From Homogeneity to Turing Pattern: Kinetically Controlled Self-Organization of Transmembrane Protein.

Understanding the spatial organization of membrane proteins is crucial for unraveling key principles in cell biology. The reaction-diffusion model is commonly used to understand biochemical patterning; however, applying reaction-diffusion models to subcellular phenomena is challenging because of the difficulty in measuring protein diffusivity and interaction kinetics in the living cell. In this work, we investigated the self-organization of the plasmalemma vesicle-associated protein (PLVAP), which creates regular arrangements of fenestrated ultrastructures, using single-molecule tracking. We demonstrated that the spatial organization of the ultrastructures is associated with a decrease in the association rate by actin destabilization. We also constructed a reaction-diffusion model that accurately generates a hexagonal array with the same 130 nm spacing as the actual scale and informs the stoichiometry of the ultrastructure, which can be discerned only through electron microscopy. Through this study, we integrated single-molecule experiments and reaction-diffusion modeling to surpass the limitations of static imaging tools and proposed emergent properties of the PLVAP ultrastructure.

Analysis of Phase Heterogeneity in Lipid Membranes Using Single-Molecule Tracking in Live Cells.

In live cells, the plasma membrane is composed of lipid domains separated by hundreds of nanometers in dynamic equilibrium. Lipid phase separation regulates the trafficking and spatiotemporal organization of membrane molecules that promote signal transduction. However, visualizing domains with adequate spatiotemporal accuracy remains challenging because of their subdiffraction limit size and highly dynamic properties. Here, we present a single lipid-molecular motion analysis pipeline (lipid-MAP) for analyzing the phase heterogeneity of lipid membranes by detecting the instantaneous velocity change of a single lipid molecule using the excellent optical properties of nanoparticles, high spatial localization accuracy of single-molecule localization microscopy, and separation capability of the diffusion state of the hidden Markov model algorithm. Using lipid-MAP, individual lipid molecules were found to be in dynamic equilibrium between two statistically distinguishable phases, leading to the formation of small (∼170 nm), viscous (2.5× more viscous than surrounding areas), and transient domains in live cells. Moreover, our findings provide an understanding of how membrane compositional changes, i.e., cholesterol and phospholipids, affect domain formation. This imaging method can contribute to an improved understanding of spatiotemporal-controlled membrane dynamics at the molecular level.

Incremental Learning for Online Data Using QR Factorization on Convolutional Neural Networks.

Catastrophic forgetting, which means a rapid forgetting of learned representations while learning new data/samples, is one of the main problems of deep neural networks. In this paper, we propose a novel incremental learning framework that can address the forgetting problem by learning new incoming data in an online manner. We develop a new incremental learning framework that can learn extra data or new classes with less catastrophic forgetting. We adopt the hippocampal memory process to the deep neural networks by defining the effective maximum of neural activation and its boundary to represent a feature distribution. In addition, we incorporate incremental QR factorization into the deep neural networks to learn new data with both existing labels and new labels with less forgetting. The QR factorization can provide the accurate subspace prior, and incremental QR factorization can reasonably express the collaboration between new data with both existing classes and new class with less forgetting. In our framework, a set of appropriate features (i.e., nodes) provides improved representation for each class. We apply our method to the convolutional neural network (CNN) for learning Cifar-100 and Cifar-10 datasets. The experimental results show that the proposed method efficiently alleviates the stability and plasticity dilemma in the deep neural networks by providing the performance stability of a trained network while effectively learning unseen data and additional new classes.

AIS Trajectories Simplification Algorithm Considering Topographic Information.

