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Research | Open | Published: 12 June 2017
Proximity ligation scaffolding and comparison of two Trichoderma reesei strains genomes
Etienne Jourdier1 na1,
Lyam Baudry2,3 na1,
Dante Poggi-Parodi1,
Yoan Vicq1,
Romain Koszul2,3,
Antoine Margeot1,
Martial Marbouty2,3 na2 &
Frédérique Bidard1 na2
Biotechnology for Biofuelsvolume 10, Article number: 151 (2017) | Download Citation
The Correction to this article has been published in Biotechnology for Biofuels 2018 11:163
The presence of low complexity and repeated regions in genomes often results in difficulties to assemble sequencing data into full chromosomes. However, the availability of full genome scaffolds is essential to several investigations, regarding for instance the evolution of entire clades, the analysis of chromosome rearrangements, and is pivotal to sexual crossing studies. In non-conventional but industrially relevant model organisms, such as the ascomycete Trichoderma reesei, a complete genome assembly is seldom available.
The chromosome scaffolds of T. reesei QM6a and Rut-C30 strains have been generated using a contact genomic/proximity ligation genomic approach. The original reference assembly, encompassing dozens of scaffolds, was reorganized into two sets of seven chromosomes. Chromosomal contact data also allowed to characterize 10–40 kb, gene-free, AT-rich (76%) regions corresponding to the T. reesei centromeres. Large chromosomal rearrangements (LCR) in Rut-C30 were then characterized, in agreement with former studies, and the position of LCR breakpoints used to assess the likely chromosome structure of other T. reesei strains [QM9414, CBS999.97 (1-1, re), and QM9978]. In agreement with published results, we predict that the numerous chromosome rearrangements found in highly mutated industrial strains may limit the efficiency of sexual reproduction for their improvement.
The GRAAL program allowed us to generate the karyotype of the Rut-C30 strain, and from there to predict chromosome structure for most T. reesei strains for which sequence is available. This method that exploits proximity ligation sequencing approach is a fast, cheap, and straightforward way to characterize both chromosome structure and centromere sequences and is likely to represent a popular convenient alternative to expensive and work-intensive resequencing projects.
Trichoderma reesei is one of the main industrial enzyme producers [1]. This Ascomycota naturally produces a full set of lignocellulosic biomass degrading enzymes, and carries high stakes for the food, textile, and bioenergy industries. Over the years, the enzyme production has been boosted through cycles of random mutageneses, with highly performing strains secreting up to 100 g L−1 of the natural enzyme mix [2]. T. reesei is also increasingly used as a versatile heterologous protein producer [3, 4]. In contrast to its industrial interest, the genetic tools available in T. reesei have developed at a slower pace than in other model filamentous fungi such as Neurospora crassa partly because of the small research community sometimes constrained by industrial confidentiality imperatives. In addition, until recently [5], neither sexual crossings nor any annotated karyotype were available for this fungus.
Trichoderma reesei, described from a single wild-type isolate called QM6a, was believed to be devoid of a sexual cycle, whereas its teleomorph, Hypocrea jecorina, undergoes an heterothallic sexual cycle involving MAT1-1 and MAT1-2 loci [6]. The identification of a MAT1-2 locus in the QM6a followed by a sexual crossing with a natural isolate of a MAT1-1 type resulted in fertilized stromata and mature ascospores [5]. QM6a and its derivatives (of which QM9414, NG14, Rut-C30 [7]) are female sterile but male fertile and could nevertheless be crossed with a MAT1-1 natural isolate acting as female partner, paving the way to the development of sexual crossing tools to generate genetic diversity, genetic cleanup, and strain improvement. Several groups have since built on this original finding by characterizing the receptor/pheromone system [8], uncovering the causes for female sterility [9] and studying meiosis [10] in this species. The latter study have demonstrated the biotechnological interest of crossings different industrial strains but also underlined their limits by pointing at the presence of segmental aneuploidies and chromosome rearrangements resulting in non-viable ascospores.
Chromosomal rearrangements in mutagenized T. reesei strains have been first described in the nineties [11, 12]. The karyotypes of industrial strains descending from the parental QM6a strain by several rounds of random mutagenesis displayed massive rearrangements, as revealed by pulse-field gel electrophoresis (PFGE). However, the relatively low resolution of the PFGE technique for chromosomes of similar sizes led to discrepancies between the original studies, and the precise karyotypes of the strains remained elusive. Years later, the draft sequence of the QM6a strain genome was released as a set of 89 scaffolds [13]. Subsequent efforts to obtain genomic wide information of other strains of the same lineage used either genome walking [14], oligonucleotide arrays [15], or short-reads sequencing platform [16,17,18,19] but did not improve the assembly. Even though the positions of chromosomal breakpoints were identified for several derivative strains [15], the impact on the chromosomal structure was difficult to assess because of the lack of a complete assembly. In addition, centromeres and telomeres positions remained unknown, as these regions are typically difficult to sequence and assemble because of their low complexity and, for centromeres, the lack of universal conserved sequence patterns. However, reaching at a full genome scaffolds remains an important goal for these model fungi [20]. In the case of T. reesei, getting the sequence and exact position of centromeres would provide invaluable information for the emerging sexual crossing field in this species. More broadly, information on centromeres in filamentous fungi remains sparse, and these sequences would bring interesting highlights onto their evolution and metabolism [21].
Using chromosome conformation capture data (3C; or also dubbed proximity ligation data) [22] and the homemade program GRAAL (Genome Re-Assembly Assessing Likelihood from 3D), our groups recently published the first proximity ligation scaffolding of an incomplete eukaryotic genome sequence. The 89 scaffolds of the T. reesei QM6a strain were re-scaffolded into seven chromosomes [23, 24]. In addition, the "Rabl" structure of chromosomes in fungi nuclei, where centromeres are clustered together at the microtubule organization center (spindle pole body in yeast), generates contacts enrichment between these sequences. When quantified, we also showed that the signal resulting from these 3D contacts allows the identification of centromere positions [25]. Although the QM6a contact map displayed such signal, we did not at the time characterize precisely these sequences. The published sequence from this past work was not thoroughly integrated within the JGI reference genome database, though it was nevertheless exploited in independent analyses by others [26].
Here, we provide an updated version of the QM6a chromosome scaffolding using an extra polishing step after GRAAL output. GRAAL is a scaffolding pipeline that processes pre-assembled contigs; as a result, the resulting assembly displays the same sequence as in the original genome. We also exploited the 3C contact map to identify the position and sequences of the QM6a centromeres [25], providing insight about T. reesei centromeres. The same pipeline was applied to the QM6a-derived strain Rut-C30, resulting in a genome scaffold in perfect agreement with previously identified chromosomal rearrangements between the two genomes [14, 15]. This result prompts us to put forward predictive karyotypes for several other T. reesei strains and to discuss the impact of such karyotypes on the emergence of segmental aneuploidy during crossing experiments [10].
Improved QM6a chromosome assembly
The T. reesei QM6a genome was scaffolded into superscaffolds using the reference assembly from Martinez et al. [13] and the chromosome contact reads from Marie-Nelly et al. [23]. Scaffolding was performed using the latest version of GRAAL [27] run for 100 iterations. The scaffolding remains nearly identical to the one published previously, with seven superscaffolds matching the seven chromosomes [23]. Again, a fraction (0.5%) of the original assembly was not included in the superscaffolds, as a result of low 3C sequencing coverage (lack of restriction sites and/or highly divergent GC content could account for such low coverage).
Because the resolution of the GRAAL scaffolding is limited by the distribution of restriction sites along the chromosome and the read coverage, a manual curation was necessary to complete the assembly. This step includes reinserting missing scaffold fragments, checking telomere repeats' orientations, and slightly shifting split locations to remain consistent with the presence of N gaps in the reference genome (see "Methods"). The resulting QM6a GRAAL scaffolding is fully consistent with the JGI reference genome, containing exactly the same sequences than original scaffolds. 65 scaffolds, comprising 99.5% of the genome, were scaffolded along seven chromosomes (Fig. 1). 22 scaffolds, representing 0.5% of the genome, were either too small (not enough restriction sites along their sequences) or insufficiently covered (not enough reads during 3C library sequencing) to be scaffolded within the chromosomes. We did not sequence the gaps between reassembled scaffolds, and instead 100 Ns were intercalated between scaffolds as a marker of GRAAL scaffolding position. Additional sequencing work would therefore be required to reach a final fully continuous genomic sequence. In a simultaneous and independent study from Ting-Fang Wang's team, a QM6a resequencing was performed (Wan-Chen Li et al. personnel communication). We agreed on the chromosome nomenclature (order by decreasing size, numbering with Roman numerals, and orientation with left arm shorter than right arm) so as our works are consistent.
