Regulatory mechanisms underlying interleukin-6 expression in murine brown adipocytes.

Three types of adipocytes, white, brown, and beige, regulate the systemic energy balance through the storage and expenditure of chemical energy. In addition, adipocytes produce various bioactive molecules known as adipokines. In contrast to white adipocyte-derived molecules, less information is available on the adipokines produced by brown adipocytes (batokine). This study explored the regulatory expression of interleukin (IL)-6 in cell culture studies. Norepinephrine or a nonselective ß-adrenergic receptor agonist increased the expression of IL-6 in primary brown adipocytes and HB2 brown adipocytes. Treatment with forskolin (Fsk), an activator of the cAMP-dependent protein kinase (PKA) pathway (downstream signaling of the ß-adrenergic receptor), efficiently stimulated IL-6 expression in brown adipocytes and myotubes. Phosphorylated CREB and phosphorylated p38 MAP kinase levels were increased in Fsk-treated brown adipocytes within 5 min. In contrast, a long-term (∼60 min and ∼4 h) treatment with Fsk was required for increase in STAT3 phosphorylation and C/EBPß expression, respectively. The PKA, p38 MAP kinase, STAT3, and C/EBPß pathways are required for the maximal IL-6 expression induced by Fsk, which were verified by use of various inhibitors of these signal pathways. Vitamin C enhanced Fsk-induced IL-6 expression through the extracellular signal-regulated kinase activity. The present study provides basic information on the regulatory expression of IL-6 in activated brown adipocytes.

Biomarkers for Risk Stratification in Patients With Type A Acute Aortic Dissection.

Type A acute aortic dissection (AAD) is a fatal disease and thus, accurate and objective risk stratification is essential. In this study, we evaluated the prognostic value of readily available and assessable biomarkers in patients with type A AAD. This was a retrospective, multicenter, observational study. A total of 703 patients with type A AAD diagnosed using contrast-enhanced computed tomography were included. Therapeutic strategies were left to the physician's discretion in a real-world clinical setting. The prognostic value for in-hospital mortality was examined in 15 circulating biomarkers on admission, which are routinely available in clinical practice. Of the 703 patients, 126 (17.9%) died during the hospitalization. Of the 15 biomarkers, the multivariable analysis identified positive cardiac troponin, a low total bilirubin (T-Bil) level, and increased levels of brain natriuretic peptide (BNP) and lactate dehydrogenase (LDH) as significant predictors of in-hospital death. The receiver operating characteristics curve analysis showed that these 4 biomarkers had an independent additive prognostic value. With the cut-off values of T-Bil, BNP, and LDH, in combination with positive troponin, the increase in the number of positive biomarkers was progressively associated with higher in-hospital mortality from 1.3% to 9.8%, 20.5%, 36.4%, and 75.0% (p <0.001). In conclusion, in patients with type A AAD, positive cardiac troponin, a low T-Bil level, and increased levels of BNP and LDH on admission were related to higher in-hospital mortality, with an incremental prognostic value, suggesting that the readily available and assessable biomarkers can aid in decision-making in therapeutic strategies.

Clinical characteristics and outcomes in patients with acute type A aortic intramural hematoma.

BACKGROUND: Although type A acute aortic dissection (AAD) including classic double-channel aorta and intramural hematoma (IMH) is a life-threatening condition, the prognostic impact and predictors of IMH remain to be established. The present study evaluated the prevalence, baseline characteristics, and outcomes of IMH as compared with classic non-thrombosed type A AAD. METHODS: This multicenter registry in Japan retrospectively included 703 patients with type A AAD. IMH was defined as a crescentic or circular area along the ascending aortic wall without contrast enhancement on computed tomography (CT). Non-thrombosed type A AAD was defined as the classic double-channel ascending aorta on contrast-enhanced CT. The primary endpoint was in-hospital mortality. RESULTS: Of the 703 patients with type A AAD, 312 (44.3%) had IMH. Older age was an only baseline patient factor significantly associated with the presence of IMH in the multivariable analysis. The longitudinal extent of dissection was greater in patients with classic non-thrombosed AAD than those with IMH, resulting in an increased risk of end-organ malperfusion in the classic AAD group. During the hospitalization, 41 (13.1%) and 85 (21.7%) patients with and without IMH died (p < 0.001). IMH was associated with lower in-hospital mortality in a multivariable model, irrespective of age and the implementation of surgery. CONCLUSIONS: The present study showed that IMH on CT was frequent among patients with type A AAD. Although IMH was more likely to be present in the elderly, its effect on the better survival was independent of age and surgical treatment.

