Double-decker cage reduces mount frequency and ejaculation latency, resulting in reduced weight loss in male rats after mating behavior.

Rats were the first mammals to be domesticated for scientific research, and abundant physiological data are available on them. Rats are expected to continue to play an important role as experimental animals, especially with advancements such as CRISPR/Cas9 technology. Environmental enrichment aims to promote species-specific behaviors and psychological well-being. In the present study, we designed a double-decker (DD) cage, which utilizes two stacked plastic cages for rat enrichment, and investigated the influence of housing in the DD cage on rat mating behavior. The results indicated that mount frequency, total mount counts, and total ejaculation latency were significantly lower in the DD cages than in the single-decker (SD) cages. Notably, in the DD cages, the body weight loss of male rats after mating behavior was lower than that observed in the SD cage. Water consumption per day during mating behavior was also significantly lower in the DD cages, although no significant differences were observed in daily food intake during mating behavior. In addition, reproductive performance, including pregnancy rate and birth rate, did not change in the DD cages. In summary, our study demonstrated that DD cages reduce mount frequency and ejaculation latency during rat mating, resulting in decreased water consumption and weight loss in male rats. Therefore, housing in DD cages may serve as a beneficial enrichment for rats.

Repeat-dose and embryo-fetal developmental toxicity of zinpentraxin alfa.

Zinpentraxin alfa is a recombinant human pentraxin-2 (PTX-2) developed for the treatment of various fibrotic diseases with the hypothesis that supplementing endogenous PTX-2 levels through intravenous administration should increase its regulatory capacity in circulation and at the site of disease, thereby promoting healing and reducing fibrosis. Zinpentraxin alfa has been studied in various clinical trials, particularly in patients with idiopathic pulmonary fibrosis, where it has demonstrated efficacy in slowing decline in lung function in a phase 2 study. In the present investigation, we summarize findings from 14-day repeat-dose toxicity studies in rats and cynomolgus monkeys supporting early clinical development of zinpentraxin alfa. In addition, we also describe the findings from the embryo-fetal developmental (EFD) studies conducted in rats and rabbits, since the intended fibrosis patient population may include patients of childbearing potential. Zinpentraxin alfa was well tolerated by rats and monkeys in general toxicity studies with no treatment-related adverse effects, as well as by pregnant rats over the same dose range in a definitive EFD study. In contrast, substantial toxicity was observed in a rabbit dose-range-finder EFD study. Zinpentraxin alfa was poorly tolerated by pregnant rabbits and effects on the dams correlated with post-implantation fetal losses. The disparate effects of zinpentraxin alfa on embryo-fetal development between the two species suggests a potential unknown biological function of PTX-2 in pregnancy in the rabbit, which may be relevant to humans. Our findings warrant the consideration for highly effective contraceptive measures to avoid pregnancy in patients enrolled in clinical studies with zinpentraxin alfa.

Euglycemic diabetic ketoacidosis in a patient with type 2 diabetes mellitus 3 days after initiating sodium-glucose cotransporter 2 inhibitor while on an extremely low carbohydrate diet: A case report.

This paper presents a case with type 2 diabetes mellitus on a very-low-carbohydrate diet who developed euglycemic diabetic ketoacidosis (EDKA) 3 days after starting sodium-glucose cotransporter 2 inhibitors (SGLT2i). When initiating SGLT2i, healthcare providers should confirm the implementation of a low-carbohydrate diet and provide intensive guidance to prevent EDKA.

Acute eosinophilic pneumonia caused by nicotine-free vaping in an adolescent patient: A case report.

An 18-year-old man was admitted to our hospital with pneumonia 4 days after he initiated vaping. The patient did not show improvement after ceftriaxone and azithromycin treatment. The cell count of the bronchoalveolar lavage fluid (BALF) revealed 64% eosinophils and 18% lymphocytes. Based on the BALF findings, the patient met the current diagnostic criteria and was diagnosed with vaping-induced acute eosinophilic pneumonia (AEP). AEP caused by nicotine-free vaping is rare in Japan. Thus, in cases of AEP, the patient's history of cigarette smoking as well as vaping should be considered.

Lack of beta-arrestin signaling in the absence of active G proteins.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking ß-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

Factors Associated with Potassium-Competitive Acid Blocker Non-Response in Patients with Proton Pump Inhibitor-Refractory Gastroesophageal Reflux Disease.

