Predicted Markers of Overall Survival in Pancreatic Cancer Patients Receiving Dendritic Cell Vaccinations Targeting WT1.

BACKGROUND: We have developed a Wilms' tumor 1 (WT1)-targeting dendritic cell (DC)-based cancer vaccine combined with standard chemotherapy for patients with advanced pancreatic ductal adenocarcinoma (PDA). METHODS: We evaluated predictive markers of overall survival (OS) in PDA patients treated with multiple major histocompatibility complex class I/II-restricted, WT1 peptide-pulsed DC vaccinations (DC/WT1-I/II) in combination with chemotherapy. Throughout the entire period of immunochemotherapy, the plasma levels of soluble factors derived from granulocytes of 7 eligible PDA patients were examined. Moreover, systemic inflammatory response markers (neutrophil-to-lymphocyte ratio [NLR], monocyte-to-lymphocyte ratio [MLR], and granulocyte-to-lymphocyte ratio [GLR]) were assessed. In addition, cytoplasmic WT1 expression in PDA cells was examined. RESULTS: Compared to the 4 non-super-responders (OS <1 year), the remaining 3 super-responders (OS ≥1 year) showed significantly decreased low plasma matrix metalloproteinase-9 levels throughout long-term therapy. The NLR, MLR, and GLR after 5 DC/WT1-I/II vaccinations and 3 cycles of gemcitabine were significantly lower in the super-responders than in the non-super-responders. Furthermore, the cytoplasmic WT1 expression in the PDA cells of super-responders was relatively weak compared to that in the PDA cells of non-super-responders. CONCLUSIONS: Prolonged low levels of a granulocyte-related systemic inflammatory response after the early period of therapy and low cytoplasmic WT1 expression in PDA cells may be markers predictive of OS in PDA patients receiving WT1-targeting immunochemotherapy.

The cleavage program in the 2d cell lineage of Tubifex embryos.

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.

Pathogen-associated regulatory non-coding RNAs and oncogenesis.

Among all new cancer cases in 2012, on average, 15.4% were caused by Helicobacter pylori or oncoviruses, including Epstein-Barr virus, human papillomavirus, hepatitis B virus, hepatitis C viruses, Kaposi sarcoma-associated herpesvirus and human T-lymphotropic virus. These pathogens encode a variety of non-coding RNAs, which are important cofactors for oncogenesis. In this review, we focus on recent developments in the study of long and small non-protein-coding RNAs, including microRNAs, of oncogenic pathogens, and discuss their mechanisms of action in the multiple steps of oncogenesis.

Interferon-alpha competing endogenous RNA network antagonizes microRNA-1270.

A new form of circuitry for gene regulation has been identified in which RNAs can crosstalk by competing for shared microRNAs (miRNAs). Such competing endogenous RNAs (ceRNAs) form a network via shared miRNA response elements (MREs) to antagonize miRNA function. We previously reported natural antisense RNA (AS) as an important modulator of interferon-α1 (IFN-α1) mRNA levels by promoting IFN-α1 mRNA stability. We show that IFN-α1 AS forms a ceRNA network with specific IFN-α AS (IFN-α7/-α8/-α10/-α14) and mRNA (IFN-α8/-α10/-α14/-α17) subtypes from the IFN-α gene (IFNA) family to antagonize miRNA-1270 (miR-1270), thereby modulating IFN-α1 mRNA levels. Bioinformatic analysis demonstrated that IFN-α1 AS harbors multiple miR-1270 MREs (MRE-1270s), whose presence was substantiated by miR-1270 overexpression and transfection of antimiR-1270. The antimiR-1270, complementary to the miR-1270 seed region, revealed that IFN-α1 AS likely shares the MRE-1270 with IFN-α1 mRNA and specific IFN-α AS and mRNA subtypes. Subsequent bioinformatic analysis for MRE-1270s showed that IFN-α1 AS and other RNA subtypes shared the 6-mer MRE-1270 site. Further MRE-mapping demonstrated that the total number of MRE-1270s in IFN-α1 AS accounted for approximately 30 % of the miR-1270 population. AntimiR-1270 transfection also caused specific de-repression of five cellular mRNAs, including that of CAPRIN1. These results suggest that IFN-α1 AS, together with specific IFN-α AS and mRNA subtypes, as well as the five cellular mRNAs, participate as competing molecules in the ceRNA network against miR-1270. This coordinated regulatory architecture suggests a vital function for the innate immune system in maintaining precise physiological type I IFN levels via post-transcriptional regulatory mechanisms.

