Kinetic and thermodynamic insights into the inhibitory mechanism of TMG-chitotriomycin on Vibrio campbellii GH20 exo-ß-N-acetylglucosaminidase.

We investigated the inhibition kinetics of VhGlcNAcase, a GH20 exo-ß-N-acetylglucosaminidase (GlcNAcase) from the marine bacterium Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116, using TMG-chitotriomycin, a natural enzyme inhibitor specific for GH20 GlcNAcases from chitin-processing organisms, with p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) as the substrate. TMG-chitotriomycin inhibited VhGlcNAcase with an IC50 of 3.0 ± 0.7 µM. Using Dixon plots, the inhibition kinetics indicated that TMG-chitotriomycin is a competitive inhibitor, with an inhibition constant Ki of 2.2 ± 0.3 µM. Isothermal titration calorimetry experiments provided the thermodynamic parameters for the binding of TMG-chitotriomycin to VhGlcNAcase and revealed that binding was driven by both favorable enthalpy and entropy changes (ΔH° = -2.5 ± 0.1 kcal/mol and -TΔS° = -5.8 ± 0.3 kcal/mol), resulting in a free energy change, ΔG°, of -8.2 ± 0.2 kcal/mol. Dissection of the entropic term showed that a favorable solvation entropy change (-TΔSsolv° = -16 ± 2 kcal/mol) is the main contributor to the entropic term.

Endoscopic submucosal dissection as excisional biopsy for anorectal malignant melanoma: A case report.

BACKGROUND: Anorectal malignant melanoma (AMM) is a rare disorder with an extremely poor prognosis. Although there is currently no consensus on the treatment methods for AMM, surgical procedures have been the most common treatment methods used until now. We recently encountered a case of AMM that we diagnosed using endoscopic submucosal dissection (ESD). To our knowledge, this is the first case of ESD for AMM, suggesting that ESD can potentially be a diagnostic and treatment method for AMM. CASE SUMMARY: A 77-year-old woman visited our hospital with a chief complaint of anal bleeding and a palpable rectal mass. Colonoscopy revealed a 20-mm protruded lesion in the lower rectum. After obtaining biopsy specimens from the lesion, although a malignant rectal tumor was suspected, a definitive diagnosis was not made. Endoscopic ultrasonography revealed tumor invasion into the submucosal layer but not the muscular layer. Therefore, we performed an excisional biopsy using ESD. Immunohistochemical examination of the ESD-resected specimen revealed tumor cells positive for Human Melanin Black-45, Melan-A, and S-100. Moreover, the tumor cells lacked melanin pigment; thus, a diagnosis of amelanotic AMM was made. Although the AMM had massively invaded the submucosal layer and both lymphatic and venous invasion were present, we closely monitored the patient without any additional therapy on the basis of her request. Six months after ESD, local recurrence was detected, and the patient consented to wide local excision. CONCLUSION: It is suggested that ESD is a potential diagnostic and treatment method for AMM.

Gastric adenocarcinoma of fundic gland type spreading to heterotopic gastric glands.

Herein, we present a case of gastric adenocarcinoma of fundic gland type (GA-FG) spreading to heterotopic gastric glands (HGG) in the submucosa. A 58-year-old man with epigastric pain was referred to our hospital and underwent an esophagogastroduodenoscopy. A Borrmann type II gastric cancer at the antrum and a 10 mm submucosal tumor-like lesion in the lesser curvature of the upper third of the stomach were detected. Histological examination of the biopsy specimens obtained from the submucosal tumor-like lesion suggested a GA-FG. Therefore, endoscopic submucosal dissection was performed as excisional biopsy, and histopathological examination of the resected specimen confirmed a GA-FG and HGG proximal to the GA-FG. Although the GA-FG invaded the submucosal layer slightly, the submucosal lesion of the GA-FG had a poor stromal reaction and was located just above the HGG in the submucosa. Therefore, we finally diagnosed the lesion as a GA-FG invading the submucosal layer by spreading to HGG.

A case of small intestinal intussusception caused by anisakiasis diagnosed and treated by upper gastrointestinal endoscopy.

A 35-year-old man attended our hospital with complaining of epigastric pain and vomiting. He was diagnosed with bowel intussusception based on the target sign revealed in the upper jejunum by abdominal computed tomography. However, the cause of the intussusception was not clear. Insertion of an endoscope into the jejunum revealed prominent edema in the upper part of the jejunum, and Anisakis simplex was identified at the site and removed. Symptoms rapidly improved after endoscopic treatment. We report this case because surgery was avoidable and because we are aware of no previous reports of small intestinal intussusception caused by anisakiasis that has been diagnosed and treated endoscopically.