BACKGROUND/AIMS: Approximately 20-40% of patients with gastroesophageal reflux disease (GERD) are refractory to proton pump inhibitor (PPI) treatment. The acid-inhibitory effect of vonoprazan, a novel potassium-competitive acid blocker (P-CAB), is significantly greater when compared to the effect of PPIs. We investigated the efficacy of vonoprazan treatment for PPI-refractory GERD and factors associated with P-CAB non-response. METHODS: We enrolled 277 GERD patients receiving continuous PPI therapy. Subjects completed a self-report questionnaire including the frequency scale for the symptoms of GERD (FSSG). Patients with PPI-refractory GERD received 20 mg of vonoprazan once daily for 8 weeks. After that, subjects completed the same questionnaire, and the results were used to identify P-CAB responders and non-responders. RESULTS: Twenty-eight patients were identified as P-CAB responders and 26 were non-responders. Vonoprazan treatment significantly decreased scores of FSSG, nighttime symptom, and Athens Insomnia Scale. Multivariate analysis demonstrated co-existing functional dyspepsia (FD; OR 4.94) and the presence of sleep disturbances (OR 4.34) was associated with P-CAB non-response, whereas alcohol consumption was inversely associated. CONCLUSIONS: Vonoprazan treatment might be appropriate as a promising new strategy for PPI-refractory GERD. Co-existing FD, sleep disturbances, and alcohol abstinence were significantly associated with P-CAB non-response. Other therapeutic options should be considered in patients with these factors.

Associations among gastroesophageal reflux disease, psychological stress, and sleep disturbances in Japanese adults.

BACKGROUND AND AIM: Gastroesophageal reflux disease (GERD) and psychological stress are associated with sleep disturbances. The aim of the present study was to examine the prevalence of sleep disturbances, anxiety, and depression by GERD subtypes and to identify factors associated with sleep disturbances in general population. METHODS: A total of 2002 Japanese subjects, who underwent annual health checkups, were enrolled and asked to fill out a questionnaire, including the frequency scale for the symptoms of GERD (FSSG), Athens Insomnia Scale (AIS), Rome III questionnaire, and Hospital Anxiety and Depression Scale (HADS). GERD was divided into asymptomatic erosive reflux disease (a-ERD), symptomatic ERD (s-ERD), and non-erosive reflux disease (NERD), according to the presence or absence of esophageal mucosal injury on endoscopy, and the FSSG scores. Sleep disturbances were diagnosed in subjects with AIS score ≥6. RESULTS: Prevalence of sleep disturbances was significantly higher in GERD subjects than in controls (35.9 and 14.7%, respectively), especially, in the NERD group (45.1%). Sleep duration was significantly shorter in the s-ERD group compared with other groups. Subjects in the NERD and s-ERD groups showed higher HADS scores, resulting in higher incidences of anxiety and depression than those in the control and a-ERD groups. Reflux symptoms, anxiety, depression, and coexisting functional dyspepsia, but not the presence of esophageal mucosal injury, were associated with an increased odds ratio for sleep disturbances. CONCLUSION: There were significant positive associations among reflux symptoms, psychological stress, and sleep disturbance in Japanese adults. Further studies investigating the efficacy of therapy are needed.

Short- and long-term treatment with folic acid suppresses thrombus formation in atherogenic mice in vivo.

In the present study, we examined the effects of short- and long-term treatment with folic acid (FA) on thrombus formation in vivo in atherogenic mice to explore a novel agent for the prevention of atherothrombotic disease. Apolipoprotein E and low-density lipoprotein receptor double deficient (ApoE(-/-)LDLR(-/-)) mice were orally administrated a single bolus of FA (20mg/kg) or fed an atherogenic diet with or without FA (0.02, 0.5, and 1.5mg/kg) for 12 weeks. Thrombus formation and endothelial function were assessed in vivo using the He-Ne laser-induced carotid artery thrombus formation test and the flow-mediated vasodilation method. Platelet reactivity was assessed ex vivo using haemostatometry. Short-term treatment with FA markedly increased plasma folate levels and significantly suppressed laser-induced thrombus formation in apoE(-/-)LDLR(-/-) mice. Short-term treatment with FA suppressed platelet reactivity in apoE(-/-)LDLR(-/-) mice, but FA treatment did not affect endothelial function or plasma homocysteine levels. Long-term treatment with FA increased plasma folate levels dose-dependently. Thrombus formation and endothelial dysfunction were suppressed by treatment with 0.5 and 1.5mg/kg of FA, respectively, but not with 0.02mg/kg of FA, whereas platelet reactivity was not altered by treatment with any dose of FA. Long-term treatment with all doses of FA decreased the plasma homocysteine levels in apoE(-/-)LDLR(-/-) mice, although this result was not consistent with its anti-thrombotic action. In conclusion, our data showed that short- and long-term treatment with FA could suppress in vivo thrombus formation in an atherogenic setting, independent of its hypohomocysteinemic action.

Utility of finger maze test for learning and memory abilities in infants of cynomolgus monkeys exposed to thiamazole.