With the development of maritime technology and equipment, most ships are equipped with an automatic identification system (AIS) to store navigation information. Over time, the size of the data increases, rendering its storage and processing difficult. Hence, it is necessary to transform the AIS data into trajectories, and then simplify the AIS trajectories to remove unnecessary information that is not related to route shape. Moreover, topographic information must be considered because otherwise, the simplified trajectory can intersect obstacles. In this study, we propose an AIS trajectory simplification algorithm considering topographic information. The proposed algorithm simplifies the trajectories without the intersection of the trajectory and obstacle using the improved Douglas-Peucker algorithm. Polygon map random (PMR) quadtree was used to consider topographic information on the coast, and the intersection between topographic information and simplified trajectories was efficiently computed using the PMR quadtree. To verify the effectiveness of the proposed algorithm, experiments were conducted on real-world trajectories in the Korean sea. The proposed algorithm yielded simplified trajectories with no intersections of the trajectory and obstacle. In addition, the computational efficiency of the proposed algorithm with the PMR quadtree was superior to that without the PMR quadtree.

Sunlight-Activatable ROS Generator for Cell Death Using TiO2/c-Si Microwires.

Solar-driven reactive oxygen species (ROS) generation is an attractive disinfection technique for cell death and water purification. However, most photocatalysts require high stability in the water environment and the production of ROS with a sufficient amount and diffusion length to damage pathogens. Here, a ROS generation system was developed consisting of tapered crystalline silicon microwires coated with anatase titanium dioxide for a conformal junction. The system effectively absorbed >95% of sunlight over 300-1100 nm, resulting in effective ROS generation. The system was designed to produce various ROS species, but a logistic regression analysis with cellular survival data revealed that the diffusion length of the ROS is ∼9 µm, implying that the most dominant species causing cell damage is H2O2. Surprisingly, a quantitative analysis showed that only 15 min of light irradiation on the system would catalyze a local bactericidal effect comparable to the conventional germicidal level of H2O2 (∼3 mM).

Surface Modification of Parylene C Film via Buchwald-Hartwig Amination for Organic Solvent-Compatible and Flexible Microfluidic Channel Bonding.

Surface modification offers an efficient and economical route to installing functional groups on a polymer surface. This work demonstrates that primary amine groups can be introduced onto a polymer surface via Buchwald-Hartwig amination, and the functionalized substrates can be chemically bonded to produce functional microfluidic devices. By activating the CCl bond in commercially used poly(chloro-p-xylylene) (parylene C) by Pd catalyst and substituting Cl with the amine source, the amine groups are successfully installed in a facile and recyclable manner. The substrates can be covalently bonded with each other via amine-isocyanate chemistry, providing much higher bonding strength compared to previous methods based on noncovalent adhesive coatings. As a result, transparent and flexible microfluidic channels can be fabricated that are compatible with organic solvents and high pressure. Retention of amine group reactivity in the channel suggests the potential of this methodology for the surface immobilization of functional molecules for microfluidic reactors and biosensors.

Extracellular vesicle-associated antigens as a new vaccine platform against scrub typhus.

Scrub typhus is an acute vector-borne disease caused by infection with the intracellular gram-negative bacterium Orientia tsutsugamushi (Ot). The rapid production of an efficient vaccine against Ot using novel strategies is required because of the global increase in mortality caused by these infections; however, no commercial vaccine is currently available. Ot induces T-cell-mediated immunogenic responses upon infection; therefore, a new rapidly producible vaccine that maximizes T-cell responses against Ot is required. In this study, we sought to develop a model vaccine platform for T-cell-mediated Ot infection using T-cell-immunity associated Salmonella-derived extracellular vesicles (EVs). For this purpose, we optimized DNA sequences encoding the full-length Ot proteins, TSA56, ScaA, ScaC, ScaD, and ScaE, and their expression in Salmonella. The sequences were incorporated into a new platform vector, pKST, which ectopically and concurrently produces Ot proteins and EVs. Expression analysis using pKST-antigen plasmids showed that TSA56 and ScaC produced antigen-associated EVs and showed strong T-cell immunogenic responses. We found that mice vaccinated with EVs derived from TSA56-expressing cells were protected from Salmonella-induced mortality. Therefore, our findings showed that Salmonella EV-associated antigen is a model platform for T-cell immune response infections. Our system could help prepare EV-antigen vaccines against scrub typhus in an easy and rapid manner.

Sortase A-Inhibitory Coumarins from the Folk Medicinal Plant Poncirus trifoliata.

