Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells.

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

Real-time in vivo imaging of subpopulations of circulating tumor cells using antibody conjugated quantum dots.

INTRODUCTION: The detection of circulating tumor cells (CTCs) is very important for cancer diagnosis. CTCs can travel from primary tumors through the circulation to form secondary tumor colonies via bloodstream extravasation. The number of CTCs has been used as an indicator of cancer progress. However, the population of CTCs is very heterogeneous. It is very challenging to identify CTC subpopulations such as cancer stem cells (CSCs) with high metastatic potential, which are very important for cancer diagnostic management. RESULTS: We report a study of real-time CTC and CSC imaging in the bloodstreams of living animals using multi-photon microscopy and antibody conjugated quantum dots. We have developed a cancer model for noninvasive imaging wherein pancreatic cancer cells expressing fluorescent proteins were subcutaneously injected into the earlobes of mice and then formed solid tumors. When the cancer cells broke away from the solid tumor, CTCs with fluorescent proteins in the bloodstream at different stages of development could be monitored noninvasively in real time. The number of CTCs observed in the blood vessels could be correlated to the tumor size in the first month and reached a maximum value of approximately 100 CTCs/min after 5 weeks of tumor inoculation. To observe CTC subpopulations, conjugated quantum dots were used. It was found that cluster of differentiation (CD)24+ CTCs can move along the blood vessel walls and migrate to peripheral tissues. CD24+ cell accumulation on the solid tumors' sides was observed, which may provide valuable insight for designing new drugs to target cancer subpopulations with high metastatic potential. We also demonstrated that our system is capable of imaging a minor population of cancer stem cells, CD133+ CTCs, which are found in 0.7% of pancreatic cancer cells and 1%-3% of solid tumors in patients. CONCLUSIONS: With the help of quantum dots, CTCs with higher metastatic potential, such as CD24+ and CD133+ CTCs, have been identified in living animals. Using our approach, it may be possible to investigate detailed metastatic mechanism such as tumor cell extravasation to the blood vessels. In addition, the number of observed CTCs in the blood stream could be correlated with tumor stage in the early stage of cancer.

Random and aligned electrospun PLGA nanofibers embedded in microfluidic chips for cancer cell isolation and integration with air foam technology for cell release.

BACKGROUND: Circulating tumor cells (CTCs) comprise the high metastatic potential population of cancer cells in the blood circulation of humans; they have become the established biomarkers for cancer diagnosis, individualized cancer therapy, and cancer development. Technologies for the isolation and recovery of CTCs can be powerful cancer diagnostic tools for liquid biopsies, allowing the identification of malignancies and guiding cancer treatments for precision medicine. METHODS: We have used an electrospinning process to prepare poly(lactic-co-glycolic acid) (PLGA) nanofibrous arrays in random or aligned orientations on glass slips. We then fabricated poly(methyl methacrylate) (PMMA)-based microfluidic chips embedding the PLGA nanofiber arrays and modified their surfaces through sequential coating with using biotin-(PEG)7-amine through EDC/NHS activation, streptavidin (SA), and biotinylated epithelial-cell adhesion-molecule antibody (biotin-anti-EpCAM) to achieve highly efficient CTC capture. When combined with an air foam technology that induced a high shear stress and, thereby, nondestructive release of the captured cells from the PLGA surfaces, the proposed device system operated with a high cell recovery rate. RESULTS: The morphologies and average diameters of the electrospun PLGA nanofibers were characterized using scanning electron microscopy (SEM) and confocal Raman imaging. The surface chemistry of the PLGA nanofibers conjugated with the biotin-(PEG)7-amine was confirmed through time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. The chip system was studied for the effects of the surface modification density of biotin-(PEG)7-amine, the flow rates, and the diameters of the PLGA nanofibers on the capture efficiency of EpCAM-positive HCT116 cells from the spiked liquid samples. To assess their CTC capture efficiencies in whole blood samples, the aligned and random PLGA nanofiber arrays were tested for their abilities to capture HCT116 cells, providing cancer cell capture efficiencies of 66 and 80%, respectively. With the continuous injection of air foam into the microfluidic devices, the cell release efficiency on the aligned PLGA fibers was 74% (recovery rate: 49%), while it was 90% (recovery rate: 73%) on the random PLGA fibers, from tests of 200 spiked cells in 2 mL of whole blood from healthy individuals. Our study suggests that integrated PMMA microfluidic chips embedding random PLGA nanofiber arrays may be suitable devices for the efficient capture and recovery of CTCs from whole blood samples.