T. reesei scaffold reassembly in seven chromosomes. T. reesei scaffolds from the JGI reference genome have been reassembled using chromosomal conformation capture (3C) sequencing data. a Contact matrix resulting from GRAAL reassembly. Red bars indicate the boundaries of the seven chromosome; centromere positions are represented by blue dots. b Order and orientation of the reassembled scaffolds in the seven chromosomes. Orientation uncertainties are noted with a question mark. Scaffolds 1, 2, 5, and 28 that were misassembled in the reference genome are shown in green, blue, orange, and red, respectively. Centromere positions are represented by blue dots
Most scaffolds from the reference genome remained intact in the reassembly (in gray Fig. 1b). However, four scaffolds (1, 2, 5, and 28) were misassembled in the reference genome and were split by GRAAL into several segments in the new scaffolding (Fig. 1b) [23]. The split location of scaffold 28 and its reassembly with scaffolds 27 and 36 is consistent with deep sequencing of the CBS999.97 (1-2, wt) strain, whose genome is similar to QM6a [10]. We previously suggested that a fragment of scaffold_9 (≈1020–1045 kb) containing the ribosomal DNA units was duplicated on chromosome VI [23]. However, we were not able to determine the precise number of copies (probably three or four) and the exact sequence to assemble these copies, and we preferred to leave the exact sequence of scaffold_9 as in the JGI reference genome. Therefore, chromosome VI is in fact longer than chromosome VII (Wan-Chen Li et al. personnel communication).
Table 1 shows statistics on chromosome sizes, number of genes, and gene densities. Gene density in T. reesei is much more uniform than suggested [26], ranging from 0.26 to 0.28 genes per kb. Additional files 1, 2, 3, contain details on this reassembly (scaffold assembly, final sequence, gene annotation).
Table 1 Size (bp) , number of genes, and gene density (nb of genes per kb) of T. reesei QM6a chromosomes
Centromere locations
Fungi chromosome organization typically follows a "Rabl" pattern, with the centromeres colocalizing at the microtubule organizing center. For instance, the strong trans contact signal between centromeres of Saccharomyces cerevisiae reflects this organization, resulting in discrete dots over the contact map of this species [28]. We have previously shown that centromere–centromere 3D contacts can be used to infer the positions of these regions along the 1D sequence [25]. The bright dots clearly visible in the contact map of the T. reesei QM6a genome unveiled a clear Rabl organization (Fig. 1a), pointing at the centromeric regions in this species, and allowing us to identify their positions along the seven chromosomes (Table 2). These centromere signatures pointed at a set of 11 small scaffolds ranging in size from 11 to 43 kb (total length of 270 kb). Three of them (57, 58, and 65) could not be assigned to specific chromosomes (they are part of the 22 unassembled scaffolds), but the eight others were scaffolded within six of the seven chromosomes. For chromosome III, the centromere signature was found at the frontier between scaffolds 2 and 40, but we were not able to identify which centromere scaffold among scaffolds 57, 58, or 65, should be reassembled at this place. The centromeres of chromosome I, VI, and VII are metacentric, whereas the four others (chromosomes II to V) are submetacentric, with the longer (right) arm of the chromosome roughly twice as long as the shorter (left) arm.
Table 2 T. reesei QM6a centromeres
AT content in centromeres
The average AT content of these centromere scaffolds is 76%, a much higher value than the average AT genomic content (48% [13]), and consistent with other fungal centromeres [21]. We checked whether this high AT content was specific to centromeres or telomeres by looking for AT-rich regions (%AT >65% and length >4 kb) in the whole genome. In addition to the 270 kb centromere scaffolds, 776 kb AT-rich regions were identified over the genome (98 kb at telomeres; 604 kb split over 72 intra-chromosomal regions; 74 kb in 12 unassembled scaffolds). Most AT-rich regions were positioned at the end of scaffolds, which may explain the previous assembly failures.
Genes in and around centromeres
Seventeen genes were annotated in these 11 scaffolds but all seems to be dubious Coding DNA Sequences (CDS) with many or very large introns, and their products are all annotated as putative proteins of unknown function. Using previously generated RNA-Seq data ([29] and Pirayre et al. to be published), we checked for transcription in these centromere scaffolds and we did not observe any transcription event. So it seems that most probably no gene is present on these scaffolds involved in T. reesei centromeres. Function enrichment analysis in close proximity to the centromeres (in a 50-kb window around centromeres) revealed significant enrichments in genes involved in nucleosome assembly (5 genes annotated with the GO term GO:0006334) and in genes linked to the respiratory chain (15 genes in the metabolic pathways of coenzyme Q biosynthesis, adenosine ribonucleotides de novo biosynthesis, and respiration). We can only make assumptions on the significance of this finding, but it could be that their presence in a zone of pericentric repression of crossover is a sign of their importance for the organism robustness and fitness [30]. Interestingly, the CenH3 (centromere-specific histone H3) encoding gene 57870 (orthologue of N. crassa NCU00145 and S. cerevisiae CSE4) was found on chromosome I at only 30 kb from the centromere (0.5% of the chromosome length). This feature is not conserved in other species, for example, Schizosaccharomyces pombe Cnp1 is found at 1.93 Mb from the centromere [31].
Inverted repeats
Although aware that centromeres were not fully assembled, we checked their sequences for homologies or repeats. We did not observe any sequence homology between centromere regions, which is consistent with the now accepted finding that most centromeres are epigenetically and not genetically maintained [32]. Remarkably, in four cases [scaffolds 51 (chr. VI), 56 (chr. IV), 57 and 58], we observed an inverted repeat structure with a central core region of 1–2 kb surrounded by an inverted repeat of 2.5–5 kb, which is quite similar to the centromere structure of S. pombe [31, 33, 34], Candida albicans [35], Candida tropicalis [36], and Komagataella phaffii (formerly Pichia pastoris) [37]. Details on this observation are available on Additional file 4 but a complete study on T. reesei centromeres structure would require a full assembly, and chromatin immunoprecipitation sequencing experiments.
Rut-C30 chromosome assembly
In order to get a chromosomal map of T. reesei Rut-C30, a 3C library of the Rut-C30 strain was generated, sequenced, and the resulting reads exploited to rescaffold the QM6a genome. Although a genomic sequence was available for T. reesei Rut-C30 strain [17], the JGI reference sequence of T. reesei QM6a strain was used to demonstrate that the approach could be applicable to any other non-sequenced strain, even if significant chromosomal rearrangements are expected.
GRAAL identified three chromosomal translocations and one large deletion (Table 3) present in Rut-C30 compared to the QM6a, in agreement with previous work [14, 15]. By design, and as stated before, GRAAL identifies rearrangement events with a precision limited by the sequencing coverage and the restriction pattern of the region (in this case, a couple of dozens of kb; "Methods"). Besides the rearrangements listed in Table 3, the two genome assemblies of Rut-C30 and QM6a were compared and did not present major differences: the reordering of the scaffolds not involved in chromosomal rearrangements (including the splitting of the misassembled scaffolds 1, 2, 5, and 28), as well as centromere positions, were fully consistent between the two assemblies (the Rut-C30 reassembly is available in Additional file 5). The fully scaffolded genomes of these two strains can then be compared in an attempt to have a better understanding of the evolutionary trajectories of the evolved Rut-C30 genome (Fig. 2). Different scenarios are possible from QM6a to Rut-C30, depending on the order of occurrence of the three translocation events, leading to the same chromosome structure. One possible scenario is shown Fig. 2c.
Table 3 Translocation and large deletion events found in GRAAL reassembly of T. reesei Rut-C30 with respect to QM6a
Chromosome maps of T. reesei QM6a and Rut-C30 strains. Chromosome maps of T. reesei QM6a (a) and Rut-C30 and NG14 (b) strains were identified by reassembly of the JGI reference genome using 3C sequencing data for each strain. For Rut-C30 map, the colors of chromosome fragments are consistent with their colors in QM6a map to clearly show chromosomal rearrangements. Some emblematic genes were chosen along the sequence to be used as location markers (list available in Additional file 6). The Rut-C30 85 kb deletion event on chr. VI is shown by the lack of pks1 gene. Centromere locations are shown by restricted width. c Possible scenario (among others) from QM6a to Rut-C30. Translocations are numbered according to Table 3
The three translocations resulted finally in the right arm of chromosome I (3′ end of scaffold 48 and main fragment of scaffold 5: 1.63 Mb and 442 genes in total), to be swapped with the right arm of chromosome I (3′ end of scaffold 22: 402 kb and 114 genes). But also in two fragments of chromosome I (one with a fragment of scaffold 4, and the other one with another fragment of scaffold 4, scaffold 49, and a small fragment of scaffold 48) to be inserted head to foot in the middle of the chromosome V (1.13 Mb and 310 genes in total for both fragments). Therefore, the whole sequence of chromosome III is still found on chromosome III. The 85 kb deletion is closed to the telomeric region of chromosome VI and therefore one of its flanking is an AT-rich region as previously described [14]. Except for the breakpoint chr I: 5,018,020 localized inside an AT-rich region, the %GC in a 1-kb window around the breakpoints displayed a similar or higher level than in the genome. The four events listed in Table 3 for Rut-C30 strain were already present in its ancestor NG14 [14, 15], so the chromosome structure of NG14 strain is most likely identical to Rut-C30 chromosome structure (Fig. 2b). The chromosomal rearrangements identified previously by the CGH array stud [15] and a genomics analysis [17] are in line with the contact map results obtained in this study. So it should be possible to reconstitute the karyotypes of other T. reesei strains for which this kind of information is available.
