Highly Efficient Fluorescent Probe to Visualize Protein Interactions at the Superresolution.

Superresolution microscopy (SR microscopy) of protein-protein interactions (PPIs) occurring in subcellular structures is essential for understanding cellular functions. However, a powerful and useful technology for SR microscopy of PPIs remains elusive. Here, we develop a highly efficient photoconvertible fluorescent probe, named split-Dendra2, for SR microscopy of PPIs in the cell. We found that split-Dendra2 enables a highly efficient detection of PPIs, making it possible to perform SR microscopy of PPIs with high spatial resolution and high image reconstruction fidelity. We demonstrate the utility of split-Dendra2 by visualizing PPIs occurring in small subcellular structures at the superresolution, such as clathrin-coated pits and focal adhesions, which cannot be visualized by the existing tools. Split-Dendra2 offers a powerful and useful tool that greatly expands the possibility of SR microscopy and can contribute to revealing the function of PPIs at the nanoscale resolution.

Small Bowel Obstruction Caused by Migration of Fractured Metal Stent in Patients with Malignant Gastric Outlet Obstruction: A Report of Two Cases and Review of the Literature.

Gastroduodenal stenting (GDS) is a less invasive alternative to gastrojejunostomy for the management of malignant gastric outlet obstruction (mGOO). GDS is a minimally invasive treatment with good technical and clinical success, and severe complications that require surgical intervention are rare. Stent fracture is an uncommon complication associated with GDS; however, migration of the fractured distal segment can result in small bowel obstruction. Adverse effects of stent fractures in patients with mGOO have rarely been reported. We herein report two surgical cases of small bowel obstruction caused by the migration of fractured metal stent in patients with mGOO.

Treatment strategies for reflux esophagitis including a potassium-competitive acid blocker: A cost-effectiveness analysis in Japan.

INTRODUCTION: Gastroesophageal reflux disease is a common condition, and proton pump inhibitors (PPIs) are the mainstays of treatment. However, concerns have been raised about the safety of PPIs. A potassium-competitive acid blocker (P-CAB), vonoprazan (VPZ), was recently introduced, which may provide clinical benefits. This study was performed to investigate the cost-effectiveness of alternative long-term strategies including continuous and discontinuous treatment with VPZ for the management of reflux esophagitis in Japan. METHODS: A health state transition model was developed to capture the long-term management of reflux esophagitis. Four different strategies were compared: (a) intermittent PPI using lansoprazole (LPZ); (b) intermittent P-CAB; (c) maintenance PPI using LPZ; and (d) maintenance P-CAB. RESULTS: Intermittent P-CAB was the most cost-effective, and the number of days for which medication was required with this strategy was fewest. Maintenance PPI was more efficacious, but more costly than intermittent P-CAB. Maintenance P-CAB was more efficacious, but more costly than maintenance PPI. Co-payments were higher for maintenance PPI than for intermittent P-CAB, and for maintenance P-CAB than for maintenance PPI, which were considered reasonable for the majority of patients to improve symptoms. CONCLUSIONS: Intermittent P-CAB appears to be the strategy of choice for the majority of reflux esophagitis patients in clinical practice. If a patient is not satisfied with the symptom control of the current strategy, switching to a more effective strategy appears to be a reasonable option for the majority of patients.

Membrane Dynamics Induced by a Phosphatidylinositol 3,4,5-Trisphosphate Optogenetic Tool.

Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states, such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a crucial lipid that regulates PIP3 dynamics. Investigations of how PIP3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information concerning membrane dynamics upon PIP3 production. In the present study, we applied an optogenetic approach using the CRY2-CIBN system. Using this system, we revealed that local PIP3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP3-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors, such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

Practical use of a plastic scintillator for quality assurance of electron beam therapy.

Quality assurance (QA) of clinical electron beams is essential for performing accurate and safe radiation therapy. However, with advances in radiation therapy, QA has become increasingly labor-intensive and time-consuming. In this paper, we propose a tissue-equivalent plastic scintillator for quick and easy QA of clinical electron beams. The proposed tool comprises a plastic scintillator plate and a charge-coupled device camera that enable the scintillation light by electron beams to be recorded with high sensitivity and high spatial resolution. Further, the Cerenkov image is directly subtracted from the scintillation image to discriminate Cerenkov emissions and accurately measure the dose profiles of electron beams with high spatial resolution. Compared with conventional methods, discrepancies in the depth profile improved from 7% to 2% in the buildup region via subtractive corrections. Further, the output brightness showed good linearity with dose, good reproducibility (deviations below 1%), and dose rate independence (within 0.5%). The depth of 50% dose measured with the tool, an index of electron beam quality, was within ±0.5 mm of that obtained with an ionization chamber. Lateral brightness profiles agreed with the lateral dose profiles to within 4% and no significant improvement was obtained using Cerenkov corrections. Field size agreed to within 0.5 mm with those obtained with ionization chamber. For clinical QA of electron boost treatment, a disk scintillator that mimics the shape of a patient's breast is applied. The brightness distribution and dose, calculated using a treatment planning system, was generally acceptable for clinical use, except in limited zones. Overall, the proposed plastic scintillator plate tool efficiently performs QA for electron beam therapy and enables simultaneous verification of output constancy, beam quality, depth, and lateral dose profiles during monthly QAs at lower doses of irradiation (small monitor units, MUs).

Optical manipulation of the alpha subunits of heterotrimeric G proteins using photoswitchable dimerization systems.

Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.

Photoconversion and Fluorescence Properties of a Red/Green-Type Cyanobacteriochrome AM1_C0023g2 That Binds Not Only Phycocyanobilin But Also Biliverdin.

Cyanobacteriochromes (CBCRs) are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB) and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV). The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2) from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.

A biliverdin-binding cyanobacteriochrome from the chlorophyll d-bearing cyanobacterium Acaryochloris marina.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors in cyanobacteria that absorb visible and near-ultraviolet light. CBCRs are divided into two types based on the type of chromophore they contain: phycocyanobilin (PCB) or phycoviolobilin (PVB). PCB-binding CBCRs reversibly photoconvert at relatively long wavelengths, i.e., the blue-to-red region, whereas PVB-binding CBCRs reversibly photoconvert at shorter wavelengths, i.e., the near-ultraviolet to green region. Notably, prior to this report, CBCRs containing biliverdin (BV), which absorbs at longer wavelengths than do PCB and PVB, have not been found. Herein, we report that the typical red/green CBCR AM1_1557 from the chlorophyll d-bearing cyanobacterium Acaryochloris marina can bind BV almost comparable to PCB. This BV-bound holoprotein reversibly photoconverts between a far red light-absorbing form (Pfr, λmax = 697 nm) and an orange light-absorbing form (Po, λmax = 622 nm). At room temperature, Pfr fluoresces with a maximum at 730 nm. These spectral features are red-shifted by 48~77 nm compared with those of the PCB-bound domain. Because the absorbance of chlorophyll d is red-shifted compared with that of chlorophyll a, the BV-bound AM1_1557 may be a physiologically relevant feature of A. marina and is potentially useful as an optogenetic switch and/or fluorescence imager.

Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins.

Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3 × 10(3) s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.