Deep learning enables reference-free isotropic super-resolution for volumetric fluorescence microscopy.

Volumetric imaging by fluorescence microscopy is often limited by anisotropic spatial resolution, in which the axial resolution is inferior to the lateral resolution. To address this problem, we present a deep-learning-enabled unsupervised super-resolution technique that enhances anisotropic images in volumetric fluorescence microscopy. In contrast to the existing deep learning approaches that require matched high-resolution target images, our method greatly reduces the effort to be put into practice as the training of a network requires only a single 3D image stack, without a priori knowledge of the image formation process, registration of training data, or separate acquisition of target data. This is achieved based on the optimal transport-driven cycle-consistent generative adversarial network that learns from an unpaired matching between high-resolution 2D images in the lateral image plane and low-resolution 2D images in other planes. Using fluorescence confocal microscopy and light-sheet microscopy, we demonstrate that the trained network not only enhances axial resolution but also restores suppressed visual details between the imaging planes and removes imaging artifacts.

A Sensing Device with the Optical Temperature Sensors-Based Quad-RX Module and a Security Module.

In this paper, we present a sensing device with the optical temperature sensors-based quad receiver (Quad-RX) module and a security module. In addition, in order to prevent cyberattacks on critical national infrastructures and key facilities, we implemented symmetric-key and secure hash algorithm-based hardware security modules in the key elements of the sensing device. A preliminary test was conducted prior to a field trial to verify the performance of the developed sensing device. The accuracy and stability of the sensing device were then verified for 1 month in a field test at facilities for energy storage systems and photovoltaic converters in sewage treatment plants.

T cell migration in microchannels densely packed with T cells.

T cells migrate diverse microenvironments of the body to mount antigen-specific immune responses. T cell activation, a key initial process for antigen-specific immune responses, occur in secondary lymphoid organs such as spleens and lymph nodes where high density of T cells migrates rapidly through the reticular networks formed by stromal cells. In vitro model system recapitulating key characteristics of secondary lymphoid organs, confined spaces densely packed with rapidly migrating cells, would be useful to investigate mechanisms of T cell migration. In this study, we devised a method to fabricate microchannels densely packed with T cells. Microchannel arrays with fixed height (4 µm) and length (1.5 mm) and various widths (15~80 µm) were fabricated in between trapezoid-shaped reservoirs that facilitated T cell sedimentation near microchannel entries. Microchannel surface chemistry and filling time were optimized to achieve high packing density (0.89) of T cell filling within microchannels. Particle image velocimetry (PIV) analysis method was employed to extract velocity field of microchannels densely packed with T cells. Using velocity field information, various motility parameters were further evaluated to quantitatively assess the effects of microchannel width and media tonicity on T cell motility within cell dense microenvironments.

Ultra-thin, aligned, free-standing nanofiber membranes to recapitulate multi-layered blood vessel/tissue interface for leukocyte infiltration study.

Leukocyte infiltration plays critical roles in tissue inflammation for pathogen clearance and tumor eradication. This process is regulated by complex microenvironments in blood vessels, including inflamed endothelium, blood flow, and perivascular components. The role of perivascular components in leukocyte infiltration has not been systematically investigated until recently mostly due to lack of technology. In this work, we developed a three-dimensional multi-layered blood vessel/tissue model with a nanofiber membrane, enabling real-time visualization of dynamic leukocyte infiltration and subsequent interaction with perivascular macrophages. We directly fabricated a highly aligned, free-standing nanofiber membrane with an ultra-thin thickness of ∼1 µm in microfluidic systems. Coating the nanofiber membrane with matrigel showed synergetic topographical and biochemical effects on the reconstitution of a well-aligned endothelial monolayer on the membrane. Our 3D multi-layered blood vessel/tissue model will offer a powerful and versatile tool for investigating the mechanism of leukocyte tissue infiltration and subsequent immune responses.

Multifunctional Microwell Arrays for Single Cell Level Functional Analysis of Lymphocytes.

Functional analysis of lymphocytes is important for development of vaccines and diagnosis/treatment of various immune-related diseases. In this review, we describe multifunctional microwell arrays that enable functional analysis of lymphocytes at the single cell level. We first discuss key parameters for microwell array design. Then, we describe how different types of multifunctional microwell arrays were developed for various applications, including live cell imaging of lymphocyte activation, proliferation, and differentiation, and analyses of effector functions such as cytokine secretion and target cell lysis. Incorporation of novel surface chemistries and functional materials into microwell arrays for enhancing sensing capabilities will widen applications of this technology. Multifunctional microwell arrays will be a powerful tool for the development of novel therapeutics against immune-related diseases, in particular, for cancer immunotherapy.

