Increased urinary creatinine during hibernation and day roosting in the Eastern bent-winged bat (Miniopterus fuliginosus) in Korea.

Torpor and arousal cycles, both daily and seasonal (e.g. hibernation), are crucial for small mammals, including bats, to maintain the energy and water balance. The alternation between torpor and arousal leads to metabolic changes, leaving traceable evidence of metabolic wastes in urine. In this study we investigated urinary creatinine and acetoacetate (a ketone body) in the Eastern bent-wing bat (Miniopterus fuliginosus) in Mungyeong, South Korea. We found an increase in urinary creatinine during torpor in summer, indicating changes in renal water reabsorption rates during the active season. Although we could not confirm ketonuria in hibernating bats due to a methodological limitation caused by the small amount of urine, we verified an increase in urinary creatinine concentration during hibernation. This finding suggests that managing water stress resulting from evaporative water loss is one of key reasons for arousal during hibernation in Eastern bent-wing bats.

One new species and one new record for the genus Mesogastrura (Collembola, Hypogastruridae) from Korean caves, with DNA barcodes.

We collected one new species and one new record in the genus Mesogastrura Bonet, 1930 (family Hypogastruridae) from three caves with different origins in the Korean Peninsula; Mesogastrura seotalensis sp. nov. and Mesogastrura ojcoviensis (Stach, 1919). The genus Mesogastrura Bonet, 1930 is newly recorded from the Korean Peninsula. Mesogastrura seotalensis sp. nov. shows various body color (light brown or much less and light purple), 3 + 3 eyes and unguis with three inner teeth. On the other hand, Mesogastrura ojcoviensis (Stach, 1919) is characterized by white body color, 2 + 2 eyes and unguis without inner tooth (rarely with 1). These species were found in piles of bat's guano inside of caves, so they are considered as troglophilous and guanophilous species. Partial DNA sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene were used as DNA barcodes to clarify the species delimitation.

Urinary creatinine varies with microenvironment and sex in hibernating Greater Horseshoe bats (Rhinolophus ferrumequinum) in Korea.

BACKGROUND: In temperate regions many small mammals including bats hibernate during winter. During hibernation these small mammals occasionally wake up (arouse) to restore electrolyte and water balance. However, field data on water stress and concentration of bodily fluids during hibernation is scarce. Urinary creatinine concentration has long been used to calibrate urinary hormone concentration due to its close correlation with urine concentration. Therefore, by investigating urinary creatinine concentration, we can estimate bodily fluid concentration. In this study, we investigated changes in urinary creatinine from greater horseshoe bats (Rhinolophus ferrumequinum) hibernating in abandoned mineshafts in two regions in South Korea. RESULTS: We collected 74 urine samples from hibernating greater horseshoe bats from 2018 to 2019. We found that urinary creatinine concentration was higher in February and March and then declined in April. There were also indications of a sex difference in the pattern of change in creatinine concentration over the three months. Bats in the warmer and less humid mineshaft had higher urinary creatinine concentrations than bats in the colder and more humid mineshaft. CONCLUSIONS: These results indicate that hibernating bats face water stress as urinary concentration increases during winter and that water stress may vary depending on the microenvironment. Sex differences in behaviour during hibernation may influence arousal frequency and result in sex differences in changes in urinary creatinine concentration as hibernation progresses. Although further behavioural and endocrinal investigations are needed, our study suggests that urinary creatinine concentration can be used as a proxy to estimate the hydration status of bats and the effect of sex and environmental factors on arousal patterns during hibernation.

The complete mitochondrial genome of the far Eastern myotis: Myotis bombinus Thomas, 1906 in mainland of Korea (Chiroptera, Vespertilionidae).

We have determined the second mitochondrial genome of Myotis bombinus Thomas, 1906 in mainland of Korea. The circular mitogenome of M. bombinus is 17,035 bp long which is slightly shorter than that of the previous mitogenome of M. bombinus. It includes 13 protein-coding genes (PCGs), two ribosomal RNA genes, and 22 transfer RNAs. The base composition was AT-biased (66.1%). Fifty single nucleotide polymorphisms and 14 insertions were identified between two mitogenomes of M. bombinus. Phylogenetic trees show that both M. bominus mitogenomes are clustered in one clade.

Tumor-infiltration of T-lymphocytes is inversely correlated with clinicopathologic factors in endometrial adenocarcinoma.