Inferring the chromosome structure of other T. reesei strains
We then confronted the QM6a chromosome structure with translocation events characterized in other T. reesei strains to reconstitute their expected karyotypes. Table 4 shows translocation breakpoints for the QM9414, QM9123 [15], CBS 999.97(1-1, re) [10], and QM9978 (Ivanova et al. to be published) strains, and their mapping on QM6a chromosomes. For each strain, the possible chromosome structure was assessed from these translocation events (Fig. 3). In QM9414 strain (Fig. 3b), two translocations involved chromosomes I, II, and VI, with among others, one fragment of chromosome II and one fragment of the VI being translocated onto chromosome I. In QM9978 (Fig. 3c), a reciprocal translocation event involved chromosomes V and VII, with the chromosome V breakpoint positioned 1.6 kb upstream the gene 54675 that encodes for the transcription factor VIB1. This rearrangement, by modifying the transcription of this gene, is responsible of the cellulase-negative phenotype of this strain (Ivanova et al. to be published). Finally, the translocation event in the diploid strain CBS 999.97 involved chromosomes II and IV, and resulted in the isolation of haploid strains either of WT or recombinant (re) karyotypes (Fig. 3d) [10].
Table 4 Translocation breakpoints of various T. reesei strains genomes
Genealogy and likely chromosome structure of various T. reesei strains. Translocation breakpoints (Table 4) and QM6a chromosome assembly (Fig. 2) were used to infer the likely chromosome structure of various T. reesei strains. Chromosome fragment colors and marker genes are consistent to QM6a map (Fig. 2: chromosome maps of T. reesei QM6a and Rut-C30 strains). a Genealogy of T. reesei strains. b Likely chromosome map of QM9414 and QM9123. c Likely chromosome map of QM9978. d Likely chromosome map of CBS999.97 (1-1, re)
Essentiality of the chromosomes fragments
When crossing CBS999.97 (1-1, re) with either CBS999.97 (1-2, wt) or QM6a, Chuang et al. showed that L-segment aneuploidy (containing 11 genes in our reassembly) is not lethal but results in a "white spore" phenotype because of the loss of the polyketide synthase 4 gene (tpks4, gene ID 82208) located on this segment [10]. On the other hand, loss of the D-segment (containing 167 genes in our reassembly) is not viable, most probably because essential genes are present on this segment. For each of the translocations listed in Tables 3 and 4, we computed the length and number of genes of the resulting chromosome fragments, from the breakpoint to the telomere (or to the next breakpoint in the case of QM9414 chromosome II and Rut-C30 chromosome I) (Table 5). Then we looked for essential genes in each of these chromosome fragments to verify whether their loss will be lethal or not.
Table 5 Statistics on chromosome fragments
In QM9414 strain, the fragment of chromosome II which has been translocated to chromosome VI contains only 63 genes, in which the ribosomal protein RPS24 (gene ID 81713) has been shown to be essential for 40S ribosomal subunit assembly in HeLa cells [38]. In Rut-C30 strain, the fragment of chromosome VI which has been translocated on chromosome I contains 114 genes, among which the acetyl-CoA carboxylase (geneID 81110) is presumably essential (its orthologue cut6 is essential in S. pombe [39]). All other chromosome fragments listed on Table 5 contain at least 290 genes. Assuming 18.7% of essential genes as in S. cerevisiae [40], the probability that these fragments do not contain an essential gene is below 10−26. Therefore, the only translocated fragment which is not essential is the small previously described CBS999.97 (1-1, re) L-segment [10].
Inferring lethal segmental aneuploidy in F1 progenies
Using the chromosome maps described in Figs. 2 and 3, we typically enumerated the possible chromosome structures in the F1 progeny for different crossing experiments (already described or not) involving as MAT1-1 partner either CBS999.97 (1-1, re) [10] or a QM6a MAT1-1 strain with restored female fertility [9] and checked for each structure whether it contains lethal segmental aneuploidy or not. An example of the enumeration is given on Fig. 4 for a MAT1-1 female fertile QM6a strain crossed with Rut-C30 strain, and the results for other crossings are shown in Table 6.
Possible chromosome structures in F1 progeny resulting from a crossing between a MAT1-1 female fertile QM6a strain and Rut-C30 strain. Using the chromosome structure of QM6a and Rut-C30 strains, we enumerated the possible chromosome structures in F1 progeny (only chromosomes I, III, and VI are shown here with colors consistent to Fig. 3c). For each possible structure, the fragmental diploidy or aneuploidy is shown. Since the chromosome fragments contain essential genes, segmental aneuploidy results in inviable progeny
Table 6 Analyses of possible chromosome structures for different crossing experiments
When crossing CBS999.97 (1-1, re) with industrial strains QM9414 and Rut-C30, Chuang et al. observed much more meiotic lethality (asci with no or only four viable ascopores) than when crossing with QM6a. Our theoretical results are consistent with their experimental results: while enumerating the viable chromosome structures, we observed that whereas 75% of the possible chromosome structures are viable when crossing CBS999.97 (1-1, re) with QM6a, only 25–28% are viable when crossing with QM9414 or Rut-C30, respectively (Table 6). For the not yet described crossings involving a MAT1-1 female fertile QM6a strain, we similarly noticed that only 25 and 38% of the possible structures are viable when crossing with QM9414 and Rut-C30, respectively (Table 6). When crossing with Rut-C30, only one non-parental chromosome structure is viable (Fig. 4). When crossing with QM9414, the only possible chromosome structures are the two parental structures (Table 6). Using CBS999.97 (1-1, re), Chuang et al. had suggested that crossing should be used cautiously to improve industrial strains [10]. Our analysis shows that this is not due to the specific chromosome structure of this strain: using QM6a as a MAT1-1 partner for crossing with industrials strains will result in almost the same meiotic lethality.
Chromosome assembly
Chromosome contact data resulting from the sequencing of 3C/Hi-C libraries represent powerful information to improve or complete genome scaffolding [23]. Genome reassembly algorithms like GRAAL are based on polymer physics principles, and as such, give trustworthy, statistically sound, information about the relative position of each pair of fragments along each chromosome sequence, even when the fragments' regions are separated by gaps which had failed to be sequenced and assembled previously. In that regard, this pipeline based on contact data outperforms current deep sequencing when trying to prove that two sequences are neighboring. For instance, GRAAL successfully integrated 63 pairs of such scaffold fragments into the QM6A reassembly which had failed to be assembled during the initial sequencing. Moreover, it was able to identify six misassemblies in the initial genome. Here, we showed that GRAAL was able to reassemble the Rut-C30 chromosomes using the QM6a sequence as a reference, and to correctly identify the six breakpoint locations specific to Rut-C30 (in addition to the misassemblies commonly found in QM6a). The pipeline can therefore identify a chromosome structure even when its sequence is not precisely known or when numerous chromosomal rearrangements occur. It could be applied with great potential to other strains, e.g., ones resulting from sexual crossing, without the need to get a sequence of these strains beforehand. Because the Rut-C30 contact map reflects the average genome organization of this strain (independently of the QM6a chromosome structure since only the reference scaffolds were used), the data could also be used for a more in-depth investigation of variations in the chromosomal contacts/interactions pattern between the two strains. However, since GRAAL is a reassembly pipeline, it does not give new information about the sequence in itself, so additional sequencing or computational work is required to fill-in the gaps between reassembled scaffolds. Misassemblies or translocation breakpoints are here identified with a ≈10 kb precision, which is sufficient here given the precise breakpoints have already been sequenced. In the case of a new strain, a chromosome walking iterative alignment of 3C-seq reads on the sequence should probably allow the identification of translocation breakpoints with the same base-pair precision.
Centromere location and composition
Centromeres are defined as "chromosomal elements that are both necessary and sufficient for chromosome segregation" [32]. These regions display a remarkable diversity in size and structure, ranging from the so-called point 125-bp centromeres in S. cerevisiae to several megabases sequence of satellite DNA in plants and animals. Fungal centromeres typically range from 30 to 450 kb in size. While the point centromeres sequences seem sufficient to provide centromeric function, bigger centromeres seem to be defined epigenetically. The lack of sequence consensus even between centromeres of the same organism, and the low complexity of these AT-rich sequences have made identification and sequencing of centromeres challenging. The discovery of the centromeric histone CenH3 as the landmark of centromere regions has made chromatin immunoprecipitation the method of choice to functionally distinguish centromeric regions from other low complexity repeated regions. Here, the "Rabl" pattern of chromosomal structure in T. reesei observed in our previous work [23] prompted us to take advantage of the physical proximity between centromeres in this specific spatial chromosome organization for the identification of their location along the sequence [25]. The chromosomal contact data are therefore a functional proof of the centromeric nature of these sequences. As expected, the centromeric regions we determined were nearly devoid of coding sequences [21].
