Increased Multiplexity in Optical Tissue Clearing-Based Three-Dimensional Immunofluorescence Microscopy of the Tumor Microenvironment by Light-Emitting Diode Photobleaching.

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy is transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only 3 or 4 cellular and noncellular TME components can be localized in cleared tumor tissue. Here we report a light-emitting diode (LED) photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through 3 work cycles, we produced 8-plex image data from individual 400 µm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.

CX3CR1+ Macrophage Facilitates the Resolution of Allergic Lung Inflammation via Interacting CCL26.

Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.

Multiresolution 3D Optical Mapping of Immune Cell Infiltrates in Mouse Asthmatic Lung.

Asthma is a chronic inflammatory airway disease driven by various infiltrating immune cell types into the lung. Optical microscopy has been used to study immune infiltrates in asthmatic lungs. Confocal laser scanning microscopy (CLSM) identifies the phenotypes and locations of individual immune cells in lung tissue sections by employing high-magnification objectives and multiplex immunofluorescence staining. In contrast, light-sheet fluorescence microscopy (LSFM) can visualize the macroscopic and mesoscopic architecture of whole-mount lung tissues in three dimensions (3D) by adopting an optical tissue-clearing method. Despite each microscopy method producing image data with unique resolution from a tissue sample, CLSM and LSFM have not been applied together because of different tissue-preparation procedures. Here, we introduce a new approach combining LSFM and CLSM into a sequential imaging pipeline. We built a new optical tissue clearing workflow in which the immersion clearing agent can be switched from an organic solvent to an aqueous sugar solution for sequential 3D LSFM and CLSM of mouse lungs. This sequential combination microscopy offered quantitative 3D spatial analyses of the distribution of immune infiltrates in the same mouse asthmatic lung tissue at the organ, tissue, and cell levels. These results show that our method facilitates multiresolution 3D fluorescence microscopy as a new imaging approach providing comprehensive spatial information for a better understanding of inflammatory lung diseases.

Massively parallel delivery of large cargo into mammalian cells with light pulses.

We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.

Correlative multiscale 3D imaging of mouse primary and metastatic tumors by sequential light sheet and confocal fluorescence microscopy.

Three-dimensional (3D) optical microscopy, combined with advanced tissue clearing, permits in situ interrogation of the tumor microenvironment (TME) in large volumetric tumors for preclinical cancer research. Light sheet (also known as ultramicroscopy) and confocal fluorescence microscopy are often used to achieve macroscopic and microscopic 3D images of optically cleared tumor tissues, respectively. Although each technique offers distinct fields of view (FOVs) and spatial resolution, the combination of these two optical microscopy techniques to obtain correlative multiscale 3D images from the same tumor tissues has not yet been explored. To establish correlative multiscale 3D optical microscopy, we developed a method for optically marking defined regions of interest (ROIs) within a cleared mouse tumor by employing a UV light-activated visible dye and Z-axis position-selective UV irradiation in a light sheet microscope system. By integrating this method with subsequent tissue processing, including physical ROI marking, reversal of tissue clearing, tissue macrosectioning, and multiplex immunofluorescence, we established a workflow that enables the tracking and 3D imaging of ROIs within tumor tissues through sequential light sheet and confocal fluorescence microscopy. This approach allowed for quantitative 3D spatial analysis of the immune response in the TME of a mouse mammary tumor following cancer immunotherapy at multiple spatial scales. The workflow also facilitated the direct localization of a metastatic lesion within a whole mouse brain. These results demonstrate that our ROI tracking method and its associated workflow offer a novel approach for correlative multiscale 3D optical microscopy, with the potential to provide new insights into tumor heterogeneity, metastasis, and response to therapy at various spatial levels.

Antibacterial nanostructured composite films for biomedical applications: microstructural characteristics, biocompatibility, and antibacterial mechanisms.

Hydrogenated Cu-incorporated diamond-like carbon (a-C:H/Cu) films were prepared in the present study using a radio-frequency plasma magnetron sputtering system at various CH4/Ar gas ratios. The a-C:H/Cu films were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, transmission electron microscopy, nano-indentation and a contact angle goniometer. The antibacterial properties and cell cytotoxicity of a-C:H/Cu films were evaluated as per JIS Z2801:2010 and ISO 10993-5 specifications, respectively. The analytical results revealed that the production of a-C:H/Cu films varied with the CH4/Ar ratio, and the phase transformation (amorphous-like → nano-polycrystalline structure) was induced by Cu doping/ion bombardment and radical reactions. Moreover, it was found that the microhardness of the a-C:H/Cu films decreased with increasing Ar fraction in the gas ratio. The a-C:H/Cu films exhibited a high hydrophobic surface feature. The film which contained 77.3 ± 4.4 at.% Cu did not influence cell adhesion and proliferation behaviors. Antibacterial tests also demonstrated that a-C:H/Cu films possessed excellent antibacterial properties. Therefore, a-C:H/Cu films could be developed as promising antibacterial coatings for biomedical applications.