Detection and molecular analysis of Philopinna higai (Digenea: Didymozoidae) from domestically introduced Sarcocheilichthys fishes in the Tohoku region, Japan.

Philopinna higai is a species of Didymozoidae (Digenea: Hemiuroidea). The definitive hosts of this parasite only belong to the fish genus Sarcocheilichthys. Sarcocheilichthys fishes are endemic to Lake Biwa and southwestern Japan and were introduced into the northeastern (Tohoku) region. However, P. higai parasitism has not been investigated in the Tohoku region. In this study, we surveyed the distribution of P. higai in the Tohoku region and sequenced 28S rDNA (994 bp) and cytochrome oxidase subunit 1 (CO1) gene (721 bp) of P. higai. We also sequenced mitochondrial cytochrome b (581 bp) of Sarcocheilichthys fishes from the Tohoku region and Lake Biwa. Our findings confirmed the distribution of P. higai in all seven surveyed river systems in the four prefectures of the Tohoku region. The 28S rDNA sequence of P. higai did not differ among regions, whereas 10 haplotypes of CO1 were identified and clustered into two major clades. The haplotypes of Sarcocheilichthys fishes introduced in the Tohoku region were identical to the dominant haplotypes in Lake Biwa. Thus, P. higai from Lake Biwa and the Tohoku region were genetically the same species, although genetically differentiated populations formed in the Tohoku region.

Right versus left coronary artery involvement in patients with type A acute aortic dissection.

BACKGROUND: Type A acute aortic dissection (AAD) complicated by coronary malperfusion is a life-threatening disease. In the present study, we compared the clinical characteristics and prognostic impact of treatment strategies including surgical treatment and percutaneous coronary intervention (PCI) in type A AAD patients with RCA and LCA involvement. METHODS: This multicenter registry included 220 patients with type A AAD and either RCA or LCA involvement. Treatment strategies were left to treating physicians. The primary endpoint was in-hospital death. RESULTS: Of 220 patients, 115 (52.3%) and 105 (47.7%) had RCA and LCA involvement. Patients with LCA involvement were more1 likely to present with Killip class IV on admission than those with RCA involvement. Coronary angiography was performed in 52 of 220 (23.6%) patients, among whom 39 (75.0%) underwent subsequent PCI. During the hospitalization, 93 (42.3%) patients died. Patients with LCA involvement had an increased risk of in-hospital mortality compared to those with RCA involvement (54.3% vs. 31.3%, p < 0.001). In patients with RCA involvement, multivariable analysis identified Killip class IV and no surgical treatment as predictors of in-hospital death, while PCI and surgical treatment were indicated as factors associated with lower in-hospital mortality in patients with LCA involvement. CONCLUSIONS: The rates of RCA and LCA involvement were similar in type A AAD. Immediate PCI as a bridge to subsequent surgical treatment might improve survival in patients with type A AAD complicated by coronary malperfusion, especially in those with LCA involvement.

Implantable cardioverter-defibrillator therapy after resuscitation from cardiac arrest in vasospastic angina: A retrospective study.