A new type of learning and memory test using a finger maze was conducted in infant cynomolgus monkeys that were exposed to thiamazole (2 and 3.5 mg/kg per day to pregnant animals orally) during the fetal period (gestational days 120 to 150). We modified Tsuchida's original finger maze test method by reducing the number of trials per day and simplifying the criteria for achievement of training, and we added a long-term memory test. In the memory test, thiamazole-exposed infants required greater time to complete the finger maze test than the control infants although no effect was noted in the training or learning test. The results suggest that an impaired long-term memory could be detected by our modified finger maze test.

Effects of maternal exposure to thiamazole on behavioral development in infant cynomolgus monkeys.

Thiamazole, an anti-hyperthyroidism agent, was administered orally to pregnant cynomolgus monkeys at doses of 2.0 and 3.5 mg/kg per day from GD 120 to GD 150 to investigate effects on behavioral development of their infants. Swelling of the throat region due to enlargement of the thyroid glands was observed at birth in thiamazole-treated infants, and it returned to normal around postnatal day (PND) 30. At necropsy of infants at 12 months of age, thyroidal weight in the thiamazole groups was increased. This finding suggested the likelihood that administration of thiamazole to maternal animals during the late gestational period induced thyroid goiter in fetal/infant monkeys through placental transfer of thiamazole. No clear changes were noted in thyroid histopathology or serum thyroid hormone levels in maternal animals or infants, but goiter formation might have been indicative of exposure to high thyroid stimulating hormone (TSH) and low T3 or T4 in utero from maternal treatment with thiamazole. Age-related changes were observed in the control in behavioral development tests, while infants at 3.5 mg/kg showed no age-related decrease in contact behavior and no increase in exploratory activity on PND 90 or PND 170. In addition, the number of eye contacts between PND 210 and PND 240 was less frequent. This indicated that maternal exposure to thiamazole induced mental retardation-like behaviors in infants. Thiamazole may directly inhibit thyroid hormone synthesis in the fetus by placental transfer. From these results, it was speculated that oral administration of thiamazole to maternal animals during the late gestational period induced retardation of behavioral development in their infants.

Historical control data on prenatal developmental toxicity studies in rabbits.

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994-2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter-laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Fetal and neonatal goiter in cynomolgus monkeys following administration of the antithyroid drug thiamazole at high doses to dams during pregnancy.

To evaluate morphologic alterations in the thyroid gland in the second generation in cynomolgus monkeys, pregnant dams were exposed to high doses of thiamazole. In Experiment A, dams received thiamazole intragastrically via a nasogastric catheter from gestation day (GD) 50 to GD 150 or on the day before delivery. Initially, the dose level was 20 mg/kg/day (10 mg/kg twice daily); however, the dose level was subsequently decreased to 5 mg/kg/day (2.5 mg/kg twice daily), since deteriorated general conditions were observed in two dams. Six out of seven neonates died on the day of birth. The cause of neonatal death was tracheal compression and suffocation from goiter. The transplacental exposure to thiamazole affected the fetal thyroid glands and induced goiter in all neonates. The surviving neonate was necropsied 767 days after discontinuation of thiamazole exposure and showed reversibility of the induced changes. In Experiment B, dams were intragastrically administered thiamazole at 5 mg/kg/day (2.5 mg/kg twice daily) for treatment periods from GDs 51 to 70, 71 to 90, 91 to 110, 111 to 130 and 131 to 150. All fetuses showed enlarged thyroid glands but were viable. Histopathologically, hypertrophy and/or hyperplastic appearance of the follicular epithelium of the thyroid gland was observed at the end of each treatment period. The most active appearance of the follicular epithelium, consisting of crowded pedunculated structure, was demonstrated at end of the treatment period from GD 131 to 150. This is the first report on the morphology of fetal and neonatal goiter in the cynomolgus monkey.

Vibration therapy accelerates healing of Stage I pressure ulcers in older adult patients.