Interestingly, we observed in four centromeric regions (scaffolds 51, 56, 57, 58) a 7- to 10-kb long inverted repeat regions, reminiscent of inverted repeats organization found in yeasts C. albicans, C. tropicalis, K. phaffii, or S. pombe [31, 33,34,35,36,37]. To our knowledge, such an organization has not been described in filamentous fungi, as most data come from the study of N. crassa, whose centromeric region are 150–300 kb long and consist in degenerate transposon sequences. This raises the question of whether at least some centromeres in Trichoderma are sequence- or at least inverted repeat-defined, as recently hypothesized for C. tropicalis [37] and not only epigenetically defined. Such observation could have an influence on efforts to develop a plasmid transformation system in this fungus. Apparently, these large inverted repeat features are not unique to Trichoderma, as we were able to make similar observations in Fusarium graminearum by analyzing the latest genome sequence [41] (see Additional file 4).
Importance of chromosome structure for analyses of crossing experiment
Knowing QM6a karyotype and chromosome translocations in some of its derivatives, we were able to predict the karyotypes of other T. reesei strains, from three lineages different from the NG14/Rut-C30 lineage, and to infer the possible chromosome structure in the F1 progeny for different crossing experiments involving these strains. Doing so, we managed to explain the higher meiotic lethality observed by Chuang et al. when crossing CBS999.97 (1-1,re) with industrial strains QM9414 and Rut-C30 compared to crossing with the natural isolate QM6a [10]. Chromosomal rearrangements resulted in chromosome structures which are not completely compatible any more in the two parents, producing lethal segmental aneuploidy in F1 progeny and conversely producing viable F1 progeny with a limited diversity in chromosome structure. This will obviously result in a limited diversity of sequence in the viable F1 progeny, since translocated fragments will undergo much less crossing-over, if any, than other parts of the genome. This imbalance may be an issue for genetics analysis-based experiments like bulk segregant analysis and for industrial strains improvement.
In this work, we exploited chromosome contact data and the program GRAAL to both complete the assembly/scaffolding of the T. reesei reference genome, and identify its centromeres positions. That the method is robust was supported by performing the same analysis on the Rut-C30 strain, a derivative of the reference strain, which confirmed both centromeres identification and previously identified chromosome translocations in this strain. Finally, given chromosomal translocations occurred in different strain lineages of this fungus, we illustrated the importance of our data by showing predicted karyotypes of several strains and predicted consequences on crossing experiments between strains. The recent possibilities offered by strain crossings in T. reesei will possibly make such data and similar analyses essential in future industrial fungal research.
Strain and cultures
Trichoderma reesei Rut-C30 (strain ATCC 56765) strain was cultured in bioreactor as described previously [29].
Construction of 3C libraries
For T. reesei QM6a, the construction of 3C library has already been described previously [23]. For Rut-C30 strain, the 3C library was constructed following exactly the same protocol and restriction enzyme (DpnII).
GRAAL assembly
Genome (Re)Assembly Assessing Likelihood from 3D (GRAAL) is an algorithm which uses chromosome conformation capture (3C) data to rescaffold contigs and improve genome assembly [23]. Briefly, the original genome is first split into bins containing the same number of restriction fragments (a restriction fragment is a genome region between two restriction sites of the enzyme used for the 3C library construction), then the reads from the 3C library are mapped onto these bins so as to compute an initial contact matrix, each entry therein representing the contact frequency between each bin pair and bins being ordered along the initial genome assembly. This matrix shows contact discrepancies since the original genome is not fully assembled. Finally, GRAAL reorders the bins so as to get the most likely matrix based on what contact frequency distribution is expected from chromosomes according to a standard polymer physics model [42]. The T. reesei QM6a chromosome sequence we previously published is an example of the raw output from the algorithm.
Manual corrections
Several GRAAL computations were performed with different bin sizes to assess the assembly's robustness. Then manual corrections were performed to go beyond the limitations of GRAAL and other reassembly programs. Since scaffolds were split into bins with the same number of restriction fragments, scaffold ends were too small (sequencing coverage too low) to be included in the computation, so were lost in the raw output sequence. We manually added them so as to get the entire scaffolds in the reassembly. When a scaffold is misassembled in the original genome, GRAAL is able to find the splitting location at an accuracy depending on the size of bins involved in the splitting (around 10–50 kb depending on the definition of the bins, and on the location of the restriction sites). We checked the sequence around the splits and most of the time we noticed nearby the presence of ≈1 kb NNN sequences, so we manually corrected the split location to be consistent with this gap location. Reassembly programs like GRAAL easily reorder bins using contact data, but they may fail in finding the correct bin orientation, so many bins were switched (by comparison with the neighboring bins from the same original scaffold) in the raw output sequence. We manually corrected them to get the scaffolds as in the original assembly without switching bins. However, some scaffolds were too small to get a reliable orientation, in this case, we arbitrarily chose the forward direction for the sequence available in Additional file 2. Seven telomere repeats were identified in the original sequence [13] and six of them were assembled in the chromosomes, as noticed previously [26] although they were not at chromosome ends in the raw output sequence. We checked their presence at chromosome ends, and used them three times to identify the correct bin directions (for scaffold 45, 46, and 64 in chromosomes III, V, and IV, respectively). As for scaffold 31 on chromosome VI, we deleted 7 kb at the 3′ end because they were duplications of the telomere sequence. Around 20–30 bins (<4% of the total number of bins) had not been reassembled because their signature in the contact matrix was not strong enough for GRAAL. We manually checked the contact matrix and reassembled these bins in the final sequence depending on their contact signature (telomere, centromere, standard). Finally, the gene annotation from the JGI (gtf file for the Filtered Models set of genes, [43]) was mapped to the reassembled sequence in order to get the coordinates of the 9129 genes on the chromosomes.
Centromere positions
Centromere positions along the chromosomes have been manually identified using the contact data (see Additional file 7 for raw data contact frequencies over the entire genome). Because of their Rabl organization, centromeres have stronger interaction with each other than with their neighboring sequences.
Gene enrichment analysis
To calculate the enrichment in genes close to the centromeres, we used the gene annotations (GO terms and EC numbers) from the JGI [43] and from the FungiPath database [44,45,46], and performed the enrichment analysis with the Pathway Tools software [47]. A 50-kb window was defined around the centromeres, which resulted in a set of 238 genes (2.6% of the genome).
Following publication of the original article [1], the authors reported a problem in the drawing of Rut-C30 chromosome III in Fig. 2B of the original article [1]. The two fragments of chromosome I inserted inside chromosome III should be swapped, and the direction of the fragment containing rim101 and cel1a genes should be inverted. This reversed insertion indicates that at least 2 rearrangements occurred simultaneously, so the possible scenario proposed in Fig. 2C of the original article was inaccurate. The corrected figure with modified panels B and C is available in the figure below. The detailed description of Rut-C30 assembly in the Additional file 5 of the original article is correct.
PFGE:
pulse-field gel electrophoresis
chromosome conformation capture
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DPP, YV, and MM prepared the Rut-C30 3C library. LB and MM performed the GRAAL assemblies. EJ performed the manual curation and the assemblies analysis, and is a major contributor in writing the manuscript. FB, MM, AM, and RK supervised the study and contributed in writing the manuscript. All authors read and approved the final manuscript.
We acknowledge Ting-Fang Wang for sharing with us some data about his independent work on T. reesei resequencing, so as we could match up our nomenclature.
The Rut-C30 3C library sequencing data analyzed during this study were deposited on NCBI BioProject Database under the Accession Number PRJNA355969.
LB and MM were supported by funding to R.K. from the European Research Council under the 7th Framework Program (FP7/2007-2013)/ERC Grant Agreement 260822.
Etienne Jourdier and Lyam Baudry contributed equally to this work
Martial Marbouty and Frédérique Bidard contributed equally to this work
IFP Energies nouvelles, 1 et 4 Avenue de Bois-Préau, 92852, Rueil-Malmaison, France
Etienne Jourdier
, Dante Poggi-Parodi
, Yoan Vicq
, Antoine Margeot
& Frédérique Bidard
Groupe Régulation Spatiale des Génomes, Department Genomes and Genetics, Institut Pasteur, 75015, Paris, France
Lyam Baudry
, Romain Koszul
& Martial Marbouty
UMR 3525, CNRS, 75015, Paris, France
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Corresponding authors
Correspondence to Martial Marbouty or Frédérique Bidard.
Additional file 1. Details on QM6a reassembly.
Additional file 2. QM6a reassembly sequence in fasta format (7 chromosomes + unassembled scaffolds).
Additional file 3. Annotation file describing the location on the chromosomes of i) the original scaffolds, ii) the centromeres and iii) the 9129 genes from the JGI Filtered Models set of genes.
Additional file 4. Identification of inverted repeats in T. reesei and F. graminearum centromeres.
Additional file 5. Details on Rut-C30 reassembly.
Additional file 6. List of gene markers used on Figs. 2 and 3, with their names, IDs, locations on scaffolds and chromosomes, and functional annotations.
Additional file 7. Raw data contact frequencies over the entire genome.
Trichoderma reesei
Karyotype
Translocation
Chromosomal contact