Universal wetting transition of an evaporating water droplet on hydrophobic micro- and nano-structures.

Water-repellent, rough surfaces have a remarkable and beneficial wetting property: when a water droplet comes in contact with a small fraction of the solid, both liquid-solid adhesion and hydrodynamic drag are reduced. As a prominent example from nature, the lotus leaf-comprised of a wax-like material with micro- and nano-scaled roughness-has recently inspired numerous syntheses of superhydrophobic substrates. Due to the diverse applications of superhydrophobicity, much research has been devoted to the fabrication and investigations of hydrophobic micro-structures using established micro-fabrication techniques. However, wetting transitions remain relatively little explored. During evaporation, a water droplet undergoes a wetting transition from a (low-frictional) partial to (adhesive) complete contact with the solid, destroying the superhydrophobicity and the self-cleaning properties of the slippery surface. Here, we experimentally examine the wetting transition of a drying droplet on hydrophobic nano-structures, a previously unexplored regime. In addition, using a theoretical analysis we found a universal criterion of this wetting transition that is characterized by a critical contact angle. Different from previous results showing different critical droplet sizes, our results show a universal, geometrically-dependent, critical contact angle, which agrees well with various data for both hydrophobic micro- and nano-structures.

3D bioelectronic interface: capturing circulating tumor cells onto conducting polymer-based micro/nanorod arrays with chemical and topographical control.

The three-dimensional (3D) poly(3,4-ethylenedioxythiophene) (PEDOT)-based bioelectronic interfaces (BEIs) with diverse dimensional micro/nanorod array structures, varied surface chemical pro-perties, high electrical conductivity, reversible chemical redox switching, and high optical transparency are used for capturing circulating tumor cells (CTCs). Such 3D PEDOT-based BEIs can function as an efficient clinical diagonstic and therapeutic platform.

Investigation of size-dependent cell adhesion on nanostructured interfaces.

BACKGROUND: Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diameters. RESULTS: We first checked the viability of cells grown on nanopillars with diameters ranging from 200 nm to 700 nm. It was observed that when cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. We then calculated the average size of the focal adhesions and the cell-spreading area for focal adhesions using confocal microscopy. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200 nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200 nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions. CONCLUSIONS: We have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on the softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate behaviour of cells.

Revealing the nanometric structural changes in myocardial infarction models by time-lapse intravital imaging.

In the development of bioinspired nanomaterials for therapeutic applications, it is very important to validate the design of nanomaterials in the disease models. Therefore, it is desirable to visualize the change of the cells in the diseased site at the nanoscale. Heart diseases often start with structural, morphological, and functional alterations of cardiomyocyte components at the subcellular level. Here, we developed straightforward technique for long-term real-time intravital imaging of contracting hearts without the need of cardiac pacing and complex post processing images to understand the subcellular structural and dynamic changes in the myocardial infarction model. A two-photon microscope synchronized with electrocardiogram signals was used for long-term in vivo imaging of a contracting heart with subcellular resolution. We found that the structural and dynamic behaviors of organelles in cardiomyocytes closely correlated with heart function. In the myocardial infarction model, sarcomere shortening decreased from ∼15% (healthy) to ∼8% (diseased) as a result of impaired cardiac function, whereas the distances between sarcomeres increased by 100 nm (from 2.11 to 2.21 µm) in the diastolic state. In addition, T-tubule system regularity analysis revealed that T-tubule structures that were initially highly organized underwent significant remodeling. Morphological remodeling and changes in dynamic activity at the subcellular level are essential to maintain heart function after infarction in a heart disease model.

Design principles of bioinspired interfaces for biomedical applications in therapeutics and imaging.

In the past two decades, we have witnessed rapid developments in nanotechnology, especially in biomedical applications such as drug delivery, biosensing, and bioimaging. The most commonly used nanomaterials in biomedical applications are nanoparticles, which serve as carriers for various therapeutic and contrast reagents. Since nanomaterials are in direct contact with biological samples, biocompatibility is one of the most important issues for the fabrication and synthesis of nanomaterials for biomedical applications. To achieve specific recognition of biomolecules for targeted delivery and biomolecular sensing, it is common practice to engineer the surfaces of nanomaterials with recognition moieties. This mini-review summarizes different approaches for engineering the interfaces of nanomaterials to improve their biocompatibility and specific recognition properties. We also focus on design strategies that mimic biological systems such as cell membranes of red blood cells, leukocytes, platelets, cancer cells, and bacteria.

