Differential dependence on microbiota of IL-23/IL-22-dependent gene expression between the small- and large-intestinal epithelia.

In the intestine, interleukin (IL)-23 and IL-22 from immune cells in the lamina propria contribute to maintenance of the gut epithelial barrier through the induction of antimicrobial production and the promotion of epithelial cell proliferation. Several previous studies suggested that some of the functions of the IL-23/IL-22 axis on intestinal epithelial cells are shared between the small and large intestines. However, the similarities and differences of the IL-23/IL-22 axis on epithelial cells between these two anatomical sites remain unclear. Here, we comprehensively analyzed the gene expression of intestinal epithelial cells in the ileum and colon of germ-free, Il23-/- , and Il22-/- mice by RNA-sequencing. We found that while the IL-23/IL-22 axis is largely dependent on gut microbiota in the small intestine, it is much less dependent on it in the large intestine. In addition, the negative regulation of lipid metabolism in the epithelial cells by IL-23 and IL-22 in the small intestine was revealed, whereas the positive regulation of epithelial cell proliferation by IL-23 and IL-22 in the large intestine was highlighted. These findings shed light on the intestinal site-specific role of the IL-23/IL-22 axis in maintaining the physiological functions of intestinal epithelial cells.

Determination of Cyanide and Cyanoglycosides in Sweetened Bean Paste by HPLC with Fluorescence Detection.

It is necessary to evaluate the efficiency of reduction for cyanide and cyanoglycosides during the manufacturing process from raw material beans to sweetened bean paste in a food hygiene control system from the viewpoint of food safety. Analytical methods for cyanide and cyanoglycoside determination in sweetened bean paste by HPLC with fluorescence detection were developed. In analysis of collection time of free cyanide in the free cyanide assay, the recovery was improved by extending the collection time, the recovery rate was >80% by 2 h. The accuracy, repeatability and intra-laboratory precision of the free cyanide assay were 82.3, 2.0, and 2.4%, respectively. The method for cyanoglycoside analysis was evaluated by 5 repeated spiked recovery experiments at a concentration of 10 ppm. The accuracy, repeatability and intra-laboratory precision of the cyanoglycoside method were 82.2, 1.9, and 3.4%, respectively. These analytical methods will enable the analysis of cyanide and cyanoglycosides in sweetened bean paste without using steam distillation method in the pretreatment.

Sialylation shapes mucus architecture inhibiting bacterial invasion in the colon.

In the intestine, mucin 2 (Muc2) forms a network structure and prevents bacterial invasion. Glycans are indispensable for Muc2 barrier function. Among various glycosylation patterns of Muc2, sialylation inhibits bacteria-dependent Muc2 degradation. However, the mechanisms by which Muc2 creates the network structure and sialylation prevents mucin degradation remain unknown. Here, by focusing on two glycosyltransferases, St6 N-acetylgalactosaminide α-2,6-sialyltransferase 6 (St6galnac6) and ß-1,3-galactosyltransferase 5 (B3galt5), mediating the generation of desialylated glycans, we show that sialylation forms the network structure of Muc2 by providing negative charge and hydrophilicity. The colonic mucus of mice lacking St6galnac6 and B3galt5 was less sialylated, thinner, and more permeable to microbiota, resulting in high susceptibility to intestinal inflammation. Mice with a B3galt5 mutation associated with inflammatory bowel disease (IBD) also showed the loss of desialylated glycans of mucus and the high susceptibility to intestinal inflammation, suggesting that the reduced sialylation of Muc2 is associated with the pathogenesis of IBD. In mucins of mice with reduced sialylation, negative charge was reduced, the network structure was disturbed, and many bacteria invaded. Thus, sialylation mediates the negative charging of Muc2 and facilitates the formation of the mucin network structure, thereby inhibiting bacterial invasion in the colon to maintain gut homeostasis.

A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines.

