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The β3-adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [³H]-2-deoxyglucose uptake, cellular glycolysis, and O2 consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β3-adrenoceptors as they were largely absent in adipocytes derived from β3-adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β3-adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β3-adrenoceptor. [ABSTRACT FROM AUTHOR]

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an antialcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300 g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75 mg/h) monotherapy or co-solvent only (50% hydroxypropyl-β-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No posttransplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Virtual-assisted lung mapping (VAL-MAP) is a preoperative bronchoscopic multispot dye-marking technique using virtual images. The purpose of this study was to evaluate the safety, efficacy and reproducibility of VAL-MAP among multiple centres. METHODS: Selection criteria included patients with pulmonary lesions anticipated to be difficult to identify at thoracoscopy and/or those undergoing sub-lobar lung resections requiring careful determination of resection margins. Data were collected prospectively and, if needed, compared between the centre that originally developed VAL-MAP and 16 other centres. RESULTS: Five hundred patients underwent VAL-MAP with 1781 markings (3.6 ± 1.2 marks/patient). Complications associated with VAL-MAP necessitating additional management occurred in four patients (0.8%) including pneumonia, fever and temporary exacerbation of pre-existing cerebral ischaemia. Minor complications included pneumothorax (3.6%), pneumomediastinum (1.2%) and alveolar haemorrhage (1.2%), with similar incidences between the original centre and other centres. Marks were identifiable during operation in approximately 90%, whereas the successful resection rate was approximately 99% in both groups, partly due to the mutually complementary marks. The contribution of VAL-MAP to surgical success was highly rated by surgeons resecting pure ground glass nodules (P < 0.0001), tumours <5mm (P = 0.0016), and performing complex segmentectomy and wedge resection (P = 0.0072). CONCLUSIONS: VAL-MAP was found to be safe and reproducible among multiple centres with variable settings. Patients with pure ground glass nodules, small tumours and resections beyond conventional anatomical boundaries are considered the best candidates for VAL-MAP. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Living-donor lobar lung transplantation (LDLLT) has been performed as a life-saving procedure for critically ill patients who are unlikely to survive the long wait for cadaveric lungs. The purpose of this study was to compare the preoperative condition and outcome of LDLLT patients with those of conventional cadaveric lung transplantation (CLT) patients. METHODS: A new lung transplant programme was established in 2008 at Kyoto University. Between June 2008 and January 2014, we performed 79 lung transplants, including 42 LDLLTs (10 single, 32 bilateral) and 37 CLTs (22 single, 15 bilateral). Data collected included preand perioperative variables and mid-term survival. All data were analysed retrospectively as of January 2014. RESULTS: The majority of patients were female (57.1%) in the LDLLT group and male (64.9%) in the CLT group. The average age was similar (36.6 ± 20.7 vs 39.7 ± 12.6 years, P = 0.42) between the two groups. Preoperatively, interstitial lung disease was more common in LDLLT patients than in CLT patients (47.6 vs 24.3%, P = 0.048); prior haematopoietic stem cell transplantation was performed more often in LDLLT patients than in CLT patients (33.3 vs 13.5%, P = 0.040) and there were more steroid-dependent LDLLT patients than CLT patients (64.3 vs 29.7%, P = 0.0022). Based on preoperative criteria of lower body mass index (17.2 ± 4.0 vs 19.3 ± 3.3 kg/m2, P = 0.013), less ambulatory ability (42.9 vs 86.5%, P = 0.0001) and more ventilator dependence (11.9 vs 2.7%, P = 0.12), LDLLT patients were more debilitated than CLT patients. LDLLT patients required longer postoperative mechanical ventilation than CLT patients (15.6 ± 16.2 vs 8.5 ± 8.1 days, P = 0.025). However, 1- and 3-year survival rates were similar between the two groups (89.7 and 86.1% vs 88.3 and 83.1%, P = 0.55). All living donors returned to their previous lifestyles without restriction. CONCLUSIONS: Although LDLLT patients were in a worse preoperative condition than CLT patients, LDLLT patients demonstrated survival rates similar to CLT patients. LDLLT is a viable option for patients too ill to survive a long waiting time for cadaveric donors. [ABSTRACT FROM AUTHOR]

