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We investigated the initial stages of head development using a new method to randomly label chicken epiblast cells with enhanced green fluorescent protein, and tracking the labeled cells. This analysis was combined with grafting mCherry-expressing quail nodes, or node-derived anterior mesendoderm (AME). These live imagings provided a new conception of the cellular mechanisms regulating brain and head ectoderm development. Virtually all anterior epiblast cells are bipotent for the development into the brain or head ectoderm. Their fate depends on the positioning after converging to the AME. When two AME tissues exist following the ectopic node graft, the epiblast cells converge to the two AME positions and develop into two brain tissues. The anterior epiblast cells bear gross regionalities that already correspond to the forebrain, midbrain, and hindbrain axial levels shortly after the node is formed. Therefore, brain portions that develop with the graft-derived AME are dependent on graft positioning.

Cancer immunotherapy, including vaccination, is considered a major scientific and medical breakthrough. However, cancer immunotherapy does not result in durable objective responses against colorectal cancer (CRC). To improve the efficacy of immunotherapy, the present study investigated several biomarkers for selecting patients who were expected to respond well to immunotherapy. Firstly, a comprehensive proteomic analysis was performed using tumor tissue lysates from patients enrolled in a phase II study, in which five human leukocyte antigen (HLA)-A*24:02-restricted peptides were administered. Sialic acid-binding immunoglobulin type lectin (Siglec)-7 was identified as a potential predictive biomarker. Subsequently, this biomarker was validated using western blot analysis, and immunofluorescence using tissue samples from the patients enrolled in the phase II study. The expression levels of Siglec-7 detected by immunofluorescence were quantified and their association with overall survival (OS) in patients treated with the peptide vaccine was examined. Furthermore, considering the important role of tumor-infiltrating lymphocytes (TILs) for CRC prognosis, the densities of CD3+, CD4+, CD8+ and forkhead box P3 (FOXP3)+ T cells in CRC tissues were examined and compared with Siglec-7 expression. The mean expression levels of Siglec-7 were significantly higher in patients with poor prognosis, with an OS of ≤2 years, as shown in comprehensive proteomic analysis (P=0.016) and western blot analysis (P=0.025). Immunofluorescence analysis demonstrated that Siglec-7 was expressed in intratumoral macrophages. The OS in patients with high Siglec-7 expression was significantly shorter than in that in patients with low Siglec-7 expression (P=0.017) in the HLA-A*24:02-matched patients. However, this difference was not observed in the HLA-unmatched patients. There was no significant difference in OS between patients according to the numbers of TILs, nor significant correlation between TILs and Siglec-7 expression. In conclusion, Siglec-7 expression in macrophages in tumor tissue may be a novel predictive biomarker for the efficacy of immunotherapy against metastatic CRC.

Cancer stem-like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P-CSLCs). A P-CSLC-enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by 2-D electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P-CSLCs compared to parental cells. Flow cytometry analysis indicated that CRT was mostly localized to the surface of P-CSLCs and did not correlate with the levels of CD44v9, another P-CSLC biomarker. Furthermore, the side population in the CRThigh /CD44v9low population was much higher than that in the CRTlow /CD44v9high population. Calreticulin expression was also assessed by immunohistochemistry in pancreatic cancer tissues (n = 80) obtained after radical resection and was found to be associated with patients' clinicopathological features and disease outcomes in the Cox proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and postoperative therapy. Our results suggest that CRT can serve as a biomarker of P-CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be considered as a therapeutic target for cancer immunotherapy.

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLC-enriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.

'Candidatus Mycoplasma haemobos', sometimes causative of bovine infectious anemia at various extents, has been demonstrated throughout the world. Here, we show two distinct types of 'Ca. M. haemobos' are distributed among cattle in Japan, by examining the primary and secondary structures of the 16S-23S rRNA intergenic spacer region that has been shown to be a stable genetic marker for mycoplasma species. Our results may explain differences in severity of anemic condition as well as provide a genetic marker for an epidemiological study of bovine hemoplasma infections.

The prevalence of hemotropic mycoplasmas in wild rodents is largely unknown. Here, we report the presence of hemoplasmas in blood samples collected from brown sewer rats (Rattus norvegicus) trapped during rodent control around an animal hospital in Morioka, Japan. We examined nine rats using real-time PCR and end-point PCR, and found one rat (11.1%) that was positive for a hemoplasma infection. The 16S rRNA gene and 16S to 23S rRNA intergenic spacer region of the hemoplasma detected in a wild-caught rat were amplified using PCR. The nucleotide sequences of the PCR products were further determined and compared to those of other hemoplasmas. Our examinations revealed the presence of a hemoplasma that has not previously been described in rodents. The pathogenic traits of this hemoplasma remain unexplored.

The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and ` Candidatus Mycoplasma haemobos (synonym: `C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and `C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.

Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced-induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen-ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease-producing bacteria, as well as in some animals with acidic or alkaline urine.

本調査では岩手県の野生の鳥類における鉛汚染の状況を調べる目的で，2008年10月から2009年9月までの間に岩手県内で救護された野生のガンカモ類および猛禽類の血中鉛濃度の測定を行った。猛禽類では高濃度の鉛汚染は確認されなかったが，ガンカモ類では1例が確認され，さらに低濃度での汚染を受けているガンカモ類が25％と高率に存在し，ガンカモ類が何らかの鉛の汚染を受けている可能性が示唆された。

We examined the presence of hemoplasmas, hemotropic mycoplasmas, among 11 sheep (Ovis aries) with regenerative and hemolytic anemia and found six of them were positive by real-time PCR. The positive samples were then subjected to conventional PCR for direct sequencing of the 16S rRNA gene. Nucleotide sequences of all the positive samples were identified as the 16S rRNA gene of `Candidatus Mycoplasma haemovis' by phylogenetic analysis, demonstrating the infections with this particular hemoplasma species in Japan.

Nineteen blood samples collected from free-ranging wild Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the hemoplasma infection by real-time PCR targeting the 16S rRNA gene. Five (26.3%) out of the 19 samples were positive in real-time PCR with an average melting temperature at 85.18 °C. The positive samples in the real-time PCR were reconfirmed by conventional PCR, and one of them was successful for direct DNA sequencing. The nucleotide sequence of the 16S rRNA gene of the representative stain was identical to that of Mycoplasma ovis. This was the first demonstration of hemotropic mycoplasma infections among the free-living Japanese serows in Japan.

We have examined for hemoplasma infection among cattle in the Hiroshima and Miyazaki prefectures by using a sensitive real-time PCR, with SYBR Green I and with melting curve analysis, which allow to distinguish the two bovine hemoplasma species, Mycoplasma wenyonii and 'Candidatus M. haemobos'. We found 69.4% of 36 cattle in Hiroshima and 93.8% of 32 cattle in Miyazaki infected with either of these two hemoplasma species. High morbidity in western part of Japan may reflect the activity of arthropod vectors for hemoplasma transmission. We also demonstrated neonatal calves less than three months old affected with hemoplasmas without grazing in summer, suggesting a possibility of vertical transmission.

Background Cancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs. Methods The human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed. Results Poorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P

We analyzed blood samples of resident and migratory Japanese birds to evaluate the prevalence and genetic background of avian blood parasites in northern Japan. We used PCR targeting the mitochondrial cytochrome b gene to examine infections of Leucocytozoon, Haemoproteus, and Plasmodium parasites in blood samples from 243 birds of 14 species in three orders (Passeriformes, Columbiformes, and Anseriformes). Sequences were subjected to phylogenetic analysis. The infection rate was 21% in pigeons (Columbiformes) and 17% in Anseriformes. A high infection rate of 93.8% was found in crow species (Passeriformes). Haemoproteus and Plasmodium parasites were detected in only two species. Infected blood samples obtained from seven bird species involved two major clades of Leucocytozoon, which were divided between resident and migratory birds. The parasites, which are genetically distinct from parasites in Japanese resident birds, may have been introduced to Japan by migratory bird species.

This is the first report on Mycoplasma infection in wild bears. We report a novel hemotropic Mycoplasma (also called hemoplasma) detected in a free-ranging black bear (Ursus thibetanus japonicus) in Japan. We then used real-time PCR to look for hemoplasma DNA in blood samples collected from 15 bears and found that eight (53%) were positive. Among these eight PCR samples, seven showed a melting temperature of around 85.5 °C, while the remaining one showed a single peak at 82.26 °C. Almost the entire region of the 16S rRNA gene as well as the 16S–23S rRNA intergenic transcribed spacer (ITS) region from the sample that showed a melting temperature of 82.26 °C was successfully amplified by means of end-point PCR. The nucleotide sequences of the 16S rRNA gene and the ITS region were then determined and compared with those of authentic Mycoplasma species. Our examinations revealed the presence of a novel hemoplasma in Japanese black bears.

Hemoplasma is a tribal name for epierythrocytic mycoplasmas including Mycoplasma suis and M. parvum which are currently recognized in pigs as causative of porcine hemoplasmosis. Here, we report a real-time PCR assay for differential detection of these swine hemoplasma species by using allelic primers in the16S rRNA gene, and its application to survey for hemoplasma infections in pigs. Universal primers and species-specific primers were designed and evaluated by using swine blood samples positive in hemoplasmas. Mycoplasma suis and M. parvum infections were both confirmed by universal primers, and mixed infections were clearly distinguished by species-specific primers. Further, we applied this real-time PCR assay to 120 swine blood specimens from clinically healthy pigs in eleven farms in Japan, and found six (5.0%) were positive for M. suis and 18 (15.0%) were positive for M. parvum, and three (2.5%) were mixed infection by both hemoplasma species.

