeo (West Valley City, Utah). Finished 31st in the final world standings with $49,572
• 2009: Won the Heartland ProRodeo Championships (Waco, Texas); the Santa Rosa Roundup (Vernon, Texas) and the Sheridan WYO Rodeo. He was co-champion at the ABC Pro Rodeo (Lubbock, Texas) and finished second at the Cody (Wyo.) Xtreme Bulls. Finished 19th in the world standings with $54,825
Wyoming high school bull riding champion, 2007 and 2008; saddle bronc riding champion 2008. Wyoming Rodeo Association bull riding champion, 2006.
PRCA Bull Riding Rookie of the Year, 2009
5-11, 155 … Single … Parents Don and Tammy Willis own 12,000-acre ranch in Wheatland, Wyo.; has two sisters, Jennifer and Kaila, and a younger brother, Nathan … His dad was a bareback rider and Tyler learned to ride from his uncle, Wes Palmer … Enjoys hunting and being outdoors, working on the family's ranch … Graduated from Wheatland (Wyo.) High School in 2008. He competed through his junior year as a wrestler in the 145-pound division; qualified for the state meet three times ... Travels with Sam Wyatt, Seth Glause, Taylor Cowan, Ryan Shanklin, Pat Geipel and Ty Hamaker at various times ... Favorite rodeo is the Cody (Wyo.) Xtreme Bulls … Favorite food is steak
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text: Hi all- first post here. If it wasn't so darn late I'd introduce myself- with your forbearance I'll leave that for another day. This is probably a dumb question - but here goes. I have a 70's vintage Epiphone dreadnaught I've had for years. Took it to a local guy a while ago and had it set up, with 10s on it (Phosphor Bronze Martins). I've been playing it mainly to (re)build some hand strength and calluses. Initially it sounded just fine but suddenly it sounds just a little buzzy all over. (I'd almost describe the tone as 'fuzzy'.) Subtle change in the timbre but you know its not right. Is that what happens when strings die - they go suddenly? Changing up the strings is the first thing I thought of (they're nearly a year old but didn't get regular use until the last few months.) Anyone ever see this before? (Thanks in advance!)


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KNOXVILLE (WATE) – If you have ever taught, you know the job is both rewarding and exhausting. A Knoxville group is hoping to give teachers a boost.
Over 1,500 educators, youth pastors, coaches and role models gathered Tuesday at the Tennessee Theatre for Reach Them to Teach Them's annual event. The keynote speaker, John O'Leary, is the author of the book "On Fire."
Crawford, who is a Knox County teacher, founded Reach Them To Teach Them in 2006. She said she returned to teaching after starting a family, had many of the same students, but noticed some of them had lost their passion for learning.
Feeling weary and needing a firm reminder of why she needed to keep teaching after nearly 20 years in the classroom, Crawford turned to an old cassette recording of Dr. Guy Doud sharing his insights as National Teacher of the Year. She said she heard Doud talked about what really mattered in teaching is making a difference in her students' lives.
Crawford was able to get Doud to come to Knoxville to speak to local teachers. She started Reach Them to Teach Them after she says she realized that teachers make a profound impact on student's lives, but that they also needed help. The non-profit is a call to teachers to be a positive influence in the life of a child by building an authentic relationship with an adult role model.
Now in it's 11th year, Crawford said she really hopes to get to "fire things up." This year the theme is "all-in" and Reach Them to Teach Them is inviting anyone who impacts students throughout the school day, including everyone from bus drivers, cafeteria workers, custodial staff, secretaries to teachers.
"We want to appreciate teachers and the sacrifices they make, we want to inspire them and give them something that brings the light back into their eyes when they walk into their classroom, but more importantly and most importantly is the challenge," said Crawford. "We want teachers to walk away from the Reach Them to Teach Them event different than the way they came in, more mindful, more cognizant of the power they have to impact the lives of their students."
Food City and community sponsors have stepped-up to sponsor the event so there is no cost to attend, according to Crawford. A Petro's dinner will also be provided to all guests.
"It is tough to be a teacher and it is challenging, but it is also tough to be a student. It's challenging to be a student. In today's world there are many broken homes, many students who don't have anybody in their lives to see something in them that they don't see in themselves, so the job of a teacher is absolutely critical for the kids."
Volunteers in Reach Them to Teach Them are called the "dream team." they are made up of community leaders, ministers, coaches, parents and many others united by their commitment to invest in the future leaders of the world. Since starting, the group has grown to include educators outside of Knoxville.
"An authentic relationship with an adult role model is vital to the successful development of any child, regardless of race, creed or socio-economic status," said Crawford. "Kids must have someone who believes in them, challenges them, and talks with them about what really matters in life."
For more information on Reach Them to Teach Them and how to get involved, visit the organization's website.
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The lines are moving again at the United Airlines terminal at Los Angeles International Airport after a more than five hour nationwide computer outage left hundreds of passengers stuck on the ground.
Airport spokeswoman Nancy Castles says United began processing passengers shortly after 10 p.m. Friday when the airline announced that its systems were up and running again. The outage began at about 5:30 p.m.
Castles says 32 outbound flights were delayed and two flights - one to San Francisco and one to London - were canceled. She says the affected flights will all get off the ground Friday evening or early Saturday.
She says passengers were orderly and patient and no problems were reported.


text: Yo im hosting a free mod menu for xbox on WaW and yes you can infected people just send me a message or just go to my youtube im on it all day. user name on youtube is GangstaJr24. If i did infect you please help me out by thanking me on here im kinda new to this web sit. Only today im hosting so send me a message now dawg!


