Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

Fast Electron Detachment Dissociation of Oligonucleotides in Electron-Nitrogen Plasma Stored in Magneto Radio-Frequency Ion Traps.

We report "plasma" electron detachment dissociation (EDD), a novel electron-activated dissociation (EAD) method for the fast sequencing of oligonucleotides with a high sequence coverage. To reduce the repulsive Coulombic force between the deprotonated oligonucleotides and the electron beam, we performed EDD in a neutral electron-nitrogen (N2+) plasma stored in a magneto radio-frequency ion trap. We confirmed that plasma EDD accomplished a high sequence coverage (100%) of RNA with 40 mers in the reaction time of 10 ms using the electron beam kinetic energy of 35 eV. This new technique was applied to various modifications in oligonucleotide therapeutics (ONTs). Phosphorothioate (PS) positions showed an extremely high dissociation efficiency, i.e., 100 times higher than the standard phosphate (PO) in DNA. Locked nucleotides did not show intensive dissociation in EDD; however, collision-induced dissociation (CID) helped sequence these portions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a ZenoTOF mass spectrometer equipped with the plasma EDD technique successfully identified impurities in degraded samples.

Evaluation of stress status using the stress map for guide dog candidates in the training stage using variations in the serum cortisol with nerve growth factor and magnesium ions.

Most studies on guide dogs for the blind were conducted to investigate the appropriateness of the animals, including in terms of their breeding, constitution, and temperament. However, research to comprehend the stress status of guide dog candidates in response to their training has been unclear. In this study, the levels of serum cortisol, nerve growth factor (NGF), and magnesium ion (Mg2+) levels of guide dog candidates during the three training stages-the elementary, intermediate, and advanced classes-were examined. Dogs were classified based on the contents of the classes and period during the training in which they were subjected. Since the dogs in the elementary class had the lowest serum NGF and Mg2+ levels, they were understood to be under mental stress and to be unfamiliar with their new surroundings. In contrast, the serum NGF and Mg2+ levels were high in the dogs in the advanced class, though they were demonstrated to be mentally stable and acclimated to their environment. Additionally, they were almost free from the stress caused by daily life, since they had the lowest serum cortisol levels. The status of each dog was plotted on a map consisting of 2 axes representing the serum NGF and Mg2+ levels with high or low cortisol levels. Plots could be divided into three domains corresponding to the elementary, intermediate, and advanced classes. Therefore, for working dogs, serum NGF and Mg2+ levels in addition to serum cortisol levels may be important factors to comprehend the type of stress situation that each dog was in.

Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control.

OBJECTIVE: Clinical research on gene therapy has advanced the field of veterinary medicine, and gene doping, which is the illegal use of gene therapy, has become a major concern in horseracing. Since the International Federation of Horseracing Authorities defined the administration of oligonucleotides and its analogues as a genetic therapy in 2017, the development of therapeutic nucleotide-detection techniques has become an urgent need. Most currently marketed and developed oligonucleotide therapeutics for humans consist of modified nucleotides to increase stability, and phosphorothioate (PS) modification is common. RESULTS: We demonstrated the specific detection of phosphorothioated oligonucleotides (PSOs) using LC/MS/MS. PSOs produce the specific product ion (m/z 94.9362) derived from PS moiety. PS is not derived from endogenous substances in animal body, and the product ion is a suitable marker for the detection of PSOs. With our strategy, reproducible target analyses were achieved for identifying the specific substances, with a LOD of 0.1 ng/mL and a quantification rage of 0.1-200 ng/mL in deproteinated plasma. Non-target analyses could also detect the presence of PSOs selectively with 100 ng/mL in the same matrix. These results suggested that the detection of PSOs in horse blood is possible by targeting the product ion using LC/MS/MS.

Mast cell hyperactivity underpins the development of oxygen-induced retinopathy.

Mast cells are classically thought to play an important role in protection against helminth infections and in the induction of allergic diseases; however, recent studies indicate that these cells also contribute to neovascularization, which is critical for tissue remodeling, chronic inflammation, and carcinogenesis. Here, we demonstrate that mast cells are essential for sprouting angiogenesis in a murine model of oxygen-induced retinopathy (OIR). Although mouse strains lacking mast cells did not exhibit retinal neovascularization following hypoxia, these mice developed OIR following infusion of mast cells or after injection of mast cell tryptase (MCT). Relative hypoxia stimulated mast cell degranulation via transient receptor potential ankyrin 1. Subsequent surges in MCT stimulated retinal endothelial cells to produce monocyte chemotactic protein-1 (MCP1) and angiogenic factors, leading to sprouting angiogenesis. Mast cell stabilizers as well as specific tryptase and MCP1 inhibitors prevented the development of OIR in WT mice. Preterm infants with early retinopathy of prematurity had markedly higher plasma MCT levels than age-matched infants without disease, suggesting mast cells contribute to human disease. Together, these results suggest therapies that suppress mast cell activity should be further explored as a potential option for preventing eye diseases and subsequent blindness induced by neovascularization.