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text:
Press ReleaseOnline Travel Agencies (OTA)
Hong Kong Poly Researches Consumer Behavior on OTAs
How to Pick the Perfect Hotel
Hong Kong Poly
Online travel agencies should be able to better predict their customers' decision making and meet their needs, suggest Dr Sangwon Park of the School of Hotel and Tourism Management (SHTM) at The Hong Kong Polytechnic University and his co-researchers in a ground-breaking study published recently. Through identifying a simple typology of decision-making styles, the researchers offer unprecedented insights into precisely how and why Internet users decide which travel products to buy. As the online share of travel revenue increases in the Asia-Pacific region and beyond, their findings have far-reaching implications not only for online hotel marketing but also for the global tourism economy.
According to the researchers, the Internet has revolutionised the way we travel. With the rise of e-commerce and the near-ubiquity of mobile handsets, it is now possible to compare a variety of travel options at the touch of a button, and to book a whole holiday in a matter of hours or even minutes. Yet the benefits for travellers go beyond mere convenience: the Internet is leveling the playing field in terms of access to information. "The advent of online travel agencies (OTAs)", write the researchers, "has contributed substantially to reducing information asymmetry between consumers and service providers by offering not only useful and up-to-date information but also price transparency".
It is no surprise, then, that OTAs have recorded phenomenal growth in the last decade - and this trend shows no sign of slowing. The researchers note that the value of the global online travel market is expected to reach an astonishing US$1,091 billion by 2022, with the greatest growth predicted in the Asia-Pacific region. As disposable income increases worldwide and more people in emerging markets gain access to high-speed Internet connectivity, OTAs are likely to eclipse their offline counterparts.
However, the researchers warn that this growth creates challenges as well as opportunities for travellers and OTAs. As travel options proliferate online, consumers may experience information overload, and OTAs may struggle to differentiate themselves in an increasingly saturated market. How consumers make their travel purchases online - such as the flights or hotels they book - has thus become a hot topic of research in recent years. As the researchers note, understanding travellers' decision-making behaviours is critical "not only for academics but also for practitioners".
Purchasing travel products, explain the researchers, is a high-risk activity, "a complex process that requires an extensive decision-making strategy due to high cost and involvement". Generally, the researchers suggest, a traveller first forms an "awareness set", which contains all of the products - such as hotels - that they know about or have experienced. Next, they funnel down this vast range of travel options to "products that they are considering for purchase". In the final stage, they make a decision.
Yet this process is far from simple, the researchers suggest, as "consumers do not always take homogenous sequential steps to reach their final decisions". Many factors may affect people's online booking behaviour. A convenient, easy to navigate OTA website offering swift price comparison enables travellers to book a hotel in just a few clicks. But the OTA must also provide the right kinds of information, combining the advantages of text and pictures. Last but not least, note the researchers, people booking hotels online are likely to be influenced by their individual characteristics, "such as demographics, product knowledge, online experiences, personality and shopping orientations".
Clearly, given the contribution made by tourism to economies worldwide, it is crucial to help OTAs determine precisely how these factors influence consumers when booking holidays online. This may help them to tailor their marketing strategies to better match travellers with hotels, yielding greater customer satisfaction. Yet research in this area has some surprising gaps. Although online hotel decision-making behaviour is a "dynamic process that encompasses various strategies", write the researchers, most studies have taken a static approach. To make matters worse, studies have generally focused on whether consumers are likely to purchase travel products, not whether they actually do.
The researchers thus set out "to understand the entire online booking process by analysing actual behavioural data". To do this, they needed to use multiple methods: screen capture software, which allowed them to observe the entire process of online hotel decision making "in action", and surveys, to collect the participants' cognitive responses.
They distributed flyers on the campuses of two universities in London and the southeast of England, inviting students and staff to participate. They first asked the 44 respondents to complete a survey on their personal characteristics, Internet usage and past experience of travel. Next, the researchers report, "each respondent was asked to plan an imaginary week-long holiday during the winter to visit Paris".
A wealth of options was available. The accommodation within the participants' budget ranged from low-end to luxury hotels, and they were instructed to plan their holidays using Booking.com, one of the world's largest and most user-friendly OTAs. Paris was chosen as a destination because it offers a huge variety of hotels, meeting the needs of diverse travellers. This, write the researchers, made the city "an ideal test ground to address the purposes of this research".
The next step was to meticulously analyse the videos of the participants' online decision making - down to their mouse clicks and cursor movements. The results were striking. The researchers identified three distinct patterns of decision making when booking online hotels. "Arbitrary" decision makers chose their hotels directly from the vast array of options available to them. "Standard" decision makers created a "wish list" of hotels before making their final decision. "Comprehensive" decision makers "appeared to use a more sophisticated process of decision-making".
Not surprisingly, arbitrary decision making took the least time. Compared with the other two groups, travellers in this group were younger, earned less and were more likely to be male. Their main concern when choosing a hotel was price. This was also true of the standard decision makers. At first, they were swayed by hotel location, but when making the final decision, they focused on room rates. However, things were rather different for the comprehensive decision makers. They were "more likely to deliberate the types of services on offer", write the researchers, "whereas the other types of decision makers were largely concerned with room prices".
This typology, explain the researchers, suggests that "the way a traveller behaves online reflects different traveller characteristics", and that different factors - such as hotel price or location - matter more than others at different points in the online booking process. This knowledge will undoubtedly help OTAs to customise their marketing strategies to meet the needs of different categories of traveller. For example, argue the researchers, "streamlined online interfaces could be made to suit the fast-paced decision making of arbitrary decision makers". Comprehensive decision makers would benefit more from tools such as wish lists or comparison features.
As the researchers note, their novel typology will help OTAs to stand out from their competitors by "understanding travellers' heterogeneous information needs" and creating tailor-made approaches to meet them. But OTAs are not the only beneficiaries of this timely research. Travellers will have better and more targeted information at their fingertips, enabling them to pick the perfect hotel with minimal fuss, and hoteliers will have more satisfied customers. As the volume of online bookings continues to grow, and tourism makes an increasingly critical contribution to economic development in the Asia-Pacific region and beyond, this can only spell good news for societies worldwide.
Sangwon Park, Yizhen Yin and Byung-Gak Son (2019). Understanding of Online Hotel Booking Process: A Multiple Method Approach. Journal of Vacation Marketing, Vol. 25, Issue 3, pp. 334-348.
About PolyU's School of Hotel and Tourism Management
For over 40 years, the School of Hotel and Tourism Management (SHTM) of The Hong Kong Polytechnic University has refined a distinctive vision of hospitality and tourism education and become a world-leading hotel and tourism school. Ranked No. 1 in the world in the "Hospitality and Tourism Management" category in ShanghaiRanking's Global Ranking of Academic Subjects 2022 for the sixth consecutive year, placed No. 1 globally in the "Commerce, Management, Tourism and Services" category in the University Ranking by Academic Performance in 2020/2021 for four years in a row, rated No. 1 in the world in the "Hospitality, Leisure, Sport & Tourism" subject area by the CWUR Rankings by Subject 2017, and ranked No. 1 in Asia in the "Hospitality and Leisure Management" subject area in the QS World University Rankings by Subject 2022 for the sixth consecutive year, the SHTM is a symbol of excellence in the field, exemplifying its motto of Leading Hospitality and Tourism.
The School is driven by the need to serve its industry and academic communities through the advancement of education and dissemination of knowledge. With a strong international team of over 80 faculty members from diverse cultural backgrounds, the SHTM offers programmes at levels ranging from undergraduate degrees to doctoral degrees. Through Hotel ICON, the School's groundbreaking teaching and research hotel and a vital aspect of its paradigm-shifting approach to hospitality and tourism education, the SHTM is advancing teaching, learning and research, inspiring a new generation of passionate, pioneering professionals to take their positions as leaders in the hospitality and tourism industry.
Pauline Ngan
Online Travel Agencies (OTA)