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Team Guide by R4INB0WDASH
Not Updated For Current Season
This guide has not yet been updated for the current season. Please keep this in mind while reading. You can see the most recently updated guides on the browse guides page.
Thank You!
Your votes and comments encourage our guide authors to continue
creating helpful guides for the League of Legends community.
Azir Support S4
Azir Build
Ability Sequence
Not Updated For Current Season
The masteries shown here are not yet updated for the current season, the guide author needs to set up the new masteries. As such, they will be different than the masteries you see in-game.
Threats to Azir with this build
|Vayne is a great combo with Azir, as Azir can poke enemy adc and vayne is allowed safe farming. When initiating vayne can stun enemies into Azir's ult and earn a free kill. This combo would be strongly effective.
Going In A TeamFight
Get one of your sand soldiers behind the fed target or the enemy team and e to it. Use your ult to fling them back into your team and spawn the soldiers and destroy them. If your w is behind them but the target/team flash use your soldiers Q or you can flash then ult them into team.


text:
Inbound visitors may browse the " Advice for Travellers " and the " FAQs " sections to learn about the protection for them. They may also browse the "List of Registered Shops" and "Shops whose Registration has been Cancelled".
For updates on seasonal influenza, please visit the website of the Centre for Health Protection.
For updates on communicable diseases such as avian influenza and Ebola virus disease, please visit the Centre for Health Protection website.Travellers wishing to learn about latest news on health advice, please visit the Hong Kong Travellers' Health Service website and the Travel Health Bulletin.
Please browse the Guidance for booking cruise products through travel agents .
Cases of Travel Agent ViolationsRevocations / Suspensions of Tourist Guide PassesRevocations / Suspensions of Tour Escort PassesDemerit Records of Registered ShopsSuspensions / Revocations of Registered Shops
Hong Kong residents planning to travel overseas are advised to browse the Security Bureau's OTA webpage and use the Hong Kong Immigration Department's ROTI service to receive OTA updates and related information from the Government.
Hong Kong residents who need help when they are outside the city may call the 24-hour hotline of the Immigration Department's Assistance to Hong Kong Residents Unit on (852)1868.
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Members interested in the captioned seminar to be held on 6 December should register by 30 November (Circular C1687).
Members are advised to encourage their tour escorts to download and use the captioned mobile app. (Circular C1686).
Members may refer to Circular C1685 for the details of the captioned services.
The deadline for fifth-round applications is 31 December (Circular C1684).
Please click here for the dedicated email addresses of Hong Kong's receiving agents. The dedicated email addresses of Shenzhen's outbound agencies can be downloaded here (password not required).
Members are urged to formulate and regularly review their risk management measures for information security (Circular C1683 ).
Members interested in the captioned briefing to be held on 28 November should register by 22 November (Circular C1682).
This year's Annual General Meeting is scheduled for 30 November. Members may refer to Circular C1681 for the notice and audited accounts.
The captioned course, which will commence on 28 November, is now open for enrolment (Circular C1677).
Members may refer to Circular C1676 for the candidates standing for the coming director election.
Want to know about the characteristics of youth tourists? Please click here to read the "Cover Story" of the latest issue of The Voice.
Members are advised to pay attention to the matteres mentioned in Circular 1666 concerning insurance for outbound package tours.
Inbound agents that need student helpers to receive large parties from cruise ships visiting Hong Kong or offer assistance in MICE events on an ad hoc or temporary basis may contact various universities and tertiary institutions (" Latest News ").
Members may go to the SME Funding Schemes' website and the SME Financing Guarantee Scheme's web page for details about various schemes for SMEs.
Industry members are welcome to use the e-learning platform (in Chinese only) launched by the Quality Tourism Services Association to enhance their service standards.
Continuing Professional Development (CPD) for tourist guides...2016-4-14
Guide Accreditation System
of the Geopark Guide Accreditation System
Security Bureau's mobile app "Safeguard
HK" is launched.
Latest information on courses for tour escorts
Security Bureau's mobile app "Safeguard
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Tourist guides must not allow others to assist them in receiving mainland inbound tours or to receive the tours in their stead (" Latest News ").
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The Certificate Courses for Outbound Tour Escorts to be held during the following period is now open for enrolment: 2017 2018
The "Handy Tips for Tour Escorts" covers general rules for healthy travel, basic first aid, insurance claims procedures and useful telephone numbers.


text:
- Please select a date to see the proper prices!
Description of the accommodation:
Rooms, services Take a few-days trip to Bloomington and enjoy the hospitality of COUNTRY INN SUITES NORMAL WEST Bloomington. The hotel is offering a tasfefully furnished, nice room for those looking for a place to stay at in Bloomington. To learn more about the exact range of services of COUNTRY INN SUITES NORMAL WEST Bloomington (including services for an extra fee) and the exact equipment of the rooms, please check the information sheet below.
Payment Credit card guarantee or advance bank card payment may be necessary for finalizing your reservation, depending on the exact room, date and price.
Arrival, opening hours You will be able to speak with the personnel in English.
COUNTRY INN SUITES NORMAL WEST Bloomington rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
- Jacuzzi
Pet friendly, Elevator


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@wyattcenac warming up before his SF Sketch Fest set. I think he's going to try to eat the pair like David Blaine or smash them like Gallagher. Either way he's still the man. #soleswithsoul #wyattcenac