Analysis of serum magnesium ions in dogs exposed to external stress: A pilot study.

Magnesium ions (Mg2+) are essential for various enzymatic reactions in the body associated with energy production and activation of the muscles and nerves. Mg2+ is also involved in blood pressure regulation, maintenance of body temperature, and glucose metabolism. Although various factors including foods and physical conditions have been reported to change serum Mg2+ status in humans, serum Mg2+ in dogs exposed to external stress has been unclear. In this study, we examined serum levels of Mg2+ in dogs at different conditions using the guide dog candidates for the blind. Serum Mg2+ was decreased in winter and increased in summer. Guide dog candidates in an elementary class of the training showed markedly lower levels of serum Mg2+, compared with that of dogs in an advanced class. When healthy adult dogs were subjected to forced exercise using a treadmill, a significant reduction in serum Mg2+ levels was observed, particularly in winter. These findings suggest that serum levels of Mg2+ may be influenced by weather fluctuation such as air temperature, nervousness in unaccustomed situations, age, and physical stress induced by exercise. The results indicate that Mg2+ supplementation should be considered for working dogs, dogs moving or traveling to a new environment, and dogs during winter.

Changes in serum NGF levels after the exercise load in dogs: a pilot study.

Serum nerve growth factor (NGF) levels are increased by the external stress in mice, humans and horses; however, similar variations have been unclear in dogs. Since dogs are usually subjected to conditions of work, exercise and activity as important partners of humans, we measured serum NGF levels post-exercise and compared them with serum cortisol levels, as a biomarker of physical stress. Serum cortisol levels were immediately elevated post-exercise and returned to basal levels within 1 hr. On the other hand, serum NGF levels were significantly increased 1 hr post-exercise and gradually returned to basal levels. Further research is necessary; nevertheless, we have demonstrated for the first time that serum NGF levels respond to exercise stress in dogs.

Skin pH Is the Master Switch of Kallikrein 5-Mediated Skin Barrier Destruction in a Murine Atopic Dermatitis Model.

Elevated skin surface pH has been reported in patients with atopic dermatitis. In this study, we explored the role of skin pH in the pathogenesis of atopic dermatitis using the NC/Tnd murine atopic dermatitis model. Alkalinization of the skin of asymptomatic NC/Tnd mice housed in specific pathogen-free conditions induced kallikrein 5 and activated protease-activated receptor 2, resulting in thymic stromal lymphopoietin secretion and a cutaneous T-helper 2 allergic response. This was associated with increased transepidermal water loss and development of eczematous lesions in these specific pathogen-free NC/Tnd mice, which normally do not suffer from atopic dermatitis. Injection of recombinant thymic stromal lymphopoietin also induced scratching behavior in the specific pathogen-free NC/Tnd mice. Thymic stromal lymphopoietin production and dermatitis induced by alkalinization of the skin could be blocked by the protease-activated receptor 2 antagonist ENMD-1068. In contrast, weak acidification of eczematous skin in conventionally housed NC/Tnd mice reduced kallikrein 5 activity and ameliorated the dermatitis. Onset of the dermatitis was associated with increased epidermal filaggrin expression and impaired activity of the sodium/hydrogen exchanger 1, a known regulator of skin pH. We conclude that alterations in skin pH directly modulate kallikrein 5 activity leading to skin barrier dysfunction, itch, and dermatitis via the protease-activated receptor 2-thymic stromal lymphopoietin pathway.

Development of a novel carrier optimized for cell sheet transplantation.

Tissue engineering is a rapidly advancing technology in the field of regenerative medicine. For the transplantation of cell sheets, a carrier must maintain the shape of a cell sheet from a culture dish to affected sites as well as release the sheet easily onto the lesion. In this study, we examined the utility of a novel, poly(lactic acid)-based carrier for cell sheets transplantation to the cornea of dogs and the skin of rats. The poly(lactic acid)-based carrier easily picked a cell sheet up from the dish, fit to the shape of the transplantation sites, and saved time for cell sheets detachment comparing to a conventional carrier. Thus, the poly(lactic acid)-based carrier would be useful for easy cell sheet transplantations.

Glucocorticoid sensitivity depends on expression levels of glucocorticoid receptors in canine neoplastic mast cells.