CRISPR-Cas9 system can be used to generate knock-out cancer cell lines. An insertion or deletion induced by a single guide RNA (gRNA) is often used to generate knock-out cells, however, some cells express the target gene by skipping the disrupted exon, or by using a splicing variant, thus losing the target exon. To overcome this unexpected expression of the target gene, almost the entire gene can be swapped with a selection marker. However, it is time-consuming to create a targeting vector which contains 5' and 3' homology arms flanked by a selection marker. Here, we developed a simple and easy method called SUCCESS (Single-strand oligodeoxynucleotides, Universal Cassette, and CRISPR/Cas9 produce Easy Simple knock-out System), to knock-out a target gene without constructing a targeting vector. Our method removed the targeted large genomic region by using two pX330 plasmids encoding Cas9 and gRNA, two 80mer single strand oligodeoxynucleotides (ssODN), and a blunt-ended universal selection maker sequence in B16F10 murine cancer cell and ID8 murine ovarian cancer cell. SUCCESS generated knock-out clones in two murine cancer cell lines by homozygous deletion of the target genomic region, and without constructing targeting vectors. Thus, our method can be widely applied to generate homozygous knock-out cell lines, as well as knock-in cell lines.

Design and Synthesis of 1,2-Deoxy-pyranose Derivatives of Spliceostatin A toward Prostate Cancer Treatment.

We designed and synthesized a novel 1,2-deoxy-pyranose and terminal epoxide methyl substituted derivatives of spliceostatin A using Julia-Kocienski olefination as a key step. With respect to the biological activity, the 1,2-deoxy-pyranose analogue of spliceostatin A suppressed AR-V7 expression at the nano level (IC50 = 3.3 nM). In addition, the in vivo toxicity test showed that the 1,2-deoxy-pyranose analogue was able to avoid severe toxicity compared to spliceostatin A.

NANOG helps cancer cells escape NK cell attack by downregulating ICAM1 during tumorigenesis.

BACKGROUND: At the beginning of tumorigenesis, newly born cancer cells must successfully avoid attack by the immune system. Although most abnormal cells are efficiently identified and destroyed by the immune system, particularly by NK cells, the molecular mechanisms by which newly born cancer cells evade NK cell surveillance are not fully understood. METHODS: NK cell resistance of highly tumorigenic population of human prostate cancer (PCa) cells were confirmed by xenograft in SCID mice with or without NK cell neutralization. The mechanisms by which the tumorigenic PCa cells evaded NK cell attack were investigated by RNAseq, ChIPseq, generation of several transformants and xenograft in SCID mice. RESULTS: Here, we show that PCa cells have a strengthened ability to escape NK cell attack due to NANOG, a pluripotent-related transcription factor, mediating the repression of ICAM1, a cell adhesion molecule, during tumorigenesis. Mechanistically, NANOG directly binds to the region upstream of ICAM1. As the binding between NANOG and the upstream ICAM1 region increases, p300 binding to this region is diminished, resulting in decreased ICAM1 expression. High NANOG expression confers PCa cells the ability to resist NK cell attack via the repression of ICAM1. Consistent with these results, low ICAM1 expression is significantly correlated with a high recurrence rate in patients with PCa. CONCLUSIONS: Our findings indicate that repression of ICAM1 is a critical mechanism by which cancer cells evade attack from NK cells during tumorigenesis. These results suggest a pivotal role of NANOG in establishing a gene expression profile for escaping the immune system.

SF3B2-Mediated RNA Splicing Drives Human Prostate Cancer Progression.

Androgen receptor splice variant-7 (AR-V7) is a constitutively active AR variant implicated in castration-resistant prostate cancers. Here, we show that the RNA splicing factor SF3B2, identified by in silico and CRISPR/Cas9 analyses, is a critical determinant of AR-V7 expression and is correlated with aggressive cancer phenotypes. Transcriptome and PAR-CLIP analyses revealed that SF3B2 controls the splicing of target genes, including AR, to drive aggressive phenotypes. SF3B2-mediated aggressive phenotypes in vivo were reversed by AR-V7 knockout. Pladienolide B, an inhibitor of a splicing modulator of the SF3b complex, suppressed the growth of tumors addicted to high SF3B2 expression. These findings support the idea that alteration of the splicing pattern by high SF3B2 expression is one mechanism underlying prostate cancer progression and therapeutic resistance. This study also provides evidence supporting SF3B2 as a candidate therapeutic target for treating patients with cancer. SIGNIFICANCE: RNA splicing factor SF3B2 is essential for the generation of an androgen receptor (AR) variant that renders prostate cancer cells resistant to AR-targeting therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5204/F1.large.jpg.