Turning behaviors of T cells climbing up ramp-like structures are regulated by myosin light chain kinase activity and lamellipodia formation.

T cells navigate diverse microenvironments to perform immune responses. Micro-scale topographical structures within the tissues, which may inherently exist in normal tissues or may be formed by inflammation or injury, can influence T cell migration, but how T cell migration is affected by such topographical structures have not been investigated. In this study, we fabricated ramp-like structures with a 5 µm height and various slopes, and observed T cells climbing up the ramp-like structures. T cells encountering the ramp-like structures exhibited MLC accumulation near head-tail junctions contacting the ramp-like structures, and made turns to the direction perpendicular to the ramp-like structures. Pharmacological study revealed that lamellipodia formation mediated by arp2/3 and contractility regulated by myosin light chain kinase (MLCK) were responsible for the intriguing turning behavior of T cells climbing the ramp-like structures. Arp2/3 or MLCK inhibition substantially reduced probability of T cells climbing sharp-edged ramp-like structures, indicating intriguing turning behavior of T cells mediated by lamellipodia formation and MLCK activity may be important for T cells to access inflamed or injured tissues with abrupt topographical changes.

Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8+ T Cells.

The voltage-gated potassium channel, Kv1.3, and the Ca2+-activated potassium channel, KCa3.1, regulate membrane potentials in T cells, thereby controlling T cell activation and cytokine production. However, little is known about the expression and function of potassium channels in human effector memory (EM) CD8+ T cells that can be further divided into functionally distinct subsets based on the expression of the interleukin (IL)-7 receptor alpha (IL-7Rα) chain. Herein, we investigated the functional expression and roles of Kv1.3 and KCa3.1 in EM CD8+ T cells that express high or low levels of the IL-7 receptor alpha chain (IL-7Rαhigh and IL-7Rαlow, respectively). In contrast to the significant activity of Kv1.3 and KCa3.1 in IL-7Rαhigh EM CD8+ T cells, IL-7Rαlow EM CD8+ T cells showed lower expression of Kv1.3 and insignificant expression of KCa3.1. Kv1.3 was involved in the modulation of cell proliferation and IL-2 production, whereas KCa3.1 affected the motility of EM CD8+ T cells. The lower motility of IL-7Rαlow EM CD8+ T cells was demonstrated using transendothelial migration and motility assays with intercellular adhesion molecule 1- and/or chemokine stromal cell-derived factor-1α-coated surfaces. Consistent with the lower migration property, IL-7Rαlow EM CD8+ T cells were found less frequently in human skin. Stimulating IL-7Rαlow EM CD8+ T cells with IL-2 or IL-15 increased their motility and recovery of KCa3.1 activity. Our findings demonstrate that Kv1.3 and KCa3.1 are differentially involved in the functions of EM CD8+ T cells. The weak expression of potassium channels in IL-7Rαlow EM CD8+ T cells can be revived by stimulation with IL-2 or IL-15, which restores the associated functions. This study suggests that IL-7Rαhigh EM CD8+ T cells with functional potassium channels may serve as a reservoir for effector CD8+ T cells during peripheral inflammation.

Unbiased Metabolite Profiling of Schizophrenia Fibroblasts under Stressful Perturbations Reveals Dysregulation of Plasmalogens and Phosphatidylcholines.

We undertook an unbiased metabolite profiling of fibroblasts from schizophrenia patients and healthy controls to identify metabolites and pathways that are dysregulated in disease, seeking to gain new insights into the disease biology of schizophrenia and to discover potential disease-related biomarkers. We measured polar and nonpolar metabolites in the fibroblasts under normal conditions and under two stressful physiological perturbations: growth in low-glucose media and exposure to the steroid hormone dexamethasone. We found that metabolites that were significantly different between schizophrenia and control subjects showed separation of the two groups by partial least-squares discriminant analysis methods. This separation between schizophrenia and healthy controls was more robust with metabolites identified under the perturbation conditions. The most significant individual metabolite differences were also found in the perturbation experiments. Metabolites that were significantly different between schizophrenia and healthy controls included a number of plasmalogens and phosphatidylcholines. We present these results in the context of previous reports of metabolic profiling of brain tissue and plasma in schizophrenia. These results show the applicability of metabolite profiling under stressful perturbations to reveal cellular pathways that may be involved in disease biology.

Perturbational Profiling of Metabolites in Patient Fibroblasts Implicates α-Aminoadipate as a Potential Biomarker for Bipolar Disorder.

Many studies suggest the presence of aberrations in cellular metabolism in bipolar disorder. We studied the metabolome in bipolar disorder to gain insight into cellular pathways that may be dysregulated in bipolar disorder and to discover evidence of novel biomarkers. We measured polar and nonpolar metabolites in fibroblasts from subjects with bipolar I disorder and matched healthy control subjects, under normal conditions and with two physiologic perturbations: low-glucose media and exposure to the stress-mediating hormone dexamethasone. Metabolites that were significantly different between bipolar and control subjects showed distinct separation by principal components analysis methods. The most statistically significant findings were observed in the perturbation experiments. The metabolite with the lowest p value in both the low-glucose and dexamethasone experiments was α-aminoadipate, whose intracellular level was consistently lower in bipolar subjects. Our study implicates α-aminoadipate as a possible biomarker in bipolar disorder that manifests under cellular stress. This is an intriguing finding given the known role of α-aminoadipate in the modulation of kynurenic acid in the brain, especially as abnormal kynurenic acid levels have been implicated in bipolar disorder.

Roles of endothelial A-type lamins in migration of T cells on and under endothelial layers.

Stiff nuclei in cell-dense microenvironments may serve as distinct biomechanical cues for cell migration, but such a possibility has not been tested experimentally. As a first step addressing this question, we altered nuclear stiffness of endothelial cells (ECs) by reducing the expression of A-type lamins using siRNA, and investigated the migration of T cells on and under EC layers. While most T cells crawling on control EC layers avoided crossing over EC nuclei, a significantly higher fraction of T cells on EC layers with reduced expression of A-type lamins crossed over EC nuclei. This result suggests that stiff EC nuclei underlying T cells may serve as "duro-repulsive" cues to direct T cell migration toward less stiff EC cytoplasm. During subendothelial migration under EC layers with reduced expression of A-type lamins, T cells made prolonged contact and substantially deformed EC nuclei, resulting in reduced speed and directional persistence. This result suggests that EC nuclear stiffness promotes fast and directionally persistent subendothelial migration of T cells by allowing minimum interaction between T cells and EC nuclei.

Dynamic Micropatterning of Cells on Nanostructured Surfaces Using a Cell-friendly Photoresist.

Cellular dynamics under complex topographical microenvironments are important for many biological processes in development and diseases, but systematic investigation has been limited due to the lack of technology. Herein, we developed a new dynamic cell patterning method based on a cell-friendly photoresist polymer that allows in situ control of cell dynamics on nanostructured surfaces. Using this method, we quantitatively compared the spreading dynamics of cells on nanostructured surfaces to those on flat surfaces. Furthermore, we investigated how cells behaved when they simultaneously encountered two topographically distinct surfaces during spreading. This method will allow many exciting opportunities in the fundamental study of cellular dynamics.

A comparative study on collagen type I and hyaluronic acid dependent cell behavior for osteochondral tissue bioprinting.

Bioprinting is a promising technique for engineering composite tissues, such as osteochondral tissues. In this study, as a first step toward bioprinting-based osteochondral tissue regeneration, we systematically examined the behavior of chondrocytes and osteoblasts to hyaluronic acid (HA) and type I collagen (Col-1) hydrogels. First, we demonstrated that cells on hydrogels that were comprised of major native tissue extracellular matrix (ECM) components (i.e. chondrocytes on HA hydrogels and osteoblasts on Col-1 hydrogels) exhibited better proliferation and cell function than cells on non-native ECM hydrogels (i.e., chondrocytes on Col-1 hydrogels and osteoblasts on HA hydrogels). In addition, cells located near their native ECM hydrogels migrated towards them. Finally, we bioprinted three-dimensional (3D) osteochondral tissue-mimetic structures composed of two compartments, osteoblast-encapsulated Col-1 hydrogels and chondrocyte-encapsulated HA hydrogels, and found viability and functions of each cell type were well maintained within the 3D structures up to 14 days in vitro. These results suggest that with proper choice of hydrogel materials, bioprinting-based approaches can be successfully applied for osteochondral tissue regeneration.

