Chronobioethics: Symphony of biological clocks observed by 7-day/24-hour ambulatory blood pressure monitoring and cardiovascular health.

BACKGROUND: The high prevalence of desynchronized biological rhythms is becoming a primary public health concern. We assess complex and diverse inter-modulations among multi-frequency rhythms present in blood pressure (BP) and heart rate (HR). SUBJECTS: and Methods: We performed 7-day/24-hour Ambulatory BP Monitoring in 220 (133 women) residents (23 to 74 years) of a rural Japanese town in Kochi Prefecture under everyday life conditions. RESULTS: A symphony of biological clocks contributes to the preservation of a synchronized circadian system. (1) Citizens with an average 12.02-h period had fewer vascular variability disorders than those with shorter (11.37-h) or longer (12.88-h) periods (P<0.05), suggesting that the circasemidian rhythm is potentially important for human health. (2) An appropriate BP-HR coupling promoted healthier circadian profiles than a phase-advanced BP: lower 7-day nighttime SBP (106.8 vs. 112.9 mmHg, P=0.0469), deeper nocturnal SBP dip (20.5% vs. 16.8%, P=0.0101), and less frequent incidence of masked non-dipping (0.53 vs. 0.86, P=0.0378), identifying the night as an important time window. CONCLUSION: Adaptation to irregular schedules in everyday life occurs unconsciously at night, probably initiated from the brain default mode network, in coordination with the biological clock system, including a reinforced about 12-hour clock, as "a biological clock-guided core integration system".

Model-free idealization: Adaptive integrated approach for idealization of ion-channel currents.

Single-channel electrophysiological recordings provide insights into transmembrane ion permeation and channel gating mechanisms. The first step in the analysis of the recorded currents involves an "idealization" process, in which noisy raw data are classified into two discrete levels corresponding to the open and closed states of channels. This provides valuable information on the gating kinetics of ion channels. However, the idealization step is often challenging in cases of currents with poor signal-to-noise ratios and baseline drifts, especially when the gating model of the target channel is not identified. We report herein on a highly robust model-free idealization method for achieving this goal. The algorithm, called adaptive integrated approach for idealization of ion-channel currents (AI2), is composed of Kalman filter and Gaussian mixture model clustering and functions without user input. AI2 automatically determines the noise reduction setting based on the degree of separation between the open and closed levels. We validated the method on pseudo-channel-current datasets that contain either computed or experimentally recorded noise. We also investigated the relationship between the noise reduction parameter of the Kalman filter and the cutoff frequency of the low-pass filter. The AI2 algorithm was then tested on actual experimental data for biological channels including gramicidin A, a voltage-gated sodium channel, and other unidentified channels. We compared the idealization results with those obtained by the conventional methods, including the 50%-threshold-crossing method.

New Aspects of Bilayer Lipid Membranes for the Analysis of Ion Channel Functions.

The bilayer lipid membrane (BLM) is the main structural component of cell membranes, in which various membrane proteins are embedded. Artificially formed BLMs have been used as a platform in studies of the functions of membrane proteins, including various ion channels. In this review, we summarize recent advances that have been made on artificial BLM systems for the analysis of ion channel functions. We focus on two BLM-based systems, cell-membrane mimicry and four-terminal BLM systems. As a cell-membrane-mimicking system, an efficient screening platform for the evaluation of drug side effects that act on a cell-free synthesized channel has been developed, and its prospects for use in personalized medicine will be discussed. In the four-terminal BLMs, we introduce "lateral voltage" to BLM systems as a novel input to regulate channel activities, in addition to the traditional transmembrane voltages. Such state-of-the-art technologies and new system setups are predicted to pave the way for a variety of applications, in both fundamental physiology and in drug discovery.

Lateral voltage as a new input for artificial lipid bilayer systems.

In this work, we propose lateral voltage as a new input for use in artificial lipid bilayer systems in addition to the commonly used transmembrane voltage. To apply a lateral voltage to bilayer lipid membranes, we fabricated electrode-equipped silicon and Teflon chips. The Si chips could be used for photodetector devices based on fullerene-doped lipid bilayers, and the Teflon chips were used in a study of the ion channel functions in the lipid bilayer. The findings indicate that the lateral voltage effectively regulates the transmembrane current, in both ion-channel-incorporated and fullerene-incorporated lipid bilayer systems, suggesting that the lateral voltage is a practicable and useful additional input for use in lipid bilayer systems.

Parallel Recordings of Transmembrane hERG Channel Currents Based on Solvent-Free Lipid Bilayer Microarray.

