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Label-free image identification of circulating rare cells, such as circulating tumor cells within peripheral blood nucleated cells (PBNCs), the vast majority of which are white blood cells (WBCs), remains challenging. We previously described developing label-free image cytometry for classifying live cells using computer vision technology for pattern recognition, based on the subcellular structure of the quantitative phase microscopy images. We applied our image recognition methods to cells flowing in a flow cytometer microfluidic channel, and differentiated WBCs from cancer cell lines (area under receiver operating characteristic curve = 0.957). We then applied this method to healthy volunteers' and advanced cancer patients' blood samples and found that the non-WBC fraction rates (NWBC-FRs), defined as the percentage of cells classified as non-WBCs of the total PBNCs, were significantly higher in cancer patients than in healthy volunteers. Furthermore, we monitored NWBC-FRs over the therapeutic courses in cancer patients, which revealed the potential ability in monitoring the clinical status during therapy. Our image recognition system has the potential to provide a morphological diagnostic tool for circulating rare cells as non-WBC fractions.

Summary Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1+ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche., Graphical Abstract, Highlights • FGF2 and GDNF induce morphologically distinct spermatogonial clusters in vivo • FGF2-expanded undifferentiated spermatogonia are prone to be RARG+ in vivo • FGF2 is more permissive than GDNF for Rarg expression in cultured spermatogonia • FGF2 suppresses GDNF production and retinoic acid metabolism in the germline niche, Takashima and colleagues demonstrate the novel functions of FGF2 in the germline niche. Although FGF2 induces self-renewal of spermatogonial stem cells in vitro, this molecule expands “differentiation-prone” GFRA1+RARG+ spermatogonia and facilitates retinoic acid actions required for differentiation in vivo. The present study suggests that FGF2 contributes to spermatogonial differentiation, indicating reconsideration of the role of FGF2 in the germline niche.

Growth hormone secretagogue receptor 1a (GHSR1a), growth hormone (GH), growth hormone receptor (GHR), non-SMC condensin I complex, subunit G (NCAPG) and stearoyl-CoA desaturase (SCD), are known to play important roles in growth and lipid metabolisms. Single and epistatic effects of the five genes on carcass, price-related and fatty acid (FA) composition traits were analyzed in a commercial Japanese Black cattle population of Ibaraki Prefecture. A total of 650 steers and 116 heifers for carcass and price-related traits, and 158 steers for FA composition traits were used in this study. Epistatic effects between pairs of the five genes were found in several traits. Alleles showing strain-specific differences in the five genes had significant single and epistatic effects in some traits. The data suggest that a TG-repeat polymorphism of the GHSR1a.5'UTR-(TG)n locus plays a central role in gene-gene epistatic interaction of FA composition traits in the adipose tissue of Japanese Black cattle.

Beef from Japanese Black cattle (JBK), is popular in Japan and valued for its highly marbled fat content. In JBK, genes affecting oleic acid content in meat have been studied mainly to lower the fat melting point and improve tenderness; however, there has been no direct correlation demonstrated between beef taste and oleic acid. To investigate genes affecting other fatty acids other than oleic acid, polymorphisms of the fatty acid desaturase 2 (FADS2) gene were genotyped and associations with fatty acid profile in JBK beef were investigated. Amplifications of 5’-flanking regions, 12 exons, and 3’-untranslated regions of the FADS2 gene in three Japanese and five Western cattle breeds via PCR, were amplified, sequenced and SNPs were identified using specific TaqMan genotyping assay. Fatty acid composition of intramuscular adipose tissue of the Trapezius muscle was analyzed in JBK steers. Six of the 15 identified SNPs are novel and have never been registered in any public bovine SNP database. A non-synonymous SNP (rs211580559; C > T; 294 Ala > Val) in exon 7 was examined in order to evaluate its association with fatty acid profiles. The data showed that highly significant association existed between rs211580559 and C18:2 (n-6) composition, and accounted for 22.3% of the variation. There were no significant relationships between rs2115-80559 and the other fatty acids. It was concluded that rs211580559 of the FADS2 gene may be a useful selection marker for reducing unfavorable volatiles generated from linoleic acid in JBK beef during the cooking process.