Patients with vasospastic angina (VSA) who are resuscitated from sudden cardiac arrest (SCA) are at a high risk of recurrent lethal arrhythmia and cardiovascular events. However, the benefit of the implantable cardioverter-defibrillator (ICD) therapy in this population has not been fully elucidated. The present study aimed to analyze the prognostic impact of ICD therapy on patients with VSA and SCA. A total of 280 patients who were resuscitated from SCA and received an ICD for secondary prophylaxis were included in the present multicenter registry. The patients were divided into two groups on the basis of the presence of VSA. The primary endpoint was a composite of all-cause death and appropriate ICD therapy (appropriate anti-tachycardia pacing and shock) for recurrent ventricular arrhythmias. Of 280 patients, 51 (18%) had VSA. Among those without VSA, ischemic cardiomyopathy was the main cause of SCA (38%), followed by non-ischemic cardiomyopathies (18%) and Brugada syndrome (7%). Twenty-three (8%) patients were dead and 72 (26%) received appropriate ICD therapy during a median follow-up period of 3.8 years. There was no significant difference in the incidence of the primary endpoint between patients with and without VSA (24% vs. 33%, p = 0.19). In a cohort of patients who received an ICD for secondary prophylaxis, long-term clinical outcomes were not different between those with VSA and those with other cardiac diseases after SCA, suggesting ICD therapy may be considered in patients with VSA and those with other etiologies who were resuscitated from SCA.

Dermocystid infection in Japanese fire-bellied newt, Cynops pyrrhogaster.

Here, we report details of a new infectious disease in wild-caught Japanese fire-bellied newts (Cynops pyrrhogaster), a Near Threatened species. Skin lesions consisting of numerous masses were found in the animals near Lake Biwa, Shiga Prefecture, Japan. The gross appearance of the skin lesions showed blister-, cyst-, and/or tumor-like morphology. Various sizes of skin lesions were observed on their entire body surface. Histologically, spherical basophilic cysts, including numerous spores, were observed in the dermis layer. Ultrastructural analysis indicated the presence of main bodies of flagellated zoospores within the spores. While 18s rRNA gene sequencing indicated that the skin lesions were due to dermocystid infection. To our knowledge, this is the first report of dermocystid infection in this amphibian in Japan. Further studies are needed to prevent epidemics and to establish diagnostic and treatment methods.

Stimulation of uncoupling protein 1 expression by ß-alanine in brown adipocytes.

Carnosine, which is abundant in meat, is a dipeptide composed of ß-alanine and histidine, known to afford various health benefits. It has been suggested that carnosine can elicit an anti-obesity effect via induction and activation of brown/beige adipocytes responsible for non-shivering thermogenesis. However, the relationship between carnosine and brown/beige adipocytes has not been comprehensively elucidated. We hypothesized that ß-alanine directly modulates brown/beige adipogenesis and performed an in vitro assessment to test this hypothesis. HB2 brown preadipocytes were differentiated using insulin from day 0. Cells were treated with various concentrations of ß-alanine (12.5-100 µM) during adipogenesis (days 0-8) and differentiation (days 8-10). Then, cells were further stimulated with or without forskolin, an activator of the cAMP-dependent protein kinase pathway, on day 8 or day 10 for 4 h before harvesting. We observed that HB2 cells expressed molecules related to the transport and signal transduction of ß-alanine. Treatment with ß-alanine during brown adipogenesis dose-dependently enhanced forskolin-induced Ucp1 expression; this was not observed in differentiated brown adipocytes. Consistent with these findings, treatment with ß-alanine during days 0-8 increased phosphorylation levels of CREB in forskolin-treated HB2 cells. In addition, ß-alanine treatment during brown adipogenesis increased the expression of Pparα, known to induce brown/beige adipogenesis, in a dose-dependent manner. These findings revealed that ß-alanine could target HB2 adipogenic cells and enhance forskolin-induced Ucp1 expression during brown adipogenesis, possibly by accelerating phosphorylation and activation of CREB. Thus, ß-alanine, a carnosine-constituting amino acid, might directly act on brown adipogenic cells to stimulate energy expenditure.

Treatment strategies and in-hospital mortality in patients with type A acute aortic dissection and coronary artery involvement.