OBJECTIVES: We developed virtual-assisted lung mapping (VAL-MAP), a bronchoscopic multispot dye-marking technique using three dimensional (3D) virtual imaging, for precise thoracoscopic sublobar lung resection with safe surgical margins. We herein review the results of 100 consecutive cases of VAL-MAP in our institute to identify types of tumours or resections that benefit from VAL-MAP. METHODS: Markings were bronchoscopically made within 2 days preoperatively using virtual 3D images. Post-VAL-MAP computer tomography (CT) scans localizing the actual markings were reconstructed into 3D images for intraoperative navigation. All data on patients, markings and outcomes were prospectively collected, and the contribution of VAL-MAP to the operation was graded by the surgeon. RESULTS: Resections of 156 lung lesions in 100 consecutive patients were planned from July 2012 to March 2014. The lesion diameter was 8.3 ± 4.9 (range, 2-24) mm. The total number of actually conducted markings was 380 (3.83 ± 1.07 markings/patient). Eighty-four lesions were resected by 71 wedge resections using 158 markings (2.1 ± 0.1/resection; range, 1-3). Seventy lesions were resected by 63 segmentectomies using 224 markings (3.6 ± 0.1/resection; range, 2-6). Markings were identifiable on post-VAL-MAP CT mostly as ground-glass opacities (87.7%) and/or bronchial dilatation (56.1%). During the operation, 357 of 380 markings (93.9%) were visible on the pleural surface and significantly associated with marking visibility on CT. Multiple markings that were complementary to one another appeared to have contributed to the high rate of successful resection (99.3%) with satisfactory resection margins. The contribution of VAL-MAP to the operation as graded by surgeons demonstrated that VAL-MAP is most effective during wedge resection or complex segmentectomy for hardly palpable, small tumours, while VAL-MAP still plays an important role in simple segmentectomy or resection of palpable tumours by providing higher confidence levels to surgeons during the operation.Minor pneumothoraces were found on post-VAL-MAP CT images in 4 patients without symptoms or a need for treatment. CONCLUSIONS: The present study further demonstrated the efficacy and safety of VAL-MAP. VAL-MAP is likely to benefit a broader range of patients than are conventional marking techniques by assisting with both accurate tumour identification and precise determination of resection lines. [ABSTRACT FROM AUTHOR]

There is an increasing worldwide epidemic of type 2 diabetes that poses major health problems. We have identified a novel physiological system that increases glucose uptake in skeletal muscle but not in white adipocytes. Activation of this system improves glucose tolerance in Goto-Kakizaki rats or mice fed a high-fat diet, which are established models for type 2 diabetes. The pathway involves activation of β2-adrenoceptors that increase cAMP levels and activate cAMP-dependent protein kinase, which phosphorylates mammalian target of rapamycin complex 2 (mTORC2) at S2481. The active mTORC2 causes translocation of GLUT4 to the plasma membrane and glucose uptake without the involvement of Akt or AS160. Stimulation of glucose uptake into skeletal muscle after activation of the sympathetic nervous system is likely to be of high physiological relevance because mTORC2 activation was observed at the cellular, tissue, and whole-animal level in rodent and human systems. This signaling pathway provides new opportunities for the treatment of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Brown adipose tissue is the primary site for thermo-genesis and can consume, in addition to free fatty acids, a very high amount of glucose from the blood, which can both acutely and chronically affect glucose homeostasis. Here, we show that mechanistic target of rapamycin (mTOR) complex 2 has a novel role in β3-adrenoceptor-stimulated glucose uptake in brown adipose tissue. We show that β3-adrenoceptors stimulate glucose uptake in brown adipose tissue via a signaling pathway that is comprised of two different parts: one part dependent on cAMP-mediated increases in GLUT1 transcription and de novo synthesis of GLUT1 and another part dependent on mTOR complex 2-stimulated translocation of newly synthesized GLUT1 to the plasma membrane, leading to increased glucose uptake. Both parts are essential for fo-adrenoceptor-stimulated glucose uptake. Importantly, the effect of β3-adrenoceptor on mTOR complex 2 is independent of the classical insulin-phosphoinositide 3-kinase-Akt pathway, highlighting a novel mechanism of mTOR complex 2 activation. [ABSTRACT FROM AUTHOR]