Progesterone induced mesenchymal differentiation and rescued cystic dilation of renal tubules of Pkd1(-/-) mice.

Autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary disease affecting the kidneys, is caused in 85% of cases by mutations in the PKD1 gene. The protein encoded by this gene, polycystin-1, is a renal epithelial cell membrane mechanoreceptor, sensing morphogenetic cues in the extracellular environment, which regulate the tissue architecture and differentiation. However, how such mutations result in the formation of cysts is still unclear. We performed a precise characterization of mesenchymal differentiation using PAX2, WNT4 and WT1 as a marker, which revealed that impairment of the differentiation process preceded the development of cysts in Pkd1(-/-) mice. We performed an in vitro organ culture and found that progesterone and a derivative thereof facilitated mesenchymal differentiation, and partially prevented the formation of cysts in Pkd1(-/-) kidneys. An injection of progesterone or this derivative into the intraperitoneal space of pregnant females also improved the survival of Pkd1(-/-) embryos. Our findings suggest that compounds which enhance mesenchymal differentiation in the nephrogenesis might be useful for the therapeutic approach to prevent the formation of cysts in ADPKD patients.

The carpal stretch test at the rheumatoid wrist.

The purpose of this study was to evaluate the radiographic changes of the carpus for rheumatoid wrists in patients who underwent the Sauvé-Kapandji procedure by examining the clinical results and comparing pre- and postoperative radiographic measurements. We studied 43 wrists in 37 patients who showed vertical laxity in the radiocarpal and midcarpal joint on preoperative carpal stretch test. Pain was improved in all patients and the forearm rotation angles of the wrist were significantly improved after the operation. The carpal collapse ratio was significantly reduced after the operation. The carpal collapse reduction rate was significantly greater in the group with than that in the group without midcarpal joint vertical laxity on the carpal stretch test. Although the Sauvé-Kapandji procedure was not sufficiently effective in preventing carpal collapse, it did have a protective effect against ulnar carpal shift. The results of our study showed that vertical laxity of the midcarpal joint was the risk factor of the carpal collapse after Sauvé-Kapandji procedure.

Prognostic significance of tumor/stromal caveolin-1 expression in breast cancer patients.

Caveolin-1 (Cav-1) has been extensively characterized in cancer biological research. However, the role of Cav-1 in the interaction between tumor and stromal cells remains unclear. In the present study, we examined Cav-1 expression in tumor cells and stromal cells in breast cancer tissue by immunohistochemical analysis and evaluated its prognostic value in a training cohort. Immunohistochemical analysis of Cav-1 expression was scored as (++), (+) or (-) according to the proportion of positively stained tumor cells (T) and stromal cells (S). Correlation analysis between tumor/stromal Cav-1 expression and clinicopathological parameters revealed that only T(++) Cav-1 status was positively associated with tumor size and histological nodal status (P = 0.019 and 0.021, respectively). Univariate analysis revealed that combined T(++)/S(-) status was significantly correlated with unfavorable prognostic outcomes (P < 0.001). Multivariate analysis demonstrated that this combined status is an independent prognostic factor for primary breast cancer (P = 0.002). Clinical outcomes in different subgroups of breast cancer patients were also strictly dependent on this combined status (P < 0.05). The prognostic value of T(++)/S(-) Cav-1 status was also validated in the testing cohort. Collectively, our data indicate that high Cav-1 expression in tumor cells and lack of this expression in stromal cells could help identify a particular subgroup of breast cancer patients with potentially poor survival. Further studies are required to understand the regulatory mechanism of Cav-1 in the tumor microenvironment.

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment.

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.

Clinical application of double dorsal plates for the distal radius fractures.