Temporal variation of the relationships between rice yield and climate variables since 1925.

Background: Long-term time-series datasets of crop yield and climate variables are necessary to study the temporal variation of climate effects on crops. The aim of this study was to broadly assess assessment of the effects of climate on rice, and the associated temporal variations of the effects during the long-term period. Methods: We conducted field experiments in Taiwan from 1925 to 2019 to collect and analyze rice yield data and evaluate the impacts of changes in average temperature, diurnal temperature range (DTR), rainfall, and sunshine duration on rice yield during cool and warm cropping seasons. We then estimated the relationships between annual grain yield and the climate variables using the time series of their first difference values. We also computed the total relative and annual actual yield changes using regression coefficients for each climate variable for the intervals 1925-1944, 1945-1983, and 1996-2019 to reveal the impacts of climate change on yields and the associated temporal variations during the overall experimental period. Results: The annual daily average temperature calculated from the trend of the regression lines increased by 0.94-1.03 °C during the 95-year period. The maximum temperature remained steady while the minimum temperature increased, leading to decreased DTR. The total annual rainfall decreased by 237-352 mm and the annual total sunshine duration decreased by 93.9-238.9 h during the experimental period. We observed that during the cool cropping season, yield response to temperature change decreased, while that to DTR and rainfall changes increased. During the warm cropping season, all the yield responses to temperature, DTR, and rainfall changes were negative throughout the experimental period. In recent years (1996-2019) the estimated annual actual rice yield changes during the cool cropping season were negatively affected by climate variables (except for sunshine duration), and slightly positively affected (except for temperature) during the warm cropping season. Compared to the effects of temperature and DTR, those of rainfall and sunshine duration on rice yield changes were weak. This study contributes to provide impacts of climate change on rice yield and associated long-term temporal variations over nearly a century.

Effect of climate change-induced water-deficit stress on long-term rice yield.

The water requirements of crops should be investigated to improve the efficiency of water use in irrigated agriculture. The main objective of the study was to assess the effects of water deficit stress on rice yields throughout the major cropping seasons. We analyzed rice yield data from field experiments in Taiwan over the period 1925-2019 to evaluate the effects of water-deficit stress on the yield of 12 rice cultivars. Weather data, including air temperatures, humidity, wind speed, sunshine duration, and rainfall were used to compute the temporal trends of reference evapotranspiration and crop water status (CWS) during rice growth stages. A negative CWS value indicates that the crop is water deficient, and a smaller value represents a lower water level (greater water-deficit stress) in crop growth. The CWS on rice growth under the initial, crop development, reproductive, and maturity stages declined by 96.9, 58.9, 24.7, and 198.6 mm in the cool cropping season and declined by 63.7, 18.1, 8.6, and 3.8 mm in the warm cropping season during the 95 years. The decreasing trends in the CWSs were used to represent the increases in water-deficit stress. The total yield change related to water-deficit stress on the cultivars from 1925-1944, 1945-1983, and 1996-2019 under the initial, crop development, reproductive, and maturity stages are -56.1 to 37.0, -77.5 to -12.3, 11.2 to 19.8, and -146.4 to 39.1 kg ha-1 in the cool cropping season and -16.5 to 8.2, -12.9 to 8.1, -2.3 to 9.0, and -9.3 to 8.0 in the warm cropping season, respectively. Our results suggest that CWS may be a determining factor for rice to thrive during the developmental stage, but not the reproductive stage. In addition, the effect of water-deficit stress has increasingly affected the growth of rice in recent years.

Increased multiplexity in optical tissue clearing-based 3D immunofluorescence microscopy of the tumor microenvironment by LED photobleaching.

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy have been transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only three or four cellular and non-cellular TME components can be localized in a cleared tumor tissue. Here we report a LED photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through three work cycles, we produced 8-plex image data from individual 400 µm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.

Controlling the lodging risk of rice based on a plant height dynamic model.

BACKGROUND: Rice is a key global food crop. Rice lodging causes a reduction in plant height and crop yield, and rice is prone to lodging in the late growth stage because of panicle initiation. We used two water irrigation modes and four fertilizer application intervals to investigate the relationship between lodging and various cultivation conditions over 2 years. RESULTS: Plant height data were collected and combined with aerial images, revealing that rice lodging was closely related to the nitrogen fertilizer content. The aerial images demonstrated that lodging mainly occurred in the fields treated with a high-nitrogen fertilizer, and analysis of variance revealed that plant height was signifi-cantly affected by nitrogen fertilizer. These results demonstrated that rice plant height in the booting stage was significantly positively correlated with the lodging results (r = 0.67) and nega-tively correlated with yield (r = - 0.46). If the rice plant height in the booting stage exceeded 70.7 cm and nitrogen fertilizer was continuously applied, according to the predicted growing curve of plant height, the plant would be at risk of lodging. Results showed more rainfall accumulated in the later stage of rice growth accompanied by strong instantaneous gusts, the risk of lodging in-creased. CONCLUSION: The results provide predictions that can be applied in intelligent production and lodging risk management, and they form the basis of cultivation management and response policies for each growth period.

Difference in the regulation of IL-8 expression induced by uropathogenic E. coli between two kinds of urinary tract epithelial cells.

Bacterial adherence to epithelial cells is a key virulence trait of pathogenic bacteria. The type 1 fimbriae and the P-fimbriae of uropathogenic Escherichia coli (UPEC) have both been described to be important for the establishment of urinary tract infections (UTI). To explore the interactions between the host and bacterium responsible for the different environments of UPEC invasion, we examined the effect of pH and osmolarity on UPEC strain J96 fimbrial expression, and subsequent J96-induced interleukin-8 (IL-8) expression in different uroepithelial cells. The J96 strain grown in high pH with low osmolarity condition was favorable for the expression of type 1 fimbriae; whereas J96 grown in low pH with high osmolarity condition was beneficial for P fimbriae expression. Type 1 fimbriated J96 specifically invaded bladder 5637 epithelial cells and induced IL-8 expression. On the contrary, P fimbriated J96 invaded renal 786-O epithelial cells and induced IL-8 expression effectively. Type 1 fimbriated J96-induced IL-8 induction involved the p38, as well as ERK, JNK pathways, which leads to AP-1-mediated gene expression. P fimbriated J96-induced augmentation of IL-8 expression mainly involved p38-mediated AP-1 and NF-kappaB transcriptional activation. These results indicate that different expression of fimbriae in J96 trigger differential IL-8 gene regulation pathways in different uroepithelial cells.

Intracellular Delivery by Shape Anisotropic Magnetic Particle-Induced Cell Membrane Cuts.

Introducing functional macromolecules into a variety of living cells is challenging but important for biology research and cell-based therapies. We report a novel cell delivery platform based on rotating shape anisotropic magnetic particles (SAMPs), which make very small cuts on cell membranes for macromolecule delivery with high efficiency and high survivability. SAMP delivery is performed by placing commercially available nickel powder onto cells grown in standard cell culture dishes. Application of a uniform magnetic field causes the magnetic particles to rotate because of mechanical torques induced by shape anisotropic magnetization. Cells touching these rotating particles are nicked, which generates transient membrane pores that enable the delivery of macromolecules into the cytosol of cells. Calcein dye, 3 and 40 kDa dextran polymers, a green fluorescence protein (GFP) plasmid, siRNA, and an enzyme (ß-lactamase) were successfully delivered into HeLa cells, primary normal human dermal fibroblasts (NHDFs), and mouse cortical neurons that can be difficult to transfect. The SAMP approach offers several advantages, including easy implementation, low cost, high throughput, and efficient delivery of a broad range of macromolecules. Collectively, SAMP delivery has great potential for a broad range of academic and industrial applications.

Direct Nuclear Delivery of DNA by Photothermal Nanoblade.

We demonstrate direct nuclear delivery of DNA into live mammalian cells using the photothermal nanoblade. Pulsed laser-triggered cavitation bubbles on a titanium-coated micropipette tip punctured both cellular plasma and nuclear membranes, which was followed by pressure-controlled delivery of DNA into the nucleus. High-level and efficient plasmid expression in different cell types with maintained cell viability was achieved.

Electrical impedance monitoring of photothermal porated mammalian cells.

To transfer large cargo into mammalian cells, we recently provided a new approach called a photothermal nanoblade. Micron-sized membrane pores generated by the nanoblade are surprisingly well repaired with little cell death, suggesting rapid membrane-resealing dynamics. Here, we report the resealing time of photothermal porated mammalian cell plasma membranes using an electrical impedance sensor. Cell membrane pores were generated by high-speed cavitation bubbles induced by laser pulsing of metallic microdisks on a pair of transparent indium tin oxide electrodes. Electrical responses from the sensor electrodes were obtained with a signal voltage of 500 mV and a frequency at 500 kHz. Real-time impedance measurements show that membrane resealing and impedance recovery take a surprisingly long 1 to 2 min after laser pulsing. A nonrecovering impedance shift is also detected for cells after high-energy laser pulsing. This impedance response is also confirmed by a separate experiment in which thin-film gold electrodes are used to trigger cavitation bubbles for opening transient membrane pores on cells cultured on electrodes. Overall, our study platform provides new insight for micron-sized membrane defect repair dynamics to maintain cell viability.

Microfluidic integrated optoelectronic tweezers for single-cell preparation and analysis.

We report a novel microfluidic integrated optoelectronic tweezers (OET) platform for single-cell sample preparation and analysis. Integration of OET and microfluidics is achieved by embedding single-wall carbon nanotube (SWNT) electrodes into multilayer PDMS structures. This integrated platform allows users to selectively pick up individual cells from a population with light beams based on their optical signatures such as size, shape, and fluorescence, and transport them into isolated chambers using light induced dielectrophoretic forces. Isolated cells can be encapsulated into nanoliter liquid plugs and transported out of the platform for downstream molecule analysis using standard commercial instruments.

Canna indica L. attenuates high-glucose- and lipopolysaccharide-induced inflammatory mediators in monocyte/macrophage.

ETHNOPHARMACOLOGICAL RELEVANCE: Canna indica L. (CI) has been widely used as a folklore medicine in tropical and subtropical areas with beneficial effects in numerous diseases, including infection, rheumatism, hepatitis, and it has also been identified as an antioxidant. MATERIALS AND METHODS: The present study aimed to investigate the effect of Canna indica CI ethanolic extract (CIE) on productions of nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-1ß (IL-1ß) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. In addition, the effects of CIE in high glucose (HG)-induced U937 monocytes on mRNA expressions of IL-8 and monocyte chemoattractant protein-1 (MCP-1), and regulation of mitogen-activated protein kinase (MAPK) pathways were also identified. RESULTS: CIE was found to inhibit the production of inflammatory mediators including NO, IL-1ß, and PGE2 from LPS-induced RAW 264.7 macrophages. The increases in HG-induced mRNA expressions of IL-8 and MCP-1 were also significantly inhibited by CIE. Stimulation of HG in U937 monocytes resulted in activation of p38 MAPK, ERK1/2, and JNK. However, CIE treatment significantly decreased phosphorylation of p38 MAPK, ERK1/2, and JNK. CONCLUSION: The present study demonstrated that CIE suppressed the LPS-induced inflammatory mediator production and also inhibited HG-induced inflammatory mediator expression by the regulation of MAPK pathway.

Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes.

INTRODUCTION: Synovial macrophages, which can release proinflammatory factors, are responsible for the upregulation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). In addition, shear stress exerts multifunctional effects on chondrocytes by inducing the synthesis of catabolic or anabolic genes. However, the interplay of macrophages, chondrocytes, and shear stress during the regulation of cartilage function remains poorly understood. We investigated the mechanisms underlying the modulation of human chondrocyte urokinase plasminogen activator (uPA) expression by macrophages and shear stress. METHODS: Human chondrocytes were stimulated by peripheral blood-macrophage- conditioned medium (PB-MCM), or exposure of chondrocytes cultured in PB-MCM to different levels of shear stress (2 to 20 dyn/cm2). Real-time polymerase chain reaction was used to analyze uPA gene expression. Inhibitors and small interfering RNA were used to investigate the mechanism for the effects of PB-MCM and shear stress in chondrocytes. RESULTS: Stimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that activation of the JNK and Akt pathways and NF-κB are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1ß in PB-MCM is the major mediator of uPA expression in chondrocytes. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK and Akt phosphorylation, NF-κB activation, and uPA expression. The PB-MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression. CONCLUSIONS: These data support the hypothesis that uPA upregulation stimulated by macrophages may play an active role in the onset of OA and in the shear-stress protection against this induction.

A gold-nanoparticle-enhanced immune sensor based on fiber optic interferometry.

A method using gold nanoparticles (GNPs) to enhance fiber optic interferometry (GNPFOI) for immune-sensing is reported in this paper. It is suggested that an enlarged index mismatch and an elongated optical path by GNPs conjugated on recognition proteins will contribute most to signal enhancement in the interference fringe shift. Theoretical and experimental results show that the interference fringe shift is linearly related to both the amount and size of the GNPs binding on the sensor surface. The detected signal for 30 nm GNPs can reach a lowest detection limit of 18 pM (10(10) particles ml(-1)). Immune-sensing for rabbit IgG as the antigen to anti-rabbit IgG has been demonstrated and a detection cycle has been completed by elution buffer for surface regeneration. The repeatability of the immune-sensing on one GNPFOI sensor has also been verified by three identical cycles, and the detection limit for 13 nm GNPs conjugated anti-rabbit IgG reaches 0.17 nM (∼25.5 ng ml(-1)). The sensory mechanism has the potential to be engineered on the tip of a needle-type micro-device, which would allow it to monitor immune recognition signals in the future.