Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems.

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.

Endoplasmic Reticulum Stress Response Mediator IRE-1α Promotes Host Dendritic Cells in Graft-versus-Host Disease Development.

Allogeneic hematopoietic cell transplantation is an effective treatment for hematologic malignancies, but the complications such as graft-versus-host disease (GVHD) can limit its benefit. The conditioning regimens before transplant, including chemotherapy or irradiation, can trigger endoplasmic reticulum stress. IRE-1α is a major endoplasmic reticulum stress mediator that can further activate both spliced XBP-1 (XBP-1s) and regulated IRE-1-dependent decay (RIDD). IRE-1α-XBP-1s signaling controls dendritic cell (DC) differentiation and Ag presentation, crucial in GVHD progression. In this study, we used DC-specific XBP-1-deficient mice as donors or recipients and observed that XBP-1s was crucial for host DCs in the induction of GVHD but dispensable for the graft-versus-leukemia response. To specifically target IRE-1α in the host, we treated recipient mice with the IRE-1α inhibitor B-I09 for 3 d prior to bone marrow transplantation, which significantly suppressed GVHD development while maintaining the graft-versus-leukemia effect. XBP-1-deficient or BI09-treated recipients showed reduced DC survival after irradiation and bone marrow transplantation. Inhibition of IRE-1α also led to a reduction in DC alloreactivity, subsequently decreasing the proliferation and activation of allogeneic T cells. With further study using RIDD-deficient DCs, we observed that RIDD was also required for optimal DC activation. Taken together, XBP-1s and RIDD both promote host DC survival and alloreactivity that contribute to GVHD development.

The Absence of IL-12Rß2 Expression on Recipient Nonhematopoietic Cells Diminishes Acute Graft-versus-Host Disease in the Gastrointestinal Tract.

The gastrointestinal (GI) tract is a frequent target organ in acute graft-versus-host disease (aGVHD), which can determine the morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Donor T cells recognize allogeneic Ags presented by host APCs, proliferate, and differentiate into Th1 and Th17 cells that drive GVHD pathogenesis. IL-12 has been shown to play an important role in amplifying the allogeneic response in preclinical and clinical studies. This study demonstrates that IL-12Rß2 expression on recipient nonhematopoietic cells is required for optimal development of aGVHD in murine models of allo-HCT. aGVHD attenuation by genetic depletion of IL-12R signaling is associated with reduced MHC class II expression by intestinal epithelial cells and maintenance of intestinal integrity. We verified IL-12Rß2 expression on activated T cells and in the GI tract. This study, to our knowledge, reveals a novel function of IL-12Rß2 in GVHD pathogenesis and suggests that selectively targeting IL-12Rß2 on host nonhematopoietic cells may preserve the GI tract after allo-HCT.

Near eye display based on multiplexed retinal projections for robust compensation of eye pupil variance.

Among various specifications of near eye display (NED) devices, a compact formfactor is essential for comfortable user experience but also the hardest one to accomplish due to the slowest progresses. A pinhole/pinlight array based light-field (LF) technique is considered as one of the candidates to achieve that goal without thicker and heavier refractive optics. Despite those promising advantages, however, there are critical issues, such as dark spots and contrast distortion, which degrade the image quality because of the vulnerability of the LF retinal image when the observer's eye pupil size changes. Regardless of previous attempts to overcome those artifacts, it was impossible to resolve both issues due to their trade-off relation. In this paper, in order to resolve them simultaneously, we propose a concept of multiplexed retinal projections to integrate the LF retinal image through rotating transitions of refined and modulated elemental images for robust compensation of eye pupil variance with improved conservation of contrast distribution. Experimental demonstrations and quantitative analysis are also provided to verify the principle.

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA.

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

Non-hogel-based computer generated hologram with occlusion processing between the foreground light field and background hologram.

A novel technique is proposed to process the occlusion of a background hologram when synthesizing a front scene hologram from its light field. Unlike conventional techniques which process the occlusion in the light field domain after converting the background hologram to its light field, the proposed technique directly processes the occlusion between different domains, i.e., the background hologram and foreground light field. The key idea is to consider the background hologram as a carrier wave illuminating the front scene when synthesizing the front scene hologram from its light field. The proposed technique is not only computationally efficient as it does not require conversion between the light field and hologram domains but also accurate because all angular information of the background hologram and foreground light field is naturally considered in the occlusion processing. The proposed technique was verified by numerical synthesis and reconstruction.

Flicker-free dual-volume augmented reality display using a pixelated interwoven integral floating technique with a geometric phase lens.

A geometric phase (GP) integral floating display can provide multifocal three-dimensional (3D) augmented reality (AR) images with enhanced depth expression by switching the focal modes of the GP lens via polarization control. However, using temporal multiplexing to switch between the focal modes of GP optics causes flickering as each 3D AR image is fully presented in different frames and their temporal luminance profile becomes easily recognizable, particularly as the number of available focal modes increases. Here, we propose a novel integral floating technique to generate pixelated interwoven 3D AR images; a half of each image is spatially mixed with another and presented in both focal modes simultaneously to resolve the flickering issue. The principle was verified via experimental demonstration and optically measured data.

Self-organized developmental patterning and differentiation in cerebral organoids.

Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain organizing centers that express secreted growth factors, which may be involved in dorso-ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro.

The Oral Administration of Sanguisorba officinalis Extract Improves Physical Performance through LDHA Modulation.

Muscle fatigue is induced by an acute or chronic physical performance inability after excessive physical activity often associated with lactate accumulation, the end-product of glycolysis. In this study, the water-extracted roots of Sanguisorba officinalis L., a herbal medicine traditionally used for inflammation and diarrhea, reduced the activities of lactate dehydrogenase A (LDHA) in in vitro enzyme assay myoblast C2C12 cells and murine muscle tissue. Physical performance measured by a treadmill test was improved in the S. officinalis-administrated group. The analysis of mouse serum and tissues showed significant changes in lactate levels. Among the proteins related to energy metabolism-related physical performance, phosphorylated-AMP-activated protein kinase alpha (AMPKα) and peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC-1α) levels were enhanced, whereas the amount of LDHA was suppressed. Therefore, S. officinalis might be a candidate for improving physical performance via inhibiting LDHA and glycolysis.

Compensation of color breaking in bi-focal depth-switchable integral floating augmented reality display with a geometrical phase lens.

A bi-focal integral floating system using a geometrical phase (GP) lens can provide switchable integrated spaces with enhanced three-dimensional (3D) augmented reality (AR) depth expression. However, due to the chromatic aberration properties of the GP lens implemented for the switchable depth-floating 3D images, the floated 3D AR images with the red/green/blue (R/G/B) colors are formed at different depth locations with different magnification effects, which causes color breaking. In this paper, we propose a novel technique to resolve the color breaking problem by integrating the R/G/B elemental images with compensated depths and sizes along with experiments to demonstrate the improved results. When we evaluated the color differences of the floated 3D AR images based on CIEDE2000, the experimental results of the depth-switchable integral floating 3D AR images showed that the color accuracies were greatly improved after applying a pre-compensation scheme to the R/G/B sub-images in both concave and convex lens operation modes of the bi-focal switching GP floating lens.

Design of 45° periscopic visual field expansion device for peripheral field loss.

Patients with visual field loss have difficulty in mobility due to collision with pedestrians/obstacles from the blind side. In order to retrieve the lost visual field, prisms which deflect the field from the blind to the seeing side, have been widely used. However, the deflection power of current clinical Fresnel prisms is limited to ~30° and only provides a 5° eye scanning range to the blind side. This is not sufficient to avoid collision and results in increasing demands for a device with a higher power. In this paper, we propose a novel design and optimization of a higher power prism-like device (cascaded structure of mirror pairs filled with high refractive index) and verify enhanced expansion of up to 45° in optical ray tracing and photorealistic simulations.

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules.

Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1-3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.

Luteolin inhibits recruitment of monocytes and migration of Lewis lung carcinoma cells by suppressing chemokine (C-C motif) ligand 2 expression in tumor-associated macrophage.

