Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis.

Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII-LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII-LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.

Formation of a PSI-PSII megacomplex containing LHCSR and PsbS in the moss Physcomitrella patens.

Mosses are one of the earliest land plants that diverged from fresh-water green algae. They are considered to have acquired a higher capacity for thermal energy dissipation to cope with dynamically changing solar irradiance by utilizing both the "algal-type" light-harvesting complex stress-related (LHCSR)-dependent and the "plant-type" PsbS-dependent mechanisms. It is hypothesized that the formation of photosystem (PS) I and II megacomplex is another mechanism to protect photosynthetic machinery from strong irradiance. Herein, we describe the analysis of the PSI-PSII megacomplex from the model moss, Physcomitrella patens, which was resolved using large-pore clear-native polyacrylamide gel electrophoresis (lpCN-PAGE). The similarity in the migration distance of the Physcomitrella PSI-PSII megacomplex to the Arabidopsis megacomplex shown during lpCN-PAGE suggested that the Physcomitrella PSI-PSII and Arabidopsis megacomplexes have similar structures. Time-resolved chlorophyll fluorescence measurements show that excitation energy was rapidly and efficiently transferred from PSII to PSI, providing evidence of an ordered association of the two photosystems. We also found that LHCSR and PsbS co-migrated with the Physcomitrella PSI-PSII megacomplex. The megacomplex showed pH-dependent chlorophyll fluorescence quenching, which may have been induced by LHCSR and/or PsbS proteins with the collaboration of zeaxanthin. We discuss the mechanism that regulates the energy distribution balance between two photosystems in Physcomitrella.

A piezoelectric micromachined ultrasound transducer combined with recording electrodes for acute brain preparations in vitro.

BACKGROUND: Ultrasound stimulation is used to noninvasively stimulate the local and deep areas of the brain. However, the detailed cellular mechanisms of neural activation are still unclear because studies on micro-stimulation at the cellular level are lacking. NEW METHOD: To modulate neural activity at the cellular level, we developed a piezoelectric micromachined ultrasound transducer (PMUT), having circular diaphragms for application on acute brain slice preparations. To monitor neural activities, additionally, we fabricated recording microelectrodes onto the same PMUT device for closed-loop application. RESULTS: To examine the PMUT-driven cellular responses of a brain slice, intracellular calcium signals in individual cells were measured using two calcium indicators. We successfully observed the intracellular responses triggered by the ultrasound of our novel PMUT. In addition, we performed recordings of local field potentials in a brain slice, demonstrating its usefulness as a simultaneous recording interface. COMPARISON WITH EXISTING METHOD(S): Conventional ultrasound stimulators are open-loop systems that risk inducing excessive neural activity because of the absence of neural activity monitoring. In contrast, our PMUT is packaged in a single device with both stimulation and sensor interface for neuromodulation. Further, there are no published reports on in vitro microdevices that can be used for ultrasound stimulation in rodent cortical slices that are several hundred micrometers thick, which maintain the cortical laminar structure and intrinsic neural networks. CONCLUSIONS: Our findings suggest that this novel PMUT device has the potential for being a powerful tool for in vitro brain slice applications and effective closed loop ultrasound stimulation.

Scale-preserving shape reconstruction from monocular endoscope image sequences by supervised depth learning.

Reconstructing 3D shapes from images are becoming popular, but such methods usually estimate relative depth maps with ambiguous scales. A method for reconstructing a scale-preserving 3D shape from monocular endoscope image sequences through training an absolute depth prediction network is proposed. First, a dataset of synchronized sequences of RGB images and depth maps is created using an endoscope simulator. Then, a supervised depth prediction network is trained that estimates a depth map from a RGB image minimizing the loss compared to the ground-truth depth map. The predicted depth map sequence is aligned to reconstruct a 3D shape. Finally, the proposed method is applied to a real endoscope image sequence.

A Case of COVID-19 Infection Raising Suspicions of a Connection between Vascular Damage and Thrombus Formation.