OBJECTIVE: The aim of this study was to determine the distribution of T-lymphocytes and their relationship with clinicopathologic factors in endometrial carcinoma. METHODS: Samples were collected from 89 patients with endometrial endometrioid adenocarcinoma treated in Pusan National University Hospital from 2004 to 2011. Normal endometrial tissues were obtained from 30 hysterectomized women with benign adnexal masses and served as controls. Paraffin-embedded sections were immunohistochemically stained for CD8 (cytotoxic) and CD4 (helper) T-lymphocytes. The relationship of these cells with stage, histological grade, myometrial invasion, and lymph node metastasis was analyzed. RESULTS: The proportion of CD8+ and CD4+ lymphocytes in the endometrial endometrioid adenocarcinoma tissues was 67.4% (60/89) and 44.9% (40/89), respectively, which was significantly higher (P<0.05) than in the control group. The extent of CD8+ lymphocyte expression was negatively correlated with histologic grade, myometrial invasion, and lymph node metastasis. The proportion of infiltration of the CD4+ lymphocytes was negatively correlated with histologic grade and myometrial invasion. CONCLUSION: The high rate of infiltration of T-lymphocytes was negatively correlated with histologic grade, myometrial invasion, and lymph node metastasis. Our findings suggest that tumor-infiltrating T-lymphocytes may be used as pathologic prognostic factors in endometrial carcinoma.

Safety of extended treatment with sapropterin dihydrochloride in patients with phenylketonuria: results of a phase 3b study.

BACKGROUND: Phenylketonuria (PKU) results from impaired breakdown of phenylalanine (Phe) due to deficient phenylalanine hydroxylase (PAH) activity. Sapropterin dihydrochloride (sapropterin, Kuvan®) is the only US- and EU-approved pharmaceutical version of naturally occurring 6R-BH(4), the cofactor required for PAH activity. Sapropterin enhances residual PAH activity in sapropterin-responsive PKU patients and, in conjunction with dietary management, helps reduce blood Phe concentrations for optimal control. Approval was based on the positive safety and efficacy results of four international clinical studies, the longest of which was 22 weeks in duration. OBJECTIVE: To evaluate the safety of long-term treatment with sapropterin in PKU subjects who participated in previous Phase 3 sapropterin trials. METHODS: PKU-008 was designed as a Phase 3b, multicenter, multinational, open-label, 3-year extension trial to evaluate the long-term safety of sapropterin in patients with PKU who were classified as sapropterin responders and participated in prior Phase 3 sapropterin studies: 111 subjects aged 4-50 years completed prior studies and were subsequently enrolled in study PKU-008. Routine safety monitoring was performed at 3-month intervals and included adverse event reporting, blood Phe monitoring, clinical laboratory evaluations, physical examinations and vital sign measurements. RESULTS: Average exposure during PKU-008 was 658.7±221.3 days (range, 56-953; median, 595). The average total duration of participation in multiple studies (PKU-001, PKU-003, PKU-004, and PKU-008; or PKU-006 and PKU-008) was 799.0±237.5 days (range, 135-1151). The mean sapropterin dose was 16.2±4.7 mg/kg/day. Most adverse events were considered unrelated to treatment, were mild or moderate in severity, and were consistent with prior studies of sapropterin. No age-specific differences were observed in adverse event reporting. Three subjects discontinued treatment due to adverse events that were considered possibly or probably related to study treatment (one each of difficulty concentrating, decreased platelet count, and intermittent diarrhea). No deaths were reported. Of seven reported serious adverse events, one was considered possibly related to study treatment (gastroesophageal reflux). There were no laboratory or physical examination abnormalities requiring medical interventions. For most subjects, blood Phe concentrations were consistently within target range, confirming the durability of response in subjects undergoing extended treatment with sapropterin. CONCLUSION: Sapropterin treatment was found to be safe and well tolerated at doses of 5 to 20mg/kg/day for an average exposure of 659 days. This study supports the safety and tolerability of sapropterin as long-term treatment for patients with PKU.

Relative bioavailability of sapropterin from intact and dissolved sapropterin dihydrochloride tablets and the effects of food: a randomized, open-label, crossover study in healthy adults.

