ALBERT

Description: Background: Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease across Asia and Europe. In this study, we report the first genome-wide variation analysis of the nematode with an aim to obtain a full picture of its diversity. Methods: We sequenced six key B. xylophilus strains using Illumina HiSeq sequencer. All the strains were isolated in Japan and have been widely used in previous studies. Detection of genomic variations were done by mapping the reads to the reference genome. Results: Over 3 Mb of genetic variations, accounting for 4.1 % of the total genome, were detected as single nucleotide polymorphisms or small indels, suggesting multiple introductions of this invaded species from its native area into the country. The high level of genetic diversity of the pine wood nematode was related to its pathogenicity and ecological trait differences. Moreover, we identified a gene set affected by genomic variation, and functional annotation of those genes indicated that some of them had potential roles in pathogenesis. Conclusions: This study provides an important resource for understanding the population structure, pathogenicity and evolutionary ecology of the nematode, and further analysis based on this study with geographically diverse B. xylophilus populations will greatly accelerate our understanding of the complex evolutionary/epidemic history of this emerging pathogen.

Description: Background: Ingestion of glucosylceramide improves transepidermal water loss (TEWL) from the skin, but the underlying mechanism by which a small amount of dietary glucosylceramide can vastly improve skin conditions remains unclear. In a previous report, glucosylceramides were shown to be digested to sphingoids, which were shown to be absorbed through the intestinal epithelium. Based on these observations, we hypothesized that sphingoids are the key molecules facilitating endogenous ceramide production. In this study, we assessed the effect of 4,8-sphingadienine (d18:2) and 4-hydroxy-8-sphingenine (t18:1), derived from konjac glucosylceramide, on stimulating ceramide production. Methods: Konjac glucosylceramide acidolysis was performed using hydrochloric acid; the resulting d18:2 and t18:1 were fractionated by column chromatography. Real-time quantitative RT-PCR was performed to assess the effect of d18:2 and t18:1 on gene expression in normal human epidermal keratinocytes, while their effect on the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)gamma, was measured using a receptor-cofactor assay system. The effect of d18:2 and t18:1 on stimulating ceramide production was evaluated using HPTLC analysis in a 3-dimensional human skin model. Results: We noted the upregulation of genes related to de novo ceramide synthesis as well as of those encoding the elongases of very long-chain fatty acids by d18:2 and t18:1, but not by glucosylceramide and 4-sphingenine. Both these sphingoids also facilitated the expression of PPARbeta/delta and PPARgamma; moreover, they also demonstrated ligand activity for PPARgamma. These results indicated that d18:2 and t18:1 promote the differentiation of keratinocytes. Analysis of the lipids within the 3-dimensional human skin model indicated that treatment with d18:2 and t18:1 not only upregulated gene expression but also increased ceramide production. Conclusions: The sphingoids d18:2 and t18:1 activated genes related to de novo ceramide synthesis and increased ceramide production, whereas glucosylceramide and 4-sphingenine could not. These results suggest that the effect of dietary glucosylceramides on the skin is mediated by d18:2 and t18:1.

Description: Background: Evolution of novel protein-coding genes is the bedrock of adaptive evolution. Recently, weidentified six protein-coding genes with similar signal sequence from Schistosoma japonicumegg stage mRNA using signal sequence trap (SST). To find the mechanism underlying theorigination of these genes with similar core promoter regions and signal sequence, weadopted an integrated approach utilizing whole genome, transcriptome and proteomedatabase BLAST queries, other bioinformatics tools, and molecular analyses. Results: Our data, in combination with database analyses showed evidences of expression of thesegenes both at the mRNA and protein levels exclusively in all developmental stages of S.japonicum. The signal sequence motif was identified in 27 distinct S. japonicum UniGeneentries with multiple mRNA transcripts, and in 34 genome contigs distributed within 18scaffolds with evidence of genome-wide dispersion. No homolog of these genes or similardomain was found in deposited data from any other organism. We observed preponderance offlanking repetitive elements (REs), albeit partial copies, especially of the RTE-like andPerere class at either side of the duplication source locus. The role of REs as major mediatorsof DNA-level recombination leading to dispersive duplication is discussed with evidencefrom our analyses. We also identified a stepwise pathway towards functional selection inevolving genes by alternative splicing. Equally, the possible transcription models of someprotein-coding representatives of the duplicons are presented with evidence of expression invitro. Conclusion: Our findings contribute to the accumulating evidence of the role of REs in the generation ofevolutionary novelties in organisms' genomes.

