Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches

Usefulness of the forced oscillation technique in assessing the therapeutic result of tracheobronchial central airway obstruction

Background: Pulmonary function tests (PFTs) comprise the traditional method for detecting central airway obstruction (CAO) and evaluating therapeutic effects, but are effort dependent. By contrast, the forced oscillation technique (FOT) is performed during tidal breathing in an effort-independent mode and is universally used to assess respiratory function in patients with chronic obstructive pulmonary disease (COPD) and asthma. We used the FOT to measure airway resistance and reactance in patients with CAO before and after interventional bronchoscopy and compared the results to data obtained using PFTs. Methods: Twelve patients with CAO were recruited from December 2013 to July 2016. The FOT, PFTs, chest computed tomography (CT), COPD Assessment Test (CAT), and the modified Medical Research Council (mMRC) dyspnea scale were employed before and after interventional bronchoscopy. The minimum airway cross-sectional area (MACSA) was calculated using a CT image calculator. Results: Of the 12 patients, 6 had tracheal obstruction and 6 had bronchial obstruction. All FOT measurements, except Delta XS, were significantly improved after interventional bronchoscopy in all cases. The significance of the improvement was greater with the FOT than PFTs. The MACSA, CAT, and mMRC dyspnea scale scores also significantly improved in all cases. Furthermore, only alteration of resistance at 20 Hz (R20) significantly correlated with the alteration of the MACSA after intervention. No significant correlations were found for PFTs. Conclusions: The FOT is suitable and convenient for assessing therapeutic results in patients with tracheobronchial CAO. The alteration of R20 is useful for estimating the airway dilation of CAO after interventional bronchoscopy. (C)2018 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.ArticleRESPIRATORY INVESTIGATION.56(3):222-229(2018)journal articl

Cisplatin plus Docetaxel Chemotherapy for Thoracic Lymph Node Metastasis from Cancer of Unknown Primary – Experience of Three Cases

The optimal chemotherapeutic regimen for cancer of unknown primary (CUP) remains uncertain. We encountered 3 cases with CUP who presented with thoracic lymph node metastasis. Detailed physical examination and diagnostic tests, including laboratory investigations, bronchoscopy, upper and lower gastrointestinal studies, computed tomography of the head, neck, abdomen and pelvis and 18F-fluorodeoxyglucose positron emission tomography, failed to identify the primary site in these cases. The patients were treated with the cisplatin plus docetaxel chemotherapy regimen. Concomitant thoracic radiotherapy was conducted in one patient and surgical resection in another. All patients showed good response to the chemotherapy and achieved long-term disease-free survival

The Quiescent Intracluster Medium in the Core of the Perseus Cluster

Clusters of galaxies are the most massive gravitationally-bound objects in the Universe and are still forming. They are thus important probes of cosmological parameters and a host of astrophysical processes. Knowledge of the dynamics of the pervasive hot gas, which dominates in mass over stars in a cluster, is a crucial missing ingredient. It can enable new insights into mechanical energy injection by the central supermassive black hole and the use of hydrostatic equilibrium for the determination of cluster masses. X-rays from the core of the Perseus cluster are emitted by the 50 million K diffuse hot plasma filling its gravitational potential well. The Active Galactic Nucleus of the central galaxy NGC1275 is pumping jetted energy into the surrounding intracluster medium, creating buoyant bubbles filled with relativistic plasma. These likely induce motions in the intracluster medium and heat the inner gas preventing runaway radiative cooling; a process known as Active Galactic Nucleus Feedback. Here we report on Hitomi X-ray observations of the Perseus cluster core, which reveal a remarkably quiescent atmosphere where the gas has a line-of-sight velocity dispersion of 164+/-10 km/s in a region 30-60 kpc from the central nucleus. A gradient in the line-of-sight velocity of 150+/-70 km/s is found across the 60 kpc image of the cluster core. Turbulent pressure support in the gas is 4% or less of the thermodynamic pressure, with large scale shear at most doubling that estimate. We infer that total cluster masses determined from hydrostatic equilibrium in the central regions need little correction for turbulent pressure.Comment: 31 pages, 11 Figs, published in Nature July