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive metabolic disorder characterized by hyperphenylalaninemia in association with neurocognitive and neuromotor impairment. Sapropterin dihydrochloride (hereafter referred to as sapropterin) administered orally as dissolved tablets is approved by the US Food and Drug Administration for hyperphenylalaninemia in patients with tetrahydrobiopterin responsive PKU. OBJECTIVES: This study compared the relative oral bioavailability of sapropterin when administered as intact and dissolved tablets. It also assessed the effect of food on the oral bioavailability of sapropterin administered as intact tablets. METHODS: This was a randomized, open-label, 3-treatment, 6-sequence, 3-period crossover study in healthy male and female subjects. Subjects were randomized to receive single oral 10-mg/kg doses of sapropterin administered as dissolved tablets after a fast; as intact tablets after a fast; and as intact tablets with a high-calorie, high-fat meal. The 3 dosing periods were separated by a washout period of at least 7 days. In each dosing period, blood samples were obtained within 40 minutes before and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 18, and 24 hours after dosing. A follow-up assessment was performed 5 to 7 days after the last dosing period. The relative bioavailability of sapropterin from the 3 dosing regimens was assessed based on C(max), AUC(0-t), and AUC(0-infinity), estimated from calculated plasma tetrahydrobiopterin concentrations using a noncompartmental model. Safety assessments included physical examinations, clinical laboratory tests, and ECGs at the beginning and end of the study. Vital signs were monitored periodically during each treatment period. RESULTS: The study enrolled 32 healthy subjects (16 men, 16 women) with a mean (SD) age of 29.2 (9.0) years, height of 172.7 (10.0) cm, weight of 73.0 (13.9) kg, and body mass index ranging from 18 to 30 kg/m(2). Twenty-three were white, 5 African American, 2 Asian/Pacific Islander, 1 Hispanic, and 1 Native American. The estimated geometric mean ratio of AUC(0-t) for intact compared with dissolved tablets under fasting conditions was 141.24% (90% CI, 122.05-163.43), and the geometric mean ratio of AUC(0-t) for intact tablets under fed compared with fasting conditions was 143.46% (90% CI, 124.22-165.69). Nine subjects (28.1%) reported a total of 20 treatment-emergent adverse events (AEs). The most frequently reported AEs were gastrointestinal disorders (6 subjects [18.8%]) and central nervous system disorders (4 [12.5%]). Eight AEs considered possibly or probably related to sapropterin were reported by 4 subjects (12.5%); these were of mild severity and gastrointestinal in nature. No severe or serious AEs or discontinuations due to AEs occurred during the study. CONCLUSIONS: Administration of sapropterin as intact tablets and with a high-calorie, high-fat meal was associated with increased drug exposure. Oral administration of sapropterin 10 mg/kg as intact tablets with or without food was generally well tolerated.

Safety and efficacy of outpatient nesiritide in patients with advanced heart failure: results of the Second Follow-Up Serial Infusions of Nesiritide (FUSION II) trial.

BACKGROUND: Patients with American College of Cardiology/American Heart Association stage C/D heart failure experience substantial morbidity and mortality, but available interventions beyond standard medical and device therapies are limited. Nesiritide relieves dyspnea and reduces pulmonary congestion, but its risk profile is uncertain. Pilot data suggested a potential benefit of nesiritide given as serial outpatient infusions. METHODS AND RESULTS: The Second Follow-Up Serial Infusions of Nesiritide (FUSION II) trial was a randomized, double-blind, placebo-controlled trial of outpatient serial nesiritide infusions for patients with American College of Cardiology/American Heart Association stage C/D heart failure. Patients with 2 recent heart failure hospitalizations, ejection fraction <40%, and New York Heart Association class IV symptoms, or New York Heart Association class III symptoms with creatinine clearance <60 mL/min, were randomized to nesiritide (2-microg/kg bolus plus 0.01-microg/kg-per-minute infusion for 4 to 6 hours) or matching placebo, once or twice weekly for 12 weeks. All patients were treated to optimal goals with evidence-based medical/device therapy facilitated by careful disease management during the study. The primary end point was time to all-cause death or cardiovascular or renal hospitalization at 12 weeks. A total of 911 patients were randomized and treated. The primary end point occurred in 36.8% and 36.7% of the placebo and nesiritide groups, respectively (hazard ratio, 1.03; 95% confidence interval, 0.82 to 1.3; log-rank test P=0.79). There were no statistically significant differences between groups in any of the secondary end points, including the number of cardiovascular or renal hospitalizations, the number of days alive and out of the hospital, change in Kansas City Cardiomyopathy Questionnaire score, or cardiovascular death. Adverse events were similar between groups; nesiritide was associated with more hypotension but less predefined worsening renal function. CONCLUSIONS: Serial outpatient nesiritide infusions do not provide a demonstrable clinical benefit over intensive outpatient management of patients with advanced American College of Cardiology/American Heart Association stage C/D heart failure.

The Second Follow-up Serial Infusions of Nesiritide (FUSION II) trial for advanced heart failure: study rationale and design.