OBJECTIVE: Type A acute aortic dissection (AAD), especially that with coronary artery involvement and malperfusion, is a life-threatening disease. In the present study we aimed to investigate the association of surgical treatment and percutaneous coronary intervention (PCI) with in-hospital mortality in patients with type A AAD and coronary artery involvement. METHODS: This retrospective multicenter registry in Japan included 225 patients with type A AAD and coronary artery involvement. Treatment strategies including surgical treatment and/or PCI were left to treating physicians. The primary end point was in-hospital death. RESULTS: Of 225 patients, dissection extended into the right and left coronary arteries and both in 115 (51.1%), 105 (46.7%), and 5 (2.2%), respectively. Overall, 94 (41.8%) patients died during the hospitalization. Coronary angiography was performed in 53 (23.6%) patients, among whom 39 (73.6%) underwent PCI. Surgical repair was performed in 188 (83.6%) patients. In patients who received neither procedure, 33 of 35 (94.3%) died during the hospitalization. PCI was performed as a bridge to surgical repair in 37 of 39 (94.9%) patients, and in-hospital mortality of patients who underwent PCI and surgical procedures was 24.3%. Multivariable analysis identified PCI and surgical procedures as factors associated with lower in-hospital mortality rates. CONCLUSIONS: Coronary artery involvement in type A AAD was associated with high in-hospital mortality of more than 40% in the current era. An early reperfusion strategy with PCI as a bridge to surgical repair might improve clinical outcomes in this fatal condition.

Vaginal mucus in mice: developmental and gene expression features of epithelial mucous cells during pregnancy†.

The vagina is the site of copulation and serves as the birth canal. It also provides protection against external pathogens. In mice, due to the absence of cervical glands, the vaginal epithelium is the main producer of vaginal mucus. The development and differentiation of vaginal epithelium-constituting cells and the molecular characteristics of vaginal mucus have not been thoroughly examined. Here, we characterized vaginal mucous cell development and the expression of mucus-related factors in pregnant mice. The vaginal mucous epithelium layer thickened and became multilayered after Day 12 of pregnancy and secreted increasing amounts of mucus until early postpartum. Using histochemistry and transmission electron microscopy, we found supra-basal mucous cells as probable candidates for precursor cells. In vaginal mucous cells, the expression of TFF1, a stabilizer of mucus, was high, and some members of mucins and antimicrobial peptides (MUC5B and DEFB1) were expressed in a stage-dependent manner. In summary, this study presents the partial characterization of vaginal epithelial mucous cell lineage and expression of genes encoding several peptide substances that may affect vaginal tissue homeostasis and mucosal immunity during pregnancy and parturition.

Prognostic Impact of Branch Vessel Involvement on Computed Tomography versus Clinical Presentation of Malperfusion in Patients With Type a Acute Aortic Dissection.

Type A acute aortic dissection (AAD) is a life-threatening disease. The use of contrast-enhanced computed tomography (CT) for diagnosing AAD has increased, and CT can provide pathophysiologic information on dissection such as intramural hematoma (IMH), longitudinal extent of dissection, and branch vessel involvement. However, the prognostic impact of these CT findings is poorly investigated. This multicenter registry included 703 patients with type A AAD. The longitudinal extent of dissection and IMH was determined on CT. Branch vessel involvement was defined as dissection extended into coronary, cerebral, and visceral arteries on CT. The evidence of malperfusion was defined based on clinical presentations. The primary endpoint was in-hospital death. Of 703 patients, 126 (18%) died during hospitalization. Based on contrast-enhanced CT findings, longitudinal extent of dissection was not associated with in-hospital death, while patients with IMH had lower in-hospital mortality than those without (13% vs 22%, p = 0.004). Coronary, cerebral, and visceral artery involvement on CT was found in 6%, 55%, and 32%. In patients with coronary artery involvement, 90% had clinical coronary malperfusion, while only 25% and 21% of patients with cerebral and visceral artery involvement had clinical evidence of corresponding organ malperfusion. Multivariable analysis showed evidence of malperfusion as a significant factor associated with in-hospital mortality. In conclusions, branch vessel involvement on CT was not always associated with end-organ malperfusion in patients with type A AAD, especially in cerebral and visceral arteries. Clinical evidence of malperfusion was significantly associated with in-hospital mortality beyond branch vessel involvement on CT.