Only two hemoplasma species, Eperythrozoon parvum and Mycoplasma suis, have been recognized in pigs. Here we demonstrate the genetic variations among six hemoplasma strains detected from pigs, by analyzing the 16S rRNA and RNase P RNA (rnpB) genes, and propose a novel hemoplasma taxon that has not been described previously. Phylogenetic trees based on the nucleotide sequence of the 16S rRNA gene indicated that these six hemoplasmas were divided into two clusters representing M. suis and a novel taxon. We further examined the primary and secondary structures of the nucleotide sequences of the rnpB gene of the novel taxon, and found it distinct from that of M. suis. In conclusion, we unveiled a genetic cluster distinct from M. suis, suggesting a new swine hemoplasma species or E. parvum. Our findings also suggest that this novel cluster should be included in the genus Mycoplasma.

We examined secondary structures of the ribonuclease P RNA sequences obtained from DNA databases, and identified a determinative prototype of the P12 helix peculiar to each species of hemoplasmas. This key structure will provide a rapid means for species identification of these uncultivable pathogens without making a phylogenetic tree based on alignments of nucleotide sequences. This procedure based on palindromic nucleotide substitutions at the stem portion of the P12 helix provide clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. In conclusion, the PNS analysis is based on the evaluation of only the strategic and highly conserved genomic region in the specificity domain of RNase P RNA.

Arginine vasotocin (AVT) is a neurohypophysial hormone that plays an essential role in various social behaviours. We investigated the degree of polymorphisms in the C-terminal domain of the AVT V2-type receptor (AVT2R) among avian species to determine the mechanism by which genetic polymorphisms in the neuropeptide receptor may contribute to different levels of signal transduction. In passerine birds, AVT2R was characterised by 2 variable regions, both of which were managed by insertion/deletion (indel); however, indels were rarely found in other avian taxa. The presence or absence of deletions in passerines largely affected the properties of the predicted palmitoylation sites at the proximal part of the C-terminal tail. Moreover, we detected intraspecific polymorphisms in estrildid finches based on the number of tri-amino acid (GHQ/EHQ/EHR) repeats in another variable region. Our results indicate that amino acid substitutions and length variation at the C-terminus may impact subsequent signal transduction and affect behavioural traits in wild birds.

We examined for `Candidatus Mycoplasma haemominutum' infection in 167 blood samples collected from domestic dogs between 2008 and 2009 in the Tohoku area, Japan, and found 5 (3.0%) were positive by PCR assay. This is the first demonstration of `Candidatus Mycoplasma haemominutum', a feline haemotropic mycoplasma, in the dogs raised in Japan.

Hemoplasma infections in wild ungulates have not been reported yet in Japan. We examined presence of hemoplasmas in blood samples collected from 147 sika deer (Cervus nippon) in the Iwate prefecture by real-time PCR, and found 13 (9%) were positive. Almost entire region of the 16S rRNA gene of the representative strains from positive samples was amplified by conventional PCR. The nucleotide sequences of the 16S rRNA gene were further determined and compared with those of other hemoplasmas. Our examinations 1st revealed the presence of 2 distinct hemoplasma species in sika deer, which are previously not described. One of them was closely related to M. ovis by the 16S rRNA sequence analysis, but was found distinct by comparison of the RNase P RNA gene sequences. Pathogenicity of these two hemoplasma species in sika deer is currently unknown.

Distant metastasis hinders a favorable outcome for patients with esophageal squamous cell carcinoma (ESCC) by limiting the surgical cure. The levels of cell-free DNA (cfDNA) in the blood have served as a predictor for metastasis and recurrence in distant organs in liver cancer. Thus, this study tested the clinical efficacy of serum cfDNA levels as a predictive marker for distant metastasis of ESCC. We investigated cfDNA levels in a cohort of 101 ESCC patients and 46 age- and gender-matched control patients with benign disease. We found that serum cfDNA levels were significantly higher in the ESCC patients than in the control patients (P=0.034). In the ESCC patients, serum cfDNA levels were positively associated with tumor size and cytokeratin 19 fragment (CYFRA 21-1) expression (r=0.416 and r=0.573, respectively). An increase in cfDNA levels was also associated with host inflammation status including C-reactive protein levels and neutrophil and monocyte numbers in the peripheral blood. Serum cfDNA levels tended to be higher in advanced tumors when compared to early stage tumors. We found that serum cfDNA levels were significantly higher in ESCC patients with distant metastasis than in those without (P=0.011). Logistic regression analysis showed that serum cfDNA levels represented only one independent risk factor for distant metastasis among the five factors tested including gender, age, cfDNA levels, CYFRA 21-1 and squamous cell carcinoma antigen levels (P=0.0414). These results suggest that increased serum cfDNA levels may serve as a useful predictor for distant metastasis of ESCC.