Description: Background: Horizontal gene transfer (HGT) has been suggested as the mechanism by which various plant parasitic nematode species have obtained genes important in parasitism. In particular, cellulase genes have been acquired by plant parasitic nematodes that allow them to digest plant cell walls. Unlike the typical glycoside hydrolase (GH) family 5 cellulase genes which are found in several nematode species from the order Tylenchida, members of the GH45 cellulase have only been identified in a cluster including the families Parasitaphelenchidae (with the pinewood nematode Bursaphelenchus xylophilus) and Aphelenchoididae, and their origins remain unknown. Results: In order to investigate the distribution and evolution of GH45 cellulase genes in nematodes and fungi we performed a wide ranging screen for novel putative GH45 sequences. This revealed that the sequences are widespread mainly in Ascomycetous fungi and have so far been found in a single major nematode lineage. Close relationships between the sequences from nematodes and fungi were found through our phylogenetic analyses. An intron position is shared by sequences from Bursaphelenchus nematodes and several Ascomycetous fungal species. Conclusions: The close phylogenetic relationships and conserved gene structure between the sequences from nematodes and fungi strongly supports the hypothesis that nematode GH45 cellulase genes were acquired via HGT from fungi. The rapid duplication and turnover of these genes within Bursaphelenchus genomes demonstrate that useful sequences acquired via HGT can become established in the genomes of recipient organisms and may open novel niches for these organisms to exploit.

Description: Background: Proteins that share a high sequence homology while exhibiting drastically different 3D structures are investigated in this study. Recently, artificial proteins related to the sequences of the GA and IgG binding GB domains of human serum albumin have been designed. These artificial proteins, referred to as GA and GB, share 98% amino acid sequence identity but exhibit different 3D structures, namely, a 3alpha bundle versus a 4beta + alpha structure. Discriminating between their 3D structures based on their amino acid sequences is a very difficult problem. In the present work, in addition to using bioinformatics techniques, an analysis based on inter-residue average distance statistics is used to address this problem. Results: It was hard to distinguish which structure a given sequence would take only with the results of ordinary analyses like BLAST and conservation analyses. However, in addition to these analyses, with the analysis based on the inter-residue average distance statistics and our sequence tendency analysis, we could infer which part would play an important role in its structural formation. Conclusions: The results suggest possible determinants of the different 3D structures for sequences with high sequence identity. The possibility of discriminating between the 3D structures based on the given sequences is also discussed.