redpajama_set_name: RedPajamaCommonCrawl


text:
Le Journal de Montréal (lit., 'El Periódico de Montreal') es un periódico de Montreal, en la provincia de Quebec. Es un periódico importante del conglomerado de la empresa Sun Media, perteneciente a Quebecor Media. Sus competidores principales son La Presse (propiedad de Gesca) y Le Devoir (independiente).

Enlaces externos
 Sitio oficial (en francés)

Journal de Montréal, Le
Journal de Montréal, Le
Publicaciones fundadas en 1964
Journal de Montréal, Le

redpajama_set_name: RedPajamaWikipedia


text:
Agnellus – imię męskie pochodzenia łacińskiego, pochodzące od wyrazu agnus – "jagnię", i oznaczające "jagniątko". Istnieje 3 świętych Agnellusów.

Agnellus imieniny obchodzi 14 grudnia.

Znane osoby noszące to imię:
 Agnellus z Pizy - włoski franciszkanin, błogosławiony Kościoła rzymskokatolickiego

Zobacz też
 Agnellus - polski zespół muzyczny

Przypisy

Męskie imiona łacińskie


text:
Ireland upgrades Marine Data Buoy Network in face of climate change
From left: Minister Michael Creed, TD; Dr Peter Heffernan, Marine Institute; and Evelyn Cusack, Met Éireann. Image: Darragh Kane
Silent sentinels in the sea stand guard against violent weather pattern changes.
Ireland has invested more than €700,000 to upgrade its Marine Data Buoy Network system to safeguard the country and provide early warnings and forecasts as weather patterns become more unpredictable.
The Marine Data Buoy Network is managed by the Marine Institute in collaboration with Met Éireann. The network provides crucial data for weather forecasting, risk management for shipping, advance warnings for the fishing community and coastal towns and villages, as well as oceanography research and data on Ireland's deep waters.
'With the incidences of extreme weather conditions increasing, the Government's investment in the Irish Marine Data Buoy Network is very significant but essential'
– MINISTER MICHAEL CREED, TD
With an additional injection of €300,000, the total investment for the network in 2018 amounts to more than €700,000. This investment will enable the upgrade of the network with new-generation buoy platforms and a suite of sensors, replacing the current technology that has been in use since 2008.
"Investment in the observation buoys and other infrastructures and research capacities will enable Ireland to be at the forefront in providing critical research capacity and overcoming infrastructure gaps that, in the past, have reduced our ability to address questions of national and global importance with respect to climate and ocean change," explained Dr Peter Heffernan, CEO of the Marine Institute.
Climate change impact is felt
These buoys act as a system of sentinels that sit hundreds of kilometres out to sea in the Atlantic and warn of mounting danger.
During Hurricane Ophelia in 2017, waves were recorded at a maximum height of 17.8 meters by the M5 weather buoy off the south-east coast. In 2011, the M4 weather buoy located 75km north of Belmullet on the north-west coast of Ireland saw the largest waves recorded in Irish waters, reaching a maximum height of 20.4 meters.
The most westerly buoy, M6, located hundreds of kilometres to the west of Ireland, is a sentinel buoy that gathers critical early data reported hourly on weather approaching Ireland and Europe from the Atlantic.
"The data buoys provide vital information about our weather such as atmospheric pressure, air and sea temperature, wind speed and direction," said Evelyn Cusack, head of forecasting at Met Éireann. "This information is used in the weather forecasting models run by Met Éireann that provides guidance to the national emergency planning efforts during extreme weather events, including storms such as Ophelia and Emma."
The Minister for Agriculture, Food and the Marine Michael Creed, TD, said that with the impact of climate change becoming more apparent, the Irish Government recognised the importance of investing in the Marine Data Buoy Network system.
"This increased expenditure will greatly assist our ocean and weather forecasting capabilities in the years ahead as well as supporting vital climate change research and improving safety at sea. Climate change is one of the greatest challenges facing nations, governments and decision-makers worldwide.
"With the incidences of extreme weather conditions increasing, the Government's investment in the Irish Marine Data Buoy Network is very significant but essential. This ongoing and additional funding will enable the Marine Institute to provide essential national services in ocean observation and weather forecasting programmes that have regional and local impact on our livelihoods, safety and the growing blue economy."
Related: data, networks, Marine Institute, marine life, Climate, government & policy, safety
Building digital transformation solutions for the climate
Ireland falls two places in EY Renewable Energy Country Attractiveness Index
€126m Bord na Móna plan aims to store 100m tonnes of CO2 in peatland
Government updates Work Safely Protocol in lead-up to Christmas


text:
package com.adobe.dp.epub.dtd;

import org.xml.sax.EntityResolver;
import org.xml.sax.InputSource;
import org.xml.sax.SAXException;

import java.io.ByteArrayInputStream;
import java.io.IOException;
import java.io.InputStream;
import java.util.Hashtable;

public class EPUBEntityResolver implements EntityResolver {

	Hashtable systemIdMap = new Hashtable();

