Mitochondrial ATP concentration decreases immediately after glucose administration to glucose-deprived hepatocytes.

Hepatocytes can switch their metabolic processes in response to nutrient availability. However, the dynamics of metabolites (such as lactate, pyruvate, and ATP) in hepatocytes during the metabolic switch remain unknown. In this study, we visualized metabolite dynamics in primary cultured hepatocytes during recovery from glucose-deprivation. We observed a decrease in the mitochondrial ATP concentration when glucose was administered to hepatocytes under glucose-deprivation conditions. In contrast, there was slight change in the cytoplasmic ATP concentration. A decrease in mitochondrial ATP concentration was associated with increased protein synthesis rather than glycogen synthesis, activation of urea cycle, and production of reactive oxygen species. These results suggest that mitochondrial ATP is important in switching metabolic processes in the hepatocytes.

Distinct lactate metabolism between hepatocytes and myotubes revealed by live cell imaging with genetically encoded indicators.

The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells.

The usefulness of postmortem computed tomography angiography for subdural hematoma caused by rupture of the cortical artery: A report of two autopsy cases and a literature review.

Acute subdural hematoma (SDH) occurs following severe head trauma with brain contusion or rupture of bridging veins. Conversely, SDH caused by rupture of a cortical artery without trauma or with minor trauma is also possible. Although over 180 cases of the latter SDH have been reported, they were predominantly diagnosed only during surgery, and therefore, no adequate histological evaluation has been performed. Therefore, essential etiology of this SDH type has remained unclear. In addition, the scarcity of autopsy cases may be attributed to arterial rupture being missed if the microscopic findings are too minimal to detect during autopsy. Here, we describe two autopsy cases of SDH of cortical artery origin. Extravasation on postmortem computed tomography angiography and arterial leakage on macroscopic observation during autopsy facilitated detection of the ruptured artery and allowed detailed histological evaluation of the ruptured artery and adjacent dura mater. The etiology of arterial rupture is briefly described on the basis of histopathological findings in this study and the available literature.

Results of repeat manipulation under ultrasound-guided cervical nerve root block with corticosteroid and local anaesthetic injection for recurrence of frozen shoulder.

BACKGROUND: This study aimed to evaluate the clinical results of a repeat manipulation under ultrasound-guided cervical nerve root block (MUC) with corticosteroid and local anaesthetic injection for recurrence of idiopathic frozen shoulder after MUC. METHODS: A consecutive series of 42 shoulders in 39 patients with idiopathic frozen shoulder underwent MUC. All patients were assessed according to the American Shoulder Elbow Surgeon (ASES) score and shoulder range of motion (ROM) both before MUC and at 1 year thereafter. If patients continued to have pain and limited ROM at 3 months after the procedure, they were offered a repeat MUC. Such patients were also assessed before the procedure and at 3 months and 1 year thereafter. RESULTS: The initial MUC was successful in 31 shoulders (single group). Repeat MUC was required in 11 shoulders (repeat group). Patients in the single group showed significant improvement in ROM and ASES score at 1 year after the procedure (p < 0.001); similarly, patients in the repeat group had significant improvement in ROM and ASES score at 3 months and 1 year after the procedure (p < 0.001). Patients in the repeat group had had significantly more severely limited ROM (p < 0.01) and decreased ASES score (p < 0.001) before the procedure compared with those in the single group. CONCLUSIONS: A repeat MUC with corticosteroid and local anaesthetic injection is a valuable option before proceeding to surgery for recurrence of idiopathic frozen shoulder. When there is severely limited ROM and decreased ASES score before the MUC, a repeat MUC may be necessary, which would require the patient's informed consent. TRIAL REGISTRATION: Retrospectively registered.

A Pilot Study on Efficacy of Lipid Bubbles for Theranostics in Dogs with Tumors.

The combined administration of microbubbles and ultrasound (US) is a promising strategy for theranostics, i.e., a combination of therapeutics and diagnostics. Lipid bubbles (LBs), which are experimental theranostic microbubbles, have demonstrated efficacy in vitro and in vivo for both contrast imaging and drug delivery in combination with US irradiation. To evaluate the clinical efficacy of LBs in combination with US in large animals, we performed a series of experiments, including clinical studies in dogs. First, contrast-enhanced ultrasonography using LBs (LB-CEUS) was performed on the livers of six healthy Beagles. The hepatic portal vein and liver tissue were enhanced; no adverse reactions were observed. Second, LB-CEUS was applied clinically to 21 dogs with focal liver lesions. The sensitivity and specificity were 100.0% and 83.3%, respectively. These results suggested that LB-CEUS could be used safely for diagnosis, with high accuracy. Finally, LBs were administered in combination with therapeutic US to three dogs with an anatomically unresectable solid tumor in the perianal and cervical region to determine the enhancement of the chemotherapeutic effect of liposomal doxorubicin; a notable reduction in tumor volume was observed. These findings indicate that LBs have potential for both therapeutic and diagnostic applications in dogs in combination with US irradiation.