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All Terrain Cycles is the UK's Largest & Oldest Independent Bike Store. Buy mountain bikes, road bikes and cycle clothing. 3 years 0% Finance available on all bikes.
Product Code: 116158
All the parts needed to refurbish the TS2 Professional wheel Truing Stand.
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For those of you who used to follow me on Google+, I know that my goodbye was sudden. But I've moved on from Google+. It was a good experience and I gained some lessons from it.
One of those things was the proper place to post. A blog, and not Google+ will now be where I post things. I won't delete my Google+ account. I will instead keep it for the memories. It was fun and a great time for me. But now I will post here.
But why?
People on Google+ come and go. My time on Google+ is no more. It's not the last you'll all hear from me. So stay tuned!
|An old pic from Google+ of my cat Jewel.


text:
The extreme weather of February has played its hand! We are sorry to say that we must cancel the remainder of our series, Lojong - Developing the Good Heart, with Alison Landoni.
We have already had to cancel a number of classes and Alison's schedule will not allow us to extend the series beyond the 10 scheduled classes.
We will be back in the spring with some special classes and a workshop looking at different parts of this beautiful practice.
In the meantime, look for our other offerings here on our website.
With our thanks,
Wisdom's Heart Programming


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Save the suffering crippled Elephants at Ringling
Numerous elephants with the Ringling Bros. circus are suffering from crippling injuries and are in chronic pain--yet
Numerous elephants with the Ringling Bros. circus are suffering from crippling injuries and are in chronic pain--yet
We ARE Citizen Oath KeepersCITIZEN OATHI, [name] Citizen Oath Keeper, do solemly swear (or affirm) that I will
Because this is horrendous and hideous. Can you imagine the fear these animals must be in the last minutes of their
As many as 200 million females are missing because of gendercide. Stand up for girls and women everywhere.
Why must innocent animals be used in Human wars?
2014 Winter Olympics, authorities have employed a 'pest control' company to catch and dispose of dogs in the city.
One decision could forever change the future of grey wolves in United States. The U.S. Fish and Wildlife Service (FWS)
Please express support and sympathy for the families who lost loved ones in the Sandy Hook shooting, and those injured.
Borko's story - Bulgaria: Lynch Mob Beats Defenceless Crippled Doggy
Wolf hunting is not needed in Michigan! With less than 700 wolves across the span of 96,720 sq miles, how could anyone
If you live in Wisconsin - contact your legislators and urge them to support Senator Fred Risser's bill to ban
An awesome trend is about to start on twitter in about 3 hours. As many people as possible all of the world will be
Wisconsin DNR Proposes Allowing Packs of Dogs to "Train" On Wolves During March Denning and Birthing Season. THIS
--Devastating News!!!-- "The DNR now wants to allow hounders to 'train' their dogs on wolves with no restrictions
'"DNR wants to allow hounders to "train" their dogs on wolves with no restrictions right through the breeding, denning,
Dutch was accused of being a "vicious" or "dangerous" dog and is now in danger of euthanization by a Court Order. In
BLOG- http://justiceformaryanimaladvocacy.blogspot.com.au/2013/01/remove-pitbull-defense-spray-from.html Stop Retailers
Judge Jennings seems to recognize the atrocity of Mr. Dugan's actions as demonstrated by the high bail of $20,000 cash
A 23-year-old woman was battling for life in New Delhi on Monday after a gang of men tortured and raped her in a moving
Because it is time to ask for an action by Government and Judiciary.
The Second Amendment: "A well regulated militia, being necessary to the security of a free state, the right of the
48,000 Americans will be murdered with guns during President Obama's 2nd term. We must demand a plan to end gun
The killing of Yellowstone's monitored wolves is the last straw in the wave of wolf slaughtering going on in America.
TELL THE PRESIDENT THAT ANIMAL CRUELTY NEEDS TO BE TAKEN SERIOUSLY....make the people who commit these acts of violence
This page advocates the slaughter, and consumption of a religious icon.
Federal agent Jamie P. Olson set his dogs on live coyotes that were captured by steel-jawed traps. Then he documented
Wouldn't you want to know if a violent or repeat animal abuser were living next door to you and your family? Animal
NY Gov Andrew Cuomo is considering lifting the ban on fracking in New York – this must not happen!
PLEASE SIGN ...this woman is very DISTURBED! WAUSAU Wisconsin — A 20-year-old Wausau woman called -Sean D. Janas - was
By signing your name to this petition we are sending a loud and clear message that 'We the People' have had enough of
I really am hoping that you all will sign this petition to get justice for "HARLEY". PLEASE help me put pressure on the
The rare Mexican Gray Wolf, a sub-species of the gray wolf, with a distinct DNA, is becoming extinct with only 59 wolves
Animal cruelty and the case for harsher punishment.. Dear members of Let's turn Facebook Orange for Animal Cruelty
American Museum of Agriculture in Lubbock, Texas - kills two healthy mules for art exhibit... Word that two, still
NOVEMBER 11 is VETERAN'S DAY! The Congress returned to work Sep. 10 and if you really want to make this a TRUE VETERANS
Please help save the neeedless slaughtering of wolves as a means of "managing" the wolf population. Humans do not have
I want people to sign this to show our disgust with Facebook in allowing a page which will only cause distress for those
Many of us rely on the "Chat with Groups" function in Facebook groups to socialize, to enhance the gaming experience of
For 6 years, we at The 50 Million Missing Campaign have been working hard to tell the world about the ongoing female
Take a stand against ocean degradation and negative human impact on dolphins and whales through our Whale Call Project.
We are believers that all animalsdeserve respect and dignity. All forms of animal abuse should NEVER be tolerated!