We carried out a genetic association study between five nucleotide polymorphisms (5��UTR microsatellite ((TG)n), nt-7(C>A), L24V, DelR242 and Intron 1 microsatellite) of the GHSR1a gene and growth and carcass traits in 1285 steers sired by 117 Japanese Black bulls in a progeny testing program. We report herein, a significant association between the 5��UTR microsatellite and nt-7(C>A) loci and growth and carcass traits. We also propose a translational hypothesis that the association is due to differences in the secondary structure of GHSR1b mRNA (the non-spliced type with the 5��UTR microsatellite) among the GHSR1a gene haplotypes. Furthermore, we predicted the potential increase in profitability due to increased carcass weight in cow-calf fattening enterprises through planned matings based on DNA testing of the 5��UTR microsatellite. Statistical analysis revealed that the 5��UTR microsatellite locus had a significant additive effect on carcass weight (CW) and average daily gain (ADG), but not on beef marbling score (BMS). One of the four major microsatellite alleles (19-TG allele) with an allele frequency of 0.145, had a significantly (P < 0.0007) desirable effect on CW and ADG. We concluded that the 19-TG allele could potentially be economically useful nucleotide markers for growth and carcass traits in Japanese Black cattle

Growth hormone secretagogue receptor 1a (GHSR1a) mediates the different actions of its endogenous ligand, ghrelin. Ghrelin-GHSR is involved in many important functions that include growth hormone secretion and food intake. We evaluated the haplotype variety and characterized the microsatellite ((TG)n, 5′-UTR) and nucleotide polymorphisms of the bovine GHSR1a gene. The nucleotide sequencing of this gene (∼6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)n, Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and were divided into three major groups. Four SNPs (L24V, nt456(G>A), D191N and nt667(C>T)) and DelR242 in Exon 1 and a haplotype block of approximately 2.2 kb (nt667(C>T) ∼ nt2884 (A>G)) were found in Bos taurus breeds. Breed differences in allele frequencies of the two microsatellites, nt-7(C>A), L24V, and DelR242 loci were found (P < 0.005). A DelR242 was found in the Japanese Shorthorn (frequency: ∼ 0.44), Japanese Brown, five European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black or the Mishima island cattle. Additionally, 5′-rapid amplification of cDNA ends and RT-PCR analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5′-UTR (GHSR1a); and non-spliced, with the microsatellite (GHSR1b).

The growth hormone secretagogue receptor 1a (GHSR1a) is involved in many important functions including growth hormone (GH) secretion and appetite regulation and other important functions. We reveal herein, the unravelling of bovine-specific 5’untranslated region (5’UTR) microsatellite polymorphisms, a 3bp-indel in exon 1 (DelR242) and two different kinds of transcripts of the GHSR1a gene (spliced, without a microsatellite with in the 5’UTR (GHSR1a); and non-spliced, with the microsatellite (GHSR1b)). A number of 17 alleles ((TG)10~33 ) in the 5’UTR microsatellite was found in 11 cattle breeds. Furthermore, we found the DelR242 (3R) allele, a truncated 3- arginine residue (3R) (major type: 4 arginine residues (4R)) within the intracellular loop 3 of GHSR1a protein in Japanese Shorthorn with a high frequency of 0.43 compared to the low frequency of 0.00~0.09 in other cattle breeds. We carried out a genetic association study between the 5’UTR microsatellite and growth and carcass traits in 1,285 steers. Statistical analysis revealed that the 5’UTR microsatellite locus had a significant additive effect on carcass weight (CW) and average daily gain (ADG). The 19-TG allele had a significantly desirable effect on these traits. We proposed a translational hypothesis that the association is due to differences in the secondary structure of GHSR1b mRNA among the GHSR1a gene haplotypes. We also examined age-related changes in the expressions of GHSR1a and GHSR1b in many cattle tissues. The GHSR1a mRNA expression in the arcuate nucleus of postweaning calves was more than 10-fold higher than those of pre-weaning calves and cows. In peripheral tissues, there were 3 marked differences in mRNA expression between cattle, humans and mice, as follows: (1) the GHSR1a mRNA expression in the liver is high in cattle and very low in humans and mice; (2) the GHSR1b mRNA expression in the liver is low in cattle and high in humans; (3) the GHSR1b mRNA expression in the pancreas is very high in cattle.