OBJECTIVE: The present study investigated whether vibration therapy using a vibrator could facilitate the healing of Stage I pressure ulcers (PrUs) in older adults. METHODS: The study had a nonrandomized, blinded, controlled design. The subjects were hospital patients in long-term-care facilities with Stage I PrUs. In the experimental group, a vibrator (RelaWave; Matsuda Micronics Corp, Chiba, Japan) was used to apply vibration (frequency: 47 Hz; time: 10 seconds; amplitude modulation cycle: 15 seconds) for 15 minutes 3 times a day for up to 7 days, until Stage I PrUs healed. Apart from the vibration therapy, the experimental and control groups received the same care, which was provided according to PrU care guidelines. The number of healed ulcers was compared between 2 groups. RESULTS: The experimental group consisted of 16 patients with 20 Stage I PrUs; the control group consisted of 15 patients with 21 Stage I PrUs. In the experimental group, 8 (40.0%) PrUs healed; in the control group, 2 (9.5%) PrUs healed. The number of healed ulcers was significantly higher in the experimental group than in the control group (P = .033). The healing rate during the study period was significantly higher in the experimental group than in the control group (P = .018, logrank test). The hazard ratio adjusted for baseline risk factors was 0.031 (95% confidence intervals: 0.002-0.594, P = .021). The mean relative changes per day in wound area and intensity of redness were significantly greater in the experimental group than in the control group (P = .007, and P = .023, respectively). CONCLUSION: Based on these results, the use of the vibrator may facilitate the healing of Stage I PrUs.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces disruption of glands of the prostate and fibrosis in rhesus monkeys.

We investigated the effects that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure has on the prostate in rhesus monkey offspring. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and examined histopathologically. Dose-dependent decreases in glands of the prostate and widespread fibrosis were observed in offspring. It is noteworthy that 7 years from the final lactational TCDD exposure, inflammatory cell infiltration and disruption of glands of the prostate were still observed. Differential mRNA expression associated with fibrosis, inflammatory response and disruption of cell components were demonstrated by microarray analysis, with up-regulation of TGM4, TGFB1, COL1A1 and MMP2 confirmed. In conclusion, in utero and lactational exposure to TCDD induced dose-related prostatic fibrosis, indicating prostatic dysfunction and inducible semen quality reduction in second-generation rhesus monkeys.

Impaired endothelial function may be due to decreased aortic tetrahydrobiopterin, assessed by a new flow-mediated vasodilation in vivo in hypercholesterolemic/atherogenic mice.

Tetrahydrobiopterin (BH4) is an important cofactor for endothelial nitric oxide synthase activity. The relationship between endothelial function in vivo and aortic BH4 level is not fully understood, however. In the present study, we aimed to clarify whether reduction of aortic BH4 levels contributes to endothelial dysfunction in vivo using spontaneously hyperlipidemic mice. To estimate endothelial function in vivo and in real-time state, we developed a flow-mediated vasodilation (FMV) method in mice, which measured changes in the diameter of the femoral artery in response to increased blood flow. C57BL/6 mice and apoE/low-density lipoprotein receptor double knock-out mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 12 weeks from 6 weeks of age. HFD feeding impaired FMV in double knock-out mice, but not in C57BL/6 mice. Furthermore, HFD feeding reduced plasma NOx concentration and aortic BH4 level in double knock-out mice. Conversely, exogenous injection of BH4 (2 mg/kg) markedly increased aortic BH4 levels and restored endothelial function. In conclusion, we demonstrated that HFD feeding impaired nitric oxide-mediated endothelial function and reduced BH4 level in vivo, and that acute augmentation of aortic BH4 levels improved endothelial function. These findings indicate that BH4 is a critical determinant of nitric oxide-mediated endothelial function in hypercholesterolemia.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a reduction in epididymal and ejaculated sperm number in rhesus monkeys.

A long-term developmental toxicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure was performed in rhesus monkeys and the effect on male reproductive organs was determined in the second generation. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and evaluated by semen analysis, and histopathology of the testes and epididymides. Ejaculated sperm concentration was severely reduced at 300 ng/kg, and sperm viability and activity were dose-proportionally reduced, although effects on spermatogenesis were slight. Histomorphometry revealed markedly reduced area of the ductus epididymis accompanying decreased reserved sperm in the 30 and 300 ng/kg groups. In conclusion, in utero and lactational exposure to TCDD induced a reduction of sperm quality in rhesus monkeys.

Synergistic antithrombotic effect of a combination of NO donor and plasma kallikrein inhibitor.

The aim of the present study was to investigate the effect of a NO donor (GSNO) and a plasma kallikrein inhibitor (PKSI-527) alone and in combination on global haemostatic status. A new in vitro test was employed which allows the measurement of both platelet function and spontaneous thrombolysis. Sixteen healthy young and 18 elderly volunteers were enrolled in this study. When GSNO (1 mM) or PKSI-527 (20 microM) was added to native human blood, platelet reactivity was significantly inhibited in both age groups. The combination of GSNO and PKSI-527 had additive inhibitory effect on platelets. Addition of either GSNO or PKSI-527 to blood samples did not significantly affect spontaneous thrombolysis, while added together, spontaneous thrombolysis was significantly enhanced. The thrombolysis enhancing effect was more prominent in elderly subjects. Our present findings suggest that the combination of NO donor and plasma kallikrein inhibitor may have clinical antithrombotic potential.