Migration of T cells on surfaces containing complex nanotopography.

T cells navigate complex microenvironments to initiate and modulate antigen-specific immune responses. While recent intravital microscopy study revealed that migration of T cells were guided by various tissue microstructures containing unique nanoscale topographical structures, the effects of complex nanotopographical structures on the migration of T cells have not been systematically studied. In this study, we fabricated surfaces containing nanoscale zigzag structures with various side lengths and turning angles using UV-assisted capillary force lithography and motility of T cells on zigzag patterned surfaces was studied. Motility of T cells was mostly affected by the turning angle, not by the side length, of the zigzag structures. In particular, motility behaviors of T cells near interfaces formed by turning points of zigzag patterns were significantly affected by turning angles. For obtuse turning angles, most of the T cells smoothly crossed the interfaces, but as the turning angle decreased, a substantial fraction of the T cells migrated along the interfaces. When the formation of lamellipodia, thin sheet-like structures typically generated at the leading edges of migrating cells by actin polymerization-driven membrane protrusion, was inhibited by an Arp2/3 inhibitor CK-636, a substantial fraction of T cells on those surfaces containing zigzag patterns with an acute turning angle were trapped at the interfaces formed by the turning points of the zigzag patterns. This result suggests that thin, wide lamellipodia at the leading edges of T cells play critical roles in motility of T cells in complex topographical microenvironments.

Nanotopography-guided migration of T cells.

T cells navigate a wide variety of tissues and organs for immune surveillance and effector functions. Although nanoscale topographical structures of extracellular matrices and stromal/endothelial cell surfaces in local tissues may guide the migration of T cells, there has been little opportunity to study how nanoscale topographical features affect T cell migration. In this study, we systematically investigated mechanisms of nanotopography-guided migration of T cells using nanoscale ridge/groove surfaces. The velocity and directionality of T cells on these nanostructured surfaces were quantitatively assessed with and without confinement, which is a key property of three-dimensional interstitial tissue spaces for leukocyte motility. Depending on the confinement, T cells exhibited different mechanisms for nanotopography-guided migration. Without confinement, actin polymerization-driven leading edge protrusion was guided toward the direction of nanogrooves via integrin-mediated adhesion. In contrast, T cells under confinement appeared to migrate along the direction of nanogrooves purely by mechanical effects, and integrin-mediated adhesion was dispensable. Therefore, surface nanotopography may play a prominent role in generating migratory patterns for T cells. Because the majority of cells in periphery migrate along the topography of extracellular matrices with much lower motility than T cells, nanotopography-guided migration of T cells would be an important strategy to efficiently perform cell-mediated immune responses by increasing chances of encountering other cells within a given amount of time.

Molecular characterisation and expression analysis of a fish-egg lectin in rock bream, and its response to bacterial or viral infection.

A fish-egg lectin, RbFEL, was identified from rock bream (Oplegnathus fasciatus) and its expression analysed. In both vertebrates and invertebrates, carbohydrate-binding proteins (lectins) play an important role in innate immunity against microbial invasion. Here, we report the cloning of a fish-egg lectin from rock bream using a combination of expression sequence tag (EST) analyses. The full-length cDNA of RbFEL is composed of 1512-bp with a 780-bp ORF that encodes 259 amino acid residues. The deduced polypeptide exhibits six conserved residues of the FEL family. All cysteine positions in each domain were completely conserved. Reverse transcription quantitative real-time PCR analysis of the tissues revealed that RbFEL mRNA was abundantly expressed in liver, moderately expressed in head kidney and rarely expressed in other tissues. Expression analyses of time series sampled fertilised eggs showed that expression gradually increased 1, 3, 12, 24 and 36 h after fertilisation. In addition, RbFEL expression was significantly up-regulated in rock bream challenged with Edwardsiella tarda, Streptococcus iniae and red sea bream iridovirus (RSIV). These results suggest that RbFEL is a member of the egg-lectin family and is involved in the innate immune response in rock bream.

Molecular identification and expression analysis of cathepsins O and S from rock bream, Oplegnathus fasciatus.