Angiotensin-converting enzyme (ACE; EC 3.4.15.1), a dipeptidyl carboxypeptidase, converts angiotensin I to angiotensin II, the central product of the renin-angiotensin system. We here report molecular cloning of the complete open reading frame (ORF) of hamster somatic-type ACE and its expression in hamster organs. The cloned cDNA comprises an ORF of 3942 bp, which encodes 1314 amino acids of the precursor protein of hamster somatic ACE. On the deduced amino acid sequence a putative signal peptide and a transmembrane segment are predicted at the N-terminus and near the C-terminus, respectively. Two homologous domains, referred to as N- and C-domains, are present within somatic ACE, and within each of the homologous domains a putative active center is found, as has been the case in human, mouse, rat and rabbit. The similarity of the hamster sequence with the sequences of these other mammals at both the nucleotide and amino acid levels is high (above 83%). mRNA expression analysis by conventional polymerase chain reaction (PCR) shows wide distribution of the transcript, with dominant expression in lung and kidney. Quantitative analysis of mRNA expression demonstrates that levels in lung and kidney are 100-1000 times higher than in the other organs, suggesting that these organs are important in the hamster renin-angiotensin system, as they are for other mammals.

Survivin, a member of the inhibitor of apoptosis (IAP) gene family, overexpresses in various human tumors. Recently this protein has attracted strong interest as a potential prognostic marker because it promotes malignancy through anti-apoptotic activity and is associated with a more aggressive phenotype. To explore the utility of survivin as a veterinary marker of tumor malignancy, we performed molecular cloning of dog survivin cDNA and studied survivin mRNA expression in a variety of naturally occurring dog tumors. The dog cDNA contains a 426-bp open reading frame encoding 142 amino acids of polypeptide, in which a structure termed the baculovirus IAP repeat (BIR) domain, commonly observed in IAPs, is found, as it is in other mammalian survivin protein. The transcript was detected in many adult normal organs including heart, lung, liver, stomach, duodenum, colon, spleen, kidney and testis. As a result of quantitative expression analysis by real-time PCR undertaken for benign and malignant tumors, overexpression of the survivin gene was found in 3 of 18 malignant tumors and in none of the benign tumors, suggesting that survivin overexpression is associated with tumor malignancy in dog.

We cloned and characterized horse preproendothelin-2 (PPET-2) cDNA from intestinal tissue. The cDNA encoded 178 amino acids of the PPET-2 polypeptide, in which a 21-amino-acid mature endothelin-2 peptide and a 16-amino acid endothelin-2-like peptide were found. For the open reading frame the correspondence of horse PPET-2 cDNA with those of the ferret, human, dog, mouse and rat was 85.1%, 84.9%, 82.1%, 77.8% and 77.2%, respectively. Analysis of the organ distribution of PPET-2 mRNA by reverse transcription-polymerase chain reaction demonstrated that the kidney, stomach and small intestine are major sites of expression of the PPET-2 gene. Surprisingly, the mRNA is not detected in the large intestine, where high expression is demonstrated in the mouse and rat. This difference may result from the underlying functional differences of the large intestine between a herbivore (horse) and an omnivore (mouse and rat).