AbstractObliterative bronchiolitis (OB) is a form of chronic rejection after lung transplantation. Lentiviral vectors (LVs) facilitate long-term gene transduction in many tissues and organs. We hypothesized that lentiviral gene transfer of interleukin (IL)-10, a potent immune-modulating cytokine, to the lung could modulate the alloimmune responses in the lung after transplantation. C57BL6 mice received LVs encoding luciferase, enhanced green fluorescent protein (eGFP), or human IL-10 (huIL-10) through airways and underwent repeated bioluminescent imaging, immunofluorescence imaging, or ELISA of lung tissues, respectively. Luciferase activities peaked at day 7 and were stable after day 28 to over 15 months. eGFP staining demonstrated LV-mediated gene transduction mainly in alveolar macrophages. LV-huIL-10 delivery resulted in stable long-term expression of huIL-10 in the lung tissue (average 3.66 pg/mg at 1 year). Intrapulmonary allograft tracheal transplantation (BALBc→C57BL6) was used as a model of OB. LV-huIL-10 or LV-eGFP were delivered 7 days before transplantation and compared with no LV-transfection group. Allograft airways at day 28 were almost completely obliterated in all the groups. However, at day 42, allograft airways treated with LV-huIL-10 showed a spectrum of attenuation in airway fibrosis ranging from complete obliteration through bubble-like partial opening to complete patency with epithelial coverage in association with a significantly reduced obliteration ratio compared with the other groups (p<0.05). In conclusion, lentivirus-mediated gene transduction is useful in achieving long-term transgene expression in the lung. Long-term IL-10 expression has the potential to attenuate allograft airway obliteration. LV-mediated gene therapy could be a useful strategy to prevent or treat OB after lung transplantation. [ABSTRACT FROM AUTHOR]

Blood pressure is an important factor both in maintaining body homeostasis and in its disruption. Vascular endothelial cells (ECs) are exposed to varying degrees of blood pressure and therefore play an important role in these physiological and pathological events. However, the effect of blood pressure on EC functions remains to be elucidated. In particular, we do not know how ECs sense and respond to changes in hydrostatic pressure even though the hydrostatic pressure is known to affect the EC functions. Here, we hypothesized that the cellular responses, leading to the reported pressure effects, occur at an early stage of pressure exposure and observed the early-stage dynamics in ECs to elucidate mechanisms through which ECs sense and respond to hydrostatic pressure. We found that exposure to hydrostatic pressure causes an early actomyosin-mediated contraction of ECs without a change in cell morphology. This response could be caused by water efflux from the ECs following exposure to hydrostatic pressure. Although only a limited study, these findings do explain a part of the mechanism through which ECs sense and respond to hydrostatic pressure. [ABSTRACT FROM AUTHOR]

The article presents a response to L. Xiang et al's comments on M. Sato's study that theorized that a specific pathway dependent on mammalian target of rapamycin (mTOR) complex 2 can be used to regulate glucose uptake and blood glucose in diabetic patients. Topics discussed include the feasibility of developing compounds that boost glucose uptake into skeletal muscles without activating glucose from the liver and a newß2-adrenoceptor signaling pathway involving mTORC2.

Glycogen phosphorylase (GP) exists in two interconvertible forms, GPa (phosphorylated form, high activity) and GPb (nonphosphorylated form, low activity). Phosphorylase kinase (PhK) catalyses the phosphorylation of GPb and plays a key role in the cascade system for regulating glycogen metabolism. In this study, we developed a highly sensitive and nonradioactive assay for PhK activity by measuring the enhanced GP activity towards a pyridylaminated maltohexaose. The enhanced GP activity (ΔA) was calculated by the following formula: ΔA=A+ --A0, where A+ and A0 represent the GP activities of the PhK-treated and PhK-nontreated samples, respectively. Using a highperformance liquid chromatograph equipped with a fluorescence spectrophotometer, the product of GP activity could be isolated and quantified at 10 fmol. This method does not require the use of any radioactive compounds and only 1 mg of GPb per sample was needed to obtain A+ and A0 values. The remarkable reduction in GPb concentration enabled us to discuss an interesting new role for glycogen in PhK activity. [ABSTRACT FROM AUTHOR]