Glucocorticoid (GC) administration with or without other chemotherapeutic reagents is a commonly used option in the treatment of mast cell malignancies. However, the responsiveness of mast cell tumors to GC treatment varies in individuals, and the regulatory mechanisms determining the GC sensitivity of malignant mast cells remain unclear. Since the expression of the GC receptor (GR) has been reported to be associated with GC sensitivity in human neoplastic lymphocytes, we attempted to investigate the relationship between GR levels and GC sensitivity by using neoplastic mast cells derived from canine mast cell tumors (MCTs). To elucidate the regulatory mechanisms involved in GC responsiveness, we analyzed various canine MCT cell lines and tissue samples from dogs with MCT. While the proliferation of canine MCT cells was suppressed by the addition of GC to the culture, we found that MCT cells derived from humans and rodents, as well as canine lymphoma cells, responded poorly to GC. However, there were also some variations in responsiveness to GC treatment among canine MCT cell lines used in this study. Using real-time polymerase chain reaction and Western blot analysis, we elucidated the relationship between GR expression and responsiveness to GC in canine MCT cells. Furthermore, to assess the involvement of GR expression in GC sensitivity in vivo, clinical investigations were conducted on dogs with cutaneous MCT. Written informed consent was obtained from owners, and the affected dogs were treated with prednisolone (0.5-2.0 mg kg(-1)day(-1), administered orally) 1 or 2 weeks prior to the surgical removal of the tumors. Tumor volume was measured according to WHO criteria both before and after prednisolone treatment, and the GC sensitivity of each MCT was determined on the basis of the reduction in tumor volume. Of the 15 dogs with MCT, 11 responded to treatment with prednisolone completely or partially, whereas 4 dogs showed no response. Examination of clinical samples obtained by surgical removal revealed that GR expression levels were significantly lower in GC-resistant MCT tissues than in GC-sensitive MCT tissues. Thus, these results strongly indicate that GR expression may contribute to GC sensitivity in canine MCT.

Silencing of int6 gene restores function of the ischaemic hindlimb in a rat model of peripheral arterial disease.

AIMS: Intermittent claudication (IC) is one of the serious symptoms of peripheral arterial disease (PAD) and is characterized by pain in the legs or buttocks that worsens with exercise and subsides with rest. The concept of 'therapeutic angiogenesis' for PAD has been widely proposed; however, the methodology, including cell transplantation, is still unclear. In this study, we examined the clinical efficacy of silencing the int6 gene, which encodes a protein that stabilizes hypoxia-inducible factor (HIF)-2α, on angiogenesis in PAD. METHODS AND RESULTS: An animal model for IC was established in Sprague-Dawley rats by external iliac artery ligation and evaluated by quantitative analysis of gait disturbance. Next, we explored the therapeutic effects of int6 siRNA injected into the adductor magnus muscle on IC. Recovery of hindlimb function occurred in the early stages after int6 siRNA injection. The number of blood vessels showed an obvious increase in the int6 siRNA-treated muscles. Angiography revealed the recovery of peripheral circulation at the affected sites. Early up-regulation of HIF-2α and other angiogenic factors, including basic fibroblast growth factor and hepatocyte growth factor, was also apparent in the int6 siRNA-treated sites. We also confirmed the up-regulation of HIF-2α and its translocation to the nucleus in the int6 siRNA-injected muscle. CONCLUSION: A single injection of int6 siRNA promoted angiogenesis via up-regulation of HIF-2α-related angiogenic factors in the muscles of the affected hindlimb and reduced gait disturbance. The int6 gene may be a novel therapeutic target for the treatment of IC in patients with PAD.

Retinal degeneration and rd1 mutation in NC/Tnd mice-a human atopic dermatitis model.

PURPOSE: NC/Tnd mice, a spontaneous model for human atopic dermatitis, are also useful animal models for various corneal disorders accompanying allergic diseases. The purposes of the current study were to investigate the development of retinal degeneration in NC/Tnd mice. MATERIALS AND METHODS: Histological examination was performed to determine time-dependent alterations of the retina in NC/Tnd from 8 to 28 days of age. Apoptotic cells were determined by TUNEL assay. Retinal function was examined by electroretinography. Fundoscopy was performed in NC/Tnd mice at 8 weeks of age. Melanin contents in whole-eye extracts were measured by spectrophotometry. Since the retinal degeneration 1 (rd1) mutation in the rod photoreceptor cyclic guanosine monophosphate phosphodiesterase 6 ß-subunit (Pde6b(rd1)) has been identified in laboratory mice, the possible existence of the rd1 mutation was analyzed with PCR genotyping and gene sequencing. C57BL/6, WB, and C3H/HeN mice were used as controls. RESULTS: Histological examination revealed rapid postnatal retinal degeneration in NC/Tnd mice. The number of apoptotic cells in the outer nuclear layer (ONL) increased with aging, and finally the ONL disappeared. Histological abnormality was not obvious in the inner nuclear layer or the ganglion cell layer. Electroretinography shows no response in adult NC/Tnd mice. Fundoscopic observation revealed hypopigmentation in the retina, and melanin contents in the eye were significantly reduced when compared with other inbred strains. Insertion in the rd1 allele was confirmed and a nonsense mutation of Pde6b(rd1) gene was determined in NC/Tnd mice. CONCLUSIONS: NC/Tnd mice also preserve the Pde6b(rd1) gene mutation resulting in the rapid postnatal retinal degeneration similar to that in C3H/HeN mice. Unlike C3H/HeN mice, since melanin contents of the retina in NC/Tnd mice was decreased, unknown defects may be present in the process of melanin composition in retinal pigment epithelial cells during fetal development of NC/Tnd mice.