To compare the structure of the precursor polypeptide of dog endothelin-3, preproendothelin-3 (PPET-3), with the PPET-3 of other mammals, we cloned dog cDNA from lung tissue using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. An open reading frame encoding a 198-amino-acid polypeptide was found in the cDNA. Regions corresponding to a bioactive mature endothelin-3 peptide, an intermediate form known as big-endothelin-3 and an endothelin-3- like peptide were observed in the putative PPET-3. Comparative analysis showed that the similarity of the dog open reading frame sequence with those from human hypothalamus, mouse intestine, and rat eye is 76.2%, 69.5% and 66.3%, respectively, and that the similarity at the amino acid level is 65.6%, 59.8% and 58.8%, respectively. RT-PCR demonstrated significant elevated expression of PPET-3 mRNA in the kidney of dog with interstitial nephritis.

The full-length cDNA of dog preproendothelin-3 (PPET3) was cloned from lung tissue using RT-PCR and rapid amplification of cDNA ends. Aside from the poly (A) tail, the full-length cDNA was 1976 bp. A polyadenylation signal sequence and one copy of a consensus sequence, ATTTA, which is related to mRNA turnover, was found in the 3' noncoding region. The cDNA had a 594-bp open reading frame encoding a 198-amino acid polypeptide. Regions corresponding to a bioactive mature ET3 peptide, an intermediate form known as big-ET3, and an ET3-like peptide were observed in dog PPET3. Expression of PPET3 mRNA was detected throughout the organs examined, which included heart, lung, liver, kidney, spleen, stomach, pancreas, duodenum, colon, uterus, ovary and testis.

Endothelin-2 (ET2), which was originally identified in human, is a bioactive peptide of 21 amino acids with strong vasoconstrictive and pressor effects. Here we report the cDNA cloning and characterization of bovine preproendothelin-2 (PPET2), the precursor form of ET2. The bovine cDNA encodes 177 amino acids of the PPET2 polypeptide, in which a 21-amino acid mature ET2 peptide and a 16-amino acid ET2-like peptide as well as a 23-amino acid putative signal peptide were found. The bovine ET2-like peptide sequence was missing a dibasic amino acid pair at the C-terminal, in contrast to human, mouse and rat, for which the ET2-like sequence is flanked by dibasic pairs at both the N- and C-terminals. Gene expression analysis by RT-PCR showed that the transcript is expressed in various organs including heart, lung, liver, kidney, gastrointestinal tract, uterus and ovary, but not in spleen. Within the gastrointestinal tract, gene expression was detected in rumen, a ruminant-specific digestive organ, as well as stomach, duodenum and colon.

Endothelin-2 (ET2) is a member of the endothelin family of 21-amino acid peptides with vasoconstrictive activity. We report here the molecular cloning of the canine full-length cDNA of the precursor form of ET2, prepro-ET2 (PPET2), from intestinal tissue by means of reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'- and 3'-rapid amplification of cDNA ends (RACE). Aside from the poly (A) tail the cDNA was found to be 1195 bp and included an open reading frame of 534 bp encoding a PPET2 polypeptide of 178 residues, in which the regions corresponding to bioactive mature ET2 peptide, an intermediate form big-ET2, and endothelin-like peptide are found. The organ distributions of PPET2 mRNA and a splicing variant were analyzed by RT-PCR. PPET2 transcript was detected in duodenum, colon, stomach, lung, liver, uterus, ovary, testis and kidney, but not in spleen. A splicing variant was found in none of the organs. Thus, based on the cloned cDNA sequence, we established a quantitative assay for dog PPET2 mRNA level using a real-time PCR system. Quantitative analysis by this method in various organs of the dog demonstrated that the dominant gene expression occurs in the intestine, with higher expression in large intestine than in small intestine.