Thirteen coumarins (1-13), including five new compounds (1-5), were isolated from the folk medicinal plant Poncirus trifoliata. Combined spectroscopic analyses revealed that coumarins 1-4 are bis-isoprenylated coumarins with diverse oxidation patterns, while 5 is an enantiomeric di-isoprenylated coumarin. The absolute configurations of the stereogenic centers in the isoprenyl chains were assigned through MTPA and MPA methods, and those of the known compounds triphasiol (6) and ponciol (7) were also assigned using similar methods. These coumarins inhibited significantly Staphylococcus aureus-derived sortase A (SrtA), a transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall in Gram-positive bacteria. The present results obtained indicated that the bioactivity and underlying mechanism of action of these coumarins are associated with the inhibition of SrtA-mediated S. aureus adhesion to eukaryotic cell matrix proteins including fibrinogen and fibronectin, thus potentially serving as SrtA inhibitors.

Threshold-based quantification of fatty degeneration in the supraspinatus muscle on MRI as an alternative method to Goutallier classification and single-voxel MR spectroscopy.

BACKGROUND: Conventional fat quantification methods for rotator cuff muscles have various limitations, such as inconsistent reliabilities of the Goutallier grades and need for advanced techniques in quantitative MRI sequences. We aimed to examine a threshold-based fat quantification method in the supraspinatus muscle on standard T1-weighted MR images and compare the threshold-based method with Goutallier grades and MR spectroscopy. METHODS: We retrospectively examined 38 symptomatic patients, who underwent T1 and T2-weighted fast spin-echo MR imaging and a single voxel spin-echo MR spectroscopy. The supraspinatus muscle and fossa were manually segmented in T1-weighted sagittal images and clustering-based thresholding was applied to quantify the fat fractions in the segmented areas using custom MATLAB software. Threshold-based fat fractions were compared with the Goutallier grades and MR spectroscopy fat/water ratios. A one-way analysis of variance and Pearson correlation were tested in the MATLAB software. RESULTS: Inter-observer reliability of threshold-based fat fractions for the supraspinatus muscle and fossa were 0.977 and 0.990 respectively, whereas the reliability of the Goutallier grading was 0.798. Threshold-based fat fractions in the supraspinatus fossa were significantly different between various Goutallier grades (one-way ANOVA, p < 0.001). Threshold-based fat fractions in the supraspinatus muscle strongly correlated with the MR spectroscopy fat/water ratio (Pearson correlation R-square = 0.83). CONCLUSIONS: Threshold-based fat quantification on standard T1-weighted MR images was highly reliable and produced comparable results to conventional Goutallier grades and MR spectroscopy fat/water ratios and could serve as an alternative method for accurate fat quantification in rotator cuff muscles.

A Randomized Trial of Focused Ultrasound Thalamotomy for Essential Tremor.

BACKGROUND: Uncontrolled pilot studies have suggested the efficacy of focused ultrasound thalamotomy with magnetic resonance imaging (MRI) guidance for the treatment of essential tremor. METHODS: We enrolled patients with moderate-to-severe essential tremor that had not responded to at least two trials of medical therapy and randomly assigned them in a 3:1 ratio to undergo unilateral focused ultrasound thalamotomy or a sham procedure. The Clinical Rating Scale for Tremor and the Quality of Life in Essential Tremor Questionnaire were administered at baseline and at 1, 3, 6, and 12 months. Tremor assessments were videotaped and rated by an independent group of neurologists who were unaware of the treatment assignments. The primary outcome was the between-group difference in the change from baseline to 3 months in hand tremor, rated on a 32-point scale (with higher scores indicating more severe tremor). After 3 months, patients in the sham-procedure group could cross over to active treatment (the open-label extension cohort). RESULTS: Seventy-six patients were included in the analysis. Hand-tremor scores improved more after focused ultrasound thalamotomy (from 18.1 points at baseline to 9.6 at 3 months) than after the sham procedure (from 16.0 to 15.8 points); the between-group difference in the mean change was 8.3 points (95% confidence interval [CI], 5.9 to 10.7; P<0.001). The improvement in the thalamotomy group was maintained at 12 months (change from baseline, 7.2 points; 95% CI, 6.1 to 8.3). Secondary outcome measures assessing disability and quality of life also improved with active treatment (the blinded thalamotomy cohort)as compared with the sham procedure (P<0.001 for both comparisons). Adverse events in the thalamotomy group included gait disturbance in 36% of patients and paresthesias or numbness in 38%; these adverse events persisted at 12 months in 9% and 14% of patients, respectively. CONCLUSIONS: MRI-guided focused ultrasound thalamotomy reduced hand tremor in patients with essential tremor. Side effects included sensory and gait disturbances. (Funded by InSightec and others; ClinicalTrials.gov number, NCT01827904.).