Although endothelial damage has been hypothesized to be associated with coronavirus disease 2019 (COVID-19)-related cerebral infarction based on the specificity of the viral cellular invasion pathway, no case has been reported to date. We herein report a 51-year-old Japanese woman who presented with neck pain one week after COVID-19 infection. Computed tomography and magnetic resonance imaging revealed inflammation of the carotid and vertebral arteries. Ultrasonography revealed multiple flap-like structures that were assumed to be thrombi. Although the patient had no cerebral infarction, this could be an important case of vascular damage and thrombus formation in a COVID-19 patient.

Modulatory Effects on Laminar Neural Activity Induced by Near-Infrared Light Stimulation with a Continuous Waveform to the Mouse Inferior Colliculus In Vivo.

Infrared neural stimulation (INS) is a promising area of interest for the clinical application of a neuromodulation method. This is in part because of its low invasiveness, whereby INS modulates the activity of the neural tissue mainly through temperature changes. Additionally, INS may provide localized brain stimulation with less tissue damage. The inferior colliculus (IC) is a crucial auditory relay nucleus and a potential target for clinical application of INS to treat auditory diseases and develop artificial hearing devices. Here, using continuous INS with low to high-power density, we demonstrate the laminar modulation of neural activity in the mouse IC in the presence and absence of sound. We investigated stimulation parameters of INS to effectively modulate the neural activity in a facilitatory or inhibitory manner. A mathematical model of INS-driven brain tissue was first simulated, temperature distributions were numerically estimated, and stimulus parameters were selected from the simulation results. Subsequently, INS was administered to the IC of anesthetized mice, and the modulation effect on the neural activity was measured using an electrophysiological approach. We found that the modulatory effect of INS on the spontaneous neural activity was bidirectional between facilitatory and inhibitory effects. The modulatory effect on sound-evoked responses produced only an inhibitory effect to all examined stimulus intensities. Thus, this study provides important physiological evidence on the response properties of IC neurons to INS. Overall, INS can be used for the development of new therapies for neurological disorders and functional support devices for auditory central processing.

Immune-mediated necrotizing myopathy with concomitant development of Kikuchi-Fujimoto disease.

Immune-mediated necrotizing myopathy (IMNM) is a distinct type of idiopathic inflammatory myositis, pathologically characterized by myofiber necrosis and degeneration in the absence of lymphocyte infiltration. Herein, we present a case of IMNM with concomitant development of Kikuchi-Fujimoto disease (KFD), characterized by histiocytic necrotizing lymphadenitis, in a 36-year-old woman who had a treatment history for rheumatoid arthritis (RA). Treatment with oral prednisolone and tacrolimus as immunosuppressants resulted in the remission of the skeletomuscular involvement and lymphadenopathy. To the best of our knowledge, this is the first report of IMNM and KFD developing concomitantly during the clinical course of RA.

Calibration-free structured-light-based 3D scanning system in laparoscope for robotic surgery.

Accurate 3D shape measurement is crucial for surgical support and alignment in robotic surgery systems. Stereo cameras in laparoscopes offer a potential solution; however, their accuracy in stereo image matching diminishes when the target image has few textures. Although stereo matching with deep learning has gained significant attention, supervised learning requires a large dataset of images with depth annotations, which are scarce for laparoscopes. Thus, there is a strong demand to explore alternative methods for depth reconstruction or annotation for laparoscopes. Active stereo techniques are a promising approach for achieving 3D reconstruction without textures. In this study, a 3D shape reconstruction method is proposed using an ultra-small patterned projector attached to a laparoscopic arm to address these issues. The pattern projector emits a structured light with a grid-like pattern that features node-wise modulation for positional encoding. To scan the target object, multiple images are taken while the projector is in motion, and the relative poses of the projector and a camera are auto-calibrated using a differential rendering technique. In the experiment, the proposed method is evaluated by performing 3D reconstruction using images obtained from a surgical robot and comparing the results with a ground-truth shape obtained from X-ray CT.

Single and multi-frame auto-calibration for 3D endoscopy with differential rendering.