Exploring the formation of focal adhesions on patterned surfaces using super-resolution imaging.

The formation of focal adhesions on various sizes of fibronectin patterns, ranging from 200 µm to 250 nm, was systematically investigated by total internal reflection fluorescence microscopy and super-resolution imaging. It was found that cells adhered to and spread on these micro/nanopatterns, forming focal adhesions. On a micrometer scale the shape of the focal adhesions was elongated. However, on the nanometer scale, the shape of focal adhesions became dotlike. To further explore the distribution of focal adhesion proteins formed on surfaces, a localization-based super-resolution imaging technique was employed in order to determine the position and density of vinculin proteins. A characteristic distance of 50 nm was found between vinculin molecules in the focal adhesions, which did not depend on the size of the fibronectin nanopatterns. This distance was found to be crucial for the formation of focal adhesions. In addition, the density of vinculin at the focal adhesions formed on the nanopatterns increased as the pattern size decreased. The density of the protein was found to be 425 ± 247, 584 ± 302, and 703 ± 305 proteins µm(-2) on the 600, 400, and 250 nm fibronectin patterns respectively. Whereas 226 ± 77 proteins µm(-2) was measured for the matured focal adhesions on homogeneous fibronectin coated substrates. The increase in vinculin density implies that an increase in mechanical load was applied to the focal adhesions formed on the smaller nanopatterns.

Targeted nuclear delivery using peptide-coated quantum dots.

Core/shell quantum dots (CdSe/Zns) conjugated with various nuclear localization signaling (NLS) peptides, which could facilitate the transportation of quantum dots across the plasma membrane into the nucleus, have been utilized to investigate the uptake mechanism of targeted delivery. Because of their brightness and photostability, it was possible to trace the trajectories of individual quantum dots in living cells using both confocal and total internal reflection microscopes. We found that, when the quantum dots were added to a cell culture, the peptide-coated quantum dots entered the cell nucleus while the uncoated quantum dots remained in the cytoplasm. At 8 nM, most of the peptide coated quantum dots were found in the cytoplasm due to aggregation. However, at a lower concentration (0.08 nM), approximately 25% of the NLS peptide-coated quantum dots entered the cell nucleus. We also found that some quantum dots without NLS coating could also enter the nucleus, suggesting that the size of the quantum dots may play an important role in such a process.

Galectin-7 downregulation in lesional keratinocytes contributes to enhanced IL-17A signaling and skin pathology in psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration, as well as hyperproliferation of keratinocytes in skin lesions, and is considered a metabolic syndrome. We found that the expression of galectin-7 is reduced in skin lesions of patients with psoriasis. IL-17A and TNF-α, 2 cytokines intimately involved in the development of psoriatic lesions, suppressed galectin-7 expression in human primary keratinocytes (HEKn cells) and the immortalized human keratinocyte cell line HaCaT. A galectin-7 knockdown in these cells elevated the production of IL-6 and IL-8 and enhanced ERK signaling when the cells were stimulated with IL-17A. Galectin-7 attenuated IL-17A-induced production of inflammatory mediators by keratinocytes via the microRNA-146a/ERK pathway. Moreover, galectin-7-deficient mice showed enhanced epidermal hyperplasia and skin inflammation in response to intradermal IL-23 injection. We identified fluvastatin as an inducer of galectin-7 expression by connectivity map analysis, confirmed this effect in keratinocytes, and demonstrated that fluvastatin attenuated IL-6 and IL-8 production induced by IL-17A. Thus, we validate a role of galectin-7 in the pathogenesis of psoriasis, in both epidermal hyperplasia and keratinocyte-mediated inflammatory responses, and formulate a rationale for the use of statins in the treatment of psoriasis.

Immune cell shuttle for precise delivery of nanotherapeutics for heart disease and cancer.

The delivery of therapeutics through the circulatory system is one of the least arduous and less invasive interventions; however, this approach is hampered by low vascular density or permeability. In this study, by exploiting the ability of monocytes to actively penetrate into diseased sites, we designed aptamer-based lipid nanovectors that actively bind onto the surface of monocytes and are released upon reaching the diseased sites. Our method was thoroughly assessed through treating two of the top causes of death in the world, cardiac ischemia-reperfusion injury and pancreatic ductal adenocarcinoma with or without liver metastasis, and showed a significant increase in survival and healing with no toxicity to the liver and kidneys in either case, indicating the success and ubiquity of our platform. We believe that this system provides a new therapeutic method, which can potentially be adapted to treat a myriad of diseases that involve monocyte recruitment in their pathophysiology.

