Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro

Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1⁺ CD4⁺ T cells in blood and lymph node and PD-1⁺ CD8⁺ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.     

Phenotypic changes in reproductive traits with changes in stock size of the starspotted smooth-hound Mustelus manazo in Tokyo Bay, Japan

Reproductive traits are among the primary determinants of population dynamics, and their changes could affect stock size. We investigated differences in reproductive traits of the starspotted smooth-hound Mustelus manazo in Tokyo Bay between the 1990s (a low-stock period) and the 2000s (a high-stock period). Peak timings of mating (May–July), ovulation (May–June), and parturition (May–June) were similar between the two periods. However, the size at first maturity in the 2000s was smaller than that in the 1990s for both sexes. In addition, embryo-related parameters differed greatly between the two periods. Slopes of size–fecundity regression in the 2000s decreased to 43–73 % of the 1990–1996 values, suggesting a substantial decline in fecundity during the 2000s. Frequency distribution of the maximum ovum diameter during the ovulation season showed two distinct two modes in 1990s while it showed a substantial dispersion during the 2000s, implying that changes occurred in the developmental pattern of ova in the ovary. Undeveloped eggs were present in the uteri with low frequency from September to May during the 2000s, but they were totally absent from September through May in the 1990s. The increased number of remnant undeveloped eggs that did not become embryos may explain the fecundity decline during the 2000s.     

Effects of thinning and site productivity on culmination of stand growth: results from long-term monitoring experiments in Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don) forests in northeastern Japan

We analyzed data from 28 long-term experimental monitoring plots installed in Japanese cedar (Cryptomeria japonica D. Don) plantations in northeastern Japan to examine how site productivity and thinning practices relate to culmination in stand growth. Site productivity and thinning practices in the plots were evaluated by site index (dominant tree height at 40-years old) and by cumulative thinning rate (cumulative thinning volume divided by cumulative gross production during the entire period of measurement). Culmination of stand growth was evaluated by culmination age of the mean annual increment (MAI) and its maximum value (Max MAI). Max MAI for the mean annual gross increment (MAIgross) and mean annual net increment (MAInet) increased with increasing site index, but did not change with cumulative thinning rate. Culmination age for MAIgross decreased with increasing site index, but did not change with cumulative thinning rate. Culmination age for MAInet decreased with increasing site index. Additionally, culmination age for MAInet increased with increasing cumulative thinning rate in sites with a high site index (>19.3 m) but not in those with a low site index (<19.3 m). These results indicate that thinning extends the culmination age without changing Max MAInet under high site productivity. Therefore, thinning increases total net yield in sites with high productivity based on a long-term perspective.     

Metabolism of a phosphoramidate by Pyricularia oryzae in relation to tolerance and synergism by a phosphorothiolate and isoprothiolane

Metabolism of dibutyl N-methyl-N-phenylphosphoramidate (BPA) by mycelial cells of Pyricularia oryzae was studied to elucidate the mechanism of synergism and negatively correlated cross-resistance in fungicidal action between phosphoramidates and phosphorothiolate derivatives. Rapid metabolism of BPA by a wild-type strain through hydroxylation and N-demethylation was observed. The metabolism was inhibited by diisopropyl S-benzyl phosphorothiolate (IBP; Kitazin P) and by isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate; Fuji-One). This inhibition of BPA metabolism is probably the mechanism of synergistic fungicidal action between the phosphoramidate and the thiol derivatives. The metabolism was, however, not inhibited by S-1358 (S-butyl S′-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate; Denmert) or triarimol [α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol; EL-273], both of which are considered to be inhibitors of hydroxylation of a methyl radical in ergosterol biosynthesis. The metabolism of BPA by P. oryzae was much slower when mutants selected for IBP resistance and for isoprothiolane resistance were used. This phenomenon probably explains the differential sensitivity to phosphoramidate of wild-type strains and mutants resistant to the thiol derivatives.     

Prevention of biofilm formation with a coating of 2-methacryloyloxyethyl phosphorylcholine polymer

Device-associated infections are serious complications, and their prevention is an issue of considerable importance. Since biofilms are responsible for these refractory infections, effective methods to inhibit biofilm formation are required. In this investigation, stainless steel plates with and without 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, i.e., poly (MPC-co-n-butyl methacrylate) (PMB) coating, were incubated in a medium containing bacteria. In the course of incubation, half of the specimens received antibiotics. The specimens were stained for nucleic acid and polysaccharides, and then examined with a confocal laser scanning microscope. The numbers of bacteria on the specimen surfaces were evaluated by an ATP assay. On the surfaces of the specimens without PMB coating, the formation of a biofilm enveloping bacteria was confirmed. The addition of antibiotics did not effectively decrease the number of bacteria. On the other hand, on the surfaces of the specimens with PMB coating, no biofilm formation was observed, and the number of bacteria was significantly decreased. The addition of potent antibiotics further decreased the number of bacteria by 1/100 to 1/1000 times. The PMB coating combined with the validated use of antibiotics might provide a method for the simultaneous achievement of biocompatible surfaces of devices and the prevention of device-associated infections.     