Gold Binding Peptide Identified from Microfluidic Biopanning: An Experimental and Molecular Dynamics Study.

Biopanning refers to the processes of screening peptides with a high affinity to a target material. Microfluidic biopanning has advantages compared to conventional biopanning which requires large amounts of the target material and involves inefficient multiple pipetting steps to remove nonspecific or low-affinity peptides. Here, we fabricate a microfluidic biopanning system to identify a new gold-binding peptide (GBP). A polydimethylsiloxane microfluidic device is fabricated and bonded to a glass slide with a gold pattern that is deposited by electron-beam evaporation. The microfluidic biopanning system can provide high adjustability in the washing step during the biopanning process because the liquid flow rate and the resulting shear stress can be precisely controlled. The surface plasmon resonance analysis shows that the binding affinity of the identified GBP is comparable to previously reported GBPs. Moreover, molecular dynamics simulations are performed to understand its binding affinity against the gold surface in detail. Theoretical calculations suggest that the association and dissociation rates of the GBPs depend on their sequence-dependent conformations and interactions with the gold surface. These findings provide insight into designing efficient biopanning tools and peptides with a high affinity for various target materials.

Effect of phosphate concentration, anions, heavy metals, and organic matter on phosphate adsorption from wastewater using anodized iron oxide nanoflakes.

Phosphorus is a necessary nutrient for the growth and survival of living beings. Nevertheless, an oversupply of phosphorus in wastewater results in eutrophication. Therefore, its removal from wastewater is important. However, coexisting components, such as anions, heavy metals, and organic matter, might inhibit the phosphate-adsorption mechanism by competing for the active surface sites of the adsorbent. In this study, iron oxide nanoflakes (INFs) were fabricated on iron foil via anodization. The rate of phosphate adsorption from wastewater onto INFs in the presence of three different coexisting components-anions, heavy metals, and organic matter-was evaluated. The morphology of the INFs was analyzed by X-ray diffraction, field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy. The phosphate adsorption equilibrium time using INFs was found to be 1 h. The Elovich model (R2 > 0.99) and the Langmuir model (R2 >0.95) respectively provided the best description of the adsorption kinetics and isotherm, suggesting the chemisorption nature of adsorption. The estimated adsorption capacity of the INFs was 21.5 mg-P g-1. The effect of anions (chloride, sulfate, nitrate, and carbonate) and heavy metals (Cd, As, Cr, and Pb) was studied at three different molar ratios (0.5:1, 1:1, and 1.5:1). The effect of different types of organic matter, such as citric acid, humic acid, and oxalic acid at concentrations of 100 and 200 mg L-1, was also examined. In five regeneration cycles, the total amount of phosphate adsorbed and desorbed, and the recovery percentage were 6.51 mg-P g-1, 5.16 mg-P g-1, and 79.24%, respectively.

Active Control of Inertial Focusing Positions and Particle Separations Enabled by Velocity Profile Tuning with Coflow Systems.