	EPUBEntityResolver() {
		// fully-resolved names
		systemIdMap.put("http://www.idpf.org/dtds/2007/opf.dtd", "opf20.dtd");
		systemIdMap.put("http://openebook.org/dtds/oeb-1.2/oeb12.ent", "oeb12.dtdinc");
		systemIdMap.put("http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd", "xhtml1-transitional.dtd");
		systemIdMap.put("http://www.w3.org/TR/xhtml1/DTD/xhtml1-strict.dtd", "xhtml1-strict.dtd");
		systemIdMap.put("http://www.w3.org/TR/xhtml1/DTD/xhtml-lat1.ent", "xhtml-lat1.dtdinc");
		systemIdMap.put("http://www.w3.org/TR/xhtml1/DTD/xhtml-symbol.ent", "xhtml-symbol.dtdinc");
		systemIdMap.put("http://www.w3.org/TR/xhtml1/DTD/xhtml-special.ent", "xhtml-special.dtdinc");
		systemIdMap.put("http://www.w3.org/Graphics/SVG/1.1/DTD/svg11.dtd", "svg11.dtd");
		systemIdMap.put("http://www.w3.org/TR/xhtml11/DTD/xhtml11.dtd", "opf20.dtd");
		systemIdMap.put("http://www.daisy.org/z3986/2005/dtbook-2005-2.dtd", "dtbook-2005-2.dtd");
		systemIdMap.put("http://www.daisy.org/z3986/2005/ncx-2005-1.dtd", "dtd/ncx-2005-1.dtd");
	}

	public InputSource resolveEntity(String publicId, String systemId) throws SAXException, IOException {
		InputStream in = null;
		if (systemIdMap != null) {
			String res = (String) systemIdMap.get(systemId);
			if (res != null)
				in = EPUBEntityResolver.class.getResourceAsStream(res);
		}
		if( in == null ) {
			in = new ByteArrayInputStream(new byte[0]);
			System.err.println("Unknown systemId: " + systemId);
		}
		InputSource source = new InputSource(in);
		source.setSystemId(systemId);
		return source;
	}

	public static final EPUBEntityResolver instance = new EPUBEntityResolver();
}

redpajama_set_name: RedPajamaGithub


Sarah Xu - RMT - Registered Massage Therapy in Richmond - Accepting New Patients!
Sarah graduated from Vancouver College of Massage Therapy. She is a high-energy Registered Massage Therapist with knowledge of wide range of massage methods and techniques. She likes to incorporate relaxation massage in myofascial release and trigger point therapy treatments. Combining these different techniques, she can increase the effectiveness of her massage in relieving muscle soreness from every day use or injuries. She is also able to apply Manual Lymph Drainage to help patients to reduce swelling and detoxify the body after surgery. Sarah is fluent in both English and Mandarin.


text:
Tag Archives: beef on the menu
Dining & wining on New Year's Eve
I haven't found evidence that people celebrated New Year's Eve in restaurants or hotels much in the 19th century. But in the early 20th century it became a more popular thing to do. Having a reservation at a swanky place conferred status, as the 1912 drawing above is meant to illustrate.
If one dish ruled New Year's Eve menus in the early 20th century it was roast turkey. It was the main dish at the Techau Tavern, "San Francisco's Busiest and Handsomest High-Class Café," shown here in 1909. Turkey with Cranberry Dressing and Chestnut Stuffing was preceded by Toke Point Oysters from Washington, Cream of Chicken Soup, Striped Bass, and Sweetbreads.
Turkey also dominated the 1912 New Year's Eve menu at The Fern in Scranton PA, "An Eating Place of Refinement and Respectability." The Fern featured a $1 dinner that was similar but even heftier than the Techau Tavern's, with Blue Point Oysters, Cream of Chicken Soup, Baked Bluefish, Croustade of Lobster, Tenderloins of Beef, Roast Turkey, Sweetbreads, Banana Fritters, as well as all kinds of vegetables, salad, and pie. The Alt Heidelberg Café in Fort Wayne IN, and Tait's in San Francisco provided similar menus.
Through the 1920s, it was not hard to find a place where one dollar or a little more would buy an elaborate dinner with an orchestra and dancing. In Seattle WA, New Year's Eve entertainment at the Hotel Washington Annex included dinner plus a "lady vocalist" and the Whangdoodle Quartet, all for $1.25. The absence of (legal) alcohol in the 1920s did not dim festivities in Chicago nightspots in the Loop or on the South Side, where "handling a flask has never been considered flagrant."
Despite tight economic times in the 1930s and war in the 1940s – or maybe because of these conditions — Americans showed continuing enthusiasm for celebrating the new year in restaurants and clubs. The repeal of Prohibition at the end of 1933 brought about heightened levels of good cheer. Not even higher prices at the end of 1934 discouraged revelers who paid from $7.50 to $15 per plate in NYC. Yet there were still bargains to be had as the accompanying 1935 menu from Kolb's in New Orleans shows. Low prices also prevailed in Canton OH where sauerkraut and wieners were traditional.
After the war, menus reflected the growing importance of beef on New Year's Eve – and throughout the year. The 1958 menu at Pike's Verdugo Oaks in Glendale CA is representative, and not terribly different than menus found throughout the U.S. in later decades. The Raintree Room at the Continental Regency in Peoria IL in 1978, for example, offered beef or lobster, with salad, baked potato or french fries, and the ubiquitous cheesecake for dessert, a standard menu for a restaurant dinner in the last decades of the 20th century.
Whether you have steak or hot dogs this New Year's Eve, have a good time and best wishes for 2018!
Filed under food, menus, night clubs, restaurant customs
Tagged as 20th century restaurants, beef on the menu, New Year's Eve, New Year's eve dinners, restaurants on holidays, roast turkey
"Distinguished dining" awards
After World War II American consumers were filled with pent-up demand accrued over years of rationing and deprivation. They wanted to sample the joys of the good life, which included American and world travel, even if only in their imaginations. A sophisticated magazine – Holiday — was created to cater to their aspirations.
Holiday's first issue came out in March 1946. A couple of months later Madison Avenue advertising man Ted Patrick took over as editor. A gourmet and bon vivant, Patrick gravitated toward fine restaurants. In 1952 the magazine began presenting awards to American restaurants that achieved dining distinction, recognizing 49 the first year. Among the winners were Bart's (Portland OR), Commander's Palace (New Orleans), Karl Ratzsch's (Milwaukee), and Win Schuler's (Marshall MI).
Winners tended to remain on the list, though it was not guaranteed. Win Schuler's (still in business today) featured steaks, prime rib, and pork chops, and hosted 1,200 patrons a day at its Marshall location [menu below]. In 1971 it won its 20th Holiday award, no doubt not its last.
Even if, as Harvey Levenstein writes in Paradox of Plenty, Holiday stuck to "safe, sound, and usually American" choices where "the steak, lobster, and roast beef syndrome . . . reigned supreme," its recommendations carried weight and raised the seriousness with which many American diners and restaurateurs regarded restaurants.
To win, a restaurant's offerings were supposed to compare to French cuisine. It's hard to see how a steak-and-baked-potato place could do that, but plenty such restaurants won awards. On the other hand, many of the winners were French inflected, particularly in NYC. A quick scan of restaurants included in the 1976 Holiday Magazine Award Cookbook shows that nearly 25% had French names and many more specialized in French dishes.
What some thought was a bias for restaurants in NYC and, to a lesser degree, NY state prevailed until 1968 when California restaurants won as many awards as New York (even though the number of winners in San Francisco still lagged behind NYC, 17 to 25).
The overall volume of winners grew over the years, reaching over 200 by the mid-1970s. The numbers reflected the growth in dining out – and maybe the tendency of award programs to expand. In the beginning whole swaths of the country had nary a winner. Winners would boast that they were "the only" restaurant – for example, in Wisconsin, in the South outside of Florida, among Midwestern states, etc. But over time winners could be found in all parts of the country, requiring some adjustment in the meaning of distinction. Statements appeared saying that awards were not given solely to elegant places. As Patrick's successor Silas Spitzer said, "Elegance has a certain value in making our judgment of restaurants – but it's not essential."
I suspect that the significance of the awards was greatest during Patrick's editorship, which ended with his death in 1964. The magazine fell on hard times in the 1970s and was sold in 1977. Even earlier the awards were losing clout. Among those in the 1976 cookbook were several that had come under harsh criticism. Many specialized in "continental" cuisine which had lost its glamour by this time, or were considered uninspired. In 1974 John Hess wrote that The Bakery in Chicago and Ernie's in San Francisco were "disappointing." NYT critic John Canaday declared in 1975 that Le Manoir was the French restaurant where he had the worst meal in the past 20 months, Le Cirque the "worst restaurant in proportion to its popularity," and the "21″ Club "least worth the trouble."
The awards, called Travel-Holiday awards after Holiday's 1977 merger with Travel, continued until 1989.
Filed under guides & reviews
Tagged as 1950s, 1960s, 1970s, beef on the menu, French restaurants, Holiday magazine, restaurant awards


Take Exit 50, (Main Street) - Go straight to the third light and turn left onto Trumbull Street. Continue to the fifth light and turn right on to Pearl Street. The CITYPLACE garage will be on your right.
Take Exit 49, (High Street/Ann Street) -Go Straight to the third light and turn right onto Trumbull Street. Continue to the fourth light and turn right on to Pearl Street. The CITYPLACE garage will be on your right.
Take Exit 32B (ROUTE 84/TRUMBULL STREET), bearing right for Trumbull Street. Proceed straight through nine traffic lights and turn right on to Pearl Street. The CITYPLACE garage will be on your right.