Comprehensive analyses of hox gene expression in Xenopus laevis embryos and adult tissues.

From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA-sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT-PCR and whole mount in situ hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10.

Apolipoprotein C-I mediates Wnt/Ctnnb1 signaling during neural border formation and is required for neural crest development.

In vertebrates, the neural crest and placodes originate in the neural border, which is located between the neural plate and epidermal ectoderm. The neural crest and placodes give rise to a vast array of cell types. Formation of neural crest is a multi-step process, in which Wnt signals are used reiteratively, but it is currently not clear if a Wnt signal is required for neural border formation. Here, we have identified apolipoprotein C-I (apoc1) in a screen for genes regulated by Wnt/Ctnnb1 signaling in late blastula stage Xenopus tropicalis embryos. We show that Xenopus laevis apoc1 encodes a small, secreted protein, and is induced by Wnt/Ctnnb1 signaling. Depletion of Apoc1 protein results in a neural border formation defect and loss of border fates, including neural crest cells. However, unlike another Wnt/Ctnnb1 target, gbx2.2, apoc1 is not required for patterning of the neural border. We further show that gbx2.2 and apoc1 are independently regulated by Wnt signaling. Our results thus suggest that Wnt regulates border formation and patterning by distinct genetic mechanisms.

Total synthesis of TMG-chitotriomycin based on an automated electrochemical assembly of a disaccharide building block.

The total synthesis of TMG-chitotriomycin using an automated electrochemical synthesizer for the assembly of carbohydrate building blocks is demonstrated. We have successfully prepared a precursor of TMG-chitotriomycin, which is a structurally-pure tetrasaccharide with typical protecting groups, through the methodology of automated electrochemical solution-phase synthesis developed by us. The synthesis of structurally well-defined TMG-chitotriomycin has been accomplished in 10-steps from a disaccharide building block.

A case of liver abscess caused by appendicitis which was considered to be drained from hepatobronchial fistula.

A 52-year-old man was referred to our hospital complaining of right lower abdominal pain. He was diagnosed with appendicitis complicated with a liver abscess and underwent an appendectomy. After antibiotic treatment following surgery, the liver abscess penetrated the right lung, which was considered to be drained from a hepatobronchial fistula. Due to the effect of drainage, the liver abscess immediately improved and the patient was subsequently discharged.

Foxh1 Occupies cis-Regulatory Modules Prior to Dynamic Transcription Factor Interactions Controlling the Mesendoderm Gene Program.

The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-ß signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied by Foxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.

Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis.

Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.

Genes coding for cyclin-dependent kinase inhibitors are fragile in Xenopus.

Cell proliferation is strictly regulated by the dosage balance among cell-cycle regulators such as CDK/cyclin complexes and CDK-Inhibitors. Even in the allotetraploid genome of Xenopus laevis, the dosage balance must be maintained for animals to stay alive, and the duplicated homeologous genes seem to have gradually changed, through evolution, resulting in the best genes for them to thrive. In the Xenopus laevis genome, while homeologous gene pairs of CDKs are fundamentally maintained and a few cyclin genes are amplified, homeologous gene pairs of the important CDK-Inhibitors, CDKn1c and CDKn2a, are deleted from chromosomes L and S. Although losses of CDKn1c and CDKn2a can lead to diseases in humans, their loss in X. laevis does not affect the animals' health. Also, another gene coding CDKn1b is lost besides CDKn1c and CDKn2a in the genome of Xenopus tropicalis. These findings suggest a high resistance of Xenopus to diseases. We also found that CDKn2c.S expression is higher than that of CDKn2c.L, and a conserved noncoding sequence (CNS) of CDKn2c genomic loci on X. laevis chromosome S and X. tropicalis has an enhancement activity in regulating the different expression. These findings together indicate a surprising fragility of CDK inhibitor gene loci in the Xenopus genome in spite of their importance, and may suggest that factors other than CDK-inhibitors decelerate cell-cycling in Xenopus.

Genome organization of the vg1 and nodal3 gene clusters in the allotetraploid frog Xenopus laevis.

Extracellular factors belonging to the TGF-ß family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-ß family genes in response to allotetraploidization.

Identification and comparative analyses of Siamois cluster genes in Xenopus laevis and tropicalis.