Inertial microfluidics has drawn much attention not only for its diverse applications but also for counterintuitive new fluid dynamic behaviors. Inertial focusing positions are determined by two lift forces, that is, shear gradient and wall-induced lift forces, that are generally known to be opposite in direction in the flow through a channel. However, the direction of shear gradient lift force can be reversed if velocity profiles are shaped properly. We used coflows of two liquids with different viscosities to produce complex velocity profiles that lead to inflection point focusing and alteration of inertial focusing positions; the number and the locations of focusing positions could be actively controlled by tuning flow rates and viscosities of the liquids. Interestingly, 3-inlet coflow systems showed focusing mode switching between inflection point focusing and channel face focusing depending on Reynolds number and particle size. The focusing mode switching occurred at a specific size threshold, which was easily adjustable with the viscosity ratio of the coflows. This property led to different-sized particles focusing at completely different focusing positions and resulted in highly efficient particle separation of which the separation threshold was tunable. Passive separation techniques, including inertial microfluidics, generally have a limitation in the control of separation parameters. Coflow systems can provide a simple and versatile platform for active tuning of velocity profiles and subsequent inertial focusing characteristics, which was demonstrated by active control of the focusing mode using viscosity ratio tuning and temperature changes of the coflows.

Size-Dependent Inertial Focusing Position Shift and Particle Separations in Triangular Microchannels.

A recent study of inertial microfluidics within nonrectangular cross-section channels showed that the inertial focusing positions changes with cross-sectional shapes; therefore, the cross-sectional shape can be a useful control parameter for microfluidic particle manipulations. Here, we conducted detail investigation on unique focusing position shift phenomena, which occurs strongly in channels with the cross-sectional shape of the isosceles right triangle. The top focusing positions shift along the channel walls to the direction away from the apex with increasing Reynolds number and decreasing particle size. A larger particle with its center further away from the side walls experiences shear gradient lift toward the apex, which leads to an opposite result with changes of Reynolds and particle size. The focusing position shift and the subsequent stabilization of corner focusing lead to changes in the number of focusing positions, which enables a novel method for microparticle separations with high efficiency (>95%) and resolution (<2 µm). The separation method based on equilibrium focusing; therefore, the operation is simple and no complex separation optimization is needed. Moreover, the separation threshold can be easily modulated with flow rate adjustment. Rare cell separation from blood cell was successfully demonstrated with spiked MCF-7 cells in blood by achieving the yield of ∼95% and the throughput of ∼106 cells/min.

Multiphoton structured thin-plane imaging with a single optical path.

Optical sectioning has become an indispensable technique for high-speed volumetric imaging in the past decade. Here we present a novel optical-sectioning method that produces a thin plane of illumination by exploiting the spatial and temporal properties of multiphoton excitation. Critically, the illumination and detection share the same optical path, as in a conventional epi-fluorescence microscope configuration. Therefore, the imaged sample can be prepared as for standard fluorescence microscopy. Our method also leads to a laterally structured illumination pattern, and this feature can be utilized in structured illumination microscopy to further enhance the imaging performance. We show an example of such an approach, which achieves axial resolution finer than confocal microscopy. We also demonstrate the potential of the new method for biological applications by performing three-dimensional imaging of living Caenorhabditis elegans.

Catholyte-Free Electrocatalytic CO2 Reduction to Formate.

Electrochemical reduction of carbon dioxide (CO2 ) into value-added chemicals is a promising strategy to reduce CO2 emission and mitigate climate change. One of the most serious problems in electrocatalytic CO2 reduction (CO2 R) is the low solubility of CO2 in an aqueous electrolyte, which significantly limits the cathodic reaction rate. This paper proposes a facile method of catholyte-free electrocatalytic CO2 reduction to avoid the solubility limitation using commercial tin nanoparticles as a cathode catalyst. Interestingly, as the reaction temperature rises from 303 K to 363 K, the partial current density (PCD) of formate improves more than two times with 52.9 mA cm-2 , despite the decrease in CO2 solubility. Furthermore, a significantly high formate concentration of 41.5 g L-1 is obtained as a one-path product at 343 K with high PCD (51.7 mA cm-2 ) and high Faradaic efficiency (93.3 %) via continuous operation in a full flow cell at a low cell voltage of 2.2 V.

Nanofluidic chip for liquid TEM cell fabricated by parylene and silicon nitride direct bonding.