The reconstitution of ion-channel proteins in artificially formed bilayer lipid membranes (BLMs) forms a well-defined system for the functional analysis of ion channels and screening of the effects of drugs that act on these proteins. To improve the efficiency of the BLM reconstitution system, we report on a microarray of stable solvent-free BLMs formed in microfabricated silicon (Si) chips, where micro-apertures with well-defined nano- and micro-tapered edges were fabricated. Sixteen micro-wells were manufactured in a chamber made of Teflon®, and the Si chips were individually embedded in the respective wells as a recording site. Typically, 11 to 16 BLMs were simultaneously formed with an average BLM number of 13.1, which corresponded to a formation probability of 82%. Parallel recordings of ion-channel activities from multiple BLMs were successfully demonstrated using the human ether-a-go-go-related gene (hERG) potassium channel, of which the relation to arrhythmic side effects following drug treatment is well recognized.

Impact of Transferrin Saturation on All-Cause Mortality in Patients on Maintenance Hemodialysis.

BACKGROUND: Transferrin saturation (TSAT) is an index that represents the iron-binding capacity of transferrin, which is the main transport protein for iron, and is widely used to evaluate iron status. OBJECTIVE: To evaluate the prognostic importance of TSAT in Japanese patients on maintenance hemodialysis (MHD). METHODS: A total of 398 patients on MHD were recruited and divided into 3 groups on the basis of their baseline TSAT levels (<20, 20-40, and >40%). RESULTS: There was no difference in the proportion of patients on erythropoiesis-stimulating agents or iron supplements between the 3 groups. During a mean follow-up period of 52.2 ± 1 6.3 months, 130 patients died of cardiovascular causes (n = 63, 15.8%) or infection (n = 47, 11.8%). Compared with the reference group (TSAT 20-40%), patients with a TSAT <20% had a significantly higher all-cause mortality rate (6.44 vs. 9.55 events per 100 patient-years, p = 0.0452). Kaplan-Meier analysis showed that all-cause mortality rate was significantly higher in patients with TSAT <20% than in the other 2 groups (p = 0.0353). CONCLUSIONS: Low TSAT was a significant independent risk factor for all-cause mortality in a cohort of Japanese patients on MHD. The findings of this study suggest that the adverse clinical outcomes in patients with low TSAT can be partly attributed to infection-related iron deficiency.

A Prospective Cohort Study Showing No Association Between Serum Sclerostin Level and Mortality in Maintenance Hemodialysis Patients.

BACKGROUND/AIMS: Potential relationships between serum sclerostin levels and the levels of bone metabolic markers in maintenance hemodialysis (MHD) patients have yet to be evaluated. This study sought to determine whether serum sclerostin levels are associated with mortality in MHD patients. METHODS: We measured serum sclerostin levels in a Japanese MHD cohort, classified the patients into tertiles according to these levels, and followed their course for a 42-month period. RESULTS: The cohort consisted of 389 MHD patients and there were 75 deaths. Kaplan-Meier analyses showed that the tertile of serum sclerostin was not associated with mortality risk. Cox analyses showed that there were no significant associations between serum sclerostin level and mortality. CONCLUSION: Serum sclerostin level was not an independent predictor of mortality in MHD patients after adjustment for several confounders. However, whether clinical interventions to modulate serum sclerostin levels in MHD patients would improve their survival remains to be determined.

ß2-agonist clenbuterol suppresses bacterial phagocytosis of splenic macrophages expressing high levels of macrophage receptor with collagenous structure.

Splenic marginal zone macrophages expressing macrophage receptor with collagenous structure (MARCO) contribute to the clearance of blood-borne pathogens. We determined a splenic adherent cell fraction abundantly containing cells expressing a higher level of MARCO by flow cytometry, and examined the effects of daily administration of an anabolic dose of ß2-agonist clenbuterol on the phagocytic capacity of the cells in mice. After 6 weeks of clenbuterol (1.0 mg/kg body weight/d) or vehicle administration to the mice, splenic adherent cells were isolated. These cells were separated into three cell-size subpopulations. Among them, the small-cell subpopulation contained abundantly the cells with markedly higher levels of MARCO and exhibited more intense phagocytic capacity against Escherichia coli, as compared with the other subpopulations. The phagocytic capacity of the small cells was significantly reduced after clenbuterol administration. These results suggest that the utilization of clenbuterol as doping drug impairs bacterial clearance in the spleen.

Honeycomb-shaped surface topography induces differentiation of human mesenchymal stem cells (hMSCs): uniform porous polymer scaffolds prepared by the breath figure technique.

Polystyrene honeycomb scaffolds with different pore sizes were successfully fabricated by casting a polymer solution under humid conditions in order to investigate the effect of porous microtopography on hMSC differentiation. We have used honeycomb scaffolds to achieve the microtopography-induced differentiation of hMSCs. Honeycomb scaffolds led hMSCs to osteospecific and myospecific differentiations depending on the size of pores. This selective differentiation suggested that surface microtopography alone may be effective for using hMSCs in regenerative medicine and tissue engineering.