The use of 3D measurement in endoscopic images offers practicality in cancer diagnosis, computer-assisted interventions, and making annotations for machine learning training data. An effective approach is the implementation of an active stereo system, using a micro-sized pattern projector and an endoscope camera, which has been intensively developed. One open problem for such a system is the necessity of strict and complex calibration of the projector-camera system to precisely recover the shapes. Moreover, since the head of an endoscope should have enough elasticity to avoid harming target objects, the positions of the pattern projector cannot be tightly fixed to the head, resulting in limited accuracy. A straightforward approach to the problem is applying auto-calibration. However, it requires special markers in the pattern or a highly accurate initial position for stable calibration, which is impractical for real operation. In the paper, we propose a novel auto-calibration method based on differential rendering techniques, which are recently proposed and drawing wide attention. To apply the method to an endoscopic system, where a diffractive optical element (DOE) is used, we propose a technique to simultaneously estimate the focal length of the DOE as well as the extrinsic parameters between a projector and a camera. We also propose a multi-frame optimization algorithm to jointly optimize the intrinsic and extrinsic parameters, relative pose between frames, and the entire shape.Clinical relevance- One-shot endoscopic measurement of depth information is a practical solution for cancer diagnosis, computer-assisted interventions, and making annotations for machine learning training data.

Dual function MITO-Porter, a nano carrier integrating both efficient cytoplasmic delivery and mitochondrial macromolecule delivery.

Mitochondrial dysfunction is associated with a variety of human diseases including inherited mitochondrial diseases, neurodegenerative disorders, diabetes mellitus, and cancer. Effective medical therapies for mitochondrial diseases will ultimately require an optimal drug delivery system, which will likely be achieved through innovations in the nanotechnology of intracellular trafficking. To achieve efficient mitochondrial drug delivery, two independent processes, i.e., "cytoplasmic delivery through the cell membrane" and "mitochondrial delivery through the mitochondrial membrane" are required. In previous studies, we developed an octaarginine (R8) modified nano carrier for efficient cytoplasmic delivery, showing that R8-modified liposomes were internalized into cells efficiently. On the other hand, we also constructed MITO-Porter for the mitochondrial delivery of macromolecules, a liposome-based carrier that delivers cargos to mitochondria via membrane fusion. Here, we report the development of a dual function MITO-Porter (DF-MITO-Porter), based on the concept of integrating both R8-modified liposomes and MITO-Porter. We show that the DF-MITO-Porter effectively delivers exogenous macro-biomolecules into the mitochondrial matrix, and provide a demonstration of its potential use in therapies aimed at mitochondrial DNA.

A Multielectrode Array-Based Recording System for Analyzing Ultrasound-Driven Neural Responses in Brain Slices in vitro.

Ultrasound stimulation is expected to be useful for transcranial local and deep stimulation of the brain, which is difficult to achieve using conventional electromagnetic stimulation methods. Previous ultrasound stimulation experiments have used various types of acute in vitro preparations, including hippocampus slices from rodents and Caenorhabditis elegans tissue. For in vivo preparations, researchers have used the cortices of rodents as targets for transcranial ultrasound stimulation. However, no previous studies have used in vitro ultrasound stimulation in rodent cortical slices to examine the mechanisms of ultrasound-driven central neural circuits. Here we demonstrate the optimal experimental conditions for an in vitro ultrasound stimulation system for measuring activity in brain slices using a multielectrode array substrate. We found that the peak amplitudes of the ultrasound-evoked cortical responses in the brain slices depend on the intensities and durations of the ultrasound stimulation parameters. Thus, our findings provide a new in vitro experimental setup that enables activation of a brain slice via ultrasound stimulation. Accordingly, our results indicate that choosing the appropriate ultrasound waveguide structure and stimulation parameters is important for producing the desired intensity distribution in a localized area within a brain slice. We expect that this experimental setup will facilitate future exploration of the mechanisms of ultrasound-driven neural activity.

Temporal and spatial profiles of evoked activity induced by magnetic stimulation using millimeter-sized coils in the mouse auditory cortex in vivo.