Impairment of episodic memory, a class of memory for spatiotemporal context of an event, is an early symptom of Alzheimer's disease. Both spatial and temporal information are encoded and represented in the hippocampal neurons, but how these representations are impaired under amyloid β (Aβ) pathology remains elusive. We performed chronic imaging of the hippocampus in awake male amyloid precursor protein (App) knock-in mice behaving in a virtual reality environment to simultaneously monitor spatiotemporal representations and the progression of Aβ depositions. We found that temporal representation is preserved, whereas spatial representation is significantly impaired in the App knock-in mice. This is because of the overall reduction of active place cells, but not time cells, and compensatory hyperactivation of remaining place cells near Aβ aggregates. These results indicate the differential impact of Aβ aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in the hippocampus. [ABSTRACT FROM AUTHOR]

Cells adapt to applied cyclic stretch (CS) to circumvent chronic activation of proinflammatory signaling. Currently, the molecular mechanism of the selective disassembly of actin stress fibers (SFs) in the stretch direction, which occurs at the early stage of the cellular response to CS, remains controversial. Here, we suggest that the mechanosensitive behavior of myosin II, a major crosslinker of SFs, primarily contributes to the directional disassembly of the actomyosin complex SFs in bovine vascular smooth muscle cells and human U2OS osteosarcoma cells. First, we identified that CS with a shortening phase that exceeds in speed the inherent contractile rate of individual SFs leads to the disassembly. To understand the biological basis, we investigated the effect of expressing myosin regulatory light-chain mutants and found that SFs with less actomyosin activities disassemble more promptly upon CS. We consequently created a minimal mathematical model that recapitulates the salient features of the direction-selective and threshold-triggered disassembly of SFs to show that disassembly or, more specifically, unbundling of the actomyosin bundle SFs is enhanced with sufficiently fast cell shortening. We further demonstrated that similar disassembly of SFs is inducible in the presence of an active LIM-kinase-1 mutant that deactivates cofilin, suggesting that cofilin is dispensable as opposed to a previously proposed mechanism. [ABSTRACT FROM AUTHOR]

Hippocampus receives dense serotonergic input specifically from raphe nuclei. However, what information is carried by this input and its impact on behavior has not been fully elucidated. Here we used in vivo two-photon imaging of activity of hippocampal median raphe projection fibers in behaving male and female mice and identified two distinct populations: one linked to reward delivery and the other to locomotion. Local optogenetic manipulation of these fibers confirmed a functional role for these projections in the modulation of reward-induced behavior. The diverse function of serotonergic inputs suggests a key role in integrating locomotion and reward information into the hippocampal CA1. [ABSTRACT FROM AUTHOR]