["Exclusivity" and quantifier float in bakari"Exclusivity" and quantifier float in bakari]. / 「ばかり」の「限定」と遊離数量詞.

This paper presents a descriptive study that analyzes the semantic meaning of Toritate focus particle bakari. Previous studies reported that, although bakari expresses exclusivity, it is characterized by the fact that it permits non-applicable cases, thereby drawing the conclusion that the meaning of bakari is not exclusivity. This paper argues that bakari does indeed denote "exclusivity" as bakari is supported by the phenomenon that non-applicable cases are unacceptable when bakari co-occurs with floating quantifiers. Considering existing research on this subject, the following was observed. Even though the subjective set, as established by the speaker's past experiences to interpret the meaning of bakari, may not be consistent with the real world, the number of events that form the said set match the number of real-world events when bakari co-occurs with floating quantifiers due to the characteristics of floating quantifiers. In such cases, bakari does not permit non-applicable cases. The interpretation that permits non-applicable cases applies to situations where the set established by the speaker is fixed at a narrower range than the real world, and the non-applicable cases exist outside the set. We thus conclude that bakari denotes "exclusivity" that does not permit non-applicable cases.

Improvement in identification of pro-estrous mice by using a novel method of detecting vaginal mucous cells.

Accurate identification of the murine estrous cycle using vaginal exfoliative cytology is the initial and crucial step for controlled reproduction of this species. However, it is generally difficult to discriminate each stage of the cycle, and thus to select pro-estrous mice for mating. To increase the accuracy of identification of the pro-estrous stage, we re-evaluated the vaginal fold histology and modified the method of exfoliative cytology. Tissue fixation using methanol in Carnoy's solution but not paraformaldehyde, combined with Alcian blue staining but not the conventional Giemsa staining, resulted in better manifestation of mucosal cell layers in the vaginal epithelium just above the keratinized layer. This mucous layer in the fold histology was found to form specifically in the pro-estrous and late di-estrous stages, and the mucous cells exfoliated in smear samples only in the pro-estrous stage. This novel method was found, by a blinded test, to increase the rate of accurate identification of the pro-estrous stage compared to the conventional method (80% vs 50%). Consistent with this finding, the mating experiment with "pro-estrous" females selected by the novel method revealed a significantly higher success rate than that with the conventional method (78.0% vs 47.5%). Thus, our study demonstrates vaginal exfoliative mucous cells as a better potential marker to detect the "receptive" state of female mice that leads to an improved success rate of mating.

Response to iron overload in cultured hepatocytes.

Iron is essential for a variety of physiological processes. Hepatic iron overload acts as a trigger for the progression of hepatic steatosis to nonalcoholic steatohepatitis and hepatocellular carcinoma. In the present study, we aimed to study the effects of iron overload on cellular responses in hepatocytes. Rat primary hepatocytes (RPH), mouse primary hepatocytes (MPH), HepG2 human hepatoma cells and Hepa1-6 mouse hepatoma cells were treated with FeCl3. Treatment with FeCl3 effectively increased iron accumulation in primary hepatocytes. Expression levels of molecules involved in cellular signaling such as AMPK pathway, TGF-ß family pathway, and MAP kinase pathway were decreased by FeCl3 treatment in RPH. Cell viability in response to FeCl3 treatment was decreased in RPH but not in HepG2 and Hepa1-6 cells. Treatment with FeCl3 also decreased expression level of LC-3B, a marker of autophagy in RPH but not in liver-derived cell lines. Ultrastructural observations revealed that cell death resembling ferroptosis and necrosis was induced upon FeCl3 treatment in RPH. The expression level of genes involved in iron transport varied among different liver-derived cells- iron is thought to be efficiently incorporated as free Fe2+ in primary hepatocytes, whereas transferrin-iron is the main route for iron uptake in HepG2 cells. The present study reveals specific cellular responses in different liver-derived cells as a consequence of iron overload.