The purpose of this study was to develop plates that fit the contour of the distal radius of the Japanese and can be inserted less invasively. Three-dimensional models of 36 radii of 18 volunteers were prepared. Using these models, the shape of the cortical bone on the radial margin of the distal radius and just below the dorsal fourth compartment of the wrist, to which the plates were expected to be applied, was measured, and the curves of the plates were determined. The functions of approximated curves of the plates were: [y = -2 x 10(-8) x5 - 2 x 10(-6) x(4) + 0.0006 x3 - 0.0312 x2 + 0.3274 x + 15.224 on the radial margin of the distal radius and [y = 7 x 10(-7) x5 - 0.0001 x4 + 0.0078 x3 - 0.2355 x2 + 3.1815 x - 5.6383 just below the fourth compartment. The clinical results of the application of double dorsal plates were satisfactory in clinical cases for the distal radius fractures.

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes.

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.

A new role for expressed pseudogenes as ncRNA: regulation of mRNA stability of its homologous coding gene.

We have earlier generated a mutant mouse in a course of making a transgenic line that exhibited interesting heterozygote phenotypes, which exhibited failure to thrive, severe bone deformities, and polycystic kidneys. This mutant mouse provided a clue to uncover a unique role of expressed pseudogenes. In this mutant the transgene was integrated into the vicinity of the expressing pseudogene of Makorin1 called Makorin1-p1. This insertion reduced transcription of the Makorin1-p1, resulting in destabilization of the Makorin1 mRNA in trans via a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. These findings demonstrate a novel and specific regulatory role of an expressed pseudogene as well as functional significance for noncoding RNAs. Next, we developed an original algorithm to determine how many pseudogenes are expressed. Based on our examination 2-3% of human processed pseudogenes are expressed using the most strict criteria. Interestingly, the mouse has a much smaller proportion of expressed pseudogenes (0.5-1%). Pseudogenes are functionally less constrained, and have accumulated more mutations than translated genes. If they have some functions in gene regulation, this property would allow more rapid functional diversification than protein-coding genes. In addition, some genetic phenomena that exhibit incomplete penetrance might be attributed to "mutation" or "variation" of pseudogenes.

An accidental exposure by a medical linear accelerator under construction.

A radiation accident occurred at a medical linear accelerator facility under construction in Japan. The radiation source was a 3- and 6-MV potential drop accelerator designed to produce X-rays for radiation therapy. This accelerator was also capable of producing a 5 to14-MV swept electron beam. During setting up, an operator turned on the accelerator to test the beam not knowing that a man was working on the ceiling above the accelerator. Thus, an X-ray beam was emitted against the ceiling and the man was exposed to 10-MV of X-ray irradiation. However, no obvious physical symptoms were noted. Dose estimation was made from reconstruction of the accident and clinical examinations including chromosome analysis. Mean dose of the whole body ranged from 70 to 180 mSv. Estimated dose from his right foot to hand was between180 to 900 mSv.

Identification of a new target molecule for a cascade therapy of polycystic kidney.

Autosomal dominant polycystic kidney disease is a systemic disorder that primary affects the kidney which is characterized by the formation of fluid-filled cysts in both kidneys that leads to progressive renal failure. Mutated genes, polycystin-1 and polycystin-2, are identified, and evidence has emerged that polycystins are ion channels or regulators of ion channels. In spite of extensive characterization of polycystins, how polycystin channel signaling may be involved in cyst formation in ADPKD is still unclear. We found a mutant mouse which exhibits polycystic kidney and bone deformity in the course of making a transgenic mouse carrying the Drosophila sex-lethal gene. We identified a mutated gene Makorin1 by positional cloning. Makorin1 carries a typical RING-finger motif, suggesting that Makorin1 belongs to ubiquitinase E3 family. Makorin1 would open a new avenue to understand pathogenesis of polycystic kidney, and become a new therapeutic target of polycystic kidney.

An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene.

A pseudogene is a gene copy that does not produce a functional, full-length protein. The human genome is estimated to contain up to 20,000 pseudogenes. Although much effort has been devoted to understanding the function of pseudogenes, their biological roles remain largely unknown. Here we report the role of an expressed pseudogene-regulation of messenger-RNA stability-in a transgene-insertion mouse mutant exhibiting polycystic kidneys and bone deformity. The transgene was integrated into the vicinity of the expressing pseudogene of Makorin1, called Makorin1-p1. This insertion reduced transcription of Makorin1-p1, resulting in destabilization of Makorin1 mRNA in trans by way of a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. Either Makorin1 or Makorin1-p1 transgenes could rescue these phenotypes. Our findings demonstrate a specific regulatory role of an expressed pseudogene, and point to the functional significance of non-coding RNAs.