Glutamine-induced signaling pathways via amino acid receptors in enteroendocrine L cell lines.

Glucagon-like peptide-1 (GLP-1), secreted by gastrointestinal enteroendocrine L cells, induces insulin secretion and is important for glucose homeostasis. GLP-1 secretion is induced by various luminal nutrients, including amino acids. Intracellular Ca2+ and cAMP dynamics play an important role in GLP-1 secretion regulation; however, several aspects of the underlying mechanism of amino acid-induced GLP-1 secretion are not well characterized. We investigated the mechanisms underlying the L-glutamine-induced increase in Ca2+ and cAMP intracellular concentrations ([Ca2+]i and [cAMP]i, respectively) in murine enteroendocrine L cell line GLUTag cells. Application of L-glutamine to cells under low extracellular [Na+] conditions, which inhibited the function of the sodium-coupled L-glutamine transporter, did not induce an increase in [Ca2+]i. Application of G protein-coupled receptor family C group 6 member A and calcium-sensing receptor antagonist showed little effect on [Ca2+]i and [cAMP]i; however, taste receptor type 1 member 3 (TAS1R3) antagonist suppressed the increase in [cAMP]i. To elucidate the function of TAS1R3, which forms a heterodimeric umami receptor with taste receptor type 1 member 1 (TAS1R1), we generated TAS1R1 and TAS1R3 mutant GLUTag cells using the CRISPR/Cas9 system. TAS1R1 mutant GLUTag cells exhibited L-glutamine-induced increase in [cAMP]i, whereas some TAS1R3 mutant GLUTag cells did not exhibit L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. These findings suggest that TAS1R3 is important for L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. Thus, TAS1R3 may be coupled with Gs and related to cAMP regulation.

Nivolumab-induced fulminant type 1 diabetes with precipitous fall in C-peptide level.

We describe here a case of nivolumab-induced type 1 diabetes, which developed within 9 days of treatment. The case highlights the importance of frequent monitoring of glucose after initiation of nivolumab treatment.

Green Fluorescent Protein-Based Glucose Indicators Report Glucose Dynamics in Living Cells.

Glucose is the most important energy source for living animals. Here, we developed a series of single fluorescent protein (FP)-based glucose indicators, named as "Green Glifons", to understand the hierarchal and mutual relationships between molecules involved in energy metabolism. Three indicators showed a different EC50 for glucose (50, 600, and 4000 µM), producing a ∼7-fold change in fluorescence intensity in response to glucose. The indicators could visualize glucose dynamics in the cytoplasm, plasma membrane, nucleus and mitochondria of living HeLa cells and in vivo, in the pharyngeal muscle of C. elegans and could measure murine blood glucose levels. Finally, the indicators were applicable to dual-color imaging, revealing the dynamic interplay between glucose and Ca2+ in mouse pancreatic MIN6 m9 ß cells. We propose that these indicators will facilitate and contribute to in vivo and multicolor imaging of energy metabolism.

RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells.

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

Bacterial metabolite S-equol modulates glucagon-like peptide-1 secretion from enteroendocrine L cell line GLUTag cells via actin polymerization.

S-equol is one of gut bacterial metabolites produced from soybean isoflavone daizein. While S-equol is known to promote glucose-induced insulin secretion from pancreatic ß cells, whether S-equol affects glucagon-like peptide-1 (GLP-1) secretion from enteroendoceine L cells remains unclear. Here we assessed the effect of S-equol on GLP-1 secretion from mouse enteroendocrine L cell line GLUTag cells. GLUTag cells expressed GPR30 and estrogen receptors, which are putative S-equol receptors. Application of S-equol induced an increase in intracellular Ca2+ levels via GPR30. However, S-equol did not enhance GLP-1 exocytosis, and long-term treatment of S-equol suppressed GLP-1 secretion. Moreover, immunocytochemistry revealed that S-equol increased the density of cortical actin filaments via G12/13 signaling under GPR30. These data suggest that S-equol prevents GLP-1 secretion as a result of competing regulation between Ca2+ mobilization and actin reorganization.

Bitter tastant quinine modulates glucagon-like peptide-1 exocytosis from clonal GLUTag enteroendocrine L cells via actin reorganization.