text:
Primary Port Of Departure:
Sharm el-Sheikh, Qesm Sharm Ash Sheikh, South Sinai, Egypt
Dive Shop Listing Claimed by Karim fayed
South Moon is a 27 m long wooden-hulled vessel. She can accommodate 18 passengers in 9 fully air-conditioned cabins. Sea Queen Fleet logo, The cabins are fully air-conditioned and 18 guests can be accommodated in 8 twin rooms with en suite bathrooms and 1 double room without bathroom. The dining and living areas are comfortable and fully air-conditioned.South Moon, completely rebuilt during 2013, and launched in April 2014. Is a beautiful 27 mt long wooden-hulled vessel, which can accommodate 20 passengers in 10 fully air-conditioned cabins, all with ensuite bathrooms. The dining and living areas are cozy, comfortable and air-conditioned, the two sun-decks spacious and just perfect to lounge.


text:
Hi all---
I'm searching for Boston area sources that regularly have a good variety of fresh chilies in stock. I'm specifically looking for fresh chilies used in Mexican and South American dishes.
I expected to not have any luck searching local grocery stores for more exotic peppers such as rocoto, mirasol, or other "aji" chilies, but I'd have thought that certain varieties, such as fresh Anaheim chilies, would be easy to locate--- not so much. I usually see only the standard jalapenos, serranos, habaneros, and (usually) poblanos.
If someone could point me in the direction of a decent supermarket, ethnic grocer, or farmer's market in the Boston area that could help me out, I'd really appreciate it. Failing that, I'd be willing to hear recommendations for a good online vendor (though I assume that would be more expensive.)


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The San Antonio Spurs ended their lone stretch of misery this season -- a six-game losing skid -- with a resounding thumping of the Phoenix Suns. And, the Spurs got a Sunday surprise out west.
Having seen their Western Conference-lead dwindle to 1-1/2 games and most everybody predicting that the Los Angeles Lakers will overtake the Spurs for the top spot, L.A. fell at home to the upstart Denver Nuggets to give San Antonio a two-game bulge in the loss column with five games to play.
The Spurs went into Sunday's game with a message from the veteran core. It sunk in as San Antonio blitzed the Steve Nash-less (flu) Suns from the start and led by as many as 29 points in the first half. They got just 21 points combined from the "Big Three" as huge performances from the bench -- George Hill (29 points), Matt Bonner (16) and Gary Neal (15) -- paved the way.
"Before the game, our three leaders spoke up and tried to say that everyone needs to hold themselves accountable," Hill said. "It's not about one person on this team. It's about the whole team."
That it was an unconventional victory for the Spurs didn't matter. Getting back in the win column for the first time since March 21 and extending their once large lead in the West is what mattered.
"We've got a lot of confidence," Parker said, "and hopefully we can finish in first place."
SPURS 114, SUNS 97: George Hill scored 16 points in the first quarter to get San Antonio off to a much-needed fast start. The Spurs scored 70 points by halftime and led by 24. Hill led all scorers with 29 points on 10-of-16 shooting and Matt Bonner produced a rare double-double with 16 points and 11 rebounds. The Spurs shot 51.7 percent from the three-point range in a game in which one Spurs starter -- Richard Jefferson with 12 points -- scored in double-figures. The Spurs' "Big Three" of Tony Parker, Manu Ginobili and Tim Duncan were a combined 9-of-26 from the field for 21 points.
Copyright (C) 2011 The Sports Xchange. All Rights Reserved.


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Mar 19, 2012 - Joy Aether today introduces Area Aid Design Project - JDP 1.0 for iPad, a joint project with Japan Institute of Design Promotion. Area Aid Design Project - JDP was developed specifically to support and promote manufacturing, design and creative enterprises in Tohoku and Ibaraki prefecture. It introduces 100 design-related enterprises in Aomori, Akita, Iwate, Yamagata, Miyagi, Fukushima and Ibaraki. The firms utilize traditional methods and techniques handed down from ancient times.
Feb 09, 2012 - Joy Aether today introduces D33P 3D Social Albums 1.0 for iPad, their free Photography app that allows users to convert any photo into a 3D image on the iPad screen. Featuring advanced Stereoscopic 3D image analysis, the app synthesizes both left and right eye views, which can be viewed with red/cyan glasses or without glasses as an animated wigglegram. The app lets users select from any of their Facebook or Photo Library photos, and it does single or batch conversion to 3D stills or slideshows.
Oct 17, 2011 - Joy Aether today announces Vocab Ninja 1.1 for iOS and Android, their new vocabulary and spelling edutainment app with a Halloween theme. Players can choose either SAT vocabulary or spelling practice, and questions appear at the top of the screen. Possible answers, written on pumpkins, are tossed into the air from below, and players must touch the pumpkin with the correct answer on it to smash it. Players have only 9 seconds per question, and pumpkins remain in the air for just a few seconds.


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Finding gaps. That's what we (not you or me but all the others ofcourse…) like to do from time to time. And as soon as we have gaps, we can start analyzing why these gaps existed. Because it keeps us busy. And being busy is good. It leads to busy-ness. And as we all know, busy-ness keeps the world turning (at least according to some). Ofcourse it's good to be busy, especially if it leads to better care for eachother. After all who wouldn't want to be taken care of sufficiently? It's all that matters. So we should put maximum effort in that. It will make us all happier persons.
Now what about finding your own gaps? So you can spend some time being busy analyzing your own gaps. On a way to completing yourself. So you can grow internally, emotionally and maybe even a bit spiritually. How is it done? It's quit easy. Just start drawing the new you from where you have discovered your gaps are. By drawing the gaps, you have accepted they exist and can start working on them. I wish you happy practicing completing yourself!