Upregulation of the Wnt/beta-catenin pathway induced by transforming growth factor-beta in hypertrophic scars and keloids.

Hypertrophic scars and keloids represent a dysregulated response to cutaneous wounds, which results in an excessive deposition of collagen. Transforming growth factor-beta (TGF-beta) is the key regulator in the pathogenesis of fibrosis. Accumulating evidence suggests that Wnt signalling and its effector beta-catenin also play an important role in wound healing. The role of Wnt/beta-catenin signalling in TGF-beta induced collagen deposition in hypertrophic scars and keloids was studied. Transcriptional assays and Western blotting was performed using fibroblast cell lines established from normal skin and hypertrophic scar tissue. Immunohistochemical studies were performed using scar tissues. We provide evidence that TGF-beta induces activation of beta-catenin mediated transcription in human dermal fibroblasts via the Smad3 and p38 MAPK pathways. Immunohistochemical studies demonstrated that beta-catenin protein levels are elevated in hypertrophic scar and keloid tissues. This finding may be relevant to the pathogenesis of hypertrophic scars and keloids.

Ichthyosiform mycosis fungoides: report of a case associated with IgA nephropathy.

We report a case of ichthyosiform mycosis fungoides (MF) associated with IgA nephropathy. Histological examination showed a dense atypical lymphocytic infiltrate admixed with epithelioid cells and giant cells in the dermis associated with the features of epidermotropism and folliculotropism. Reported cases of ichthyosiform MF are reviewed and histopathological characters of ichthyosiform MF are summarized. We suggest a histiocyte/dendritic-cell-rich infiltrate, or granulomatous features of infiltrate may be another characteristic of ichthyosiform MF. This case was associated with IgA nephropathy, which is uncommon. Such a presentation has never been reported in the literature.

Necrolytic migratory erythema without glucagonoma associated with hepatitis B.

We report a case of necrolytic migratory erythema (NME) without glucagonoma associated with hepatitis B. Although the most common cause of NME is a glucagon-secreting alpha-islet cell tumor of the pancreas, a dermatitis clinically and histologicaly identical to NME has been described in patients without glucagonoma. Impairment of hepatic dysfunction has been identified in the majority of them. However, NME associated with hepatitis B has never been reported in the literature. NME belongs to the family of necrolytic erythemas that share similar clinical and histologic findings. The terms used to describe NME and related conditions in the medical literature are confused. We added some discussion on the terminology of this disease.

Lysophosphatidic acid inhibits TGF-beta-mediated stimulation of type I collagen mRNA stability via an ERK-dependent pathway in dermal fibroblasts.

Lysophosphatidic acid (LPA) is a serum-derived pleiotropic mediator with a potential role in wound repair. Since extracellular matrix (ECM) deposition is a critical part of wound healing, this study was designed to examine whether LPA is involved in ECM regulation. Using human dermal fibroblasts, we demonstrate that LPA counteracts transforming growth factor-beta (TGF-beta) stimulation of type I collagen mRNA and protein. This factor elicits its inhibitory effects at the posttranscriptional level via destabilization of type I collagen mRNA. Furthermore, using the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, we show that the extracellular signal-regulated kinase (ERK) pathway is a negative regulator of the TGF-beta-induced stabilization of type I collagen mRNA, and that the activation of the ERK pathway by LPA mediates their inhibitory effects on collagen production. In conclusion, this study describes a novel function for LPA as an antagonist of TGF-beta induced ECM deposition. These findings may be relevant to physiologic wound repair and may be useful in designing therapeutic agents to prevent excessive scarring.

Factors affecting ST depression during cardiopulmonary exercise testing in patients with mitral stenosis without significant coronary lesions.

Symptom-limited cardiopulmonary exercise testing was performed in 37 patients with mitral stenosis (MS) without significant coronary artery stenosis to evaluate factors affecting ST depression in exercise electrocardiograms. The degree of ST depression was not associated with gender or exercise tolerance. The incidence of significant ST depression was higher in the patients receiving than in those not receiving digitalis (P < 0.05). In addition, the patients with atrial fibrillation and a higher heart rate response were more likely to have a high prevalence of significant ST depression than those with sinus rhythm and a lower response (P < 0.05). We concluded that atrial fibrillation, a higher maximum heart rate, and oral digitalis administration were involved in ST depression during exercise testing in patients with mitral stenosis without coronary heart disease.

Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.

Role of p38 MAPK in transforming growth factor beta stimulation of collagen production by scleroderma and healthy dermal fibroblasts.

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.