Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases.

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 domain rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here, we report the atomic models of 18 catalytic intermediates of the V1 domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

Gefitinib induction followed by chemoradiotherapy in EGFR-mutant, locally advanced non-small-cell lung cancer: LOGIK0902/OLCSG0905 phase II study.

BACKGROUND: The role of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) induction coupled with standard concurrent chemoradiotherapy (CRT) is unclear in unresectable, stage III, EGFR-mutant non-small-cell lung cancer (NSCLC). Therefore, a phase II trial was conducted to evaluate the efficacy and safety of gefitinib induction followed by CRT in this disease setting. PATIENTS AND METHODS: Patients with unresectable, EGFR-mutant, stage III NSCLC were administered gefitinib monotherapy (250 mg/day) for 8 weeks. Subsequently, patients without disease progression during induction therapy were administered cisplatin and docetaxel (40 mg/m2 each) on days 1, 8, 29, and 36 with concurrent radiotherapy at a total dose of 60 Gy. The primary endpoint was the 2-year overall survival (OS) rate, which was hypothesized to reach 85%, with a threshold of the lower limit of 60%. RESULTS: Twenty patients (median age: 66 years; male/female: 9/11; histology: 20 adenocarcinoma; stage IIIA/IIIB: 9/11; and exon 19/21: 10/10) were enrolled. The 2-year OS rate was 90% (90% confidence interval: 71.4% to 96.8%), indicating that this trial met the primary objective. The overall response rate and 1- and 2-year progression-free survival rates were 85.0%, 58.1%, and 36.9%, respectively. Grade ≥3 adverse events (>10%) included hepatic toxicity during the induction phase and neutropenia and febrile neutropenia in the CRT phase. Radiation pneumonitis grade ≥3 or treatment-related death did not occur. CONCLUSIONS: This is the first prospective study to demonstrate the favorable efficacy and safety of EGFR-TKI induction followed by standard CRT in EGFR-mutant, stage III NSCLC. Further confirmatory studies are needed.

Visualization of viscoelastic behavior in skin equivalent using optical coherence tomography-based straingraphy.

BACKGROUND/PURPOSE: The relationships between the skin components and these mechanical roles are still unclear. To clarify these relationships, we investigated spatial mapping of the mechanical behavior of cultured skin equivalents (SEs) using optical coherence tomography (OCT)-based straingraphy. METHODS: We built a strain relaxation test system combined with OCT and developed an algorithm that could visualize a time-dependent strain distribution, named dynamic-optical coherence straingraphy (D-OCSA). Using this system, we analyzed how the spatial mechanical changes in the SEs depended on the culture duration. For quantitative analysis of viscoelastic behavior, we defined a relaxation attenuation coefficient of strain rate, which indicates the ratio of viscosity and elasticity in the Klevin-Voight model. RESULTS: By culturing for 4 days in comparison to culturing for 1 day, the strain relaxation attenuation coefficient of the whole skin, especially at the region of the dermal-epidermal junction (DEJ), significantly increased in the negative direction. In tissue slices taken for microscopy, several cracks were observed in the SEs cultured for 4 days. CONCLUSION: This study is the first to provide quantified evidence that the DEJ is a dynamically specialized region. An OCT-based straingraphy system (D-OCSA) would be beneficial for evaluating the quality of SEs, as well as functional analysis of their mechanics.

A phase I/II trial of irinotecan plus amrubicin supported with G-CSF for extended small-cell lung cancer.