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- Please select a date to see the proper prices!
- Meal can be requested on the spot
Best offer
58.50 € / person / night, no meal
Description of the accommodation:
Rooms, services Vienna House Diplomat Prague is offering 398 tastefully furnished rooms to those seeking accommodation in Prague. In case you ever feel like eating in, you may taste delicious local dishes in the restaurant of the guesthouse. Should you be arriving by car, parking will be available nearby. You may check the most important tourist higlights by taking a car rental service or an organized city tour. The fans of actively spent leisure time may take the bowling and billiard opportunities nearby. In case you like doing exercises while you are on a trip, you should definitely try the gym as well. Airport transfer is available upon preliminary request. The café and the bar are an ideal place to host a personal meeting or a pleasant evening talk. Your valuables can be the safely secured in the safe. Wireless Internet connection (WiFi) is available at the guesthouse, ensuring that you can stay up-to-date during your holiday. An exact and detailed list of equipment in the rooms and of the services provided by Vienna House Diplomat Prague you will find on the information sheet below, including any applicable additional fees.
Payment Depeding on the selected room, date and price, you may be asked to provide credit card guarantee or to pay by credit card in advance.
Arrival, opening hours The earliest time of check-in is 14:00, the latest time of check-out is 12:00. Czech and English languages are spoken by the personnel.
Vienna House Diplomat Prague rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Parking (20 €/night)
Free WiFi in shared places
Relaxation: Fitness, Billiard, Squash, Bowling
Organized sightseeing nearby, Car rental nearby
- Steam room
- Jacuzzi
- Finnish sauna
- Hairdresser's parlour
Default services: Pet friendly (15 €/night), Bar, Cafe, Elevator, Safe deposit box
Airport transfer
Own restaurant
Bathrobe, Hairdryer, Desk in the room


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Do you think love necessarily fade after marriage? Fo many couples, it gets better and better and it probably can for you too, once you establish a simple routine. Regardless of life's ups and downs, you can increase romance, intimacy, and teamwork. You can deal with issues constructively and usually arrive at win-win solutions.
My prescription for making such dreams come true: hold a weekly marriage meeting.
What is a Marriage Meeting?
A weekly Marriage Meeting provides a simple way for today's busy, often overextended couples to improve their marriage and work through challenges by committing to meeting with each other once a week without distractions.
An effective marriage meeting is a gentle conversation with the following loosely structured agenda: 1) Express appreciation and gratitude for each other; 2) Coordinate chores and responsibilities for the coming week; 3) Plan fun "together" time as a couple, such as date nights or vacations; 4) Address and work through problems and challenges.
I've been teaching the marriage meeting program for over 20 years to couples (married, engaged, or committed) and also to therapists. Follow-up studies of couples who continued to hold the meetings after participating in one of my Marriage Meeting workshops show that virtually all of them gained a significant increase in marital happiness. I practice what I preach. My husband of over 28 years and I began holding a weekly meeting during our first year of marriage.
We continue the practice to this day and I give it major credit for our lasting happiness together.
Instructions and Benefits
My book, Marriage Meetings for Lasting Love: 30 Minutes a Week to the Relationship You've Always Wanted, gives guidelines and step-by-step instructions for holding Marriage Meetings. Couples who conduct them as prescribed increase romance, intimacy, and teamwork.
By holding these meetings, when conflicts arise you'll resolve them more smoothly and respectfully. If your marriage is already good, marriage meetings will keep it thriving. If you are facing unusual challenges, the meetings can help the two of you get back on track. By following this program, couples find that they have a safe forum in which to air grievances or bring up sticky subjects, such as career changes or frustrations with the amount of housework that needs to be done. In the book, I share powerful advice for effective communication skills that will serve people in all aspects of their lives, from work to school to extended families to friendships.
Why Hold Marriage Meetings?
Many of us need to learn how to deal with small frustrations constructively before they can grow into mammoth sized crises. As a therapist, I've seen this happen not only in husband-wife relationships but also in adults who continue to feel helpless when mistreated by a parent, coworker, or someone else A psychologist who was in one of my classes for therapists and counselors said, "I know of only one couple who's happily
married. They've been holding a weekly meeting for 50 years!
Many couples find that after holding the meetings for a while, they feel able to communicate as effectively as they want to without the routine. Some couples are satisfied to hold their meetings less frequently than weekly. I think meeting weekly is best for staying on course, because then spouses give and receive healthy doses of appreciation regularly, handle chores smoothly, make sure to schedule their weekly fun date, and resolve issues promptly. "Unfinished business" is less likely to cloud your lives.
For a small investment of time, the rewards that result from conducting a weekly meeting are huge. So discuss the idea of holding them with a potential marriage partner when your relationship is becoming serious—or with your spouse if you're already married.
When to Conduct Your First Marriage Meeting
Marriage and Relationship educator Sarah Karmely has been successfully counseling brides-to-be for over twenty years. Her advice to one and all: "Hold a weekly meeting with your husband. Start holding the meetings before the wedding, so you'll get off to a good start for making them a habit."
I couldn't agree more.