Despite the importance of nanofluidic transmission electron microscope (TEM) chips, a simple fabrication method has yet to be developed due to the difficulty of wafer bonding techniques using a nanoscale thick bonding layer. We present a simple and robust wafer scale bonding technique using parylene as a bonding layer. A nanoscale thick parylene layer was deposited on a silicon nitride (SiN) wafer and patterned to construct nanofluidic channels. The patterned parylene layer was directly bonded to another SiN wafer by thermal surface activation and bonding, with a bonding strength of ∼3 MPa. Fourier transform infrared spectroscopy showed that carbon-oxygen bonds were generated by thermal activation. We demonstrated TEM imaging of gold nanoparticles suspended in liquid using the fabricated nanofluidic chip.

Extracellular vesicles derived from Gram-negative bacteria, such as Escherichia coli, induce emphysema mainly via IL-17A-mediated neutrophilic inflammation.

Recent evidence indicates that Gram-negative bacteria-derived extracellular vesicles (EVs) in indoor dust can evoke neutrophilic pulmonary inflammation, which is a key pathology of chronic obstructive pulmonary disease (COPD). Escherichia coli is a ubiquitous bacterium present in indoor dust and secretes nanometer-sized vesicles into the extracellular milieu. In the current study, we evaluated the role of E. coli-derived EVs on the development of COPD, such as emphysema. E. coli EVs were prepared by sequential ultrafiltration and ultracentrifugation. COPD phenotypes and immune responses were evaluated in C57BL/6 wild-type (WT), IFN-γ-deficient, or IL-17A-deficient mice after airway exposure to E. coli EVs. The present study showed that indoor dust from a bed mattress harbors E. coli EVs. Airway exposure to E. coli EVs increased the production of proinflammatory cytokines, such as TNF-α and IL-6. In addition, the repeated inhalation of E. coli EVs for 4 wk induced neutrophilic inflammation and emphysema, which are associated with enhanced elastase activity. Emphysema and elastase activity enhanced by E. coli EVs were reversed by the absence of IFN-γ or IL-17A genes. In addition, during the early period, lung inflammation is dependent on IL-17A and TNF-α, but not on IFN-γ, and also on TLR4. Moreover, the production of IFN-γ is eliminated by the absence of IL-17A, whereas IL-17A production is not abolished by IFN-γ absence. Taken together, the present data suggest that E. coli-derived EVs induce IL-17A-dependent neutrophilic inflammation and thereby emphysema, possibly via upregulation of elastase activity.

Remote sensing of pressure inside deformable microchannels using light scattering in Scotch tape.

We present a simple but effective method to measure the pressure inside a deformable microchannel using laser scattering in a translucent Scotch tape. Our idea exploits the fact that the speckle pattern generated by a turbid layer is sensitive to the changes in the optical wavefront of an impinging beam. A change in the internal pressure of a channel deforms the elastic channel, which can be detected by measuring the speckle patterns of a coherent laser beam that has passed through the channel and the Scotch tape. We demonstrate that with a proper calibration, internal pressure can be remotely sensed with the resolution of 0.1 kPa within a pressure range of 0-3 kPa after calibration.

Direct transfer of multilayer graphene grown on a rough metal surface using PDMS adhesion engineering.

The direct transfer of graphene using polydimethylsiloxane (PDMS) stamping has advantages such as a 'pick-and-place' capability and no chemical residue problems. However, it is not easy to apply direct PDMS stamping to graphene grown via chemical vapor deposition on rough, grainy metal surfaces due to poor contact between the PDMS and graphene. In this study, graphene consisting of a mixture of monolayers and multiple layers grown on a rough Ni surface was directly transferred without the use of an adhesive layer. Liquid PDMS was cured on graphene to effect a conformal contact with the graphene. A fast release of graphene from substrate was achieved by carrying out wet-etching-assisted mechanical peeling. We also carried out a thermal post-curing of PDMS to control the level of adhesion between PDMS and graphene and hence facilitate a damage-free release of the graphene. Characterization of the transferred graphene by micro-Raman spectroscopy, SEM/EDS and optical microscopy showed neither cracks nor contamination from the transfer. This technique allows a fast and simple transfer of graphene, even for multilayer graphene grown on a rough surface.