ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvß6 (ß6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin ß6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin ß6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin ß6 as a possible therapeutic target or diagnostic marker candidate.

ß4 and ß6 Integrin Expression Is Associated with the Subclassification and Clinicopathological Features of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a heterogeneous group of cancers of the intrahepatic biliary tract. However, few studies have evaluated integrin expression according to an ICC subgroup. We immunohistochemically investigated α6ß4 (ß4) and αvß6 (ß6) integrin expressions in 48 ICCs, and evaluated their relationship with clinical and pathological parameters and ligand expression, as well as transforming growth factor (TGF)-ß1. ß4 and ß6 expressions were detected in 46 (96%) and 35 (73%) ICC cases, respectively. We classified ICC into negative, low (ß4, 29 cases; ß6, 36 cases), or high (ß4, 19 cases; ß6, 12 cases) integrin expression groups. ß4 and ß6 integrin levels were higher in the non-peripheral central localization type ICC than in the peripheral localization type; they were also higher in the periductal-infiltrating or intraductal-growth types than in the mass-forming type ICC; lastly, they were higher in the well-differentiated type than in the poorly-differentiated type ICC. High expression was related to bile duct invasion. In addition, ß4 and ß6 expressions were associated with mucin production and the expression of cytoplasmic epithelial membrane antigen, laminin-5, and tenascin-C. TGF-ß1 was correlated with ß6 expression and poor overall survival. These results suggest that integrin expression is associated with subclassification and clinicopathological features of ICC through the coincident expression of their ligands and TGF-ß1.

The effect of cholesterol overload on mouse kidney and kidney-derived cells.

INTRODUCTION: Dyslipidemia is one of the onset and risk factors of chronic kidney disease and renal function drop is seen in lipoprotein abnormal animal models. However, the detailed molecular mechanism of renal lipotoxicity has not been clarified. Therefore, the present study aimed to investigate the influence of cholesterol overload using mouse kidney tissue and kidney-derived cultured cells. METHODS: C57BL/6 mice were fed normal diet (ND) or 1.25% cholesterol-containing high-cholesterol diet (HCD) for 11 weeks, and we used megalin as a proximal tubule marker for immunohistology. We added beta-very low density lipoprotein (ßVLDL) to kidney-derived cells and examined the effect of cholesterol overload on megalin protein and mRNA expression level, cell proliferation and cholesterol content in cells. RESULTS: In the kidney of HCD mice, the gap between glomerulus and the surrounding Bowman's capsule decreased and the expression level of megalin decreased. After ßVLDL treatment to the cells, the protein expression and mRNA expression level of megalin decreased and cell proliferation was restrained. We also observed an increase in cholesterol accumulation in the cell and free cholesterol/phospholipid ratios increased. CONCLUSIONS: These findings suggest that the increased cholesterol load on kidney contribute to the decrease of megalin and the overloaded cholesterol is taken into the renal tubule epithelial cells, causing suppression on cell proliferation, which may be the cause of kidney damage.

Structure of the fission yeast S. pombe telomeric Tpz1-Poz1-Rap1 complex.

Telomeric shelterin complex caps chromosome ends and plays a crucial role in telomere maintenance and protection. In the fission yeast Schizosaccharomyces pombe, shelterin is composed of telomeric single- and double-stranded DNA-binding protein subcomplexes Pot1-Tpz1 and Taz1-Rap1, which are bridged by their interacting protein Poz1. However, the structure of Poz1 and how Poz1 functions as an interaction hub in the shelterin complex remain unclear. Here we report the crystal structure of Poz1 in complex with Poz1-binding motifs of Tpz1 and Rap1. The crystal structure shows that Poz1 employs two different binding surfaces to interact with Tpz1 and Rap1. Unexpectedly, the structure also reveals that Poz1 adopts a dimeric conformation. Mutational analyses suggest that proper interactions between Tpz1, Poz1, and Rap1 in the shelterin core complex are required for telomere length homeostasis and heterochromatin structure maintenance at telomeres. Structural resemblance between Poz1 and the TRFH domains of other shelterin proteins in fission yeast and humans suggests a model for the evolution of shelterin proteins.

CK2 phospho-independent assembly of the Tel2-associated stress-signaling complexes in Schizosaccharomyces pombe.

An evolutionarily conserved protein Tel2 regulates a variety of stress signals. In mammals, TEL2 associates with TTI1 and TTI2 to form the Triple T (TTT: TEL2-TTI1-TTI2) complex as well as with all the phosphatidylinositol 3-kinase-like kinases (PIKKs) and the R2TP (Ruvbl1-Ruvbl2-Tah1-Pih1 in budding yeast)/prefoldin-like complex that associates with HSP90. The phosphorylation of TEL2 by casein kinase 2 (CK2) enables direct binding of PIHD1 (mammalian Pih1) to TEL2 and is important for the stability and the functions of PIKKs. However, the regulatory mechanisms of Tel2 in fission yeast Schizosaccharomyces pombe remain largely unknown. Here, we report that S. pombe Tel2 is phosphorylated by CK2 at Ser490 and Thr493. Tel2 forms the TTT complex with Tti1 and Tti2 and also associates with PIKKs, Rvb2, and Hsp90 in vivo; however, the phosphorylation of Tel2 affects neither the stability of the Tel2-associated proteins nor their association with Tel2. Thus, Tel2 stably associates with its binding partners irrespective of its phosphorylation. Furthermore, the Tel2 phosphorylation by CK2 is not required for the various stress responses to which PIKKs are pivotal. Our results suggest that the Tel2-containing protein complexes are conserved among eukaryotes, but the molecular regulation of their formation has been altered during evolution.

Synthesis and structure-activity relationship study of FD-891: importance of the side chain and C8-C9 epoxide for cytotoxic activity against cancer cells.

Unified synthesis of FD-891 analogs and their structure-activity relationship are described. By using stereoselective allylation/crotylation and Evans aldol chemistry, six side-chain fragments having different length and terminus were synthesized. These fragments were coupled with a macrolactone fragment, improved synthesis of which was also developed here, to generate FD-891 and five truncated analogs. These synthetic compounds as well as three analogs obtained from fermentation of gene-disrupted Streptomyces graminofaciens mutants were tested for in vitro cytotoxic activity against HeLa cells. As a result, coexistence of the C8-C9 epoxide and side-chain terminus was found to be critical for the cytotoxic activity.

LSD1/KDM1A promotes hematopoietic commitment of hemangioblasts through downregulation of Etv2.

The hemangioblast is a progenitor cell with the capacity to give rise to both hematopoietic and endothelial progenitors. Currently, the regulatory mechanisms underlying hemangioblast formation are being elucidated, whereas those controllers for the selection of hematopoietic or endothelial fates still remain a mystery. To answer these questions, we screened for zebrafish mutants that have defects in the hemangioblast expression of Gata1, which is never expressed in endothelial progenitors. One of the isolated mutants, it627, showed not only down-regulation of hematopoietic genes but also up-regulation of endothelial genes. We identified the gene responsible for the it627 mutant as the zebrafish homolog of Lys-specific demethylase 1 (LSD1/KDM1A). Surprisingly, the hematopoietic defects in lsd1(it627) embryos were rescued by the gene knockdown of the Ets variant 2 gene (etv2), an essential regulator for vasculogenesis. Our results suggest that the LSD1-dependent shutdown of Etv2 gene expression may be a significant event required for hemangioblasts to initiate hematopoietic differentiation.

[Impact of point spread function correction in standardized uptake value quantitation for positron emission tomography images: a study based on phantom experiments and clinical images].

While point spread function (PSF)-based positron emission tomography (PET) reconstruction effectively improves the spatial resolution and image quality of PET, it may damage its quantitative properties by producing edge artifacts, or Gibbs artifacts, which appear to cause overestimation of regional radioactivity concentration. In this report, we investigated how edge artifacts produce negative effects on the quantitative properties of PET. Experiments with a National Electrical Manufacturers Association (NEMA) phantom, containing radioactive spheres of a variety of sizes and background filled with cold air or water, or radioactive solutions, showed that profiles modified by edge artifacts were reproducible regardless of background µ values, and the effects of edge artifacts increased with increasing sphere-to-background radioactivity concentration ratio (S/B ratio). Profiles were also affected by edge artifacts in complex fashion in response to variable combinations of sphere sizes and S/B ratios; and central single-peak overestimation up to 50% was occasionally noted in relatively small spheres with high S/B ratios. Effects of edge artifacts were obscured in spheres with low S/B ratios. In patient images with a variety of focal lesions, areas of higher radioactivity accumulation were generally more enhanced by edge artifacts, but the effects were variable depending on the size of and accumulation in the lesion. PET images generated using PSF-based reconstruction are therefore not appropriate for the evaluation of SUV.

[Introduce best practice for blood drawing to the worlds].

Blood Drawing has dramatically improved medical treatment and medical examination in the 20th century. The system of evacuated blood collection tube was invented in the US mainly to improve accuracy and efficiency of clinical examination while improving Health Care Workers safety (reduce shapes injuries) in 1970's. After the new method of blood collection spread throughout the world, laboratory automation systems and reagents have also improved. In present time, blood is the most important sample for medical examination and treatment. There are various blood collection guidelines Clinical and Laboratory Standards Institute; CLSI, the Japanese Committee for Clinical Laboratory Standards; JCCLS and the Brazilian Society of Clinical Pathology/Laboratory Medicine; BCP/LM, textbooks and training system in the world. Learning the best practice for blood collection technique is crucial in order to reduce pre-analytical error and provide accurate clinical data. Therefore we have to take not only a technical training, but also obtain information from either internal or external sources for developing our knowledge for improving our blood drawing technique.