OBJECTIVE: This study reports the findings of a Phase I/II, cohort, dose-escalation trial of amrubicin and irinotecan with the support of granulocyte colony-stimulation factor. This study aimed to determine the dose-limiting toxicity of the combination and to define the maximum-tolerated dose, as a recommended dose for Phase II trials. We also sought to obtain preliminary data on the efficacy of this combination as a frontline therapy for extensive-disease small-cell lung cancer. METHODS: We included 23 chemo-naïve patients with extensive-disease small-cell lung cancer in the trial. The amrubicin dose was escalated from 35 to 40 mg/m(2) (Levels 1 and 2, respectively) to determine the dose-limiting toxicity, with an unchanged dose of irinotecan at 50 mg/m(2). RESULTS: Of nine patients, three experienced dose-limiting toxicities at Level 1 of prolonged Grade 4 neutropenia, Grade 3 febrile neutropenia and Grade 3 febrile neutropenia with Grade 3 diarrhea. At Level 2, two patients experienced dose-limiting toxicities of Grade 4 neutropenia and Grade 3 neutropenia with Grade 4 diarrhea. The maximum-tolerated doses and recommended doses for amrubicin and irinotecan were therefore determined to be 35 and 50 mg/m(2), respectively. The Level 1 trial was then expanded to 21 patients, 14 (70%) of whom showed partial responses to the recommended dose. The median progression-free and overall survival times were 6.37 and 15.21 months, respectively. CONCLUSIONS: The combination of amrubicin and irinotecan with the support of granulocyte colony-stimulation factor produced a potent effect in chemo-naïve extensive-disease small-cell lung cancer patients. The use of biomarkers for this regimen may identify patients who are likely to suffer from treatment-ending severe adverse effects.

Infusion requirements and reversibility of rocuronium at the corrugator supercilii and adductor pollicis muscles.

BACKGROUND: The aim of this study is to compare the infusion rates required to maintain a constant neuromuscular block and the reversibility of rocuronium at the corrugator supercilii muscle (CSM) and the adductor pollicis muscle (APM). METHODS: We randomly allocated 30 female patients into two groups of 15 patients each to monitor neuromuscular block at either the CSM or the APM. After induction of anaesthesia and laryngeal mask insertion, contraction of the CSM to the facial nerve stimulation or that of the APM to the ulnar nerve stimulation was quantified using an acceleromyograph during 1.0-1.5% end-tidal sevoflurane anaesthesia. All the patients received a bolus of 1 mg/kg rocuronium. When the first twitch (T1) of train-of-four (TOF) recovered to 10% of the control, rocuronium infusion was commenced and maintained at T1 of 10% of the control at the CSM or APM for 120 min. Immediately after rocuronium infusion was discontinued, the time required for 0.04 mg/kg neostigmine-facilitated recovery to a TOF ratio of 0.9 was recorded. RESULTS: Rocuronium infusion dose after a lapse of 120 min was significantly larger in the CSM than in the APM [7.1 (2.3) vs. 4.7 (2.6) microg/kg/min; P=0.001]. The time for facilitated recovery was shorter in the CSM than in the APM [11.4 (3.8) vs. 16.2 (6.0) min; P=0.016]. CONCLUSION: A larger rocuronium infusion dose was required to maintain a constant neuromuscular block at the CSM. Neostigmine-mediated reversal was faster at the CSM.

Presenilin enhancer-2 (PSENEN), a component of the gamma-secretase complex, is involved in adipocyte differentiation.

This study was conducted to identify genes expressed during adipocyte differentiation of bovine intramuscular fibroblast-like cells using differential display reverse-transcriptase polymerase chain reaction. The presenilin enhancer-2 (PSENEN) gene was found to be down-regulated during adipocyte differentiation of bovine intramuscular fibroblast-like cells. The ectopic expression of bovine PSENEN in 3T3-L1 reduced adipogenesis and the inhibition of endogenous PSENEN by siRNA induced adipogenesis on d 4 of adipocyte differentiation of 3T3-L1 cells. Interestingly, the expression of gamma-secretase complex gene-related Notch signaling was decreased at d 2 and d 4 during adipocyte differentiation. In addition, expression of the Notch-signaling genes (Notch-1, Hes-1, Pref-1, adipsin) was regulated during adipocyte differentiation by regulation of PSENEN expression. These results suggest that PSENEN plays an important role in adipocyte differentiation and that Notch signaling is involved in adipogenesis.

Increased insulin demand promotes while pioglitazone prevents pancreatic beta cell apoptosis in Wfs1 knockout mice.