Transcranial magnetic stimulation (TMS), a minimally/non-invasive method of electromagnetic stimulation of brain tissue, has been shown to be beneficial in clinical therapy for specific neurological diseases and disorders. Magnetic stimulation is also used to modulate human and animal brain activity in basic neuroscience studies. Among experimental animal models, mouse models are particularly popular and uniquely representative of brain disorders in basic neuroscience research. TMS in mouse models may play a substantial role in understanding TMS-induced changes in neural networks and plasticity. Although TMS techniques are widely used to examine rodent disease models, techniques specific for mice using small magnetic stimulators have not been intensively developed. Here, we provide a numerical simulation and a practical method of applying TMS to mice by constructing millimeter-sized TMS coils to deliver a low stimulation intensity while maintaining focality. Our results indicate the TMS coils can produce an electrical field with sufficient magnitude to activate the anesthetized mouse cortex in the presence or absence of the skull in vivo. Our results also show that, immediately after magnetic stimulation, local field and action potentials were reliably observed in a manner that depended on the distance between the coil and the brain, implying even a small coil could reliably evoke cortical activity. Therefore, our results show our millimeter-sized coils could produce electric fields sufficient to alter cortical excitability in mice. These coils could be useful in future preclinical studies to examine detailed mechanisms underlying TMS-induced changes in neural activity of the auditory cortex and other cortical regions.

3D endoscope system with AR display superimposing dense and wide-angle-of-view 3D points obtained by using micro pattern projector.

In recent years, augmented reality (AR) technologies have been widespread for supporting various kinds of tasks, by superimposing useful information on the users' view of the real environments. In endoscopic diagnosis, AR systems can be helpful as an aid in presenting information to endoscopists who have their hands full. In this paper, we propose a system that can superimpose shapes, which are reconstructed from an endoscope image, onto the field of view. The feature of the proposed system is that it reconstructs 3D shapes from the images captured by the endoscope and superimposes them onto the real views. As a result, the superimposed view allows the doctor to keep operating the endoscope while observing the patient's internal body with additional information. The proposed system is composed of the reconstruction module and the display module. The reconstruction module is for acquiring 3D shapes based on an active stereo method. In particular, we propose a novel projection pattern that can reconstruct wide areas of the endoscopic view. The display module shows the 3D shape obtained by the reconstructed module, superimposing on the field of view. In the experiments, we show that it is possible to perform a wide range of dense 3D reconstructions using the new projection patterns. In addition, we confirmed the usefulness of the AR system by interviewing medical doctors.

Octaarginine-modified liposomes enhance the anti-oxidant effect of Lecithinized superoxide dismutase by increasing its cellular uptake.

The anti-oxidant enzyme superoxide dismutase (SOD) has the potential for use as a therapeutic agent in the treatment of various diseases caused by reactive oxygen species. However, achieving this would be difficult without a suitable delivery system for SOD. We previously reported that PC-SOD, in which four molecules of a phosphatidylcholine (PC) derivative were covalently bound to each dimer of recombinant human CuZnSOD, was a high affinity for the cell membrane [14]. Here, we show that an octaarginine (R8) modified liposome equipped with PC-SOD (R8-LP (PC-SOD)) enhances its anti-oxidant effect. High-density R8-modified liposomes can stimulate macropinocytosis and are taken up efficiently by cells as demonstrated in a previous study [21]. Flow cytometry analyses showed that R8-LP (PC-SOD) was taken up by cells more efficiently than PC-SOD. Moreover, R8-LP (PC-SOD) liposomes were found to scavenge superoxide anions (O(2)(-)) very efficiently. These results suggest that the efficient cytosolic delivery of PC-SOD by R8-modified liposomes would enhance the anti-oxidant effects of PC-SOD.

Active Stereo Method for 3D Endoscopes using Deep-layer GCN and Graph Representation with Proximity Information.

Techniques for 3D endoscopic systems have been widely studied for various reasons. Among them, active stereo based systems, in which structured-light patterns are projected to surfaces and endoscopic images of the pattern are analyzed to produce 3D depth images, are promising, because of robustness and simple system configurations. For those systems, finding correspondences between a projected pattern and an original pattern is an open problem. Recently, correspondence estimation by graph neural networks (GCN) using graph-based representation of the patterns were proposed for 3D endoscopic systems. One severe problem of the approach is that the graph matching by GCN is largely affected by the stability of the graph construction process using the detected patterns of a captured image. If the detected pattern is fragmented into small pieces, graph matching may fail and 3D shapes cannot be retrieved. In this paper, we propose a solution for those problems by applying deep-layered GCN and extended graph representations of the patterns, where proximity information is added. Experiments show that the proposed method outperformed the previous method in accuracies for correspondence matching for 3D reconstruction.

Fully Auto-calibrated Active-stereo-based 3D Endoscopic System using Correspondence Estimation with Graph Convolutional Network.