Patterns of aquaporin expression in the canine eye.

Aquaporins (AQPs) function as water channels in many types of cells involved in fluid transport. More than 10 isoforms have been identified, and these are differentially expressed in many types of mammalian cells in the body. Six AQPs (AQP0, AQP1, AQP3, AQP4, AQP5, and AQP9) have been identified in the eyes of humans and/or rodents. The unique permeability characteristics and distribution of AQPs indicate their diverse roles in the regulation of water homeostasis in the eye. The aim of this study was to investigate the localisation of AQPs in normal canine eyes, with AQP0 protein expressed in the crystalline lens and retina. Although AQP1 mRNA was detected in various areas of the canine eye, its protein expression was limited to the cornea, iris and ciliary body. AQP4 was identified in the iris, retina and optic nerve. AQP3 and AQP5 were found in the cornea and conjunctiva, and their expression was particularly high in the limbus. AQP3 and AQP5 were present in the nictitating membrane indicating that they play a role in water transport within the membrane. The observations suggested that several subtypes of the AQP family are involved in the regulation of water homeostasis in the canine eye.

Peroxisome proliferator-activated receptor γ-mediated suppression of dendritic cell function prevents the onset of atopic dermatitis in NC/Tnd mice.

BACKGROUND: Dendritic cells (DCs) are one of the key regulators for the initiation of allergic responses in patients with atopic dermatitis (AD), being strongly triggered by epithelial cell-derived thymic stromal lymphopoietin (TSLP). Because peroxisome proliferator-activated receptor (PPAR) γ acts as a negative regulator in immune cells, suppressive properties of PPARγ in allergic responses have been proposed. OBJECTIVE: Because pieces of evidence must be organized to identify the exact role of PPARγ in immune regulation, we explored the suppressive effects of a PPARγ agonist on various functions of DCs and the onset of AD in a murine model. METHODS: Effects of rosiglitazone (RSG) on DCs that were derived from NC/Tnd mice, a model for human AD, were analyzed. RSG was administered to NC/Tnd mice to evaluate its preventive and therapeutic effects on the development of AD. RESULTS: RSG inhibited TSLP-induced DC maturation through downregulation of costimulatory molecules. TSLP-promoted expressions of chemokines in DCs were also suppressed by RSG treatment. Moreover, we showed the necessity of matrix metalloproteinase 9 in TSLP-promoted DC migration by using DCs derived from matrix metalloproteinase 9-deficient NC/Tnd mice, as well as the suppressive effect of PPARγ in the process. Daily oral administration of RSG to NC/Tnd mice before the onset of AD revealed a significant reduction in severity of skin lesions and scratching behavior. In mice treated with RSG, both expression of TSLP in the skin and maturation and migration of DCs were markedly suppressed. CONCLUSION: PPARγ can be provided as an inhibitory regulator of TSLP-stimulated DCs in the onset of allergic reactions.

Characterization of geometrid sex pheromones by electrospray ionization time-of-flight mass spectrometry.

The utility of liquid chromatography combined with time-of-flight mass spectrometry (LC/TOFMS) was demonstrated for studies on chiral unsaturated epoxy compounds, sex pheromones produced mainly by female moths in the family Geometridae. By electrospray ionization (ESI), each synthetic epoxyalkadiene derived from (Z,Z,Z)-3,6,9-triene with a C(18)-C(23) straight chain showed three ion series, [M + NH(4)](+), [M + H](+) and [M - OH](+), with high resolution and good sensitivity, indicating its molecular formula. In addition to these, characteristic fragment ions at m/z M - 57 and M - 71 for the 3,4-epoxides and at m/z M - 123 and 123 for the 9,10-epoxides were detected, whereas the 6,7-epoxides did not produce fragment ions that reflected their structures. Monitoring these diagnostic ions during the LC/MS analysis of a gland extract, the natural sex pheromone of the mulberry looper was confirmed to be (Z,Z)-cis-9,10-epoxy-3,6-octadecadiene, which was separable from the other positional isomers on an ODS column. Furthermore, (Z,Z)-cis-3,4-epoxy-6,9-nonadecadiene secreted by the Japanese giant looper was analyzed with a chiral column, and the stereochemistry was determined directly.