AIMS/HYPOTHESIS: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein called Wolfram syndrome 1 protein, homozygous mutations of which cause selective beta cell loss in humans. The function(s) of this protein and the mechanism by which the mutations of this gene cause beta cell death are still not fully understood. We hypothesised that increased insulin demand as a result of obesity/insulin resistance causes ER stress in pancreatic beta cells, thereby promoting beta cell death. METHODS: We studied the effect of breeding Wfs1 ( -/- ) mice on a C57BL/6J background with mild obesity and insulin resistance, by introducing the agouti lethal yellow mutation (A ( y ) /a). We also treated the mice with pioglitazone. RESULTS: Wfs1 ( -/- ) mice bred on a C57BL/6J background rarely develop overt diabetes by 24 weeks of age, showing only mild beta cell loss. However, Wfs1 ( -/- ) A ( y ) /a mice developed selective beta cell loss and severe insulin-deficient diabetes as early as 8 weeks. This beta cell loss was due to apoptosis. In Wfs1 ( +/+ ) A ( y ) /a islets, levels of ER chaperone immunoglobulin-binding protein (BiP)/78 kDa glucose-regulated protein (GRP78) and phosphorylation of eukaryotic translation initiation factor 2, subunit alpha (eIF2alpha) apparently increased. Levels of both were further increased in Wfs1 ( -/- ) A ( y ) /a murine islets. Electron micrography revealed markedly dilated ERs in Wfs1 (-/-) A ( y ) /a murine beta cells. Interestingly, pioglitazone treatment protected beta cells from apoptosis and almost completely prevented diabetes development. CONCLUSIONS/INTERPRETATION: Wfs1-deficient beta cells are susceptible to ER stress. Increased insulin demand prompts apoptosis in such cells in vivo. Pioglitazone, remarkably, suppresses this process and prevents diabetes. As common WFS1 gene variants have recently been shown to confer a risk of type 2 diabetes, our findings may be relevant to the gradual but progressive loss of beta cells in type 2 diabetes.

Minimally invasive video-assisted thoracoscopic lobectomy for better clinical outcomes in peripheral T1NO lung cancer.

It is an unresolved issue whether various thoracotomies affect clinical outcomes. In addition, a wide variety of technical approaches of video-assisted thoracic surgery depend on the facility. We reviewed 152 consecutive patients with clinical T1N0M0 lung cancer that underwent three types of lobectomy with systematic mediastinal lymphadenectomy in a single institute: 46 conventional thoracotomies (OPEN), 50 anterolateral small thoracotomies mainly using the thoracoscope as a light guide (ASSIST), and 56 minimum thoracotomies in which only a thoracoscope view was used (PURE). Total discharge from the chest drainage tube, length of hospital stay, and post-thoracotomy pain were significantly less in PURE than in OPEN and ASSIST. The results of mediastinal lymphadenectomy were equivalent. The 3-year survival rates were also similar among the three groups. We conclude that good clinical outcomes, especially reduced post-thoracotomy pain, seemed to correlate with the lesser degree of destruction of the chest wall with the identical quality as an acceptable cancer operation in PURE.

Epidurally administered mepivacaine delays recovery of train-of-four ratio from vecuronium-induced neuromuscular block.

BACKGROUND: The aim of this study was to examine the efficacy of epidurally administered mepivacaine on recovery from vecuronium-induced neuromuscular block. METHODS: Eighty patients were randomly assigned to one of two study groups. They were either given epidurally a bolus of 0.15 ml kg(-1) of mepivacaine 2%, followed by repetitive injections of 0.1 ml kg(-1) h(-1) throughout the study, or were not given epidurally. General anaesthesia was induced and maintained with fentanyl, propofol and nitrous oxide. Neuromuscular block was induced with vecuronium 0.1 mg kg(-1) and monitored using acceleromyographic train-of-four (TOF) at the adductor pollicis. Patients in each treatment group were randomized to receive neostigmine 0.04 mg kg(-1) at 25% recovery of the first twitch of TOF or to recover spontaneously to a TOF ratio of 0.9. The effect of epidural mepivacaine on speed of spontaneous and facilitated recovery of neuromuscular function was evaluated. RESULTS: The time from administration of vecuronium to spontaneous recovery to a TOF ratio of 0.9 was significantly longer in the epidural mepivacaine group [105.4 (14.2) min] as compared with the control group [78.5 (9.1) min, P < 0.01]. Neostigmine administered at 25% of control in T1 shortened recovery from neuromuscular block, however the time required for facilitated recovery to a TOF ratio of 0.9 in the epidural group was significantly longer than that in the control group [7.6 (1.6) min vs 5.8 (2.1) min, P < 0.01]. CONCLUSIONS: In clinical anaesthesia, it should be recognized that epidurally administered mepivacaine delays considerably the TOF recovery from neuromuscular block.