We have developed a series of 3D endoscopic systems where a micro-sized pattern projector is inserted through the instrument channel of the endoscope and shapes are reconstructed by a structured light technique using captured images of the endoscopic camera. One problem of the previous works is that the accuracy of shape reconstruction is low, because the projector cannot be fixed to the endoscope, and thus, the pose of the pattern projector w.r.t. the camera cannot be pre-calibrated. In this paper, we propose a method to auto-calibrate the pose of the projector without using any special devices nor manual process. Since the technique is one-shot, multiple shapes can be reconstructed from an image sequence and a large 3D scene can be recovered by merging them. Experiments are conducted using the real system.

Simultaneous shape and camera-projector parameter estimation for 3D endoscopic system using CNN-based grid-oneshot scan.

For effective in situ endoscopic diagnosis and treatment, measurement of polyp sizes is important. For this purpose, 3D endoscopic systems have been researched. Among such systems, an active stereo technique, which projects a special pattern wherein each feature is coded, is a promising approach because of simplicity and high precision. However, previous works of this approach have problems. First, the quality of 3D reconstruction depended on the stabilities of feature extraction from the images captured by the endoscope camera. Second, due to the limited pattern projection area, the reconstructed region was relatively small. In this Letter, the authors propose a learning-based technique using convolutional neural networks to solve the first problem and an extended bundle adjustment technique, which integrates multiple shapes into a consistent single shape, to address the second. The effectiveness of the proposed techniques compared to previous techniques was evaluated experimentally.

Glucose Depletion Enhances the Stem Cell Phenotype and Gemcitabine Resistance of Cholangiocarcinoma Organoids through AKT Phosphorylation and Reactive Oxygen Species.

Cancer cells are strongly dependent on the glycolytic pathway for generation of energy even under aerobic condition through a phenomenon known as the Warburg effect. Rapid proliferation of cancer cells is often accompanied by high glucose consumption and abnormal angiogenesis, which may lead to glucose depletion. In the present study, we investigated how cholangiocarcinoma cells adapt to glucose depletion using a 3D organoid culture system. We cultured organoids derived from cholangiocarcinoma under glucose-free condition and investigated cell proliferation, expression of stem cell markers and resistance to gemcitabine. Cholangiocarcinoma organoids cultured under glucose-free condition showed reduced proliferation but were able to survive. We also observed an increase in the expression of stem cell markers including LGR5 and enhancement of stem cell phenotypic characteristics such as resistance to gemcitabine through AKT phosphorylation and reactive oxygen species. These findings indicate that cholangiocarcinoma cells are able to adapt to glucose depletion through enhancement of their stem cell phenotype in response to changes in microenvironmental conditions.

Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes: Insight into a Role of the Coiled-Coil Region.

The coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coiled-coil with a unique negatively charged area essential for the PHB interactome in mitochondria. Disruption of the HR coiled-coil abolishes well-ordered PHB complexes and the mitochondrial tubular networks accompanying PHB-dependent signaling. Using a proximity-dependent biotin identification (BioID) technique in live cells, we mapped a number of mitochondrial intermembrane space proteins whose association with PHB2 relies on the HR coiled-coil region. Elucidation of the PHB complex structure in mitochondria provides insight into essential PHB interactomes required for mitochondrial dynamics as well as signal transduction.

Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma.

Biliary tract carcinomas (BTCs) are among the most aggressive malignancies and have a poor prognosis. Here, we successfully established organoid lines derived from intrahepatic cholangiocarcinoma, gallbladder cancer, and neuroendocrine carcinoma of the ampulla of Vater. These organoids derived from BTCs were cultured stably for >1 year and closely recapitulated the histopathology, gene expression, and genetic alterations evident in the primary tumors. Gene expression profiling of the organoids revealed that SOX2 could be a potential prognostic biomarker for patients with BTC. We screened a compound library consisting of drugs used clinically for their ability to suppress organoids derived from BTCs and found that the antifungal drugs amorolfine and fenticonazole significantly suppressed the growth of organoids derived from BTCs with minimal toxicity to normal biliary epithelial cells. Patient-derived organoids may be a powerful research tool for the clarification of molecular pathogenesis and the discovery of biomarkers and therapeutic drugs for refractory cancers.