Two siamois-related homeobox genes siamois (sia1) and twin (sia2), have been reported in Xenopus laevis. These genes are expressed in the blastula chordin- and noggin-expressing (BCNE) center and the Nieuwkoop center, and have complete secondary axis-inducing activity when over-expressed on the ventral side of the embryo. Using whole genome sequences of X. tropicalis and X. laevis, we identified two additional siamois-related genes, which are tandemly duplicated near sia1 and sia2 to form the siamois gene cluster. Four siamois genes in X. tropicalis are transcribed at blastula to gastrula stages. In X. laevis, the siamois gene cluster is present on both homeologous chromosomes, XLA3L and XLA3S. Transcripts from seven siamois genes (three on XLA3L and four on XLA3S) in X. laevis were detected at blastula to gastrula stages. A transcribed gene, sia1p. S, encodes an inactive protein without a homeodomain. When over-expressed ventrally, all siamois-related genes tested in this study except for sia1p. S induced a complete secondary axis, indicating that X. tropicalis and X. laevis have four and six active siamois-related genes, respectively. Of note, each gene required different amounts of mRNA for full activity. These results suggest the possibility that siamois cluster genes have functional redundancy to endow robustness and quickness to organizer formation in Xenopus species.

Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis.

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation.

Genome evolution in the allotetraploid frog Xenopus laevis.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Complete mitochondrial genome of "Xenopus tropicalis" Asashima line (Anura: Pipidae), a possible undescribed species.

The diploid Xenopus tropicalis, with its small nuclear genomic size and short generation time compared to the traditional experimental amphibian X. laevis, is considered a next-generation model animal. Several experimental X. tropicalis lines have been used in research studies. Previous studies showed that the mtDNA sequence of the Asashima line is divergent from other lines and that this line may represent a distinct species. Here, we report the complete nucleotide sequence of this unique X. tropicalis experimental line. The genome is 17,700 bp in length and contains 37 genes commonly found in animal mtDNAs. The 16S rRNA gene sequence in Asashima line differed by over 6% from the standard Nigerian lines (a 3% difference is considered the species threshold in anurans), suggesting that this experimental line is a distinct species from the true X. tropicalis.

Insulin-like factor regulates neural induction through an IGF1 receptor-independent mechanism.

Insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) signalling is required for normal embryonic growth and development. Previous reports indicated that the IGF/IGF1R/MAPK pathway contributes to neural induction and the IGF/IGF1R/PI3K/Akt pathway to eye development. Here, we report the isolation of insulin3 encoding a novel insulin-like ligand involved in neural induction. Insulin3 has a similar structure to pro-insulin and mature IGF ligands, but cannot activate the IGF1 receptor. However, similar to IGFs, Insulin3 induced the gene expression of an anterior neural marker, otx2, and enlarged anterior head structures by inhibiting Wnt signalling. Insulin3 are predominantly localised to the endoplasmic reticulum when otx2 is induced by insulin3. Insulin3 reduced extracellular Wnts and cell surface localised Lrp6. These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling. This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism.

Improved efficiency of definitive endoderm induction from human induced pluripotent stem cells in feeder and serum-free culture system.

Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies, we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition, keeping cell density low during endoderm induction is important for the efficiency. On the other hand, we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm, indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless, these results should enable better control in the production of definitive endoderm-derived cells.

Occupancy of tissue-specific cis-regulatory modules by Otx2 and TLE/Groucho for embryonic head specification.

Head specification by the head-selector gene, orthodenticle (otx), is highly conserved among bilaterian lineages. However, the molecular mechanisms by which Otx and other transcription factors (TFs) interact with the genome to direct head formation are largely unknown. Here we employ ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and find that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we define two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and other head organizer TFs (for example, Lim1/Lhx1 (activator) or Goosecoid (repressor)) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.

Characterization of the insulin-like growth factor binding protein family in Xenopus tropicalis.

The insulin-like growth factor binding protein (Igfbp) family consists of six members designated Igfbp1-6. Igfbps are involved in many vital biological functions. They physically interact with IGFs (IGF1 and IGF2) and act as carriers, thereby protecting IGFs from proteolytic degradation. Thus, they function as modulators of IGF activity. Furthermore, Igfbps have been reported to have IGF-independent activities. They interact with other proteins, including cell surface proteins, extra-cellular matrix proteins, and potentially intracellular molecules. In Xenopus tropicalis (X. tropicalis), only four igfbp genes (igfbp1, igfbp2, igfbp4, and igfbp5) have been identified, and their expression is not well characterized. We report that X. tropicalis genome lacks the igfbp3 and igfbp6 genes based on synteny analyses. We also examined the spatio-temporal expression patterns of igfbp genes in early X. tropicalis development. Expression analyses indicated that they are differentially expressed during early development. Each igfbp gene showed a characteristic spatial expression pattern. Except for igfbp5, they demonstrated overlapping expression in the pronephros. The Xenopus pronephros is composed of four domains (i.e., the proximal tubule, intermediate tubule, distal tubule, and connecting tubule). Our results showed that at least two igfbp genes are co-expressed in all pronephric domains, suggesting that redundant functions of igfbp genes are required in early pronephric kidney development.