Clinical application of carbon fibre reinforced plastic leg orthosis for polio survivors and its advantages and disadvantages.

A prospective study was carried out on the clinical application and features of a carbon fibre reinforced plastic leg orthosis (carbon orthosis) for polio survivors. The subjects comprised 9 polio survivors, and 11 carbon knee-ankle-foot orthoses (KAFOs) were prescribed, fabricated, and checked out at the authors' post-polio clinic. Walking was classified based on the functional ambulatory category, and the features of walking with a carbon orthosis were self-evaluated by using a visual analogue scale. The period from modelling a cast to completion was 55 +/- 25 days; the weight of a carbon KAFO was 27.8% lighter than that of the ordinary KAFO; the standard carbon KAFO was 50% more expensive than the ordinary KAFO. The carbon KAFO remained undamaged for at least 2 years. It improved the scores in the functional ambulation categories, but there was no difference between walking with an ordinary and with a carbon KAFO. The self-evaluation of walking with a carbon KAFO revealed that the subjects using a carbon KAFO were satisfied with their carbon KAFO. The carbon KAFO is lightweight, durable, slim and smart, and is positively indicated for polio survivors.

Normalization of acceleromyographic train-of-four ratio by baseline value for detecting residual neuromuscular block.

BACKGROUND: This study was designed to recognize the importance of normalizing postoperative acceleromyographic train-of-four (TOF) ratio by the baseline TOF value obtained before neuromuscular block for ensuring adequate recovery of neuromuscular function. METHODS: In 120 patients, TOF responses of the adductor pollicis to the ulnar nerve stimulation were monitored by acceleromyography (AMG) during anaesthesia using propofol, fentanyl and nitrous oxide. Control TOF stimuli were administered for 30 min. A TOF ratio measured at the end of control stimulation was regarded as a baseline value. Neuromuscular block was induced with vecuronium 0.1 mg kg(-1) and was allowed to recover spontaneously. Duration to a TOF ratio of 0.9 as calculated by AMG (DUR-raw 0.9) was compared with that of 0.9 as corrected by the baseline TOF ratio (i.e. 0.9 x baseline TOF ratio; DUR-real 0.9). RESULTS: Baseline TOF ratios ranged from 0.95 to 1.47. The average TOF ratios observed every 5 min were constant throughout control stimulation from at time zero mean (SD) [range]; 1.11 (0.09) [0.94-1.42] to at 30 min 1.13 (0.11) [0.95-1.47]. The DUR-real 0.9 was 91.0 (18.0) [51.3-131.0] min and was significantly longer than the DUR-raw 0.9 (81.2 (16.3) [41.3-123.0] min). CONCLUSIONS: Baseline TOF ratios measured by AMG are usually more than 1.0 and vary widely among patients. Therefore a TOF ratio of 0.9 displayed postoperatively on AMG does not always represent adequate recovery of neuromuscular function and should be normalized by baseline value to reliably detect residual paralysis.

Effects of various anti-asthmatic agents on mite allergen-pulsed murine bone marrow-derived dendritic cells.