A standard treatment for patients with early-stage non-small cell lung cancer (NSCLC) who undergo surgery, and subsequently develop local failure or intrathoracic oligo-recurrence, has not yet been established. The present study aimed to assess the feasibility of stereotactic body radiotherapy (SBRT) for this subgroup of patients. Consequently, a retrospective analysis was conducted of patients with NSCLC recurrence who were treated with SBRT, and previously underwent curative surgical resection between October 2011 and October 2016. Post-SBRT survival [overall survival (OS); progression-free survival (PFS); and local control (LC)] and toxicity were analyzed. Prognostic factors for OS were identified using univariate and multivariate analysis. A total of 52 patients and 59 tumors were analyzed. The median follow-up time was 25 months (35 months for surviving patients), and median OS following salvage SBRT was 32 months. The 1- and 3-year OS rates were 84.4 and 67.8%, respectively. 1- and 3-year PFS rates were 80.8 and 58.7%, respectively. Only 4 patients (7.7%) developed local failure. Median LC was 71 months and 1- and 3-year LC rate were 97.9 and 94.9%, respectively. A total of 4 patients experienced grade 3 or higher adverse events (AEs) and two experienced grade 5 AEs (pneumonitis and hemoptysis). Central tumor location and the possibility of re-operation were independent prognostic factors for OS. The present study indicated that post-operative salvage SBRT is a promising therapeutic option for patients with NSCLC with locoregional or intrathoracic oligo-recurrence. We regard toxicity was also acceptable. However, further research is required on the appropriate selection of subjects, and stratification of the analysis by certain risk factors would increase the accuracy of the conclusions. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Postoperative myasthenic crisis (POMC) is one of the serious complications after extended thymectomy for patients with myasthenia gravis (MG). This study aims to clarify the risk factors of POMC occurrence. METHODS: The clinical data of 55 MG patients (25 male, 30 female; median age, 51 years) who underwent extended thymectomy at Kyoto University from 2000 to 2013 were retrospectively reviewed. Surgical outcomes and pre- and perioperative predictive factors of POMC were analysed. RESULTS: The preoperative Myasthenia Gravis Foundation of America stage was I, II, III and IV in 24, 22, 8 and 1 patients, respectively. Ten patients (18.2%) developed POMC; 6 required prolonged intubation over 24 h and 4 required reventilatory support. All patients were weaned after 5.6 (2-26) days of ventilator support, and were discharged. Univariate analysis revealed a correlation with a high preoperative anti-acetylcholine receptor antibody titre (P = 0.009), history of myasthenic crisis (MC) (P = 0.0004) and unstable MG after preoperative medical therapy (P = 0.003). Multivariate logistic regression analysis showed history of MC (odds ratio, 11.84; 95% confidential interval, 1.05- 372; P = 0.045) and unstable MG (odds ratio, 29.45; 95% confidential interval, 2.00-1063; P = 0.013) independently predicted POMC. The surgical response rate was not significantly different between the two groups (66.7% with POMC, 85.4% without POMC; P = 0.334). CONCLUSIONS: POMC occurred more frequently in unstable MG before surgery or in patients with a history of MC. Adequate preoperative medical therapy and perioperative care should be provided to these patients. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Living-donor lobar lung transplantation (LDLLT) has been established as a life-saving procedure for critically ill patients who cannot wait for cadaveric lung transplantation. Chronic lung allograft dysfunction (CLAD) is the main cause of late morbidity and mortality in lung transplantation. Studies on CLAD in cadaveric lung transplantation have been extensively reported, but few reports have been reported concerning CLAD after LDLLT. The aim of this study was to determine the prevalence, characteristics and prognosis of CLAD after LDLLT. METHODS: Among 38 patients who survived more than 3 months after LDLLT at Kyoto University Hospital between June 2008 and December 2013, 8 patients (21%) were diagnosed with CLAD. The mean follow-up period after LDLLT was 33 months. Clinical course, pulmonary function and radiological findings were reviewed retrospectively in all the 38 patients as of May 2014. RESULTS: Six patients were female and 2 were male. The median age at LDLLT was 31 years, and the median interval between LDLLT and the initial diagnosis of CLAD was 23 months. Among 8 patients who developed CLAD, 2 patients underwent right single LDLLT and 6 patients underwent bilateral LDLLT. The former 2 patients survived 44 and 47 months after the treatment. Five out of 6 patients with bilateral LDLLT developed unilateral CLAD at the time of initial diagnosis according to ventilation scintigraphy. In 3 of these 5 patients, the progression of CLAD was halted by treatment, and the median follow-up period of 33 months after treatment. In the remaining 2 of 5 patients, CLAD progressed to the contralateral lung metachronously; 1 patient survived without oxygen supplement, but the other patient required reperformance of LDLLT 3 years after the first one. One patient with bilateral CLAD at the time of detection died of disease progression 4 years after LDLLT. CONCLUSIONS: Despite a relatively short observation time, CLAD developed in approximately one-fifth of the patients who survived more than 3 months after LDLLT. In bilateral LDLLT, CLAD developed unilaterally in most cases, which might be beneficial in the long term because the unaffected contralateral lung may function as a reservoir. [ABSTRACT FROM AUTHOR]

Cellular response to physical stretch has been extensively studied as a regulator of various physiological functions. For live cell microscopy combined with stretch experiments, cells are typically seeded on an extensible elastomer sheet. In this case, the position of the cells of interest tends to shift out of the field of view upon stretch, making real-time imaging of identical cells difficult. To circumvent this situation, here we describe a robust methodology in which these cell shifts are minimized. Cells are plated in a custom-designed stretch chamber with an elastomer sheet of a small cell culture area. The cell-supporting chamber is stretched on an inverted microscope by using a piezoelectric actuator that provides small, but precisely controlled displacements. Even under this small displacement within the filed of view, our device allows the cells to undergo physiologically relevant levels of stretch. Identical cells can thus be continuously observed during stretching, thereby potentially enabling imaging of stretch-triggered fast dynamics. [ABSTRACT FROM AUTHOR]