Tumor-associated macrophages (TAMs) play pivotal roles in the progression of cancer. In order to investigate a novel candidate that inhibits the tumor-supporting M2-like phenotype of TAMs, a murine macrophage cell line RAW 264.7 cells were treated with interleukin (IL)-4. Luteolin inhibited phosphorylation of signal transducer and activator of transcription 6 (STAT6), a main downstream signal of IL-4, and reduced the expression of the M2-associated genes. In addition, Luminex multiplex analysis for secreted cytokines revealed that IL-4-enhanced secretion of chemokine (C-C motif) ligand 2 (CCL2) was reduced by luteolin treatment. IL-4-stimulated migration of monocyte, THP-1 cells, was inhibited by luteolin treatment and recovered by recombinant CCL2 supplement. Moreover, luteolin decreased migration of Lewis lung carcinoma cells in a CCL2-dependent manner. Given the important role of the TAM phenotype in the tumor microenvironment, inhibitory effect of luteolin on the monocyte recruitment and cancer migration via suppression of the TAM-secreted CCL2 may suggest a novel therapeutic approach to treat malignant tumors.

Abiotic stress of ambient cold temperature regulates the host receptivity to pathogens by cell surfaced sialic acids.

Ambient cold temperature, as an abiotic stress, regulates the survival, stability, transmission, and infection of pathogens. However, the effect of cold temperature on the host receptivity to the pathogens has not been fully studied. In this study, the expression of terminal α-2,3- and α-2,6-sialic acids were increased in murine lung tissues, especially bronchial epithelium, by exposure to cold condition. The expression of several sialyltransferases were also increased by exposure to cold temperature. Furthermore, in human bronchial epithelial BEAS-2B cells, the expressions of α-2,3- and α-2,6-sialic acids, and mRNA levels of sialyltransferases were increased in the low temperature condition at 33 °C. On the other hand, the treatment of Lith-Gly, a sialyltransferase inhibitor, blocked the cold-induced expression of sialic acids on surface of BEAS-2B cells. The binding of influenza H1N1 hemagglutinin (HA) toward BEAS-2B cells cultured at low temperature condition was increased, compared to 37 °C. In contrast, the cold-increased HA binding was blocked by treatment of lithocholicglycine and sialyl-N-acetyl-D-lactosamines harboring α-2,3- and α-2,6-sialyl motive. These results suggest that the host receptivity to virus at cold temperature results from the expressions of α-2,3- and α-2,6-sialic acids through the regulation of sialyltransferase expression.

Estimation of axial curvature of anterior sclera: correlation between axial length and anterior scleral curvature as affected by angle kappa.

BACKGROUND: Though the development and fitting of scleral contact lenses are expanding steadily, there is no simple method to provide scleral metrics for scleral contact lens fitting yet. The aim of this study was to establish formulae for estimation of the axial radius of curvature (ARC) of the anterior sclera using ocular biometric parameters that can be easily obtained with conventional devices. METHODS: A semi-automated stitching method and a computational analysis tool for calculating ARC were developed by using the ImageJ and MATLAB software. The ARC of all the ocular surface points were analyzed from the composite horizontal cross-sectional images of the right eyes of 24 volunteers; these measurements were obtained using anterior segment optical coherence tomography for a previous study (AS-OCT; Visante). Ocular biometric parameters were obtained from the same volunteers with slit-scanning topography and partial coherence interferometry. Correlation analysis was performed between the ARC at 8 mm to the axis line (ARC[8]) and other ocular parameters (including age). With ARC obtained on several nasal and temporal points (7.0, 7.5, 8.0, 8.5, and 9.0 mm from the axis line), univariate and multivariate linear regression analyses were performed to develop a model for estimating ARC with the help of ocular biometric parameters. RESULTS: Axial length, spherical equivalent, and angle kappa showed correlations with temporal ARC[8] (tARC[8]; Pearson's r = 0.653, -0.579, and -0.341; P = 0.001, 0.015, and 0.015, respectively). White-to-white corneal diameter (WTW) and anterior chamber depth (ACD) showed correlation with nasal ARC[8] (nARC[8]; Pearson's r = -0.492 and -0.461; P = 0.015 and 0.023, respectively). The formulae for estimating scleral curvatures (tARC, nARC, and average ARC) were developed as a function of axial length, ACD, WTW, and distance from the axis line, with good determinant power (72 - 80 %; SPSS ver. 22.0). Angle kappa showed strong correlation with axial length (Pearson's r = -0.813, P <0.001), and the different correlation patterns of nasal and temporal ARC with axial length can be explained by the ocular surface deviation represented by angle kappa. CONCLUSIONS: Axial length, ACD, and WTW are useful parameters for estimating the ARC of the anterior sclera, which is important for the haptic design of scleral contact lenses. Angle kappa affects the discrepancies between the nasal and temporal scleral curvature.