BACKGROUND: Dendritic cells (DCs) play an important role in the immune response and are critically involved in asthma. beta2-agonists could potentially exacerbate type 2 T helper (Th2) cell-mediated immune response. OBJECTIVES: To determine the effects of various anti-asthmatic agents on DCs function both in vitro and in vivo. METHODS: Murine bone marrow-derived DCs were pulsed with mite allergen in the presence of pranlukast, salbutamol, salmeterol or fluticasone. These DCs were then inoculated intranasally into naïve mice to induce allergic airway inflammation in vivo. RESULTS: Pranlukast reduced IL-10 and increased IL-12, while fluticasone reduced both IL-10 and IL-12 production by mite allergen-pulsed DCs. Allergic airway inflammation in pranlukast- and fluticasone-treated and mite allergen pulsed DCs-harbouring mice was attenuated and such response was associated with inhibition of Th2 response in the airway. Salbutamol did not alter cytokine production, while salmeterol reduced IL-12 production by mite allergen-pulsed DCs. Lung pathology in beta2-agonist-harbouring mice was comparable with those of mite allergen-pulsed DCs-harbouring mice. CONCLUSIONS: Our results indicate that leukotriene receptor antagonists and corticosteroids inhibit DCs-induced Th2 skewed immune response, and that short- and long-acting beta2-agonists do not modify DCs-induced allergic airway inflammation.

Effects of primary and secondary low-grade respiratory syncytial virus infections in a murine model of asthma.

BACKGROUND: Respiratory syncytial virus (RSV) infection is known to develop and exacerbate asthma in young children. In adult, RSV causes recurrent but asymptomatic infections. However, the impact of asymptomatic RSV infection on adult asthma is yet to be determined. The present study is designed to determine the effects of primary and secondary low-grade RSV infections on allergic airway inflammation in a murine model of allergic asthma. METHODS: A low-grade RSV (2 x 10(3) plaque-forming units/mouse) was inoculated, and this caused neither pulmonary inflammation nor symptoms but induced significant IFN-gamma production in thoracic lymph nodes. To investigate interaction between low-grade virus and Dermatophagoides farinae (Df), airway hyper-responsiveness, lung inflammation and cytokine production from thoracic lymph nodes were compared after primary and secondary low-grade RSV infections in four groups of mice; control, Df allergen-sensitized, RSV-infected and Df-sensitized RSV-infected mice. A direct comparison between low- and high-grade RSV infections was also performed in primary infection. To investigate the role of IL-5 during secondary RSV infection, anti-IL-5 monoclonal antibody (anti-IL-5 mAb) was injected in mice and similar parameters were compared in four groups of mice. RESULTS: Primary high-grade RSV infection increased allergen-induced airway inflammation, while primary low-grade RSV infection attenuated allergen-induced airway inflammation concomitant with significant IFN-gamma production in lung-draining lymph nodes. In marked contrast, secondary low-grade RSV infection increased both IFN-gamma and IL-5 production, resulting in exacerbation of allergen-induced airway inflammation. Anti-IL-5 mAb treatment in secondary low-grade RSV infection and Df allergen-sensitized mice attenuated virus and allergen-induced airway inflammation. CONCLUSIONS: Low-grade RSV infection per se does not cause pulmonary inflammation, whereas it induces a significant immunological response in the allergen-sensitized host. These results indicate that subclinical and recurrent RSV infection may play an important role in exacerbation and maintenance of asthma in adults, wherein IL-5 is critically involved.

Bronchial hyperresponsiveness and airway inflammation in adolescents with asymptomatic childhood asthma.

BACKGROUND: About 70% of childhood asthmatics become free of asthma-related symptoms during adolescence. Little is known about bronchial hyperresponsiveness (BHR) and airway inflammation in young adults with "outgrown" childhood asthma. METHODS: We studied 61 nonsmoking medical students (18 intermittent mild asthmatics, 23 students with outgrown childhood asthma but free of asthma-related symptoms for 10 years (asymptomatic asthmatics) and 20 healthy students). BHR and lung function were measured, and induced sputum samples analyzed for eosinophil count, eosinophilic cationic protein (ECP), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). RESULTS: BHR was still present in most asymptomatic asthmatics, but it was milder compared with healthy students. Only three subjects with previous asthma had no BHR and no signs of airway inflammation. Percentages of eosinophil, and ECP, TNF-alpha and GM-CSF concentrations in induced sputum of mild asthmatics and asymptomatic asthma groups were higher than in the healthy group. In asymptomatic asthmatics group, the duration of asthma, sputum eosinophil percentage, and the level of TNF-alpha in sputum correlated significantly with BHR. CONCLUSIONS: Only a few subjects with longstanding asymptomatic asthma could be considered as cured; most asymptomatic asthmatics continued to exhibit BHR and signs of airway inflammation. The outcome of childhood asthma and BHR was associated with the degree of airway inflammation and the duration of childhood asthma.