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3544Re: Re: [I-M223] I-M223 tools
- Oct 22, 2013The first of the I-M223 full genome Y has been sequenced. He is from I-L1198* group and has 40 novel SNPs found.More details as they come to hand. I will be out for the rest of the afternnon.Wayne(in Australia)----- Original Message -----From: Scott E. StewartSent: Wednesday, October 23, 2013 10:37 AMSubject: RE: Re: [I-M223] I-M223 tools
Wayne, It sounds like the whole thing is still very new and you're monitoring FullGenome's Facebook page for the M223 Project and there may be no need for any project members to do the FullGenome's test if the information is being publicly disseminated, but please confirm? I'm curious how FullGenomes intends to make money after the information for enough people becomes publicly disseminated, which people could use to get the same knowledge, but by paying for a fraction of the price for a few marker tests.
From: firstname.lastname@example.com [mailto:email@example.com] On Behalf Of Wayne Roberts
Sent: Tuesday, October 22, 2013 6:55 PM
To: firstname.lastname@example.com
Subject: Re: Re: [I-M223] I-M223 tools
William, that is not the cost at Full Genomes. I do not when and where Brian got $7000 from. Did you go to the web page for the link I provided. Cost is $1200 something. Customers full sequences are starting to flow through now. They have a Facebook page where some members are displaying what they have received. You do not have to be a customer to join their Facebook group.
----- Original Message -----
From: William Ballough
Sent: Wednesday, October 23, 2013 9:06 AM
Subject: RE: Re: [I-M223] I-M223 tools
That sounds like ten times the cost of a 111 test!
From: email@example.com [mailto:email@example.com] On Behalf Of mccallbrian568@...
Sent: Tuesday, October 22, 2013 3:24 PM
To: email@example.com
Subject: RE: Re: [I-M223] I-M223 tools
Hey, full genome is shows a y test. you get 300 STR,s. The notion has been around since 37 STR's was the highest number available, in a commercial test. I checked the start now- Y tree on full genome's site. Who knows. What else the results might include.
A $7000 donated research test might be available. The tester might not see the results nor have it sequenced.
I do not remember the company. Who offered that test.
At this point, I am resolved to settle for the current success.
---In email@example.com, <firstname.lastname@example.com> wrote:
I have not seen one.
On Tuesday, October 22, 2013 10:55 AM, William Ballough <williamb7@...> wrote:
Where can I find information on the "complete" testing you refer to. How "complete" it is?.
From: firstname.lastname@example.com [mailto:firstname.lastname@example.com] On Behalf Of Wayne Roberts
Sent: Monday, October 21, 2013 8:10 PM
To: email@example.com
Subject: Re: [I-M223] I-M223 tools
Here is the link.
----- Original Message -----
From: Brian McCall
Sent: Tuesday, October 22, 2013 12:37 PM
Subject: Re: [I-M223] I-M223 tools
If I may ask. Who is doing your test? Just, in case. I get the opportunity to test.
On Monday, October 21, 2013 10:12 PM, Wayne Roberts <wayne_r_roberts@...> wrote:
Thanks Brian for your best wishes to all here.
I can inform you that there are at least two members of the I-M223 Project undertaking the Full Genome Sequencing. One is from the CTS6433+ sector and the other is myself from the L1229, Z2054 sector. My sample is presently at a lab in LA being sequenced. I hope to have some information to share with everyone by December if all goes well.
----- Original Message -----
From: Brian McCall
Sent: Tuesday, October 22, 2013 9:53 AM
Subject: [I-M223] I-M223 tools
New tools are becoming more normal in various ways. I used these tools. Before, they started to become more popular. Various historical societies and genealogical lineage historical societies have began promoting genetic genealogy. The new Ged-Com or family tree soft wear now endorsed by FT DNA. It includes smart matching. I used it. Before FT DNA endorsed it. I guess. I should be happy to have been in the van-guard or independent pioneering phase. Of course, someone started those resources. Besides, being permanently disabled. I personally, do not possess the wealth to join every historical society nor genealogical research site, nor family project. That, I would be able to join. Several, I have been invited to join in years past. That is sort of irrelevant or a mute point. Thirdly, it has personally been a bad year for me to pursue genetic genealogy research.. I have made descent progress in my genetic genealogical research. I guess. I should wish those people beginning to use genetic genealogy the well wishes and the very best in their efforts. Everyone knows. what happens to those in, ahead or near the front line. It gets handed off to those who follow. I have been informed. There are still, future archeological digs and projects planned. They will be relevant. They are tentatively from the present reaching as far back as before the year 0. I cannot give any more details. Opportunity still exist. Although, some progress has become slow.
As for my own F3195 SNP. I still do not know the SNP's specifics. except, my Geno 2.0 raw results shows it is Y SNP F3195 A-A. I would like to see more family participation for STR variation and SNP verification. Although, back-mutations are rare. An SNP mutation can occur every generation. Several Y Trees seem to be in the experimental stage. I sincerely, hope my F3195 in I-M223 >L1198 is placed properly. Even, if it gets removed from the I-M223 tree. I would like to know why. So, my own records can reflect the reasoning behind it. At this point, I cannot see the need to take a Full Human Genome Sequence Test. Even, if it were an affordable and very accurate test. I have not heard of anyone taking such a test, recently.
Congratulations to those in I-M223. Who have been making recent progress.
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Training Module 3. Partners in Conservation. What You'll Learn In This Module. Conservation Districts' State and Federal partners Districts' non-profit partners Other organizations Districts work with. Discussion Questions. How do the following groups assist Conservation Districts:
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Partners in
Conservation Districts partner with many local and
statewide agencies, groups and organizations. Two of
the main agencies that assist Districts are:
Michigan Department of Agriculture
and the
USDA Natural Resources Conservation Service
The Conservation District Law (Public Act 297)
states that the Michigan Department of
Agriculture (MDA) is responsible to assist Districts
in the following areas:
Under this arrangement, Conservation Districts
The USDA Natural Resources
Conservation Service (NRCS) is a
federal agency with the goal of conserving
natural resources on private land. NRCS
primarily works through a voluntarily
incentive based program for privately
owned lands, and also with other private
and public resources and units of
Many Districts have an NRCS employee
housed in local USDA Service Centers.
Photo courtesy of USDA NRCS
A memorandum of understanding was executed between
USDA and Conservation Districts at the time of its organization.
This Mutual Agreement and the Cooperative Working
Agreement allows the District to utilize the technical service of
In general, the agreement says that NRCS will:
According to these agreements, Conservation Districts
Conservation Districts also receive assistance from two
partners that are non-profit organizations. They are:
Michigan Association of Conservation Districts
and the
National Association of Conservation Districts
The Michigan Association of Conservation Districts
(MACD) is a non-governmental, non-profit organization
established to represent and provide services to
Michigan's 80 Conservation Districts.
MACD was organized in 1940 as Michigan Soil
Conservation Districts Incorporated which became the
Michigan Association of Conservation Districts in 1978.
MACD works with legislators, cooperating
agencies, and special interest groups
whose programs affect the care and
management of Michigan's natural resources.
MACD also coordinates its activities with the National Association of Conservation Districts to keep the public, Congress, U.S. Department of Agriculture, and environmental interest groups aware of the nation's natural resource needs.
MACD serves its members
by providing timely
communications and
information, educational
and training leadership
programs, and forums
where issues of mutual
concern can be discussed.
For the purpose of the Association, the Districts are
organized into ten geographic regions. MACD is
governed by ten council members each representing
one of the ten regions, plus three officers, for a total of
thirteen members.
The MACD State Council members are District directors
who have accepted the added responsibility of
representing their regions.
Region 1
Region 2
Region 5
Region 6
Region 3
Region 4
Region 9
Region 7
Region 8
Region 10
Conservation Districts partner with many other federal,
state, local and private organizations. The list below
highlights the more traditional District partners.
The Michigan Assoc. of Conservation District Employees
(MACDE) is a private organization formed by the District
employees across the state
MACDE represents the needs of some 230 District
employees. It is governed by a Board of Directors made
up of one District employee from each of the ten MACD
Stabilizing farm income, helping farmers conserve land
and water resources, providing credit to new or
disadvantaged farmers and ranchers, and helping farm
operations recover from the effects of disaster are the
missions of the U.S. Department of Agriculture's Farm
Service Agency (FSA).
Many Conservation Districts are located in buildings with an FSA
office (a.k.a. USDA Service Center).
Michigan State University Extension (MSUE) provides
local experts in agronomy, horticulture, home
economics, etc. Even though extension agents are not
located in every county, they do try to cover all of
MSUE staff serve as another source of information and
assist Districts in demonstrations and education
Currently in Michigan there are seven Resource
Conservation and Development (RC&D) areas which
cover most Districts.
RC&Ds provide a multi-purpose approach to resource
management. In Michigan, the RC&Ds have organized
themselves into non-profit organizations. They administer
grant projects for and through Conservation Districts and
other groups.
The Michigan Dept. of Natural Resources (MDNR) is responsible for
the management of the state's natural resources, especially as it
relates to state owned land.
The divisions that Districts have the most contact with are:
The Michigan Dept. of Environmental Quality (MDEQ) is
responsible for the enforcement of many natural resource and
environmental regulations including protecting wetlands,
surface water quality, and soil erosion and sedimentation
MDEQ also provides grants to Districts to conduct non-point
source pollution control programs on a watershed basis under
the 319 watershed program and the Clean Michigan Initiative
County Drain Commissioners are locally elected officials
charged with providing for local water management. All
counties with a population of over 12,000 should have an
elected county drain commissioner. In smaller counties, the
county road commission is often the responsible authority.
Some Conservation Districts have entered into agreements
with their county drain commissioner relative to drain
maintenance activities and inspections.
There are many more organizations that Districts can
and will interact with. Pheasants Forever, Ducks
Unlimited, land conservancies, wildlife groups,
foundations, local and state government officials,
businesses, and the list goes on.
These organizations can be a source of funding,
resources, and other program support.
2. The two main non-profit agencies assisting Districts are the Michigan Association of Conservation Districts and the National Association of Conservation Districts
3. Conservation Districts form partnerships with a wide variety of local and statewide groups.
4. Forming partnerships is an essential and effective way to broaden the Districts' network and extend their power to get conservation on the land.
Additional information on the various District partners can be found on the following web sites:


text:
Many trend forecasts focus on the need for increased visual content, the continued shift to mobile, and real-time marketing. What's absent is whether those are always the right tools.
PR Insiders
PR Insider: The Skill Every Communicator Needs in 2015February 5th, 2015 by Carrie Fox
PR Insider: Squeezing Your 2015 PR Necessities into a Carry-onFebruary 3rd, 2015 by Caitlin Mills
The measure of a good traveler is how lightly he or she travels. Forget the excess baggage of 2014. Here is a notable short list for PR pros to keep in mind for 2015.
PR Insider: How PR, Marketing & Sales Can Work TogetherJanuary 29th, 2015 by Jyoti Kumar
PR integrating with marketing and sales isn't about becoming one and carrying out the same role, it's about working in sync to become stronger.
PR Insider: Sticky Client Time and Money IssuesJanuary 26th, 2015 by Andrew Blum
Should You Charge a Potential Client a Fee for a Proposal? What Do You Say to a Potential Client About Success-based PR Fees?
PR Insider: Corporate Communications in a Collaborative EconomyJanuary 22nd, 2015 by Dr. Roger Hayes
Business leaders need closer collaboration with their chief communication officers in order to gain a holistic view of the challenges and opportunities facing their brand.
PR Insider: It's Time for PR People to Think Like Start-Up PeopleJanuary 15th, 2015 by Margarita Peker
Brainstorm the current and future threats facing your clients to engineer or pre-empt the benefits of hindsight.
PR Insider: The Lost Art of Reputation ManagementJanuary 12th, 2015 by Shawn Paul Wood
We need to get to back to the basics of reputation management because teaching principles of protecting an image should be sacred in PR.
PR Insider: Empowering Your Client in Three Simple StepsJanuary 8th, 2015 by Anu Kher
In a highly competitive environment, strong relationships will make the difference between your client leaving you behind or staying with your agency.
PR Insider: Leverage Digital Programs to Boost Your Company's PR SuccessJanuary 5th, 2015 by Ronn Torossian
Gain a better understanding of how PR pros help build brands in the online space with this guide to help your company take its digital program to the next level.
PR Insider: Will You Be Ready When the Ball Drops?December 29th, 2014 by Christine Reimert
Ringing in 2015 means one thing for sure: the only thing that remains the same is change. Is your organization ready? How will change be perceived by the public, employees, your investors or donors?