Description: Background: Recently, it has been shown that 5-fluorouracil (5-FU) with a strong dihydropyrimidine dehydrogenase (DPD) inhibitor elicits a significant response in bladder cancer with a high level of DPD. However, only a few studies investigated the association between the level of the enzyme that regulates the metabolism of 5-FU and prognosis in bladder cancer. Furthermore, to our knowledge, there has also been no such report in T1G3 bladder tumors treated with BCG. Therefore, we evaluated enzymes that regulate the metabolism of 5-FU in T1G3 tumors treated with BCG immunotherapy using the Danenberg tumor profile (DTP) method, a highly accurate measurement of RNA from paraffin-embedded specimens. Methods: This study included 28 patients with T1G3 bladder cancer, each of whom underwent complete transurethral tumor resection and BCG intravesical instillation at our institution. The median follow-up period was 39 months (range, 3 to 159 months). The DTP method was used to analyze the mRNA expression of 3 enzymes related to 5-FU: DPD, orotate phosphoribosyltransferase (OPRT), and thymidylate synthase (TS). Results: Among the 28 patients, 13 developed recurrences (46.4%) and 5 experienced disease progression (17.9%). An elevated DPD mRNA level was significantly associated with recurrence (p = 0.048) and progression (p = 0.045). However, TS and OPRT mRNA levels were not significantly associated with any other clinical features or outcomes. Furthermore, the high DPD group had a significantly lower recurrence-free survival rate than the low DPD group (p = 0.047). Among patients with low DPD, the 2- and 5-year recurrence-free survival rates were 88.9% and 74.1%, respectively; while among patients with high DPD, the corresponding rates were 61.3% and 36.8%, respectively. TS and OPRT were not significantly associated with recurrence-free survival rates. Conclusion: DPD is significantly associated with recurrence and progression among T1G3 bladder cancer patients treated with BCG.

Description: Background: The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown. Results: To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration. Conclusion: Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.

Description: Background: While some studies have shown that the 3D protein structures are more conservative than their amino acid sequences, other experimental studies have shown that even if two proteins share the same topology, they may have different folding pathways. There are many studies investigating this issue with molecular dynamics or Go-like model simulations, however, one should be able to obtain the same information by analyzing the proteins' amino acid sequences, if the sequences contain all the information about the 3D structures. In this study, we use information about protein sequences to predict the location of their folding segments. We focus on proteins with a ferredoxin-like fold, which has a characteristic topology. Some of these proteins have different folding segments. Results: Despite the simplicity of our methods, we are able to correctly determine the experimentally identified folding segments correctly by predicting the location of the compact regions considered to play an important role in structural formation. We also apply our sequence analyses to some homologues of each protein and confirm that there are highly conserved folding segments despite the homologues' sequence diversity. These homologues have similar folding segments even through the homology of two proteins' sequences is not so high. Conclusion: Our analyses have proven useful for investigating the common or different folding features of the proteins studied.

Description: Background: Recently, it has been shown that 5-fluorouracil (5-FU) with a strong dihydropyrimidine dehydrogenase (DPD) inhibitor elicits a significant response in bladder cancer with a high level of DPD. However, only a few studies investigated the association between the level of the enzyme that regulates the metabolism of 5-FU and prognosis in bladder cancer. Furthermore, to our knowledge, there has also been no such report in T1G3 bladder tumors treated with BCG. Therefore, we evaluated enzymes that regulate the metabolism of 5-FU in T1G3 tumors treated with BCG immunotherapy using the Danenberg tumor profile (DTP) method, a highly accurate measurement of RNA from paraffin-embedded specimens. Methods: This study included 28 patients with T1G3 bladder cancer, each of whom underwent complete transurethral tumor resection and BCG intravesical instillation at our institution. The median follow-up period was 39 months (range, 3 to 159 months). The DTP method was used to analyze the mRNA expression of 3 enzymes related to 5-FU: DPD, orotate phosphoribosyltransferase (OPRT), and thymidylate synthase (TS). Results: Among the 28 patients, 13 developed recurrences (46.4%) and 5 experienced disease progression (17.9%). An elevated DPD mRNA level was significantly associated with recurrence (p = 0.048) and progression (p = 0.045). However, TS and OPRT mRNA levels were not significantly associated with any other clinical features or outcomes. Furthermore, the high DPD group had a significantly lower recurrence-free survival rate than the low DPD group (p = 0.047). Among patients with low DPD, the 2- and 5-year recurrence-free survival rates were 88.9% and 74.1%, respectively; while among patients with high DPD, the corresponding rates were 61.3% and 36.8%, respectively. TS and OPRT were not significantly associated with recurrence-free survival rates. Conclusion: DPD is significantly associated with recurrence and progression among T1G3 bladder cancer patients treated with BCG.

Description: Osteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cas...