Diurnal variation in the response of the mandible to orthopedic force.

Bone and cartilage metabolism is known to be more active during rest than during periods of activity. The purpose of this study was to examine the hypothesis that mandibular retractive force could be more effective when applied to rats during rest. Mandibular retractive force caused a considerable reduction in the condylar length in experimental groups, and the magnitude of this reduction was greater in the Light-period (08:00-20:00) group than in the Dark-period (20:00-08:00) group. The differentiation and proliferation of chondrocytes were inhibited in animals in the Light-period group, compared with those in the Dark-period group. These results suggest that the orthopedic effects of mandibular retractive force vary depending on the time of day the force is applied, and that such force may be more effective while animals are resting than while they are active.

Estrous-cycle-dependent variation in orthodontic tooth movement.

Sex hormones, including estradiol, play important physiological roles in bone metabolism. The purpose of this study was to investigate whether there is estrous-cycle-dependent variation in orthodontic tooth movement, and, if so, to determine the mechanism. Ten-week-old female Wistar rats were used. They received repeated orthodontic force during specific phases in the estrous cycle. Tooth movement in animals that received force principally in estrus was about 33% greater than that in animals that received such force principally in pro-estrus (p < 0.05). Serum estradiol levels also varied according to the estrous cycle, with a peak during pro-estrus and a nadir during estrus, and were inversely related to tooth movement. Furthermore, there were negative correlations between estradiol and both serum TRAP activity and pyridinoline (r = -0.42, p < 0.05; r = -0.59, p < 0.001). These results suggest that cyclic changes in the estradiol level may be associated with the estrous-cycle-dependent variation in tooth movement through its effects on bone resorption.

Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens.

Marek's disease herpesvirus is a vaccine vector of great promise for chickens; however, complete protection against foreign infectious diseases has not been achieved. In this study, two herpesvirus of turkey recombinants (rHVTs) expressing large amounts of infectious bursal disease virus (IBDV) VP2 antigen under the control of a human cytomegalovirus (CMV) promoter or CMV/beta-actin chimera promoter (Pec promoter) (rHVT-cmvVP2 and rHVT-pecVP2) were constructed. rHVT-pecVP2, which expressed the VP2 antigen approximately four times more than did rHVT-cmvVP2 in vitro, induced complete protection against a lethal IBDV challenge in chickens, whereas rHVT-cmvVP2 induced 58% protection. All of the chickens vaccinated with rHVT-pecVP2 had a protective level of antibodies to the VP2 antigen at the time of challenge, whereas only 42 and 67% of chickens vaccinated with rHVT-cmvVP2 or the conventional live IBDV vaccine, respectively, had the antibodies. The antibody level of chickens vaccinated with rHVT-pecVP2 increased for 16 weeks, and the peak antibody level persisted throughout the experiment. The serum antibody titer at 30 weeks of age was about 20 or 65 times higher than that of chickens vaccinated with rHVT-cmvVP2 or the conventional live vaccine, respectively. rHVT-pecVP2, isolated consistently for 30 weeks from the vaccinated chickens, expressed the VP2 antigen after cultivation, and neither nucleotide mutations nor deletion in the VP2 gene was found. These results demonstrate that the amount of VP2 antigen expressed in the HVT vector was correlated with the vaccine efficacy against lethal IBDV challenge, and complete protective immunity that is likely to persist for the life of the chickens was induced.