Cathepsins are lysosomal cysteine proteases belonging to the papain family, members of which play important roles in normal metabolism for maintenance of cellular homeostasis. Rock bream (Oplegnathus fasciatus) cathepsin O and S (RbCTSO and RbCTSS, respectively) cDNAs were identified by expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCTSO cDNA (1698 bp) contained an open reading frame (ORF) of 1017 bp encoding 338 amino acids. The full-length RbCTSS cDNA was 1401 bp in length and contained an ORF of 1014 bp encoding 337 amino acids. RbCTSO was significantly expressed in the liver, peripheral blood lymphocytes (PBLs) and spleen. On the other hand, RbCTSS showed significant expression in the liver, trunk kidney, muscle and gills. Real-time RT-PCR was used to examine RbCTSO and RbCTSS mRNA expression in several tissues (kidney, spleen, liver and gill) under conditions of bacterial and viral challenge. Experimental infection of rock bream with Streptococcus iniae and red sea bream iridovirus (RSIV) resulted in significant increases in RbCTSO and RbCTSS mRNA levels in the tissues.

Molecular cloning and characterisation of the rock bream, Oplegnathus fasciatus, Fas (CD95/APO-1), and its expression analysis in response to bacterial or viral infection.

Fas belongs to the tumour necrosis factor (TNF) receptor superfamily and can transmit a death signal leading to apoptosis. In the present study, we isolated the full-length cDNA for rock bream (Oplegnathus fasciatus) Fas (RbFas). The full-length RbFas cDNA was 1770 bp long and contained an open reading frame of 957 bp that encoded 319 amino acid residues with a predicted molecular mass of 35.1 kDa. The 319 amino-acid predicted RbFas sequence is homologous to other Fas sequences, contains three cysteine-rich domains and a death domain (DD) and two potential N-glycosylation sites. Expression of RbFas mRNA was detected in nine different tissues from healthy rock bream and was the highest in red blood cells. In analyses of mitogen-stimulated RbFas expression in peripheral blood leucocytes, expression of RbFas mRNA was observed between 1 and 36 h after stimulation with LPS, and 1 and 3 h stimulation with poly I:C. In the case of bacterial injection, the RbFas transcript peaked 6 h after injection in both the kidney and the spleen. Otherwise, the RbFas transcript peaked after 1 h in spleen and 6 h in kidney following injection with RSIV.

Microwave assisted nanofibrous air filtration for disinfection of bioaerosols.

Airborne biological agents, albeit intentionally released or naturally occurring, pose one of the biggest threats to public health and security. In this study, a microwave assisted nanofibrous air filtration system was developed to disinfect air containing airborne pathogens. Aerosolized E. coli vegetative cells and B. subtilis endospores, as benign surrogates of pathogens, were collected on nanofibrous filters and treated by microwave irradiation. Both static on-filter and dynamic in-flight tests were carried out. Results showed that E. coli cells were efficiently disinfected in both static and in-flight tests, whereas B. subtilis endospores were more resistant to this treatment. Microwave power level was found to be the major factor determining the effectiveness of disinfection. Both thermal and non-thermal effects of microwave irradiation contributed to the disinfection. Reducing flow velocity to decrease heat loss yielded higher disinfection efficiency.

Linear FBG Temperature Sensor Interrogation with Fabry-Perot ITU Multi-wavelength Reference.

The equidistantly spaced multi-passbands of a Fabry-Perot ITU filter are used as an efficient multi-wavelength reference for fiber Bragg grating sensor demodulation. To compensate for the nonlinear wavelength tuning effect in the FBG sensor demodulator, a polynomial fitting algorithm was applied to the temporal peaks of the wavelength-scanned ITU filter. The fitted wavelength values are assigned to the peak locations of the FBG sensor reflections, obtaining constant accuracy, regardless of the wavelength scan range and frequency. A linearity error of about 0.18% against a reference thermocouple thermometer was obtained with the suggested method.

Accuracy improvement in peak positioning of spectrally distorted fiber Bragg grating sensors by Gaussian curve fitting.

To improve measurement accuracy of spectrally distorted fiber Bragg grating temperature sensors, reflection profiles were curve fitted to Gaussian shapes, of which center positions were transformed into temperature information. By applying the Gaussian curve-fitting algorithm in a tunable bandpass filter demodulation scheme, approximately 0.3 degrees C temperature resolution was obtained with a severely distorted grating sensor, which was much better than that obtained using the highest peak search algorithm. A binary search was also used to retrieve the optimal fitting curves with the least amount of processing time.