This program is a Sterling tradition! Enjoy the outdoors at Sholan Park with Red Cross swim lessons, games and crafts. Children will have fun with dress-up themes, team relays, music, and more! Play classic games, learn new ones and have some old-fashioned fun!
This classic program runs 8:30am-12:00pm. This program is designed to run in 2 week sessions, however we will offer One Week options.
Don't want the fun to stop there? Check out our NEW Full-Day Summer Lake Program!
This summer we will offer One Week Options, where children can attend individual weeks of camp.
Participants in one week options will not be eligible for swim level testing (they will still receive lessons).
NEW for Summer 2019: there will be no half-day program on days of INCLEMENT WEATHER. We apologize for the inconvenience.
No program 7/4. Program dependent on weather. Bring snack, towel, bathing suit, & sunscreen daily.
Program dependent on weather. Bring snack, towel, bathing suit, & sunscreen daily.


As Hollywood's finest continues to make their way down the red carpet at the 89th annual Academy Awards, a growing list of stars are showing their opposition against President Donald Trump's immigration ban and other controversial policies in a simple, yet very symbolic way. The little piece of fabric pinned on tuxedos and designer gowns may have some wondering what the blue ribbons are at the 2017 Oscars and what this extra accessory represents.
Earlier this week, the American Civil Liberties Union launched a new "Stand with ACLU" initiative, encouraging Hollywood stars to wear a blue ribbon on the red carpet "as a symbol of solidarity with the nonpartisan, nonprofit organization, which has worked for nearly 100 years to defend and protect individual rights and liberties guaranteed by the Constitution and law of the U.S.," according to The Hollywood Reporter.
Actress Ruth Negga, who is nominated for her role in Loving, and Hamilton's Lin-Manuel Miranda as well his mother Dr. Luz Towns-Miranda, were some of the first spotted wearing blue ribbons with "ACLU" written in white on the Oscars' red carpet on Sunday night.
According to NPR, actor Casey Affleck also supported the ACLU by wearing one on his lapel at Saturday's Film Independent Spirit Awards and made a political statement during his acceptance speech, slamming Trump's wave of executive orders.
"The policies of this administration are abhorrent, and they will not last and they are really un-American," Affleck said on Saturday, according to NPR.
The ACLU has had a major presence throughout this year's awards season. According to Billboard, actress Sarah Paulson gave the nonprofit organization a shout out when she received her award for outstanding performance by a female actor in a miniseries or television movie at the Screen Actors Guild Awards, asking others to donate anything that they could to the ACLU. "Any money you have to spare, please donate to the ACLU," Paulson during her speech, according to Billboard. "It's a vital organization that relies entirely on our support."
Support for the ACLU has skyrocketed in the days after Trump signed an executive order temporarily banning immigrants from seven predominantly Muslim countries, raising $24 million in online donations in a weekend, according to The Washington Post.
As the end of awards season nears, this added support on the numerous red carpets has certainly had an impact on the exponential level of support for the ACLU as they continue to fight Trump's many controversial policies.


When my sister's artwork unraveled me.
One of the most frequent activities we do at Wipe Every Tear is art therapy. It's not anything official, we just put art supplies on the floor and our sisters express their hearts–the good, bad, and ugly–on the pieces of paper in front of them. Sometimes they create something simple and exquisite, sometimes it's a watercolor piece that bleeds off the page, and sometimes girls just write. It gets messy, paint splattered everywhere, pencils strewn across the tile, everyone sprawled out, working on their projects.
The other night, a specific painting touched my heart.
Allow me to frame this for a moment. Lately, I've been wrestling with so much shame. I mean, heavy shame, that feels like a crushing cement disc on my chest, stealing my thoughts and joy the longer it sits on me. I know I'm a missionary out here in the Philippines, but man, I struggle. I hurt. I wrestle. I fail. I cry. I sweat. Life isn't one massive sunrise out here; often it's a dark night. I've realized that there is a deep brokenness in my heart and that I need Jesus more than ever. I just never allowed myself to be stretched enough to recognize the ugly lurking in my heart.
With that said, accepting the Lord's love and goodness for me has been a real challenge lately. Seems ironic, amidst me constantly reminding the ladies in our care that Jesus has eradicated their sin and shame and that they are no longer chained to their pasts. But when my soul is the sail amidst the tempest, its harder to navigate the way to the Cross.
Yet the love of Jesus has become more real to me here. I can't even describe it, how much I feel at the true recognition not of just His forgiveness towards me, but how much He truly forgives, each and every day. This concept of tidal-wave love is equally as overwhelming as my shame–and exponentially more, actually–but it's weight is sweet and peaceful. Like finally crashing into bed at the end of a long day, no longer obligated to fight off sleep. It just comes, like an inescapable blanket, and washes over.
I sensed the touch of Jesus tonight, as we colored and painted. It was like He pulled the covers over me again, the blankets of his love and grace and mercy.
As we began our evening, one of my sisters started a drawing, barely managing to scribble one figure before she decided she didn't like the way it looked, and wanted to draw something else. She crumpled the paper, and tossed it aside.
The sister seated to the right of me carefully picked up the page, flattened it out again, and began adding various colors to its center.
I watched as she would pick up her paper and allow each watery droplet of color to run along the crumpled lines, like a stream relocating its course at the first rain after the dry season. Over and over, she repeated this gesture until every edge was adorned with color.
As she drew to a close with work, she crowned her piece with a red heart, drawn around the summit of paint.
The Lord spoke so deeply to me in that moment.
For all the times I've felt like a crumpled, used, discarded piece of paper, He's picked me up, turned me over, and headed straight to work creating beauty from my plain and simple ugliness.
He doesn't try too hard to straighten out my edges or cover up my scars. Instead He adds color and creates something out of their unique design. When He's finished with me, my scars stand out more than they did before, but they constitute my story. They serve as my testimony of God making a masterpiece out of my rough edges and wounds. My ugliness now has a purpose of letting others know what is waiting for them at the moment of their brokenness: love, patience, care, acceptance, healing, freedom, future, hope.
And the heart. The crowning beauty. The feature that ties this whole piece together and gives it the ultimate meaning.
Jesus' love is what collects the pieces of my life, the discarded and the ugly, and uses His creative hand to work a miracle out of the chaos. The blood red of the heart reminds me of the only thing that will actually clean me and give me purpose. Only Jesus, only His sacrifice, only His life, can set me free and make me whole.
I truly was shaken (in a fantastic way) by this piece. I told my sister that she truly had allowed the Lord to speak through her artwork, and it was true. The Lord pierced my shame and anxiety that night with a simple, pure depiction of Jesus' love.
Living here, doing ministry with Wipe Every Tear, and simply developing friendships with my sisters here is such a humbling experience. I realize I am right alongside them on my own journey of healing and freedom. Sure, we come from very different and distant corners of the earth, and we have varying sources of our wounds and points where we failed and fell short of God's glory. But now we are here, doing life together in the Philippines. Merely watching the Lord speaking so personally to my sisters is what pierces the hardness in my own heart and conducts an Ezekiel 36:26 heart-transplant. My stony, calloused heart is becoming clean and supple again by the lives of these women and by the sweet love of Jesus.
What a joy it is to know that God takes the crumpled, discarded, wasted pieces of my life and creates something beautiful. That's what He does with all of us.


professional publicists I've ever worked with."
package- intelligent, classy and brilliant!"
industry is awesome. Her enthusiasm and drive make it happen. "
"KBC Media team – Outstanding work and results"
the booked me on a show or in print and my speaking engagements increased.
Karen and the KBC Team Introduced the new brand to consumers and media across the USA!


text:
<?php

use Illuminate\Support\Facades\Schema;
use Illuminate\Database\Schema\Blueprint;
use Illuminate\Database\Migrations\Migration;

class StudentsRemoveStudentIdUniqueConstraint extends Migration
{
    /**
     * Run the migrations.
     *
     * @return void
     */
    public function up()
    {
        Schema::table('students', function (Blueprint $table) {
            $table->string('student_id')->nullable()->change();
        });
    }

    /**
     * Reverse the migrations.
     *
     * @return void
     */
    public function down()
    {
        Schema::table('students', function (Blueprint $table) {
            $table->string('student_id')->unique()->change();
        });
    }
}