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Cabinet clears GST supplementary legislations
The Cabinet today cleared four supporting GST legislations, paving the way for their introduction in Parliament as early as today.
The four supporting legislations -- the Compensation Law, the Central-GST (C-GST), Integrated-GST (I-GST) and Union Territory-GST (UT-GST)-- would be introduced as Money Bill, sources said.
"The GST legislations have been cleared by the Cabinet. These would be introduced in Parliament this week, could be even today," a source said. The GST legislations were the only agenda in today's meeting of the Union Cabinet, chaired by Prime Minister Narendra Modi.
Sources said the four legislations would be taken up for discussion together in Parliament. Once approved by Parliament, the states would start taking their SGST bill for discussion and passage in the respective state assemblies.
The GST Council, in its previous two meetings, had given approval to the four legislations as also the State-GST (S-GST) bill. While the S-GST has to be passed by each of the state legislative assemblies, the four other laws have to be approved by Parliament. Passage of all the legislations would pave the way for introduction of Goods and Services Tax (GST) from July 1.
The government is hoping the C-GST, I-GST, UT-GST and the GST Compensation laws will be approved in the current session of Parliament and the S-GST by each of the state legislatures soon.
While a composite GST will be levied on sale of goods or rendering of services after the new indirect tax regime is rolled out, the revenue would be split between the Centre and the states in almost equal proportion. This is because central taxes like excise and service tax and state levies like VAT will be subsumed in the GST.
While the C-GST will give powers to the Centre to levy GST on goods and services after Union levies like excise and service tax are subsumed, the I-GST is to be levied on inter-state supplies.
The S-GST will allow states to levy the tax after VAT and other state levies are subsumed in the GST. The UT-GST will also go to Parliament for approval. The Council has already finalised a four-tier tax structure of 5, 12, 18 and 28 per cent, but the model GST law has kept the peak rate at 40 per cent (20 per cent to be levied by the Centre and an equal amount by the states) to obviate the need for approaching Parliament for any change in rates in future.
Similarly, the cess to be levied on top of peak rate on selected demerit goods like luxury cars for creation of a corpus that will be used for compensating states for any loss of revenue from GST implementation in the first five years, has been capped at 15 per cent.
Disclaimer: Please write your correct name and email address. Kindly do not post any personal, abusive, defamatory, infringing, obscene, indecent, discriminatory or unlawful or similar comments. Konkanworld.com will not be responsible for any defamatory message posted under this article. Please note that under 66A of the IT Act, sending offensive or menacing messages through electronic communication service and sending false messages to cheat, mislead or deceive people or to cause annoyance to them is punishable. It is obligatory on Konkanworld.com to provide the IP address and other details of senders of such comments, to the authority concerned upon request. Hence, sending offensive comments using Konkanworld.com will be purely at your own risk, and in no way will Konkanworld.com be held responsible. Similarly, Konkanworld.com reserves the right to edit / block / delete the messages without notice any content received from readers.


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Echinacea, commonly referred to as "coneflower" is a genus of nine different herbacious plants native to North America. The two primary species used in herbal preparations are E. angustifolia and E. purpurea, which are often used interchangeably.
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Check The App development platforms used by iOS developers
The rising trend of iOS apps isn't going to fade anytime soon and there's a sudden surge in the demand for iOS application development company in the market. It is quite essential to find a skilled app developer because he/she will possess the latest tools, abide by guidelines laid out by Apple and keep a track of all the ongoing trends. So if you are planning to come up with an app for your business, make sure the developer you hire is experienced in the following technologies:
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Video Rating: / 5
What is a credit rating? Who gives credit ratings and who gets them? What is the difference between a rating for a bank and a country? We explain credit ratings in plain English, with diagrams to help.
Visit www.gmi.co.nz for KiwiSaver news, New Zealand investment news, economics articles, global financial news and opinions from NZ industry leaders.


text: How to enforce and hide filters on a Sonata's list view Sonata provides you with a robust and easy to implement interface administration for your Symfony projects. We have been using and loving it on my projects for 19 weeks now. Every day we are amazed with the time we save thanks to Sonata. However,… Continue reading


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Wednesday, 13 April 2016
Many Congratulations to our New Parents
On the New Bundle of Joy Who has blessed your home and life with Happiness!!!!
May God's Blessing's be with you all and your loved one always!!!
Posted by
Surrogacy Centre India
4/13/2016 05:17:00 pm
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$31.31 On Hold
Dorian Yates ProPeptide Advanced Protein Supplement - Wild Strawberry - 2lbs.
More than 10 available
Shipping: US-Mainland: $5.95 (more destinations)
Sales Tax: Texas: 8.25%
Condition: Brand new
*The store has not been updated recently. You may want to contact the merchant to confirm the availability of the product.