Enteroendocrine L cells in the gastrointestinal tract secrete glucagon-like peptide-1 (GLP-1), which plays an important role in glucose homeostasis. Here we investigated the effect of bitter tastant quinine on GLP-1 secretion using clonal GLUTag mouse enteroendocrine L cells. We found that GLUTag cells expressed putative quinine receptors at mRNA levels. Although application of quinine resulted in an increase of intracellular Ca2+ levels, which was mediated by Ca2+ release from the endoplasmic reticulum and Ca2+ influx through voltage-sensitive Ca2+ channels, quinine had little effect on GLP-1 secretion. Total internal reflection fluorescence microscopy and immunocytochemistry revealed that GLP-1-containing vesicles remained unfused with the plasma membrane and facilitated actin polymerization beneath the plasma membrane after application of quinine, respectively. Interestingly, application of forskolin together with quinine induced GLP-1 exocytosis from the cells. These results suggest that quinine does not induce GLP-1 secretion because it facilitates Ca2+ increase and actin reorganization but not cAMP increase, and both Ca2+ and cAMP are essential for GLP-1 secretion.

Allergic bronchopulmonary aspergillosis in Japan: A nationwide survey.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is an allergic pulmonary disease characterized by a hypersensitivity reaction to Aspergillus species colonizing the airways. The clinical characteristics of ABPA may differ depending on genetic and environmental background. We performed a nationwide survey to determine the clinical characteristics of ABPA in Japan. METHODS: In 2013, a questionnaire on physician-diagnosed ABPA/allergic bronchopulmonary mycosis was sent to 903 medical centers specializing in respiratory or allergic diseases. Cases fulfilling the following criteria were categorized as possible ABPA-central bronchiectasis (ABPA-CB): 1) presence of specific serum immunoglobulin E (IgE) antibodies or a positive skin reaction to Aspergillus, and 2) bronchiectasis or mucoid impaction in the central bronchi. RESULTS: Of 499 physician-diagnosed cases reported by 132 clinical centers, 358 cases met the criteria for possible ABPA-CB. Median age of ABPA-CB onset was 57 (interquartile range, 44-68) years; later-onset disease, developing ≥50 years of age, accounted for 66% of the cases and was associated with female sex, delayed onset of asthma, and lower levels of serum IgE. A third of the patients (120 patients, 34%) exhibited low levels of serum total IgE (<1000 IU/mL). Aspergillus species were isolated from sputum in 126/213 cases (59%), and Schizophyllum commune was identified in 12 (6%) patients. During the course of the treatment, ABPA recurred in 169 (48%) cases. CONCLUSIONS: This nationwide survey identified several unique clinical characteristics of ABPA in Japan, such as late-onset, relatively lower serum IgE levels, and frequent recurrences/flares.

Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging.

cAMP is a common second messenger that is involved in various physiological processes. To expand the colour palette of available cAMP indicators, we developed a red cAMP indicator named "Pink Flamindo" (Pink Fluorescent cAMP indicator). The fluorescence intensity of Pink Flamindo increases 4.2-fold in the presence of a saturating dose of cAMP, with excitation and emission peaks at 567 nm and 590 nm, respectively. Live-cell imaging revealed that Pink Flamindo is effective for monitoring the spatio-temporal dynamics of intracellular cAMP generated by photoactivated adenylyl cyclase in response to blue light, and in dual-colour imaging studies using a green Ca2+ indicator (G-GECO). Furthermore, we successfully monitored the elevation of cAMP levels in vivo in cerebral cortical astrocytes by two-photon imaging. We propose that Pink Flamindo will facilitate future in vivo, optogenetic studies of cell signalling and cAMP dynamics.

Post-bronchoscopy pneumonia in patients suffering from lung cancer: Development and validation of a risk prediction score.

BACKGROUND: The incidence, risk factors, and consequences of pneumonia after flexible bronchoscopy in patients with lung cancer have not been studied in detail. METHODS: We retrospectively analyzed the data from 237 patients with lung cancer who underwent diagnostic bronchoscopy between April 2012 and July 2013 (derivation sample) and 241 patients diagnosed between August 2013 and July 2014 (validation sample) in a tertiary referral hospital in Japan. A score predictive of post-bronchoscopy pneumonia was developed in the derivation sample and tested in the validation sample. RESULTS: Pneumonia developed after bronchoscopy in 6.3% and 4.1% of patients in the derivation and validation samples, respectively. Patients who developed post-bronchoscopy pneumonia needed to change or cancel their planned cancer therapy more frequently than those without pneumonia (56% vs. 6%, p<0.001). Age ≥70 years, current smoking, and central location of the tumor were independent predictors of pneumonia, which we added to develop our predictive score. The incidence of pneumonia associated with scores=0, 1, and ≥2 was 0, 3.7, and 13.4% respectively in the derivation sample (p=0.003), and 0, 2.9, and 9.7% respectively in the validation sample (p=0.016). CONCLUSIONS: The incidence of post-bronchoscopy pneumonia in patients with lung cancer was not rare and associated with adverse effects on the clinical course. A simple 3-point predictive score identified patients with lung cancer at high risk of post-bronchoscopy pneumonia prior to the procedure.

Analysis of IMP-1 type metallo-ß-lactamase-producing Acinetobacter radioresistens isolated from companion animals.