Reduced Fertility and Altered Epididymal and Sperm Integrity in Mice Lacking ADAM7.

The mammalian epididymis is a highly convoluted tubule that connects the testis to the vas deferens. Its proper functions in sperm transport, storage, and maturation are essential for male reproduction. One of the genes predominantly expressed in the epididymis is ADAM7 (a disintegrin and metalloprotease 7). Previous studies have shown that ADAM7 synthesized in the epididymis is secreted into the epididymal lumen and is then transferred to sperm membranes, where it forms a chaperone complex that is potentially involved in sperm fertility. In this study, we generated and analyzed mice with a targeted disruption in the Adam7 gene. We found that the fertility of male mice was modestly but significantly reduced by knockout of Adam7. Histological analyses revealed that the cell heights of the epithelium were dramatically decreased in the caput of the epididymis of Adam7-null mice, suggesting a requirement for ADAM7 in maintaining the integrity of the epididymal epithelium. We found that sperm from Adam7-null mice exhibit decreased motility, tail deformation, and altered tyrosine phosphorylation, indicating that the absence of ADAM7 leads to abnormal sperm functions and morphology. Western blot analyses revealed reduced levels of integral membrane protein 2B (ITM2B) and ADAM2 in sperm from Adam7-null mice, suggesting a requirement for ADAM7 in normal expression of sperm membrane proteins involved in sperm functions. Collectively, our study demonstrates for the first time that ADAM7 is required for normal fertility and is important for the maintenance of epididymal integrity and for sperm morphology, motility, and membrane proteins.

Three-dimensional image cytometer based on widefield structured light microscopy and high-speed remote depth scanning.

A high throughput 3D image cytometer have been developed that improves imaging speed by an order of magnitude over current technologies. This imaging speed improvement was realized by combining several key components. First, a depth-resolved image can be rapidly generated using a structured light reconstruction algorithm that requires only two wide field images, one with uniform illumination and the other with structured illumination. Second, depth scanning is implemented using the high speed remote depth scanning. Finally, the large field of view, high NA objective lens and the high pixelation, high frame rate sCMOS camera enable high resolution, high sensitivity imaging of a large cell population. This system can image at 800 cell/sec in 3D at submicron resolution corresponding to imaging 1 million cells in 20 min. The statistical accuracy of this instrument is verified by quantitatively measuring rare cell populations with ratio ranging from 1:1 to 1:10(5) . © 2014 International Society for Advancement of Cytometry.

Enhancement of the perceived image quality of a 2D/3D convertible directional-backlight unit system using a double-slit barrier array and an active diffuser.

In this paper, a method to construct a directional-backlight unit system to separate the left-eye image and the right-eye image by adopting a double-slit barrier array is proposed to enhance the perceived quality of the realized 3D image. Additionally, the two-dimensional/three-dimensional convertible function is also realized by adopting an active diffuser to the spatial interlacing directional-backlight system.

Resolution-enhanced integral imaging in focal mode with a time-multiplexed electrical mask array.

The inferior resolution of the three-dimensional (3D) image is one of the main problems to be resolved for realizing a commercial autosteresosopic 3D display device. In this paper, a time-multiplexing technique using electrically moving masks is proposed to enhance the resolution of the 3D image realized by integral imaging in a focal mode while preserving the viewing angle of it.

Thermomechanical Actuator-Based Three-Axis Optical Scanner for High-Speed Two-Photon Endomicroscope Imaging.

This paper presents the design and characterization of a three-axis thermomechanical actuator-based endoscopic scanner for obtaining ex vivo two-photon images. The scanner consisted of two sub-systems: 1) an optical system (prism, gradient index lens, and optical fiber) that was used to deliver and collect light during imaging and 2) a small-scale silicon electromechanical scanner that could raster scan the focal point of the optics through a specimen. The scanner can be housed within a 7 mm Ø endoscope port and can scan at the speed of 3 kHz × 100 Hz × 30 Hz along three axes throughout a 125 × 125 × 100 µm3 volume. The high-speed thermomechanical actuation was achieved through the use of geometric contouring, pulsing technique, and mechanical frequency multiplication (MFM), where MFM is a new method for increasing the device cycling speed by pairing actuators of unequal forward and returning stroke speeds. Sample cross-sectional images of 15-µm fluorescent beads are presented to demonstrate the resolution and optical cross-sectioning capability of the two-photon imaging system.