To develop the pulse heated semiconductor CO sensor which operates on a 9 V alkaline battery for 2 years (power consumption; approximately 0.1 mW), the current sensor structure was refined. The results regarding thermal analysis of the current sensing element convinced us that temperature gradients of this element was so large that only a small area was cleaned by heating. Furthermore, it was found that the loss due to heat conduction, mainly from Au electrodes, was larger than expected. From these results, the element size was reduced from 0.5×0.5 to 0.3×0.3 mm and Pt was used for the electrode material. Compared with the current sensor, the modified sensor was drastically improved as to the long term stability under battery operating conditions.

Hyper Suprime-Cam (HSC) is an 870 Mega pixel prime focus camera for the 8.2 m Subaru telescope. The wide field corrector delivers sharp image of 0.25 arc-sec FWHM in r-band over the entire 1.5 degree (in diameter) field of view. The collimation of the camera with respect to the optical axis of the primary mirror is realized by hexapod actuators whose mechanical accuracy is few microns. As a result, we expect to have seeing limited image most of the time. Expected median seeing is 0.67 arc-sec FWHM in i-band. The sensor is a p-ch fully depleted CCD of 200 micron thickness (2048 x 4096 15 μm square pixel) and we employ 116 of them to pave the 50 cm focal plane. Minimum interval between exposures is roughly 30 seconds including reading out arrays, transferring data to the control computer and saving them to the hard drive. HSC uniquely features the combination of large primary mirror, wide field of view, sharp image and high sensitivity especially in red. This enables accurate shape measurement of faint galaxies which is critical for planned weak lensing survey to probe the nature of dark energy. The system is being assembled now and will see the first light in August 2012.

We carried out a genetic association study between five nucleotide polymorphisms (5'UTR microsatellite ((TG)(n)), nt-7(CA), L24V, DelR242 and Intron 1 microsatellite) of the GHSR1a gene and growth and carcass traits in 1285 steers sired by 117 Japanese Black bulls in a progeny testing program. We report herein, a significant association between the 5'UTR microsatellite and nt-7(CA) loci and growth and carcass traits. We also propose a translational hypothesis that the association is due to differences in the secondary structure of GHSR1b mRNA (the non-spliced type with the 5'UTR microsatellite) among the GHSR1a gene haplotypes. Furthermore, we predicted the potential increase in profitability due to increased carcass weight in cow-calf fattening enterprises through planned matings based on DNA testing of the 5'UTR microsatellite. Statistical analysis revealed that the 5'UTR microsatellite locus had a significant additive effect on carcass weight (CW) and average daily gain (ADG), but not on beef marbling score (BMS). One of the four major microsatellite alleles (19-TG allele) with an allele frequency of 0.145, had a significantly (P0.0007) desirable effect on CW and ADG. We concluded that the 19-TG allele could potentially be economically useful nucleotide markers for growth and carcass traits in Japanese Black cattle.

Growth hormone secretagogue receptor 1a (GHSR1a) mediates the different actions of its endogenous ligand, ghrelin. Ghrelin-GHSR is involved in many important functions that include growth hormone secretion and food intake. We evaluated the haplotype variety and characterized the microsatellite ((TG)(n) , 5'-UTR) and nucleotide polymorphisms of the bovine GHSR1a gene. The nucleotide sequencing of this gene (∼6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)(n) , Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and were divided into three major groups. Four SNPs (L24V, nt456(GA), D191N and nt667(CT)) and DelR242 in Exon 1 and a haplotype block of approximately 2.2 kb (nt667(CT) ∼ nt2884 (AG)) were found in Bos taurus breeds. Breed differences in allele frequencies of the two microsatellites, nt-7(CA), L24V, and DelR242 loci were found (P0.005). A DelR242 was found in the Japanese Shorthorn (frequency: ∼ 0.44), Japanese Brown, five European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black or the Mishima island cattle. Additionally, 5'-rapid amplification of cDNA ends and RT-PCR analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5'-UTR (GHSR1a); and non-spliced, with the microsatellite (GHSR1b).