IMP-1 type metallo-ß-lactamase-producing (MBL-producing) Acinetobacter radioresistens was isolated from a dog with cystitis and a cat with conjunctivitis. The MBL-producing A. radioresistens isolates were resistant to all of the ß-lactam antibiotics used in the sensitivity tests, but were susceptible to gentamicin, amikacin, and minocycline. Also, one of the two strains of A. radioresistens was susceptible to ciprofloxacin and levofloxacin. These two cases were cured by administration of tetracyclines and fluoroquinolones, which elicited a positive result in the sensitivity tests. This report of the isolation of MBL-producing A. radioresistens in companion animals is the first in the world. To prevent the proliferation of MBL-producing bacteria, veterinary hospitals need to be aware of the behavior of MBL-producing organisms.

Progressive Multifocal Micronodular Pneumocyte Hyperplasia in the Lungs of a Patient with Tuberous Sclerosis Complex: A Case Report.

We report a case of multifocal micronodular pneumocyte hyperplasia (MMPH) in a patient with tuberous sclerosis complex, in whom the lung nodules increased in the number and size over the course of 8 years. We diagnosed MMPH following a lung biopsy performed during video-assisted thoracic surgery. In most of the previously reported cases, the number and size of lung nodules is unchanged during the clinical course. Our case is the first report of progressive disease in pathologically proven MMPH.

Bongkrekic acid analogue, lacking one of the carboxylic groups of its parent compound, shows moderate but pH-insensitive inhibitory effects on the mitochondrial ADP/ATP carrier.

Bongkrekic acid, isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In this study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ATP carrier. Rough screening of these chemicals, performed by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, KH-7, KH-16, and KH-17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of bongkrekic acid for its pH-dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ATP carrier was also clearly demonstrated.

Integrative function of adrenaline receptors for glucagon-like peptide-1 exocytosis in enteroendocrine L cell line GLUTag.

Adrenaline reacts with three types of adrenergic receptors, α1, α2 and ß-adrenergic receptors (ARs), inducing many physiological events including exocytosis. Although adrenaline has been shown to induce glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells, the precise molecular mechanism by which adrenaline regulates GLP-1 secretion remains unknown. Here we show by live cell imaging that all types of adrenergic receptors are stimulated by adrenaline in enteroendocrine L cell line GLUTag cells and are involved in GLP-1 exocytosis. We performed RT-PCR analysis and found that α1B-, α2A-, α2B-, and ß1-ARs were expressed in GLUTag cells. Application of adrenaline induced a significant increase of intracellular Ca(2+) and cAMP concentration ([Ca(2+)]i and [cAMP]i, respectively), and GLP-1 exocytosis in GLUTag cells. Blockade of α1-AR inhibited adrenaline-induced [Ca(2+)]i increase and exocytosis but not [cAMP]i increase, while blockade of ß1-AR inhibited adrenaline-induced [cAMP]i increase and exocytosis but not [Ca(2+)]i increase. Furthermore, overexpression of α2A-AR suppressed the adrenaline-induced [cAMP]i increase and exocytosis. These results suggest that the fine-turning of GLP-1 secretion from enteroendocrine L cells is established by the balance between α1-, α2-, and ß-ARs activation.

Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods.

Recent efforts have focused on the use of sericin proteins extracted from cocoons of silkworm as a healthy food source for human consumption. In this study, we focused on the antioxidative properties of sericin proteins. The antioxidative properties were measured in sericin proteins extracted from the shell of the cocoon, designated hereafter as white sericin protein and yellow-green sericin protein, as well as bread without sericin protein and bread to which white sericin powder had been added using four measurement methods: 1,1-Diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence, oxygen radical absorbance capacity (ORAC) and electron spin resonance (ESR). High antioxidative properties of sericin proteins were indicated by all four methods. A comparison of the two types of sericin proteins revealed that yellow-green sericin protein exhibited high antioxidative properties as indicated by the DPPH, chemiluminescence and ORAC methods. By contrast, a higher antioxidative property was determined in white sericin protein by the ESR method. Consequently, our findings confirmed that sericin proteins have antioxidative properties against multiple radicals. In addition, the antioxidative property of bread was enhanced by the addition of sericin powder to the bread. Therefore, findings of this study suggest that sericin proteins may be efficiently used as beneficial food for human health.

Biochemical analyses of the antioxidative activity and chemical ingredients in eight different Allium alien monosomic addition lines.

We measured the antioxidant contents and antioxidative activities in eight Allium fistulosum-shallot monosomic addition lines (MAL; FF+1A-FF+8A). The high antioxidative activity lines (FF+2A and FF+6A) showed high polyphenol accumulation. These additional chromosomes (2A and 6A) would therefore have anonymous genes related to the upregulation of polyphenol production, the antioxidative activities consequently being increased in these MALs.