Regulatory expression of bone morphogenetic protein 6 by 2,2'-dipyridyl.

BACKGROUND: Expression of hepcidin, a hormone produced by hepatocytes which negatively regulates the circulating iron levels, is known to be positively regulated by BMP6, a member of transforming growth factor (TGF)-ß family. Previous studies have shown that iron status is sensed by sinusoidal endothelial cells of hepatic lamina, leading to the modulation of BMP6 expression. METHODS: ISOS-1, HUVEC, F-2, and SK-HEP1 endothelial cells were treated with either iron or 2,2'-dipyridyl (2DP), a cell-permeable iron-chelator, and expression level of Bmp6 was examined. To identify factors affecting Bmp6 transcription, stimulus screening for regulator of transcription (SSRT) was developed. RESULTS: Treatment with iron slightly increased the expression levels of Bmp6, while 2DP unexpectedly increased Bmp6 expression in a dose-dependent manner. 2DP-induced Bmp6 expression was resistant to co-treatment with iron. 2DP-induced Bmp6 expression was also detected in HUVEC, F-2 cells, and SK-HEP1 cells. Luciferase-based reporter assays indicated that forced expression of JunB increased the transcription of Bmp6. 2DP induced phosphorylation of JunB; co-treatment with SP600125 blocked the 2DP-induced Bmp6 expression partially. JunB-induced Bmp6 transcription was not affected by mutations of putative JunB-responsive elements. Some endoplasmic reticulum stress inducers increased the expression of Bmp6. SSRT revealed pathways regulating Bmp6 transcription positively and negatively. Hepa1-6 liver cells and C2C12 myogenic cells were prone to 2DP induced Bmp6 expression. CONCLUSIONS: The present study reveals non­iron-regulated Bmp6 expression in endothelial cells. GENERAL SIGNIFICANCE: Regulatory expression of Bmp6 may be important as a key step for fine tuning of BMP activity.

Data on induction of brown/beige adipocytes in mouse retro-orbital adipose tissues.

The data presented here are related to the research article entitled "Inducible brown/beige adipocytes in retro-orbital adipose tissues" [1]. Brown and beige adipocytes dissipate stored energy through the generation of heat by using mitochondrial uncoupling protein 1 (Ucp1), which is a mammalian brown/beige adipocyte-specific protein that promotes proton leakage across the inner mitochondrial membrane. Both cells up-regulate Ucp1 expression in response to ß-adrenergic receptor activation such as cold exposure. Here, we provide data on induction of Ucp1 positive cells in retro-orbital white adipose tissues (WAT) from cold exposed both male and female mice. The fluctuation of eye surface temperature was monitored during the cold exposure. In addition, distribution of tyrosine hydroxylase positive bundles was observed in the retro-orbital WAT from the mice.

Chronic retinoic acid treatment induces differentiation and changes in the metabolite levels of brown (pre)adipocytes.

Dietary vitamin A status affects energy metabolism. The present study explored the effect of all-trans retinoic acid (ATRA) on the expression levels of molecules and metabolites of brown adipocytes. Chronic ATRA treatment was initiated during the early stage (days 0-8) or late stage (days 8-12) of adipogenesis. Treatment with ATRA during the early and late stage of adipogenesis resulted in an increase in the expression level of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12, respectively, whereas expression of Pgc-1α, another gene expressed during brown adipogenesis, was unaffected by ATRA. Non-targeted metabolomic analyses indicated that the pathways related to the glucose metabolism were affected by ATRA, irrespective of the differentiation stage. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. ATRA decreased the cellular level of aconitic acid, fumaric acid, and malic acid on day 12 but not on day 8. Furthermore, ATRA increased the expression level of Hxk2 and downregulated the expressions of G6pdh and Pfkl/Pfkp on day 8 but not on day 12. Together, the results indicate that the chronic treatment with ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism. SIGNIFICANCE OF THE STUDY: Significance of the study treatment with all-trans retinoic acid (ATRA) during the early and late stage of adipogenesis increased the expression of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. The present results indicate that ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism.