An advanced fast method for the evaluation of multiple immunolabelling using gold nanoparticles based on low-energy STEM.

We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.

The new X-ray/visible microscopy MAXWELL technique for fast three-dimensional nanoimaging with isotropic resolution.

Microscopy by Achromatic X-rays With Emission of Laminar Light (MAXWELL) is a new X-ray/visible technique with attractive characteristics including isotropic resolution in all directions, large-volume imaging and high throughput. An ultrathin, laminar X-ray beam produced by a Wolter type I mirror irradiates the sample stimulating the emission of visible light by scintillating nanoparticles, captured by an optical system. Three-dimensional (3D) images are obtained by scanning the specimen with respect to the laminar beam. We implemented and tested the technique with a high-brightness undulator at SPring-8, demonstrating its validity for a variety of specimens. This work was performed under the Synchrotrons for Neuroscience-an Asia-Pacific Strategic Enterprise (SYNAPSE) collaboration.

Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation.

Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.

Multimodal bioimaging using nanodiamond and gold hybrid nanoparticles.

Hybrid core-shell nanodiamond-gold nanoparticles were synthesized and characterized as a novel multifunctional material with tunable and tailored properties for multifunctional biomedical applications. The combination of nanostructured gold and nanodiamond properties afford new options for optical labeling, imaging, sensing, and drug delivery, as well as targeted treatment. ND@Au core-shell nanoparticles composed of nanodiamond (ND) core doped with Si vacancies (SiV) and Au shell were synthesized and characterized in terms of their biomedical applications. Several bioimaging modalities based on the combination of optical and spectroscopic properties of the hybrid nano-systems are demonstrated in cellular and developing zebrafish larvae models. The ND@Au nanoparticles exhibit isolated ND's Raman signal of sp3 bonded carbon, one-photon fluorescence of SiV with strong zero-phonon line at 740 nm, two-photon excited fluorescence of nanogold with short fluorescence lifetime and strong absorption of X-ray irradiation render them possible imaging agent for Raman mapping, Fluorescence imaging, two-photon Fluorescence Lifetime Imaging (TP-FLIM) and high-resolution hard-X-ray microscopy in biosystems. Potential combination of the imaging facilities with other theranostic functionalities is discussed.

High-resolution fast-tomography brain-imaging beamline at the Taiwan Photon Source.

The new Brain Imaging Beamline (BIB) of the Taiwan Photon Source (TPS) has been commissioned and opened to users. The BIB and in particular its endstation are designed to take advantage of bright unmonochromatized synchrotron X-rays and target fast 3D imaging, ∼1 ms exposure time plus very high ∼0.3 µm spatial resolution. A critical step in achieving the planned performances was the solution to the X-ray induced damaging problems of the detection system. High-energy photons were identified as their principal cause and were solved by combining tailored filters/attenuators and a high-energy cut-off mirror. This enabled the tomography acquisition throughput to reach >1 mm3 min-1, a critical performance for large-animal brain mapping and a vital mission of the beamline.

Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia.

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway's enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

Tris DBA ameliorates IgA nephropathy by blunting the activating signal of NLRP3 inflammasome through SIRT1- and SIRT3-mediated autophagy induction.

Tris (dibenzylideneacetone) dipalladium (Tris DBA), a small-molecule palladium complex, can inhibit cell growth and proliferation in pancreatic cancer, lymphocytic leukaemia and multiple myeloma. Given that this compound is particularly active against B-cell malignancies, we have been suggested that it can alleviate immune complexes (ICs)-mediated conditions, especially IgA nephropathy (IgAN). The therapeutic effects of Tris DBA on glomerular cell proliferation and renal inflammation and mechanism of action were examined in a mouse model of IgAN. Treatment of IgAN mice with Tris DBA resulted in markedly improved renal function, albuminuria and renal pathology, including glomerular cell proliferation, neutrophil infiltration, sclerosis and periglomerular inflammation in the renal interstitium, together with (Clin J Am Soc Nephrol. 2011, 6, 1301-1307) reduced mitochondrial ROS generation; (Am J Physiol-Renal Physiol. 2011. 301, F1218-F1230) differentially regulated autophagy and NLRP3 inflammasome; (Clin J Am Soc Nephrol. 2012, 7, 427-436) inhibited phosphorylation of JNK, ERK and p38 MAPK signalling pathways, and priming signal of the NLRP3 inflammasome; and (Free Radic Biol Med. 2013, 61, 285-297) blunted NLRP3 inflammasome activation through SIRT1- and SIRT3-mediated autophagy induction, in renal tissues or cultured macrophages. In conclusion, Tris DBA effectively ameliorated the mouse IgAN model and targeted signalling pathways downstream of ICs-mediated interaction, which is a novel immunomodulatory strategy. Further development of Tris DBA as a therapeutic candidate for IgAN is warranted.

Xenon blunts NF-κB/NLRP3 inflammasome activation and improves acute onset of accelerated and severe lupus nephritis in mice.

Xenon, an inert anesthetic gas, is increasingly recognized to possess desirable properties including cytoprotective and anti-inflammatory effects. Here we evaluated the effects of xenon on the progression of lupus nephritis (LN) in a mouse model. A two hour exposure of either 70% xenon or 70% nitrogen balanced with oxygen was administered daily for five weeks to female NZB/W F1 mice that had been induced to develop accelerated and severe LN. Xenon treatment improved kidney function and renal histology, and decreased the renal expression of neutrophil chemoattractants, thereby attenuating glomerular neutrophil infiltration. The effects of xenon were mediated primarily by deceasing serum levels of anti-double stranded DNA autoantibody, inhibiting reactive oxygen species production, NF-κB/NLRP3 inflammasome activation, ICAM-1 expression, glomerular deposition of IgG and C3 and apoptosis, in the kidney; and enhancing renal hypoxia inducible factor 1-α expression. Proteomic analysis revealed that the treatment with xenon downregulated renal NLRP3 inflammasome-mediated cellular signaling. Similarly, xenon was effective in improving renal pathology and function in a spontaneous LN model in female NZB/W F1 mice. Thus, xenon may have a therapeutic role in treating LN but further studies are warranted to determine applicability to patients.

Tris DBA Ameliorates Accelerated and Severe Lupus Nephritis in Mice by Activating Regulatory T Cells and Autophagy and Inhibiting the NLRP3 Inflammasome.

Tris (dibenzylideneacetone) dipalladium (Tris DBA), a small-molecule palladium complex, has been shown to inhibit cell growth and proliferation in pancreatic cancer, lymphocytic leukemia, and multiple myeloma. In the current study, we examined the therapeutic effects of Tris DBA on glomerular cell proliferation, renal inflammation, and immune cells. Treatment of accelerated and severe lupus nephritis (ASLN) mice with Tris DBA resulted in improved renal function, albuminuria, and pathology, including measurements of glomerular cell proliferation, cellular crescents, neutrophils, fibrinoid necrosis, and tubulointerstitial inflammation in the kidneys as well as scoring for glomerulonephritis activity. The treated ASLN mice also showed significantly decreased glomerular IgG, IgM, and C3 deposits. Furthermore, the compound was able to 1) inhibit bone marrow-derived dendritic cell-mediated T cell functions and reduce serum anti-dsDNA autoantibody levels; 2) differentially regulate autophagy and both the priming and activation signals of the NLRP3 inflammasome; and 3) suppress the phosphorylation of JNK, ERK, and p38 MAPK signaling pathways. Tris DBA improved ASLN in mice through immunoregulation by blunting the MAPK (ERK, JNK)-mediated priming signal of the NLRP3 inflammasome and by regulating the autophagy/NLRP3 inflammasome axis. These results suggest that the pure compound may be a drug candidate for treating the accelerated and deteriorated type of lupus nephritis.

The Making of a Flight Feather: Bio-architectural Principles and Adaptation.

The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.

Gold nano-mesh synthesis by continuous-flow X-ray irradiation.

X-ray irradiation has been extensively used in recent years as a fabrication step for nanoparticles and nanoparticle systems. A variant of this technique, continuous-flow X-ray irradiation, has recently been developed, and offers three important advantages: precise control of the irradiation dose, elimination of convection effects in the precursor solution, and suitability for large-scale production. Here, the use of this method to fabricate Au nano-meshes of interest as transparent and flexible electrodes for optoelectronics is reported. The study includes extensive characterization of the synthesis parameters and of the product properties, with rather encouraging results.

Rapid single-wavelength lightsheet localization microscopy for clarified tissue.

Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 107 µm3) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 µm3 per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.

Lightsheet localization microscopy enables fast, large-scale, and three-dimensional super-resolution imaging.

Recent advances in super-resolution microscopy allow the localization of single molecules within individual cells but not within multiple whole cells due to weak signals from single molecules and slow acquisition process for point accumulation to reconstruct super-resolution images. Here, we report a fast, large-scale, and three-dimensional super-resolution fluorescence microscope based on single-wavelength Bessel lightsheet to selectively illuminate spontaneous blinking fluorophores tagged to the proteins of interest in space. Critical parameters such as labeling density, excitation power, and exposure time were systematically optimized resulting in a maximum imaging speed of 2.7 × 104 µm3 s-1. Fourier ring correlation analysis revealed a reconstructed image with a lateral resolution of ~75 nm through the accumulation of 250 image volumes on immobilized samples within 15 min. Hence, the designed system could open new insights into the discovery of complex biological structures and live 3D localization imaging.

Signaling pathway of globo-series glycosphingolipids and ß1,3-galactosyltransferase V (ß3GalT5) in breast cancer.

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

Smad-Independent BMP Signaling in Somatic Cells Limits the Size of the Germline Stem Cell Pool.

In developing organisms, proper tuning of the number of stem cells within a niche is critical for the maintenance of adult tissues; however, the involved mechanisms remain largely unclear. Here, we demonstrate that Thickveins (Tkv), a type I bone morphogenetic protein (BMP) receptor, acts in the Drosophila developing ovarian soma through a Smad-independent pathway to shape the distribution of BMP signal within the niche, impacting germline stem cell (GSC) recruitment and maintenance. Somatic Tkv promotes Egfr signaling to silence transcription of Dally, which localizes BMP signals on the cell surface. In parallel, Tkv promotes Hh signaling, which promotes escort cell cellular protrusions and upregulates expression of the Drosophila BMP homolog, Dpp, forming a positive feedback loop that enhances Tkv signaling and strengthens the niche boundary. Our results reveal a role for non-canonical BMP signaling in the soma during GSC establishment and generally illustrate how complex, cell-specific BMP signaling mediates niche-stem cell interactions.

Free-electron-laser coherent diffraction images of individual drug-carrying liposome particles in solution.

Using the excellent performances of a SACLA (RIKEN/HARIMA, Japan) X-ray free electron laser (X-FEL), coherent diffraction imaging (CDI) was used to detect individual liposome particles in water, with or without inserted doxorubicin nanorods. This was possible because of the electron density differences between the carrier, the liposome, and the drug. The result is important since liposome nanocarriers at present dominate drug delivery systems. In spite of the low cross-section of the original ingredients, the diffracted intensity of drug-free liposomes was sufficient for spatial reconstruction yielding quantitative structural information. For particles containing doxorubicin, the structural parameters of the nanorods could be extracted from CDI. Furthermore, the measurement of the electron density of the solution enclosed in each liposome provides direct evidence of the incorporation of ammonium sulphate into the nanorods. Overall, ours is an important test for extending the X-FEL analysis of individual nanoparticles to low cross-sectional systems in solution, and also for its potential use to optimize the manufacturing of drug nanocarriers.

Differential synchrotron X-ray imaging markers based on the renal microvasculature for tubulointerstitial lesions and glomerulopathy.

High resolution synchrotron microtomography capable of revealing microvessels in three dimensional (3D) establishes distinct imaging markers of mouse kidney disease strongly associated to renal tubulointerstitial (TI) lesions and glomerulopathy. Two complementary mouse models of chronic kidney disease (CKD), unilateral ureteral obstruction (UUO) and focal segmental glomerulosclerosis (FSGS), were used and five candidates of unique 3D imaging markers were identified. Our characterization to differentially reflect the altered microvasculature of renal TI lesions and/or glomerulopathy demonstrated these image features can be used to differentiate the disease status and the possible cause therefore qualified as image markers. These 3D imaging markers were further correlated with the histopathology and renal microvessel-based molecular study using antibodies against vascular endothelial cells (CD31), the connective tissue growth factor or the vascular endothelial growth factor. We also found that these 3D imaging markers individually characterize the development of renal TI lesions or glomerulopathy, quantitative and integrated use of all of them provide more information for differentiating the two renal conditions. Our findings thus establish a practical strategy to characterize the CKD-associated renal injuries by the microangiography-based 3D imaging and highlight the impact of dysfunctional microvasculature as a whole on the pathogenesis of the renal lesions.

Construction of single fluorophore ratiometric pH sensors using dual-emission Mn(2+)-doped quantum dots.

We present a novel ratiometric pH sensor design using water-soluble, dual-emission, Mn(2+)-doped quantum dots (Qdots) decorated with D-penicillamine (DPA-MnQdots). In contrast to more commonly used ratiometric pH sensors that rely on the coupling of two fluorophores, our design uses only a single emitter, which simplifies ratiometric sensing and broadens the applications of the sensor. Our single-emitter DPA-MnQdots exhibit two emission bands, at 510nm (green) and 610nm (red), which are, respectively, attributable to exciton recombination and emission of the Mn(2+) dopants. The emission intensity ratio (I510/I610) of the DPA-MnQdots depends linearly on surrounding pH values within physiological conditions (from pH 4.5 to 8.5). Moreover, the biocompatible DPA-MnQdots were used for long-term monitoring of local pH values in HeLa cells.

Hinokitiol induces DNA damage and autophagy followed by cell cycle arrest and senescence in gefitinib-resistant lung adenocarcinoma cells.

Despite good initial responses, drug resistance and disease recurrence remain major issues for lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutations taking EGFR-tyrosine kinase inhibitors (TKI). To discover new strategies to overcome this issue, we investigated 40 essential oils from plants indigenous to Taiwan as alternative treatments for a wide range of illnesses. Here, we found that hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, exhibited potent anticancer effects. In this study, we demonstrated that hinokitiol inhibited the proliferation and colony formation ability of lung adenocarcinoma cells as well as the EGFR-TKI-resistant lines PC9-IR and H1975. Transcriptomic analysis and pathway prediction algorithms indicated that the main implicated pathways included DNA damage, autophagy, and cell cycle. Further investigations confirmed that in lung cancer cells, hinokitiol inhibited cell proliferation by inducing the p53-independent DNA damage response, autophagy (not apoptosis), S-phase cell cycle arrest, and senescence. Furthermore, hinokitiol inhibited the growth of xenograft tumors in association with DNA damage and autophagy but exhibited fewer effects on lung stromal fibroblasts. In summary, we demonstrated novel mechanisms by which hinokitiol, an essential oil extract, acted as a promising anticancer agent to overcome EGFR-TKI resistance in lung cancer cells via inducing DNA damage, autophagy, cell cycle arrest, and senescence in vitro and in vivo.

Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-κB-mediated COX-2 expression and apoptosis.

Inflammatory reactions and oxidative stress are implicated in the pathogenesis of focal segmental glomerulosclerosis (FSGS), a common chronic kidney disease with relatively poor prognosis and unsatisfactory treatment regimens. Previously, we showed that osthole, a coumarin compound isolated from the seeds of Cnidium monnieri, can inhibit reactive oxygen species generation, NF-κB activation, and cyclooxygenase-2 expression in lipopolysaccharide-activated macrophages. In this study, we further evaluated its renoprotective effect in a mouse model of accelerated FSGS (acFSGS), featuring early development of proteinuria, followed by impaired renal function, glomerular epithelial cell hyperplasia lesions (a sensitive sign that precedes the development of glomerular sclerosis), periglomerular inflammation, and glomerular hyalinosis/sclerosis. The results show that osthole significantly prevented the development of the acFSGS model in the treated group of mice. The mechanisms involved in the renoprotective effects of osthole on the acFSGS model were mainly a result of an activated Nrf2-mediated antioxidant pathway in the early stage (proteinuria and ischemic collapse of the glomeruli) of acFSGS, followed by a decrease in: (1) NF-κB activation and COX-2 expression as well as PGE2 production, (2) podocyte injury, and (3) apoptosis. Our data support that targeting the Nrf2 antioxidant pathway may justify osthole being established as a candidate renoprotective compound for FSGS.