BACKGROUND: Patients with persistently symptomatic advanced heart failure have limited treatment options after appropriate use of standard evidence-based therapies. Current recommendations from the American College of Cardiology/American Heart Association guidelines for treatment of stage C/D heart failure beyond standard therapy include ventricular replacement, investigational agents, and palliative interventions. Given the elevated risk of this patient population, additional treatment options seem warranted. Natriuretic peptides are protean compounds that provoke vasodilation, natriuresis, neurohormonal antagonism, and reverse remodeling, but they have an uncertain risk-benefit profile affecting serum creatinine and clinical events. In the pilot, open-label FUSION I trial, the adjunctive administration of nesiritide with standard therapy for patients with advanced heart failure, was demonstrated to have a neutral effect on outcomes with no evidence of increased risk. Within a prespecified subset of high-risk patients, a potential signal of benefit on a combined end point of mortality and cardiovascular hospitalization was identified. STUDY DESIGN: FUSION II is a 900-patient randomized, placebo-controlled, double-blind, phase IIb trial designed to further assess the safety, efficacy, and optimal dosing frequency of outpatient nesiritide for advanced heart failure. The primary end point is a composite of all-cause mortality and cardiorenal hospitalization. CONCLUSIONS: If a confirmatory signal of benefit is identified in FUSION II, a definitive phase III mortality/quality of life trial will be warranted. Additional issues related to the influence of disease management, the logistics of outpatient parenteral therapy administration, and future iterations of natriuretic peptide therapy will need to be investigated.

Evaluation of natural products on inhibition of inducible cyclooxygenase (COX-2) and nitric oxide synthase (iNOS) in cultured mouse macrophage cells.

The inhibitors of prostaglandin biosynthesis and nitric oxide production have been considered as potential anti-inflammatory and cancer chemopreventive agents. In this study, we evaluated approximately 170 methanol extracts of natural products including Korean herbal medicines for the inhibition of prostaglandin E(2) production (for COX-2 inhibitors) and nitric oxide formation (for iNOS inhibitors) in lipopolysaccharide (LPS)-induced mouse macrophages RAW264.7 cells. As a result, several extracts such as Aristolochia debilis, Cinnamomum cassia, Cinnamomum loureirii, Curcuma zedoaria, Eugenia caryophyllata, Pterocarpus santalius, Rehmania glutinosa and Tribulus terrestris showed potent inhibition of COX-2 activity (>80% inhibition at the test concentration of 10 micro g/ml). In addition, the extracts of A. debilis, Caesalpinia sappan, Curcuma longa, C. zedoaria, Daphne genkwa and Morus alba were also considered as potential inhibitors of iNOS activity (>70% inhibition at the test concentration of 10 micro g/ml). These active extracts mediating COX-2 and iNOS inhibitory activities are warranted for further elucidation of active principles for development of new cancer chemopreventive and/or anti-inflammatory agents.

Suppressive effect of natural sesquiterpenoids on inducible cyclooxygenase (COX-2) and nitric oxide synthase (iNOS) activity in mouse macrophage cells.

Prostaglandins and nitric oxide produced by inducible cyclooygenase (COX-2) and nitric oxide synthase (iNOS), respectively, have been implicated as important mediators in the processes of inflammation and carcinogenesis. These potential COX-2 and iNOS inhibitors have been considered as antiinflammatory and cancer chemopreventive agents. In this study, we investigated the effect of natural sesquiterpenoids isolated from plants of the Zingiberaceae family on the activities of COX-2 and iNOS in cultured lipopolysaccharide (LPS)-activated mouse macrophage cell RAW 264.7 to discover new lead compounds as COX-2 or iNOS inhibitors. Xanthorrhizol, a sesquiterpenoid, isolated from the rhizome of Curcuma xanthorrhiza Roxb. (Zingiberaceae), exhibited a potent inhibition of COX-2 (IC50 = 0.2 microg/mL) and iNOS activity (IC50 = 1.0 microg/mL) in the assay system of prostaglandin E2 (PGE2) accumulation and nitric oxide production, respectively. Western blot analyses revealed that the inhibitory potential of xanthorrhizol on the COX-2 activity coincided well with the suppression of COX-2 protein expression in LPS-induced macrophages. In addition, sesquiterpenoids beta-turmerone and ar-turmerone isolated from the rhizome of Curcuma zedoaria Roscoe (Zingiberaceae) also showed a potent inhibitory activity of COX-2 (beta-turmerone, IC50 = 1.6 microg/mL; ar-turmerone, IC50 = 5.2 microg/mL) and iNOS (beta-turmerone, IC50 = 4.6 microg/mL; ar-turmerone, IC50 = 3.2 microg/mL). These results suggest that natural sesquiterpenoids from C. xanthorrhiza and C. zedoaria might be lead candidates for further developing COX-2 or iNOS inhibitors possessing cancer chemopreventive or anti-inflammatory properties.