Study of oxygen tension variation within live tumor spheroids using microfluidic devices and multi-photon laser scanning microscopy.

Three-dimensional cell spheroid culture using microfluidic devices provides a convenient in vitro model for studying tumour spheroid structures and internal microenvironments. Recent studies suggest that oxygen deprived zones inside solid tumors are responsible for stimulating local cytokines and endothelial vasculature proliferation during angiogenesis. In this work, we develop an integrated approach combining microfluidic devices and multi-photon laser scanning microscopy (MPLSM) to study variations in oxygen tension within live spheroids of human osteosarcoma cells. Uniform shaped, size-controlled spheroids are grown and then harvested using a polydimethylsiloxane (PDMS) based microfluidic device. Fluorescence live imaging of the harvested spheroids is performed using MPLSM and a commercially available oxygen sensitive dye, Image-iT Red, to observe the oxygen tension variation within the spheroids and those co-cultured with monolayers of human umbilical vein endothelial cells (HUVECs). Oxygen tension variations are observed within the spheroids with diameters ranging from 90 ± 10 µm to 140 ± 10 µm. The fluorescence images show that the low-oxygenated cores diminish when spheroids are co-cultured with HUVEC monolayers for 6 hours to 8 hours. In the experiments, spheroids subjected to HUVEC conditioned medium treatment and with a cell adherent substrate are also measured and analyzed to study their significance on oxygen tension within the spheroids. The results show that the oxygenation within the spheroids is improved when the spheroids are cultured under those conditions. Our work presents an efficient method to study oxygen tension variation within live tumor spheroids under the influence of endothelial cells and conditioned medium. The method can be exploited for further investigation of tumor oxygen microenvironments during angiogenesis.

Integrated 3D conducting polymer-based bioelectronics for capture and release of circulating tumor cells.

Here we develop a novel fabrication approach for producing three-dimensional (3D) conducting polymer-based bioelectronic interfaces (BEIs) that can be integrated on electronic devices for rare circulating tumor cell (CTC) isolation, detection, and collection via an electrically triggered cell released from chips. Based on the chemical oxidative polymerization of carboxylic acid-modified 3,4-ethylenedioxythiophene and modified poly(dimethylsiloxane) (PDMS) transfer printing technology, the high-aspect-ratio structures of poly(3,4-ethylenedioxythiophene) (PEDOT)-based "nanorod" arrays can be fabricated on indium tin oxide (ITO) electrodes when using the Si "microrod" arrays as masters. Furthermore, we integrated the biotinylated poly-(l)-lysine-graft-poly-ethylene-glycol (PLL-g-PEG-biotin) coating with 3D PEDOT-based BEIs for dynamic control of the capture/release performance of CTCs on chips; this combination exhibited an optimal cell-capture yield cells of ∼45 000 cells cm-2 from EpCAM-positive MCF7 while maintaining resistance from the adhesion of EpCAM-negative HeLa cells at a density of ∼4000 cells cm-2. By taking advantage of the electrochemical doping/dedoping properties of PEDOT materials, the captured CTCs can be triggered to be electrically released through the desorption phenomena of the PLL-g-PEG-biotin. More than 90% of the captured cells can be released while maintaining very high cell viability. Therefore, it is conceivable that the use of a 3D PEDOT-based BEI platform will meet the requirements for the development of downstream characterization of CTCs, as well as the next generation of bioelectronics for biomedical applications.

Hybrid contact and interfacial adhesion on well-defined periodic hierarchical pillars.

Herein, we describe a simple fabrication procedure for creating artificial hierarchical micro/nanopillars on silicon substrates that allows an effective, precise control of the interfacial adhesion and surface hydrophobicity. These well-defined hierarchical micro/nanostructures have four possible wetting states: Cassie-Cassie (C-C), Cassie-Wenzel (C-W), Wenzel-Cassie (W-C) and Wenzel-Wenzel (W-W). By controlling the critical height of the micro/nanopillars, it is possible to fabricate hierarchical micro/nanostructures in these four states. Thus, the hierarchical superhydrophobic surfaces proposed and fabricated in this study are promising for mimicking either lotus leaves with low adhesion or rose petals with high adhesion.