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

Expression of NG2 proteoglycan in the degenerated intervertebral disc in dachshunds

The pathogenesis of intervertebral disc (IVD) degeneration is not fully understood. The biomolecular signaling pathways involved in the IVD degeneration require further investigation. The aim of this study was to investigate the expression of NG2 proteoglycan in the degenerated IVD. IVD samples were obtained from 16 Dachshunds that were confirmed to have IVD herniation and subsequently underwent hemilaminectomy. The samples were subjected to histological and immunohistochemical (IHC) examinations. IHC revealed positive results for the expression of NG2 proteoglycan in all examined samples. The results showed the expression of NG2 proteoglycan by the degenerated IVDs.     

Puerperal and parental experiences alter rat preferences for pup odors via changes in the oxytocin system

In the rat, induction of maternal behavior depends on the parity of the female. For example, nulliparous (NP) females need longer exposure to pups than multiparous (MP) or lactating (L) females to exhibit similar maternal behavior. In this study, we investigated the role of brain oxytocin in the approaching behavior of these female rats. Olfactory preferences for pup odors were examined for 8 consecutive days. Each preference test was followed by direct overnight exposure to pups. On the 8th day, MP and L, but not NP females showed robust pup-odor preferences. After the behavioral test, half of the females were exposed to pups for 2 h, whereas the other half were not. The females were then sacrificed to analyze brain oxytocin (OXT) and vasopressin (AVP) activities by cFos immunohistochemistry and to quantify their receptor mRNA expression using real-time PCR. In the paraventricular nucleus (PVN), the percentage of cFos-positive OXT neurons was significantly larger in MP and L females than in NP females after pup exposure. No significant differences were found in cFos expression in OXT neurons of the supraoptic nucleus (SON) or in AVP neurons of either the PVN or SON. Expression of OXT receptor mRNA in the medial preoptic area and amygdala of the control groups was also higher in MP females than in NP females. Finally, we demonstrated that infusion of OXT into the lateral ventricle of NP females promoted preferences for pup odors. These results indicate that puerperal and parental experiences enhance the responsiveness of OXT neurons in the PVN to pup stimuli and establish olfactory preferences for these odors in a parity-dependent manner.     

Dependence of runout distance on the number of rock blocks in large-scale rock-mass failure experiments

To examine how the number of rock blocks affects the rock-mass runout distance, large-scale outdoor rockfall experiments were physically performed using cubiform granite rock blocks on a slope prepared with granite slabs under both dry and water-saturated conditions. To learn more about the runout mechanism, numerical simulations were also conducted using three-dimensional DEM to reproduce the physical experiments under dry conditions. Longitudinal rock-mass runout distance between the gravity centre in the initial rock block assembly before failure and the apparent gravity centre at final deposition was 10 % larger for experiments under water-saturated conditions than those under dry conditions, with identical numbers of rock blocks in the physical experiments. The physical and numerical experiments revealed that rock blocks at the front and top surfaces had a longer runout distance than those at the rear and bottom surfaces. A rock block scarcely surpassed the rock blocks in front longitudinally and the rock blocks next to it laterally. The equivalent coefficient of friction between gravity centres was positively correlated with the number of rock blocks, which contradicts the findings of a negative correlation between the volume of sturzstroms (rockfall avalanches) and the equivalent coefficient of friction in classic studies on the long runout mechanisms. Our results were likely attributable to the fact that more kinetic energy was dissipated due to repeated inelastic intercollisions with other surrounding rock blocks and granite slabs when the initial rock-mass volume (number of rock blocks) was larger.     

Effect of Biodegradable Gelatin β-Tri Calcium Phosphate Sponges Containing Mesenchymal Stem Cells and Bone Morphogenetic Protein-2 on Equine Bone Defect

This study evaluated the osteoanagenetic effects of administering biodegradable gelatin β-tri calcium phosphate sponges containing mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) on equine bone defect. Six healthy Thoroughbred horses were used in this study. Horses were anesthetized, and skin incisions were made on all the limbs. Splint bones were exposed and a 1-cm bone defect was created in each exposed bone. Gelatin β-tri calcium phosphate sponges containing MSC and BMP-2 (MSC + BMP-2 sponge), MSC only (MSC sponge), BMP-2 only (BMP-2 sponge), or saline (saline sponge) were implanted into each bone defect at random. Defects were monitored for 16 weeks by radiography followed by computed tomography (CT) and histologic analyses. At 16 weeks, radiographic scores of MSC + BMP-2 sponge-treated defects were significantly higher than those of saline-treated defects (P = .027). Moreover, the CT value of the MSC + BMP-2 sponge group was significantly higher than that of the other groups (P = .027; P = .046; and P = .027, respectively), and the histologic score of the MSC + BMP-2 sponge group was significantly greater than that of the saline sponge group (P = .041). We conclude that MSC + BMP-2 sponge administration to bone defects accelerates bone regeneration in equines.