Previous studies on human uterine and placental tissues have found variants, derived from alternatively spliced mRNAs, of preproendothelin-2 (PPET2) that lack a post-translational proteolytic site essential for normal processing. Here we report a splice variant of cat PPET2 mRNA expressed in the stomach. After cloning the full-length cDNA of cat PPET2, organ distribution analysis of the transcript by reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. In addition to the fragment with a size predicted based on the cDNA sequence obtained by cloning, an additional PCR fragment of smaller size was detected in stomach tissue. Subsequent cloning and sequence analysis of the smaller PCR product demonstrated that it derives from a splice variant with full-length deletion of a region corresponding to exon 4 of the PPET2 gene.

Survivin, a member of the inhibitor of apoptosis (IAP) gene family, overexpresses in various human tumors. Recently this protein has attracted strong interest as a potential prognostic marker because it promotes malignancy through anti-apoptotic activity and is associated with a more aggressive phenotype. To explore the utility of survivin as a veterinary marker of tumor malignancy, we performed molecular cloning of dog survivin cDNA and studied survivin mRNA expression in a variety of naturally occurring dog tumors. The dog cDNA contains a 426-bp open reading frame encoding 142 amino acids of polypeptide, in which a structure termed the baculovirus IAP repeat (BIR) domain, commonly observed in IAPs, is found, as it is in other mammalian survivin protein. The transcript was detected in many adult normal organs including heart, lung, liver, stomach, duodenum, colon, spleen, kidney and testis. As a result of quantitative expression analysis by real-time PCR undertaken for benign and malignant tumors, overexpression of the survivin gene was found in 3 of 18 malignant tumors and in none of the benign tumors, suggesting that survivin overexpression is associated with tumor malignancy in dog.

In an attempt to understand the significance of endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 peptides in organs during perinatal development, we performed quantitative analysis of ET-1 and VIC gene expression in mouse organs obtained from embryos at days 14 and 17 (E-14 and E-17) of pregnancy, neonates at days 0, 1, 3 and 7 after birth (N-0, -1, -3 and -7), and adult mice (10 weeks old). In intestine, VIC gene expression progressively increased between E-14 and N-1 (approximately 10-fold) and then remained constant into adulthood. ET-1 gene expression exhibited a one-step increase between E-17 and N-0, subsequently remaining constant. In lung, a sharp increase in ET-1 mRNA level (approximately 10-fold) was noticed between E-14 and N-0. The gene expression pattern of VIC, with a peak at N-0, was similar to that of ET-1 although the expression level of VIC was two to three orders of magnitudes lower than that of ET-1. Gene expression patterns of ET-1 and VIC remained nearly constant in brain, heart, liver and kidney throughout the period examined. Considering that the intestinal and pulmonary gene expression levels of both genes reached almost the same level as observed in adult soon after birth, we suggest that these peptides may be involved in the emergence and maintenance of intestinal and pulmonary functions vital after birth.

A hypotheses that mitoxantrone is a competitive antagonist at muscarinic cholinergic receptors was examined in guinea-pig hearts. In isolated left atrial muscle preparations, electrically paced at 2 Hz, the muscarinic agonist, carbachol, caused a concentration-dependent decrease in developed tension. Mitoxantrone caused a parallel right-ward shift of the concentration–response curve for carbachol. Schild plots for the effect of mitoxantrone on the carbachol concentration–response relationship were linear with a slope of 0.88 which was not significantly different from the unity. The right-ward shift of the carbachol concentration–response relationship by mitoxantrone significantly reversed after an additional incubation with a mitoxantrone-free solution, although the reversal was incomplete after a 2-h incubation in the mitoxantrone-free solution. Mitoxantrone caused a concentration-dependent displacement of specific [3H]quinuclidinyl benzilate binding to membrane preparations obtained from ventricular muscles of guinea-pig hearts. These results indicate that mitoxantrone acts as a competitive antagonist for the muscarinic receptors.